Apoptosis In Melanoma Cell Lines Research Articles

Arginine Deprivation as a Targeted Therapy for Cancer

Certain cancers may be auxotrophic for a particular amino acid, and amino acid deprivation is one method to treat these tumors. Arginine deprivation is a novel approach to target tumors which lack argininosuccinate synthetase (ASS) expression. ASS is a key enzyme which converts citrulline to arginine. Tumors which usually do not express ASS include melanoma, hepatocellular carcinoma, some mesotheliomas and some renal cell cancers. Arginine can be degraded by several enzymes including arginine deiminase (ADI). Although ADI is a microbial enzyme from mycoplasma, it has high affinity to arginine and catalyzes arginine to citrulline and ammonia. Citrulline can be recycled back to arginine in normal cells which express ASS, whereas ASS(-) tumor cells cannot. A pegylated form of ADI (ADI-PEG20) has been formulated and has shown in vitro and in vivo activity against melanoma and hepatocellular carcinoma. ADI-PEG20 induces apoptosis in melanoma cell lines. However, arginine deprivation can also induce ASS expression in certain melanoma cell lines which can lead to in vitro drug resistance. Phase I and II clinical trials with ADI-PEG20 have been conducted in patients with melanoma and hepatocellular carcinoma, and antitumor activity has been demonstrated in both cancers. This article reviews our laboratory and clinical experience as well as that from others with ADI-PEG20 as an antineoplastic agent. Future direction in utilizing this agent is also discussed.

GSK-3β Inhibition Enhances Sorafenib-induced Apoptosis in Melanoma Cell Lines

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.

Sodium arsenite alters cell cycle progression and induces apoptosis in melanoma cell lines

Arsenic trioxide is used to treat acute promyelocytic leukemia and is in clinical trials for use with other cancers. This study examines arsenic as a drug for melanoma. A375 and SK-Mel-2 melanoma cell lines were sensitive to clinically achievable concentrations of arsenite. SK-Mel-3 and SK-Mel-28 cell lines were resistant to arsenite but were sensitized by pretreatment with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. Arsenic resistance was also overcome by co-treatment with MK571, an inhibitor of MRP-family transport proteins. Arsenite alone or with BSO or MK571 induced caspase 3 and poly(ADP-ribose) polymerase (PARP) cleavage suggesting apoptotic cell death. Apoptosis in arsenite-sensitive cell lines was associated with mitotic arrest and cyclin B stabilization. In A375 cells, active caspase 3 co-localized with mitotic marker phospho-histone H3. Degradation or inhibition of cyclin B is needed for mitotic exit. Roscovitine, a cyclin B/CDK1 inhibitor, lowered the mitotic index of mitotic arsenite-treated cells and abrogated caspase 3 and PARP cleavage. Arsenite with or without BSO or MK571 did not arrest SK-Mel-3 and SK-Mel-28 cells in mitosis, but induced GADD45α, suggesting G2 arrest. Arsenite’s ability to induce apoptosis in these cells warrants further study of its use with melanoma. (Support by PHS grants R01ES011314 and T32ES011564.)

WP760, a melanoma selective drug

Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma. The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels. WP760 induced G(2)/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27(Kip1) protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated. Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.

New thymidylate synthase inhibitors induce apoptosis in melanoma cell lines

Malignant melanoma is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the past years a huge variety of new compounds has been developed with potential chemotherapeutic activity which needs to be tested in vitro and in vivo. We investigated the in vitro action of three new experimental antifolate substances (MR7, MR21 and MR36) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. The response of two melanoma cell lines (SK-MEL-2 derived from malignant melanoma metastasis and SK-MEL-28 derived from primary malignant melanoma) was examined after treatment with these substances. The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by the TUNEL technique and Comet Assay. Western-blot analysis showed a down-regulation of Bcl-2 protein level and PARP cleavage, otherwise p53 and Bax expressions were not modulated. Moreover, these antifolate-induced apoptosis was accompanied by both pro-caspase-9 and -8 activations. These results were supported by the use of the pan-caspases inhibitor Z–VAD–FMK that almost completely decreased the amount of apoptosis in both the melanoma cell lines treated with antifolate. In conclusion our results show that TS inhibitors are able to induce apoptosis through a caspase-mediated pathway, but without the involvement of the p53/Bax signalling.

526. Induction of Apoptosis in Melanoma Cell Lines by the Noxa Gene

Background: Melanoma is the most aggressive form of skin cancers and has shown a dramatic increase in incidence over the past decades. Although new therapies have become available, unfortunately advanced tumors are invariably resistant to conventional treatment due to a dysregulation of apoptosis. In the present study, we demonstrate the feasibility of electroporation of the BH3-only gene Noxa to induce apoptosis in melanoma cell lines.

Inhibition of melanoma growth by hemocyanin occurs via early apoptotic pathways

Background We hypothesized that keyhole limpet hemocyanin (KLH) would reduce cellular proliferation and effect apoptosis of melanoma cell lines in vitro. Methods Two human melanoma cell lines (HTB68 and HTB72) were subjected to a dose-response treatment regimen of KLH (0.4 μg to 100 μg/well). Cell viability was tested by MTT assay (SIGMA, St Louis, MO) at 72 hours. Apoptosis and necrosis were measured by the Annexin V FITC assay (Biovision Inc, Mountain View, CA). Results Melanoma cell proliferation was significantly reduced in the HTB68 cell line treated with 6.3 μg or higher doses of KLH. A significant reduction in cell growth was also observed in the HTB72 cells at 50 and 100 μg of KLH. KLH increased early apoptotic activity, whereas both late apoptosis and necrosis were decreased by the addition of KLH. Conclusions KLH significantly reduces cellular proliferation in vitro in melanoma, via early apoptotic pathways. The results warrant in vivo studies into the effects of KLH in melanoma.

Modulation of Vitamin D‐induced growth inhibition in melanoma cell lines: implications for an important function of vitamin D receptor (VDR) and 1,25‐dihydroxyvitamin D3‐24‐hydroxylase (24‐OHase) expression, histonedeacetylation, and calpain activity

Increasing evidence indicates an important role of the skin vitamin D system for tumorigenesis and progression of malignant melanoma. We have now characterized the vitamin D system in melanoma cell lines, detecting strong RNA expression of vitamin D receptor (VDR), 25‐hydroxyvitamin D‐1α‐hydroxylase (1αOHase), vitamin D‐25‐hydroxylase (25OHase) and 1,25‐dihydroxyvitamin D‐24‐hydroxylase (24OHase) in various melanoma cell lines (MEWO, SKMEL28, BU47HOM) using real time PCR (LightCycler) and specific hybridisation probes. We then have analyzed effects of 1,25‐dihydroxyvitamin D3 and analogs (EB 1089, MC 1288) on proliferation and apoptosis as well as on expression of VDR, 1αOHase), 25OHase, and 24OHase in various melanoma cell lines (BUHOM, MEWO, SKMEL)in vitro. Using a tetrazolium salt (WST‐1) based colorimetric assay, we detected dose‐dependent inhibition of cell growth (up to 40%) induced by calcitriol or its analogs. Treatment of melanoma cells with calcitriol resulted in decreased immunoreactivity of proliferation markers including Ki‐67 and PCNA. Flow cytometry experiments (bcl‐2, bcl‐xl, bcl‐xs, bcl‐x, bax, CD95) and analysis of annexin Pi expression revealed no induction of apoptosis by calcitriol (10−7 M) or its analogs, while increased levels of bcl‐2 protein were detected. Additionally, we show modulation of vitamin D‐induced inhibition of cell proliferation by calpain inhibitors I and II (Calbiochem) as well as by inhibitors of histonedeacetylation (sodium butyrate, trichostatin A). In conclusion our findings indicate that (i) vitamin D analogs suppress proliferation but do not induce apoptosis in melanoma cell lines, (ii) induction of VDR and 24‐OHase expression as well as histonedeacetylation and calpain activity modulate vitamin D‐induced growth inhibition, (iii) new calcitriol analogs exerting less systemic side effects may be interesting candidates for the treatment of metastasizing malignant melanoma.

Depsipeptide (FR901228) inhibits proliferation and induces apoptosis in primary and metastatic human uveal melanoma cell lines.

Uveal melanoma (UM) is the most common primary malignant ocular tumor in adults. No effective chemotherapy regimens are available for either intraocular or metastatic uveal melanoma. Therefore, the ability of the histone deacetylase inhibitors (HDACIs), depsipeptide, sodium butyrate (NaB) and trichostatin A (TSA), to induce apoptosis and inhibit cell growth of UM cell lines in vitro was examined. Three primary and two metastatic UM cell lines were treated in vitro with different concentrations of histone deacetylase inhibitors (HDACIs). Cell proliferation was studied in 24-well plates. Induction of apoptosis was studied by flow cytometry. Changes in gene expression of Fas/FasL, p21(Waf/Cip1), and p27(Kip1) were studied by RT-PCR. Western blot analysis was used to study histone acetylation, Fas/FasL, p21(Waf/Cip1), p27(Kip1) and caspase-3 protein levels. Real-time PCR was used to study changes in bcl-2/bax gene expression. A dose-dependent increase in histone acetylation was observed in all cell lines. This corresponded to significant inhibition of cell growth and induction of apoptosis in all melanoma cell lines in a concentration-dependent manner. Western blot analysis revealed dose-dependent increases in the amount of caspase-3, Fas/FasL, p21(Waf/Cip1), and p27(Kip1) proteins. However, no changes in bcl-2/bax gene expression were detected by real-time PCR. HDACIs are potent inhibitors of primary and metastatic UM cell growth in vitro. The apoptosis is probably mediated through the Fas/FasL signaling pathway, whereas bcl-2 appears not to be involved. These data support further clinical evaluation of depsipeptide and other HDACIs in patients with primary and metastatic UM.

Induction of Apoptosis in Melanoma Cell Lines by p53 and its Related Proteins

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361). Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

In vitro and in vivo effects of a cyclic peptide with affinity for the ανβ3 integrin in human melanoma cells

Expression of the integrin ανβ3 has been shown to be associated with increasing metastatic potential in malignant melanoma. It also has a functional role on vascular endothelial cells during angiogenesis. The cyclic oligopeptide cRGDfV is known to bind with high affinity to ανβ3. We have investigated the cellular effects of cRGDfV on a panel of human melanoma cell lines in vitro and also on the A375 melanoma cell line growing as xenografts in nude mice. cRGDfV is a potent inhibitor of ανβ3-mediated cell adhesion, however, we have found no convincing evidence that integrin ligation by cRGDfV induces apoptosis in melanoma cell lines. However, cRGDfV when administered subcutaneously into nude mice did inhibit the growth of A375 melanoma xenografts. Histological examination of the tumours indicated that this effect was primarily one of angiogenesis inhibition. The results suggest that agents which target the ανβ3 integrin may have a useful role as anti-angiogenesis agents in clinical oncology, but that they may not exert a direct effect on αvβ3-expressing tumour cells.