Effects of betulinic acid alone and in combination with irradiation in human melanoma cells

Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alone and in combination with ionizing radiation in a panel of established human melanoma cell lines as well as in normal human melanocytes. Betulinic acid strongly and consistently suppressed the growth and colony-forming ability of all human melanoma cell lines investigated. In combination with ionizing radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human melanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the expression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermore, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiotherapy. We conclude that the strictly additive mode of growth inhibition in combination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting different target cell populations.     

Mcl-1 antisense therapy chemosensitizes human melanoma in a SCID mouse xenotransplantation model

It is well established that high expression of the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL can significantly contribute to chemoresistance in a number of human malignancies. Much less is known about the role the more recently described Bcl-2 family member Mcl-1 might play in tumor biology and resistance to chemotherapy. Using an antisense strategy, we here address this issue in melanoma, a paradigm of a treatment-resistant malignancy. After in vitro proof of principle supporting an antisense mechanism of action with specific reduction of Mcl-1 protein as a consequence of nuclear uptake of the Mcl-1 antisense oligonucleotides employed, antisense and universal control oligonucleotides were administered systemically in combination with dacarbazine in a human melanoma SCID mouse xenotransplantation model. Dacarbazine, available now for more than three decades, still remains the most active single agent for treatment of advanced melanoma. Mcl-1 antisense oligonucleotides specifically reduced target protein expression as well as the apoptotic threshold of melanoma xenotransplants. Combined Mcl-1 antisense oligonucleotide plus dacarbazine treatment resulted in enhanced tumor cell apoptosis and led to a significantly reduced mean tumor weight (mean 0.16 g, 95% confidence interval 0.08-0.26) compared to the tumor weight in universal control oligonucleotide plus dacarbazine treated animals (mean 0.35 g, 95% confidence interval 0.2-0.44) or saline plus dacarbazine treated animals (mean 0.39 g, 95% confidence interval 0.25-0.53). We thus show that Mcl-1 is an important factor contributing to the chemoresistance of human melanoma in vivo. Antisense therapy against the Mcl-1 gene product, possibly in combination with antisense strategies targeting other antiapoptotic Bcl-2 family members, appears to be a rational and promising approach to help overcome treatment resistance of malignant melanoma.     

Apoptosis regulators and responses in human melanocytic and keratinocytic cells

Apoptosis in keratinocytes is required for epidermal turnover, stratum corneum formation, and removal of ultraviolet-damaged premalignant cells. Its role in melanocyte homeostasis and transformation, on the other hand, has not been defined, although apoptosis resistance is a commonly recognized feature of melanoma. We examined the expression of apoptosis regulators in melanocytes, keratinocytes, melanoma, and HaCat cells. Melanocytic cells expressed relatively high levels of Bcl-2, Bcl-X(L), Mcl-1, C-IAP-1, C-IAP-2, XIAP, Livin, and Apaf-1. The only apoptotic regulator that was differentially expressed in melanoma cells and not melanocytes was Survivin, whereas Bax was expressed in melanocytes but not in most melanoma lines. Keratinocytic cells, on the other hand, expressed high levels of FLIP and were relatively deficient in Bcl-2 family proteins. Levels of p53 were highest in HaCat cells and some of the melanoma lines, and barely detectable in melanocytes and keratinocytes. Next, susceptibility of these cells types to apoptosis induced by ultraviolet B, the tyrosine analog 4-tert-butylphenol, and cytotoxic drugs was examined. Melanocytes were relatively resistant to ultraviolet B, whereas keratinocytes were unresponsive to 4-tert-butylphenol. Melanocytes and keratinocytes were generally less susceptible than melanoma lines and HaCat cells to etoposide, cisplatin, and staurosporine. Induction of apoptosis in these cell types was generally associated with decreased levels of Mcl-1, XIAP, and Livin, and increased levels of p53, whereas levels of other apoptotic regulators were unaltered. These results provide insights into the potential roles of apoptosis in the function and transformation of epidermal melanocytes and keratinocytes.     

BCL2 and BCLxL are key determinants of resistance to antitubulin chemotherapeutics in melanoma cells

Malignant melanoma is refractory to various chemotherapeutics including antitubulin agents such as paclitaxel. Previous studies have suggested a link between βIII‐tubulin overexpression and paclitaxel resistance through alterations in the properties of the mitotic spindle. We found that paclitaxel treatment induced temporary mitotic arrest in 7 melanoma cell lines irrespective of the βIII‐tubulin level, suggesting that βIII‐tubulin had no significant influence on spindle properties. On the other hand, the amount of BCL2, an anti‐apoptotic protein, was well correlated with paclitaxel resistance. Treatment of the paclitaxel‐resistant cell lines with ABT‐737, an inhibitor of BCL2 and BCLxL, or simultaneous knock‐down of BCL2 and BCLxL dramatically increased the cells’ sensitivity, while knock‐down of MCL1, another member of the BCL2 family, had only a minimal effect. Our results suggest that the paclitaxel sensitivity of melanoma cells is attributable to apoptosis susceptibility rather than a change in spindle properties and that BCL2 and BCLxL play a pivotal role in the former.     

The phosphotidyl inositol 3-kinase/Akt signal pathway is involved in interleukin-6-mediated Mcl-1 upregulation and anti-apoptosis activity in basal cell carcinoma cells

Dysregulation of interleukin-6 has been reported to be associated with various types of tumors, and interleukin-6 plays an important part in regulating apoptosis in many types of cells. Previously, Mcl-1 was shown to be significantly increased in interleukin-6-overexpressed basal cell carcinoma cells and conferred on them anti-apoptotic activity. The aim of this study was to investigate which signaling pathway is involved in the anti-apoptotic effect of interleukin-6 on basal cell carcinoma cells. Here we show that the addition of recombinant 100 ng per ml interleukin-6 to basal cell carcinoma cells induced a 2.3-fold increase in the level of Mcl-1 protein in basal cell carcinoma cells. Transfection with dominant-negative STAT3 (STAT3F) into inter-leukin-6-treated basal cell carcinoma cells caused a decrease of phosphotyrosyl STAT3 but did not alter Mcl-1 protein levels; however, AG490, a Janus tyrosine kinase inhibitor, was capable of inhibiting the interleukin-6-induced elevation of Mcl-1 protein. Next, interleukin-6 stimulation elicited extracellular signal-regulated kinase activation in basal cell carcinoma cells, and the mitogen-activated protein kinase inhibitor, PD98059, could affect this response without affecting the interleukin-6-medi-ated Mcl-1 upregulation. Use of the two phosphotidyl inositol 3-kinase inhibitors, LY294002 and wortmannin, to check whether this pathway is involved in Mcl-1 upregulation by interleukin-6, we found that the phosphotidyl inositol 3-kinase inhibitors completely attenuated the interleukin-6-induced Mcl-1 upregulation. Furthermore, in the interleukin-6-overexpressing basal cell carcinoma cell clone, dominant-negative Akt also significantly reduced the increased level of Mcl-1. Interestingly, Janus tyrosine kinase inhibitor, AG490, treatment strongly blocked the phosphotidyl inositol 3-kinase pathway activation, as evidenced by the decrease in phospho-Akt level. Blockage of phosphotidyl inositol 3-kinase/Akt pathway abolished the interleukin-6-mediated anti-apoptotic activity in ultraviolet B treated cells. Unexpectedly, without ultraviolet B irradiation, STAT3F transfection also induced a significant apoptosis in basal cell carcinoma/interleukin-6 cells. Taken together, our data suggest that both the phosphotidyl inositol 3-kinase/Akt and STAT3 pathways are potentially involved in interleukin-6-mediated cell survival activity in basal cell carcinoma cells; however, the upregulation of the anti-apoptotic Mcl-1 protein by interleukin-6 is mainly through the Janus tyrosine kinase/phosphotidyl inositol 3-kinase/Akt, but not the STAT3 pathway.     

Notch and NOXA-related pathways in melanoma cells

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.     

FGF-2 blocks TGF-beta1-mediated suppression of Bcl-2 in normal melanocytes

Normal melanocytes require growth support provided by the adjacent basement membrane. In contrast, nevus cells and melanoma cells survive in the dermis, and in vitro on a soft collagen gel. Transforming growth factor-beta1 (TGF-beta1) produced by melanocytes themselves induces apoptosis in normal melanocytes cultured on collagen gel, an effect that can be counteracted by fibroblast growth factor-2 (FGF-2). The purpose of this study was to investigate the mechanisms by which FGF-2 counteracts the apoptotic signals from TGF-beta1 in melanocytes cultured on collagen gel. We report that FGF-2 did not interfere with the signal transduction from the TGF-beta1 receptors to SMAD2/3 proteins. Instead, TGF-beta1 decreased the level of Bcl-2 in normal melanocytes cultured on collagen gel, and FGF-2 reversed the TGF-beta1-mediated reduction in the level of Bcl-2. In nevus and melanoma cells, TGF-beta1 was unable to induce a decrease in the level of Bcl-2, and treatment with FGF-2 did not cause an increase in the level of Bcl-2 in nevus or melanoma cells. In conclusion, our results suggest that a reduction in the level of the anti-apoptotic Bcl-2 is involved in the execution of apoptosis induced by TGF-beta1 in normal melanocytes cultured on collagen gel and that FGF-2 can prevent TGF-beta1 from causing this reduction.     

The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis

Defective cytochrome c release and the resulting loss of caspase-3 activation was recently shown to be essential for the susceptibility of human melanoma cells to CD95/Fas-induced apoptosis. Cytochrome c release from mitochondria is regulated by the relative amounts of apoptosis-promoting and apoptosis-inhibiting Bcl-2 proteins in the outer membrane of these organelles. The assignment of Bax/Bcl-2 ratios by quantitative Western blotting in 11 melanoma cell populations revealed a relation to the susceptibility to CD95-mediated apoptosis. We could show that a low Bax/Bcl-2 ratio was characteristic for resistant cells and a high Bax/Bcl-2 ratio was characteristic for sensitive cells. Low Bax expression was not a consequence of mutations in the p53 coding sequence. The Bax/Bcl-2 ratio was also in clear correlation with sensitivity to another cell death inducer, N-acetylsphingosine. Furthermore, Bcl-2 overexpression abolished apoptosis triggered by both apoptotic stimuli, confirming the critical role of the Bax/Bcl-2 ratio as a rheostat that determines the susceptibility to apoptosis in melanoma cells by regulating mitochondrial function. Interestingly, some chemotherapeutics lead to the activation of death pathways by CD95L upregulation, ceramide generation, direct activation of upstream caspases, or upregulation of proapoptotic genes. Taken together, these signals enter the apoptotic pathway upstream of mitochondria, resulting in activation of this central checkpoint. We therefore assumed that apoptosis deficiency of malignant melanoma can be circumvented by drugs directly influencing mitochondrial functions. For this purpose we used betulinic acid, a cytotoxic agent selective for melanoma, straightly perturbing mitochondrial functions. In fact, betulinic acid induced mitochondrial cytochrome c release and DNA fragmentation in both CD95-resistant and CD95-sensitive melanoma cell populations, independent of the Bax/Bcl-2 ratio.     

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.     

溶瘤腺病毒介导IL-24增强黑素瘤细胞MV3放疗敏感性的研究

目的:研究溶瘤腺病毒介导IL-24(ZD55-IL-24)对黑素瘤细胞MV3放疗敏感性的影响。方法:将对数生长的MV3细胞分为ZD55-IL-24联合放疗组、ZD55-IL-24组、放疗组、PBS对照组。应用免疫细胞化学法检测黑素瘤MV3细胞中Bax和Bcl-2凋亡相关蛋白的表达:应用Western blot法检测E1A蛋白及Bax、Bcl-2、Caspase-3凋亡相关蛋白的表达。结果:免疫细胞化学法检测联合治疗组Bax染色强度最大,Bcl-2染色强度最小。ZD55-IL-24联合放疗作用的MV3细胞高效表达E1A,较其它组Bax的表达量增加,Bcl-2的表达量降低,Caspase-3活化更明显。结论:ZD55-IL-24联合放疗有明显的协同杀伤黑素瘤细胞的作用。     

Effect of proteasome inhibitors on proliferation and apoptosis of human cutaneous melanoma-derived cell lines

BACKGROUND: Cutaneous malignant melanoma is an aggressive type of skin cancer which causes disproportionate mortality in young and middle-aged adults. Once disseminated, melanoma can be considered an incurable disease, highly resistant to standard antineoplastic treatment, such as chemotherapy or radiation therapy. The proteasome represents a novel target for cancer therapy that can potentially be used in melanoma. OBJECTIVES: To assess the effect of four structurally different proteasome inhibitors on human cutaneous melanoma-derived cell lines. METHODS: Sixteen human cutaneous melanoma-derived cell lines which are original were obtained from patients who were treated by two of the authors. Cells were cultured, exposed to proteasome inhibitors (bortezomib, ALLN, MG-132 and epoxomicin) and then assayed for cell cycle and cell death analyses. RESULTS: Proteasome inhibitors inhibited the in vitro growth of melanoma cells, and this effect was due to a reduction in cell proliferation rate and an induction of both caspase-dependent and caspase-independent cell death. Moreover, release of apoptosis-inducing factor was observed in the presence of the broad-specificity caspase inhibitor BAF (Boc-D-fmk). In addition, the four different proteasome inhibitors induced caspase 2 processing. CONCLUSIONS: This study provides information regarding the in vitro effects of proteasome inhibitors on melanoma cell lines, and the molecular mechanisms involved. It also gives support to the future use of such inhibitors in the treatment of patients with melanoma, either administered alone or in combination with other drugs.     

Hair follicle apoptosis and Bcl-2

Hair follicle (HF) morphogenesis and cycling are characterized by a tightly controlled balance of proliferation, differentiation and apoptosis. The members of the bcl-2 family of proto-oncogenes are important key players in the apoptosis control machinery of most cell types. Bcl-2, an apoptosis inhibitor, and Bax, an apoptosis promoter, show tightly regulated, hair cycle-dependent expression patterns: during catagen, the distal ORS of the HF remains strongly positive for Bcl-2 and Bax; in contrast, the proximal epithelial part of the HF loses most Bcl-2 expression while it remains strongly positive for Bax. In Bcl-2 null mice, skin becomes markedly hypopigmented during the first postnatal anagen probably due to increased melanocyte apoptosis. Reportedly, these mice also show a retardation of the first anagen development after birth. Transgenic mice overexpressing Bcl-2 under the control of the keratin-1 promoter display multifocal epidermal hyperplasia and aberrant expression of keratin-6, while alterations of HF cycling have not been investigated. Surprisingly, Bcl-2 overexpression under the control of the keratin-14 promoter leads to accelerated catagen progression and increased chemotherapy-induced apoptosis, HF dystrophy and alopecia. Transgenic mice overexpressing Bcl-X(L), another anti-apoptotic bcl-2 family member, under the control of the K14 promoter, reportedly also display accelerated catagen development. These and other Bcl-2 transgenic and null mice are now available to further dissect the as yet unclear, and likely complex, role of Bcl-2 in HF growth and pigmentation.     

Activation of mitochondrial apoptosis pathways in cutaneous squamous cell carcinoma cells by diclofenac/hyaluronic acid is related to upregulation of Bad as well as downregulation of Mcl-1 and Bcl-w

Actinic keratosis (AK) is characterized by high prevalence and the risk to proceed to squamous cell carcinoma (SCC). Cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE (2) ) synthesis has been reported in AK and SCC, and the COX inhibitor diclofenac in hyaluronic acid (diclofenac/HA) was approved for AK therapy. Its mode of action, however, remained to be unravelled. In the present study, diclofenac resulted in reduced PGE (2) levels in apoptosis-sensitive cutaneous SCC cell lines (SCL-II, SCC-12, SCC-13) whereas no PGE (2) and no COX-2 expression was detectable in a SCC cell line resistant to apoptosis induction (SCL-I). Activation of mitochondrial apoptosis pathways was evident in SCC cells owing to loss of the mitochondrial membrane potential and release of the mitochondrial factors cytochrome c and apoptosis-inducing factor. Characteristic proapoptotic changes at the level of Bcl-2 proteins occurred in sensitive cells, as upregulation of Bad and downregulation of Mcl-1 and Bcl-w. In contrast, Bad was already high, and Mcl-1 and Bcl-w were already low in resistant SCL-I, even without treatment, which may be explained by the lack of PGE (2) . An antiapoptotic downregulation of proapoptotic Bcl-2 proteins Noxa and Puma was, however, also seen in SCL-I, suggesting here pathways independent of COX-2. The regulations of Mcl-1 and Bad were also reproduced in SCC cells by the more selective COX-2 inhibitor celecoxib, thus further underlining the specific role of COX-2. The findings illuminate the mode of action of diclofenac/HA in SCC cells as well as principles of their resistance, which may allow further adaptation and improvement of the new therapy.     

Higher susceptibility to apoptosis following ultraviolet B irradiation of xeroderma pigmentosum fibroblasts is accompanied by upregulation of p53 and downregulation of Bcl-2

Apoptosis plays an important part as a defence mechanism in eliminating damaged cells. Among the complex factors which regulate apoptosis, the p53 tumour suppressor protein which is induced by DNA damage has been suggested to play a crucial part. Cells from xeroderma pigmentosum (XP) patients, which are defective in nucleotide excision repair, express higher levels of p53 and are highly susceptible to cell death after ultraviolet (UV) irradiation. To examine the relationships between DNA damage, p53 and apoptosis, normal and XP group A fibroblasts were exposed to UVB, and expressions of molecules involved in apoptosis were examined. Apoptosis of XP and normal cells was clearly detected at 48 h after irradiation with UVB at doses of 5 and 40 mJ/cm2, respectively. Cells were positive by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) staining under these exposure conditions. At 6 h after irradiation, p53 protein expression was induced in normal and XP cells at minimal doses of 10 and 2.5 mJ/cm2, respectively. Bcl-2 protein, an inhibitor of apoptosis, was downregulated prior to cell death following UVB exposure at doses that induced apoptosis in both cell types. These results suggest that DNA damage due to UVB induces apoptosis by upregulating proapoptotic molecules such as p53, and by downregulating anti-apoptotic molecules such as Bcl-2.     

Interleukin-18 prevents apoptosis via PI3K/Akt pathway in normal human keratinocytes

Interleukin-18 (IL-18) is a pleiotropic cytokine expressed in both immune and non-immune cells. In the present study, we demonstrate an anti-apoptotic role of IL-18 in normal human neonatal foreskin epidermal keratinocytes (NHEK-F). Cultured NHEK-F spontaneously produced the active form of IL-18. Treatment of NHEK-F cells with anti-IL-18 receptor alpha-chain neutralizing antibody increased apoptosis and caspase-3 activity. Exogenous IL-18 augmented phosphorylation of Akt and activation of NF-kappaB. The promotion of Akt phosphorylation by IL-18 was abolished by LY294002, a PI3K inhibitor, but not SN50, an NF-kappaB inhibitor, indicating that IL-18 functions via the PI3K/Akt pathway and independently of NF-kappaB. In addition, IL-18 was found to augment expression of anti-apoptotic proteins, Bcl-2, XIAP and glucose regulated protein78/BiP, while anti-IL-18 receptor alpha-chain neutralizing antibody suppressed expression of Bcl-2, XIAP, glucose regulated protein94 and protein disulfide isomerase. Taken together, these results indicate that IL-18 plays an important role in keratinocyte survival.     

The role of apoptosis in therapy and prophylaxis of epithelial tumours by nonsteroidal anti-inflammatory drugs (NSAIDs)

Summary In addition to having anti-inflammatory activities, nonsteroidal anti-inflammatory drugs (NSAIDs) also inhibit neoplastic cell proliferation by inducing apoptosis. Diclofenac is the anti-neoplastic compound in diclofenac 3% gel (Solaraze^TM) used for topical treatment of actinic keratosis (AK). Main target of NSAIDs seems to be the inhibition of cyclo-oxygenase-2 (COX-2), which is overexpressed in several epithelial tumours and catalyses the synthesis of prostaglandins. The precise mechanism of action of diclofenac in cutaneous cells is still unclear, but induction of apoptosis is a key effect of anti-neoplastic drugs, including NSAIDs.
In this paper we give an overview of the anti-tumoural activities of NSAIDs with emphasis on induction of apoptosis. Cyclo-oxygenase-2-mediated synthesis of prostaglandin E₂ (PGE₂) leads to activation of mitogen-activated protein kinase (MAPK), as well as phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Induction of the anti-apoptotic Bcl-2 and Mcl-1, as well as activation of the caspase-8 inhibitor cFLIP have been reported. In addition, altered lipid concentrations in the cytoplasmic membrane may modulate death receptor activities. Downregulation of both the intrinsic mitochondrial and the extrinsic pathways have been reported.
Our data demonstrate induced apoptosis and activation of the caspase cascade in three of four cutaneous squamous cell carcinoma (SCC) cell lines, after treatment with diclofenac plus hyaluronic acid and diclofenac alone; one cell line remained nonresponsive. The effects were less pronounced in normal keratinocytes and cytotoxic effects were not seen. Detailed analysis of apoptosis pathways employed by diclofenac in these cells may help to improve therapeutic strategies and to overcome possible mechanisms that are involved in nonresponsiveness.     

Synergistic inhibition of cell proliferation by combined targeting with kinase inhibitors and dietary xanthone is a promising strategy for melanoma treatment

α‐Mangostin is a dietary xanthone that displays various biological activities, and numerous reports have shown its efficacy in cancer prevention and inhibition. As most agents have been shown to be ineffective as single‐agent therapy for malignant melanoma (MM), the principle of targeted chemotherapy for MM is to use effective inhibitors and combination methods. In this study, we tested the cytotoxicity of several kinase inhibitors, including the glycogen synthase kinase (GSK)‐3 inhibitor CHIR99021, and rapamycin, in combination with a dietary xanthone, α‐mangostin, by screening from a kinase inhibitor library for melanogenesis in SK‐MEL‐2 MM cells, and verified these by clone formation efficiency, terminal dUTP nick end labelling, and expression of apoptosis‐related proteins. We also explored the molecular mechanisms for the apoptosis‐inducing effects reported. We found a marked synergistic effect of CHIR99021 or rapamycin in combination with α‐mangostin, which we verified through apoptosis‐related methods. These data provide a strong rationale for the use of α‐mangostin as an adjunct to GSK‐3 inhibitor or mammalian target of rapamycin inhibitor treatment. The intrinsic mechanism behind α‐mangostin might be inhibition of phosphatidylinositol 3‐kinase/AKT signalling and autophagy, and induction of reactive oxygen species generation.