Apoptosis In Melanoma Research Articles

Abstract 253: The cancer testis antigen MAGED2 protects human melanoma cells from TRAIL-induced apoptosis by suppressing TRAIL-R2 expression

Abstract Melanoma-associated antigen D2 (MAGED2), a member of the MAGE-II cancer testis antigen family, is expressed by almost all normal tissues and many different cancer tissues. Although it has been reported that MAGE proteins have a pro-survival role in cancer cells such as malignant mast cells, the potential biological significance of MAGE proteins in melanoma remains to be established. Here, we show that MAGED2 is involved in the protection of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppressing the expression of TRAIL-R2, the major TRAIL death receptor that mediates TRAIL-induced apoptosis in melanoma. siRNA knockdown of MAGED2 sensitised melanoma cells to apoptosis induced by TRAIL. This was associated with enhanced activation of the caspase cascade and formation of the TRAIL death-inducing signalling complex (DISC), suggesting that MAGED2 acts at or above the DISC to protect melanoma cells against TRAIL-induced apoptosis. Indeed, inhibition of MAGED2 by siRNA resulted in up-regulation of TRAIL-R2 on the melanoma cell surface, which appeared to be specific, because knockdown of MAGED2 did not impinge on the expression of other TNF receptor family members, nor did it up-regulate TRAIL-R2 in melanocytes. Up-regulation of TRAIL-R2 by inhibition of MAGED2 was mediated by a p53-responsive transcriptional increase, in that p53 was also up-regulated in melanoma cells with MAGED2 knocked down, and that knockdown of MAGED2 did not up-regulate TRAIL-R2 in melanoma cells deficient in p53. In summary, our results identify MAGED2 as an important mechanism that suppresses TRAIL-R2 expression, thus protecting melanoma cells from TRAIL-induced apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 253. doi:1538-7445.AM2012-253

Abstract 260: FK506 binding protein (FKBP) 51 controls “TNF-related apoptosis inducing ligand” (TRAIL) response in melanoma

Abstract FKBP51 is a cochaperone that regulates several biological processes in the cell, through protein-protein interaction. Very recently, we have found that FKBP51 is hyperexpressed in malignant melanoma and plays a crucial role in its chemo- and radio-resistance. Downmodulation of FKBP51, by gene silencing, produces an increase of melanoma apoptosis induced by several cytotoxic stimuli. To investigate the mechanism of the increased apoptosis sensitivity of FKBP51-silenced melanoma, we performed a study of protein expression profiles in melanoma cells that were depleted or not of FKBP51, using ionizing radiation (IR) as apoptotic stimulus. Among the multiple signaling pathways that were found modulated in FKBP51-silenced cells, a marked upregulation of TRAIL receptors was found. In particular, the death receptors were significantly increased in FKBP51-depleted cells, while the decoy receptors, that do not transduce death signal, were decreased in the same cells. Among TRAIL receptors, TRAIL R2 (DR5) transduces most efficiently the death signal. Aim of the present study was to validate the increase of DR5 at mRNA and protein level in FKBP51-silenced melanoma cells and investigate the effect of FKBP51 silencing on TRAIL-induced apoptosis. Summary of key findings FKBP51-silencing increased DR5 transcript by 6,1 folds (range 2,8/14,7 folds), in melanoma cells, measured by Real-time PCR. Flow cytometry confirmed the increased expression of DR5 (by more than 100%) in FKBP51 silenced cells, compared with non silenced melanoma cells. The study of apoptosis was in accordance with the expression of death receptors. Indeed, FKBP51 silencing increased TRAIL-induced apoptosis, as suggested by both propidium iodide incorporation and annexin V staining of melanoma cells. Low levels of apoptosis were measured in melanoma cells stimulated with 100ng/ml TRAIL (18 + 6%). By contrast, 39 + 14% of apoptosis was measured in FKBP51-depleted melanoma cells, following TRAIL stimulation (P= 0.01). FKBP51 regulated the acetylation pattern of melanoma as suggested by the reduced amount af acetylated protein in FKBP51 silenced-melanoma cells (D), most interestingly, reduced levels of acetylated Yin Yang 1 transcription factor (YY1) involved in control of DR5 tanscription, were found in FKBP51-silenced melanoma, as suggested by western blot assay. Conclusions FKBP51 gene silencing produces a marked increase in TRAIL sensitivity of melanoma, due to upregulation of TRAIL death receptors. FKBP51 decreases the acetylation pattern of melanoma, and in particular of the DR5 transcriptional repressor YY1, suggesting that epigenetic modification of DNA could be responsible for reduced TRAIL-receptor transcription and resistance to apoptosis of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 260. doi:1538-7445.AM2012-260

Abstract 4893: XIAP downregulation accompanies inhibition of melanoma xenograft growth by mebendazole

Abstract Background: Melanoma's intrinsic resistance to therapy has been attributed to modulation of cellular survival pathways including apoptosis. We originally reported that mebendazole (MBZ), which acts as a microtubule-disrupting agent, is a potent inducer of apoptosis in melanoma cells in vitro via a Bcl-2 dependent mechanism (Molecular Cancer Research, 2008). Here, we show that MBZ is also able to effectively modulate another key anti-apoptotic factor, XIAP, to promote melanoma apoptosis both in vitro and in vivo. Methodology: Safety and efficacy of mebendazole was assessed using a human melanoma xenograft model established from M-14 cells. The effect of MBZ on XIAP expression and induction of apoptotic pathways was evaluated by immunoblotting and co-immunoprecipitation techniques along with densitometric quantification. The growth inhibitory effects of MBZ were determined in a panel of melanoma cell lines with the sulphorhodamine (SRB) assay. siRNA techniques were used to reduce expression of XIAP. Results: Oral MBZ is as effective as the current standard of care temozolomide (TMZ) in reducing melanoma growth in vivo, with no notable toxicities observed. Inhibition of melanoma growth in vivo is accompanied by MBZ specific reduced expression of XIAP and cleavage of caspase 9. In response to MBZ treatment, sensitive cell lines display reduced levels of XIAP expression whereas resistant cell lines do not. Moreover, down-regulation of XIAP expression by siRNA in the MBZ-resistant melanoma cell line UACC1097 was accompanied by a reduction in the IC50 for MBZ from >10 μM to 3.7 μM. Exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating inhibition of apoptosis by XIAP. Conclusions: In addition to demonstrating efficacy and safety in vivo, our preclinical studies of the promising anti-melanoma agent mebendazole provide insight into characterization of an apoptotic promoting mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4893. doi:1538-7445.AM2012-4893

Benzyl Isothiocyanate (BITC) Induces G2/M Phase Arrest and Apoptosis in Human Melanoma A375.S2 Cells through Reactive Oxygen Species (ROS) and both Mitochondria-Dependent and Death Receptor-Mediated Multiple Signaling Pathways

Benzyl isothiocyanates (BITC), a member of the isothiocyanate (ITC) family, inhibits cell growth and induces apoptosis in many types of human cancer cell lines. The present study investigated mechanisms underlying BITC-induced apoptosis in A375.S2 human melanoma cancer cells. To observe cell morphological changes and viability, flow cytometric assays, cell counting, and a contrast-phase microscopic examination were carried out in A375.S2 cells after BITC treatment. Cell cycle distribution and apoptosis were assessed with the analysis of cell cycle by flow cytometric assays, DAPI staining, propidium iodide (PI), and annexin V staining. Apoptosis-associated factors such as reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and caspase-3 activity were evaluated by flow cytometric assays. Abundance of cell cycle and apoptosis associated proteins was determined by Western blotting. AIF and Endo G expression was examined by confocal laser microscope. Results indicated that (1) BITC significantly reduced cell number and induced cell morphological changes in a dose-dependent manner in A375.S2 cells; (2) BITC induced arrest in cell cycle progression at G(2)/M phase through cyclin A, CDK1, CDC25C/Wee1-mediated pathways; (3) BITC induced apoptosis and increased sub-G(1) population; and (4) BITC promoted the production of ROS and Ca(2+) and loss of ΔΨ(m) and caspase-3 activity. Furthermore, BITC induced the down-regulation of Bcl-2 expression and induced up-regulation of Bax in A375.S2 cells. Moreover, BITC-induced cell death was decreased after pretreatment with N-acetyl-l-cysteine (NAC, a ROS scavenger) in A375.S2 cells. In conclusion, the results showed that BITC promoted the induction of G(2)/M phase arrest and apoptosis in A375.S2 human melanoma cells through ER stress- and mitochondria-dependent and death receptor-mediated multiple signaling pathways. These data suggest that BITC has potential as an agent for the treatment of melanoma.

Contributors

To investigate the role of circHIPK3 in melanoma.Bioinformatics analysis and experiments including RT-qPCR, Pearson's correlation analysis, luciferase reporter, Western blot, and RIP assays were applied to explore the function and mechanism of circHIPK3 in melanoma.CircHIPK3 expression was strikingly upregulated while miR-215–5p was downregulated in melanoma tissues and cell lines. Pearson's correlation analysis unveiled circHIPK3 expression was positively correlated with Ki-67 (a marker of proliferation), which implied that circHIPK3 may play a vital role in the progression of melanoma. In mechanism, luciferase reporter and RIP assays validated that circHIPK3 was able to bind with miR-215–5p. Moreover, we confirmed that overexpression of circHIPK3 could facilitate cell proliferation and depress cell apoptosis in melanoma while overexpression of miR-215–5p exerted opposite effects. Besides, our findings indicated that miR-215–5p overexpression significantly reversed the circHIPK3 overexpressing-mediated promotive effect on cell proliferation and inhibitory effect on cell apoptosis. Furthermore, we found that miR-215–5p could directly target YY1. Upregulation of YY1 could notably offset the inhibitory effect of circHIPK3 downregulation on cell proliferation and the promotive effect on cell apoptosis.Our study corroborated that circHIPK3 regulated melanoma cell behaviors via the miR-215–5p/YY1 axis, which might provide a novel insight for the treatment of melanoma patients.

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34+ cells

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

Salmonella‐mediated tumor‐targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.

The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC50) was approximately 0.8μM and by 5μM both cell lines were completely growth-arrested within 72h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1μM canertinib accumulated the cells in the G1-phase of the cell cycle within 24h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1μM canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses ⩽5μM canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.

Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: a role of combined treatment versus monotherapy

Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20-40 μM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5-20 μM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation.

A DC-81-indole conjugate agent suppresses melanoma A375 cell migration partially via interrupting VEGF production and stromal cell-derived factor-1α-mediated signaling

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1α (SDF-1α) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF-1α-enhanced melanoma migration. IN4CPBD not only abolished the SDF-1α-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF-1α-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency.

A novel form of melanoma apoptosis resistance: Melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.

Abstract LB-19: The Smac mimetic LBW242 induces apoptosis in melanoma in a TLR-3-dependent and TNFR-independent manner

Abstract Toll-like Receptor-3 (TLR-3), a pattern recognition receptor family member, has been shown to activate immune system pathways; however, it has also been shown to induce apoptosis in certain cancer cells. Combination of the synthetic TLR-3 ligand, poly I:C, with the Smac mimetic LBW242, an inhibitor of apoptosis protein (IAP) antagonist, has been shown to potently induce apoptosis in melanoma cells which are insensitive to either single agent. To better elucidate the mechanism responsible for this combination induced cell death, we performed a pooled shRNA screen. We identified receptor interacting protein kinase-1 (RIPK1) and as an important component of this process. Constitutive knockdown of RIPK1 prevents the LBW242 and poly I:C combination induced apoptosis. The IAP proteins cIAP1 and cIAP2 have been shown previously to regulate Caspase-8 activation downstream of Tumor Necrosis Factor Receptor-1 (TNFR1). Interestingly, we found that the combination induced apoptosis was independent of TNF signaling. Utilizing both gain of function and loss of function studies, we found that expression of TLR-3 is necessary for the combination induced apoptosis in melanoma cells. Taken together, our data indicate that the combination of the Smac mimetic LBW242 and TLR-3 agonist poly I:C induces a novel type of TLR-3 dependent apoptosis in melanoma cells that is independent of TNFR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-19. doi:10.1158/1538-7445.AM2011-LB-19

Structure−Activity Study To Develop Cationic Lipid-Conjugated Haloperidol Derivatives as a New Class of Anticancer Therapeutics

Haloperidol (HP), a neuroleptic drug, shows high affinity toward σ receptors (SR). HP and reduced-HP at higher concentration were known to induce apoptosis in SR-overexpressing carcinomas and melanomas. Herein, we report the development of cationic lipid-conjugated haloperidol as a new class of anticancer therapeutics. In comparison to HP, the C-8 carbon chain analogue (HP-C8) showed significantly high, SR-assisted antiproliferative activity against cancer cells via caspase-3-mediated apoptosis and down-regulation of pAkt. Moreover, melanoma tumor aggressiveness in HP-C8-treated mice was significantly lower than that in HP-treated mice. HP-C8 simultaneously reduced Akt-protein level and increased Bax/Bcl-2 ratio in vascular endothelial cells, thereby indicating a possible protein kinase down-regulatory and apoptosis inducing role in tumor-associated vascular cells. In conclusion, we developed σ receptor-targeting cationic lipid-modified HP derivatives as a promising class of anticancer therapeutic that concurrently affects cancer and tumor environment associated angiogenic vascular cells through induction of apoptosis and Akt protein down-regulation.

Endothelial nitric oxide synthase uncoupling as a key mediator of melanocyte malignant transformation associated with sustained stress conditions

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH4, and increased by the inhibitor of BH4 synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. The eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. The inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. The maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation.

17β-Estradiol-Linked Nitro-l-arginine as Simultaneous Inducer of Apoptosis in Melanoma and Tumor-Angiogenic Vascular Endothelial Cells

Aggressive melanoma is commonly associated with rapid angiogenic growth in tumor mass, tumor cells acquiring apoptosis resistance, inhibition of cellular differentiation etc. Designing a single anticancer molecule which will target all these factors simultaneously is challenging. In the pretext of inciting anticancer effect through inhibiting nitric oxide synthase (NOS) via estrogen receptors (ER) in ER-expressing skin cancer cells, we developed an estrogen-linked L-nitro-arginine molecule (ESAr) for inciting anticancer effect in melanoma cells. ESAr showed specific anticancer effect through diminishing aggressiveness and metastatic behavior in melanoma cells and tumor. In comparison, ESAr showed significantly higher antiproliferative effect than parent molecule L-nitroarginine methyl ester (L-NAME, a NOS inhibitor) through induction of prominent apoptosis in melanoma cells. ESAr-pretreated aggressive melanoma cells could not form tumor possibly because of transformation/differentiation into epithelial-type cells. Furthermore, its antiangiogenic effect was demonstrated through ESAr-induced antiproliferation in HUVEC cells and apoptosis-induction in tumor-associated vascular endothelial cells, thereby significantly restricting severe growth in melanoma tumor. The targeting moiety, estrogen, at the therapeutic concentration of ESAr has apparently no effect in tumor-growth reduction. Albeit, no specific NOS-inhibition was observed, but ESAr could simultaneously induce these three cancer-specific antiaggressiveness factors, which the parent molecule could not induce. Our data rationalize and establish a new use of estrogen as a ligand for potentially targeting multiple cellular factors for treating aggressive cancers.

Sensitization of human melanoma cells by tamoxifen to apoptosis induction by pancratistatin, a nongenotoxic natural compound

The objective of this study was to determine the efficacy of the natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis, in human melanoma cells. Melanoma is an aggressive form of skin cancer that is commonly fatal if not diagnosed in its early stage of development. Melanoma is resistant to many treatments, thus drastically limiting chemotherapy options for this cancer. We have shown that exposure to PST induces apoptosis in human melanoma within 72 h using Hoechst staining. Interestingly tamoxifen (TAM), an estrogen receptor antagonist, sensitizes these cells to apoptosis induction by PST as observed with Hoechst and annexin-V staining. This cotreatment did not affect the viability of normal noncancerous human fibroblasts. Both of these compounds have been shown to target the mitochondria synergistically, as indicated by higher levels of reactive oxygen species generation from isolated mitochondria. PST alone and in combination with TAM shows depolarization of the mitochondrial membrane potential as shown by JC-1 staining. Melanoma drug resistance was not observed after posttreatment recuperation, as cells displayed apoptotic morphology up to 96 h after drug-free media replacement. Our results indicate that TAM alone does not induce apoptosis in this cell line, but sensitizes the mitochondria, thereby enhancing the effect of PST exposure. In conclusion, combination of two nongenotoxic compounds offers a novel treatment regime for this notoriously resilient form of skin cancer.

Suppression of the proinflammatory response of metastatic melanoma cells increases TRAIL‐induced apoptosis

Melanoma is the most lethal form of human skin cancer. However, only limited chemotherapy is currently available for the metastatic stage of the disease. Since chemotherapy, radiation and sodium arsenite treatment operate mainly through induction of the intrinsic mitochondrial pathway, a strongly decreased mitochondrial function in metastatic melanoma cells, could be responsible for low efficacy of the conventional therapy of melanoma. Another feature of metastatic melanoma cells is their proinflammatory phenotype, linked to endogenous expression of the inflammatory cytokines, such as TNFα IL6 and IL8, their receptors, and constitutive NF-κB- and STAT3-dependent gene expression, including cyclooxygenase-2 (PTGS2/COX2). In the present study, we treated melanoma cells with immunological (monoclonal antibody against TNFα or IL6), pharmacological (small molecular inhibitors of IKKβ-NF-κB and JAK2-STAT3) or genetic (specific RNAi for COX-2) agents that suppressed the inflammatory response in combination with induction of apoptosis via TRAIL. As a result of these combined treatments, exogenous TRAIL via interactions with TRAIL-R2/R1 strongly increased levels of apoptosis in resistant melanoma cells. The present study provides new understanding of the regulation of TRAIL-mediated apoptosis in melanoma and will serve as the foundation for the potential development of a novel approach for a therapy of resistant melanomas.

Chemo-resistant melanoma sensitized by tamoxifen to low dose curcumin treatment through induction of apoptosis and autophagy

Melanoma is the deadliest form of skin cancer, which is notoriously aggressive and chemo-resistant, and for which there is little effective treatment available if it goes undetected. Curcumin from the tumeric spice (Curcuma longa) has long been used in Southeast Asian medicine to alleviate ailments and cure an array of diseases and disorders. It possesses anti-inflammatory, anti-oxidant and most importantly anti-carcinogenic activity. There have been contradictory reports discussing the efficacy of curcumin-induced death on melanoma. In this report we show that curcumin does induce apoptosis in A375 and the relatively resistant G361 malignant human melanoma cell lines at higher doses. Tamoxifen is an estrogen receptor (ER) blocker that is used for ER positive breast cancer treatment. Recently, tamoxifen has been shown to directly target the mitochondria. Given that curcumin is a pro oxidant and tamoxifen can act on mitochondria, we ask whether the combinatorial treatment could result in synergistic induction of apoptosis in chemo-resistant melanoma. Our results show a corresponding increase in phosphatidyl serine flipping, mitochondria depolarization and reactive oxygen species (ROS) generation by the combined treatment at lower doses. Interestingly, there was significant induction of autophagy along with apoptosis following the combined treatment. Importantly, non-cancerous cells are unaffected by the combination of these non-toxic compounds. However, once exposed to low doses of this co treatment, melanoma cells still retain signals to commit suicide even after removal of the drugs. This combination provides a non-toxic option for combinatorial chemotherapy with great potential for future use.

The Efficacy of Dandelion Root Extract in Inducing Apoptosis in Drug‐Resistant Human Melanoma Cells

Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25–29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible), and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE) specifically and effectively induces apoptosis in human melanoma cells without inducing toxicity in noncancerous cells. Characteristic apoptotic morphology of nuclear condensation and phosphatidylserine flipping to the outer leaflet of the plasma membrane of A375 human melanoma cells was observed within 48 hours. DRE-induced apoptosis activates caspase-8 in A375 cells early on, demonstrating employment of an extrinsic apoptotic pathway to kill A375 cells. Reactive Oxygen Species (ROS) generated from DRE-treated isolated mitochondria indicates that natural compounds in DRE can also directly target mitochondria. Interestingly, the relatively resistant G361 human melanoma cell line responded to DRE when combined with the metabolism interfering antitype II diabetic drug metformin. Therefore, treatment with this common, yet potent extract of natural compounds has proven novel in specifically inducing apoptosis in chemoresistant melanoma, without toxicity to healthy cells.

FK506 Binding Proteins as Targets in Anticancer Therapy

FK506 binding proteins (FKBPs) are the intracellular ligands of FK506 and rapamycin, two natural compounds with powerful and clinically efficient immunosuppressive activity. In recent decades, a relevant role for immunosuppressants as anticancer agents has emerged. Especially, rapamycin and its derivatives are used, with successful results, across a variety of tumors. Of note, rapamycin and FK506 bind to FKBP12, and the resulting complexes interfere with distinct intracellular signaling pathways driven, respectively, by the mammalian target of rapamycin and calcineurin phosphatase. These pathways are related to T-cell activation and growth. Hyperactivation of the mammalian target of rapamycin (mTOR), particularly in cancers that have lost the tumor suppressor gene PTEN, plays an important pathogenetic role in tumor transformation and growth. The signaling pathway involving calcineurin and nuclear factors of activated T-lymphocytes is also involved in the pathogenesis of different cancer types and in tumor metastasis, providing a rationale for use of FK506 in anticancer therapy. Recent studies have focused on FKBPs in apoptosis regulation: Targeting of FKBP12 promotes apoptosis in chronic lymphocytic leukemia, FKBP38 knockdown sensitizes hepatoma cells to apoptosis, and FKBP51 silencing overcomes resistance to apoptosis in acute lymphoblastic leukemia, prostate cancer, melanoma, and glioma. Interestingly, derivatives of FK506 that have the same FKBP12-binding properties as FK506 but lack functional immunosuppressant activity, exert the same apoptotic effect as FK506 in chronic lymphocytic leukemia.These findings suggest that a direct FKBP inhibition represents a further mechanism of immunosuppressants.' anticancer activity. In this review, we focus on the role of FKBP members in apoptosis control and summarize the data on the antitumor effect of selective targeting of FKBP.