Apoptosis In Human Melanoma Cells Research Articles

Andrographolide inhibits proliferation and induces cell cycle arrest and apoptosis in human melanoma cells.

Andrographolide (Andro), a natural compound isolated from Andrographis paniculata, has been demonstrated to have anticancer efficacy in several types of tumors. In the present study, the anticancer effects and mechanism of Andro in human malignant melanoma were investigated. Cell viability analysis was performed using an MTT assay and the effect of Andro on the cell cycle and apoptosis of human malignant melanoma cells was determined by flow cytometry. Western blot analysis was performed to evaluate the protein expression levels of human malignant melanoma cells following treatment with Andro. The results revealed that Andro potently inhibited cell proliferation by inducing G2/M cell-cycle arrest in human malignant melanoma C8161 and A375 cell lines. In addition, treatment with Andro induced apoptosis, which was associated with the cleavage of poly(adenosine diphosphate-ribose) polymerase and activation of caspase-3. It was observed that Andro induced activation of the c-Jun N-terminal kinase and p38 signaling pathway, which may be connected with cell cycle arrest and apoptosis. In conclusion, the results demonstrated that Andro may be a promising and effective agent for antitumor therapy against human malignant melanoma.

Ingenol-3-Angelate Suppresses Growth of Melanoma Cells and Skin Tumor Development by Downregulation of NF-κB-Cox2 Signaling.

BackgroundA recent focus in skin cancer prevention intervenes though modulating molecular links between inflammation and cell growth signaling, such as NF-κB. This study elucidates the effect of a non-tumor promoting phorbol ester, ingenol-3-angelate (I3A), on the growth of human melanoma cells and on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation and 7,12-Dimethylbenz(a)anthracene (DMBA)-induced skin carcinoma in mice.Material/MethodsCell viability was assessed by MTT assay, cell proliferation by clonogenic assay, apoptosis and cell cycle arrest was analyzed by flow cytometry, protein expression was studied by IHC and Western blotting, and gene expression by qPCR.ResultsI3A suppressed the survival and proliferation of human melanoma cells with estimated IC50 values around 38 and 46 μM for A2058 and HT144 cell, respectively. I3A activated the protein levels of PKCδ and PKCɛ, which induced apoptosis by activating caspase-9 and caspace-3 followed by lowering of mitochondrial membrane potential and enhancing DNA fragmentation. I3A induced G1 phase cell cycle arrest as well as G2/M phase arrest in both cell lines. I3A inhibited the levels of NF-κB p65 protein as well as phosphorylation of p65 and its nuclear translocation. I3A suppressed the gene expression of NF-κB, COX-2 and iNOS. I3A inhibited TPA-induced inflammation and epidermal hyperplasia in female ICR mice by downregulating NF-κB and iNOS. I3A suppressed the growth of skin tumor in DMBA-induced mice in dose-dependent manner.ConclusionsThe mechanism of I3A induces apoptosis in human melanoma cells and suppresses skin inflammation and carcinoma via downregulation of NF-κB-iNOS-COX-2 signaling.

Pharmacological activation of TRPV4 produces immediate cell damage and induction of apoptosis in human melanoma cells and HaCaT keratinocytes

BackgroundTRPV4 channels are calcium-permeable cation channels that are activated by several physicochemical stimuli. Accordingly, TRPV4 channels have been implicated in the regulation of osmosensing, mechanotransduction, thermosensation, and epithelial/endothelial barrier functions. Whether TRPV4 is also mechanistically implicated in melanoma cell proliferation is not clear. Here, we hypothesized that TRPV4 is expressed in human melanoma and that pharmacological activation interferes with cell proliferation.Methodology/Principal findingsTRPV4 functions were studied in melanoma cell lines (A375, SK-MEL-28, MKTBR), immortalized non-cancer keratinocytes (HaCaT), and murine 3T3 fibroblasts by patch-clamp, qRT-PCR, intracellular calcium measurements, cell proliferation, and flow cytometric assays of apoptosis and cell cycle. The selective TRPV4-activator, GSK1016790A, elicited non-selective cation currents with TRPV4-typical current-voltage-relationship in all cell lines. GSK1016790A-induced currents were blocked by the TRPV4-blocker, HC067047. TRPV4 mRNA expression was demonstrated by qRT-PCR. In A375 cells, TRPV4 activation was frequently paralleled by co-activation of calcium/calmodulin-regulated KCa3.1 channels. Light microscopy showed that TRPV4-activation produced rapid cellular disarrangement, nuclear densification, and detachment of a large fraction of all melanoma cell lines and HaCaT cells. TRPV4-activation induced apoptosis and drastically inhibited A375 and HaCaT proliferation that could be partially prevented by HC067047.Conclusions/SignificanceOur study showed that TRPV4 channels were functionally expressed in human melanoma cell lines and in human keratinocytes. Pharmacological TRPV4 activation in human melanoma cells and keratinocytes caused severe cellular disarrangement, necrosis and apoptosis. Pharmacological targeting of TRPV4 could be an alternative or adjuvant therapeutic strategy to treat melanoma progression and other proliferative skin disorders.

Antiproliferative Activity and Induction of Apoptosis in Human Melanoma Cells by Houttuynia cordata Thunb Extract.

To analyze the apoptotic effect of Houttuynia cordata Thunb (HCT) extract on human melanoma A375 cells and its underlying mechanisms. The effects of HCT on cell death were determined using the MTT assay. Hoechst 33342 staining was conducted to confirm the detection of cell apoptosis. Caspase-3 and caspase-8 mRNA and cleaved protein levels were investigated by RT-PCR and western blotting, respectively. The release of high mobility group box 1 (HMGB1) and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by ELISA. Caspase-3 and caspase-8 specific inhibitors suppressed HCT-induced cell death. HCT increased caspase-3 and caspase-8 mRNA, protein levels, and caspase activities in a concentration- and time-dependent manner. HCT induced MAPK phosphorylation in a time-dependent fashion. Pretreatment of cells with a selective inhibitor of p38 MAPK reduced apoptosis and reversed the levels of HMGB1 release in response to HCT treatment. HCT induces A375 programmed cell death by activating the caspase-dependent pathway and by p38 phosphorylation associated with HMGB1 reduction.

Juglone potentiates TRAIL‑induced apoptosis in human melanoma cells via activating the ROS‑p38‑p53 pathway.

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑based cancer therapy offers promise as TRAIL can kill cancer cells without apparent toxicity towards normal cells. However, intrinsic or acquired resistance to TRAIL inseveral types of cancer cell has become a major challenge in TRAIL‑based cancer therapy. Juglone is a natural compound isolated from walnut trees. In the present study, it was demonstrated that juglone sensitized melanoma cells to TRAIL‑induced cytotoxicity by MTT and crystal violet assays. Flow cytometry analysis indicated that juglone potentiated TRAIL‑induced cell death. Western blot assay demonstrated that the expressions of cleaved poly(ADP‑ribose) polymerase (PARP) and cleaved caspase 3 were markedly increased in the juglone combined with TRAIL group. Exposure to TRAIL alone did not induce the production of reactive oxygen species (ROS), activation of p38 orincrease of p53 in the TRAIL‑resistant melanoma cells, as determined by flow cytometry and western blot analysis. However, exposure to TRAIL in combination with juglone markedly increased the production of ROS, activated p38 and increased p53, compared with the cells treated with either juglone or TRAIL alone. Pretreatment with N‑acetyl cysteine, a ROS scavenger, significantly reduced the cytotoxicity of juglone in combination with TRAIL, which further supported that ROS was involved in the juglone‑induced sensitization of TRAIL. In conclusion, juglone potentiated TRAIL‑induced apoptosis in melanoma cells, and these effects were partially mediated through the ROS‑p38‑p53 pathway. These findings suggested that juglone may be a potential sensitizer for TRAIL therapy in the treatment of melanoma.

The NAMPT/E2F2/SIRT1 axis promotes proliferation and inhibits p53-dependent apoptosis in human melanoma cells

Melanoma is the most common primary malignant neoplasm in adults, causing more deaths than any other skin cancer, necessitating the development of new target-based approaches. Current evidence suggests SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD+ biosynthetic enzyme, together comprise a novel systemic regulatory network to play a pivotal role in cell proliferation and apoptosis. Nevertheless, how the regulation of this cofactor interfaces with signal transduction network remains poorly understood in melanoma. Here, we report NAMPT is highly expressed in melanomaassociated with poor overall survival in patients. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and melanoma cell proliferation capacity, and NAMPT knockdown induced apoptosis through the activity of the tumor suppressor p53. Next, we demonstrate NAMPT regulates the transcription factor E2F family member 2 (E2F2) in the apoptosis process. Downstream, E2F2 control the mRNA and protein levels of SIRT1. Finally, we find NAMPT mediates the apoptosis resistance of melanoma cells through NAMPT-E2F2-SIRT1 axis, more than NAD+-driven transcriptional program. Accordingly, our results demonstrated that NAMPT is a prognostic marker in melanoma, and the identificationofNAMPT-E2F2-SIRT1 pathway establishes another link between NAMPT and apoptosis events in melanoma, with therapeutic implications for this deadly cancer.

Mitochondria-Associated Apoptosis in Human Melanoma Cells Induced by Cardanol Monoene from Cashew Nut Shell Liquid.

Cardanol monoene (CM) is the major phenolic component extracted from cashew nut shell liquid (CNSL), which has been relevant to wide range of biological effects. In this study, we found that CM could inhibit the M14 human melanoma cells proliferation in a dose dependent and time dependent manner, and the IC50 values were determined to be 23.15 ± 2.42 μM and 12.30 ± 1.67 μM after 24 and 48 h treatment, respectively. The flow cytometric analysis demonstrated that CM induced M14 cell cycle arrest at the S phase, along with the collapse of mitochondrial membrane potential (ΔΨm) and the accumulation of reactive oxygen species (ROS) level in cells, but the apoptotic cells reduced when treated with Z-VAD-FMK (pan-caspase inhibitor). Western blotting showed that the expressions of p53, cytosol cytochrome C, cleaved-caspase-3, and cleaved-PARP were up-regulated, and the expression level of Bax/Bcl-2 ratio increased significantly. The 2527 significant differentially expressed genes were obtained by RNA-seq, which were assigned to 270 KEGG pathways. These results indicated that CM induced M14 cells apoptosis via the ROS triggered mitochondrial-associated pathways, which supports the potential application of CM for the therapy of melanoma cancer.

Abstract 3967: High microsomal PGE2synthase-1 levels associate with low CD8 T cells and poorer melanoma patient survival

Abstract COX-2 and its product PGE2 are reported to enhance carcinogenesis and tumor progression and to support immunosuppression, as reported by others in melanoma and other cancers. As most COX-2 inhibitors result in cardiotoxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is now also an alternative consideration for therapeutic targeting. Our previously published results (Kim, et al., 2016) showed that mPGES1 protein increased with melanoma patient clinical stage and that its intense expression was associated with reduced patient survival. We also reported that mPGES1 inhibition attenuated cell survival and increased apoptosis in human melanoma cells and significantly suppressed tumor growth in melanoma xenografts. This current study was designed to evaluate whether expression of mPGES1 contributes to immune evasion. Tumors in a Stage III melanoma TMA analysis demonstrated that high mPGES1 expression (intensity 3) was significantly associated with low CD8 levels (lower than median CD8 percent value) (r=-0.2722, p=0.0128). Importantly, stage III melanoma patients with this high mPGES1 and low CD8 signature had significantly increased risk of death as determined by Kaplan-Meier method and Univariate Cox proportional hazards regression models, and compared with patients with low mPGES1/low CD8 and low mPGES1/high CD8. Furthermore, the differential levels of several chemokines including CXCL1 and CXCL2 to regulate T cell migration were correlated the mPGES1 expression in the TCGA melanoma data set. Collectively, our study continues to resolve the potential role of mPGES1 in regulation of immune evasion and is designed as a preclinical approach to develop a rational therapeutic strategy targeting PGE2-driven inflammatory mediators as useful adjuvants for immune-based therapy. Citation Format: Sun-Hee Kim, Jason Roszik, Weiyi Peng, Suhendan Ekmekcioglu, Elizabeth A. Grimm. High microsomal PGE2synthase-1 levels associate with low CD8 T cells and poorer melanoma patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3967. doi:10.1158/1538-7445.AM2017-3967

Design, synthesis, and identification of a novel napthalamide-isoselenocyanate compound NISC-6 as a dual Topoisomerase-IIα and Akt pathway inhibitor, and evaluation of its anti-melanoma activity

Synthesis and anti-melanoma activity of novel naphthalimide isoselenocyanate (NISC) and naphthalimide selenourea (NSU) analogs are described. The novel agents were screened for growth inhibition of different human melanoma cell lines including those having BRAFV600E mutation (UACC903, 1205Lu, and A375M) and BRAFWT (CHL-1). In general, the NISC analogs (4a-d) were more effective in inhibiting the cell viability than the NSU analogs (7a-b). Overall, NISC-6 (4d), having a six-carbon alkyl chain, was identified as the most cytotoxic compound in both BRAFV600E mutated and BRAFWT cells. NISC-6 docked strongly into the binding sites of Akt1 and human topoisomerase IIα (Topo-IIα), and the docking results were supported by experimental findings showing NISC-6 to inhibit of both Akt pathway and Topo-IIα activity in a dose dependent manner. Furthermore, NISC-6 effectively induced apoptosis in human melanoma cells, inhibited tumor growth by ∼69% in a melanoma mouse xenograft model, and showed excellent compliance with the Lipinski’ rule of five, suggesting both its efficacy and drug-like behavior under physiological conditions.

Chaetocin induces apoptosis in human melanoma cells through the generation of reactive oxygen species and the intrinsic mitochondrial pathway, and exerts its anti-tumor activity in vivo.

Chaetocin is a small-molecule natural product produced by Chaetomium species fungi, and it has a potent anti-proliferative pharmacological activity on various cancer cells. However, the effect of chaetocin on anti-melanoma pharmacological role has not been investigated. Therefore, in this study, we explored the effect of chaetocin on cell proliferation in the human melanoma Sk-Mel-28 and A375 cells and the growth of tumor xenografts in nude mice. The results indicated that chaetocin treatment significantly suppressed cell proliferation and induced apoptosis in the Sk-Mel-28 and A375 cells in a dose- and time-dependent manner. Furthermore, chaetocin treatment resulted in an increased level of cellular reactive oxygen species (ROS), and pre-incubation of cells with N-acetylcysteine (NAC) significantly abrogated chaetocin-induced apoptosis in the melanoma cells. A significant reduction of mitochondrial membrane potential and the release of cytochrome c were observed after chaetocin treatment. Additionally, chaetocin treatment significantly up-regulated the protein levels of Bax, cleaved caspase-9/-3, simultaneously down-regulated the protein levels of Bcl-2, procaspase-9/-3, and activated caspase-9/-3 activity in the melanoma cells. The in vivo data demonstrated that chaetocin treatment significantly inhibited the growth of melanoma tumor xenografts in nude mice, which was closely associated with apoptosis induction, a reduced level of PCNA (proliferating cell nuclear antigen) expression, and activation of capase-9/-3 in tumor xenografts. These are the first data to demonstrate that chaetocin exerts a proapoptotic activity on human melanoma cells through ROS generation and the intrinsic mitochondrial pathway. Therefore, chaetocin might represent an effective candidate for melanoma chemotherapy.

NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma.

Inflammasomes are mediators of inflammation, and constitutively activated NLRP3 inflammasomes have been linked to IL-1β-mediated tumorigenesis in human melanoma. Whereas NLRP3 regulation of caspase-1 activation requires the adaptor protein ASC, caspase-1 activation by another danger-signaling sensor NLRP1 does not require ASC because NLRP1 contains a C-terminal CARD domain that facilitates direct caspase-1 activation via CARD-CARD interaction. We hypothesized that NLRP1 has additional biological activities besides IL-1β maturation and investigated its role in melanoma tumorigenesis. NLRP1 expression in melanoma was confirmed by analysis of 216 melanoma tumors and 13 human melanoma cell lines. Unlike monocytic THP-1 cells with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm. Knocking down NLRP1 revealed a tumor promoting property of NLRP1 both in vitro and in vivo. Mechanistic studies showed that caspase-1 activity, IL-1β production, IL-1β secretion, and NF-kB activity were reduced by knocking down of NLRP1 in human metastatic melanoma cell lines 1205Lu and HS294T, indicating that NLRP1 inflammasomes are active in metastatic melanoma. However, unlike previous reports showing that NLRP1 enhances pyroptosis in macrophages, NLRP1 in melanoma behaved differently in the context of cell death. Knocking down NLRP1 increased caspase-2, -9, and -3/7 activities and promoted apoptosis in human melanoma cells. Immunoprecipitation revealed interaction of NLRP1 with CARD-containing caspase-2 and -9, whereas NLRP3 lacking a CARD motif did not interact with the caspases. Consistent with these findings, NLRP1 activation but not NLRP3 activation reduced caspase-2, -9, and -3/7 activities and provided protection against apoptosis in human melanoma cells, suggesting a suppressive role of NLRP1 in caspase-3/7 activation and apoptosis via interaction with caspase-2 and -9. In summary, we showed that NLRP1 promotes melanoma growth by enhancing inflammasome activation and suppressing apoptotic pathways. Our study demonstrates a tumor-promoting role of NLRP1 in cancer cells.

Development of efficient adeno-associated virus (AAV)-mediated gene delivery system with a phytoactive material for targeting human melanoma cells

We exploited the emerging potential of gene therapy strategies to design a powerful therapeutic system that combines two key components—AAV vector and [6]-gingerol. In this study, we created an AAV2 construct expressing the proapoptotic protein BIM, which uses HSPG as its primary receptor, to target HSPG-overexpressing melanoma cells. This combination treatment showed promising results in vitro, inducing apoptosis in human melanoma cells. This new platform technology will make a significant contribution to numerous therapeutic applications, most notably for melanoma, including overcoming resistance to conventional anticancer therapies.

Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming.

Transcriptional activation of genes belonging to the plastidial MEP-derived isoprenoid pathway by elicitation with methyl jasmonate and coronatine enhanced the content of bioactive abietane diterpenes in Salvia sclarea hairy roots. We have shown that aethiopinone, an abietane diterpene synthesized in Salvia sclarea roots is cytotoxic and induces apoptosis in human melanoma cells. To develop a production platform for this compound and other abietane diterpenes, hairy root technology was combined with the elicitation of methyl jasmonate (MeJA) or the phytotoxin coronatine (Cor). Both MeJA and Cor induced a significant accumulation of aethiopinone, but prolonged exposure to MeJA irremediably caused inhibition of hairy root growth, which was unaffected by Cor treatment. Considering together the fold increase in aethiopinone content and the final hairy root biomass, the best combination was a Cor treatment for 28days, which allowed to obtain up to 105.34±2.30mg L-1 of this compound to be obtained, corresponding to a 24-fold increase above the basal content in untreated hairy roots. MeJA or Cor elicitation also enhanced the synthesis of other bioactive abietane-quinone diterpenes. The elicitor-dependent steering effect was due to a coordinated transcriptional activation of several biosynthetic genes belonging to the plastidial MEP-derived isoprenoid pathway. High correlations between aethiopinone content and MeJA or Cor-elicited level of gene transcripts were found for DXS2 (r 2=0.99), DXR (r 2=0.99), and GGPPS (r 2=0.98), encoding enzymes acting upstream of GGPP, the common precursor of diterpenes and other plastidial-derived terpenes, as well as CPPS (r 2=0.99), encoding the enzyme involved in the first cyclization steps leading to copalyl-diphosphate, the precursor of abietane-like diterpenes. These results point to these genes as possible targets of metabolic engineering approaches to establish a more efficient production platform for such promising anti-proliferative plant-derived compounds.

ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo

Inflammation plays a key role in tumor promotion and development. Indeed, cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer. An emerging class of compounds with significant anti-inflammatory properties is the hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs). They consist of a traditional NSAID to which an H2S-releasing moiety is covalently attached. We have recently demonstrated that H2S donors inhibit melanoma cell proliferation. In the current study, we evaluated the potential beneficial effects of a new H2S-releasing derivative of naproxen, ATB-346 [2-(6-methoxynapthalen-2-yl)-propionic acid 4-thiocarbamoyl phenyl ester] which inhibits COX activity but also releases H2S.We used cell culture and a mouse melanoma model to evaluate the effect of ATB-346 on: i) in vitro growth of human melanoma cells; ii) in vivo melanoma development in mice.Cell culture studies demonstrated that ATB-346 reduced the in vitro proliferation of human melanoma cells and this effect was associated to induction of apoptosis and inhibition of NF-κB activation. Moreover, ATB-346 had novel Akt signaling inhibitory properties. Daily oral dosing of ATB-346 (43μmol/kg) significantly reduced melanoma development in vivo.This study shows that ATB-346, a novel H2S-NSAID, inhibits human melanoma cell proliferation by inhibiting pro-survival pathways associated with NF-κB and Akt activation. Furthermore, oral treatment with ATB-346 inhibits melanoma growth in mice. In conclusion, the combination of inhibition of cyclooxygenase and delivery of H2S by ATB-346 may offer a promising alternative to existing therapies for melanoma.

Cellular inactivation of nitric oxide induces p53-dependent apoptosis in human melanoma cells

Purpose: To examine the role of endogenous nitric oxide (NO • ) and influence of p53 status during apoptosis induced by a selective iNOS inhibitor, N-[(3-aminomethyl) benzyl] acetamidine (1400W), and/or an NO • scavenger carboxy-PTIO (c-PTIO) in two isogenic human melanoma cell lines, wild-type p53 (A375) and p53 mutant (SK mel-28) cells. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Annexin V/propidium iodide assay were used to test for antiproliferation and apoptosis, respectively. Griess and reverse transcription-polymerase chain reaction (RT-PCR) reactions were carried out to assay NO • production and the mRNA levels of inhibitors of apoptosis (IAP). Results: c-PTIO and 1400W, alone or in combination, inhibited cell growth and promoted apoptosis via sub-G1 cell cycle arrest mediated by decrease in NO • . Apoptosis was delayed and greatly reduced in magnitude in SK mel-28 cells, underscoring the importance of p53 modulation of the response. In both cell types, apoptosis induced by iNOS inhibition and/or NO • depletion was blocked by an exogenous NO • donor, sodium nitroprusside. It was also found that inhibitors of apoptosis family (survivin, XIAP and cIAP1) were significantly depressed, which appear to play an important role in the regulation of p53- mediated apoptotic response under these conditions. Conclusion: The data obtained provide insight into the mechanism of cell proliferation action of endogenous NO • , based on p53 status, and indicate manipulation of iNOS may offer exciting opportunities to improve the effectiveness of melanoma treatment. Keywords: Apoptosis, Human melanoma cells, Inducible nitric oxide synthase, p53

Vitamin E δ-tocotrienol triggers endoplasmic reticulum stress-mediated apoptosis in human melanoma cells

Malignant melanoma is the leading cause of death from skin cancer. Drug toxicity and resistance represent a serious challange for melanoma treatments. Evidence demonstrates that natural compounds may play a crucial role in cancer prevention, growth and progression. Vitamin E tocotrienols (TT) were shown to possess antitumor activity. Here, we analyzed the effects of δ-TT on melanoma cell growth and the involvement of the endoplasmic reticulum (ER) stress in this activity. The experiments were performed on human melanoma cell lines, BLM and A375. δ-TT exerted a significant proapoptotic effect on both cell lines, involving the intrinsic apoptosis pathway; importantly, this compound did not affect the viability of normal human melanocytes. In melanoma cells, δ-TT exerted its antitumor effect through activation of the PERK/p-eIF2α/ATF4/CHOP, IRE1α and caspase-4 ER stress-related branches. Salubrinal, an inhibitor of the ER stress, counteracted the cytotoxic activity of δ-TT. In vivo experiments performed in nude mice bearing A375 xenografts evidenced that δ-TT reduces tumor volume and tumor mass; importantly, tumor progression was significantly delayed by δ-TT treatment. In conclusion, δ-TT exerts a proapoptotic activity on melanoma cells, through activation of the ER stress-related pathways. δ-TT might represent an effective option for novel chemopreventive/therapeutic strategies for melanoma.

Effects of an ar-turmerone derivative on the proliferation and apoptosis of A375 human melanoma cells and their mechanisms

Objective To investigate the effects of an ar-turmerone derivative (ATD)on the proliferation and apoptosis of A375 human melanoma cells. Methods Both A375 cells and human skin fibroblasts (HSFs)were cultured with different concentrations (5, 10, 20, 40 and 80 μmol/L)of ATD, vincristine and ar-turmerone, separately, for 48 hours in vitro. Subsequently, cell counting kit-8 (CCK-8)was used to evaluate cell proliferation, inverted microscopy to observe cell morphology after acridine orange/ethidium bromide (AO/EB)staining, and a colorimetric method to estimate caspase-3 activity. DNA fragmentation assay and flow cytometry were performed to assess cell apoptosis, and flow cytometry was conducted to analyze cell cycle. Results ATD, vincristine and Ar-turmerone all inhibited the proliferation of A375 cells in a dose-dependent manner (ATD: R2 = 0.99, F = 340.96, P < 0.05; vincristine: R2 = 0.99, F = 349.19, P < 0.05; ar-turmerone: R2 = 0.89, F = 25.41, P < 0.05). The fifty percent inhibitory concentra-tions (IC50s)of ATD, vincristine and ar-turmerone against A375 cells were 15.96±0.02 μmol/L, 77.00±0.04 μmol/L and 356.95±0.01 μmol/L respectively. When the drug concentrations were 5 and 10 μmol/L, the proliferation of HSFs was inhibited by 8%±0.06% and 25%±0.02% respectively by ATD, by 49%±0.09% and 34%±0.07% respectively by ar-turmerone, and by 33%±0.04% and 29%±0.08% respectively by vincristine, and the proliferation of A375 cells was inhibited by 26%±0.06% and 39%±0.02% respectively by ATD, by 6%±0.09% and 10%±0.07% respectively by ar-turmerone, and by 8%±0.04% and 17%±0.08% respectively by vincristine, with the inhibitory effects of the three drugs being significantly different from that of dimethyl sulfoxide (all P < 0.05). ATD showed stronger inhibitory effects on the proliferation of A375 cells, but weaker cytotoxic effects on HSFs compared with ar-turmerone and vincristine (all P < 0.05). Meanwhile, ATD, vincristine and ar-turmerone all induced the apoptosis of A375 cells (P < 0.05), and caspase-3 activity increased with the increase in drug concentrations (ATD: R2 = 0.98, F = 162.30, P < 0.05; vincristine: R2 = 0.96, F = 94.39, P < 0.05; ar-turmerone : R2 = 0.95, F = 57.35, P < 0.05). The effect of ATD on caspase-3 activity was strongest, followed by that of vincristine and ar-turmerone. As flow cytometry showed, all the three drugs induced cell apoptosis to different degrees, and ATD showed a relatively strong effect on cell apoptosis, especially late apoptosis, compared with the other two drugs. In the ATD group, the number of A375 cells in G1 phase gradually increased, while that in G2 phase and S phase significantly decreased with the increase in drug concentrations. Conclusions ATD exhibited proliferation-inhibiting and apoptosis-inducing effects on A375 cells, and the effects were stronger than those of vincristine and ar-turmerone. It is quite possible that ATD affects cell proliferation and differentiation by activating caspase-3 and arresting cell cycle in the G1 phase. Key words: Melanoma, experimental; Cell line, tumor; Turmerone; Vincristine; Cell proliferation; Apoptosis; Ar-turmerone derivatives

Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells.

Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma.

Silvestrol induces early autophagy and apoptosis in human melanoma cells.

BackgroundSilvestrol is a cyclopenta[b]benzofuran that was isolated from the fruits and twigs of Aglaia foveolata, a plant indigenous to Borneo in Southeast Asia. The purpose of the current study was to determine if inhibition of protein synthesis caused by silvestrol triggers autophagy and apoptosis in cultured human cancer cells derived from solid tumors.MethodsIn vitro cell viability, flow cytometry, fluorescence microscopy, qPCR and immunoblot was used to study the mechanism of action of silvestrol in MDA-MB-435 melanoma cells.ResultsBy 24 h, a decrease in cyclin B and cyclin D expression was observed in silvestrol-treated cells relative to control. In addition, silvestrol blocked progression through the cell cycle at the G2-phase. In silvestrol-treated cells, DAPI staining of nuclear chromatin displayed nucleosomal fragments. Annexin V staining demonstrated an increase in apoptotic cells after silvestrol treatment. Silvestrol induced caspase-3 activation and apoptotic cell death in a time- and dose-dependent manner. Furthermore, both silvestrol and SAHA enhanced autophagosome formation in MDA-MB-435 cells. MDA-MB-435 cells responded to silvestrol treatment with accumulation of LC3-II and time-dependent p62 degradation. Bafilomycin A, an autophagy inhibitor, resulted in the accumulation of LC3 in cells treated with silvestrol. Silvestrol-mediated cell death was attenuated in ATG7-null mouse embryonic fibroblasts (MEFs) lacking a functional autophagy protein.ConclusionsSilvestrol potently inhibits cell growth and induces cell death in human melanoma cells through induction of early autophagy and caspase-mediated apoptosis. Silvestrol represents a natural product scaffold that exhibits potent cytotoxic activity and could be used for the further study of autophagy and its relationship to apoptosis in cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1988-0) contains supplementary material, which is available to authorized users.

Pentoxifylline triggers autophagy via ER stress response that interferes with Pentoxifylline induced apoptosis in human melanoma cells

Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor is known to inhibit the growth of various cancer cells including melanoma. Here in this study, we have found that PTX induces autophagy in human melanoma cell lines (A375 and MeWo). Induction of autophagy is associated with the increase in Atg5 expression as knockdown of Atg5 effectively inhibited PTX mediated autophagy. A decrease in mTOR activation was also observed after PTX treatment. We observed that autophagy was activated as a downstream effector mechanism of ER stress induced by PTX. ER stress response was confirmed by upregulation of IRE-1α, GRP78 and CHOP expression. PTX treatment also resulted in an increase in intracellular calcium (Ca2+) level. Ca2+ is the central player as blocking Ca2+ by intracellular calcium chelator (BAPTA-AM) effectively inhibited the PTX induced ER stress response as well as autophagy. Moreover, silencing of CHOP also resulted in autophagy inhibition with a decrease in Atg5 expression. Collectively, PTX triggers ER stress response followed by induction of autophagy via involvement of Ca2+→CHOP→Atg5 signalling cascade. Interestingly, inhibition of intracellular calcium level by BAPTA-AM significantly increased PTX mediated cell death by augmenting intrinsic apoptotic pathway. Inhibition of autophagy by the ATG5 siRNA and pharmacological inhibitor, chloroquine also enhances PTX induced cell death. Taken together, our results clearly indicate that activation of ER stress response and autophagy provides resistance to PTX mediated apoptosis, and thus, interferes with the anticancer activity of PTX in human melanoma cells.