Melanoma Cell Death Research Articles

Toxicity of oxidized phosphatidylcholines in cultured human melanoma cells

The oxidized phospholipids (oxPL) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are generated from 1-palmitoyl-2-arachidonoyl-phosphatidylcholine under conditions of oxidative stress. These oxPL are components of oxidized low density lipoprotein. They are cytotoxic in cells of the arterial wall thus playing an important role in the development and progression of atherosclerosis. The toxic lipid effects include inflammation and under sustained exposure apoptosis. The aim of this study was to find out whether such toxic effects, especially apoptosis, are also elicited by oxPL in melanocytic cells in order to assess their potential for therapeutic intervention.FACS analysis after staining with fluorescent markers was performed to identify the mode of lipid-induced cell death. Activation of sphingomyelinase which generates apoptotic ceramide was measured using an established fluorescence assay. Ceramide profiles were determined by mass spectrometry.We found that 50μM POVPC induce cell death in human melanoma cells isolated from different stages of tumor progression but affect primary human melanocytes to a much lesser extent. In contrast, 50μM PGPC was only apoptotic in two out of four cell lines used in this study. The toxicity of both compounds was associated with efficient lipid uptake into the tumor cells and activation of acid sphingomyelinase. In several but not all melanoma cell lines used in this study, activation of the sphingomyelin degrading enzyme correlated with an increase in the concentration of the apoptotic mediator ceramide. The individual patterns of the newly formed ceramide species were also cell line-specific.PGPC and POVPC may be considered potential drug candidates for topical skin cancer treatment. They are toxic in malignant cells. The respective oxidized phospholipids are naturally formed in the body and resistance to these compounds is not likely to occur.

Regulation of viability, differentiation and death of human melanoma cells carrying neural stem cell biomarkers: a possibility for neural trans-differentiation.

During embryonic development, melanoblasts, the precursors of melanocytes, emerge from a subpopulation of the neural crest stem cells and migrate to colonize skin. Melanomas arise during melanoblast differentiation into melanocytes and from young proliferating melanocytes through somatic mutagenesis and epigenetic regulations. In the present study, we used several human melanoma cell lines from the sequential phases of melanoma development (radial growth phase, vertical growth phase and metastatic phase) to compare: (i) the frequency and efficiency of the induction of cell death via apoptosis and necroptosis; (ii) the presence of neural and cancer stem cell biomarkers as well as death receptors, DR5 and FAS, in both adherent and spheroid cultures of melanoma cells; (iii) anti-apoptotic effects of the endogenous production of cytokines and (iv) the ability of melanoma cells to perform neural trans-differentiation. We demonstrated that programed necrosis or necroptosis, could be induced in two metastatic melanoma lines, FEMX and OM431, while the mitochondrial pathway of apoptosis was prevalent in a vast majority of melanoma lines. All melanoma lines used in the current study expressed substantial levels of pluripotency markers, SOX2 and NANOG. There was a trend for increasing expression of Nestin, an early neuroprogenitor marker, during melanoma progression. Most of the melanoma lines, including WM35, FEMX and A375, can grow as a spheroid culture in serum-free media with supplements. It was possible to induce neural trans-differentiation of 1205Lu and OM431 melanoma cells in serum-free media supplemented with insulin. This was confirmed by the expression of neuronal markers, doublecortin and β3-Tubulin, by significant growth of neurites and by the negative regulation of this process by a dominant-negative Rac1N17. These results suggest a relative plasticity of differentiated melanoma cells and a possibility for their neural trans-differentiation without the necessity for preliminary dedifferentiation.

Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner.

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.

Gα13 mediates human cytomegalovirus-encoded chemokine receptor US28-induced cell death in melanoma.

US28, a constitutively active G-protein-coupled receptor encoded by the human cytomegalovirus, leads to mechanistically unknown programmed cell death. Here we show that expression of wild-type US28 in human melanoma cells leads to apoptotic cell death via caspase 3 activation along with reduced cell proliferation. Reduced tumor growth upon US28 expression was observed in a xenograft mouse model. The signaling mute US28R129A showed a reduced antiproliferative effect. On evaluating different G-proteins coupled to US28 for signal transduction, Gα13 was identified as the main G-protein executing the apoptotic effect. Silencing of Gα13 but not Gαq resulted in a substantial increase in cell survival. Overexpression of Gα13 but not Gαq and their GTPase deficient forms Gα13Q226L and GαqQ209L, respectively, confirmed the requirement of Gα13 for US28 mediated cell death. Increasing expression of Gα13 alone induced cell death underscoring its relay function for US28 mediated decreased cell viability. Further reduced expression of Gα13 in melanoma cell lines isolated from advanced lesions and melanoma tissue was observed. These findings identified Gα13 as crucial for US28-induced cell death, substantiating that the effect of US28 on cell fate depends on preferred G-protein binding.

Polyphenols from green tea inhibit the growth of melanoma cells through inhibition of class I histone deacetylases and induction of DNA damage.

Melanoma is the leading cause of skin cancer-related deaths. We have examined the effect of green tea polyphenols (GTPs), a natural mixture of epicatechin monomers, on melanoma cancer cell growth and the molecular mechanism underlying these effects using different human melanoma cell lines as an in vitro model. Treatment of melanoma cell lines (A375, Hs294t, SK-Mel28 and SK-Mel119) with GTPs significantly inhibited the cell viability as well as colony formation ability of melanoma cells in a dose-dependent manner. These effects of GTPs were associated with a significant inhibition of histone deacetylase (HDAC) activity, reduction in the levels of class I HDAC proteins, enhancement of histone acetyltransferase (HAT) activity and induction of DNA damage, as detected by Comet assay, in melanoma cells. GTPs-induced decrease in the levels of class I HDAC proteins is mediated through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of melanoma cells. Treatment of A375 and Hs294t cells with GTPs resulted in a decrease in the levels of cyclins and cyclin dependent kinases of G1 phase of cell cycle whereas upregulated the levels of tumor suppressor proteins (Cip1/WAF1/p21, p16 and p53).

Abstract A50: Crotamine derived fusion short-peptides and their effect on melanoma and breast cancer cells cultured in vitro

Abstract The search for molecules with therapeutic potential against cancer is a crucial issue to reduce the amount of death in human population around the world. Previously, we showed that crotamine, a natural polypeptide from the venom of the rattlesnake Crotalus durissus terrificus, has ability to induce cancer cells apoptosis in vitro, as well as, delay and inhibit melanomas formation in vivo in mice. In order to reduce crotamine length and toxicity, while maintaining its anti-cancer properties, two short peptides were designed and synthetized. Such short peptides represent an intermediate potential step toward to the development of drugs targeted cancer death. The aim of present study was to analyze pro-apoptotic activity of both of these peptides (1 and 2), composed by crotamine (crot.) and BAX (pro-apoptotic protein from BCL2 family) both derived sequences in melanoma and breast cancer cells in vitro. Melanoma and breast cancer cells (MEL-85, A2058, B16-F10 and SKBR3) were used. The MTT assay was performed after addition of both of these peptides used in different concentrations into cancer cells. The expression of proteins (PARP, Cleaved - Caspase 3, Caspase 3, Phospho - BAD) was evaluated by western blotting. Flow cytometry was performed to evaluate the cell cycle. The mitochondria potential was also verified before and after peptides addition. Morphologic changes, such as, detachment from substrate, cell contraction, loss of inter-cell elongations and several others effects were observed in cancer cells after 24 hours of incubation with peptides. At that time, the peptides exhibit cytotoxic activity as verified by MTT assay. Cell cycle analyses demonstrated that they decrease the timing of G2/M phases, while increasing a sub-G0/G1 phase. Unexpectedly, both peptides increase mitochondria potential of cancer cells, when compared with control cultures (without addition of peptides). Increase of expression pro-apoptotic proteins, such as, cleaved Caspase 3 and PARP was evidenced in cancer cells after addition of peptide -1, while Phospho-BAD was increased after addition of peptide - 2. Our data suggest that both peptides are efficient to trigger cell death in melanoma and breast cancer cells in vitro. Crot alone was not able to induce such changes in cancer cells. Citation Format: Nicole Caroline Mambelli, Paulo Luis De-Sá-Júnior, Jr., Adilson Kleber Ferreira, Ricardo Alexandre Azevedo, Diana Aparecida Câmara, Irina Kerkis, Sr.. Crotamine derived fusion short-peptides and their effect on melanoma and breast cancer cells cultured in vitro. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A50.

Atmospheric‐Pressure Plasma‐ and TRAIL‐Induced Apoptosis in TRAIL‐Resistant Colorectal Cancer Cells

The ideal anti‐cancer treatments are those that can selectively kill cancer cells without harming normal cells. Tumor necrosis factor (TNF)‐related apoptosis inducing ligand (TRAIL) is an example of a molecule that can selectively kill cancer cells but not normal cells. However, some tumors are resistant to the TRAIL treatment. We have recently shown that atmospheric cold non‐equilibrium gas plasmas (AGPs) selectively induced cell death in melanoma and colorectal cancer cell lines by activating TNF and caspases 3/7 apoptosis pathways. Here, we demonstrated that AGPs can be used to make TRAIL‐resistant colorectal cancer cells sensitive to the TRAIL treatment. AGPs plus TRAIL promoted the induction of the death receptor 5 (DR5), thereby enhancing the activation and apoptosis of ROS‐dependent caspases 3/7. This study revealed the mechanisms involved in AGP‐assisted TRAIL‐induced cancer cell death and the associated synergistic effects that AGPs has on TRAIL‐resistant colorectal cancer cells.

Calpain-3 impairs cell proliferation and stimulates oxidative stress-mediated cell death in melanoma cells.

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.

Prenylation inhibition-induced cell death in melanoma: reduced sensitivity in BRAF mutant/PTEN wild-type melanoma cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells.

Piperine promotes ultraviolet (UV)-B-induced cell death in B16F10 mouse melanoma cells through modulation of major regulators of cell survival

Piperine elevates intracellular ROS formation and impairs calcium homeostasis. It acts as a potent UVB photosensitizer, causing cell death and attenuation of major regulators of survival signalling pathways, offering a possible, practical therapeutic strategy for melanoma.

MD11-mediated delivery of recombinant eIF3f induces melanoma and colorectal carcinoma cell death

The f subunit of the eukaryotic initiation factor 3 (eIF3f) is downregulated in several cancers and in particular in melanoma and pancreatic cancer cells. Its enforced expression by transient gene transfection negatively regulates cancer cell growth by activating apoptosis. With the aim to increase the intracellular level of eIF3f proteins and activate apoptosis in cancer cell lines, we developed a protein transfer system composed of a cell-penetrating peptide sequence fused to eIF3f protein sequence (MD11-eIF3f). To determine whether exogenously administered eIF3f proteins were able to compensate the loss of endogenous eIF3f and induce cancer cell death, we analyzed the therapeutic action of MD11-eIF3f in several tumor cells. We identified four cell lines respondent to eIF3f-treatment and we evaluated the antitumor properties of the recombinant proteins using dose- and time-dependent studies. Our results demonstrate that this protein delivery approach represents an innovative and powerful strategy for cancer treatment.

A polyphenolic fraction from Caesalpinia spinosa induces immunogenic cell death of melanoma cells and enhances tumor control through the activation of the immune system

A polyphenolic fraction from Caesalpinia spinosa induces immunogenic cell death of melanoma cells and enhances tumor control through the activation of the immune system

Concurrent MEK and autophagy inhibition is required to restore cell death associated danger-signalling in Vemurafenib-resistant melanoma cells

Vemurafenib (PLX4032), an inhibitor of BRAFV600E, has demonstrated significant clinical anti-melanoma effects. However, the majority of treated patients develop resistance, due to a variety of molecular mechanisms including MAPK reactivation through MEK. The induction of a cancer cell death modality associated with danger-signalling resulting in surface mobilization of crucial damage-associated-molecular-patterns (DAMPs), e.g. calreticulin (CRT) and heat shock protein-90 (HSP90), from dying cells, is emerging to be crucial for therapeutic success. Both cell death and danger-signalling are modulated by autophagy, a key adaptation mechanism stimulated during melanoma progression. However, whether melanoma cell death induced by MAPK inhibition is associated with danger-signalling, and the reliance of these mechanisms on autophagy, has not yet been scrutinized.Using a panel of isogenic PLX4032-sensitive and resistant melanoma cell lines we show that PLX4032-induced caspase-dependent cell death and DAMPs exposure in the drug-sensitive cells, but failed to do so in the drug-resistant cells, displaying heightened MEK activation. MEK inhibitor, U0126, treatment sensitized PLX4032-resistant cells to death and re-established their danger-signalling capacity. Only melanoma cells exposing death-induced danger-signals were phagocytosed and induced DC maturation. Although the PLX4032-resistant melanoma cells displayed higher basal and drug-induced autophagy, compromising autophagy, pharmacologically or by ATG5 knockdown, was insufficient to re-establish their PLX4032 sensitivity. Interestingly, autophagy abrogation was particularly efficacious in boosting cell death and ecto-CRT/ecto-HSP90 in PLX4032-resistant cells upon blockage of MEK hyper-activation by U0126.Thus combination of MEK inhibitors with autophagy blockers may represent a novel treatment regime to increase both cell death and danger-signalling in Vemurafenib-resistant metastatic melanoma.

Abstract B37: Prenylation inhibition induces cell death in NRAS mutant melanoma cells

Abstract While targeted therapeutic approaches are now available for the treatment of BRAF mutant melanomas, non-BRAF mutant melanoma still lacks this modality. In order to explore the potential of prenylation inhibition we investigated the response of NRAS or BRAF mutant and double wild-type melanoma to zoledronic acid treatment. Effect of zoledronic acid on proliferation, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were characterized in vitro. In vivo subcutaneous xenograft and spleen to liver colonization models were used to evaluate the influence of zoledronic acid treatment on primary tumor growth and metastatic potential of melanoma cells. Zoledronic acid significantly decreased proliferation in NRAS mutant cells as compared to BRAF mutant and double wild-type cells, in vitro. Similarly, TUNEL assay revealed apoptosis induction in an oncogenic mutation specific manner. In line with this finding, activation of ribosomal protein S6 was decreased. Unexpected activation of the downstream effector Erk1/2 and increase in migration was found in BRAF mutant cells. Zoledronic acid failed to show significant synergism with cytotoxic cisplatin or DTIC treatment in vitro. In vivo zoledronic acid did not inhibit the subcutaneous growth of melanoma cells irrespective of oncogenic mutations. In contrast, the lower dose of zoledronic acid resulted in significantly higher liver metastasis in double wild-type cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target melanoma cells. Citation Format: Tamas Garay, Istvan Kenessey, Viktoria Laszlo, Jozsef Tovari, Walter Berger, Laszlo Kovacs, Jozsef Timar, Balazs Hegedus. Prenylation inhibition induces cell death in NRAS mutant melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B37. doi: 10.1158/1557-3125.RASONC14-B37

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.

Mastoparan induces apoptosis in B16F10-Nex2 melanoma cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo

Mastoparan is an α-helical and amphipathic tetradecapeptide obtained from the venom of the wasp Vespula lewisii. This peptide exhibits a wide variety of biological effects, including antimicrobial activity, increased histamine release from mast cells, induction of a potent mitochondrial permeability transition and tumor cell cytotoxicity. Here, the effects of mastoparan in malignant melanoma were studied using the murine model of B16F10-Nex2 cells. In vitro, mastoparan caused melanoma cell death by the mitochondrial apoptosis pathway, as evidenced by the Annexin V-FITC/PI assay, loss of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species, DNA degradation and cell death signaling. Most importantly, mastoparan reduced the growth of subcutaneous melanoma in syngeneic mice and increased their survival. The present results show that mastoparan induced caspase-dependent apoptosis in melanoma cells through the intrinsic mitochondrial pathway protecting the mice against tumor development.

Abstract 1242: Green tea polyphenols inhibit the growth and induce death of melanoma cells by initiating DNA damage and inhibition of class I histone deacetylases

Abstract Melanoma remains a leading cause of death from skin diseases due to its propensity to metastasis, and is increasing rapidly in children. Although, melanoma is less common than other types of skin cancers, however, it causes the majority (75%) of skin cancer-related deaths. The average survival of patients with advanced stage of melanoma is less than a year because no effective strategies are available for metastatic melanoma. Since melanoma is a highly malignant cancer, an approach that can inhibit its growth and progression may facilitate the development of an effective strategy for its treatment or prevention. As green tea polyphenols (GTPs) have been shown to have anti-skin carcinogenic effects, we have examined the chemotherapeutic effect of GTPs on the growth of different melanoma cell lines and the molecular mechanisms underlying these effects using an in vitro model. Treatment of melanoma cells (A375 and Hs294t) with GTPs (0, 20, 40 and 60 µg/ml) for 24 and 48 h significantly inhibited histone deacetylase (HDAC) activity, reduced the levels of class 1 HDAC proteins (HDAC 1, 2, 3 and 8) and enhanced histone acetyltransferase (HAT) activity in a dose-dependent manner. These effects of GTPs on melanoma cells were associated with DNA damage, which was evident by Comet assay, and significant reduction in the viability of melanoma cells and induction of cell death. Concomitant treatment of melanoma cells with a proteasome inhibitor, MG132, prevented GTPs-induced degradation of class I HDACs, suggesting that GTPs reduced the levels of HDAC proteins in melanoma cells through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of melanoma cells (A375 and Hs294t). Treatment of A375 and Hs294t cells with GTPs resulted in an alteration of cell cycle regulatory proteins of G0/G1 phase, and that is a decrease in the levels of cyclin D1, D2 and E, and cyclin dependent kinases, CDK2, CDK4 and CDK6 in a dose-dependent manner. Simultaneously, it was also observed that the levels of p21 and p53 proteins were also increased in the cells after the treatment with GTPs, which may have a role in G0/G1 phase arrest and DNA damage. Together, our study provides new insights into the role of class I HDACs in the chemotherapeutic effects of green tea polyphenols on melanoma cells. Citation Format: Ram Prasad, Santosh K. Katiyar. Green tea polyphenols inhibit the growth and induce death of melanoma cells by initiating DNA damage and inhibition of class I histone deacetylases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1242. doi:10.1158/1538-7445.AM2014-1242

Curcumin induces apoptosis through mitochondrial pathway and caspases activation in human melanoma cells.

Melanoma is the most malignant skin cancer and is highly resistant to chemotherapy and radiotherapy. Curcumin is a component of turmeric, the yellow spice derived from the rhizome of Curcuma longa. It has been demonstrated to modulate multiple cell signaling pathways, including apoptosis, proliferation, angiogenesis and inflammation. In this study, we studied the signaling pathways involved in melanoma cell death after treatment with curcumin using western blotting. Colorimetric assays (MTT) assessed cell viability. Flow cytometry and DNA laddering evaluated cell apoptosis. Fluorescent microscopy was used to evaluate of Hoechst 33342 staining of nuclei. The result demonstrated that curcumin could induce apoptosis and inhibit proliferation in melanoma cells. Curcumin stimulated the expression of pro-apoptotic Bax, and inhibited the activation of anti-apoptotic Mcl-1 and Bcl-2. During curcumin treatment, caspase-8 and Caspase-3 were cleaved in time and dose-dependent manners. Curcumin treatment also altered the expressions of apoptosis associated proteins NF-κB, p38 and p53. Curcumin induced DNA double strand breaks, which were indicated by phosphorylated H2AX. Our data suggested that curcumin could be used as a novel and effective approach for the treatment of melanoma.

Mitochondrial division inhibitor-1 induces mitochondrial hyperfusion and sensitizes human cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer treatment, but some cancer cell types are resistant to TRAIL cytotoxicity. Therefore, overcoming this resistance is necessary for effective TRAIL therapy. Mitochondrial morphology is important for the maintenance of cell function and survival, and is regulated by the delicate balance between fission and fusion. However, the role of mitochondrial morphology dynamics in TRAIL-induced apoptosis is unknown. Here we show that mitochondrial division inhibitor-1 (mdivi-1), an inhibitor of dynamin-related protein1 (Drp1), modulates mitochondrial morphology and TRAIL-induced apoptosis in human cancer cells. mdivi-1 treatment (≥12.5 µM) caused dose- and time‑dependent cell death in malignant melanoma, lung cancer and osteosarcoma cells, while sparing normal cells. mdivi-1 also sensitized cancer cells to TRAIL-induced apoptosis. This potentiation of apoptosis occurred through a caspase-depependent mechanism including the mitochondrial and endoplasmic reticulum (ER) stress pathways. Mdivi-1 potentiated mitochondrial oxidative stress, a major cause of mitochondrial and ER stresses, as evidenced by increases in mitochondrial reactive oxygen species levels, mitochondrial mass, and cardiolipin oxidation. Live cell fluorescence imaging using MitoTracker Red CMXRos revealed that Mdivi-1 caused substantial mitochondrial hyperfusion. Moreover, silencing of Drp1 expression also caused mitochondrial hyperfusion and sensitized cancer cells to TRAIL-induced apoptosis. Our results suggest that cancer cells are more vulnerable than normal cells to a perturbation in mitochondrial morphology dynamics and that this higher susceptibility can be exploited to selectively kill cancer cells and sensitize to TRAIL.

FTY720 and cisplatin synergistically induce the death of cisplatin-resistant melanoma cells through the downregulation of the PI3K pathway and the decrease in epidermal growth factor receptor expression.

Sphingosine kinase (SK), a key enzyme in sphingosine-1-phosphate (S1P) synthesis, is known to be overexpressed in various types of cancer cells. The effects of anticancer agents on SK1/S1P signaling have not yet been fully assessed in melanoma cells. In the present study, we investigated the effects of the combination of FTY720, an S1P receptor antagonist, and cisplatin, a DNA-damaging agent, on the induction of the death of human melanoma cells, as well as the molecular mechanisms involved. The viability of various human melanoma cell lines was examined following treatment with anticancer drugs. The cisplatin-resistant SK-Mel-28 and cisplatin-sensitive A375 cell lines were selected for this analysis. Protein expression and apoptotic rates were evaluated by western blot analysis following treatment with cisplatin and/or FTY720. Following treatment with a combination of FTY720 and cisplatin, cell viability significantly decreased and the expression of apoptosis-associated cleaved poly(ADP-ribose) polymerase (PARP) was significantly higher in comparison to treatment with cisplatin alone in the SK-Mel-28 cells. In addition, the combination of FTY720 and cisplatin reduced the protein expression of SK1 and the phosphorylation levels of phosphoinositide 3-kinase (PI3K), Akt and mTOR in the SK-Mel-28 cells; the expression of epidermal growth factor receptor (EGFR) was also markedly reduced. These findings suggest that FTY720 and cisplatin synergistically induce cell death through the downregulation of the PI3K/Akt/mTOR pathway and the decrease in EGFR expression in SK-Mel-28 cells. Thus, the combination of FTY720 and cisplatin may have therapeutic potential for chemotherapy-resistant melanoma, and the effects are likely exerted through the downregulation of S1P signaling.