Biflorin inhibits the proliferation of gastric cancer cells by decreasing MYC expression

Journal Pre-proof Biflorin inhibits the proliferation of gastric cancer cells by decreasing MYC expression

Gleyce S. Barbosa-Jobim, Évelyn Costa-Lira, Ana Carolina L. Ralph, Luciana Gregório, Telma L.G. Lemos, Rommel R. Burbano, Danielle Q. Calcagno, Marília A.C. Smith, Raquel C. Montenegro, Marne C. Vasconcellos PII:

S0887-2333(19)30431-X

DOI:

https://doi.org/10.1016/j.tiv.2019.104735

Reference:

TIV 104735

To appear in:

Toxicology in Vitro

Received date:

4 June 2019

Revised date:

2 November 2019

Accepted date:

17 November 2019

Please cite this article as: G.S. Barbosa-Jobim, É. Costa-Lira, A.C.L. Ralph, et al., Biflorin inhibits the proliferation of gastric cancer cells by decreasing MYC expression, Toxicology in Vitro(2018), https://doi.org/10.1016/j.tiv.2019.104735

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© 2018 Published by Elsevier.

Journal Pre-proof Biflorin inhibits the proliferation of gastric cancer cells by decreasing Myc expression Gleyce S. Barbosa-Jobima, Évelyn Costa-Liraa, Ana Carolina L. Ralpha, Luciana Gregóriob, Telma L. G. Lemosb, Rommel R. Burbanod, Danielle Q. Calcagnoc, Marília A. C. Smithc, Raquel C. Montenegrod, Marne C. Vasconcellosa* a

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Faculty of Pharmaceutical Sciences, Federal University of Amazonas, 6200 Rodrigo Octavio Avenue, Manaus, Amazonas, Brazil. b Department of Organic and Inorganic Chemistry, Mister Hull Avenue, Federal University of Ceara, Fortaleza, Ceara, Brazil. c Department of Morphology, Federal University of São Paulo, 740 Botucatu Street, Sao Paulo, Sao Paulo, Brazil. d Institute of Biological Sciences, Federal University of Pará, 01 Augusto Correia Street, Belem, Para, Brazil. e Laboratory of Pharmacogenetics, Drug Research and Development Center (NPDM), Federal University of Ceará, Fortaleza, Brazil. * Corresponding author: Marne C. Vasconcellos [email protected] Telephone number: +55.92.984391434 Address: 6200 Rodrigo Octavio Avenue, Coroado I Zip Code: 69080-900

Abstract

Gastric cancer is the third leading cause of cancer-related death worldwide. To evaluate the anticancer potential and molecular mechanism of biflorin, a prenyl-

Journal Pre-proof ortho-naphthoquinone obtained from Capraria biflora L. roots, we used ACP02, a gastric cancer cell line established from a primary diffuse gastric adenocarcinoma. In this study, biflorin was shown to be a potent cytotoxic agent against ACP02 by Alamar Blue and Trypan Blue assays. Morphological analysis indicated cell death with features of necrosis. Furthermore, a decrease in colony formation, migration and invasion of ACP02 cells was observed after treatment with biflorin (1.0, 2.5 and 5.0 μM). Regarding the underlying molecular mechanism of biflorin in ACP02 cells, we observed a decrease in MYC expression and telomere length using FISH. Our findings suggest a novel molecular target of biflorin in ACP02 cells, which may be a significant therapeutic approach for gastric cancer management.

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Key words: gastric cancer; MYC; hTERT; cell proliferation; telomeres; cytotoxicity.

Journal Pre-proof 1. Introduction Gastric cancer is the fifth most common cancer worldwide and the third leading cause of cancer-related death in several developing countries1. In Brazil, it is estimated that 13,540 new cases of gastric cancer in men and 7,750 in women are diagnosed, and this disease is the second leading cause of death by cancer in men and the fifth in women2. Despite advances in diagnostic techniques and neoadjuvant chemoradiotherapy and surgery, the five-year survival rate for gastric cancer remains poor, particularly when the disease is at a more advanced stage3-5. While

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the curative treatment for gastric cancer is complete resection followed by adjuvant chemoradiotherapy, if necessary, the standard treatment for metastatic patients is chemotherapy and palliative treatment6. There is not much consensus on optimal systemic treatments, and despite ongoing research into classical chemotherapy

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agents (whether used as single agents or in combination therapy), there has been a

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lack of efficacy of these treatments7. Therefore, efforts to develop new drugs that target molecular abnormalities in signal transduction pathways specific to gastric

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cancer are needed and relevant.

Naphthoquinones are ubiquitous molecules that have been extensively

anti‐inflammatory,

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investigated for their wide range of biological properties, including their antibacterial

and

anticancer

activities8.

Biflorin,

an

ο-

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naphthoquinone isolated from the roots of Capraria biflora L., has shown promise as a leading drug for the treatment of cancer. Over the past years, Vasconcellos and

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coworkers9-15 have shown the cytotoxic, genotoxic, antimutagenic and protective effects of biflorin in various tumor cells in vitro and in vivo against the Sarcoma 180, Ehrlich ascites carcinoma and melanoma tumor models, with the latter showing increased survival rates in animals. The multistep carcinogenesis of gastric cancer is still not well understood. Chromosome instability can lead to loss of tumor suppressor genes, activation of oncogenes and multiple mutations16,17. Trisomy of chromosome 8, where the MYC oncogene is located, was found to be one of the most important molecular alterations leading to gain of function in gastric cancer18. MYC genes can activate and repress the transcription of target genes through distinct mechanisms and contribute to tumorigenesis by promoting cell growth, metastasis, angiogenesis and genomic instability and differentiation19-21. High MYC

Journal Pre-proof amplification has frequently been observed in primary tumors from individuals from northern Brazil, indicating that cell models are suitable to study gastric cancer molecular pathways and treatment22-24. Pathways that incorporate MYC gene-family members (c-MYC, MYCN, MYCL) are a hallmark of many cancer types, and their association with other survival pathways, such as human telomerase reverse transcriptase (hTERT)25, leads to an increase in the proliferative capacity of neoplastic cells, indicating high lethality and therapeutic failure. Considering the lack of information and the need to understand the importance of new mechanisms of action of promising cytotoxic drugs, this study evaluated the

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role of biflorin in cell viability, clonogenicity, motility, differentiation, telomere length and MYC amplification in the ACP02 gastric cancer cell line.

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2. Materials and Methods

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2.1 Chemicals

Doxorubicin hydrochloride (Adriamycin, CAS no. 25316-40-9), Alamar BlueTM (CAS

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no. 153796-08-8 St. Louis, MO, USA) and penicillin/streptomycin (USB, Cleveland, OH, USA) were purchased from Sigma Aldrich, Co. DMSO (dimethylsulfoxide) (CAS

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no. 67-68-5), DMEM (Dulbecco’s modified Eagle’s medium) growth medium and fetal calf serum were purchased from Gibco® (Invitrogen, Carlsbad, CA, USA). Crystal

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violet (CAS no. 548-62-9), methanol (Cas no. 67-56-1), glacial acetic acid (CAS no. 64-19-7), KCl (CAS no. 74-47-40-7), and 12-O-tetradecanoylphorbol-13-acetate

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(TPA) (CAS no. 16561-29-8) were purchased from Merck.

2.2 Isolation of biflorin

C. biflora was identified by Dr. Edson Paula Nunes (Botany Professor of Federal University of Ceará, Biology Department, Prisco Bezerra Herbarium). A voucher specimen (number: 30848) was then deposited in the Herbarium Prisco Bezerra of the Biology Department of the Federal University of Ceará. Biflorin (Fig. 1) was obtained as previously described26 and was always supplied at greater than 95% purity. Stock solutions were made by dissolving biflorin in DMSO (CAS no. 67-68-5) immediately prior to use. The DMSO concentrations never exceeded 0.1% in culture and resulted in 100% cell viability when used as a control27,28.

Journal Pre-proof 2.2 Cell lines and cell culture ACP02 (human gastric adenocarcinoma), AGP01 (human gastric ascites), PG100 (human gastric adenocarcinoma) and MRC5 (normal human fibroblast) cell lines were obtained from João de Barros Barreto, University Hospital (HUJBB) in Pará State, Brazil. The cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2. The cells were split every 3 days and were diluted 1 day before each experiment.

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2.3 Cytotoxicity

ACP02, AGP01, PG100 and MRC5 cells were seeded in 96-well plates (0.5x104 cells per well) and treated with biflorin, and the Alamar BlueTM assay was

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performed29. Biflorin (1.25 to 20 µM) was added to each well and incubated for 24, 48 or 72 h. Doxorubicin (0.078 to 5 μM) was used as a positive control. Control

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groups received the same amount of DMSO (0.1%). Two hours before the end of the incubations, 10 µL of alamarBlueTM was added to each well. The fluorescent signal

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was monitored using a multiplate reader with a 530-560 nm excitation wavelength and a 590 nm emission wavelength. The fluorescent signal generated from the

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assay was proportional to the number of living cells in the sample, according to the

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manufacturer’s instructions.

2.4 Analysis of morphological changes

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Untreated and biflorin-treated ACP02 cells were examined for morphological changes by light microscopy (Leica DM500, Heerburgg, Swiss). To evaluate morphology, cells were harvested, transferred to cytospin slides, fixed with ethanol for 1 min, and stained with hematoxylin–eosin. TPA (0.16 μM) was used as the positive control9. 2.5 Clonogenic assay (clone formation assay) After biflorin treatment, cells (400 cells/per well) were incubated in complete medium at 37 °C in a humidified atmosphere containing 5% CO2 for 7 days. Colonies were washed with PBS, fixed with methanol (p.a.), stained with 0.1% crystal violet, and counted, and the survival is expressed as a percentage relative to that of the negative control treatment30.

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2.6 In vitro migration assay The capacity of ACP02 cells to spread and migrate was assessed using a Boyden chamber transwell assay, which evaluates the expansion of a cell population through a chamber with an 8 μm pore polycarbonate filter. The cells were seeded into cell culture dishes for 24 h at 37 ºC and treated with 0.1% DMSO (control group) and biflorin (1.0 μM, 2.5 μM and 5.0 μM) for 12 h. Next, cells were detached with trypsin and plated in 24-well plates at a concentration of 2×10 5 cells/well in serum-free DMEM. DMSO (0.1%; control group) and biflorin (1.0 μM, 2.5 μM or 5.0 μM)

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dissolved in serum-free DMEM were added to the wells to complete the 24 h of treatment in an incubator at 37 °C with 5% CO2. In the lower part of the chamber, DMEM supplemented with 10% FBS was applied to promote chemoattraction. At the

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end of the incubation, the cells on the upper surface of the membrane were removed with a cotton swab. The cells that migrated to the bottom of the chamber were fixed

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with 4% paraformaldehyde for 2 min and permeabilized with methanol for 20 min before being stained with Giemsa for 15 min. Seven representative images from

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each well were taken to estimate the relative migration of cells. The cells were

2.7 Matrigel assays

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counted using ImageJ software. The experiments were performed in triplicate.

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To analyze the invasion capacity of ACP02 cells treated with biflorin against a negative control (0.1% DMSO), a Matrigel assay was performed. First, a layer of

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1:10 v/v Matrigel (#356230; BD Biosciences, Oxford, UK) was prepared, added to the Boyden chamber and incubated overnight at 37 ºC. The cells were seeded into cell culture dishes, incubated for 24 h at 37 ºC and treated with 0.1% DMSO (control group) or biflorin (1.0 μM, 2.5 μM or 5.0 μM) for 12 h. Then, the cells were detached with trypsin and plated in 24-well plates at a concentration of 2×105 cells/well on top of the Matrigel in serum-free DMEM. DMSO (0.1%; control group) and biflorin (1.0 μM, 2.5 μM or 5.0 μM) dissolved in serum-free DMEM were added to the wells to complete the 24 h of treatment in an incubator at 37 °C with 5% CO2. In the lower part of the chamber, DMEM supplemented with 10% FBS was applied. At the end of the incubation, the cells on the upper surface of the membrane were removed with a cotton swab. The cells that migrated to the bottom were fixed with 4% paraformaldehyde for 2 min and permeabilized with methanol for 20 min before

Journal Pre-proof staining with Giemsa for 15 min. Seven representative images from each well were taken to estimate the relative migration of cells. The cells were counted using ImageJ software. The experiments were performed in triplicate.

2.8 NBT reducing activity ACP02 cells (2.5 x 104 cells/mL) were treated with biflorin at 1, 2.5 and 5 μM in DMEM containing 10% FBS for 24 and 72 h, after which the NBT (nitro blue tetrazolium) reducing activity was determined by the method described by Kohroki and collaborators31. Briefly, after the incubation, the cells were harvested, mixed with

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freshly prepared solution containing 1 μg/mL TPA and 2 mg/mL NBT, and incubated for 30 min at 37 °C. The reaction was stopped by the addition of cold PBS (3 mL). The suspension was centrifuged, resuspended in PBS and transferred to Cytospin

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slides, and the cells were then fixed with ethanol for 1 min and stained with eosin. Two hundred cells were counted using light microscopy. TPA at 0.16 μM was used

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as the positive control.

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2.9 Fluorescent in situ hybridization analysis For FISH analysis, metaphasic and/or tumoral interphasic nuclei, before and after

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treatment with biflorin (1, 2.5 or 5.0 μM), were hybridized with a probe specific for a region of the MYC gene (8q24.12–q24.13) (ONPONC0824; Bioagency®). The

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hybridization was visualized by a fluorescence microscope (Olympus BX41) with a double filter (FITC/TRITC) and a capture and image analysis system (Applied

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Spectral Imaging®). Two hundred interphasic nuclei and/or metaphase events were evaluated per slide, and the fluorescent signals were verified. Untreated normal lymphocyte nuclei were used as negative controls, whereas untreated ACP02 cells were used as positive controls for MYC amplification32,33. For fluorescence in situ hybridization with probes to telomeric sequences (TELOFISH), the method from Link (2016)30 and Pennarun et al. (2010)35 was used. Nuclei obtained from temporary culture of human lymphocytes, using the protocol established by Preston et al. (1987)36, were fixed (3:1 methanol:acetic acid) on slides. The slides were hybridized with a telomeric probe, pan-telomeric Star FISH, following the manufacturer's recommendations. A measurement of telomere length, determined in each interphase nucleus, was acquired by the Applied Spectral

Journal Pre-proof Imaging image analysis system. The images were processed using the TFL-TELO software following the manufacturer’s protocol37.

2.10 Quantitative reverse transcription real-time PCR (qRT-PCR) Total RNA was extracted with Tri-reagent (Life Technologies, USA) according to the manufacturer's instructions. RNA concentration and quality were determined using a NanoDrop spectrophotometer (Kisher, Germany). Complementary DNA was synthesized using the High-Capacity cDNA Reverse Archive kit (Life Technologies, Poland) following the manufacturer's instructions. Real-time qRT-PCR primers and

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Taqman probes targeting MYC (Hs00153408_m1) and hTERT (Hs00972656_m1) were purchased as Assays-on-demand Products for Gene Expression (Life Technologies, USA) and applied by following the manufacturer's instructions. B2M

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(Hs00984230_m1) and ACTB (Hs03023943_g1) were selected as internal controls for RNA input and reverse transcription efficiency with the TaqMan Human

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Endogenous Control (Life Technologies, USA). All real-time qRT-PCRs for the target gene and internal control were performed in triplicate on the same plate. Gene

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expression values were calculated using the ΔΔCt method38, where for each sample,

2.11 Statistical analysis

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the nontreated corresponding sample was designated as a calibrator.

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For cytotoxicity, the IC50 values and their 95% confidence intervals (95% CI) were obtained by nonlinear regression. In the clonogenic assay, the statistical analyses

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were performed by one-way ANOVA. For migration and invasion, a one-way Student’s t test was performed. For NBT, statistical analyses were performed by twoway ANOVA. For the FISH assay, two-way ANOVA was used. GRAPHPAD 5.0 was used for all analyses (Intuitive Software for Science, San Diego, CA). Results were considered statistically significant at p< 0.05.

3. Results

3.1 Biflorin displayed in vitro cytotoxicity in gastric cancer cell lines To determine the capacity of biflorin to kill gastric cell lines in vitro, the cell lines ACP02 (human gastric adenocarcinoma), AGP01 (human gastric ascites), PG100 (human gastric adenocarcinoma) and MRC5 (normal human fibroblast) at 0.5x104

Journal Pre-proof cells/plate were treated with biflorin for 24 or 72 h and analyzed by alamarBlue assay. Table 1 shows the obtained IC50 values. The IC50 values ranged from 0.73 µM in MRC5 to 7.08 µM in PG100. Doxorubicin showed IC50 values from 0.1 µM in MRC5 to 5.89 µM in AGP01. All subsequent experiments were carried out in ACP02 gastric cancer cells. For mechanistic purposes, the experiments were conducted after 24 h or 72 h of incubation at concentrations of 1.0, 2.5, or 5.0 µM biflorin.

3.2 Biflorin inhibits the formation of gastric cell colonies in vitro Neoplastic cells normally grow near other cells after the arrival of stimulating growth

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factors; after one cell begins to grow, the others form truly close colonies. To determine the inhibition of proliferation, we performed clonogenic assays over 7 days using 1.0, 2.5 or 5.0 μM biflorin (Figs. 2-3) at 400 cells/per well, and the results

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showed a progressive inhibition of the formation of cell colonies.

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3.3 Biflorin inhibits the migration and invasion of gastric cells in vitro Gastric cancer cells become metastatic cells and generally migrate and

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progressively invade other tissues, which is part of the metastasis process. After treatment with 1.0, 2.5 or 5.0 μM biflorin, ACP02 cells plated in Boyden chambers

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with or without Matrigel presented a significant reduction in migration and invasion

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(Fig. 4) compared to the negative control cells.

3.4 Biflorin induces the differentiation of gastric cells in vitro

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To confirm the ability of biflorin to induce cell differentiation, we performed NBT reduction assays. After 24 h, the cells exposed to biflorin at all doses began to differentiate. Biflorin-induced cell differentiation peaked at 72 h of incubation (Fig. 5).

3.5 Molecular cytogenetic analysis MYC amplification and telomere size were evaluated by FISH. Of the 200 cells analyzed after biflorin treatment (2.5 or 5 µM), the cells that had been incubated for 24 h or 72 h showed a significant increase in the percentage of cells exhibiting 1–2 signals and a proportional reduction in those exhibiting 3-5 signals in the MYC amplification assay (Tab. 2). For telomeres, we observed a significant elongation reduction in biflorin-treated cells (5.0 µM - 24 or 72 h) (Tab. 3).

Journal Pre-proof 3.6 Relative quantification (RQ) of MYC and hTERT mRNA The MYC gene expression was assessed by qRT-PCR in AGP02 cells after biflorin treatment (1.0, 2.5 and 5.0 µM). The MYC and hTERT mRNA levels were significantly reduced after treatment with biflorin (2.5 μM and 5.0 μM) compared with the negative control (p < 0.005), as shown in Fig. 6.

4. Discussion

Gastric cancer is one of the most aggressive cancers in the world. Patients

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with inoperable, recurrent or metastatic tumors are incurable, and the prognosis is only a few months of life with the best supportive care. Despite progress in recent decades, metastatic gastric cancer remains an incurable disease. The drugs used

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for treatment are not efficient. New strategies for the future include tailored interventions based on new cytotoxic drugs, targeted therapies, identification of

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predictive or prognostic markers and integration of molecular determinants that may

drugs is a constant search.

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help to select patients likely to benefit from treatment39. Therefore, screening for new Biflorin, an -naphthoquinone isolated from C. biflora, has been shown to be

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cytotoxic to several cancer cell lines in vitro and in vivo models9-15. In this work, the ACP02 cell line was chosen as a model to evaluate the potential of biflorin against

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gastric tumors. Several studies8,12,40 have shown the cytotoxicity of biflorin towards

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multiple cancer cell lines; however, this is the first time that these effects were shown in gastric cancer cells. Biflorin displayed cell growth inhibition in both normal and cancer cells, but Vasconcellos and collaborators9-15 demonstrated that biflorin is not genotoxic and has protective effects against free radicals in normal V79 cells. Furthermore, mutagenic models using Saccharomyces cerevisiae and Salmonella typhimurium revealed that biflorin is not mutagenic, which indicates that biflorin is safe at the tested concentrations. Metastasis is a hallmark of cancer and a major cause of gastric cancer death and also has a significant influence on therapeutic response. In addition to growth inhibition, biflorin affected the migration of ACP02 gastric cells. Quinones have demonstrated similar activities6,41,42. β-lapachone, a natural 1,2-naphthoquinone, showed various anticancer activities in vitro and in vivo. The biflorin and β-lapachone

Journal Pre-proof structures differ only on the side chain formed from the heterocyclic ring; biflorin is three carbon units longer than β-lapachone and thus is more liposoluble6. Moreover, biflorin has a heterocyclic ring linked to the 4,5 naphthoquinone carbon skeleton ring, while β-lapachone has a 3,4 carbon ring6. Kim et al. (2007)43 showed that βlapachone inhibited the migration of both HepG2 and Hep3B cells in a woundhealing model. These findings were associated with a dramatic increase in erg-1 and thrombospondin-1 protein levels and a downregulation of Snail and upregulation of E-cadherin expression. The same experiments were performed by Manu et al. (2011)44 using plumbagin on breast and gastric cancer cells, and the results showed

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that plumbagin could inhibit CXCL12-induced migration and invasion when CXCR4 expression was suppressed. Chia et al. (2010)45 tested the Chinese herbal cocktail Tien-Hsien liquid, which could inhibit the migration and invasion of MDA-MB-231,

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H1299, PC-3, and CT-26 cancer cells, as determined by Boyden chamber transwell assays. An anthraquinone, aloe-emodin, showed the same inhibitory effects and was

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associated with the suppression of MMP-2 and MMP-9 protein expression46. All these findings suggest that quinone is a scaffold that can inhibit cell migration as

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observed in biflorin-treated ACP02 cells.

Cell differentiation is essential for normal growth, and dedifferentiation is a key

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process in the development of malignant tumors. Therefore, drug induction of redifferentiation of tumor cells is an important mechanism of some anticancer

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chemotherapies, and discovery of differentiation-inducing factors is a critical strategy for drug development47. The changes in morphology first indicated that biflorin

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induces cell redifferentiation. The ability of treated cells to reduce NBT implies functional redifferentiation, and the results demonstrated a time- and dosedependent effect on NBT reduction, which was maximal at 72 h with 5.0 µM (Fig. 5). Other studies with gastric cell lines demonstrated different pathways responsible for these phenomena. Ling et al. (2006)48 showed that a substance extracted from garlic (diallyl disulfide) induced cell differentiation of MGC803 cells by ERK pathway inhibition. In a study with SGC7901 cells, Zhang et al. (2011)47 showed that cells treated with melatonin demonstrated greater morphological differentiation that untreated cells, but the authors correlated the results to the negative balance of alkaline phosphatase and lactate dehydrogenase, enzymes involved in differentiation of gastric tissue. Another work with SGC7901 cells demonstrated that p27, but not p21, is an important protein involved on cell differentiation of gastric cancer49.

Journal Pre-proof MYC is a oncogene commonly deregulated in gastric cancer that is mainly involved in cell cycle regulation, differentiation and growth arrest 50. MYC is an important regulator of cell growth, and enhanced expression of MYC contributes to almost every aspect of tumor cell biology, including angiogenesis, dedifferentiation, cell adhesion reduction and metastasis promotion51,52. Our group found that biflorintreated cells (2.5 and 5.0 µM) showed a significant reduction in MYC amplification (Tab. 2) and significantly reduced expression of MYC (Fig 6). Bretones et al. (2015)53 suggested that inhibition of MYC expression and downregulation or inactivation of MYC in cycling cells resulted in cell cycle arrest and impaired cell

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cycle progression. Calcagno et al. (2006)54 and (2013)55 demonstrated the association of the increase in the number of C-MYC alleles with poor prognosis in human neoplasias, as well as increased C-MYC mRNA expression with the

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presence of lymph node metastasis and advanced gastric cancer. These findings suggested that C-MYC deregulation is a strong factor for malignancy in GC. A recent

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study by Pinto et al. (2019)56 showed that MYC inhibition led to apoptosis in a gastric cancer cell line. All these results suggest that MYC inhibition can lead to apoptosis

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and thus can be a biomarker to inhibit in gastric cancer. Telomeres, the DNA-protein complexes present at the end of eukaryotic

as

degradation

of

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chromosomes, are protective against events that promote genomic instability, such the

chromosome

terminal

regions

and

inappropriate

a

specialized

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recombination. These chromosomal regions are specific to the action of telomerase, ribonucleoprotein

reverse

transcriptase

that

helps

maintain

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telomeres57. Telomerase activity is suppressed in most normal human somatic cells, and these cells gradually lose part of the telomeres every cell cycle due to a lack of telomerase activity58. Increased telomerase activity and telomere enhancement are found in most cancer cells and are associated with immortalization and unlimited cell division. This specific expression in affected carcinogenic tissues identifies telomeres as targets for anticancer therapy59. In this study, we observed that biflorin (5.0 µM) reduced telomere length after 24 and 72 h of treatment (Tab. 3). β-lapachone, a naphthoquinone, was shown to decrease telomerase activity in human leukemia cells (U937, K562, HL60, and THP-1)60 and in human prostate DU145 carcinoma cells61. In summary, our study showed that biflorin has cytotoxic and antiproliferative activity associated with the inhibition of migration and invasion of the ACP02 gastric

Journal Pre-proof cell line and reduced MYC expression and telomere length. These findings provide important new insights into the possible molecular mechanisms of this group of substances, and biflorin could be considered a new therapeutic approach for gastric cancer as a promising antitumor molecule or lead molecule for new drug design with minor toxicological effects14.

5. Conclusions

In conclusion, we have shown that biflorin affects cell survival, clonogenicity, and

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motility, which are hallmarks of cancer. In addition, biflorin is associated with a reduction in MYC amplification, and telomere length is also affected. This length reduction confirms that this substance may reduce cell immortalization in the ACP02

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cell line.

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Acknowledgments

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The authors are grateful to the Brazilian agencies CNPq, FAPEAM and CAPES for

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fellowships and financial support.

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There is no conflict of interest to declare.

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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[4] Jiang, Y., Ajani, J.A. 2010. Multidisciplinary management of gastric cancer. Curr. Opin. Gastroenterol. 26, 640-646.

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[6] Montenegro, R.C., Burbano, R.R., da Silva, M.N., Lemos, T.G., Vasconcellos, M.C. 2013. Biflorin, A Naphthoquinone, Inhibitsegfr in Breast Cancer Cells. Med chem 3, 179-182.

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[7] Kiyozumi, Y., Iwatsuki, M., Yamashita, K., Koga, Y., Yoshida, N., Baba, H. 2018. Update on targeted therapy and immune therapy for gastric cancer. J. Cancer Metastasis Treat. 4, 31.

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Figures

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Figure 1. Chemical structure of biflorin (6,9-dimethyl-3-(4-methyl-3-pentenyl)naphtha[1,8bc]-pyran-7,8-dione).

2.5

5.0

B

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A

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Figure 2. Morphological alterations in ACP02 cells stained with hematoxylin/eosin 72 h after biflorin treatment. (C) Untreated cells - 400x; (1) cell treated with 1.0 µM biflorin - 200x; (2.5) cells treated with 2.5 µM biflorin - 200x; (5.0) cells treated with 5.0 µM biflorin - 400x. Black arrows: necrotic cells.

Figure 3. A – Effect of biflorin on the numbers of colonies and B – Morphological analysis of ACP02 colonies stained with crystal violet. Experiments were performed during 10 days of treatment. (C) Untreated cells; (1), (2.5) and (5.0), cells treated with biflorin at 1.0, 2.5 and 5.0 µM - 400 x. *p < 0.05 compared to the negative control. ANOVA + Bonferroni post-test.

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Migration (mm)

0.03

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Control

1.0

0h

2.5

24h

5.0

48h

Figure 4. Migration of ACP02 cells after scratching the cell layer; 0, 24 and 48 h - 50x. The vehicle 0.1% DMSO was used as the (C) negative control; (1.0), (2.5) and (5.0), cells treated with biflorin at 1.0, 2.5 and 5.0 µM. The experiments were performed in triplicate. *p < 0.05 compared to the negative control. Student’s t test.

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72 h

80

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60

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Number of cells (%)

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MYC

hTERT

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RQ of mRNA expression

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Figure 5. ACP02 differentiation analysis by NBT tests after 24 and 72 h of treatment; 24 h (control) untreated cells, (1.0), (2.5) and (5.0), cells treated with biflorin at 1.0, 2.5 and 5.0 µM, (TPA) cells treated with TPA at 0.16 µM; 72 h (control) untreated cells, 1.0, 2.5 and 5.0, cells treated with biflorin at 1.0, 2.5 and 5.0 µM, (TPA) cells treated with 0.16 µM TPA. *p < 0.05. ANOVA.

0.0

hTERT

MYC

C 1.0 2.5 5.0

hTERT

*

*

0.5

MYC

24h

*

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* *

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48h

*

*

*

72h

Figure 6. Relative mRNA quantification of MYC and hTERT gene levels in ACP02 cells after treatment with biflorin at 1.0, 2.5 and 5.0 µM after 24, 48 and 72 h. The experiments were performed in triplicate. *p < 0.05 compared to DMSO (negative control). ANOVA + Bonferroni post-test.

Journal Pre-proof Tables Table 1 - Cytotoxic activity of biflorin and doxorubicin in the gastric malignant cell lines ACP02, AGP01, and PG100 and in normal fibroblast cell line MRC5. IC50 values are expressed in M. *p < 0,05 MRC5 x other cell lines; #p < 0,05 biflorin x doxorubicin. Nonlinear regression.

ACP02 AGP01 PG100 MRC5

Biflorin 24 h 3.46 (2.83 – 4.22) 5.63# (3.08 – 10.30) 7.08 (6.26 – 8.00) 4.91 (4.27 – 5.66)

Biflorin 72 h 1.92 (1.7 – 2.19) 3.44# (3.32 – 3.56) 6.35 (5.67 – 7.11) 0.73 (0.62 – 0.85)

Doxorubicin 24h 1.12 (0.84-1.50) < 10 0.45* (0.38 - 0.52) 0.98 (0.75 -1.27)

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Cell Lines

Doxorubicin 72h 1.30 (1.21 – 1.41) 5.89 (4.76 - 7.30) 0.43 (0.37 - 0.51) 0.10 (0.086 - 0.13)

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Table 2 - FISH analysis of the MYC copy number in the gastric cancer cell line ACP02. The FISH analysis was performed on 200 nuclei. HA: High amplification.*p > 0.05 compared to the negative control. ANOVA. Nuclei exhibiting MYC signals, no. (200 nuclei)a 4 signals

≥5 signals

HA

69 (34,5)

82 (41)

29 (14.5)

8 (4)

72 (36)

79 (39.5)

23 (11.5)

9 (4.5)

11 (5.5)

65 (32.5)

81 (40.5)

27 (13.5)

11 (5.5)

6 (3)

15 (7.5)

66 (33)

78 (39)

25 (12.5)

10 (5)

2.5 24h

6 (3)

19 (9.5)*

72 (36)

75 (37.5)

20 (10)*

8 (4)

2.5 72h

8 (4)*

21 (10.5)*

77 (38,5)

74 (37)

17(8.5)

3 (1.5)

5.0 24h

9 (4.5)*

31 (17.5) *

81 (40.5)

69 (34,5)

9 (4.5)*

1 (0.5)

5.0 72h

9 (4.5)*

37 (18.5)*

89 (44.5)*

60 (30)*

4 (2)*

1 (0.5)

C 24 h

3 (1.5)

9 (4.5)

C 72 h

4 (2)

13 (6.5)

1.0 24h

5 (2.5)

1.0 72h

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2 signals

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3 signals

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Table 3 - Telomere fluorescence intensity in human chromosomes as measured by a pan telomeric probe. #indirect relative measurement of the size of telomeres (units of TFL-TELO software). *p > 0.05 compared to the negative control. ANOVA. Treatment (µM)

Allarms (mean#)

24 h

0.0

12.54

72 h

0.0 1.0

11.96 11.10

1.0 2.5

10.87 10.90

24h

2.5 5.0

8.30 7.00*

72h

5.0

6.52*

24h 72h 24h 72 h

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HIGHLIGHTS

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

Biflorin was shown to be a powerful cytotoxic agent. Biflorin affected cell survival, clonogenicity and motility. Biflorin was associated with a reduction in MYC amplification and changes in telomere length. Biflorin reduced cell immortalization in the ACP02 cell line.

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  