Isocostunolide, a sesquiterpene lactone, induces mitochondrial membrane depolarization and caspase-dependent apoptosis in human melanoma cells

Isocostunolide is a sesquiterpene lactone isolated from the roots of Inula helenium. Its chemical structure was determined by NMR and FAB-MS spectra. No biological activities of this compound have yet been reported. In this study, we found isocostunolide could effectively induce cytotoxicity in three cancer cell lines (A2058, HT-29, and HepG2), with an IC(50) of 3.2, 5.0, and 2.0 micro g/mL, respectively. DNA flow cytometric analysis indicated that isocostunolide actively induced apoptosis of cancer cells accompanied by a marked loss of G0/G1 phase cells. To address the mechanism of the apoptotic effect of isocostunolide, we analyzed the induction of apoptosis-related proteins in A2058. The levels of pro-caspase-8, Bid, pro-caspase-3, and poly(ADP-ribose) polymerase (PARP) decreased. However, the level of Fas was increased markedly in a dose-dependent manner. Furthermore, this compound markedly induced a depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol. The findings suggest that isocostunolide may activate a mitochondria-mediated apoptosis pathway. To address this, we found that isocostunolide-induced loss of mitochondrial membrane potential occurred via modulation of the Bcl-2 family proteins. The production of intracellular reactive oxygen species (ROS) in A2058 was not elicited. In summary, for the first time, we have isolated and characterized isocostunolide from I. helenium. This compound induces apoptosis through a mitochondria-dependent pathway in A2058 cells.     

Identification of a caspase-2 isoform that behaves as an endogenous inhibitor of the caspase cascade

Procaspase-2 is one of the aspartate-specific cysteine proteases that are activated in response to various apoptotic stimuli. Two isoforms of human procaspase-2 have been described initially. Overexpression of the long isoform (caspase-2L) promotes cell death whereas the short isoform (caspase-2S) antagonizes some apoptotic pathways. In the present study, we identified two additional CASP-2 mRNAs, designated CASP-2L-Pro and CASP-2s-Pro. The proteins encoded by these isoforms corresponded to the prodomain of procaspase-2L and -2S, in which the last alpha-helix of their caspase recruitment domains was deleted. Caspase-2L-Pro mRNA and protein were detected in a series of human tissues and cell lines. Yeast 2-hybrid assays and immunoprecipitation studies indicated that caspase-2L-Pro can interact with procaspase-2L and the adaptor protein RAIDD/CRADD, but not with FADD/MORT1 or APAF-1 adaptor proteins. The addition of recombinant caspase-2L-Pro negatively interfered with cytochrome c/dATP-mediated activation of the caspase cascade in a cell-free system. In transient expression studies of human B lymphoma Namalwa cells, overexpression of caspase-2L-Pro weakly induced apoptosis, which was prevented by a D83A/E87A double mutation. In stable selected CASP-2L-Pro-transfected Namalwa cells, overexpression of caspase-2L-Pro delayed apoptotic DNA fragmentation induced by death receptor agonists (anti-Fas antibodies, tumor necrosis factor-alpha) and DNA topoisomerase I- (camptothecin) and II- (etoposide) inhibitors, and prevented etoposide-induced activation of the caspase cascade. These inhibitory effects were not observed in stable transfected cells expressing the D83A/E87A double mutant. Altogether, these data indicated that the caspase-2L-Pro isoform functions as an endogenous apoptosis inhibitory protein that antagonizes caspase activation and cell death.     

Rottlerin sensitizes glioma cells to TRAIL-induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP

In the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, treatment with TRAIL in combination with subtoxic doses of rottlerin induced rapid apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in these cells, treatment with rottlerin efficiently recovered TRAIL-induced activation of caspases. Treatment with rottlerin significantly decreased Cdc2 activity through the downregulation of cyclin A, cyclin B, and Cdc2 proteins, whereas the sensitizing effect of rottlerin on TRAIL-induced apoptosis was independent of PKCdelta activity. Furthermore, treatment with rottlerin downregulated the protein levels of survivin and X-chromosome-linked IAP (XIAP), two major caspase inhibitors. Forced expression of Cdc2 together with cyclin B attenuated rottlerin-potentiated TRAIL-induced apoptosis by over-riding the rottlerin-mediated downregulation of survivin and XIAP protein levels. Taken together, inhibition of Cdc2 activity and the subsequent downregulation of survivin and XIAP by subtoxic doses of rottlerin contribute to amplification of caspase cascades, thereby overcoming resistance of glioma cells to TRAIL-mediated apoptosis. Since rottlerin can sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells but not human astrocytes to TRAIL-induced apoptosis, this combined treatment may offer an attractive strategy for safely treating resistant gliomas.     

Caffeic acid phenethyl ester decreases cholangiocarcinoma growth by inhibition of NF‐κB and induction of apoptosis

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF‐κB), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF‐κB DNA‐binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 μM. CAPE decreases the expression of NF‐κB1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU‐positive cells treated with CAPE at 20 μM compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz‐ChA‐1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl‐2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz‐ChA‐1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2‐fold in CAPE compared to vehicle‐treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH. © 2009 UICC     

Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2

Two hepatoma cell lines were incubated for 72 h with ATRA and its analog 13cisRA and according to MTT assay, Hep3B cells were highly susceptible whereas HepG2 cells were more resistant to the treatment. At the high concentration of 166 microM, retinoids were able to induce apoptosis in both cell lines and the highest effect was observed in HepG2 cells treated with ATRA. TUNEL-based photometric ELISA showed that at the same retinoid concentration tested by flow cytometry, both cell lines showed apoptosis whereas plasma membranes were not significantly disrupted. Inhibitors of apoptosis Bcl-xL and survivin were downregulated in Hep3B cells by treatment with both retinoids. Bax, a pro-apoptotic protein, was not significantly upregulated in Hep3B cells, but was slightly increased in HepG2 cells treated with 13cisRA. Both procaspase-3 and procaspase-8 were cleaved in Hep3B cells, suggesting apoptosis could be triggered through the extrinsic pathway. In the case of HepG2 cells, lack of caspase activation suggests a mechanism dependent on other kind of proteases.     

Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells

The aim of this study was to investigate the mechanism of flavonoid-induced apoptosis in HL-60 leukaemic cells. Thus, the effect of structurally related flavonoids on cell viability, DNA fragmentation and caspase activity was assessed. Loss of membrane potential and reactive oxygen species generation were also monitored by flow cytometry. The structurally related flavonoids, such as apigenin, quercetin, myricetin, and kaempferol were able to induce apoptosis in human leukaemia HL-60 cells. Treatment with flavonoids (60 microM) caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP). Furthermore, these flavonoids induced loss of mitochondrial transmembrane potential, elevation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of procaspase-9 processing. The potency of these flavonoids on these features of apoptosis were in the order of: apigenin > quercetin > myricetin > kaempferol in HL-60 cells treated with 60 microM flavonoids. These results suggest that flavonoid-induced apoptosis is stimulated by the release of cytochrome c to the cytosol, by procaspase-9 processing, and through a caspase-3-dependent mechanism. The induction of apoptosis by flavonoids may be attributed to their cancer chemopreventive activity. Furthermore, the potency of flavonoids for inducing apoptosis may be dependent on the numbers of hydroxyl groups in the 2-phenyl group and on the absence of the 3-hydroxyl group. This provides new information on the structure-activity relationship of flavonoids.     

Caffeic acid phenylethyl ester and MG132, two novel nonconventional chemotherapeutic agents,induce apoptosis of human leukemic cells by disrupting mitochondrial function

The ability to modulate balance between cell survival and death is recognized for its great therapeutic potential. Therefore, research continues to focus on elucidation of cell machinery and signaling pathways that control cell proliferation and apoptosis. Conventional chemotherapeutic agents often have a cytostatic effect over tumor cells. New natural or synthetic chemotherapeutic agents have a wider spectrum of interesting antitumor activities that merit in-depth studies. In the present work, we aimed at characterizing the molecular mechanism leading to induction of cell death upon treatment of the lymphoblastoid cell line PL104 with caffeic acid phenylethyl ester (CAPE), MG132 and two conventional chemotherapeutic agents, doxorubicine (DOX) and vincristine (VCR). Our results showed several apoptotic hallmarks such as phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane, nuclear fragmentation, and increase sub-G1 DNA content after all treatments. In addition, all four drugs downregulated survivin expression. CAPE and both chemotherapeutic agents reduced Bcl-2, while only CAPE and MG132 significantly increased Bax level. CAPE and VCR treatment induced the collapse of mitochondrial membrane potential (?ψm). All compounds induced cytochrome c release from mitochondrial compartment to cytosol. However, only MG132 caused the translocation of Smac/DIABLO. Except for VCR treatment, all other drugs increased reactive oxygen species (ROS) production level. All treatments induced activation of caspases 3/7, but only CAPE and MG132 led to the activation of caspase 9. In conclusion, our results indicate that CAPE and MG132 treatment of PL104 cells induced apoptosis through the mitochondrial intrinsic pathway, whereas the apoptotic mechanism induced by DOX and VCR may proceed through the extrinsic pathway.     

The p75(NTR) tumor suppressor induces caspase-mediated apoptosis in bladder tumor cells

p75(NTR) was identified as a tumor and metastasis suppressor that functions in part via induction of apoptosis in tumor cells. To examine p75(NTR)-dependent apoptosis in tumor cells, we demonstrated that a dose-dependent increase in p75(NTR) expression was associated with a concomitant increase in the mitochondrial proapoptotic effector proteins Bad, Bax and Bik and a decrease in the mitochondrial prosurvival effector proteins phospho-Bad, Bcl-2 and Bcl-x(L). Significantly, p75(NTR)-dependent induction of cytochrome c release from the mitochondria occurred during CHX potentiation of apoptosis. Furthermore, p75(NTR) expression largely suppressed expression of IAP-1 and induced cleavage of procaspase-9 and procaspase-7 but not of procaspases 2, 3, 6, 8 and 10. A specific peptide inhibitor of procaspase-9 cleavage also inhibited cleavage of procaspase-7, indicating that caspase-7 is downstream of caspase-9. As end points of apoptosis, we observed p75(NTR)-dependent annexin V binding to the plasma membrane, an indicator of early apoptotic events, and Hoechst staining of DNA nuclear fragmentation, an indicator of late apoptotic events, whereas control tumor cells that lack expression of the p75(NTR) protein did not exhibit either of these apoptotic markers. Together, these results delineate the mitochondria-mediated apoptotic pathway of the p75(NTR) tumor-suppressor gene product.     

IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis

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

5-aza-2'-deoxycytidine upregulates caspase-9 expression cooperating with p53-induced apoptosis in human lung cancer cells

Treating lung cancer cell lines using low-dose 5-aza-2'-deoxycytidine (DAC) caused an accumulation of procaspase-9 through mRNA upregulation, but the cells did not undergo apoptosis. However, when cells were treated with DAC and infected with a low dose of a recombinant wild-type p53 adenovirus vector (Ad-p53), a synergistic growth inhibitory effect was observed. Combination treatment induced Apaf-1 and procaspase-9 expression in which cytochrome c releases by Ad-p53 triggered the mitochondrial pathway of apoptosis. Selective blockage of caspase-9 activities by Z-LEHD-FMK completely attenuated DAC-induced enhancement of apoptosis mediated by Ad-p53 infection, and ectopic overexpression of procaspase-9 sensitized cells to Ad-p53-induced apoptosis in p53-null cells. In addition, DAC sensitized lung cancer cells to cisplatin and paclitaxel. Induction of the mitochondrial pathway of apoptosis using a slightly toxic dose of DAC may therefore be a strategy for treating lung cancer, and DAC treatment may have clinical implications when combined with chemotherapy or apoptosis-inducing gene therapy.     

The role of caspase family protease, caspase-3 on cisplatin-induced apoptosis in cisplatin-resistant A431 cell line

Purpose: Cisplatin (cis-diamminedichloroplatinum(II), CDDP) has been reported to induce apoptosis in cancer cells, the mechanism of the apoptosis in cancer cells induced by CDDP is still unclear. Recent studies have revealed that caspase family of cystine proteases play an important role in the regulation of several apoptotic processes. In this study, whether apoptosis induced by CDDP could be mediated by the activation of caspase-3, a caspase family protease, was investigated. Methods: The CDDP-resistant subline A431/CDDP2 from the previously established human epidermoid carcinoma cell line A431 was used. The parent A431 cells (A431/P) and the A431/CDDP2 were exposed to CDDP with or without a caspase family protease inhibitor (Z-Asp-CH₂-DCB), and cellular sensitivity to CDDP was determined. DNA fragmentation was then analyzed, and the caspase-3 protein levels determined by Western blotting following exposure of the cells to CDDP with or without Z-Asp-CH₂-DCB. Results: In the A431/P cells, the cytotoxicity of CDDP was clearly reduced by Z-Asp-CH₂-DCB compared with its cytotoxicity in A431/CDDP2 cells. Furthermore, quantitative analysis of DNA fragmentation revealed that Z-Asp-CH₂-DCB inhibited DNA fragmentation induced by CDDP in A431/P cells, but not in A431/CDDP2 cells. Western blotting analysis demonstrated a marked reduction in procaspase-3 protein levels in A431/P cells treated with Z-Asp-CH₂-DCB. In the A431/CDDP2 cells, procaspase-3 protein levels were no different with and without Z-Asp-CH₂-DCB. Conclusions: These findings suggest that caspase-3 may mediate apoptosis induced by CDDP, and its induction could represent a novel approach to the effective treatment of malignant tumors. Received: 11 November 1999 / Accepted: 20 March 2000     

Fas signaling in thyroid carcinomas is diverted from apoptosis to proliferation.

PURPOSE: The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis. Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. EXPERIMENTAL DESIGN: We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens. RESULTS: All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor kappaB (NF-kappaB) inhibitor IkappaB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-kappaB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase, NF-kappaB, and activator protein-1 pathways in thyroid carcinoma cells in vitro; and protected cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. CONCLUSIONS: In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.     

Antiproliferation and radiosensitization of caffeic acid phenethyl ester on human medulloblastoma cells

Purpose: To investigate antiproliferative and radiosensitizing effects of caffeic acid phenethyl ester (CAPE) on medulloblastoma (MB) Daoy cells. Methods and materials: Daoy cells were treated with CAPE in different concentrations and assessed for cell viability, apoptosis, cell cycles, cyclin B1 expressions, radiosensitization and chemosensitization. Human astroglia SVGp12 cells were treated with CAPE to present the possible protection or complication effects in normal tissues. Results: CAPE inhibited the growth of Daoy cells in a time- and concentration-dependent manner in MTT and Trypan blue exclusion assays. Flow cytometry revealed that CAPE significantly decreased G2/M fraction, and increased the S phase fraction. Western blot demonstrated a down-regulated cyclin B1 protein expression. Pretreatment with CAPE markedly decreased the viability of irradiated Daoy cells. The sensitizer enhancement ratios (SERs) were increased in CAPE-treated Daoy cells. CAPE in doxorubicin and cisplatin did not show chemosensitizing effect. Conclusions: These findings demonstrate the antiproliferative and radiosensitizing effects of CAPE for Daoy cells, which might bring improvement to the treatment of MB. For clinical application, in vivo models are expected.     

The role of Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis

Ectopic overexpression of Apaf-1 (2.5-fold) in human acute myelogenous leukemia HL-60 cells (HL-60/Apaf-1 cells) induced apoptosis and sensitized HL-60/Apaf-1 cells to etoposide- and paclitaxel-induced apoptosis (C. Perkins et al., Cancer Res., 58: 4561-4566, 1998). In this report, we demonstrate that in HL-60/Apaf-1 cells, the activity of caspase-9 and -3 induced by Apaf-1 overexpression was associated with a significant increase (5-fold) in the cytosolic accumulation of cytochrome c (cyt c), loss of mitochondrial membrane potential (deltapsim), and an increase in the reactive oxygen species. These were also associated with the processing of procaspase-8 and Bid (cytosolic, proapoptotic BH3 domain containing protein). Transient transfection of Apaf-1 into the Apaf-1-containing mouse embryogenic fibroblasts (MEFs; Apaf-1+/- MEFs) or Apaf-1-/- MEFs also induced the processing of procaspase-9 and procaspase-8, Bid cleavage, and apoptosis. These events were secondary to the activity of the downstream caspases induced by Apaf-1. This conclusion is supported by the observation that in HL-60/Apaf-1 cells, ectopic expression of dominant negative caspase-9, its inhibitory short isoform caspase-9b, or XIAP or treatment with the caspase inhibitor zVAD (50 microM) inhibited Apaf-1-induced caspase-8 and Bid cleavage, mitochondrial deltapsim, release of cyt c, and apoptosis. In contrast, a transient transfection of dominant negative caspase-8 or CrmA or exposure to caspase-8 inhibitor zIETD-fmk inhibited the processing of procaspase-8 and Bid but did not inhibit the cytosolic accumulation of cyt c in either the untreated HL-60/Apaf-1 cells or the etoposide-treated HL-60/Apaf-1 and HL-60/neo cells. These results indicate that Apaf-1 overexpression lowers the apoptotic threshold by activating caspase-9 and caspase-3. This triggers the mitochondrial deltapsim and cyt c release into the cytosol through a predominant mechanism other than cleavage of caspase-8 and/or Bid. This mechanism may involve a cytosolic mitochondrial permeability transition factor, which may be processed and activated by the downstream effector caspases, thereby completing an amplifying feedback loop, which triggers the mitochondrial events during apoptosis.     

The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells

Mitogen-activated protein kinase (MAPK) is activated in the majority of melanomas, and its activity is essential for cell survival. In this report, we examined the effects of a novel raf inhibitor BAY 43-9006 on melanoma cell viability and intracellular signaling and found that it induces apoptosis through a caspase-independent mechanism. At concentrations that suppress extracellular signal-regulated kinase (ERK) phosphorylation, BAY 43-9006 dephosphorylates Bad on Ser(75) and Ser(99), activates Bak and Bax, and reduces the mitochondrial transmembrane potential. BAY 43-9006 (sorafenib) down-modulates the levels of Bcl-2 and Bcl-X(L) in a MAPK-independent manner in A2058 and SKMEL5 melanoma cells but not in the more resistant A375 cells. Of the three lines tested, only A375 cells were rescued from BAY 43-9006-induced apoptosis by knocking down Bad. BAY 43-9006 induced poly(ADP-ribose) polymerase cleavage and the mitochondrial release of cytochrome c and SMAC. However, the pan-caspase inhibitor Z-VAD-fmk had only a modest protective effect against the drug, suggesting that BAY 43-9006-induced apoptosis is largely caspase independent. BAY 43-9006 but not the MAP/ERK kinase inhibitors PD98059 or U0126 induced the nuclear translocation of apoptosis-inducing factor (AIF) in A2058 and SKMEL5 cells, and the introduction of a small interfering RNA (siRNA) for AIF partially protected these cells from BAY 43-9006-induced apoptosis. The AIF siRNA had little effect in A375 cells, in which drug-induced AIF release was negligible. These data indicate that in sensitive cell lines, BAY 43-9006-induced apoptosis is independent of Bad dephosphorylation and caspase activation and largely mediated through the nuclear translocation of AIF.