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mTOR pathway
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Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway Qing Xiao, Jiayi Ying, Zhuhui Qiao, Yiwen Yang, Xiaoxi Dai, Zhongyi Xu, Chengfeng Zhang* , Leihong Xiang* Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 December 2019 Received in revised form 4 February 2020 Accepted 14 February 2020
Background: : Melanoma is one of the most aggressive, therapy-resistant skin cancers in the world. Hydrogen sulfide (H2S), a newly discovered gasotransmitter, plays a crucial role in the progression and development of many types of cancers. However, the effect of H2S on human skin melanoma remains to be elucidated. Objective: : We aimed to explore the effect of exogenous H2S on melanoma cells and its underlying mechanisms. Methods: : In this study, human skin melanoma cell lines, including A375 and SK-MEL-28, were treated with a donor of H2S (NaHS). CCK-8, scratch assay, flow cytometric analysis, western blotting and transmission electron microscopy (TEM) were performed to explore the effects of H2S on cell behaviors. Results: : Treatment with NaHS inhibited cell proliferation, migration and division, while it could induce cell apoptosis and autophagy in melanoma cell lines. Moreover, NaHS significantly decreased the expression of p-PI3K, p-Akt and mTOR proteins. Furthermore, insulin-like growth factor-1 (IGF-1), the activator of PI3K/AKT/mTOR pathway, could reverse the cell behaviors caused by NaHS. Conclusion: : Our results demonstrated that exogenous hydrogen sulfide could inhibit human melanoma cell development via suppression of the PI3K/AKT/mTOR pathway. Hydrogen sulfide might serve as a potential therapeutic option for melanoma. © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Keywords: Hydrogen sulfide Melanoma PI3K/AKT/ mTOR pathway
1. Introduction Melanoma, also known as malignant melanoma, is the most dangerous type of skin cancer [1], since it is much more likely to invade nearby tissues and spread to other parts of the body. Most deaths from skin cancers are caused by melanoma. Globally, in 2015 there were 3.1 million new diagnosis of melanoma and
Abbreviations: H2S, hydrogen sulfide; NaHS, Sodium hydrosulfide; TEM, transmission electron microscopy; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p-PI3K, phosphorylated-PI3K; p-Akt, phosphorylated-Akt; mTOR, mammalian target of rapamycin; IGF-1, insulin-like growth factor-1; NO, nitric oxide; CO, carbon monoxide; CSE, cystathionine g-lyase; CBS, cystathionine βsynthase; 3-MST, 3-mercaptopyruvate sulfurtransferase; NF-kB, nuclear factor-kB; RNA, ribonucleic acid; FBS, fetal bovine serum; ATG7, autophagy-related protein 7; shRNA, short hairpin RNA; DAPI, 4'6-diamidino-2-phenylindole; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; LC3, microtubule-associated protein light chain 3; ER, endoplasmic reticulum. * Corresponding authors at: Department of Dermatology, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai, 200040, PR China. E-mail addresses: [email protected] (C. Zhang), fl[email protected] (L. Xiang).
59,800 deaths [2]. The incidence of melanoma is still rising rapidly in recent years. The prognosis of patients is often poor mainly due to the high metastasis rate and drug resistance. Although immunetargeted strategies have shown marked progress in some patients, their effects on overall survival remain variable [3]. Therefore, new therapeutic approaches are urgently required to optimize current treatment for melanoma. Gasotransmitter hydrogen sulfide (H2S), the third gaseous signaling molecule along with nitric oxide (NO) and carbon monoxide (CO), can be produced enzymatically in mammals under normal conditions by the catalysis of two pyridoxal-50 -phosphatedependent enzymes, cystathionine g-lyase (CSE), cystathionine βsynthase (CBS) and a pyridoxal-50 -phosphate-independent enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST) [4]. Recently, a growing body of evidence has revealed that H2S exerts a variety of physiological and pathological functions in cell growth, proliferation and differentiation [5]. This gas has been found to relax blood vessels [6], shield neurons and cardiac muscles from oxidative stress [7] and possess different properties on cancer across a wide range of doses. A relatively high concentration of exogenous H2S could exhibit antitumor effects on human colon
https://doi.org/10.1016/j.jdermsci.2020.02.004 0923-1811/ © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Q. Xiao, et al., Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2020.02.004
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cancer cells and HepG2 cancer cells mainly through induction of cell cycle arrest, facilitation of uncontrolled intracellular acidification and acceleration of apoptosis [8]. Overexpression of CSE in human melanoma cells could induce spontaneous apoptosis by suppressing the activity of nuclear factor-kB (NF-kB) and decreasing the expression of anti-apoptotic proteins [9]. However, there has been little research about the effect and mechanism of exogenous H2S on melanoma. In this study, we aimed to explore the effect of exogenous H2S on melanoma cells and its underlying mechanisms, in order to provide new insight into the alternative strategies for melanoma treatment. To this end, we first observed the effect of H2S on melanoma cell proliferation, migration, apoptosis, division and autophagy. We then blocked autophagy by ATG7 shRNA in melanoma cell lines to examine whether autophagy plays a role in NaHS-modulated cell behaviors. Further, we investigated the underlying molecular mechanisms involved in the above cell behaviors induced by H2S treatment. 2. Materials and methods 2.1. Reagents Sodium hydrosulfide (NaHS, H2S donor) was ordered from Sigma Aldrich (Shanghai, China), IGF-1 was ordered from Novoprotein, rapamycin was from Selleck. Primary antibodies against LC3, mTOR, Akt, phospho-Akt, PI3k, Bcl-xL, Cleaved Caspase-3 were purchased from Cell Signaling Technology, antiSQSTM1/p62 and anti-ATG7 were purchased from Abcam, phospho- PI3k was from Affinity and anti-β-actin from Proteintech. Secondary antibodies were goat anti-rabbit IgG and goat antimouse IgG also from Cell Signaling Technology. To knockdown ATG7 gene expression, melanoma cells were transfected with specific short hairpin ATG7 RNA (shRNA, 50 -ATGATCCCTGTAACTTAGCCCA -30 and 50 -CACGGAAGCAAACAACTTCAAC -30 ). The shRNA and negative control shRNA (shNC) were purchased from GeneChem (Shanghai, China). 2.2. Cell culture and treatment Human melanoma cell lines A375 and SK-MEL-28 were purchased from Shanghai Institute of Biochemistry and Cell Biology, CAS (Cell Bank/Stem Cell Technology platform), cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) (Gibco, CA) at 37 C in a 5% CO2 incubator. NaHS and IGF-1 were solubilized in sterile phosphate buffer saline (PBS). Rapamycin was solubilized in DMEM. A375 and SK-MEL-28 cells were treated with NaHS (0, 0.5, 1, 2, 5, 10 mM) for 24 h or with IGF-1 (100 ng/mL) for 24 h or rapamycin (100 nM) for 12 h. Cells were transfected with ATG7 shRNA or shNC using the transfection reagent for 72 h according to the manufacturer's protocol. 2.3. Western blotting Protein lysates extracted from melanoma cell lines were separated by 15 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretic transfer of proteins from gels onto polyvinylidene difluoride membrane (Thermo Fisher Scientific, MA, USA) at 100 V for one and a half hour at 4 C. After blocking in 5% bovine serum albumin in Tris Buffered Saline Tween (TBST) to inhibit nonspecific binding, the membranes were incubated with primary antibodies overnight at 4 C, washed three times with TBST, and then incubated with peroxidase-coupled secondary antibodies for 1 h at room temperature. Membranes were washed again in TBST three times
at room temperature. Protein bands were visualized by incubation using an enhanced chemiluminescence (Thermo Fisher Scientific, MA, USA) and were exposed to X-ray film. Western blotting was repeated at least three times for each experiment. 2.4. Cell proliferation analysis The Cell Counting Kit (CCK-8) (Dojindo, Kumamoto, Japan) was employed to evaluate the effect of NaHS, sh-ATG7 and IGF-1 on cell proliferation at indicated time points. Cells were seeded in 96-well plates in quintuplicate at densities of 5*103 cells per well and subjected to various treatments before analysis by the CCK-8 assay. Absorbances were measured at 450 nm wavelength on a Microplate Reader (Model 680, Bio-Rad). Cell proliferation was calculated using the formula: Cell proliferation% = (450 sample background)/ (450 control–background) 100 %. 2.5. Wound healing assay 1*106 melanoma cells were seeded and grown per well into 6well culture plate. After allowing them to completely attach, cells were scratched with a sterile 200 ml pipette tip in 3 separate places in each well. All the wound sizes were verified to make sure they were all the same width. After washing by PBS for three times, we changed the culture medium to DMEM without FBS. Cells were subjected to different treatments as: negative control, NaHS (2 mM), IGF-1 (100 ng/mL), IGF-1 with NaHS for 24 h. The wound was viewed and captured under a light microscope at respective intervals (0 h and 24 h) with a Zeiss microscope system (Zeiss Axivert 400C, Germany). The data are representative of 3 independent experiments. 2.6. Cell apoptosis analysis A375 and SK-MEL-28 were cultured in DMEM with 10 % FBS and then subjected to various treatments as: negative control, NaHS (2 mM), IGF-1 (100 ng/mL), IGF-1 with NaHS for 24 h, then harvested by trypsinization, then washed twice with PBS, and resuspended in binding buffer. Annexin V-FITC and Propidium iodide (PI) solution (Beyotime Institute of Biotechnology) were added for 20 min at room temperature in the dark before the samples were analyzed by flow cytometry (Beckman Coulter, Miami, FL). 2.7. Cell cycle assay 2*105 cells were seeded and grown per well into a 6-well culture plate with DMEM containing 10 % FBS, and were allowed to attach overnight. The cells were treated with distinct treatments as: negative control, NaHS (2 mM), IGF-1 (100 ng/mL), IGF-1 with NaHS for 24 h, then harvested by trypsinization, washed in cold PBS for 2 times and fixed in precooled anhydrous ethanol in a 4 ℃ refrigerator overnight. On the next day, centrifuged at 4 C and resuspended in cold PBS. Bovine pancreatic RNAase (Sigma– Aldrich) was added and incubated at 37 C for half an hour, then 250 mg/ml propidium iodide (Sigma–Aldrich) was added and the cell suspension was filtered with 350-mesh nylon membrane for testing. In each group, 50,000 cells were analyzed by flow cytometry (Beckman Coulter, Miami, FL). 2.8. Confocal microscopy for GFP-mRFP-LC3 A375 and SK-MEL-28 were seeded in 35-mm confocal dishes. When cells reached 70 % confluence, tandem fluorescent mRFPGFP-LC3 adenoviruses (Hanheng, Shanghai, China) were transfected into the cells for 2 h. Tandem fluorescent mRFP-GFP-LC3 allows to measure the extent of autophagosome and autolysosome
Please cite this article in press as: Q. Xiao, et al., Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2020.02.004
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formation simultaneously, because LC3 puncta labeled with both GFP and mRFP represent autophagosomes before the fusion with lysosomes, then the GFP fluorescence is quenched in the acidic pH of the lysosomal compartment, so those labeled with mRFP alone represent autolysosomes. After 24 h, the cells were treated with or without 2 mM NaHS for 24 h. Then the melanoma cells were fixed with 4% paraformaldehyde and cell nuclei were visualized by staining with 4,6-diamidino-2-phenylindole (DAPI). The images were observed with a confocal laser scanning microscope (Leica TCS SP5, Germany) to indicate the formation of GFP-LC3 and mRFPLC3 puncta, indicative of autophagosomes and/or autolysosomes. The number of GFP and mRFP puncta per cell was manually quantified. At least 20 cells per dish were randomly analyzed. The experiments were performed at least three times. 2.9. Transmission electron microscopy (TEM) Cells were fixed in 4% glutaraldehyde overnight at 4 C and rinsed with precooled PBS for 3 times, then cells were post-fixed in 1% osmium tetroxide (OsO4) at 4 C. After that, PBS washed again, samples were dehydrated with graded alcohol and embedded in epon-araldite resin. Ultrathin sections were obtained by an ultramicrotome. Sections were then stained with uranyl acetate and lead citrate. The ultrastructures of cells were observed and imaged under JEM-1220 TEM (JEOL, Tokyo, Japan). 2.10. Statistical analysis The results were expressed as mean standard deviation on at least three independent experiments. Statistical analysis was calculated by two-tailed Student’s t-test (SPSS 17.0 SPSS Inc, Chicago, IL), with *P < 0.05; **P < 0.01; ***P < 0.001 considered to be statistically significant. 3. Results 3.1. NaHS suppressed cell proliferation, migration, division and induced apoptosis of melanoma cell lines To investigate the functional role of NaHS in melanoma cell lines, cell proliferation was determined by CCK8 assay. As shown in Fig. 1a and b, cell proliferation was inhibited in both cell lines after treatment with NaHS (0, 0.5, 1, 2, 5, 10 mM) in a dose-dependent manner (A375: 100 %3.69, 96.73 % 3.42, 88.43 % 1.98, 82.92 % 2.99, 76.44 % 2.87, 70.37 % 3.74; SK-MEL-28: 100 % 4.95, 94.31 % 2.52, 84.20 % 2.72, 82.01 % 3.68, 80.93 % 4.79, 65.60 % 6.2, respectively). The wound healing assay was performed to evaluate the effect of H2S on melanoma cell migration and the results revealed that cell motilities of both A375 and SK-MEL-28 (Fig. 1c) cells were significantly attenuated when subjected to NaHS (2 mM) exposure for 24 h compared with the control. Moreover, the cell cycle of both A375 and SK-MEL-28 changed after the administration of 2 mM NaHS. As illustrated in Fig. 1d, there were more cells in G1/G0 phase and fewer in S phase compared with those without treatment. Flow cytometric analysis also exhibited that NaHS (2 mM) induced apoptosis of melanoma cells (Fig. 1e), which was further confirmed by western blotting showing that NaHS treatment (0, 0.5, 1, 2, 5 mM) upregulated protein level of cleaved Caspase-3 and decreased Bcl-xL expression in a dose-depended manner in both cell lines (Fig. 1f). Therefore, by donating H2S, NaHS induces cell cycle arrest and apoptosis in human melanoma cells. 3.2. NaHS promotes autophagy in melanoma cell lines Human skin melanoma cell line, A375 and SK-MEL-28 were treated under 2 mM NaHS for 24 h, and then western blotting,
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confocal microscopy analysis and transmission electron microscopy (TEM) assays were performed to determine whether NaHS could affect autophagy. Lipidation of the microtubule-associated protein light chain 3 (LC3) leading to its conversion to LC3-II is an early key step in autophagosome formation. As shown in Fig. 2a and b, LC3 conversion was readily detected by immunoblotting in both two cell lines after treatment with NaHS (2 mM). Moreover, the expression of the autophagy adaptor protein p62 was reduced after NaHS treatment in both cell lines (Figs. 2a), further indicating the induction of autophagy. Confocal microscopy for mRFP-GFP-LC3 analysis demonstrated that the numbers of both autophagosomes (yellow dots) and autolysosomes (free red dots) were significantly increased upon 2 mM NaHS exposure compared with untreated cells (Fig. 2c and d). Furthermore, TEM delineated increased numbers of intracellular double membrane vacuolar structures containing various kinds of cytoplasmic components in those cells treated with 2 mM NaHS (Fig. 2f). 3.3. Suppression of autophagy by ATG7 shRNA enhanced H2Sregulated cell apoptosis, but not cell proliferation, migration or cell cycle The Atg7 protein is essential for the conversion of the cytosolic form of LC3, LC3-I, into its lipidated form, LC3-II—an obligatory step of autophagy. To investigate the impact of autophagy on H2S-regulated cell behaviors, we transfected both melanoma cell lines with shRNA against ATG7. The efficacy of shRNA treatment was confirmed by western blotting analysis, which showed significant reductions of ATG7 level and LC3 conversion in both cell lines (Fig. 3a). CCK8 and migration assay revealed that knockdown of ATG7 inhibited proliferation (Fig. 3b and c) and migration (Fig. 3d) of melanoma cells, while apoptosis was induced by suppression of autophagy in melanoma cells (Fig. 3f). Next, we analyzed the cell cycle distribution. As shown in Fig. 3e, cells in G1/G0 phase were significantly increased and slightly reduced in S phase in melanoma cell lines without autophagy. Moreover, NaHS (2 mM) combined with ATG7 shRNA further contributed to a substantial amount of cell apoptosis compared with the individual treatment of NaHS (2 mM, Figs. 3f), whereas decreased cell proliferation, migration, mitosis induced by NaHS were not recovered by suppressing autophagy. Taken together, these results indicated that exogenous H2S inhibited human melanoma progression not completely by promoting autophagy. 3.4. NaHS inhibited melanoma cell development via suppression of PI3K/AKT/ mTOR signaling pathway The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is one of the major pathways modulating cell proliferation, growth, survival, metabolism and angiogenesis. This pathway is frequently activated in many human cancer types [10]. Hence, we further investigated whether PI3K/AKT/mTOR signaling pathway was involved in the regulation of H2S in melanoma cell lines. Indeed, western blotting analysis exhibited that NaHS (2 mM) dramatically diminished expression of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt) and mTOR, which resembled rapamycin (100 nM, 12 h) treatment (Figs. 4a) in A375 and SK-MEL-28 cells. Given that rapamycin mediates tumor cell growth and development by inhibiting mTOR expression, we speculate that H2S might regulate melanoma cell functions through inhibiting the PI3K/ AKT/mTOR signaling pathway. Thus, A375 and SK-MEL-28 cells were treated with or without NaHS and then exposed to IGF-1 (100 ng/mL), an activator of PI3K-AKT pathway in tumor progression.
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Fig. 1. NaHS suppressed cell proliferation, migration, division and induced apoptosis of melanoma cell lines. CCK-8 assay was used to assess A375(a) and SK-MEL-28 (b) cell proliferation. (c)The wound healing assay was preformed to evaluate cell migration. Melanoma cells were treated with 2 mM NaHS for 24 h then images were captured under a light microscope at 0 and 24 h respectively (Scale bar: 300 mm). Flow cytometry was used to analyze the cell cycle (d) and cell apoptosis (e) after NaHS treatment for 24 h. The percentage of cell cycle distribution and apoptotic cells was calculated and shown in histogram separately on the right. (f) Western blotting detected expression of Bcl-xL and Cleaved Caspase-3 apoptosis-related protein in A375 and SK-MEL-28 cells treated with graded NaHS, β-actin was used as an internal control. The data express the mean standard deviation of three experiments. NS: nonsignificant difference, *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control (NC). 201 285 mm (300 300 DPI).
Western blotting analysis showed that IGF-1 (100 ng/mL) could reverse the inhibition of PI3K-AKT by NaHS (2 mM) treatment, which manifested by inducing phosphorylation of Akt (Fig. 4b). Moreover, the H2S-induced cell behaviors, including decreased
proliferation, migration, cell cycle and increased apoptosis, were mostly reversed by IGF-1 (100 ng/mL) exposure (Fig. 4c – f). Collectively, these data indicated that NaHS could inhibit melanoma via suppression of PI3K/AKT/ mTOR signaling pathway.
Please cite this article in press as: Q. Xiao, et al., Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2020.02.004
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Fig. 2. NaHS promotes autophagy in melanoma cell lines. (a) Western blotting detected expression of LC3-I, LC3-II and p62 protein in the concentration of 2 mM NaHS in A375 and SK-MEL-28 cells. β-actin was used as an internal control. (b) Optical densitometric analysis of LC3-II/LC3-I and P62 relative expression were shown in the bar chart. (c) A375 and SK-MEL-28 cells transfected with GFP-mRFP-LC3 adenovirus for 2 h, LC3 puncta dots (yellow and red) were observed under a laser scanning confocal microscope, the nuclei (blue) were stained with DAPI. (Scale bar: 10 mm) (d) The quantification of the average number of autophagosomes (yellow dots) and autolysosomes (Free red dots) per cell (e) The ratio of cells presenting typical LC3 puncta dots. (f) The ultrastructural features of autophagy and autophagic vesicles were imaged by TEM (Scale bars: 1 and 0.5 mm). The data express the mean standard deviation of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control (NC). 208 276 mm (300 300 DPI).
4. Discussion Melanoma is a malignant tumor of melanocytes, which are the cells that make the pigment melanin and are derived from the neural crest [1]. It is a lethal disease with increasing incidence, afflicting over 68,000 people in the United States annually and casing 8000 annual deaths. In addition, malignant melanoma is the fifth most frequently diagnosed cancer in men and the sixth most common in women [2]. The prognosis of patients is often poor mainly due to the high distant metastasis rate and drug resistance
[3]. Therefore, molecular targets hold considerable clinical significance for early detection and development of novel therapeutic approaches for patients with melanoma. H2S is now widely considered the third gaseous signaling molecule and plays important biological and pharmacological roles in the progression of many types of cancer [8]. In recent years, an increasing number of researches have revealed that endogenously produced or exogenously treatment H2S could exert two obviously opposite functions on the growth of cancer cells [11–13]. The immunohistochemical analysis performed on over 100 human
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Fig. 3. Suppression of autophagy by ATG7 shRNA enhanced H2S-regulated cell apoptosis in melanoma. Cells were transfected with ATG7 shRNA or control for 3 days. (a) Western blot analysis of ATG7 and LC3 protein expression in A375 and SK-MEL- 28 cells respectively, β-actin was used as an internal control. Cells transfected with the shRNA against ATG7 or control were subjected to the CCK-8(b and c) and migration assay(d). Representative fields of migrated cells are shown. (Scale bar: 300 mm). Flow cytometry was used to analyze the cell cycle(e) and cell apoptosis (f) The percentage of apoptotic cells and cell cycle distribution was calculated and shown in histogram separately on the right. The data express the mean standard deviation of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control shRNA (shNC). NS: nonsignificant difference. 200 297 mm (300 300 DPI).
samples showed that CSE expression increased from nevi to primary melanoma, decreased in metastatic tissue and was silent in lymph node metastases, which also suggested the involvement of H2S in the progression of melanoma [9]. Endogenous H2S or relatively low concentrations of exogenous H2S for a relatively short time might promote or maintain cancer cell growth, while
overexpression of H2S generating enzymes or treatment with relatively high levels of H2S donor for a relatively long duration may exhibit anti-cancer effects. It has been reported that 25–50 mM NaHS could enhance proliferation, viability, migration, and invasion of human thyroid carcinoma cells, whereas NaHS at the concentration of 200 mM
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Fig. 4. NaHS inhibited melanoma via suppression of PI3K/AKT/ mTOR signaling pathway. Western blot analysis of PI3K/ p-PI3K, Akt/p-Akt and mTOR expression in A375 and SK-MEL-28 cells treated with NaHS(2 mM,24 h), rapamycin(100 nM,12 h), IGF-1(100 ng/mL,24 h) alone or NaHS in combination with IGF-1(24 h), β-actin was used as an internal control (a and b). Cells treated with NaHS(2 mM,24 h), IGF-1(100 ng/mL,24 h) alone or NaHS in combination with IGF-1(24 h) were subjected to the CCK-8(c and d) and migration assay(e). Representative fields of migrated cells are shown. (Scale bar: 300 mm). Flow cytometry was used to analyze the cell cycle (f) and cell apoptosis (g). The percentage of apoptotic cells and cell cycle distribution was calculated and shown in histogram separately on the right. The data express the mean standard deviation of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NaHS group. 199 297 mm (300 300 DPI).
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might exert inhibitory effect [14]. Likewise, growth and differentiation of human keratinocytes can be promoted by exogenous H2S [15], while a high level of NaHS possessed controversy traits, meanwhile induced apoptosis and cell cycle arrest in SGC-7901 gastric cancer cells [16] and hepatocellular carcinoma [17]. In this study, we used 2 mM NaHS as a donor of H2S gas to treat human melanoma cell lines for 24 h and it attenuated cell proliferation and migration, arrested cell cycle but induced cell autophagy. Cleaved Caspase-3 activation and decrease of antiapoptotic protein (Bcl-xL) further confirmed that H2S triggers apoptosis of melanoma cells. The difference between their results and ours may be attributed to diversity in H2S treatment modes and cell lines. Since different types of cancer cells hold distinct drug susceptibilities and physiological traits, further researches are needed to parse out the reasonable strategy for H2S application on different types of human cancers, in order to achieve optimal anticancer efficacy. In combination with previous studies, our results suggested that H2S can be multitasking by regulating different downstream effectors in different cancers and it functions primarily as a metastasis suppressor in melanoma. Autophagy is a highly regulated process that degrades damaged cytoplasm components and organelles in lysosomes to provide substrates in times of nutrient deprivation and thus maintain cellular homeostasis [18]. It was found that autophagy is playing an increasingly important role in cell growth and development modified by H2S. Recent studies indicated that H2S could significantly inhibit the autophagic activity in the rats with middle cerebral artery occlusion [19] and reduce renal tissue fibrosis by downregulating autophagy in diabetic rats [20], whereas, autophagy in hepatocellular carcinoma cells and human keratinocytes could be promoted by NaHS [15,17]. While defective autophagy has been reported to be responsible for carcinogenesis, this cellular process maintains the survival of cancer cells in the presence of stress and nutrient deprivation [21–23]. Most malignant melanomas are under high endoplasmic reticulum (ER) stress, an inducer of autophagy, and metabolic energy balance of invasive melanoma cells may be correlated with autophagy conditions [24,25]. The absolute levels of some autophagosomal proteins, including LC3B, are higher in tumor tissues and increased LC3B expression is associated with a low grade of differentiation and poorer overall survival [26,27]. Therefore, knockdown essential genes for autophagy induction, or treatment with autophagy inhibitor 3methyladenine, have been reported to attenuate the cell invasion and migration [28]. Here we also demonstrated that H2S concurrently exhibited anti-tumor and pro-autophagic effects on melanoma cells. Intriguingly, shRNA-mediated abrogation of autophagy only enhanced the apoptotic process caused by H2S, suggesting that exogenous H2S might exert an anti-tumor effect independent of autophagy. Further studies are needed to elucidate the exact relationship between H2S and autophagy in regulating melanoma progression. To better understand the mechanism by which this effect was achieved, we evaluated the possible involvement of the PI3K/AKT/ mTOR signaling pathway, which plays a critical role in cell motility, survival, growth and angiogenesis. PI3K first activates the threonine/serine kinase AKT, which then phosphorylate mTOR via a cascade of molecules [29,30]. The PI3K/AKT/mTOR pathway is one of the most common dysregulated signaling pathways in melanoma survival and progression. It has been reported to be involved in primary and secondary resistance to targeted therapy [31]. In addition, other reports proved that exogenous H2S exerted multiple biological effects by inhibiting the PI3K/AKT/mTOR pathway in hepatocellular carcinoma cells [15] and thyroid cancer cells [14]. In this study, we found administration of NaHS decreased the expression of phosphorylated PI3K, AKT and mTOR, while IGF-1, which has been widely known to activate PI3K/AKT
and drive the progression and metastatic spread of multiple tumors [32,33,34], could potentiate the PI3K-AKT pathway inhibited by NaHS. Meanwhile, the impact of NaHS on the melanoma cell phenotype could be mostly reversed by IGF-1. High levels of circulating IGF-1 and IGF-1R expression are relevant to a higher risk of several common cancers, including melanoma [34]. Moreover, evidence suggested that miR-425 could inhibit melanoma growth, migration, invasion and stem-cell like properties via suppression of PI3K-Akt pathway by targeting IGF-1 [28]. Collectively, these results implied that PI3K/AKT/mTOR signaling pathway may be involved in exogenous H2S-regulated biological functions of melanoma cells. A limitation of the present study is that we did not confirm the modulatory effects of H2S on melanoma cells in vivo. Previous studies indicated that exogenous H2S donors (DATS) significantly inhibit tumor growth of mice subcutaneously injected with B16F10 cells by 67 % as compared with control groups [9]. Itraconazole inhibited melanoma growth and extended survival of athymic nude mice subcutaneously implanted with A375 melanoma cells [35]. Additionally, implantation of miR-23a-overexpressed cells resulted in increased overall survival and led to a decreased number of metastases in the lung and liver, which were consistent with their results in vitro [28]. Therefore, it would be particularly interesting to decipher whether NaHS can play a similar role by suppressing melanoma malignant progression in vivo. Another shortage is that we did not evaluate the level or the expression of hydrogen sulfide producing enzymes-CSE and CBS in melanoma. Further studies are required to further clarify whether endogenous H2S is synthesized in melanoma cells and define its potential role in the regulation of various cell behaviors. In conclusion, the present study demonstrated that exogenous H2S could inhibit human melanoma cell development by regulating the PI3K/AKT/mTOR signaling pathway. Our findings provide evidence for the underlying signal pathways and biochemical mechanisms that exist in the melanoma cells. Hydrogen sulfide releasing drugs could be a potential candidate for the treatment of melanoma. Authors' contributions Chengfeng Zhang and Jiayi Ying designed the experiments and analyzed the results. Qing Xiao, Zhuhui Qiao and Yiwen Yang carried out experiments. Qing Xiao, Xiaoxi Dai and Zhongyi Xu wrote the paper. Jiayi Ying provided experimental materials and contributed to data analyses. Leihong Xiang supervised the design of experiments and data analyses. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by grants from Natural Science Foundation of China (Nos. 81573064, 81872544 and 81903243). References [1] A.M. Eggermont, A. Spatz, C. Robert, Cutaneous melanoma, Lancet. 383 (9919) (2014) 816–827. [2] Global, regional, and national life expectancy, all-cause mortality, and causespecific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015, Lancet. 388 (2016) 1459–1544. [3] J.K. Winkler, K. Buder-Bakhaya, A. Dimitrakopoulou-Strauss, Malignant melanoma: current status, Radiologe 57 (2017) 814–821. [4] M.D. Hartle, M.D. Pluth, A practical guide to working with H2S at the interface of chemistry and biology, Chem. Soc. Rev. 45 (22) (2016) 6108–6117.
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Q. Xiao et al. / Journal of Dermatological Science xxx (2019) xxx–xxx [5] J. Chen, J. Gao, W. Sun, et al., Involvement of exogenous H2S in recovery of cardio protection from ischemic post-conditioning via increase of autophagy in the aged hearts, Int. J. Cardiol. 220 (2016) 681–692. [6] Y. Sun, Y. Huang, R. Zhang, et al., Hydrogen sulfide upregulates KATP channel expression in vascular smooth muscle cells of spontaneously hypertensive rats, J. Mol. Med. 93 (4) (2015) 439–455. [7] Y. Kimura, Y.I. Goto, H. Kimura, Hydrogen sulfide protects neurons from oxidative stress, Neurosci. Res. 71 (2011) 88. [8] D. Wu, W. Si, M. Wang, S. Lv, Y. Li, Hydrogen sulfide in cancer: friend or foe? Nitric Oxide 50 (2015) 38–45. [9] E. Panza, P. DeCicco, C. Armogida, G. Scognamiglio, V. Gigantino, G. Botti, D. Germano, M. Napolitano, A. Papapetropoulos, M. Bucci, G. Cirino, A. Ianaro, Role of the cystathionine g lyase/hydrogen sulfide pathway in human melanoma progression Pigment, Cell Melanoma Res. 28 (1) (2015) 61–72. [10] K. Sheppard, K.M. Kinross, B. Solomon, R.B. Pearson, W.A. Phillips, Targeting PI3 Kinase/AKT/mTOR signaling in Cancer, Crit. Rev. Oncog. 17 (1) (2012) 69–95. [11] Z.W. Lee, X.Y. Teo, E.Y. Tay, C.H. Tan, T. Hagen, P.K. Moore, L.W. Deng, Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance, Br. J. Pharmacol. 171 (18) (2014) 4322–4336. [12] Z. Ma, Q. Bi, Y. Wang, Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines, Oral Dis. 21 (2) (2015) 156–162. [13] C. Szabo, M.R. Hellmich, Endogenously produced hydrogen sulfide supports tumor cell growth and proliferation, Cell Cycle 12 (18) (2013) 2915–2916. [14] D. Wu, J. Li, et al., Exogenous hydrogen sulfide regulates the growth of human thyroid carcinoma cells, Oxid. Med. Cell. Longev. (2019). [15] X. Xie, H. Dai, B. Zhuang, et al., Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes, Biochem. Biophys. Res. Commun. 472 (3) (2016) 437–443. [16] K. Ma, Y. Liu, Q. Zhu, C.H. Liu, J.L. Duan, B.K. Tan, Y.Z. Zhu, H2S donor, Spropargyl-cysteine, increases CSE in SGC-7901 and cancer-induced mice: evidence for a novel anti-cancer effect of endogenous H2S? PLoS One 6 (6) (2011)e20525. [17] S.S. Wang, Y.H. Chen, N. Chen, L.J. Wang, D.X. Chen, H.L. Weng, et al., Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/AKT/mTOR signaling pathway, Cell Death Dis. 8 (3) (2017) e2688. [18] B. Ravikumar, S. Sarkar, J.E. Davies, M. Futter, M. Garcia-Arencibia, Z.W. GreenThompson, et al., Regulation of mammalian autophagy in physiology and pathophysiology, Physiol. Rev. 90 (4) (2010) 1383–1435. [19] W.-W. Jiang, H.Y. Bai-Sheng Huang, et al., Sodium hydrosulfide attenuates cerebral ischemia/ reperfusion injury by suppressing overactivated autophagy in rats, FEBS Open Bio 7 (11) (2017) 1686–1695.
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[20] L. Li, et al., Hydrogen sulfide reduced renal tissue fibrosis by regulating autophagy in diabetic rats, Mol. Med. Rep. 16 (2017) 1715–1722. [21] R. Mathew, V. Karantza-Wadsworth, E. White, Role of autophagy in cancer, Nat. Rev. Cancer 7 (12) (2007) 961. [22] M.C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Self- eating and self-killing: crosstalk between autophagy and apoptosis, Nat. Rev. Mol. Cell Biol. 8 (9) (2007) 741–752. [23] X.H. Ma, S. Piao, D. Wang, et al., Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma, Clin. Cancer Res. 17 (10) (2011) 3478–3489. [24] D.T. Rutkowski, R.J. Kaufman, That which does not kill me makes me stronger: adapting to chronic ER stress, Trends Biochem. Sci. 32 (10) (2007) 469. [25] P. Hersey, X.D. Zhang, Adaptation to ER stress as a driver of malignancy and resistance to therapy in human melanoma, Pigment Cell Melanoma Res. 21 (3) (2008) 358. [26] R. Lazova, V. Klump, J. Pawelek, Autophagy in cutaneous malignant melanoma, J. Cutan. Pathol. 37 (2) (2010) 256–268. [27] C. Miracco, G. Cevenini, A. Franchi, et al., Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions, Hum. Pathol. 41 (4) (2010) 503–512. [28] G. Weinan, W. Huina, Y. Yuqi, et al., Down-regulated miR-23a contributes to the metastasis of cutaneous melanoma by promoting autophagy, Theranostics. 7 (8) (2017) 2231–2249. [29] J. Rodon, R. Dienstmann, V. Serra, et al., Development of PI3K inhibitors: lessons learned from early clinical trials, Nat. Rev. Clin. Oncol. 10 (3) (2013) 143–153. [30] A. Perl, Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases, Nat. Rev. Rheumatol. 12 (2016) 169–182. [31] P. Liu, Y. Hu, L. Ma, et al., miR-425 inhibits melanoma metastasis through repression of PI3K-Akt pathway by targeting IGF-1, Biomed. Pharmacother. 75 (2015) 51–57. [32] A. Grimberg, Mechanisms by which IGF-I may promote cancer, Cancer Biol. Ther. 2 (6) (2003) 630–635. [33] N. Shimada, S. Iwakami, M. Moriyama, H. Moriyama, et al., Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor, PLoS One 9 (2014)e86459. [34] R. Kucera, I. Treskova, J. Vrzalova, S. Svobodova, O. Topolcan, R. Fuchsova, M. Rousarova, V. Treska, T. Kydlicek, Evaluation of IGF1 serum levels in malignant melanoma and healthy subjects, Anticancer Res. 34 (9) (2014) 5217–5220. [35] G. Liang, M. Liu, Q. Wang, et al., Itraconazole exerts its anti-melanoma effect by suppressing Hedgehog, Wnt, and PI3K/mTOR signaling pathways, Oncotarget 8 (17) (2017) 28510–28525.
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Journal of Dermatological Science xxx (2019) xxx–xxx
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Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway Qing Xiao, Jiayi Ying, Zhuhui Qiao, Yiwen Yang, Xiaoxi Dai, Zhongyi Xu, Chengfeng Zhang* , Leihong Xiang* Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 December 2019 Received in revised form 4 February 2020 Accepted 14 February 2020
Background: : Melanoma is one of the most aggressive, therapy-resistant skin cancers in the world. Hydrogen sulfide (H2S), a newly discovered gasotransmitter, plays a crucial role in the progression and development of many types of cancers. However, the effect of H2S on human skin melanoma remains to be elucidated. Objective: : We aimed to explore the effect of exogenous H2S on melanoma cells and its underlying mechanisms. Methods: : In this study, human skin melanoma cell lines, including A375 and SK-MEL-28, were treated with a donor of H2S (NaHS). CCK-8, scratch assay, flow cytometric analysis, western blotting and transmission electron microscopy (TEM) were performed to explore the effects of H2S on cell behaviors. Results: : Treatment with NaHS inhibited cell proliferation, migration and division, while it could induce cell apoptosis and autophagy in melanoma cell lines. Moreover, NaHS significantly decreased the expression of p-PI3K, p-Akt and mTOR proteins. Furthermore, insulin-like growth factor-1 (IGF-1), the activator of PI3K/AKT/mTOR pathway, could reverse the cell behaviors caused by NaHS. Conclusion: : Our results demonstrated that exogenous hydrogen sulfide could inhibit human melanoma cell development via suppression of the PI3K/AKT/mTOR pathway. Hydrogen sulfide might serve as a potential therapeutic option for melanoma. © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Keywords: Hydrogen sulfide Melanoma PI3K/AKT/ mTOR pathway
1. Introduction Melanoma, also known as malignant melanoma, is the most dangerous type of skin cancer [1], since it is much more likely to invade nearby tissues and spread to other parts of the body. Most deaths from skin cancers are caused by melanoma. Globally, in 2015 there were 3.1 million new diagnosis of melanoma and
Abbreviations: H2S, hydrogen sulfide; NaHS, Sodium hydrosulfide; TEM, transmission electron microscopy; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p-PI3K, phosphorylated-PI3K; p-Akt, phosphorylated-Akt; mTOR, mammalian target of rapamycin; IGF-1, insulin-like growth factor-1; NO, nitric oxide; CO, carbon monoxide; CSE, cystathionine g-lyase; CBS, cystathionine βsynthase; 3-MST, 3-mercaptopyruvate sulfurtransferase; NF-kB, nuclear factor-kB; RNA, ribonucleic acid; FBS, fetal bovine serum; ATG7, autophagy-related protein 7; shRNA, short hairpin RNA; DAPI, 4'6-diamidino-2-phenylindole; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; LC3, microtubule-associated protein light chain 3; ER, endoplasmic reticulum. * Corresponding authors at: Department of Dermatology, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai, 200040, PR China. E-mail addresses: [email protected] (C. Zhang), fl[email protected] (L. Xiang).
59,800 deaths [2]. The incidence of melanoma is still rising rapidly in recent years. The prognosis of patients is often poor mainly due to the high metastasis rate and drug resistance. Although immunetargeted strategies have shown marked progress in some patients, their effects on overall survival remain variable [3]. Therefore, new therapeutic approaches are urgently required to optimize current treatment for melanoma. Gasotransmitter hydrogen sulfide (H2S), the third gaseous signaling molecule along with nitric oxide (NO) and carbon monoxide (CO), can be produced enzymatically in mammals under normal conditions by the catalysis of two pyridoxal-50 -phosphatedependent enzymes, cystathionine g-lyase (CSE), cystathionine βsynthase (CBS) and a pyridoxal-50 -phosphate-independent enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST) [4]. Recently, a growing body of evidence has revealed that H2S exerts a variety of physiological and pathological functions in cell growth, proliferation and differentiation [5]. This gas has been found to relax blood vessels [6], shield neurons and cardiac muscles from oxidative stress [7] and possess different properties on cancer across a wide range of doses. A relatively high concentration of exogenous H2S could exhibit antitumor effects on human colon
https://doi.org/10.1016/j.jdermsci.2020.02.004 0923-1811/ © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Q. Xiao, et al., Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2020.02.004
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cancer cells and HepG2 cancer cells mainly through induction of cell cycle arrest, facilitation of uncontrolled intracellular acidification and acceleration of apoptosis [8]. Overexpression of CSE in human melanoma cells could induce spontaneous apoptosis by suppressing the activity of nuclear factor-kB (NF-kB) and decreasing the expression of anti-apoptotic proteins [9]. However, there has been little research about the effect and mechanism of exogenous H2S on melanoma. In this study, we aimed to explore the effect of exogenous H2S on melanoma cells and its underlying mechanisms, in order to provide new insight into the alternative strategies for melanoma treatment. To this end, we first observed the effect of H2S on melanoma cell proliferation, migration, apoptosis, division and autophagy. We then blocked autophagy by ATG7 shRNA in melanoma cell lines to examine whether autophagy plays a role in NaHS-modulated cell behaviors. Further, we investigated the underlying molecular mechanisms involved in the above cell behaviors induced by H2S treatment. 2. Materials and methods 2.1. Reagents Sodium hydrosulfide (NaHS, H2S donor) was ordered from Sigma Aldrich (Shanghai, China), IGF-1 was ordered from Novoprotein, rapamycin was from Selleck. Primary antibodies against LC3, mTOR, Akt, phospho-Akt, PI3k, Bcl-xL, Cleaved Caspase-3 were purchased from Cell Signaling Technology, antiSQSTM1/p62 and anti-ATG7 were purchased from Abcam, phospho- PI3k was from Affinity and anti-β-actin from Proteintech. Secondary antibodies were goat anti-rabbit IgG and goat antimouse IgG also from Cell Signaling Technology. To knockdown ATG7 gene expression, melanoma cells were transfected with specific short hairpin ATG7 RNA (shRNA, 50 -ATGATCCCTGTAACTTAGCCCA -30 and 50 -CACGGAAGCAAACAACTTCAAC -30 ). The shRNA and negative control shRNA (shNC) were purchased from GeneChem (Shanghai, China). 2.2. Cell culture and treatment Human melanoma cell lines A375 and SK-MEL-28 were purchased from Shanghai Institute of Biochemistry and Cell Biology, CAS (Cell Bank/Stem Cell Technology platform), cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) (Gibco, CA) at 37 C in a 5% CO2 incubator. NaHS and IGF-1 were solubilized in sterile phosphate buffer saline (PBS). Rapamycin was solubilized in DMEM. A375 and SK-MEL-28 cells were treated with NaHS (0, 0.5, 1, 2, 5, 10 mM) for 24 h or with IGF-1 (100 ng/mL) for 24 h or rapamycin (100 nM) for 12 h. Cells were transfected with ATG7 shRNA or shNC using the transfection reagent for 72 h according to the manufacturer's protocol. 2.3. Western blotting Protein lysates extracted from melanoma cell lines were separated by 15 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretic transfer of proteins from gels onto polyvinylidene difluoride membrane (Thermo Fisher Scientific, MA, USA) at 100 V for one and a half hour at 4 C. After blocking in 5% bovine serum albumin in Tris Buffered Saline Tween (TBST) to inhibit nonspecific binding, the membranes were incubated with primary antibodies overnight at 4 C, washed three times with TBST, and then incubated with peroxidase-coupled secondary antibodies for 1 h at room temperature. Membranes were washed again in TBST three times
at room temperature. Protein bands were visualized by incubation using an enhanced chemiluminescence (Thermo Fisher Scientific, MA, USA) and were exposed to X-ray film. Western blotting was repeated at least three times for each experiment. 2.4. Cell proliferation analysis The Cell Counting Kit (CCK-8) (Dojindo, Kumamoto, Japan) was employed to evaluate the effect of NaHS, sh-ATG7 and IGF-1 on cell proliferation at indicated time points. Cells were seeded in 96-well plates in quintuplicate at densities of 5*103 cells per well and subjected to various treatments before analysis by the CCK-8 assay. Absorbances were measured at 450 nm wavelength on a Microplate Reader (Model 680, Bio-Rad). Cell proliferation was calculated using the formula: Cell proliferation% = (450 sample background)/ (450 control–background) 100 %. 2.5. Wound healing assay 1*106 melanoma cells were seeded and grown per well into 6well culture plate. After allowing them to completely attach, cells were scratched with a sterile 200 ml pipette tip in 3 separate places in each well. All the wound sizes were verified to make sure they were all the same width. After washing by PBS for three times, we changed the culture medium to DMEM without FBS. Cells were subjected to different treatments as: negative control, NaHS (2 mM), IGF-1 (100 ng/mL), IGF-1 with NaHS for 24 h. The wound was viewed and captured under a light microscope at respective intervals (0 h and 24 h) with a Zeiss microscope system (Zeiss Axivert 400C, Germany). The data are representative of 3 independent experiments. 2.6. Cell apoptosis analysis A375 and SK-MEL-28 were cultured in DMEM with 10 % FBS and then subjected to various treatments as: negative control, NaHS (2 mM), IGF-1 (100 ng/mL), IGF-1 with NaHS for 24 h, then harvested by trypsinization, then washed twice with PBS, and resuspended in binding buffer. Annexin V-FITC and Propidium iodide (PI) solution (Beyotime Institute of Biotechnology) were added for 20 min at room temperature in the dark before the samples were analyzed by flow cytometry (Beckman Coulter, Miami, FL). 2.7. Cell cycle assay 2*105 cells were seeded and grown per well into a 6-well culture plate with DMEM containing 10 % FBS, and were allowed to attach overnight. The cells were treated with distinct treatments as: negative control, NaHS (2 mM), IGF-1 (100 ng/mL), IGF-1 with NaHS for 24 h, then harvested by trypsinization, washed in cold PBS for 2 times and fixed in precooled anhydrous ethanol in a 4 ℃ refrigerator overnight. On the next day, centrifuged at 4 C and resuspended in cold PBS. Bovine pancreatic RNAase (Sigma– Aldrich) was added and incubated at 37 C for half an hour, then 250 mg/ml propidium iodide (Sigma–Aldrich) was added and the cell suspension was filtered with 350-mesh nylon membrane for testing. In each group, 50,000 cells were analyzed by flow cytometry (Beckman Coulter, Miami, FL). 2.8. Confocal microscopy for GFP-mRFP-LC3 A375 and SK-MEL-28 were seeded in 35-mm confocal dishes. When cells reached 70 % confluence, tandem fluorescent mRFPGFP-LC3 adenoviruses (Hanheng, Shanghai, China) were transfected into the cells for 2 h. Tandem fluorescent mRFP-GFP-LC3 allows to measure the extent of autophagosome and autolysosome
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formation simultaneously, because LC3 puncta labeled with both GFP and mRFP represent autophagosomes before the fusion with lysosomes, then the GFP fluorescence is quenched in the acidic pH of the lysosomal compartment, so those labeled with mRFP alone represent autolysosomes. After 24 h, the cells were treated with or without 2 mM NaHS for 24 h. Then the melanoma cells were fixed with 4% paraformaldehyde and cell nuclei were visualized by staining with 4,6-diamidino-2-phenylindole (DAPI). The images were observed with a confocal laser scanning microscope (Leica TCS SP5, Germany) to indicate the formation of GFP-LC3 and mRFPLC3 puncta, indicative of autophagosomes and/or autolysosomes. The number of GFP and mRFP puncta per cell was manually quantified. At least 20 cells per dish were randomly analyzed. The experiments were performed at least three times. 2.9. Transmission electron microscopy (TEM) Cells were fixed in 4% glutaraldehyde overnight at 4 C and rinsed with precooled PBS for 3 times, then cells were post-fixed in 1% osmium tetroxide (OsO4) at 4 C. After that, PBS washed again, samples were dehydrated with graded alcohol and embedded in epon-araldite resin. Ultrathin sections were obtained by an ultramicrotome. Sections were then stained with uranyl acetate and lead citrate. The ultrastructures of cells were observed and imaged under JEM-1220 TEM (JEOL, Tokyo, Japan). 2.10. Statistical analysis The results were expressed as mean standard deviation on at least three independent experiments. Statistical analysis was calculated by two-tailed Student’s t-test (SPSS 17.0 SPSS Inc, Chicago, IL), with *P < 0.05; **P < 0.01; ***P < 0.001 considered to be statistically significant. 3. Results 3.1. NaHS suppressed cell proliferation, migration, division and induced apoptosis of melanoma cell lines To investigate the functional role of NaHS in melanoma cell lines, cell proliferation was determined by CCK8 assay. As shown in Fig. 1a and b, cell proliferation was inhibited in both cell lines after treatment with NaHS (0, 0.5, 1, 2, 5, 10 mM) in a dose-dependent manner (A375: 100 %3.69, 96.73 % 3.42, 88.43 % 1.98, 82.92 % 2.99, 76.44 % 2.87, 70.37 % 3.74; SK-MEL-28: 100 % 4.95, 94.31 % 2.52, 84.20 % 2.72, 82.01 % 3.68, 80.93 % 4.79, 65.60 % 6.2, respectively). The wound healing assay was performed to evaluate the effect of H2S on melanoma cell migration and the results revealed that cell motilities of both A375 and SK-MEL-28 (Fig. 1c) cells were significantly attenuated when subjected to NaHS (2 mM) exposure for 24 h compared with the control. Moreover, the cell cycle of both A375 and SK-MEL-28 changed after the administration of 2 mM NaHS. As illustrated in Fig. 1d, there were more cells in G1/G0 phase and fewer in S phase compared with those without treatment. Flow cytometric analysis also exhibited that NaHS (2 mM) induced apoptosis of melanoma cells (Fig. 1e), which was further confirmed by western blotting showing that NaHS treatment (0, 0.5, 1, 2, 5 mM) upregulated protein level of cleaved Caspase-3 and decreased Bcl-xL expression in a dose-depended manner in both cell lines (Fig. 1f). Therefore, by donating H2S, NaHS induces cell cycle arrest and apoptosis in human melanoma cells. 3.2. NaHS promotes autophagy in melanoma cell lines Human skin melanoma cell line, A375 and SK-MEL-28 were treated under 2 mM NaHS for 24 h, and then western blotting,
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confocal microscopy analysis and transmission electron microscopy (TEM) assays were performed to determine whether NaHS could affect autophagy. Lipidation of the microtubule-associated protein light chain 3 (LC3) leading to its conversion to LC3-II is an early key step in autophagosome formation. As shown in Fig. 2a and b, LC3 conversion was readily detected by immunoblotting in both two cell lines after treatment with NaHS (2 mM). Moreover, the expression of the autophagy adaptor protein p62 was reduced after NaHS treatment in both cell lines (Figs. 2a), further indicating the induction of autophagy. Confocal microscopy for mRFP-GFP-LC3 analysis demonstrated that the numbers of both autophagosomes (yellow dots) and autolysosomes (free red dots) were significantly increased upon 2 mM NaHS exposure compared with untreated cells (Fig. 2c and d). Furthermore, TEM delineated increased numbers of intracellular double membrane vacuolar structures containing various kinds of cytoplasmic components in those cells treated with 2 mM NaHS (Fig. 2f). 3.3. Suppression of autophagy by ATG7 shRNA enhanced H2Sregulated cell apoptosis, but not cell proliferation, migration or cell cycle The Atg7 protein is essential for the conversion of the cytosolic form of LC3, LC3-I, into its lipidated form, LC3-II—an obligatory step of autophagy. To investigate the impact of autophagy on H2S-regulated cell behaviors, we transfected both melanoma cell lines with shRNA against ATG7. The efficacy of shRNA treatment was confirmed by western blotting analysis, which showed significant reductions of ATG7 level and LC3 conversion in both cell lines (Fig. 3a). CCK8 and migration assay revealed that knockdown of ATG7 inhibited proliferation (Fig. 3b and c) and migration (Fig. 3d) of melanoma cells, while apoptosis was induced by suppression of autophagy in melanoma cells (Fig. 3f). Next, we analyzed the cell cycle distribution. As shown in Fig. 3e, cells in G1/G0 phase were significantly increased and slightly reduced in S phase in melanoma cell lines without autophagy. Moreover, NaHS (2 mM) combined with ATG7 shRNA further contributed to a substantial amount of cell apoptosis compared with the individual treatment of NaHS (2 mM, Figs. 3f), whereas decreased cell proliferation, migration, mitosis induced by NaHS were not recovered by suppressing autophagy. Taken together, these results indicated that exogenous H2S inhibited human melanoma progression not completely by promoting autophagy. 3.4. NaHS inhibited melanoma cell development via suppression of PI3K/AKT/ mTOR signaling pathway The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is one of the major pathways modulating cell proliferation, growth, survival, metabolism and angiogenesis. This pathway is frequently activated in many human cancer types [10]. Hence, we further investigated whether PI3K/AKT/mTOR signaling pathway was involved in the regulation of H2S in melanoma cell lines. Indeed, western blotting analysis exhibited that NaHS (2 mM) dramatically diminished expression of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt) and mTOR, which resembled rapamycin (100 nM, 12 h) treatment (Figs. 4a) in A375 and SK-MEL-28 cells. Given that rapamycin mediates tumor cell growth and development by inhibiting mTOR expression, we speculate that H2S might regulate melanoma cell functions through inhibiting the PI3K/ AKT/mTOR signaling pathway. Thus, A375 and SK-MEL-28 cells were treated with or without NaHS and then exposed to IGF-1 (100 ng/mL), an activator of PI3K-AKT pathway in tumor progression.
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Fig. 1. NaHS suppressed cell proliferation, migration, division and induced apoptosis of melanoma cell lines. CCK-8 assay was used to assess A375(a) and SK-MEL-28 (b) cell proliferation. (c)The wound healing assay was preformed to evaluate cell migration. Melanoma cells were treated with 2 mM NaHS for 24 h then images were captured under a light microscope at 0 and 24 h respectively (Scale bar: 300 mm). Flow cytometry was used to analyze the cell cycle (d) and cell apoptosis (e) after NaHS treatment for 24 h. The percentage of cell cycle distribution and apoptotic cells was calculated and shown in histogram separately on the right. (f) Western blotting detected expression of Bcl-xL and Cleaved Caspase-3 apoptosis-related protein in A375 and SK-MEL-28 cells treated with graded NaHS, β-actin was used as an internal control. The data express the mean standard deviation of three experiments. NS: nonsignificant difference, *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control (NC). 201 285 mm (300 300 DPI).
Western blotting analysis showed that IGF-1 (100 ng/mL) could reverse the inhibition of PI3K-AKT by NaHS (2 mM) treatment, which manifested by inducing phosphorylation of Akt (Fig. 4b). Moreover, the H2S-induced cell behaviors, including decreased
proliferation, migration, cell cycle and increased apoptosis, were mostly reversed by IGF-1 (100 ng/mL) exposure (Fig. 4c – f). Collectively, these data indicated that NaHS could inhibit melanoma via suppression of PI3K/AKT/ mTOR signaling pathway.
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Fig. 2. NaHS promotes autophagy in melanoma cell lines. (a) Western blotting detected expression of LC3-I, LC3-II and p62 protein in the concentration of 2 mM NaHS in A375 and SK-MEL-28 cells. β-actin was used as an internal control. (b) Optical densitometric analysis of LC3-II/LC3-I and P62 relative expression were shown in the bar chart. (c) A375 and SK-MEL-28 cells transfected with GFP-mRFP-LC3 adenovirus for 2 h, LC3 puncta dots (yellow and red) were observed under a laser scanning confocal microscope, the nuclei (blue) were stained with DAPI. (Scale bar: 10 mm) (d) The quantification of the average number of autophagosomes (yellow dots) and autolysosomes (Free red dots) per cell (e) The ratio of cells presenting typical LC3 puncta dots. (f) The ultrastructural features of autophagy and autophagic vesicles were imaged by TEM (Scale bars: 1 and 0.5 mm). The data express the mean standard deviation of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control (NC). 208 276 mm (300 300 DPI).
4. Discussion Melanoma is a malignant tumor of melanocytes, which are the cells that make the pigment melanin and are derived from the neural crest [1]. It is a lethal disease with increasing incidence, afflicting over 68,000 people in the United States annually and casing 8000 annual deaths. In addition, malignant melanoma is the fifth most frequently diagnosed cancer in men and the sixth most common in women [2]. The prognosis of patients is often poor mainly due to the high distant metastasis rate and drug resistance
[3]. Therefore, molecular targets hold considerable clinical significance for early detection and development of novel therapeutic approaches for patients with melanoma. H2S is now widely considered the third gaseous signaling molecule and plays important biological and pharmacological roles in the progression of many types of cancer [8]. In recent years, an increasing number of researches have revealed that endogenously produced or exogenously treatment H2S could exert two obviously opposite functions on the growth of cancer cells [11–13]. The immunohistochemical analysis performed on over 100 human
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Fig. 3. Suppression of autophagy by ATG7 shRNA enhanced H2S-regulated cell apoptosis in melanoma. Cells were transfected with ATG7 shRNA or control for 3 days. (a) Western blot analysis of ATG7 and LC3 protein expression in A375 and SK-MEL- 28 cells respectively, β-actin was used as an internal control. Cells transfected with the shRNA against ATG7 or control were subjected to the CCK-8(b and c) and migration assay(d). Representative fields of migrated cells are shown. (Scale bar: 300 mm). Flow cytometry was used to analyze the cell cycle(e) and cell apoptosis (f) The percentage of apoptotic cells and cell cycle distribution was calculated and shown in histogram separately on the right. The data express the mean standard deviation of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with negative control shRNA (shNC). NS: nonsignificant difference. 200 297 mm (300 300 DPI).
samples showed that CSE expression increased from nevi to primary melanoma, decreased in metastatic tissue and was silent in lymph node metastases, which also suggested the involvement of H2S in the progression of melanoma [9]. Endogenous H2S or relatively low concentrations of exogenous H2S for a relatively short time might promote or maintain cancer cell growth, while
overexpression of H2S generating enzymes or treatment with relatively high levels of H2S donor for a relatively long duration may exhibit anti-cancer effects. It has been reported that 25–50 mM NaHS could enhance proliferation, viability, migration, and invasion of human thyroid carcinoma cells, whereas NaHS at the concentration of 200 mM
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Fig. 4. NaHS inhibited melanoma via suppression of PI3K/AKT/ mTOR signaling pathway. Western blot analysis of PI3K/ p-PI3K, Akt/p-Akt and mTOR expression in A375 and SK-MEL-28 cells treated with NaHS(2 mM,24 h), rapamycin(100 nM,12 h), IGF-1(100 ng/mL,24 h) alone or NaHS in combination with IGF-1(24 h), β-actin was used as an internal control (a and b). Cells treated with NaHS(2 mM,24 h), IGF-1(100 ng/mL,24 h) alone or NaHS in combination with IGF-1(24 h) were subjected to the CCK-8(c and d) and migration assay(e). Representative fields of migrated cells are shown. (Scale bar: 300 mm). Flow cytometry was used to analyze the cell cycle (f) and cell apoptosis (g). The percentage of apoptotic cells and cell cycle distribution was calculated and shown in histogram separately on the right. The data express the mean standard deviation of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NaHS group. 199 297 mm (300 300 DPI).
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might exert inhibitory effect [14]. Likewise, growth and differentiation of human keratinocytes can be promoted by exogenous H2S [15], while a high level of NaHS possessed controversy traits, meanwhile induced apoptosis and cell cycle arrest in SGC-7901 gastric cancer cells [16] and hepatocellular carcinoma [17]. In this study, we used 2 mM NaHS as a donor of H2S gas to treat human melanoma cell lines for 24 h and it attenuated cell proliferation and migration, arrested cell cycle but induced cell autophagy. Cleaved Caspase-3 activation and decrease of antiapoptotic protein (Bcl-xL) further confirmed that H2S triggers apoptosis of melanoma cells. The difference between their results and ours may be attributed to diversity in H2S treatment modes and cell lines. Since different types of cancer cells hold distinct drug susceptibilities and physiological traits, further researches are needed to parse out the reasonable strategy for H2S application on different types of human cancers, in order to achieve optimal anticancer efficacy. In combination with previous studies, our results suggested that H2S can be multitasking by regulating different downstream effectors in different cancers and it functions primarily as a metastasis suppressor in melanoma. Autophagy is a highly regulated process that degrades damaged cytoplasm components and organelles in lysosomes to provide substrates in times of nutrient deprivation and thus maintain cellular homeostasis [18]. It was found that autophagy is playing an increasingly important role in cell growth and development modified by H2S. Recent studies indicated that H2S could significantly inhibit the autophagic activity in the rats with middle cerebral artery occlusion [19] and reduce renal tissue fibrosis by downregulating autophagy in diabetic rats [20], whereas, autophagy in hepatocellular carcinoma cells and human keratinocytes could be promoted by NaHS [15,17]. While defective autophagy has been reported to be responsible for carcinogenesis, this cellular process maintains the survival of cancer cells in the presence of stress and nutrient deprivation [21–23]. Most malignant melanomas are under high endoplasmic reticulum (ER) stress, an inducer of autophagy, and metabolic energy balance of invasive melanoma cells may be correlated with autophagy conditions [24,25]. The absolute levels of some autophagosomal proteins, including LC3B, are higher in tumor tissues and increased LC3B expression is associated with a low grade of differentiation and poorer overall survival [26,27]. Therefore, knockdown essential genes for autophagy induction, or treatment with autophagy inhibitor 3methyladenine, have been reported to attenuate the cell invasion and migration [28]. Here we also demonstrated that H2S concurrently exhibited anti-tumor and pro-autophagic effects on melanoma cells. Intriguingly, shRNA-mediated abrogation of autophagy only enhanced the apoptotic process caused by H2S, suggesting that exogenous H2S might exert an anti-tumor effect independent of autophagy. Further studies are needed to elucidate the exact relationship between H2S and autophagy in regulating melanoma progression. To better understand the mechanism by which this effect was achieved, we evaluated the possible involvement of the PI3K/AKT/ mTOR signaling pathway, which plays a critical role in cell motility, survival, growth and angiogenesis. PI3K first activates the threonine/serine kinase AKT, which then phosphorylate mTOR via a cascade of molecules [29,30]. The PI3K/AKT/mTOR pathway is one of the most common dysregulated signaling pathways in melanoma survival and progression. It has been reported to be involved in primary and secondary resistance to targeted therapy [31]. In addition, other reports proved that exogenous H2S exerted multiple biological effects by inhibiting the PI3K/AKT/mTOR pathway in hepatocellular carcinoma cells [15] and thyroid cancer cells [14]. In this study, we found administration of NaHS decreased the expression of phosphorylated PI3K, AKT and mTOR, while IGF-1, which has been widely known to activate PI3K/AKT
and drive the progression and metastatic spread of multiple tumors [32,33,34], could potentiate the PI3K-AKT pathway inhibited by NaHS. Meanwhile, the impact of NaHS on the melanoma cell phenotype could be mostly reversed by IGF-1. High levels of circulating IGF-1 and IGF-1R expression are relevant to a higher risk of several common cancers, including melanoma [34]. Moreover, evidence suggested that miR-425 could inhibit melanoma growth, migration, invasion and stem-cell like properties via suppression of PI3K-Akt pathway by targeting IGF-1 [28]. Collectively, these results implied that PI3K/AKT/mTOR signaling pathway may be involved in exogenous H2S-regulated biological functions of melanoma cells. A limitation of the present study is that we did not confirm the modulatory effects of H2S on melanoma cells in vivo. Previous studies indicated that exogenous H2S donors (DATS) significantly inhibit tumor growth of mice subcutaneously injected with B16F10 cells by 67 % as compared with control groups [9]. Itraconazole inhibited melanoma growth and extended survival of athymic nude mice subcutaneously implanted with A375 melanoma cells [35]. Additionally, implantation of miR-23a-overexpressed cells resulted in increased overall survival and led to a decreased number of metastases in the lung and liver, which were consistent with their results in vitro [28]. Therefore, it would be particularly interesting to decipher whether NaHS can play a similar role by suppressing melanoma malignant progression in vivo. Another shortage is that we did not evaluate the level or the expression of hydrogen sulfide producing enzymes-CSE and CBS in melanoma. Further studies are required to further clarify whether endogenous H2S is synthesized in melanoma cells and define its potential role in the regulation of various cell behaviors. In conclusion, the present study demonstrated that exogenous H2S could inhibit human melanoma cell development by regulating the PI3K/AKT/mTOR signaling pathway. Our findings provide evidence for the underlying signal pathways and biochemical mechanisms that exist in the melanoma cells. Hydrogen sulfide releasing drugs could be a potential candidate for the treatment of melanoma. Authors' contributions Chengfeng Zhang and Jiayi Ying designed the experiments and analyzed the results. Qing Xiao, Zhuhui Qiao and Yiwen Yang carried out experiments. Qing Xiao, Xiaoxi Dai and Zhongyi Xu wrote the paper. Jiayi Ying provided experimental materials and contributed to data analyses. Leihong Xiang supervised the design of experiments and data analyses. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by grants from Natural Science Foundation of China (Nos. 81573064, 81872544 and 81903243). References [1] A.M. Eggermont, A. Spatz, C. Robert, Cutaneous melanoma, Lancet. 383 (9919) (2014) 816–827. [2] Global, regional, and national life expectancy, all-cause mortality, and causespecific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015, Lancet. 388 (2016) 1459–1544. [3] J.K. Winkler, K. Buder-Bakhaya, A. Dimitrakopoulou-Strauss, Malignant melanoma: current status, Radiologe 57 (2017) 814–821. [4] M.D. Hartle, M.D. Pluth, A practical guide to working with H2S at the interface of chemistry and biology, Chem. Soc. Rev. 45 (22) (2016) 6108–6117.
Please cite this article in press as: Q. Xiao, et al., Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2020.02.004
G Model DESC 3572 No. of Pages 9
Q. Xiao et al. / Journal of Dermatological Science xxx (2019) xxx–xxx [5] J. Chen, J. Gao, W. Sun, et al., Involvement of exogenous H2S in recovery of cardio protection from ischemic post-conditioning via increase of autophagy in the aged hearts, Int. J. Cardiol. 220 (2016) 681–692. [6] Y. Sun, Y. Huang, R. Zhang, et al., Hydrogen sulfide upregulates KATP channel expression in vascular smooth muscle cells of spontaneously hypertensive rats, J. Mol. Med. 93 (4) (2015) 439–455. [7] Y. Kimura, Y.I. Goto, H. Kimura, Hydrogen sulfide protects neurons from oxidative stress, Neurosci. Res. 71 (2011) 88. [8] D. Wu, W. Si, M. Wang, S. Lv, Y. Li, Hydrogen sulfide in cancer: friend or foe? Nitric Oxide 50 (2015) 38–45. [9] E. Panza, P. DeCicco, C. Armogida, G. Scognamiglio, V. Gigantino, G. Botti, D. Germano, M. Napolitano, A. Papapetropoulos, M. Bucci, G. Cirino, A. Ianaro, Role of the cystathionine g lyase/hydrogen sulfide pathway in human melanoma progression Pigment, Cell Melanoma Res. 28 (1) (2015) 61–72. [10] K. Sheppard, K.M. Kinross, B. Solomon, R.B. Pearson, W.A. Phillips, Targeting PI3 Kinase/AKT/mTOR signaling in Cancer, Crit. Rev. Oncog. 17 (1) (2012) 69–95. [11] Z.W. Lee, X.Y. Teo, E.Y. Tay, C.H. Tan, T. Hagen, P.K. Moore, L.W. Deng, Utilizing hydrogen sulfide as a novel anti-cancer agent by targeting cancer glycolysis and pH imbalance, Br. J. Pharmacol. 171 (18) (2014) 4322–4336. [12] Z. Ma, Q. Bi, Y. Wang, Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines, Oral Dis. 21 (2) (2015) 156–162. [13] C. Szabo, M.R. Hellmich, Endogenously produced hydrogen sulfide supports tumor cell growth and proliferation, Cell Cycle 12 (18) (2013) 2915–2916. [14] D. Wu, J. Li, et al., Exogenous hydrogen sulfide regulates the growth of human thyroid carcinoma cells, Oxid. Med. Cell. Longev. (2019). [15] X. Xie, H. Dai, B. Zhuang, et al., Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes, Biochem. Biophys. Res. Commun. 472 (3) (2016) 437–443. [16] K. Ma, Y. Liu, Q. Zhu, C.H. Liu, J.L. Duan, B.K. Tan, Y.Z. Zhu, H2S donor, Spropargyl-cysteine, increases CSE in SGC-7901 and cancer-induced mice: evidence for a novel anti-cancer effect of endogenous H2S? PLoS One 6 (6) (2011)e20525. [17] S.S. Wang, Y.H. Chen, N. Chen, L.J. Wang, D.X. Chen, H.L. Weng, et al., Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/AKT/mTOR signaling pathway, Cell Death Dis. 8 (3) (2017) e2688. [18] B. Ravikumar, S. Sarkar, J.E. Davies, M. Futter, M. Garcia-Arencibia, Z.W. GreenThompson, et al., Regulation of mammalian autophagy in physiology and pathophysiology, Physiol. Rev. 90 (4) (2010) 1383–1435. [19] W.-W. Jiang, H.Y. Bai-Sheng Huang, et al., Sodium hydrosulfide attenuates cerebral ischemia/ reperfusion injury by suppressing overactivated autophagy in rats, FEBS Open Bio 7 (11) (2017) 1686–1695.
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[20] L. Li, et al., Hydrogen sulfide reduced renal tissue fibrosis by regulating autophagy in diabetic rats, Mol. Med. Rep. 16 (2017) 1715–1722. [21] R. Mathew, V. Karantza-Wadsworth, E. White, Role of autophagy in cancer, Nat. Rev. Cancer 7 (12) (2007) 961. [22] M.C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Self- eating and self-killing: crosstalk between autophagy and apoptosis, Nat. Rev. Mol. Cell Biol. 8 (9) (2007) 741–752. [23] X.H. Ma, S. Piao, D. Wang, et al., Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma, Clin. Cancer Res. 17 (10) (2011) 3478–3489. [24] D.T. Rutkowski, R.J. Kaufman, That which does not kill me makes me stronger: adapting to chronic ER stress, Trends Biochem. Sci. 32 (10) (2007) 469. [25] P. Hersey, X.D. Zhang, Adaptation to ER stress as a driver of malignancy and resistance to therapy in human melanoma, Pigment Cell Melanoma Res. 21 (3) (2008) 358. [26] R. Lazova, V. Klump, J. Pawelek, Autophagy in cutaneous malignant melanoma, J. Cutan. Pathol. 37 (2) (2010) 256–268. [27] C. Miracco, G. Cevenini, A. Franchi, et al., Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions, Hum. Pathol. 41 (4) (2010) 503–512. [28] G. Weinan, W. Huina, Y. Yuqi, et al., Down-regulated miR-23a contributes to the metastasis of cutaneous melanoma by promoting autophagy, Theranostics. 7 (8) (2017) 2231–2249. [29] J. Rodon, R. Dienstmann, V. Serra, et al., Development of PI3K inhibitors: lessons learned from early clinical trials, Nat. Rev. Clin. Oncol. 10 (3) (2013) 143–153. [30] A. Perl, Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases, Nat. Rev. Rheumatol. 12 (2016) 169–182. [31] P. Liu, Y. Hu, L. Ma, et al., miR-425 inhibits melanoma metastasis through repression of PI3K-Akt pathway by targeting IGF-1, Biomed. Pharmacother. 75 (2015) 51–57. [32] A. Grimberg, Mechanisms by which IGF-I may promote cancer, Cancer Biol. Ther. 2 (6) (2003) 630–635. [33] N. Shimada, S. Iwakami, M. Moriyama, H. Moriyama, et al., Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor, PLoS One 9 (2014)e86459. [34] R. Kucera, I. Treskova, J. Vrzalova, S. Svobodova, O. Topolcan, R. Fuchsova, M. Rousarova, V. Treska, T. Kydlicek, Evaluation of IGF1 serum levels in malignant melanoma and healthy subjects, Anticancer Res. 34 (9) (2014) 5217–5220. [35] G. Liang, M. Liu, Q. Wang, et al., Itraconazole exerts its anti-melanoma effect by suppressing Hedgehog, Wnt, and PI3K/mTOR signaling pathways, Oncotarget 8 (17) (2017) 28510–28525.
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