H1 histamine receptor antagonists induce genotoxic and caspase-2-dependent apoptosis in human melanoma cells

Previously, we found that the H1 histamine receptor antagonist diphenhydramine induces apoptosis in human acute T-lymphocytic leukemia cells. Since histamine has been shown to act as a growth factor in malignant melanoma cells, we decided to evaluate the in vitro effect of diphenhydramine and other H1 histamine receptor antagonists, such as terfenadine, astemizol and triprolidine on four malignant human melanoma cell lines. These antagonists were found to induce apoptotic cell death in all four melanoma cell lines. Apoptosis was determined by assessment of phosphatidylserine exposure on the surface of the cells and nuclear fragmentation. Importantly, H1 antagonist treatments did not adversely affect the viability of human melanocytes and murine fibroblasts at the same doses and duration of exposure. Treatment of melanoma cells with terfenadine induced DNA damage and caspases 2, 3, 6, 8 and 9 activation. Furthermore, the general caspase inhibitor (z-VAD-FMK) and a selective inhibitor of caspase-2 (z-VDVAD-FMK) protected melanoma cells from terfenadine-induced apoptosis. In contrast, the caspase-8 inhibitor (z-IETD-FMK) was ineffective. In addition, we found that mitochondria are involved in TEF-induced apoptosis, characterized by the dissipation of the mitochondrial transmembrane potential, the release of cytochrome c into the cytosolic compartment and caspase-9 activation. On the basis of these results we conclude that H1 histamine receptor antagonists induce apoptosis in human melanoma cells but not in normal melanocytes and embryonic murine fibroblasts; this apoptosis appears to be caspase-2-dependent and involves the mitochondrial pathway. The present results may contribute to the elaboration of novel therapeutic strategies for the treatment of malignant human melanoma.     

CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity

The CACNA2D2 gene, a new subunit of the Ca(2+)-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca(2+) signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca(2+) contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca(2+) were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca(2+) contents.     

Mechanism of 4-HPR-induced apoptosis in glioma cells: evidences suggesting role of mitochondrial-mediated pathway and endoplasmic reticulum stress

N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid is under clinical evaluation as a therapeutic agent in a variety of cancers. Its mechanism(s) of action involves multiple overlapping pathways that still remain unclear. In glioma cells its mechanism of action is not well elucidated. Here, we show that 4-HPR and not all-trans retinoic acid and 9-cis retinoic acid effectively induce apoptosis in glioma cells. 4-HPR-induced apoptosis is associated with hydroperoxide production and loss of mitochondrial membrane potential (Delta Psi(m)). Ultrastructural changes further indicate 4-HPR-induced mitochondrial swelling, endoplasmic reticulum (ER) dilation as well as close proximity of mitochondria and ER. As suggested by dilated ER, 4-HPR treatment increased the free cytosolic Ca(2+) as well as mitochondrial Ca(2+). Chelation of extracellular Ca(2+) by EGTA did not prevent Ca(2+) elevation, thus suggesting involvement of intracellular calcium stores in the release. Buffering of intracellular calcium by BAPTA-AM did not prevent 4-HPR-induced apoptosis; however, blocking the release of Ca(2+) from ER by heparin inhibited apoptosis, indicating the role of depletion of Ca(2+) from ER stores in apoptosis. 4-HPR treatment also resulted in an increase in Bax levels along with its translocation to mitochondria that promote mitochondrial membrane permeabilization. 4-HPR-induced apoptosis was further associated with the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol and nucleus, respectively, along with caspase-3 and caspase-7 activation. However, AIF nuclear translocation, peripheral chromatin condensation and apoptosis were not completely prevented by general caspase inhibitors, thus suggesting involvement of a caspase-dependent and caspase-independent pathway in 4-HPR-induced apoptosis. Taken together, these results suggest the role of mitochondrial-mediated pathway and ER stress as a key event in 4-HPR-induced apoptosis in glioma cells.     

Elevated levels of intracellular Ca2+ and apoptosis in human lung cancer cells given heat-shock.

The chronological changes in intracellular Ca(2+)concentrations ([Ca(2+)](i)) were analysed during heat-induced apoptosis in human lung cancer cell lines LK-2 (squamous cell carcinoma) and LU65A (large cell carcinoma). In LK-2 cells, increased [Ca(2+)](i) levels were maintained at levels between 250-350 nm 9 h after heat-shock. Treatment with BAPTA, an intracellular Ca(2+) chelator, prior to heat-shock, decreased the frequency of heat-induced apoptosis in LK-2, while thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, did not change the number of apoptotic cells, regardless of the presence or absence of Ca(2+)-supplemented medium. In LU65A cells, treatment with BAPTA or thapsigargin did not alter the apoptotic rates. Western blotting demonstrated that, although expression of Bax and Bcl-2 were not changed by heat-shock, p53 expression was elevated in LK-2, but not LU65A cells. Immunohistochemistry showed that p53 was localized predominantly in the cytoplasms of LK-2 cells, suggesting that p53 protein is not functional in LK-2. Heat-shock also elevated activities of caspase-3, -8 and -9 in both cell lines. It is concluded that a temporal increase in [Ca(2+)](i) is the important initiating factor in hyperthermia-induced apoptosis in LK-2 cells and that, in these two lung cancer cell lines, apoptosis may occur through 'cross-talk' between p53-independent mitochondrial and death receptor pathways.     

In vitro and in vivo effects of a cyclic peptide with affinity for the alpha(nu)beta3 integrin in human melanoma cells

Expression of the integrin alpha(nu)beta3 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 alpha(nu)beta3. 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 alpha(nu)beta3-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 alpha(nu)beta3 integrin may have a useful role as anti-angiogenesis agents in clinical oncology, but that they may not exert a direct effect on alphavbeta3-expressing tumour cells.     

Relationships between chemotaxis, chemotactic modulators, and cyclic nucleotide levels in tumor cells

Like many freely moving cells, Walker 256 carcinosarcoma cells respond to chemotactic stimuli. Since cyclic nucleotides are involved in the chemotaxis of other cells, we have examined the action of several nucleosides and nucleotides as chemoattractants and as modulators of tumor cell movement. We have also studied the effect of chemoattractants and prostaglandins on intracellular cyclic nucleotide levels and the effect of prostaglandins as modulators of chemotaxis. Of the agents studied, only the cyclic nucleotides and prostaglandins were found to modulate cellular motility. Neither cyclic adenosine 3':5'-monophosphate (cAMP) nor cyclic guanosine 3':5'-monophosphate (cGMP) was a chemoattractant, but cGMP and N6,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate at low concentrations (approximately 10(-10) M to 10(-8) M) enhanced chemotaxis by 80 +/- 15%, and both cAMP and N6,O2-dibutyryl cyclic adenosine 3':5'-monophosphate had an inhibitory effect at concentrations greater than 10(-6) M. Chemotaxis was suppressed by 21 to 100% in media depleted of Ca2+ and/or Mg2+, but in the presence of 10(-8) M cGMP, there was partial recovery of the chemotactic response. In response to chemotactic stimulation, there was a 28 to 60% rise in intracellular cAMP within 30 sec. This returned to basal levels within 2 min. Intracellular cGMP levels became elevated approximately 3- to 3.5-fold after this time. Incubation of cells with prostaglandins A1 and F2 alpha stimulated chemotaxis at lower concentrations (10(-7) and 10(-9) M, respectively) and resulted in elevation of cGMP, while incubation with prostaglandin E2 resulted in inhibition of chemotaxis and a rise in cAMP levels.     

Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells

Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (the statins) that inhibit the synthesis of mevalonic acid are in wide use for treatment of hypercholesterolemia. Although antitumor effects on a variety of cell types have been reported for statins, the effect of simvastatin (one of the statins) on human melanoma cell lines is not known. Here, we report antitumor effects of simvastatin on human melanoma cell lines. We treated human melanoma cell lines, A375M, G361, C8161, GAK, and MMAc with simvastatin in various concentrations for 1 to 3 days. To investigate the antitumor effect of simvastatin, we analyzed cell viability, morphologic changes, reversibility of inhibition by geranylgeranyl pyrophosphate and farnesyl pyrophosphate, apoptosis and the cell cycle. Simvastatin treatment reduced cell viability in all five melanoma cell lines. The different melanoma cell lines, however, displayed different sensitivities to simvastatin. The addition of geranylgeranyl pyrophosphate to A375M and G361 cells in the presence of simvastatin completely restored the viability of cells, but the addition of farnesyl pyrophosphate did not. DNA fragmentation assay showed that simvastatin induced apoptosis in A375M and G361 cells. Simvastatin caused a G1 arrest in G361 and MMAc cells. Consistent with the cell cycle arrest, simvastatin caused an increase in the mRNA levels of p21 and p27 on G361 and MMAc cells.We conclude that simvastatin has an antitumor effect on human melanoma cells in vitro via apoptosis and cell cycle arrest. These results suggest that simvastatin may be an effective anticancer drug for malignant melanoma.     

Molecular mechanisms of apoptosis induced by magnolol in colon and liver cancer cells.

Magnolol has been reported to have anticancer activity. In this study we found that treatment with 100 microm magnolol induced apoptosis in cultured human hepatoma (Hep G2) and colon cancer (COLO 205) cell lines but not in human untransformed gingival fibroblasts and human umbilical vein endothelial cells. Our investigation of apoptosis in Hep G2 cells showed a sequence of associated intracellular events that included (a) increased cytosolic free Ca(2+); (b) increased translocation of cytochrome c (Cyto c) from mitochondria to cytosol; (c) activation of caspase 3, caspase 8, and caspase 9; and (d) downregulation of bcl-2 protein. Pretreatment of the cells with the phospholipase C inhibitor 1-[6-[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione (U73122) or the intracellular chelator of Ca(2+) 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM) inhibited the subsequent magnolol augmentation of [Ca(2+)](i) and also the activation of caspase-8 and caspase-9, so that the occurrence of apoptosis in those cells was greatly reduced. Pretreatment of the cells with ZB4 (which disrupts the Fas response mechanism) also decreased the subsequent magnolol-induced caspase-8 activation and reduced the occurrence of apoptosis. We interpreted these findings to indicate that the above-listed sequence of intracellular events led to the apoptosis seen in Hep G2 cells and that [Ca(2+)](i), Cyto c, and Fas function as intracellular signals to coordinate those events.     

Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.     

Bax gene expression alters Ca(2+) signal transduction without affecting apoptosis in an epithelial cell line

Bax is an oncogene that has proapoptotic properties but not all cells that express Bax undergo apoptosis. Bax may have a function unrelated to apoptosis. To elucidate the role of Bax in cell signaling, an epithelial cell line called SMG-C6 was transfected with the human bax gene. Stable transfectants were studied for their response to carbachol, a muscarinic receptor agonist, by measuring the increase in intracellular free Ca(2+) and Ca(2+) influx. Carbachol-mediated release of Ca(2+) from intracellular stores was significantly higher in Bax transfectants compared to control transfectants (empty vector). Ca(2+) influx was also increased in Bax transfectants. Bax had no affect on the storage operated channels. However, the concentration of Ca(2+) in the intracellular stores (i.e., mitochondria and granules) was 40-50% lower in the Bax transfectants. There was no significant difference in thapsigargin-mediated apoptosis in Bax transfectants compared to wild-type and control transfectants. Measurement of glutathione was reduced in the Bax transfectant. Restoration of glutathione levels with glutathione monoethyl ester partially normalized Ca(2+) mobilization and storage capacity in the mitochondria to control levels. This study shows that sub-apoptotic levels of Bax can reduce Ca(2+) content in intracellular stores and Ca(2+) homeostasis. Bax may mediate these effects by reducing the levels of antioxidants resulting in mild oxidative stress.     

Vitamin D and cellular Ca2+ signaling in breast cancer

The hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) regulates a variety of signaling pathways via intracellular Ca(2+). Modulation of apoptosis is emerging as a promising strategy for treatment and prevention of cancer. Cellular Ca(2+) has been implicated in triggering of apoptosis, however, the vitamin D/Ca(2+)-dependent targets involved in apoptotic signaling have not been identified. Here, we review our studies on mechanisms of 1,25(OH)(2)D(3)-induced Ca(2+) signaling and Ca(2+)-mediated apoptosis in breast cancer cells. The results obtained demonstrate that 1,25(OH)(2)D(3) regulates Ca(2+) entry from the extracellular space, Ca(2+) mobilization from the intracellular stores and intracellular Ca(2+) buffering. In breast cancer cells, 1,25(OH)(2)D(3) induces the apoptotic Ca(2+) signal, a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) reaching elevated, but not cytotoxic levels. This increase in [Ca(2+)](i) is associated with activation of Ca(2+)-dependent μ-calpain and Ca(2+)/calpain-dependent caspase-12. Activation of these proteases appears to be sufficient for the execution of apoptosis in cancer cells. Normal mammary epithelial cells resist induction of apoptosis with 1,25(OH)(2)D(3) due to their large Ca(2+)-buffering capacity. The results indicate that the 1,25(OH)(2)D(3)-induced cellular Ca(2+) signal can act as an apoptotic initiator that directly recruits Ca(2+)-dependent apoptotic effectors capable of executing apoptosis. These findings provide a novel rationale for evaluating the role of vitamin D in prevention and treatment of breast cancer.     

Asiatic acid,a triterpene,induces apoptosis through intracellular Ca2+ release and enhanced expression of p53 in HepG2 human hepatoma cells

Asiatic acid (AA), a triterpene, decreased viability and induced apoptosis of HepG2 human hepatoma cells in a dose-dependent manner. AA also markedly increased intracellular Ca(2+) level, which was blocked by TMB-8 and dantrolene, intracellular Ca(2+) release blockers, but not by EGTA, an extracellular Ca(2+) chelator. Moreover, AA-induced apoptosis was significantly suppressed by treatment with TMB-8 and dantrolene, suggesting that intracellular Ca(2+) release may play an essential role in the AA-induced apoptosis. In addition, AA profoundly increased protein level of p53, which was also inhibited by BAPTA/AM, an intracellular Ca(2+) chelator, TMB-8 and dantrolene. Treatment with A23187, a Ca(2+) ionophore, or thapsigargin, a Ca(2+)-ATPase inhibitor, alone enhanced p53 nuclear accumulation, indicating that p53 accumulation is dependent on intracellular Ca(2+) increase. Furthermore, the viability of Hep3B, p53-null cells, was much higher than that of HepG2, p53-wild type cells, when treated with AA. Taken together, these results suggest that AA induced apoptosis through increased intracellular Ca(2+), which, in turn, enhanced p53 expression in HepG2 cells. These results further suggest that AA may be a valuable agent for the therapeutic intervention of human hepatomas.     

Molecular mechanism of black tea polyphenols induced apoptosis in human skin cancer cells: involvement of Bax translocation and mitochondria mediated death cascade

Theaflavins (TF) and thearubigins (TR) are the most exclusive polyphenols of black tea. Even though few previous reports showed the anticancer effects of TF through apoptosis, the potential effect of TR has not been appraised. This study investigated the induction of apoptosis in human skin cancer cells after treatment of TF and TR. We report that both TF and TR could exert inhibition of A431 (human epidermoid carcinoma) and A375 (human malignant melanoma) cell proliferation without adversely affecting normal human epidermal keratinocyte cells. Growth inhibition of A375 cells occurred through apoptosis, as evident from cell cycle arrest at G(0)/G(1) phase, increase in early apoptotic cells, externalization of phosphatidylserine and DNA fragmentation. In our pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether cell death is being mediated by mitochondria. In our system, Bax translocation to mitochondria persuaded depolarization of mitochondrial membrane potential, cytochrome c release in cytosol and induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apoptosis also explained that TF and TR augmented Bax:Bcl2 ratio, up-regulated the expression of p53 as well as p21 and inhibited phosphorylation of the cell survival protein Akt. Furthermore, TF and TR elicited intracellular reactive oxygen species generation in A375 cells. These observations raise speculations that TF as well as TR might exert chemopreventive effect through cell cycle arrest and induction of apoptogenic signals via mitochondrial death cascade in human skin cancer cells.     

Functional genomics of calcium channels in human melanoma cells

Ca(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca(2+) channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X(7) channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X(7), RyR2 does not function as a calcium channel. On the other hand, the P2X(7) receptor has an antiapoptotic function in melanoma cells, since ATP-activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.     

Evidence for a role of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase in thapsigargin and Bcl-2 induced changes in Xenopus laevis oocyte maturation

Thapsigargin (Tg), a selective inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), causes depletion of intracellular Ca(2+) stores, hence activation of capacitative Ca(2+) entry (CCE). Incubation of Xenopus laevis oocytes with Tg resulted in an increased rate of progesterone-induced meiotic maturation. Non-mitochondrial (45)Ca(2+) uptake by SERCA-containing microsomes prepared from control wild-type oocytes microinjected with sterile water was inhibited essentially 100% by Tg. However, overexpression of Bcl-2, an oncogene known to protect against Tg-induced apoptosis in certain cell types, resulted in only 40% inhibition of microsomal (45)Ca(2+) uptake by Tg while non-inhibited (45)Ca(2+) uptake remained unchanged. Moreover Bcl-2 overexpression also protected against inhibition of CCE. I(Cl(Ca)) was similar in Bcl-2-overexpressing and control oocytes when intracellular Ca(2+) store depletion was induced by microinjection of inositol 1,4,5-trisphosphate (InsP(3)) and other means and when CCE was induced by means independent of SERCA inhibition. Our data indicate that Bcl-2 affects neither the InsP(3) receptor nor Ca(2+) entry itself. At the end of a 24-h period after progesterone addition to the medium, only 25% of Bcl-2-overexpressing oocytes had matured compared to 85% of control oocytes. Our data suggest that SERCA participates in Xenopus oocyte maturation by controlling cytosolic Ca(2+) and/or intracellular Ca(2+) stores, hence CCE. An observed progesterone-dependent protein kinase-catalysed phosphorylation of SERCA is further indication of its role in oocyte maturation.     

Role of oxidation-triggered activation of JNK and p38 MAPK in black tea polyphenols induced apoptotic death of A375 cells

Theaflavins (TF) and thearubigins (TR) are the major polyphenols of black tea. Our previous study revealed that TF- and TR-induced apoptosis of human malignant melanoma cells (A375) is executed via a mitochondria-mediated pathway. In our present study we observed the role of the three most important MAPK (ERK, JNK, and p38) in TF- and TR-induced apoptosis. TF and TR treatment of A375 cells led to sustained activation of JNK and p38 MAPK but not ERK, suggesting that JNK and p38 are the effector molecules in this polyphenol-induced cell death. This idea was further supported by subsequent studies in which JNK and p38 activation was inhibited by specific inhibitors. Significant inhibition was found in TF- and TR-treated A375 cell death pretreated with JNK- or p38-specific inhibitors only. Further, we have found that TF and TR treatment induces a time-dependent increase in intracellular reactive oxygen species generation in A375 cells. Interestingly, treatment with the antioxidant N -acetyl cystein inhibits TF- and TR-induced JNK and p38 activation as well as induction of cell death in A375 cells. We also provide evidence demonstrating the critical role of apoptosis signal-regulating kinase 1 in TF- and TR-induced apoptosis in A375 cells. Taken together our results strongly suggest that TF and TR induce apoptotic death of A375 cells through apoptosis signal-regulating kinase 1, MAPK kinase, and the JNK–p38 cascade, which is triggered by N -acetyl cystein intracellular oxidative stress. ( Cancer Sci  2009; 100: 1971–1978)     

P73 regulates cisplatin-induced apoptosis in ovarian cancer cells via a calcium/calpain-dependent mechanism

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca(2+)](i)) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca(2+)-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca(2+)](i) increase in OV2008 cells was not effected by the elimination of extracellular Ca(2+), but this was attenuated by the depletion of internal Ca(2+) store, indicating that mobilization of intracellular Ca(2+]) stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca(2+)-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca(2+)/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.     

Mu-calpain activation in beta-lapachone-mediated apoptosis

Beta-lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Recently, our laboratory showed that beta-lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+) from endoplasmic reticulum stores, and that BAPTA-AM (an intracellular Ca(2+) chelator) blocked these early increases and partially inhibited all aspects of beta-lap-induced apoptosis. We now show that exposure of NQO1-expressing breast cancer cells to beta-lap stimulates a unique proteolytic apoptotic pathway involving mu-calpain activation. No apparent activation of m-calpain was noted. Upon activation, mu-calpain translocated to the nucleus concomitant with specific nuclear proteolytic events. Apoptotic responses in beta-lap-exposed NQO1-expressing cells were significantly delayed and survival enhanced by exogenous over-expression of calpastatin, a natural inhibitor of mu- and m-calpains. Furthermore, purified mu-calpain cleaved PARP to a unique fragment (approximately 60 kDa), not previously reported for calpains. We provide evidence that beta-lap-induced, mu-calpain-stimulated apoptosis does not involve any known apoptotic caspases; the activated fragments of caspases were not observed after beta-lap exposures, nor were there any changes in the pro-enzyme forms as measured by Western blot analyses. The ability of beta-lap to trigger an apparently novel, p53-independent, calpain-mediated apoptotic cell death further support the development of this drug for improved breast cancer therapy.