Apoptosis In Melanoma Research Articles

Molecular Probes for Malignant Melanoma Imaging

Malignant melanoma represents a serious public health problem and is a deadly disease when it is diagnosed at late stage. Though (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) has been widely used clinically for melanoma imaging, other approaches to specifically identify, characterize, monitor and guide therapeutics for malignant melanoma are still needed. Consequently, many probes targeting general molecular events including metabolism, angiogenesis, hypoxia and apoptosis in melanoma have been successfully developed. Furthermore, probes targeting melanoma associated targets such as melanocortin receptor 1 (MC1R), melanin, etc. have undergone active investigation and have demonstrated high melanoma specificity. In this review, these molecular probes targeting diverse melanoma biomarkers have been summarized. Some of them may eventually contribute to the improvement of personalized management of malignant melanoma.

The Calcium-binding Protein S100B Down-regulates p53 and Apoptosis in Malignant Melanoma

The S100B-p53 protein complex was discovered in C8146A malignant melanoma, but the consequences of this interaction required further study. When S100B expression was inhibited in C8146As by siRNA (siRNA(S100B)), wt p53 mRNA levels were unchanged, but p53 protein, phosphorylated p53, and p53 gene products (i.e. p21 and PIDD) were increased. siRNA(S100B) transfections also restored p53-dependent apoptosis in C8146As as judged by poly(ADP-ribose) polymerase cleavage, DNA ladder formation, caspase 3 and 8 activation, and aggregation of the Fas death receptor (+UV); whereas, siRNA(S100B) had no effect in SK-MEL-28 cells containing elevated S100B and inactive p53 (p53R145L mutant). siRNA(S100B)-mediated apoptosis was independent of the mitochondria, because no changes were observed in mitochondrial membrane potential, cytochrome c release, caspase 9 activation, or ratios of pro- and anti-apoptotic proteins (BAX, Bcl-2, and Bcl-X(L)). As expected, cells lacking S100B (LOX-IM VI) were not affected by siRNA(S100B), and introduction of S100B reduced their UV-induced apoptosis activity by 7-fold, further demonstrating that S100B inhibits apoptosis activities in p53-containing cells. In other wild-type p53 cells (i.e. C8146A, UACC-2571, and UACC-62), S100B was found to contribute to cell survival after UV treatment, and for C8146As, the decrease in survival after siRNA(S100B) transfection (+UV) could be reversed by the p53 inhibitor, pifithrin-alpha. In summary, reducing S100B expression with siRNA was sufficient to activate p53, its transcriptional activation activities, and p53-dependent apoptosis pathway(s) in melanoma involving the Fas death receptor and perhaps PIDD. Thus, a well known marker for malignant melanoma, S100B, likely contributes to cancer progression by down-regulating the tumor suppressor protein, p53.

Induction of apoptosis in human melanoma by the BRAF inhibitor PLX4720: The key to therapeutic success?

8559 Background: A high proportion of Patients with metastatic melanoma have shown clinical responses when treated with the PLX4032/NP22657 inhibitor against the mutated V600E form of BRAF. The present study examined an analogue of this inhibitor for its ability to inhibit viability and induce apoptosis in a large panel of established and short term cultured melanoma. Methods: The oncocarta mutation panel was used to assess muations in 31 melanoma lines and short-term cultures. Each line was tested for viability in MTS assays and apoptosis by subG1 PI fractions over 72 hours. Western blots were used to measure Bcl-2 family protein expression. Results: There were 7 lines with no detectable mutations, 10 with mutations in NRAS or KRAS,13 with BRAF mutations and one with mutated cKit.Apoptosis induction was highest in the lines with mutated BRAF and correlated inversely with cell viability except for 2 lines with mutated CDK4 (R24c) which had low viability and low apoptosis.Inhibition of caspases indicated that apoptosis was the main mechanism for reduced cell viabilty. Induction of apoptosis correlated with upregulation of the proapoptotic BH3 protein Bim as reported in previous studies on the MEK inhibitor UO126. In particular there was selective upregulation of the short form of Bim and this correlated with induction of apoptosis. There were no significant changes in the the other BH3 only proteins PUMA or NOXA Combination of a MEK inhibitor with the BRAF inhibitor had strong potentiating effects on cell lines that were relatively resistant to the BRAF inhibitor. Conclusions: These studies confirm that the BRAF inhibitor PLX4720 induces apoptosis in human melanoma with mutated BRAF at concentrations achievable in vivo. Melanoma lines with mutated CDK4 or EGRF receptors had lower cell viability. Apoptosis was correlated with the induction of the proapoptotic Bim, particularly Bim short and may be a useful biomarker to assess apoptotic responses and guide drug doses and combinations in the clinic. No significant financial relationships to disclose.

Abstract 2538: A novel therapeutic strategy for malignancy: Specific disruption of FAK and IGF-1R interaction with a small molecule compound

Abstract Introduction: Pancreatic cancer and other aggressive malignancies are associated with poor patient survival rates. FAK and IGF-1R are two important tyrosine kinases important for cell survival signaling and found to be upregulated in several malignancies. As a novel strategy, we targeted FAK and IGF-1R protein interactions with a small molecule compound that altered crucial signaling pathways, and hypothesized that this approach would significantly inhibit cell proliferation and lead to apoptosis both alone and in combination with chemotherapy. Methods: The compound C31 (INT2-31) was identified from a high throughput screen to bind to FAK and disrupt protein-protein interactions. The effects of this compound when administered alone or together with 5-FU, gemcitabine or dacarbazine chemotherapy on cell signaling, viability and apoptosis in human pancreatic (Miapaca-2, Panc-1) esophageal (KYSE 70, 140) and melanoma (A375, C8161, SK-Mel28) cancer cells or on in vivo growth of direct human cancer xenografts was evaluated. Statistical analysis via t-test with significance defined as p<0.05. Results: INT2-31 caused a disruption of protein interactions between FAK and IGF-1R in whole cancer cell lysates starting at a concentration of 2.5 μM. It also caused a dose dependent inhibition of cell viability and induction of apoptosis starting at doses of 0.5 µM and 5µM, respectively. These effects were associated with a decrease in phosphorylation of Akt and ERK1/ERK2. INT2-31 treatment, when administered via subcutaneous or intraperitoneal injection, at 15 to 50mg/kg daily, resulted in a significant decrease of in vivo tumor growth in multiple cells lines in the subcutaneous or orthotopic pancreatic model as well as inhibition of growth of direct cancer xenografts. For instance, the size of a direct esophageal cancer subcutaneous xenograft was significantly decreased in INT2-31 treated animals vs control (236 +131 vs 931 +375 cm3, respectively, p<0.005). Tumor Ki67 staining from INT2-31 treated animals demonstrated a significant decrease in positive cells (52% vs 32%, control vs INT2-31, p<0.05). Furthermore, treatment with 5-FU chemotherapy resulted in a synergistic decrease in cell growth and increase in apoptosis when combined with INT2-31 compared to 5-FU or INT2-31 alone. Conclusions: A novel small molecule compound that disrupts the protein interactions of FAK and IGF-1R results in significantly decreased cancer cell proliferation and sensitizes cells to chemotherapy. These effects appear to be mediated through downregulation of p-AKT and p-ERK. This compound deserves further study as a novel treatment strategy in patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2538.

Nanosecond pulsed electric fields stimulate apoptosis without release of pro-apoptotic factors from mitochondria in B16f10 melanoma

Nanosecond pulsed electric fields (nsPEFs) eliminates B16f10 melanoma in mice, but cell death mechanisms and kinetics of molecular events of cell death are not fully characterized. Treatment of B16f10 cells in vitro resulted in coordinate increases in active caspases with YO-PRO-1 uptake, calcium mobilization, decreases in mitochondria membrane potential with decreases in forward light scatter (cell size), increases in ADP/ATP ratio, degradation of actin cytoskeleton and membrane blebbing. However, there was no mitochondrial release of cytochrome c, AIF or Smac/DIABLO or generation of reactive oxygen species. Phosphatidylserine externalization was absent and propidium iodide uptake was delayed in small populations of cells. The results indicate that nsPEFs rapidly recruit apoptosis-like mechanisms through the plasma membrane, mimicking the extrinsic apoptosis pathway without mitochondrial amplification yet include activation of initiator and executioner caspases. nsPEFs provide a new cancer therapy that can bypass cancer-associated deregulation of mitochondria-mediated apoptosis in B16f10 melanoma.

MicroRNA‐15b represents an independent prognostic parameter and is correlated with tumor cell proliferation and apoptosis in malignant melanoma

MicroRNAs (miRNAs) are small noncoding RNAs ( approximately 22 bp) that posttranscriptionally regulate gene expression. MiRNAs possess oncogenic or tumor suppressor activity in various tumors but little is known about miRNA expression pattern in malignant melanoma. We determined the expression level of 16 potentially relevant miRNAs (miR-15a, miR-15b, miR-16, miR-34a, miR-210, let-7I, miR-23a, miR-23b, miR-24, miR-27a, miR-27b, miR-100, miR-137, miR-222, miR-373-1, miR-373*) by real-time PCR in 6 preparations of normal melanocytes vs. 10 melanoma cell lines and in formalin fixed paraffin embedded tissue of 11 melanocytic nevi versus 16 melanomas. MiR-15b and miR-210 were significantly upregulated, miR-34a was significantly downregulated in melanomas compared with melanocytic nevi. These 3 miRNAs were analyzed in a total of 128 primary melanomas from patients with detailed clinical follow-up information. High expression of miR-15b (but not miR-210 upregulation and miR-34a downregulation) was significantly associated with poor recurrence free survival and overall survival by univariate Kaplan-Meier and multivariate Cox analyses. Downregulation of miR-15b in two melanoma cell lines with high miR-15b expression by transfection with anti-miR-15b siRNA was associated with reduced tumor cell proliferation, whereas apoptosis was increased. In summary, miRNA expression levels show distinct differences comparing benign and malignant melanocytic cell proliferations and can provide independent prognostic informations. MiR-15b appears to represent a particular important miRNA in melanoma that is associated with poor prognosis and tumorigenesis.

Nucleoside analog ARC targets Mcl-1 to induce apoptosis in leukemia cells

Nucleoside analog ARC targets Mcl-1 to induce apoptosis in leukemia cells

Cytochalasin D, LY294002 and olomoucine synergize in promoting death of melanoma cells through activation of caspase-3 and apoptosis

Many of the current anticancer therapies rely on the induction of apoptosis, and several mechanisms that protect cells against apoptosis may be upregulated in tumors. A growing body of evidence suggests that single drugs with a clearly defined intracellular target may be less efficient in arresting tumor growth and induction of apoptosis than multitargeted strategies. To prove that this is also the case for melanoma, we studied five cell lines, which represent different stages of tumor progression. We tested cell viability, terminal dUTP nick-end labeling and activation of caspase-3 upon exposure to cytochalasin D, LY294002 and olomoucine, added either alone or in various combinations. The obtained data were compared with effects caused by staurosporine. The results show that whereas staurosporine efficiently induced apoptosis in all tested melanoma cell lines, the other drugs had only moderate effects when administered alone. In contrast, the combinations of drugs were more effective in inducing caspase-3 activity and reducing cell viability. In particular, the triple combination of cytochalasin D+LY294002+olomoucine was almost as effective as staurosporine in inducing caspase-3 activity and apoptosis. These results prove that it is possible to design new pharmacological strategies that will effectively induce caspase-3 activity and apoptosis in melanoma. The possible explanations of the observed synergy between the tested drugs are also discussed.

Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells

Telomeres are essential for the maintenance of genomic integrality, they enablecomplete DNA replication, organize subnuclear domains, and protect chromosome endsagainst fusion. Loss of telomere function in human cells results in the formation ofdicentric chromosomes and other abnormalities such as end-to-end fusions.TRF2 is aubiquitously expressed protein which binds directly to the tandem array of duplextelomeric repeats and is involved in the telomere structure and chromosome endprotection. Inhibition of TRF2 induces end-to-end chromosome fusions and growth arrestor apoptosis in human melanoma cells.Lack of TRF2 in ALT cells also caused PMLdependentp53 activation and loss of telomeric DNA.TRF2 over-expression is found inseveral human tumors such as breast carcinomas, liver hepatocarcinomas, lungcarcinomas and gastric carcinomas. This suggests that TRF2 may have a role intumorigenisis.Cancers of the gastrointestinal tract share numerous common geneticfeatures despite differing widely in incidence and outcome.However, the role andmechanisms that lead to over-expressed TRF2 in colorectal carcinoma are not wellknown. Sp1 is a member of the multigene family with sequence-specific DNA-bindingC-terminal zinc-finger motifs, which plays a critical role in the regulation of expressionof mammalian genes lacking a TATA box, by binding to GC/GT boxes to activatetranscription.Cancer-related Sp1 targets include PDEGF, p21, VEGF, TGF-β, cyclinD1, E2F1, c-fos, transforming growth factor α and osteopontin (OPN).The promoterof TRF2 is GC-rich sequence (70-75%), which lacks an obvious TATA box. It is unclearwhether or not TRF2 is regulated via Sp1, which may in turn be regulated to genomicstability. We tested the expression of TRF2 in colorectal carcinoma tissues and coloncarcinoma SW480 cell line, and studied the role of Sp1 in regulating TRF2 expression incolon carcinoma. Results showed that TRF2 was over-expressed in colorectal carcinomatissues and SW480 cell line. It was also shown that Sp1 transcription factor is involved inthe up-regulation of TRF2 expression.

The induction of tumor apoptosis in B16 melanoma following STAT3 siRNA delivery with a lipid-substituted polyethylenimine

Persistent activation of signal transducer and activator of transcription 3 (STAT3) has been shown to impart several oncogenic features in many solid and blood tumors. In this study, we investigated the potential of nanoparticles based on polyethylenimine (PEI) modified with stearic acid (StA), to deliver siRNA for efficient STAT3 downregulation in B16 melanoma cells. The B16 cells were targeted with ∼6–200 n m of siRNA complexes for 36 h. Compared to the PEI complexes, the PEI-StA complexes showed higher potency in STAT3 silencing in B16 cells accompanied by a significant induction of IL-6 secretion and a reduction of VEGF production. Moreover, with PEI-StA complexes, the level of the cellular Caspase 3 activity (an indicator of apoptotic activity) was found to be 2.5 times higher than that of PEI complexes. When the cells were treated with 50 n m of siRNA complexes on a daily basis, the cell viability was dramatically reduced reaching only to 16% after the third daily dose of PEI-StA complexes, as compared to the 69% viability observed with the PEI complexes at an equivalent time period. Consistently, in vivo results indicated significant regression in tumor growth and tumor weight after siRNA/PEI-StA treatment as compared to the siRNA/PEI. This was accompanied with significant increase in IL-6 levels and Caspase 3 activity, and a significant decrease in VEGF level and STAT3 activity in the tumor tissue. The lipid-modified PEI is a promising carrier for siRNA delivery and downregulation of STAT3 by polymer-mediated siRNA delivery is an effective strategy for cancer treatment especially when an optimum delivery system can potentiate the silencing activity of siRNA.

Autophagy Links Pattern Recognition Receptors to Tumor Cell Apoptosis

Pathogen nucleic acids are detected by the infected body’s immune cells via specific innate immune receptors, resulting in reactions such as secretion of T helper type 1 (Th1)-like and proinflammatory cytokines. Two studies have now reported the induction of cell death in melanoma cells, but not healthy skin cells, via stimulation of the cytoplasmic receptor MDA5 by the synthetic double-stranded RNA (dsRNA) agonist poly I:C, and one of the studies elucidated the mechanism for this response. Polyethyleneimine (PEI)-formulated poly I:C engages an intracellular process called autophagy via MDA5 to result in the induction of prodeath programs that ultimately lead to apoptosis of melanoma cells. Together with previous work, these reports demonstrate that immune detection of foreign nucleic acids involves diverse signaling pathways and thus yields different cellular outcomes that may overcome the inherent resistance of cancer cells to current cancer treatments.

MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines

This study will determine whether MTT assays accurately assess the effect of STI571 (Gleevec; Abl kinase inhibitor) on the viability of cancer cells containing highly active Abl kinases. Growth kinetics, tritiated thymidine, fluorescent caspase, MTT, and Cell Titer Glo (CTG) assays were used to determine the effect of STI571 on growth, proliferation, apoptosis, and viability of melanoma and breast cancer cells. STI571 inhibited growth and proliferation, and increased apoptosis. However, MTT assays indicated that STI571 increased cell viability. In contrast, STI571 induced a dose-dependent decrease in viability using CTG assays. Doses of STI571 (1-10 microM) required to inhibit endogenous Abl kinases interfere with the MTT assay, and therefore MTT cannot be used to determine the effect of STI571 on viability using these doses. Additionally, caution should be utilized when interpreting the results of MTT assays used to screen kinase inhibitors for anti-cancer activity, as drug effectiveness may be minimized.

Mitogen-Activated Protein Kinase Inhibition Induces Translocation of Bmf to Promote Apoptosis in Melanoma

Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF have been identified in approximately 85% of melanomas, and components of this pathway have been developed as molecular targets for therapeutic intervention. We and others have shown that inhibition of this pathway with specific small molecule MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors induces a wide range of apoptotic responsiveness in human melanoma cells both in vitro and in vivo. To define the molecular mechanism underlying variable apoptotic sensitivity of melanoma cells to MEK inhibition, we examined the expression and subcellular localization of Bcl-2 family members in a comprehensive set of human melanoma cell lines. Whereas the proapoptotic protein Bim was activated and localized to the mitochondrial membrane in all cell lines regardless of apoptotic sensitivity, Bmf activation and cytosolic translocation was exclusive to sensitive cells. In resistant cells, Bmf remained sequestered to the cytoskeleton through dynein light chain 2 (DLC2) binding. Overexpression of Bmf in resistant cells did not enhance apoptosis, whereas expression of mutant BmfA69P, which has decreased binding to DLC2, promoted cell death. Expression of BmfA69P mutants possessing the Bcl-2 homology 3 (BH3) domain mutation L138A, which impairs BH3 interactions, did not enhance apoptosis in resistant cells. RNA interference targeting Bim and Bmf provided protection from apoptosis induced by MEK inhibition. These results show a novel role for Bmf in promoting apoptosis and provide insight into the mechanism of apoptotic resistance to MEK inhibition in melanoma.

The role of GRP78, p-ERK and Bcl-2 proteins in endoplasmic reticulum stress-induced apoptosis and anti-apoptosis in melanoma

The generation of anti-apoptotic factors is complex. Previous studies have demonstrated that endoplasmic reticulum (ER) stress results in both apoptosis and anti-apoptosis in some tumours. ER stress may induce apoptosis via multiple pathways. Adaptation of melanoma to ER stress may result in anti-apoptosis and resistance to therapy. The anti-apoptosis mechanisms include RAS/RAF/MEK/ERK activation, increased GRP78 and Mcl-1 protein. In the present study, we sought to better characterise the correlation of the ER stress protein GRP78 expression with ERK activation and Bcl-2 family proteins. The expression of GRP78, Bcl-2 and Mcl-1 in formalin-fixed sections of 135 naevi, primary and metastatic melanoma by immunohistochemistry was examined. p-ERK expression was assessed in 82 of the 135 cases. The correlations of GRP78 with p-ERK, Bcl-2 and Mcl-1 were analysed. The immunoreactive score (IRS) of GRP78 and Mcl-1 were related to melanocytic tumour progression. The intensity of GRP78 and Mcl-1 increased with the progression of melanocytic tumours. In contrast, Bcl-2 showed high expression in melanoma ≤1mm and less expression in melanoma metastases. GRP78 expression increased with increased p-ERK expression (<i>p</i><0.01). GRP78 expression increased with increased expression of Mcl-1 (<i>p</i><0.0001). The findings of this study suggest that activated ERK may enhance GRP78 levels and are consistent with previous results from melanoma cell lines. GRP78 and Mcl-1 expression may play a role in protecting cells from ER stress-induced apoptosis. Bcl-2 is increased in thin melanoma and this suggests that Bcl-2 plays its anti-apoptotic role in the early stage of melanoma progression.

Synthesis and antitumor activity of novel enediyne-linked pyrrolo[2,1- c][1,4]benzodiazepine hybrids

A series of novel pyrrolo[2,1- c][1,4]benzodiazepine (PBD) hybrids linked with enediyne is described. These compounds were prepared by linking C-8 of DC-81 ( 1) with an enediyne ( 10– 16) through carbon chain linkers to afford PBD hybrid agents 17– 23 in good yields. Most of the hybrids on human cancer cell lines exhibited higher cytotoxicity, and an increase in the sub-G1 population than 1. In a previous article, we have demonstrated that DC-81-indole conjugate agents ( 3–6) are potent inducers of cell apoptosis in melanoma. In the present article, we investigated whether DC-81-enediyne agents possess more cytotoxicity than 6 on human 293T cells. Our data revealed that treatment of 293T cells with DC-81-enediyne resulted in a significant increase of annexin V binding, caspase-3 degradation, and p53 arrest to identify apoptotic cells than 6. These results suggest that the DC-81-enediyne agents are more efficient in inducing apoptosis than DC-81-indole in 293T cells.

Involvement of endoplasmic reticulum stress in Docetaxel-induced JNK-dependent apoptosis of human melanoma

Our previous studies revealed that Docetaxel-induced apoptosis of melanoma cells is entirely dependent on activation of the JNK signalling pathway. Here, we show that Docetaxel-induced apoptosis is mediated by induction of ER stress. This was shown by Docetaxel-induced activation of proteins involved in ER stress signalling namely GRP78, ATF6, IRE1alpha, and PERK/eIF2alpha. Knockdown of IRE1alpha by siRNA markedly inhibited Docetaxel-induced JNK activation and downstream targets of JNK indicating that activation of IRE1alpha was upstream of activation of the JNK. Co-immunoprecipitation experiments showed that activation of JNK is due to activation of ASK1 through formation of an IRE1alpha-TRAF2-ASK1 complex. ER stress mediated activation of the JNK pathway is downstream of activation of PKCdelta in that downregulation of PKCdelta expression using specific PKCdelta siRNA significantly inhibited Docetaxel-induced activation of IRE1alpha and the JNK pathway. These findings provide new insights to understand the mode of action of taxanes in treatment of human melanoma.

IFN-α and Bortezomib Overcome Bcl-2 and Mcl-1 Overexpression in Melanoma Cells by Stimulating the Extrinsic Pathway of Apoptosis

We hypothesized that IFN-alpha would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-alpha induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-alpha was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-alpha displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-alpha for malignant melanoma.

The biology and management of uveal melanoma

Uveal melanoma is the most common primary intraocular malignancy in adults. Overall mortality rate remains high because of the frequent development of metastatic disease, especially hepatic metastasis. While traditional systemic chemotherapies provide only marginal benefit to patients, local treatments for hepatic metastases, such as immunoembolization, have improved patient prognoses. Progress has also been made in identifying potential targets in the pathways involved in apoptosis, proliferation, invasion, metastasis, and angiogenesis of uveal melanoma. Among these pathways, the c-Kit, c-Met, and IGF-1R signal pathways and the PTEN-related PI3K-Akt pathway are the most important targets. Clinical trials using blockades of these pathways in conjunction with strategies to facilitate apoptosis is a direction for future clinical trials. Application of these approaches in the adjuvant setting after primary therapy for high-risk uveal melanoma patients is also a future consideration to improve the clinical outcome of this disease.

Mcl-1 cleavage and sustained phosphorylation of c-Jun -N-terminal kinase mediate melanoma apoptosis induced by 2-acetyl furanonaphthoquinone : Roles of bcl-2 and p53

2-acetyl furanonaphthoquinone (FNQ) is a naturally occurring drug with enhanced toxicity versus glucose-starved tumor cells , which frequently show topoisomerase II drug resistance. Since loss of p53 tumor suppressor function or overexpression of the anti-apoptotic bcl-2 gene can decrease susceptibility to some cancer therapies , we now investigated the effect of FNQ against genetically matched C8161 melanoma cell lines transduced to express unequal levels of Bcl-2 , or engineered to harbour a functional wt p53 for comparison with dominant-negative mutant p53 R175H .Cells with differing p53 genotype showed susceptibility to FNQ. However , this response was attenuated in those overexpressing mutant p53 , although a brief p53 induction was early seen in FNQ –treated wt p53 cells . Cells susceptible to FNQ showed cleavage of anti-apoptotic Mcl-1, sustained activation of the c-Jun N-terminal Kinase ( p-JNK) , and apoptosis-associated PARP fragmentation , all of which were counteracted in bcl-2 overexpressing cells. Suppression of JNK activation with the specific inhibitor , SP600125 also prevented FNQ –mediated cell death. Our data suggests that Bcl-2 , persistent JNK phosphorylation and cleavage of anti-apoptotic Mcl-1 are key events controlling susceptibility to FNQ.

Tumour malignancy loss and cell differentiation are associated with induction ofgefgene in human melanoma cells

Gene therapy is a new method used to induce cancer cell differentiation. Our group previously showed that transfection of the gef gene from Escherichia coli, related to cell-killing functions, may be a novel candidate for cancer gene therapy. Its expression leads to cell cycle arrest unrelated to the triggering of apoptosis in MS-36 melanoma cells. To determine the basis of the antiproliferative effect of the gef gene in this cell line. Transmission electron microscopy, apoptosis analysis by confocal microscopy, flow cytometry and immunocytochemical analysis were used. Ultrastructural analysis showed a strikingly different morphology after treatment with dexamethasone and expression of the gef gene, with large accumulations of pigment throughout the cell cytoplasm and presence of melanosomes in different stages of development. High mitochondrial turnover and myeloid bodies, characteristics of neurone cells, were also observed. In addition, both immunocytochemical and indirect immunofluorescence analysis demonstrated a significant decrease in HMB-45, Ki-67 and CD44 antigen expression and an increase in S100 and p53 expression in gef gene-transfected MS-36 melanoma cells that were correlated with the duration of dexamethasone treatment. In the present work, we report that gef gene not only reduces cell proliferation in transfected melanoma MS-36TG cell line but also induces morphological changes clearly indicative of melanoma cell differentiation and a reduction in tumour malignancy. These findings support the hypothesis that the gef gene offers a new approach to differentiation therapy in melanoma.