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A novel stilbene-like compound that inhibits melanoma growth by regulating melanocyte differentiation and proliferation
Accepted Manuscript A novel stilbene-like compound that inhibits melanoma growth by regulating melanocyte differentiation and proliferation
Noah A. Stueven, Nicholas M. Schlaeger, Aaron P. Monte, ShengPing L. Hwang, Cheng-chen Huang PII: DOI: Reference:
S0041-008X(17)30416-7 doi:10.1016/j.taap.2017.10.008 YTAAP 14072
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
23 March 2017 12 October 2017 13 October 2017
Please cite this article as: Noah A. Stueven, Nicholas M. Schlaeger, Aaron P. Monte, Sheng-Ping L. Hwang, Cheng-chen Huang , A novel stilbene-like compound that inhibits melanoma growth by regulating melanocyte differentiation and proliferation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2017), doi:10.1016/j.taap.2017.10.008
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ACCEPTED MANUSCRIPT A Novel Stilbene-like Compound That Inhibits Melanoma Growth by Regulating Melanocyte Differentiation and Proliferation Noah A. Stueven1 , Nicholas M. Schlaeger1 , Aaron P. Monte2 , Sheng-Ping L. Hwang3 , and Cheng-chen Huang1¶ Biology Department, University of Wisconsin-River Falls, River Falls, WI 54022
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Department of Chemistry and Biochemistry, University of Wisconsin-La Crosse, La Crosse, WI
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Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan 115
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To whom correspondence should be addressed.
Biology Department
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Cheng-chen Huang, Ph. D.
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University of Wisconsin- River Falls
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410 South Third Street River Falls, WI 54022 Phone: 715-425-4276 Fax:
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email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Melanoma is the most aggressive form of skin cancer. Current challenges to melanoma therapy include the adverse effects from immunobiologics, resistance to drugs targeting the MAPK pathway, intricate interaction of many signal pathways, and cancer
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heterogeneity. Thus combinational therapy with drugs targeting multiple signaling
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pathways becomes a new promising therapy. Here, we report a family of stilbene-like
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compounds called A11 that can inhibit melanoma growth in both melanoma-forming zebrafish embryos and mouse melanoma cells. The growth inhibition by A11 is a result of
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mitosis reduction but not apoptosis enhancement. Meanwhile, A11 activates both MAPK
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and Akt signaling pathways. Many A11-treated mouse melanoma cells exhibit morphological changes and resemble normal melanocytes. Furthermore, we found that
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A11 causes down-regulation of melanocyte differentiation genes, including Pax3 and MITF.
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Together, our results suggest that A11 could be a new melanoma therapeutic agent by
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inhibiting melanocyte differentiation and proliferation.
Key words: A11, melanoma, melanocyte differentiation, MAPK, Akt, mitf
Running title: Novel Therapy for Melanoma
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ACCEPTED MANUSCRIPT Introduction Melanoma accounts for the highest mortality rate among skin cancers. One of the reasons is that melanomas are highly metastatic. Melanomas in the early stages, Stage I and II, are local and appear like a mole. However, they can quickly turn into Stage III, which show local metastasis in
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nearby lymph nodes, or turn into Stage IV, which is classified with distant metastasis to the lungs,
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brain, liver, and other organs. While many treatment options are available for non-metastatic
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melanoma, the prognosis for metastatic melanomas have been extremely poor and is still one of the major challenges in cancer therapy. According to the 2009 AJCC (American Joint Committee
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on Cancer) Melanoma Staging Database, the 5-yr survival rate is less than 10% for stage IV
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distant metastatic melanoma (Balch et al., 2009; Dickson and Gershenwald, 2011).
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Melanomas are derived from melanocytes that have become cancerous, typically due to
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mutations. The common mutations found in melanoma include mutations in the BRAF, NRAS, and CKIT genes (Vennepureddy et al., 2016). In fact, approximately 70% of malignant
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melanomas have a valine600 to glutamic acid (V600E) mutation in the BRAF gene. These
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mutations consequently caused constitutively activation of the MAPK signaling pathway leading to the activation of the transcription factor MITF which promotes the proliferation and survival
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of melanocytes (Levy et al., 2006). The discovery of the MAPK pathway in melanoma opens a new direction for melanoma therapy. Several drugs have been developed to target the molecules in the MAPK pathway, including vemurafenib and dabrafenib, inhibitors of BRAF, and trametinib, an inhibitor of MEK. All of them showed high tumor response rates and striking overall survival rates (Vennepureddy et al., 2016). Unfortunately, in addition to the many adverse effects including causing cutaneous squamous cell carcinoma, drug resistance was
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ACCEPTED MANUSCRIPT developed toward these targeted therapy drugs in many patients. One plausible hypothesis for the drug resistance with supporting evidence is that other signaling pathway(s) become activated to compensate the inactivation of MAPK (Tang et al., 2016).
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Immunotherapy, although not specific to melanoma, is another approach that has shown
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significant effectiveness. Ipilimumab, which was approved by FDA in 2011 for the treatment of
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metastatic melanoma, is an antibody that can enhance the function of our immune system by blocking the immune checkpoints. Nivolumab is another FDA approved biologic that has similar
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mechanism as ipilimumab. These drugs showed substantial benefits but tend to cause immune-
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related adverse events (IrAEs) in many organs, including skin, gastrointestinal system, liver, and endocrine systems. Recently, therapies combining drugs tackling different molecules have shown
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potential to reduce adverse effects while generating synergistic therapeutic effects
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(Vennepureddy et al., 2016). New drugs targeting other signaling pathway, such as the wnt
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pathway, are also being developed (Prasad et al., 2015).
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Another cause to drug resistance comes from the intratumoral heterogeneity. Cancer stem cells have been identified in many cancers and are shown to be responsible for chemotherapy
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resistance primarily due to their low proliferation rate (Dean et al., 2005). In melanomas, some cells have the BRAF V600E single mutation but some have BRAF V600E and NRAS double mutations (Nazarian et a., 2010). Melanoma cells also show a wide range of MITF expression levels which seems to correlate with their proliferative and metastatic ability (Carriera et al., 2006; Hoek et al., 2006; Hoek and Goding, 2010). The discovery of melanoma subpopulations suggests the challenges as well as new ideas for melanoma therapy. More detail about molecular
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ACCEPTED MANUSCRIPT heterogeneity of melanomas and therapeutic directions could be found in the review article by Somasundaram et al., 2012.
In this study, we report the identification and characterization of a novel compound named A11
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that can inhibit melanoma growth. This inhibition of A11 is seen on melanoma forming zebrafish
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embryos as well as in the mouse melanoma cell line B16F10. A11 decreased mitotic index but
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did not cause apoptosis in melanoma cells. Molecularly, while a MEK inhibitor (MEK-I) used in our study exhibited expected inhibition of melanoma growth and MAPK activation, which is
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consistent with the current melanoma therapy, A11 surprisingly inhibited melanoma growth by
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elevating MAPK activity. We also found that A11 caused lower expression of melanocyte differentiation genes, including Pax3 and MITF. Furthermore, when compared with the
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morphology of B16F10, A11-treated B16F10 and primary melanocytes, A11-treated melanoma
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cells underwent dramatic morphological changes so that they resembled normal melanocytes. These results suggest that A11 might down regulate melanocyte differentiation genes and turn
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the highly proliferative cancerous melanocytes into less proliferative normal melanocytes. A11
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could be a novel therapeutic agent for melanoma therapy.
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ACCEPTED MANUSCRIPT Material and Methods Zebrafish Husbandry and in vitro Fertilization The zebrafish stocks used in this study are maintained following standard procedures (Westerfield, 2000) and bred by in vitro fertilization. In brief, mature male and female zebrafish
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were separated by a mesh in a breeding tank the night before breeding. Soon after the light is
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turned on the next morning, fish were anesthetized in ~0.16% tricaine for 1-2 min. The females
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were placed on a paper towel to dry the body surface briefly before being transferred into a 6 cm dish. The eggs were expelled onto the dish by gently pressing the lateral and ventral side of the
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belly with fingers. The males were also dried briefly and then placed ventral side up on a sponge
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well. Under a dissecting microscope, the lateral sides of the belly were pressed gently with a pair of blunt forceps while holding a capillary tube close to the anus to collect sperm. The sperm were
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immediately mixed with the eggs and ~1 ml of egg water was added (more details in Westerfield,
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2000). The melanoma zebrafish was obtained from crosses between the Et(kita:GalTA4,UAS:mCherry)hzm1 (Distel et al., 2009) and Tg(UAS:eGFP-HRAS_GV12)io006
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(Anelli et al., 2009) transgenic fish (Santoriello et al., 2010), provided by Dr. Maria Mione at
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Karlsruhe Institute for Technology, Karlsruhe, Germany.
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Chemical Treatment of Zebrafish Embryos Zebrafish embryos were collected and arrayed with 5 embryos per well of a 96-well plate in 200 μl of egg water (distilled water containing 60 μg/ml sea salt from Coralife, USA) which were later replaced with the same volume of egg water containing desired concentration of compounds.
Cell culture
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ACCEPTED MANUSCRIPT The mouse melanoma cell line B16F10 (ATCC CRL-6475) was purchased from ATCC (American Tissue Culture Center) and maintained in DMEM supplemented with 10% fetal bovine serum. The human primary epidermal melanocytes (PCS-200-013) was also purchased from ATCC and maintained in Dermal Cell Basal Medium (PCS-200-030) supplemented with
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the Adult Melanocyte Growth Kit (PCS-200-042). Trypsinization and subculture were performed
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following the protocols found on ATCC website. For chemical treatment, approximately 14,000
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melanoma cells and 30,000 normal melanocytes were seeded in each well of the 24-well plate with 0.5 ml of culture medium. Cells were set up triplicate and allowed to grow two days for
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melanoma or three days for normal melanocytes before chemical treatment. After 24 hours of
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chemical incubation, cells were first washed with PBS once and trypsinized. Equal volume of cell suspension and trypan blue was mixed in an eppendorf tube before the cells were counted
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with a hemacytometer.
Immunohistochemistry
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Zebrafish embryos were fixed in 4% paraformaldehyde at least overnight in 4 o C. The fixed
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embryos were washed twice with PBS, then once with H2 O, 5 min/each, and permeabilized with -20 o C acetone for 7 minutes followed by washes in H2 O and then PBS. The embryos were
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incubated with 3% BSA in PBST (PBS with 0.1% tween 20) for at least 1 hour at room temperature before overnight incubation in 4 o C with the phosphor-Histone H3 (Ser10) Rabbit mAb-Alexa Fluor 555 conjugate in PBST (Cell Signaling Technology). The next day, the embryos were washed with PBST at least 4 times, 15 min/each, then stored in ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology). Apoptotic cells were detected by TUNEL assay using the TMR In Situ Cell Death Detection Kit from Roche. For the
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ACCEPTED MANUSCRIPT immunostaining on cultured cells, we grew around 100 cells in each well of the Teflon-coated slides (HTC Super-cured, 4-well, 10mm from Cell-Line/Thermo Fisher), and the cells grow for two days before chemical treatment. After 24 hours of chemical incubation, cells were briefly washed with PBS then fixed with 4% PFA followed by blocking and antibody incubation. The
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stained embryos or cultured cells were analyzed using a fluorescence compound microscope.
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test was performed to determine the statistical significance.
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The number of positively stained melanocytes was manually counted and graphed. Student’s T-
Western blot
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Cells were prepared in a 6-well plate. To harvest the proteins, we first added 400 l of lysis buffer (0.2% NP-40, 100mM Tris, 150 mM NaCl, 8mM of EDTA, pH 7.4) with protease
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inhibitor cocktail added freshly (P8340, Sigma) to the well and scrapped off the cells using cell
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scrapper. The cells then were transferred to a microcentrifuge tube. The cells were lysed for 30
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min in 4C on a shaker. The lysate was then centrifuged at 12,000 rpm, 4C, for 20 min. After the centrifugation, protein supernatant was transferred to a clean tube and stored in -80C. Protein
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concentration was determined using the Pierce BCA Assay Kit (23227, Thermo Scientific). A brief summary of the assay and a set of sample data were available in Supplemental Figure 1. 10-
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20 g of proteins were loaded into 12% SDS-PAGE gel (Biorad) for gel electrophoresis and later for western blot using the Pierce Fast Western Blot Kit (35050, Thermo Scientific). Antibodies were purchased from Cell Signaling Technology. The anti-MITF antibody (MITF D5G7V rabbit antibody) recognizes all the MITF isoforms and thus detected the total MITF proteins.
Reverse transcription-qPCR
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ACCEPTED MANUSCRIPT Twenty embryos from each treatment were subjected to RNA extraction with Trizol following the commercial instruction (Invitrogen, Carlsbad, CA). RNA concentration and quality was determined using the Genesys 10S UV-VIS spectrophotometer (Thermo Scientific, USA). Up to 5 μg of total RNA were then used to generate the cDNA library using the Superscript III kit
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(Invitrogen, Carlsbad, CA). Quantitative PCR (RT-qPCR) was set up using the Power
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SYBRGreen PCR kit (Biorad, Foster City, CA) and run on the Mx300P QPCR thermocycler
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(Agilent Technologies, Santa Clara, CA). Data was analyzed and graphed using the MxPro QPCR software provided by the manufacturer to calculate the mRNA quantity of the genes of
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interest using the -actin gene as a normalizer. Results of experimental groups were then
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compared to the H2 O treated group which was set to 1 as the baseline. Primers used for RTqPCR are:
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GAPDH L, 5’ TGCACCACCAACTGCTTAGC 3’
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GAPDH R, 5’ TCTTCTGGGTGGCAGTGATG 3’ Pax3 L, 5’ TCGGCCTTGCGTCATTTC 3’
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Pax3 R, 5’ CAGGATCTTAGAGACGCAACCA 3’
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MITF L, 5’ TGCCTTGTTTATGGTGCCTTCT 3’ MITF R, 5’ TCCCTCTACTTTCTGTAATTCCAATTC 3’
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tyrosinase L, 5’ ACGACCTCTTTGTATGGATGCA 3’ tyrosinase R, 5’ TTTCAGAGCCCCCAAGCA 3’ DCT L, 5’ CCGGCCCCGACTGTAATC 3’ DCT R, 5’ GGGCAGTCAGGGAATGGATAT 3’
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ACCEPTED MANUSCRIPT Results A11 inhibits melanoma formation in transgenic melanoma fish embryo We reported that A11 could inhibit the pigment formation in wild type zebrafish embryo (Fig. 1A; Huang et al., 2013; Martinson et al., in preparation). Here we first tested whether A11 had a
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similar effect on the melanoma forming fish, kita-eGFP-HRAS_GV12 (Santoriello et al., 2010).
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These melanoma zebrafish were created by crossing two transgenic lines,
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Et(kita:GalTA4,UAS:mCherry)hzm1 (Distel et al., 2009) and Tg(UAS:eGFP-HRAS_GV12)io006 (Anelli et al., 2009). The resulting embryos exhibited hyperpigmentation as early as 3 days post
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fertilization due to the overexpression of HRAS and consequently overproliferation of
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melanocytes. As in wild type embryos, A11 caused nearly 100% pigment inhibition in the melanoma forming embryos incubated from 24 to 48 hours post fertilization (hpf) (Figure 1B).
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When the treated embryos were withdrawn from the compounds at 48 hpf and allowed to
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develop in water until 72 hpf (see Materials and Methods for details), pigment recovery occurred quickly and completely for MEK-I and PTU but slower in A11-treated melanoma embryos
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(Figure 1B). A closer examination on the pigment recovering melanoma embryos revealed that
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A11 seemed to cause fewer melanocytes (Figure 1C). We went on to examine the mitosis using the anti-phosphorylated histone H3 antibody, which is a known mitosis marker (Hendzel et al.,
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1997) and cell death using TUNEL assay in A11-treated embryos. The results showed that A11 significantly decreased the number of mitotic melanocytes in the melanoma forming embryos. A11, however, does not increase the number of apoptotic melanocytes (Figure 2). MEK-I, an inhibitor of MEK kinase used as a comparison, also caused significant reduction of melanocyte proliferation but had no effect on cell death.
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ACCEPTED MANUSCRIPT A11 inhibits cell proliferation in B16F10 mouse melanoma cell To test whether A11 also inhibited the proliferation of mammalian melanoma cells, we treated the B16F10 mouse melanoma cells with A11. B16F10 mouse melanoma cells are highly pigmented and proliferative and have been used for in many areas of melanoma and melanocyte
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studies. This experiment started with seeding the same number of melanoma cells in 24-well
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plates with 500 l of complete DMEM medium containing A11 or other compounds. After 24
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hours of incubation, cells were trypsinized, mixed with trypan blue for viability check, and
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counted. The results showed that A11 at 40 M indeed caused near 50% less cells than the control (Figure 3A). As expected, MEK-I showed inhibition in melanoma proliferation.
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Surprisingly, kojic acid, a known tyrosinase enzyme inhibitor commonly used for depigmentation (or skin-lightening), also inhibited melanoma cell proliferation, albeit to a much
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less degree than A11 and MEK-I. To test whether A11 inhibited melanoma proliferation, we
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examined the number of mitotic cells in compound-treated B16F10 cells using anti-
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phosphorylated histone H3 antibody (Figure 3B). The results showed that while control B16F10 cells had about 10 mitotic cells out of 500 cells, A11-treated melanoma cells only showed 2
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mitotic cells (Figure 3C). In conclusion, A11 reduces melanoma proliferation. In addition,
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several lines of evidence suggest that A11 does not cause melanoma cell death. First, A11 does not cause any observed cell toxicity as seen in hydroquinone treated cells. Hydroquinone is a tyrosinase inhibitor that produces strong depigmentation and has been used as a reference standard for depigmentation evaluation (Brenner and Hearing, 2008). Once in the cells, hydroquinone also produces toxic quiones and reactive oxygen species which can cause cell death (Briganti et al., 2003;). In our experiments, hydroquinone at 50 M caused near all the cells to die and float in the medium within less than 12 hours (Figure 3D). In contrast, the A11-
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ACCEPTED MANUSCRIPT treated melanoma cells appeared healthy, strongly adhered to the culture dish, and with only a few cells floating in the wells (also see Figure 6). Second, by observations, trypan blue staining showed indistinguishable level of cell death between A11-treated and control cells.
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A11 activates MAPK and Akt pathways in B16F10 melanoma
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The MAPK signaling pathway is known to activate the MITF transcription factor and promote
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melanoma proliferation and survival (Wellbrock and Arozarena, 2015). We examined the MAPK activity in A11-treated B16F10 cells. In this experiment, we chose to use sub-dosage of each
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compound, 5 M of each, and set up a combined treatment to see if there is any additive or
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synergistic relationship between A11 and MEK-I. The treated cells were counted after the treatment then lysed for western blot analysis. At 5 M, MEK-I alone did not seem to inhibit
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melanoma proliferation while A11 showed slight inhibition. MEK-I and A11 combined
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treatments clearly showed more enhanced inhibition than A11 alone (Figure 4A). On the western
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blot, we found that MEK-I clearly showed the expected inhibition on MAPK but were surprised to find that A11 actually strongly activated MAPK (Figure 4B). In the cells treated with MEK-I
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and A11 combined, the MAPK activity is completely inhibited, indicating A11’s function in
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activating MAPK is upstream of MEK-I. We then examined the Akt signaling pathway which is also known to promote melanocyte proliferation and survival (Wu et al, 2003; Davies, 2012). We found that Akt was activated both by A11 and MEK-I. However, the activation of Akt was approximately the same between the single and combined treatments. These results suggested a novel and complex mechanism of A11 in inhibiting melanoma growth.
A11 suppresses the expression of melanoma differentiation genes
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ACCEPTED MANUSCRIPT B16F10 cell line is composed of heterogeneous melanocytes, including differentiated melanocytes and melanoma stem cells (Somasundaram et al., 2012). It is also found that the expression level of MITF gene correlates with the differentiation status and proliferation ability of melanocytes, with high level of MITF expression in the terminally differentiated and low
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proliferative B16F10 cells, medium level of MITF in highly proliferative but less differentiated,
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and low MITF in melanoma stem cells (Hoek and Goding, 2010). To understand how A11
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inhibits melanoma proliferation, we examined the MITF expression level in A11-treated B16F10 cells by western blot and RT-qPCR. Interestingly, we found that MITF protein level is
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slightly lower in the A11-treated melanoma cells than that in the DMSO control (Figure 5A).
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The result of decreased MITF protein level by A11, albeit subtle, is supported by the RT-qPCR which showed decreased MITF gene expression (Figure 5B). We went on to examine the
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upstream genes of melanocyte differentiation Pax3, which is a known transcriptional activator of
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MITF, and found that A11 also reduces Pax3 expression. These results could explain the decrease of melanocyte proliferation by A11 and lead us to hypothesize that the genes involved
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in melanin synthesis which are typically expressed in differentiated melanocytes would also
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show reduction by A11. As expected, the expression of tyrosinase gene which is the key enzyme in the melanin synthesis was also decreased by A11 (Figure 5B). The expression of DCT
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(dopachrome tautomerase) gene, another enzyme in melanin synthesis pathway, showed slightly, but not statistical significant, decrease by A11. All these results suggest that A11 inhibits melanocyte proliferation by regulating melanocyte differentiation. Interestingly, MEK-I seemed to induce high level of MITF expression evident by the western blot and RT-qPCR results (Figure 5A, B). This result is consistent with the finding that overexpression of MITF in murine BRAF-transformed melanocytes inhibits proliferation (Wellbrock and Marais, 2005).
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A11 on normal human melanocytes To test whether A11 has any effect on normal melanocyte, we obtained a primary culture of human melanocytes derived from human foreskin. We found that A11 also inhibited the
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proliferation in normal melanocytes even though the inhibition was not as strong as in the mouse
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melanoma cells (Figure 6A). MEK-I, however, did not seem to inhibit the proliferation of normal
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melanocyte. When we compared the morphology of normal melanocytes, control B16F10 melanoma, and A11-treated B16F10, we noticed that A11 caused dramatic morphological
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changes in the B16F10 melanoma cells. Normal melanocytes have thinner and longer cell bodies
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with several long cellular processes but melanoma cells are more rounded with shorter cellular process. Also, B16F10 are more pigmented than normal melanocytes. In A11-treated B16F10
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melanoma cells, we found that approximately 50% of the cells developed thinner cell bodies
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with long cellular processes, a phenotype that is intermediate between melanoma and normal melanocytes (Figure 6B). This morphology change of melanoma by A11 seems to suggest that
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A11 might turn melanoma cells back to less proliferative quasi-normal melanocytes.
A11 analogs show different degrees of melanoma inhibition
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Finally, we synthesized nearly fifty A11 analogs and tested their inhibition on melanoma growth. While some analogs seemed to lose the inhibition ability, such as SK0392 and CL-4, several A11 analogs could still inhibit melanoma growth, albeit less potent than A11 (Figure 7A). FSK0392 (fluorinated SK0392), however, showed strong toxicity to melanoma cells killing all the cells at 25 M in less than 24 hours of treatment. In addition, A4, but not all other analogs, caused the same morphological change as A11, suggesting these analogs retain some of the A11 molecular
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ACCEPTED MANUSCRIPT functions but lose others. Furthermore, western blot results showed MAPK activity was significantly higher in the A11 analog-treated cells than in the control. Also, these analogs did show different degrees of MAPK activation which seemed to correlate with the growth inhibition potency, i.e. the higher pMAPK the stronger melanoma growth inhibition, as A9 showing lower
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MAPK activation and little growth inhibition than A4, FSK0392, and SK0516 (Figure 7B).
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ACCEPTED MANUSCRIPT Discussion A11 could be a new therapeutic agent for melanoma While the major current melanoma drugs either try to enhance the immune function (immunotherapy) or inhibit the MAPK pathway (targeted therapy), our studies suggest a novel
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mechanism of A11 in inhibiting melanoma growth. Unlike the BRAF inhibitor, such as
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vemurafenib, or MEK inhibitor, such as trametinib, A11 does not inhibit MAPK. Instead, A11
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(and its analogs) greatly elevated MAPK activity. However, when the elevation in MAPK activity associated with A11 treatment was prevented by the simultaneous administration of
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MEK-I, melanoma cell proliferation was still significantly reduced. This result strongly suggests
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that A11 must influence other cell signaling pathway(s) to inhibit melanoma growth. Since drug resistance is commonly seen in patients receiving BRAF or MEK inhibitors, and more and more
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evidence suggests the involvement or activation of other pathways to support the growth of
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melanoma (Prasad et al., 2015), A11 could be a new melanoma drug for combinational therapies. Wnt signaling was found to be activated in 30% of melanomas (Rimm et al., 1999; Prasad et al.,
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2015). It will be interesting to see if A11 suppresses the Wnt signaling pathway. Furthermore,
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our results might shed light on the role of Akt in melanoma development and therapies. The PI3Akt signaling was believed to promote melanoma survival, proliferation and migration (Davies,
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2012) but our result clearly showed that both A11 and MEK-I activate Akt and inhibit melanoma
Skin-lightening and melanocyte growth inhibition by A11 In another manuscript by our group (Martinson et al., in prep), we showed that A11 reduced pigment in both zebrafish and mammalian cells and could be a potent and safe skin-lightening
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ACCEPTED MANUSCRIPT agent for cosmetic industry. After finding that A11 did not inhibit tyrosinase enzyme activity like other skin-lightening compound, we discovered that A11 actually inhibited melanocyte mitosis. Here we further confirmed the proliferation inhibition of A11 using both the melanoma forming zebrafish embryos and the mouse melanoma cell culture B16F10, and normal human
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melanocytes. Our result also showed that A11 only inhibited the proliferation and did not seem
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to enhance apoptosis. Through our other studies, we detected little or no toxicity of A11 (Huang
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et al., 2013; Martinson et al., in prep), suggesting that A11 might be a safe compound for both skin-lightening and melanoma therapy at the same time. So far, there is no other compound that
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clearly demonstrates this dual function except for the study by Inoue et al., 2013. In that study,
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the authors showed that hydroquinone, but not arbutin which is a natural derivative of hydroquinone, caused down-regulation of genes involved in melanocyte differentiation using
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mouse embryonic stem cells. As hydroquinone is known to be very toxic to cells, the down-
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regulation of gene expression by hydroquinone may not be a true differentiation regulation.
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A11 regulates melanocyte differentiation
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The slow pigment recovery by A11 in both normal and melanoma zebrafish embryos raised interesting questions to us. One early hypothesis was maybe it was due to cell death caused by
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A11 which was soon ruled out by the TUNEL result (Figure 2). Meanwhile, the reduced number of DCT expressing cells in A11-treated wild type embryos from our previous study (Martinson et al., in prep) suggests that A11 might inhibit melanocyte differentiation. In this study, we further explored this hypothesis using melanoma cells. Consistent with the fish results, we found A11treated melanoma cells have lower expression of the melanocyte differentiation genes, including Pax3 and MITF, and consequently lower expression of the melanogenesis genes, such as
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ACCEPTED MANUSCRIPT tyrosinase. Although many studies showed that activation of MAPK leads to upregulation of MITF and melanocyte differentiation and melanogenesis. There are reports of MAPK activation leading to down-regulation of MITF and melanogenesis (Kim et al., 2008; Nishio et al., 2016). The differentiation regulation by A11 can explain almost all the phenotypes we saw on zebrafish,
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including reducing existing pigment, slow pigment recovery, and a decrease of dct expressing
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cells. However, it is still not clear at which level of differentiation A11 does regulate.
This melanocyte differentiation regulation by A11 is further supported, albeit indirectly, by the
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observation of morphological changes in A11-treated B16F10 cells. As described in the result,
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we found that A11 seemed to change the more rounded, less dendritic melanoma cells into stretched and more dendritic morphology of normal melanocytes. It will be very interesting to
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perform the cancer colony forming and metastasis assays to see if the A11-treated melanomas
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actually are losing cancer properties. If these hypotheses turned out to be true, A11 will open a new avenue of cancer therapy to turn cancer cells back to normal cells. But then many questions
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will emerge regarding the therapeutic efficacy and adverse effects of A11 for melanoma. Is the
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differentiation inhibition by A11 reversible, i.e. will the A11-treated cells return to cancer cells after A11 withdrawal? How long will the inhibition last? Would A11 turn cancer melanoma into
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less differentiated melanoblasts or even melanocyte stem cells causing chemotherapy resistance? These are certainly interesting questions awaits further research.
MITF protein level and melanoma proliferation The MITF rheostat model describes the correlation of MITF expression level and melanoma proliferation (Hoek and Goding, 2010). More specifically, melanoma stem cells express low
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ACCEPTED MANUSCRIPT level of MITF and stay at the G1 phase of cell cycle but maintain invasive potential. Medium level of MITF generates either proliferating cells or differentiated pigment-producing cells. More importantly, these melanoma cells are able to switch phenotypes by regulating the expression of MITF. The phenotype switching behaviors of melanoma cells, which depend on the molecular
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signals, pose great challenges to melanoma therapy. If A11 can inhibit MITF expression and
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melanocyte differentiation, it might limit the heterogeneity of melanoma cells and increase the
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therapeutic efficacy. On the other hand, our result that showed increased MITF protein level and Pax3 gene expression by MEK-I might suggest the increase of melanoma heterogeneity by
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MEK-I and offer an explanation to why there is high resistance to drugs targeting MAPK
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signaling pathway. However, as melanoma stem cells express low level of MITF, it raises a question whether A11 might increase melanoma stem cell population by inhibiting MITF
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expression and consequently causing more therapeutic challenges. It will be interesting to know
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Potential pathway for A11
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whether melanoma stem cell population is increased upon A11 treatment.
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To search for other potential cell signaling pathway that might be regulated by A11, we utilized the SEA (Similarity Ensemble Approach) computer software (Keiser et al., 2007) available at
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www.sea.bkslab.org. The software predicts the aryl hydrocarbon receptor (AhR) to be a potential binding molecule for A11. AhR is a nuclear receptor that once activated can function as a transcription factor to activate numbers of metabolism-related enzymes, such as cytochrome P450 and many others in the toxic and carcinogenic responses (review by Mulero-Navarro and Fernandez-Salguero, 2016). Many studies have shown that AhR inhibits cell proliferation and survival. Studies also showed that AhR may regulate several growth factor signaling pathways,
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ACCEPTED MANUSCRIPT including IGF (insulin growth factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), and KGF (keratinocyte growth factor) to inhibit or induce cell proliferation depending on the cell types (Yin et al., 2016). More interestingly, AhR was recently found to interact with Wnt and BMP signaling pathways to regulate stem cell differentiation and regeneration (Wang et al.,
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proliferation by inhibiting the AhR signaling pathway.
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ACCEPTED MANUSCRIPT Acknowledgements This project is supported by a Faulty Research Grant (PRJ82VD) to C.-C.H. and a Summer Scholar Fellowship to N.S. both from University of Wisconsin-River Falls and a WiSys Innovation Grant (133-4-061600-AAB5817) from the WiSys Technology Foundation. Thanks
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Dr. Michael J. Martin for his helpful comments and suggestions. The
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Et(kita:GalTA4,UAS:mCherry)hzm1 and Tg(UAS:eGFP-H-RAS_GV12)io006 transgenic fish
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lines were generated by Maria Mione, Karlsruhe Institute for Technology (KIT), maintained by European Zebrafish Resource Center of the Karlsruhe Institute of Technology and
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obtained from the Taiwan Zebrafish Core Facility at Academia Sinica.
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References Anelli, V., Santoriello, C., Distel, M., Koster, R.W., Ciccarelli, F.D., and Mione, M. (2009). Global repression of cancer gene expression in a zebrafish model of melanoma is linked to epigenetic regulation. Zebrafish 6, 417-424. Balch, C.M. et al., (2009). Final version of 2009 AJCC melanoma staging and classification. J Clin. Oncol. 27, 6199-6206. Brenner, M. and Hearing V.J. (2008). Modifying skin pigmentation—approaches through intrinsic biochemistry and exogenous agents. Drug Discovery Today: Disease Mechanisms 5, e189-e199. Briganti, S., Camera, E., and Picardo, M. (2003). Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Res. 16, 101-110. Carreira, S., Goodall, J., Denat, L., Rodriguez, M., Nuciforo, P., Hoek, K.S., Testori, A., Larue, L., and Goding, C.R., 2006. Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes and Development 20, 3426-3439. Davies, M.A. (2012). The role of the PI3K-Akt pathway in melanoma. Cancer J. 18, 142-147. Dean, M., Fojo, T., and Bates, S. (2005). Tumour stem cells and drug resistance. Nat Rev Cancers 5, 275-284. Dickson, P.V. and Gershenwald, J.E. (2011). Staging and prognosis of cutaneous melanoma. Surg. Oncol. Clin. N Am. 20, 1-17. Distel, M., Wullimann, M.F., and Koster, R.W. (2009). Optimized Gal4 geneticsfor permanent gene expression mapping in zebrafish. Proc. Natl. Acad. Sci. USA 105, 1255-1260. Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., BazettJones, D.P., and Allis, C.D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348-60. Hoek, K.S., Schlegel, N.C., Brafford, P., Sucker, A., Ugurel, S., Kumar, R., Weber, B.L., Nathanson, K.L., Phillips, D.J., Herlyn, M., Schadendorf, D., and Dummer, R (2006). Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature. Pigment Cell Res. 19, 290-302. Hoek, K.S. and Goding, C.R., (2010). Cancer stem cells versus phenotype-switching in melanoma. Pigment Cell Melanoma Res. 23, 746-759. Huang, C-C., Monte, A., Cook, J.M., Kabir, M.S., and Peterson, K.P. (2013). Zebrafish heart failure models for the evaluation of chemical probes and drugs. Assay and Drug Dev. 11, 561-572. Inoue, Y., Hasegawa, S., Yamada, T., Date, Y., Mizutani, H., Nakata, S., Matsunaga, K., and Akamatsu, H., (2013). Analysis of the effects of hydroquinone and arbutin on the differentiation of melanocytes. Biol. Pharm. Bull. 36, 1722-1730. Keiser MJ, Roth BL, Armbruster BN, Ernsberger P, Irwin JJ, Shoichet BK. Relating protein pharmacology by ligand chemistry. Nat. Biotech. 25, 197-206 (2007). Kim, J.H., Baek, S.H., Kim, D.H., Choi, T.Y., Yoon, T.J., Hwang, J.S., Kim, M.R., Kwon, H.J, and Lee, C.H. (2008). Down regulation of melanin synthesis by haginin A and its application to in vivo lightening model. J Invest. Dermatol. 128, 1227-1235. Levy, C., Khaled, M., and Fisher D.E. (2006). Mitf: master regulator of melanocyte development and melanoma oncogene. Trends Mol. Med. 12, 406-414.
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Martinson, K., Stueven, N., Monte, A., and Huang, C.-C. (2016). A11 part I: novel stilbene-like compounds that reduce melanin through inhibiting melanocyte differentiation and proliferation without inhibiting tyrosinase. Xx,xxxx-xxxx. Mulero-Navarro, S. and Fernandez-Salguero, P.M. (2016). New trends in aryl hydrocarbon receptor biology. Front. Cell Dev. Biol. 4, 45. Nishio, T., Usami, M., Awaji, M., Shinohara, S., and Sato, K. (2016). Dual effects of acetylsalicylic acid on ERK signaling and mitf transcription lead to inhibition of melanogenesis. Mol. Cell. Biochem. 412, 101-110. Prasad, C.P., Mohapatra, P., and Anderson, T. (2015). Therapy for BRAFi-resistant melanomas: is wnt5a the answer? Cancers 7, 1900-1924. Rimm, D.L., Caca, K., Hu, G., Harrison, F.B., Fearon, E.R. (1999). Frequent nuclear/cytoplasmic localization of b-catenin without exon 3 mutations in malignant melanoma. Am. J. Pathol. 154, 325-329. Santoriello, C., Gennaro, E., Anelli, V., Distel, M., Kelly, A., Koster, R.W., Hurlstone, A., and Mione, M. (2010). Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish. PLoS ONE 5, e15170. Singh, K.P., Bennett, J.A., Casado, F.L., Walrath, J.L., Welle, S.L., and Gasiewicz, T.A. (2014). Loss of aryl hydrocarbon receptor promotes gene changes associated with premature hematopoietic stem cell exhaustion and development of a myeloproliferative disorder in aging mice. Stem Cells Dev. 23, 95-106. Somasundaram, R., Villanueva, J., and Herlyn, M. (2012). Intratumoral heterogeneity as a therapy resistance mechanism: role of melanoma subpopulations. Adv. Pharmacol. 65, 335-359. Tang, T., Eldabaje, R., and Yang L. (2016). Current status of biological therapies for the treatment of metastatic melanoma. Anticancer Res. 36, 3229-3242. Vennepureddy, A., Thumallapally, N., Nehru, V.M., Atallah, J.-P., and Terjanian, T. (2016). Novel drugs and combination therapies for the treatment of metastatic melanoma. J Clin. Med. Res. 8, 63-75. Wang, Q., Kurita, H., Carreira, V., Ko, C.I., Fan, Y., Zhang, X., Biesiada, J., Medvedovic, M., and Puga, A. (2016). Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions. Toxicol. Sci. 149, 346-357. Wellbrock, C. and Arozarena, I. (2015). Microphthalmia-associated transcription factor in melanoma development and MAPK-kinase pathway targeted therapy. Pigment Cell Res. 28, 390-406. Wellbrock, C. and Marais, R. (2005). Elevated expression of MITF counteracts B-RAFstimulated melanocyte and melanoma cells proliferation. J. Cell Biol. 170, 703-708. Westerfield, M. (2007). The zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). Eugene: University of Oregon Press. White, R.M. and Zon, L.I, (2008). Melanocytes in development, regeneration, and cancer. Cell Stem Cell 3, 242-252. Wu, H., Goel, V., and Haluska, F.G. (2003). PTEN signaling pathways in melanoma. Oncogene 22, 3113-3122.
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ACCEPTED MANUSCRIPT Figure 1. Suppression of melanoma formation and recovery by A11. (A) Wild type zebrafish embryos in H2 O or 10 uM of A11 compound from 24-72 hpf. A11 causes significant reduction of pigment but no other morphological defect. (B, left) The melanoma forming zebrafish embryos, kita-eGFP-HRAS_GV12, were treated with skin-
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lightening compounds from 24 to 48 hpf. Strong pigment inhibition was apparent by the tested
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compounds, A11 or MEK-I both at 10 M or PTU at 0.0003% throughout the study unless
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specified. (B, right) The treated embryos then were washed with egg water twice and allowed to
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develop in egg water until 72 hpf. Melanocytes recovered quickly in MEK-I-, H2 O-, and PTUtreated embryos, but not in those treated with A11. These experiments were done with twenty
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embryos for each treatment group and repeated three times. The phenotypic penetrance for each group was near 100%. (C) A closer examination of the mid-trunk regions revealed that the
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number of recovered melanocytes in A11-treated melanoma fish embryo is significantly lower
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than that in control (H2 O). In comparison with wild type embryo, the A11-treated embryo
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seemed to recover the normal level of melanocytes.
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ACCEPTED MANUSCRIPT Figure 2. Mitotic index is lower in the A11-treated melanoma forming zebrafish embryos. (A) Immunostaining using anti-phosphorylated histone H3 antibody labeled the cells at metaphase of mitosis (green fluorescence). Mitotic melanocytes (Arrow) could be readily identified under fluorescence microscope. (B) Quantitative data showed that A11- and MEK-I-
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treated melanoma forming zebrafish embryos have significant fewer mitotic melanocytes (n=5).
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* P<0.05, ** P<0.01 by Student’s T-test. (C) TUNEL-labeled apoptotic melanocyte in zebrafish
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indistinguishable level of melanocyte apoptosis (n=5).
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embryo (arrow). (D) A11- or MEK-I treated melanoma forming embryos showed
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ACCEPTED MANUSCRIPT Figure 3. A11 inhibits mouse melanoma cell growth. (A) Same number of mouse melanoma cells B16F10 were grown in 24-well culture plate for two days before they were treated with different compounds. After 24 hours of treatment, cells were trypsinized and counted. A11 and MEK-I (20 M) treated cells showed significantly lower
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proliferation. * P<0.05, ** P<0.01 by Student’s T-test. (B) Immunostaining using anti-
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phosphorylated histone H3 to label mitotic cells (red). The cells were co-labeled with DAPI to
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mark the nuclei in blue and phalloidin to mark the f-actin in green. (C) Quantitative data showed
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that significant lower number of mitotic cells in 40 M A11- and 10 M MEK-I-treated melanoma cells. FSK0392 which is an analog of A11 also showed proliferation inhibition at 5
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M. Error bars represent standard deviation. * P<0.05 by Student’s T-test. (D) Melanoma cells treated with A11 appeared healthy and adhered to dish well whereas hydroquinone caused cells
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ACCEPTED MANUSCRIPT Figure 4. A11 activates MAPK and Akt in mouse melanoma cells. (A) The proliferation of B16F10 melanoma cells was not affected by 5 M MEK-1 but significantly decreased by A11 or A11 and MEK-I combined. * P<0.05, ** P<0.01 by Student’s T-test.
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(B) Western blotting for the proteins from B16F10 cells treated with different compounds using
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anti-phosphorylated Akt (pAkt), total Akt, total MAPK, phosphorylated MAPK (pMAPK), or -
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actin (as an internal control). As expected, MEK-I inhibited MAPK phosphorylation completely.
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A11 caused high level of pMAPK. Interestingly, both MEK-I and A11 showed high level of pAkt. When cells were treated both MEK-I and A11, MEK-I abolished the MAPK activation by
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ACCEPTED MANUSCRIPT Figure 5. A11 inhibits melanoma differentiation. (A) Western blot showed slightly lower level of MITF proteins in A11-treated melanoma cells. MEK-I however, increased MITF protein in melanoma. (B) RT-qPCR revealed that A11 caused lower expression of two melanocyte differentiation genes, MITF and Pax3, as well as a
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differentiation gene, tyrosinase. The DCT expression in A11- treated cells, however, was slightly
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lower, but not statistically different from the control. MEK-I had no effect on Pax3 expression
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tyrosinase gene expression. * P<0.05, by Student’s T-test.
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but induced high level of MITF gene expression. PTU also caused reduction of DCT and
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ACCEPTED MANUSCRIPT Figure 6. A11 also inhibits the proliferation in normal human melanocytes. (A) Same number of normal human melanocytes were seeded in 24-well plate and allowed to grow for three days before treated with different compounds. After 24 hours of chemical treatment, the cells were trypsinized and counted. A11 caused significant reduction of cell
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number but MEK-I and PTU did not. * P<0.05 by Student’s T-test. (B) Normal human
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melanocytes developed long processes while B16F10 mouse melanoma cells were more rounded
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and pigmented. A11-treated B16F10 melanoma cells exhibited intermediate phenotypes.
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ACCEPTED MANUSCRIPT Figure 7. A11 analogs also inhibit melanoma growth corresponding with high level of pMAPK. (A) Representative data of melanoma inhibition by A11 analogs. Some compounds were tested at several concentrations, for example A11 were tested at 2, 10, 20, 40 M. While most analogs
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showed slightly lower inhibition potency than A11, SK0392, FSK0392 (fluorinated SK0392) at 5
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M), and CL4 at 40 M did not seem to inhibit melanoma growth at all. FSK0392, however,
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caused near 100% cell death at 25 M. * P<0.05 by Student’s T-test. (B) Western blot revealed
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significantly high level of pMAPK (Top row) by several A11 analogs, including A4, FSK0392, and SK0516 but only slightly by A9 which showed less melanoma growth inhibition. The
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ACCEPTED MANUSCRIPT Highlights of ‘A Novel Stilbene-like Compound That Inhibits Melanoma Growth by Regulating Melanocyte Differentiation and Proliferation’ by Stueven et al., This project identified a stilbene-like compound named A11 for melanoma treatment.
A11 shows different molecular mechanisms than current melanoma drugs.
A11 could be a good candidate for melanoma combinational treatment.
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Noah A. Stueven, Nicholas M. Schlaeger, Aaron P. Monte, ShengPing L. Hwang, Cheng-chen Huang PII: DOI: Reference:
S0041-008X(17)30416-7 doi:10.1016/j.taap.2017.10.008 YTAAP 14072
To appear in:
Toxicology and Applied Pharmacology
Received date: Revised date: Accepted date:
23 March 2017 12 October 2017 13 October 2017
Please cite this article as: Noah A. Stueven, Nicholas M. Schlaeger, Aaron P. Monte, Sheng-Ping L. Hwang, Cheng-chen Huang , A novel stilbene-like compound that inhibits melanoma growth by regulating melanocyte differentiation and proliferation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2017), doi:10.1016/j.taap.2017.10.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A Novel Stilbene-like Compound That Inhibits Melanoma Growth by Regulating Melanocyte Differentiation and Proliferation Noah A. Stueven1 , Nicholas M. Schlaeger1 , Aaron P. Monte2 , Sheng-Ping L. Hwang3 , and Cheng-chen Huang1¶ Biology Department, University of Wisconsin-River Falls, River Falls, WI 54022
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Department of Chemistry and Biochemistry, University of Wisconsin-La Crosse, La Crosse, WI
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Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan 115
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To whom correspondence should be addressed.
Biology Department
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Cheng-chen Huang, Ph. D.
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University of Wisconsin- River Falls
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410 South Third Street River Falls, WI 54022 Phone: 715-425-4276 Fax:
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email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Melanoma is the most aggressive form of skin cancer. Current challenges to melanoma therapy include the adverse effects from immunobiologics, resistance to drugs targeting the MAPK pathway, intricate interaction of many signal pathways, and cancer
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heterogeneity. Thus combinational therapy with drugs targeting multiple signaling
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pathways becomes a new promising therapy. Here, we report a family of stilbene-like
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compounds called A11 that can inhibit melanoma growth in both melanoma-forming zebrafish embryos and mouse melanoma cells. The growth inhibition by A11 is a result of
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mitosis reduction but not apoptosis enhancement. Meanwhile, A11 activates both MAPK
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and Akt signaling pathways. Many A11-treated mouse melanoma cells exhibit morphological changes and resemble normal melanocytes. Furthermore, we found that
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A11 causes down-regulation of melanocyte differentiation genes, including Pax3 and MITF.
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Together, our results suggest that A11 could be a new melanoma therapeutic agent by
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inhibiting melanocyte differentiation and proliferation.
Key words: A11, melanoma, melanocyte differentiation, MAPK, Akt, mitf
Running title: Novel Therapy for Melanoma
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ACCEPTED MANUSCRIPT Introduction Melanoma accounts for the highest mortality rate among skin cancers. One of the reasons is that melanomas are highly metastatic. Melanomas in the early stages, Stage I and II, are local and appear like a mole. However, they can quickly turn into Stage III, which show local metastasis in
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nearby lymph nodes, or turn into Stage IV, which is classified with distant metastasis to the lungs,
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brain, liver, and other organs. While many treatment options are available for non-metastatic
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melanoma, the prognosis for metastatic melanomas have been extremely poor and is still one of the major challenges in cancer therapy. According to the 2009 AJCC (American Joint Committee
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on Cancer) Melanoma Staging Database, the 5-yr survival rate is less than 10% for stage IV
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distant metastatic melanoma (Balch et al., 2009; Dickson and Gershenwald, 2011).
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Melanomas are derived from melanocytes that have become cancerous, typically due to
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mutations. The common mutations found in melanoma include mutations in the BRAF, NRAS, and CKIT genes (Vennepureddy et al., 2016). In fact, approximately 70% of malignant
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melanomas have a valine600 to glutamic acid (V600E) mutation in the BRAF gene. These
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mutations consequently caused constitutively activation of the MAPK signaling pathway leading to the activation of the transcription factor MITF which promotes the proliferation and survival
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of melanocytes (Levy et al., 2006). The discovery of the MAPK pathway in melanoma opens a new direction for melanoma therapy. Several drugs have been developed to target the molecules in the MAPK pathway, including vemurafenib and dabrafenib, inhibitors of BRAF, and trametinib, an inhibitor of MEK. All of them showed high tumor response rates and striking overall survival rates (Vennepureddy et al., 2016). Unfortunately, in addition to the many adverse effects including causing cutaneous squamous cell carcinoma, drug resistance was
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ACCEPTED MANUSCRIPT developed toward these targeted therapy drugs in many patients. One plausible hypothesis for the drug resistance with supporting evidence is that other signaling pathway(s) become activated to compensate the inactivation of MAPK (Tang et al., 2016).
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Immunotherapy, although not specific to melanoma, is another approach that has shown
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significant effectiveness. Ipilimumab, which was approved by FDA in 2011 for the treatment of
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metastatic melanoma, is an antibody that can enhance the function of our immune system by blocking the immune checkpoints. Nivolumab is another FDA approved biologic that has similar
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mechanism as ipilimumab. These drugs showed substantial benefits but tend to cause immune-
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related adverse events (IrAEs) in many organs, including skin, gastrointestinal system, liver, and endocrine systems. Recently, therapies combining drugs tackling different molecules have shown
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potential to reduce adverse effects while generating synergistic therapeutic effects
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(Vennepureddy et al., 2016). New drugs targeting other signaling pathway, such as the wnt
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pathway, are also being developed (Prasad et al., 2015).
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Another cause to drug resistance comes from the intratumoral heterogeneity. Cancer stem cells have been identified in many cancers and are shown to be responsible for chemotherapy
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resistance primarily due to their low proliferation rate (Dean et al., 2005). In melanomas, some cells have the BRAF V600E single mutation but some have BRAF V600E and NRAS double mutations (Nazarian et a., 2010). Melanoma cells also show a wide range of MITF expression levels which seems to correlate with their proliferative and metastatic ability (Carriera et al., 2006; Hoek et al., 2006; Hoek and Goding, 2010). The discovery of melanoma subpopulations suggests the challenges as well as new ideas for melanoma therapy. More detail about molecular
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ACCEPTED MANUSCRIPT heterogeneity of melanomas and therapeutic directions could be found in the review article by Somasundaram et al., 2012.
In this study, we report the identification and characterization of a novel compound named A11
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that can inhibit melanoma growth. This inhibition of A11 is seen on melanoma forming zebrafish
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embryos as well as in the mouse melanoma cell line B16F10. A11 decreased mitotic index but
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did not cause apoptosis in melanoma cells. Molecularly, while a MEK inhibitor (MEK-I) used in our study exhibited expected inhibition of melanoma growth and MAPK activation, which is
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consistent with the current melanoma therapy, A11 surprisingly inhibited melanoma growth by
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elevating MAPK activity. We also found that A11 caused lower expression of melanocyte differentiation genes, including Pax3 and MITF. Furthermore, when compared with the
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morphology of B16F10, A11-treated B16F10 and primary melanocytes, A11-treated melanoma
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cells underwent dramatic morphological changes so that they resembled normal melanocytes. These results suggest that A11 might down regulate melanocyte differentiation genes and turn
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the highly proliferative cancerous melanocytes into less proliferative normal melanocytes. A11
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could be a novel therapeutic agent for melanoma therapy.
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ACCEPTED MANUSCRIPT Material and Methods Zebrafish Husbandry and in vitro Fertilization The zebrafish stocks used in this study are maintained following standard procedures (Westerfield, 2000) and bred by in vitro fertilization. In brief, mature male and female zebrafish
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were separated by a mesh in a breeding tank the night before breeding. Soon after the light is
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turned on the next morning, fish were anesthetized in ~0.16% tricaine for 1-2 min. The females
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were placed on a paper towel to dry the body surface briefly before being transferred into a 6 cm dish. The eggs were expelled onto the dish by gently pressing the lateral and ventral side of the
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belly with fingers. The males were also dried briefly and then placed ventral side up on a sponge
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well. Under a dissecting microscope, the lateral sides of the belly were pressed gently with a pair of blunt forceps while holding a capillary tube close to the anus to collect sperm. The sperm were
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immediately mixed with the eggs and ~1 ml of egg water was added (more details in Westerfield,
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2000). The melanoma zebrafish was obtained from crosses between the Et(kita:GalTA4,UAS:mCherry)hzm1 (Distel et al., 2009) and Tg(UAS:eGFP-HRAS_GV12)io006
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(Anelli et al., 2009) transgenic fish (Santoriello et al., 2010), provided by Dr. Maria Mione at
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Karlsruhe Institute for Technology, Karlsruhe, Germany.
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Chemical Treatment of Zebrafish Embryos Zebrafish embryos were collected and arrayed with 5 embryos per well of a 96-well plate in 200 μl of egg water (distilled water containing 60 μg/ml sea salt from Coralife, USA) which were later replaced with the same volume of egg water containing desired concentration of compounds.
Cell culture
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ACCEPTED MANUSCRIPT The mouse melanoma cell line B16F10 (ATCC CRL-6475) was purchased from ATCC (American Tissue Culture Center) and maintained in DMEM supplemented with 10% fetal bovine serum. The human primary epidermal melanocytes (PCS-200-013) was also purchased from ATCC and maintained in Dermal Cell Basal Medium (PCS-200-030) supplemented with
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the Adult Melanocyte Growth Kit (PCS-200-042). Trypsinization and subculture were performed
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following the protocols found on ATCC website. For chemical treatment, approximately 14,000
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melanoma cells and 30,000 normal melanocytes were seeded in each well of the 24-well plate with 0.5 ml of culture medium. Cells were set up triplicate and allowed to grow two days for
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melanoma or three days for normal melanocytes before chemical treatment. After 24 hours of
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chemical incubation, cells were first washed with PBS once and trypsinized. Equal volume of cell suspension and trypan blue was mixed in an eppendorf tube before the cells were counted
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Immunohistochemistry
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Zebrafish embryos were fixed in 4% paraformaldehyde at least overnight in 4 o C. The fixed
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embryos were washed twice with PBS, then once with H2 O, 5 min/each, and permeabilized with -20 o C acetone for 7 minutes followed by washes in H2 O and then PBS. The embryos were
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incubated with 3% BSA in PBST (PBS with 0.1% tween 20) for at least 1 hour at room temperature before overnight incubation in 4 o C with the phosphor-Histone H3 (Ser10) Rabbit mAb-Alexa Fluor 555 conjugate in PBST (Cell Signaling Technology). The next day, the embryos were washed with PBST at least 4 times, 15 min/each, then stored in ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology). Apoptotic cells were detected by TUNEL assay using the TMR In Situ Cell Death Detection Kit from Roche. For the
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stained embryos or cultured cells were analyzed using a fluorescence compound microscope.
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test was performed to determine the statistical significance.
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The number of positively stained melanocytes was manually counted and graphed. Student’s T-
Western blot
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Cells were prepared in a 6-well plate. To harvest the proteins, we first added 400 l of lysis buffer (0.2% NP-40, 100mM Tris, 150 mM NaCl, 8mM of EDTA, pH 7.4) with protease
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inhibitor cocktail added freshly (P8340, Sigma) to the well and scrapped off the cells using cell
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scrapper. The cells then were transferred to a microcentrifuge tube. The cells were lysed for 30
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min in 4C on a shaker. The lysate was then centrifuged at 12,000 rpm, 4C, for 20 min. After the centrifugation, protein supernatant was transferred to a clean tube and stored in -80C. Protein
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concentration was determined using the Pierce BCA Assay Kit (23227, Thermo Scientific). A brief summary of the assay and a set of sample data were available in Supplemental Figure 1. 10-
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20 g of proteins were loaded into 12% SDS-PAGE gel (Biorad) for gel electrophoresis and later for western blot using the Pierce Fast Western Blot Kit (35050, Thermo Scientific). Antibodies were purchased from Cell Signaling Technology. The anti-MITF antibody (MITF D5G7V rabbit antibody) recognizes all the MITF isoforms and thus detected the total MITF proteins.
Reverse transcription-qPCR
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ACCEPTED MANUSCRIPT Twenty embryos from each treatment were subjected to RNA extraction with Trizol following the commercial instruction (Invitrogen, Carlsbad, CA). RNA concentration and quality was determined using the Genesys 10S UV-VIS spectrophotometer (Thermo Scientific, USA). Up to 5 μg of total RNA were then used to generate the cDNA library using the Superscript III kit
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(Invitrogen, Carlsbad, CA). Quantitative PCR (RT-qPCR) was set up using the Power
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SYBRGreen PCR kit (Biorad, Foster City, CA) and run on the Mx300P QPCR thermocycler
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(Agilent Technologies, Santa Clara, CA). Data was analyzed and graphed using the MxPro QPCR software provided by the manufacturer to calculate the mRNA quantity of the genes of
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interest using the -actin gene as a normalizer. Results of experimental groups were then
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compared to the H2 O treated group which was set to 1 as the baseline. Primers used for RTqPCR are:
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GAPDH L, 5’ TGCACCACCAACTGCTTAGC 3’
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GAPDH R, 5’ TCTTCTGGGTGGCAGTGATG 3’ Pax3 L, 5’ TCGGCCTTGCGTCATTTC 3’
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Pax3 R, 5’ CAGGATCTTAGAGACGCAACCA 3’
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MITF L, 5’ TGCCTTGTTTATGGTGCCTTCT 3’ MITF R, 5’ TCCCTCTACTTTCTGTAATTCCAATTC 3’
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tyrosinase L, 5’ ACGACCTCTTTGTATGGATGCA 3’ tyrosinase R, 5’ TTTCAGAGCCCCCAAGCA 3’ DCT L, 5’ CCGGCCCCGACTGTAATC 3’ DCT R, 5’ GGGCAGTCAGGGAATGGATAT 3’
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ACCEPTED MANUSCRIPT Results A11 inhibits melanoma formation in transgenic melanoma fish embryo We reported that A11 could inhibit the pigment formation in wild type zebrafish embryo (Fig. 1A; Huang et al., 2013; Martinson et al., in preparation). Here we first tested whether A11 had a
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similar effect on the melanoma forming fish, kita-eGFP-HRAS_GV12 (Santoriello et al., 2010).
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These melanoma zebrafish were created by crossing two transgenic lines,
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Et(kita:GalTA4,UAS:mCherry)hzm1 (Distel et al., 2009) and Tg(UAS:eGFP-HRAS_GV12)io006 (Anelli et al., 2009). The resulting embryos exhibited hyperpigmentation as early as 3 days post
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fertilization due to the overexpression of HRAS and consequently overproliferation of
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melanocytes. As in wild type embryos, A11 caused nearly 100% pigment inhibition in the melanoma forming embryos incubated from 24 to 48 hours post fertilization (hpf) (Figure 1B).
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When the treated embryos were withdrawn from the compounds at 48 hpf and allowed to
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develop in water until 72 hpf (see Materials and Methods for details), pigment recovery occurred quickly and completely for MEK-I and PTU but slower in A11-treated melanoma embryos
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(Figure 1B). A closer examination on the pigment recovering melanoma embryos revealed that
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A11 seemed to cause fewer melanocytes (Figure 1C). We went on to examine the mitosis using the anti-phosphorylated histone H3 antibody, which is a known mitosis marker (Hendzel et al.,
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1997) and cell death using TUNEL assay in A11-treated embryos. The results showed that A11 significantly decreased the number of mitotic melanocytes in the melanoma forming embryos. A11, however, does not increase the number of apoptotic melanocytes (Figure 2). MEK-I, an inhibitor of MEK kinase used as a comparison, also caused significant reduction of melanocyte proliferation but had no effect on cell death.
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ACCEPTED MANUSCRIPT A11 inhibits cell proliferation in B16F10 mouse melanoma cell To test whether A11 also inhibited the proliferation of mammalian melanoma cells, we treated the B16F10 mouse melanoma cells with A11. B16F10 mouse melanoma cells are highly pigmented and proliferative and have been used for in many areas of melanoma and melanocyte
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studies. This experiment started with seeding the same number of melanoma cells in 24-well
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plates with 500 l of complete DMEM medium containing A11 or other compounds. After 24
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hours of incubation, cells were trypsinized, mixed with trypan blue for viability check, and
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counted. The results showed that A11 at 40 M indeed caused near 50% less cells than the control (Figure 3A). As expected, MEK-I showed inhibition in melanoma proliferation.
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Surprisingly, kojic acid, a known tyrosinase enzyme inhibitor commonly used for depigmentation (or skin-lightening), also inhibited melanoma cell proliferation, albeit to a much
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less degree than A11 and MEK-I. To test whether A11 inhibited melanoma proliferation, we
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examined the number of mitotic cells in compound-treated B16F10 cells using anti-
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phosphorylated histone H3 antibody (Figure 3B). The results showed that while control B16F10 cells had about 10 mitotic cells out of 500 cells, A11-treated melanoma cells only showed 2
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mitotic cells (Figure 3C). In conclusion, A11 reduces melanoma proliferation. In addition,
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several lines of evidence suggest that A11 does not cause melanoma cell death. First, A11 does not cause any observed cell toxicity as seen in hydroquinone treated cells. Hydroquinone is a tyrosinase inhibitor that produces strong depigmentation and has been used as a reference standard for depigmentation evaluation (Brenner and Hearing, 2008). Once in the cells, hydroquinone also produces toxic quiones and reactive oxygen species which can cause cell death (Briganti et al., 2003;). In our experiments, hydroquinone at 50 M caused near all the cells to die and float in the medium within less than 12 hours (Figure 3D). In contrast, the A11-
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ACCEPTED MANUSCRIPT treated melanoma cells appeared healthy, strongly adhered to the culture dish, and with only a few cells floating in the wells (also see Figure 6). Second, by observations, trypan blue staining showed indistinguishable level of cell death between A11-treated and control cells.
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A11 activates MAPK and Akt pathways in B16F10 melanoma
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The MAPK signaling pathway is known to activate the MITF transcription factor and promote
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melanoma proliferation and survival (Wellbrock and Arozarena, 2015). We examined the MAPK activity in A11-treated B16F10 cells. In this experiment, we chose to use sub-dosage of each
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compound, 5 M of each, and set up a combined treatment to see if there is any additive or
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synergistic relationship between A11 and MEK-I. The treated cells were counted after the treatment then lysed for western blot analysis. At 5 M, MEK-I alone did not seem to inhibit
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melanoma proliferation while A11 showed slight inhibition. MEK-I and A11 combined
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treatments clearly showed more enhanced inhibition than A11 alone (Figure 4A). On the western
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blot, we found that MEK-I clearly showed the expected inhibition on MAPK but were surprised to find that A11 actually strongly activated MAPK (Figure 4B). In the cells treated with MEK-I
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and A11 combined, the MAPK activity is completely inhibited, indicating A11’s function in
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activating MAPK is upstream of MEK-I. We then examined the Akt signaling pathway which is also known to promote melanocyte proliferation and survival (Wu et al, 2003; Davies, 2012). We found that Akt was activated both by A11 and MEK-I. However, the activation of Akt was approximately the same between the single and combined treatments. These results suggested a novel and complex mechanism of A11 in inhibiting melanoma growth.
A11 suppresses the expression of melanoma differentiation genes
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ACCEPTED MANUSCRIPT B16F10 cell line is composed of heterogeneous melanocytes, including differentiated melanocytes and melanoma stem cells (Somasundaram et al., 2012). It is also found that the expression level of MITF gene correlates with the differentiation status and proliferation ability of melanocytes, with high level of MITF expression in the terminally differentiated and low
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proliferative B16F10 cells, medium level of MITF in highly proliferative but less differentiated,
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and low MITF in melanoma stem cells (Hoek and Goding, 2010). To understand how A11
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inhibits melanoma proliferation, we examined the MITF expression level in A11-treated B16F10 cells by western blot and RT-qPCR. Interestingly, we found that MITF protein level is
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slightly lower in the A11-treated melanoma cells than that in the DMSO control (Figure 5A).
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The result of decreased MITF protein level by A11, albeit subtle, is supported by the RT-qPCR which showed decreased MITF gene expression (Figure 5B). We went on to examine the
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upstream genes of melanocyte differentiation Pax3, which is a known transcriptional activator of
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MITF, and found that A11 also reduces Pax3 expression. These results could explain the decrease of melanocyte proliferation by A11 and lead us to hypothesize that the genes involved
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in melanin synthesis which are typically expressed in differentiated melanocytes would also
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show reduction by A11. As expected, the expression of tyrosinase gene which is the key enzyme in the melanin synthesis was also decreased by A11 (Figure 5B). The expression of DCT
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(dopachrome tautomerase) gene, another enzyme in melanin synthesis pathway, showed slightly, but not statistical significant, decrease by A11. All these results suggest that A11 inhibits melanocyte proliferation by regulating melanocyte differentiation. Interestingly, MEK-I seemed to induce high level of MITF expression evident by the western blot and RT-qPCR results (Figure 5A, B). This result is consistent with the finding that overexpression of MITF in murine BRAF-transformed melanocytes inhibits proliferation (Wellbrock and Marais, 2005).
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A11 on normal human melanocytes To test whether A11 has any effect on normal melanocyte, we obtained a primary culture of human melanocytes derived from human foreskin. We found that A11 also inhibited the
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proliferation in normal melanocytes even though the inhibition was not as strong as in the mouse
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melanoma cells (Figure 6A). MEK-I, however, did not seem to inhibit the proliferation of normal
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melanocyte. When we compared the morphology of normal melanocytes, control B16F10 melanoma, and A11-treated B16F10, we noticed that A11 caused dramatic morphological
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changes in the B16F10 melanoma cells. Normal melanocytes have thinner and longer cell bodies
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with several long cellular processes but melanoma cells are more rounded with shorter cellular process. Also, B16F10 are more pigmented than normal melanocytes. In A11-treated B16F10
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melanoma cells, we found that approximately 50% of the cells developed thinner cell bodies
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with long cellular processes, a phenotype that is intermediate between melanoma and normal melanocytes (Figure 6B). This morphology change of melanoma by A11 seems to suggest that
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A11 might turn melanoma cells back to less proliferative quasi-normal melanocytes.
A11 analogs show different degrees of melanoma inhibition
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Finally, we synthesized nearly fifty A11 analogs and tested their inhibition on melanoma growth. While some analogs seemed to lose the inhibition ability, such as SK0392 and CL-4, several A11 analogs could still inhibit melanoma growth, albeit less potent than A11 (Figure 7A). FSK0392 (fluorinated SK0392), however, showed strong toxicity to melanoma cells killing all the cells at 25 M in less than 24 hours of treatment. In addition, A4, but not all other analogs, caused the same morphological change as A11, suggesting these analogs retain some of the A11 molecular
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ACCEPTED MANUSCRIPT functions but lose others. Furthermore, western blot results showed MAPK activity was significantly higher in the A11 analog-treated cells than in the control. Also, these analogs did show different degrees of MAPK activation which seemed to correlate with the growth inhibition potency, i.e. the higher pMAPK the stronger melanoma growth inhibition, as A9 showing lower
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MAPK activation and little growth inhibition than A4, FSK0392, and SK0516 (Figure 7B).
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ACCEPTED MANUSCRIPT Discussion A11 could be a new therapeutic agent for melanoma While the major current melanoma drugs either try to enhance the immune function (immunotherapy) or inhibit the MAPK pathway (targeted therapy), our studies suggest a novel
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mechanism of A11 in inhibiting melanoma growth. Unlike the BRAF inhibitor, such as
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vemurafenib, or MEK inhibitor, such as trametinib, A11 does not inhibit MAPK. Instead, A11
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(and its analogs) greatly elevated MAPK activity. However, when the elevation in MAPK activity associated with A11 treatment was prevented by the simultaneous administration of
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MEK-I, melanoma cell proliferation was still significantly reduced. This result strongly suggests
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that A11 must influence other cell signaling pathway(s) to inhibit melanoma growth. Since drug resistance is commonly seen in patients receiving BRAF or MEK inhibitors, and more and more
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evidence suggests the involvement or activation of other pathways to support the growth of
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melanoma (Prasad et al., 2015), A11 could be a new melanoma drug for combinational therapies. Wnt signaling was found to be activated in 30% of melanomas (Rimm et al., 1999; Prasad et al.,
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2015). It will be interesting to see if A11 suppresses the Wnt signaling pathway. Furthermore,
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our results might shed light on the role of Akt in melanoma development and therapies. The PI3Akt signaling was believed to promote melanoma survival, proliferation and migration (Davies,
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2012) but our result clearly showed that both A11 and MEK-I activate Akt and inhibit melanoma
Skin-lightening and melanocyte growth inhibition by A11 In another manuscript by our group (Martinson et al., in prep), we showed that A11 reduced pigment in both zebrafish and mammalian cells and could be a potent and safe skin-lightening
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ACCEPTED MANUSCRIPT agent for cosmetic industry. After finding that A11 did not inhibit tyrosinase enzyme activity like other skin-lightening compound, we discovered that A11 actually inhibited melanocyte mitosis. Here we further confirmed the proliferation inhibition of A11 using both the melanoma forming zebrafish embryos and the mouse melanoma cell culture B16F10, and normal human
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melanocytes. Our result also showed that A11 only inhibited the proliferation and did not seem
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to enhance apoptosis. Through our other studies, we detected little or no toxicity of A11 (Huang
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et al., 2013; Martinson et al., in prep), suggesting that A11 might be a safe compound for both skin-lightening and melanoma therapy at the same time. So far, there is no other compound that
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clearly demonstrates this dual function except for the study by Inoue et al., 2013. In that study,
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the authors showed that hydroquinone, but not arbutin which is a natural derivative of hydroquinone, caused down-regulation of genes involved in melanocyte differentiation using
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mouse embryonic stem cells. As hydroquinone is known to be very toxic to cells, the down-
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regulation of gene expression by hydroquinone may not be a true differentiation regulation.
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A11 regulates melanocyte differentiation
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The slow pigment recovery by A11 in both normal and melanoma zebrafish embryos raised interesting questions to us. One early hypothesis was maybe it was due to cell death caused by
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A11 which was soon ruled out by the TUNEL result (Figure 2). Meanwhile, the reduced number of DCT expressing cells in A11-treated wild type embryos from our previous study (Martinson et al., in prep) suggests that A11 might inhibit melanocyte differentiation. In this study, we further explored this hypothesis using melanoma cells. Consistent with the fish results, we found A11treated melanoma cells have lower expression of the melanocyte differentiation genes, including Pax3 and MITF, and consequently lower expression of the melanogenesis genes, such as
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ACCEPTED MANUSCRIPT tyrosinase. Although many studies showed that activation of MAPK leads to upregulation of MITF and melanocyte differentiation and melanogenesis. There are reports of MAPK activation leading to down-regulation of MITF and melanogenesis (Kim et al., 2008; Nishio et al., 2016). The differentiation regulation by A11 can explain almost all the phenotypes we saw on zebrafish,
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including reducing existing pigment, slow pigment recovery, and a decrease of dct expressing
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cells. However, it is still not clear at which level of differentiation A11 does regulate.
This melanocyte differentiation regulation by A11 is further supported, albeit indirectly, by the
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observation of morphological changes in A11-treated B16F10 cells. As described in the result,
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we found that A11 seemed to change the more rounded, less dendritic melanoma cells into stretched and more dendritic morphology of normal melanocytes. It will be very interesting to
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perform the cancer colony forming and metastasis assays to see if the A11-treated melanomas
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actually are losing cancer properties. If these hypotheses turned out to be true, A11 will open a new avenue of cancer therapy to turn cancer cells back to normal cells. But then many questions
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will emerge regarding the therapeutic efficacy and adverse effects of A11 for melanoma. Is the
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differentiation inhibition by A11 reversible, i.e. will the A11-treated cells return to cancer cells after A11 withdrawal? How long will the inhibition last? Would A11 turn cancer melanoma into
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less differentiated melanoblasts or even melanocyte stem cells causing chemotherapy resistance? These are certainly interesting questions awaits further research.
MITF protein level and melanoma proliferation The MITF rheostat model describes the correlation of MITF expression level and melanoma proliferation (Hoek and Goding, 2010). More specifically, melanoma stem cells express low
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ACCEPTED MANUSCRIPT level of MITF and stay at the G1 phase of cell cycle but maintain invasive potential. Medium level of MITF generates either proliferating cells or differentiated pigment-producing cells. More importantly, these melanoma cells are able to switch phenotypes by regulating the expression of MITF. The phenotype switching behaviors of melanoma cells, which depend on the molecular
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signals, pose great challenges to melanoma therapy. If A11 can inhibit MITF expression and
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melanocyte differentiation, it might limit the heterogeneity of melanoma cells and increase the
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therapeutic efficacy. On the other hand, our result that showed increased MITF protein level and Pax3 gene expression by MEK-I might suggest the increase of melanoma heterogeneity by
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MEK-I and offer an explanation to why there is high resistance to drugs targeting MAPK
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signaling pathway. However, as melanoma stem cells express low level of MITF, it raises a question whether A11 might increase melanoma stem cell population by inhibiting MITF
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expression and consequently causing more therapeutic challenges. It will be interesting to know
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Potential pathway for A11
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whether melanoma stem cell population is increased upon A11 treatment.
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To search for other potential cell signaling pathway that might be regulated by A11, we utilized the SEA (Similarity Ensemble Approach) computer software (Keiser et al., 2007) available at
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www.sea.bkslab.org. The software predicts the aryl hydrocarbon receptor (AhR) to be a potential binding molecule for A11. AhR is a nuclear receptor that once activated can function as a transcription factor to activate numbers of metabolism-related enzymes, such as cytochrome P450 and many others in the toxic and carcinogenic responses (review by Mulero-Navarro and Fernandez-Salguero, 2016). Many studies have shown that AhR inhibits cell proliferation and survival. Studies also showed that AhR may regulate several growth factor signaling pathways,
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ACCEPTED MANUSCRIPT including IGF (insulin growth factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), and KGF (keratinocyte growth factor) to inhibit or induce cell proliferation depending on the cell types (Yin et al., 2016). More interestingly, AhR was recently found to interact with Wnt and BMP signaling pathways to regulate stem cell differentiation and regeneration (Wang et al.,
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2016; Singh et al., 2014). So it is possible that A11 might regulate melanoma differentiation and
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proliferation by inhibiting the AhR signaling pathway.
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ACCEPTED MANUSCRIPT Acknowledgements This project is supported by a Faulty Research Grant (PRJ82VD) to C.-C.H. and a Summer Scholar Fellowship to N.S. both from University of Wisconsin-River Falls and a WiSys Innovation Grant (133-4-061600-AAB5817) from the WiSys Technology Foundation. Thanks
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Dr. Michael J. Martin for his helpful comments and suggestions. The
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Et(kita:GalTA4,UAS:mCherry)hzm1 and Tg(UAS:eGFP-H-RAS_GV12)io006 transgenic fish
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lines were generated by Maria Mione, Karlsruhe Institute for Technology (KIT), maintained by European Zebrafish Resource Center of the Karlsruhe Institute of Technology and
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obtained from the Taiwan Zebrafish Core Facility at Academia Sinica.
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References Anelli, V., Santoriello, C., Distel, M., Koster, R.W., Ciccarelli, F.D., and Mione, M. (2009). Global repression of cancer gene expression in a zebrafish model of melanoma is linked to epigenetic regulation. Zebrafish 6, 417-424. Balch, C.M. et al., (2009). Final version of 2009 AJCC melanoma staging and classification. J Clin. Oncol. 27, 6199-6206. Brenner, M. and Hearing V.J. (2008). Modifying skin pigmentation—approaches through intrinsic biochemistry and exogenous agents. Drug Discovery Today: Disease Mechanisms 5, e189-e199. Briganti, S., Camera, E., and Picardo, M. (2003). Chemical and instrumental approaches to treat hyperpigmentation. Pigment Cell Res. 16, 101-110. Carreira, S., Goodall, J., Denat, L., Rodriguez, M., Nuciforo, P., Hoek, K.S., Testori, A., Larue, L., and Goding, C.R., 2006. Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes and Development 20, 3426-3439. Davies, M.A. (2012). The role of the PI3K-Akt pathway in melanoma. Cancer J. 18, 142-147. Dean, M., Fojo, T., and Bates, S. (2005). Tumour stem cells and drug resistance. Nat Rev Cancers 5, 275-284. Dickson, P.V. and Gershenwald, J.E. (2011). Staging and prognosis of cutaneous melanoma. Surg. Oncol. Clin. N Am. 20, 1-17. Distel, M., Wullimann, M.F., and Koster, R.W. (2009). Optimized Gal4 geneticsfor permanent gene expression mapping in zebrafish. Proc. Natl. Acad. Sci. USA 105, 1255-1260. Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., BazettJones, D.P., and Allis, C.D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348-60. Hoek, K.S., Schlegel, N.C., Brafford, P., Sucker, A., Ugurel, S., Kumar, R., Weber, B.L., Nathanson, K.L., Phillips, D.J., Herlyn, M., Schadendorf, D., and Dummer, R (2006). Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature. Pigment Cell Res. 19, 290-302. Hoek, K.S. and Goding, C.R., (2010). Cancer stem cells versus phenotype-switching in melanoma. Pigment Cell Melanoma Res. 23, 746-759. Huang, C-C., Monte, A., Cook, J.M., Kabir, M.S., and Peterson, K.P. (2013). Zebrafish heart failure models for the evaluation of chemical probes and drugs. Assay and Drug Dev. 11, 561-572. Inoue, Y., Hasegawa, S., Yamada, T., Date, Y., Mizutani, H., Nakata, S., Matsunaga, K., and Akamatsu, H., (2013). Analysis of the effects of hydroquinone and arbutin on the differentiation of melanocytes. Biol. Pharm. Bull. 36, 1722-1730. Keiser MJ, Roth BL, Armbruster BN, Ernsberger P, Irwin JJ, Shoichet BK. Relating protein pharmacology by ligand chemistry. Nat. Biotech. 25, 197-206 (2007). Kim, J.H., Baek, S.H., Kim, D.H., Choi, T.Y., Yoon, T.J., Hwang, J.S., Kim, M.R., Kwon, H.J, and Lee, C.H. (2008). Down regulation of melanin synthesis by haginin A and its application to in vivo lightening model. J Invest. Dermatol. 128, 1227-1235. Levy, C., Khaled, M., and Fisher D.E. (2006). Mitf: master regulator of melanocyte development and melanoma oncogene. Trends Mol. Med. 12, 406-414.
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Martinson, K., Stueven, N., Monte, A., and Huang, C.-C. (2016). A11 part I: novel stilbene-like compounds that reduce melanin through inhibiting melanocyte differentiation and proliferation without inhibiting tyrosinase. Xx,xxxx-xxxx. Mulero-Navarro, S. and Fernandez-Salguero, P.M. (2016). New trends in aryl hydrocarbon receptor biology. Front. Cell Dev. Biol. 4, 45. Nishio, T., Usami, M., Awaji, M., Shinohara, S., and Sato, K. (2016). Dual effects of acetylsalicylic acid on ERK signaling and mitf transcription lead to inhibition of melanogenesis. Mol. Cell. Biochem. 412, 101-110. Prasad, C.P., Mohapatra, P., and Anderson, T. (2015). Therapy for BRAFi-resistant melanomas: is wnt5a the answer? Cancers 7, 1900-1924. Rimm, D.L., Caca, K., Hu, G., Harrison, F.B., Fearon, E.R. (1999). Frequent nuclear/cytoplasmic localization of b-catenin without exon 3 mutations in malignant melanoma. Am. J. Pathol. 154, 325-329. Santoriello, C., Gennaro, E., Anelli, V., Distel, M., Kelly, A., Koster, R.W., Hurlstone, A., and Mione, M. (2010). Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish. PLoS ONE 5, e15170. Singh, K.P., Bennett, J.A., Casado, F.L., Walrath, J.L., Welle, S.L., and Gasiewicz, T.A. (2014). Loss of aryl hydrocarbon receptor promotes gene changes associated with premature hematopoietic stem cell exhaustion and development of a myeloproliferative disorder in aging mice. Stem Cells Dev. 23, 95-106. Somasundaram, R., Villanueva, J., and Herlyn, M. (2012). Intratumoral heterogeneity as a therapy resistance mechanism: role of melanoma subpopulations. Adv. Pharmacol. 65, 335-359. Tang, T., Eldabaje, R., and Yang L. (2016). Current status of biological therapies for the treatment of metastatic melanoma. Anticancer Res. 36, 3229-3242. Vennepureddy, A., Thumallapally, N., Nehru, V.M., Atallah, J.-P., and Terjanian, T. (2016). Novel drugs and combination therapies for the treatment of metastatic melanoma. J Clin. Med. Res. 8, 63-75. Wang, Q., Kurita, H., Carreira, V., Ko, C.I., Fan, Y., Zhang, X., Biesiada, J., Medvedovic, M., and Puga, A. (2016). Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions. Toxicol. Sci. 149, 346-357. Wellbrock, C. and Arozarena, I. (2015). Microphthalmia-associated transcription factor in melanoma development and MAPK-kinase pathway targeted therapy. Pigment Cell Res. 28, 390-406. Wellbrock, C. and Marais, R. (2005). Elevated expression of MITF counteracts B-RAFstimulated melanocyte and melanoma cells proliferation. J. Cell Biol. 170, 703-708. Westerfield, M. (2007). The zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). Eugene: University of Oregon Press. White, R.M. and Zon, L.I, (2008). Melanocytes in development, regeneration, and cancer. Cell Stem Cell 3, 242-252. Wu, H., Goel, V., and Haluska, F.G. (2003). PTEN signaling pathways in melanoma. Oncogene 22, 3113-3122.
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ACCEPTED MANUSCRIPT Figure 1. Suppression of melanoma formation and recovery by A11. (A) Wild type zebrafish embryos in H2 O or 10 uM of A11 compound from 24-72 hpf. A11 causes significant reduction of pigment but no other morphological defect. (B, left) The melanoma forming zebrafish embryos, kita-eGFP-HRAS_GV12, were treated with skin-
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lightening compounds from 24 to 48 hpf. Strong pigment inhibition was apparent by the tested
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compounds, A11 or MEK-I both at 10 M or PTU at 0.0003% throughout the study unless
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specified. (B, right) The treated embryos then were washed with egg water twice and allowed to
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develop in egg water until 72 hpf. Melanocytes recovered quickly in MEK-I-, H2 O-, and PTUtreated embryos, but not in those treated with A11. These experiments were done with twenty
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embryos for each treatment group and repeated three times. The phenotypic penetrance for each group was near 100%. (C) A closer examination of the mid-trunk regions revealed that the
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number of recovered melanocytes in A11-treated melanoma fish embryo is significantly lower
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than that in control (H2 O). In comparison with wild type embryo, the A11-treated embryo
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seemed to recover the normal level of melanocytes.
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ACCEPTED MANUSCRIPT Figure 2. Mitotic index is lower in the A11-treated melanoma forming zebrafish embryos. (A) Immunostaining using anti-phosphorylated histone H3 antibody labeled the cells at metaphase of mitosis (green fluorescence). Mitotic melanocytes (Arrow) could be readily identified under fluorescence microscope. (B) Quantitative data showed that A11- and MEK-I-
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treated melanoma forming zebrafish embryos have significant fewer mitotic melanocytes (n=5).
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* P<0.05, ** P<0.01 by Student’s T-test. (C) TUNEL-labeled apoptotic melanocyte in zebrafish
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indistinguishable level of melanocyte apoptosis (n=5).
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embryo (arrow). (D) A11- or MEK-I treated melanoma forming embryos showed
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ACCEPTED MANUSCRIPT Figure 3. A11 inhibits mouse melanoma cell growth. (A) Same number of mouse melanoma cells B16F10 were grown in 24-well culture plate for two days before they were treated with different compounds. After 24 hours of treatment, cells were trypsinized and counted. A11 and MEK-I (20 M) treated cells showed significantly lower
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proliferation. * P<0.05, ** P<0.01 by Student’s T-test. (B) Immunostaining using anti-
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phosphorylated histone H3 to label mitotic cells (red). The cells were co-labeled with DAPI to
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mark the nuclei in blue and phalloidin to mark the f-actin in green. (C) Quantitative data showed
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that significant lower number of mitotic cells in 40 M A11- and 10 M MEK-I-treated melanoma cells. FSK0392 which is an analog of A11 also showed proliferation inhibition at 5
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M. Error bars represent standard deviation. * P<0.05 by Student’s T-test. (D) Melanoma cells treated with A11 appeared healthy and adhered to dish well whereas hydroquinone caused cells
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ACCEPTED MANUSCRIPT Figure 4. A11 activates MAPK and Akt in mouse melanoma cells. (A) The proliferation of B16F10 melanoma cells was not affected by 5 M MEK-1 but significantly decreased by A11 or A11 and MEK-I combined. * P<0.05, ** P<0.01 by Student’s T-test.
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(B) Western blotting for the proteins from B16F10 cells treated with different compounds using
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anti-phosphorylated Akt (pAkt), total Akt, total MAPK, phosphorylated MAPK (pMAPK), or -
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actin (as an internal control). As expected, MEK-I inhibited MAPK phosphorylation completely.
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A11 caused high level of pMAPK. Interestingly, both MEK-I and A11 showed high level of pAkt. When cells were treated both MEK-I and A11, MEK-I abolished the MAPK activation by
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A11. Proliferation in cells treated with A11 or both A11 and MEK-I was reduced.
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ACCEPTED MANUSCRIPT Figure 5. A11 inhibits melanoma differentiation. (A) Western blot showed slightly lower level of MITF proteins in A11-treated melanoma cells. MEK-I however, increased MITF protein in melanoma. (B) RT-qPCR revealed that A11 caused lower expression of two melanocyte differentiation genes, MITF and Pax3, as well as a
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differentiation gene, tyrosinase. The DCT expression in A11- treated cells, however, was slightly
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lower, but not statistically different from the control. MEK-I had no effect on Pax3 expression
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tyrosinase gene expression. * P<0.05, by Student’s T-test.
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but induced high level of MITF gene expression. PTU also caused reduction of DCT and
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ACCEPTED MANUSCRIPT Figure 6. A11 also inhibits the proliferation in normal human melanocytes. (A) Same number of normal human melanocytes were seeded in 24-well plate and allowed to grow for three days before treated with different compounds. After 24 hours of chemical treatment, the cells were trypsinized and counted. A11 caused significant reduction of cell
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number but MEK-I and PTU did not. * P<0.05 by Student’s T-test. (B) Normal human
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ACCEPTED MANUSCRIPT Figure 7. A11 analogs also inhibit melanoma growth corresponding with high level of pMAPK. (A) Representative data of melanoma inhibition by A11 analogs. Some compounds were tested at several concentrations, for example A11 were tested at 2, 10, 20, 40 M. While most analogs
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showed slightly lower inhibition potency than A11, SK0392, FSK0392 (fluorinated SK0392) at 5
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M), and CL4 at 40 M did not seem to inhibit melanoma growth at all. FSK0392, however,
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caused near 100% cell death at 25 M. * P<0.05 by Student’s T-test. (B) Western blot revealed
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significantly high level of pMAPK (Top row) by several A11 analogs, including A4, FSK0392, and SK0516 but only slightly by A9 which showed less melanoma growth inhibition. The
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ACCEPTED MANUSCRIPT Highlights of ‘A Novel Stilbene-like Compound That Inhibits Melanoma Growth by Regulating Melanocyte Differentiation and Proliferation’ by Stueven et al., This project identified a stilbene-like compound named A11 for melanoma treatment.
A11 shows different molecular mechanisms than current melanoma drugs.
A11 could be a good candidate for melanoma combinational treatment.
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