TIMP-1-GPI in combination with hyperthermic treatment of melanoma increases sensitivity to FAS-mediated apoptosis

Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8°C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes. Elfriede Noessner, Peter J. Nelson are equal contributors.     

Conditional expression of exogenous Bcl-X(S) triggers apoptosis in human melanoma cells in vitro and delays growth of melanoma xenografts

The Bcl-2-related proteins Bcl-X(L) and Bcl-X(S) represent alternative splice products and exert opposite activities in the control of apoptosis, but their significance for melanoma is not yet clear. Applying the tetracycline-inducible expression system Tet-On, we found overexpression of Bcl-X(S) by itself sufficient to induce apoptosis in vitro in stably transfected human melanoma cell lines. Combination with proapoptotic agents such as etoposide, pamidronate, and ceramide resulted in additive proapoptotic effects, whereas Bcl-X(L) protected from apoptosis caused via CD95/Fas stimulation. In nude mice growth of melanoma xenotransplants derived from stably transfected cells was significantly reduced after induction of Bcl-X(S) by doxycycline. Our results indicate that Bcl-X proteins are of major importance for control of apoptosis in malignant melanoma.     

Bim plays a crucial role in synergistic induction of apoptosis by the histone deacetylase inhibitor SBHA and TRAIL in melanoma cells

The wide variation in sensitivity of cancer cells to TRAIL- or histone deacetylase (HDAC) inhibitor – induced apoptosis precludes successful treatment of cancer with these agents. We report here that TRAIL and SBHA synergistically induce apoptosis of melanoma cells as revealed by quantitative analysis using the normalized isobologram method. This is supported by enhanced activation of caspase-3 and cleavage of its substrates, PARP and ICAD. Co-treatment with SBHA and TRAIL did not enhance formation of the death-inducing signaling complex (DISC) and processing of caspase-8 and Bid, but potentiated activation of Bax and release of Cytochrome C and Smac/DIABLO from mitochondria into the cytosol. SBHA down-regulated Bcl-X_L, Mcl-1 and XIAP, but up-regulated Bax, Bak, and the BH3-only protein Bim_EL. Up-regulation of the latter by SBHA was attenuated by the presence of TRAIL, which was inhibitable by the pan-caspase inhibitor z-VAD-fmk. Inhibition of Bim by siRNA attenuated conformational changes of Bax, mitochondrial apoptotic events, and activation of caspase-3, leading to marked inhibition of the synergy between SBHA and TRAIL. Thus, Bim plays an essential role in synergistic induction of apoptosis by SBHA and TRAIL in melanoma. This work was supported by the NSW State Cancer Council, the Melanoma and Skin Cancer Research Institute Sydney, the Hunter Melanoma Foundation, NSW, and the National Health and Medical Research Council, Australia. X.D. Zhang is a Cancer Institute NSW Fellow.     

Induction of apoptosis by ionizing radiation and CI-1033 in HuCCT-1 cells

CI-1033 is a quinazoline-based HER family tyrosine kinase inhibitor that is currently being evaluated as a potential anticancer agent. The present study examines the molecular mechanism by which CI-1033 induces apoptosis either as a single agent or in combination with radiation. Although CI-1033 alone did not induce apoptosis, the simultaneous exposure of cells to CI-1033 and radiation induced significant levels of apoptosis. The sequential treatment of cells with CI-1033 followed by radiation induced an even greater effect with 62.6% of cells undergoing apoptosis but this enhanced effect was not seen if cells were treated first with radiation and then CI-1033. The combination treatment induces apoptosis of HuCCT-1 via upregulation of FasL and Bid cleavage. These data suggest that modulation of the Fas-FasL pathway and activation of Bid could be useful for increasing the anti-tumor effect of CI-1033 in this type of cancer.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Role of the pro-survival molecule Bfl-1 in melanoma

Bfl-1 is a pro-survival Bcl-2 family member overexpressed in a subset of chemoresistant tumours, including melanoma. Here, we characterised the expression and regulation of Bfl-1 in normal and malignant melanocytes and determined its role in protecting these cells from chemotherapy-induced apoptosis. Bfl-1 was mitochondrially resident in both resting and apoptotic cells and experienced regulation by the proteasome and NFκB pathways. siRNA-mediated knockdown enhanced sensitivity towards various relevant drug treatments, with forced overexpression of Bfl-1 protective. These findings identify Bfl-1 as a contributor towards therapeutic resistance in melanoma cells and support the use of NFκB inhibitors alongside current treatment strategies.     

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.Abbreviations NAC N-acetyl-l-cysteine - ROS reactive oxygen species - _m mitochondrial membrane potentialJae Seung Kang and Daeho Cho contributed equally to this work.     

Application of microwave-irradiation technique in deglycosylation of ginsenosides for improving apoptosis induction in human melanoma SK-MEL-2 cells

To increase the contents of medicinally effective ginsenosides, we used high-temperature and high-pressure thermal processing of ginseng by exposing it to microwave irradiation. To determine the anti-melanoma effect, the malignant melanoma SK-MEL-2 cell line was treated with an extract of microwave-irradiated ginseng. Microwave irradiation caused changes in the ginsenoside contents: the amounts of ginsenosides Rg1, Re, Rb1, Rb2, Rc, and Rd were disappeared, while those of less polar ginsenosides, such as Rg3, Rg5, and Rk1, were increased. In particular, the contents of Rk1 and Rg5 markedly increased. Melanoma cells treated with the microwave-irradiated ginseng extract showed markedly increased cell death. The results indicate that the microwave-irradiated ginseng extract induced melanoma cell death via the apoptotic pathway and that the cytotoxic effect of the microwave-irradiated ginseng extract is attributable to the increased contents of specific ginsenosides.     

Role of caspases-3 and -7 in Apaf-1 proteolytic cleavage and degradation events during cisplatin-induced apoptosis in melanoma cells

Apoptosis protease-activating factor-1 (Apaf-1), the central element in the mitochondrial pathway of apoptosis, is frequently absent or poorly expressed in metastatic melanomas, a tumor type showing a low degree of spontaneous apoptosis and a poor response to conventional therapies. In the present study, we used the Apaf-1-positive Me665/2/21 melanoma cell line to investigate the fate of Apaf-1 during cisplatin-induced apoptosis. As novel findings described for the first time in melanoma cells, we observed that Apaf-1 was markedly decreased during apoptosis, already at early stages of cell damage; concurrently, an immunoreactive N-terminal fragment of congruent with 26 kDa was evident. In spite of the remarkable decrease of Apaf-1 in apoptotic cells, caspase-9 was found to be processed and enzymatically active. Both Apaf-1 depletion and its proteolytic cleavage were markedly prevented in presence of the caspase-3/-7 inhibitor ac-DEVD-CHO. In presence of ac-DEVD-CHO, caspase-9 activity was also inhibited, along with a partially different pattern of caspase-9 processing forms. Unexpectedly, the inhibition afforded by ac-DEVD-CHO on several components, that is, caspase-3/-7 and caspase-9 activities, and Apaf-1 proteolytic degradation, did not abrogate the apoptotic morphology and cell detachment, nor the proteolytic degradation of crucial targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. Together, our results suggest that caspase-3 and -7, proved to be dispensable for the above apoptosis-associated events, play a role on Apaf-1 handling and possibly on apoptosome function.     

siRNA targeting PLK-1 induces apoptosis of synoviocytes in rheumatoid arthritis

Polo-like kinase-1 (PLK-1) is a member of the PLK family and participates in the control of cell mitosis. Here, we show that immunoreactive PLK-1 is strongly expressed in synoviocytes and some infiltrative mononuclear cells in synovial tissues from patients with rheumatoid arthritis (RA), while patients with osteoarthritis and injury show little or no expression of PLK-1 in synovial tissues. Western blot analysis shows that PLK is expressed and its expression is enhanced by IL-1beta in RA synoviocytes. IL-1beta also enhanced the cell growth of RA synoviocytes. Moreover, siRNA targeted against PLK-1 significantly decreases the expression of PLK-1 of RA synoviocytes stimulated by IL-1beta and suppresses the proliferation of these synoviocytes through apoptosis. These findings suggest that PLK-1 plays a critical role in the proliferation of RA synoviocytes leading to bone destruction, and siRNA against PLK-1 is potentially useful for the treatment of RA.     

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 (ΔΨ_m), 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.     

Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.     

Molecular mechanism underlying differential apoptosis between human melanoma cell lines UACC903 and UACC903(+6) revealed by mitochondria-focused cDNA microarrays

Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecular pathways underlying this difference. Using our recently developed mitochondria-focused cDNA microarrays, we identified 154 differentially expressed genes including proapoptotic (BAK1 [6p21.3], BCAP31, BNIP1, CASP3, CASP6, FAS, FDX1, FDXR, TNFSF10 and VDAC1) and antiapoptotic (BCL2L1, CLN3 and MCL1) genes. Expression of these pro- and anti-apoptotic genes was higher in UACC903(+6) than in UACC903 before UV treatment and was altered after UV treatment. qRT-PCR and Western blots validated microarray results. Our bioinformatic analysis mapped these genes to differential molecular pathways that predict resistance and sensitivity of UACC903 and UACC903(+6) to apoptosis respectively. The pathways were functionally confirmed by the FAS ligand-induced cell death and by siRNA knockdown of BAK1 protein. These results demonstrated the differential molecular pathways underlying survival and apoptosis of UACC903 and UACC903(+6) cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Qiuyang Zhang, Jun Wu, and AnhThu Nguyen made equal contributions to this work.     

Induction of apoptosis and endothelin-1 secretion in primary human lung endothelial cells by HIV-1 gp120 proteins

Pulmonary hypertension associated with human immunodeficiency virus (HIV) infection also involves injury to the lung endothelium. However, the pathogenesis of HIV-induced pulmonary hypertension is not known; we hypothesized that HIV or secreted viral proteins could play a role in vascular injury and the increased frequency of pulmonary hypertension observed in HIV-infected patients. Here, we report that exposure of HIV-1 gp120 proteins to primary human lung microvascular endothelial cells causes apoptosis, as assessed by TUNEL assay, Annexin-V staining, and DNA laddering. Using ribonuclease protection assay and Western blotting we find that gp120-induced apoptosis of lung endothelial cells involves a down-regulation in Bcl-xl mRNA and proteins. In addition, gp120 significantly increases secretion of the potent vasoconstrictor endothelin-1 by human lung endothelial cells. These data suggest that secreted HIV gp120 proteins induce lung endothelial cell injury and could contribute to the development of HIV-associated pulmonary hypertension.     

Dickkopf-1 activates cell death in MDA-MB435 melanoma cells

Dickkopf-1 (DKK-1) is known inhibitor of the canonical Wnt pathway. Recent studies strongly suggested that activation of DKK-1 expression results in inhibition of cell tumorigenicity. Reduced levels of DKK-1 in melanomas were recently shown. However, it is not known if DKK-1 activation in melanoma cells will inhibit cell tumorigenicity. In the present study, we overexpressed DKK-1 in melanoma cell line MDA-MB435. We show that while DKK-1 did not affect cell growth in soft agar, weak but significant inhibition of tumorigenicity in nude mice in vivo was observed. Analysis of resulting tumors revealed activation of cell death. In tumors originating from cells transduced with DKK-1, tumor mass was permeated with areas of necrosis. In tumors, originated from control cells, areas of necrosis were limited to the central region, a common feature of large tumors growing in nude mice. TUNEL assay revealed that in tumors originating from cells transduced with DKK-1 apoptotic cells were detected along the border of necrotic and viable areas of the tumors indicating significant increase in apoptotic process. Thus, our results indicate that activation of DKK-1 in melanoma cells leads to activation of apoptosis in vivo and, thus, is incompatible with tumor growth in nude mice.     

Markers of mitochondrial dysfunction during the diclofenac-induced apoptosis in melanoma cell lines

Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered.     

Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors. In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model. We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4′,6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways). Further, the anticancer effect in vivo was confirmed through a xenograft model. Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner. In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis. Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins. Additionally, shikonin was overexpressed in MAPK pathways. Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, pathologic changes were not observed in the liver and kidney of mice. Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.     

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, IRE1α, and PERK/eIF2α. Knockdown of IRE1α by siRNA markedly inhibited Docetaxel-induced JNK activation and downstream targets of JNK indicating that activation of IRE1α 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 IRE1α-TRAF2-ASK1 complex. ER stress mediated activation of the JNK pathway is downstream of activation of PKCδ in that downregulation of PKCδ expression using specific PKCδ siRNA significantly inhibited Docetaxel-induced activation of IRE1α and the JNK pathway. These findings provide new insights to understand the mode of action of taxanes in treatment of human melanoma.