Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.     

Ectopic expression of Hsp70 confers resistance and silencing its expression sensitizes human colon cancer cells to curcumin-induced apoptosis

We have shown earlier that heat shock renders human colon cancer cells resistant to curcumin-induced apoptosis, but the contribution of individual heat shock proteins (hsps) to this resistance has not been tested. High expression of hsp27 and hsp70 in breast, endometrial and gastric cancers has been associated with metastasis, poor prognosis and resistance to chemo- or radiotherapy. In this study, SW480 cells were transfected with hsp70 cDNA in either the sense or antisense orientation and stable clones were selected and tested for their sensitivity to curcumin. The cells were protected from curcumin-induced cell death by hsp70 while cells harboring antisense hsp70 (Ashsp70) were highly sensitive to curcumin. Curcumin-induced nuclear condensation was less in hsp70 but more in Ashsp70 cells when compared with control vector-transfected cells. Loss of mitochondrial transmembrane potential induced by curcumin was further accelerated by antisense hsp70 expression and hsp70 restored it partly. Ashsp70 cells released more cytochrome c, AIF and Smac from mitochondria upon curcumin treatment than control cells. hsp70 partly prevented the release of AIF but not the other proteins. Activation of caspases 3 and 9 induced by curcumin was also inhibited by hsp70, whereas more activation could be seen in Ashsp70 cells, although caspase 8 activation was unaffected by changes in hsp70 expression. Curcumin-induced cleavage of PARP and DFF45 was inhibited by hsp70 but enhanced in Ashsp70 cells. The present study demonstrates the potential of hsp70 in protecting SW480 cells from curcumin-induced apoptosis and highlights that silencing the expression of hsp70 is an effective approach to augment curcumin-based therapy in cancers that are resistant due to hsp70 expression.     

Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species,down-regulation of Bcl-XL and IAP,the release of cytochrome c and inhibition of Akt

Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. The mechanism by which curcumin initiates apoptosis remains poorly understood. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human renal Caki cells. Treatment of Caki cells with 50 microM curcumin resulted in the activation of caspase 3, cleavage of phospholipase C-gamma1 and DNA fragmentation. Curcumin-induced apoptosis is mediated through the activation of caspase, which is specifically inhibited by the caspase inhibitor, benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone. Curcumin causes dose-dependent apoptosis and DNA fragmentation of Caki cells, which is preceded by the sequential dephosphorylation of Akt, down-regulation of the anti-apoptotic Bcl-2, Bcl-XL and IAP proteins, release of cytochrome c and activation of caspase 3. Cyclosporin A, as well as caspase inhibitor, specifically inhibit curcumin-induced apoptosis in Caki cells. Pre-treatment with N-acetyl-cysteine, markedly prevented dephosphorylation of Akt, and cytochrome c release, and cell death, suggesting a role for reactive oxygen species in this process. The data indicate that curcumin can cause cell damage by inactivating the Akt-related cell survival pathway and release of cytochrome c, providing a new mechanism for curcumin-induced cytotoxicity.     

Bax and Bak genes are essential for maximum apoptotic response by curcumin, a polyphenolic compound and cancer chemopreventive agent derived from turmeric, Curcuma longa

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.     

Rapid induction of mitochondrial events and caspase-independent apoptosis in Survivin-targeted melanoma cells

The inhibitor of apoptosis (IAP) protein Survivin is expressed in most cancers and is a key factor in maintaining apoptosis resistance. Although several IAPs have been shown to act as direct inhibitors of caspases, the precise antiapoptotic function of Survivin remains controversial. To clarify the mechanism by which Survivin protects cells, we investigated the kinetics of apoptosis and apoptotic events following Survivin inhibition utilizing a melanoma cell line harboring a tetracycline-regulated Survivin dominant-negative mutant (Survivin-T34A). Blocking Survivin resulted in both caspase activation and apoptosis; however, the level of apoptosis was only partially reduced by caspase inhibition. Survivin blockade also resulted in mitochondrial events that preceded caspase activation, including depolarization and release of cytochrome c and Smac/DIABLO. Levels of other IAPs were not altered in Survivin-targeted cells, although modest cleavage of XIAP and Livin was observed. The earliest proapoptotic event observed in Survivin-targeted cells was nuclear translocation of mitochondrial apoptosis-inducing factor (AIF), known to trigger both apoptotic mitochondrial events and caspase-independent DNA fragmentation. These findings suggest that a key antiapoptotic function of Survivin relates to inhibition of mitochondrial and AIF-dependent apoptotic pathways, and its expression in melanoma and other cancers likely protects against both caspase-independent and -dependent apoptosis.     

Involvement of Bcl-2 family members, phosphatidylinositol 3'-kinase/AKT and mitochondrial p53 in curcumin (diferulolylmethane)-induced apoptosis in prostate cancer

Curcumin (diferulolylmethane), an active ingredient derived from the rhizome of the plant Curcuma longa, has anticancer activity in vitro and in vivo. Although curcumin possesses chemopreventive properties against several types of cancer, the molecular mechanisms by which it inhibits cell growth and induces apoptosis are not clearly understood. Our data revealed that curcumin inhibited growth and induced apoptosis in androgen-dependent and -independent prostate cancer cells, but had no effect on normal human prostate epithelial cells. Curcumin downregulated the expression of Bcl-2, and Bcl-XL and upregulated the expression of p53, Bax, Bak, PUMA, Noxa, and Bim. Curcumin upregulated the expression of p53 as well as its phosphorylation at serine 15, and acetylation in a concentration-dependent manner. Acetylation of histone H3 and H4 was increased in cells treated with curcumin, suggesting histone modification may regulate gene expression. Treatment of LNCaP cells with curcumin resulted in translocation of Bax and p53 to mitochondria, production of reactive oxygen species, drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO and Omi/HtrA2), activation of caspase-3 and induction of apoptosis. Furthermore, curcumin inhibited expression of phosphatidyl-inositol-3 kinase (PI3K) p110 and p85 subunits, and phosphorylation of Ser 473 AKT/PKB. Downregulation of AKT by inhibitors of PI3K (Wortmannin and LY294002) and AKT, or by dominant negative AKT increased curcumin-induced apoptosis, whereas transfection of constitutively active AKT attenuated this effect. Similarly, wild-type phosphatase and tensin homolog deleted from chromosome 10 (PTEN) enhanced curcumin-induced apoptosis and, in contrast, inactive PTEN (G129E and G129R) inhibited curcumin-induced apoptosis. Overexpression of constitutively active AKT inhibited curcumin-induced p53 translocation to mitochondria, and Smac release to cytoplasm, whereas inhibition of AKT by dominant negative AKT enhanced curcumin-induced p53 translocation to mitochondria and Smac release. Our study establishes a role for AKT in modulating the direct action of p53 on the caspase-dependent mitochondrial death pathway and suggests that these important biological molecules interact at the level of the mitochondria to influence curcumin sensitivity. These properties of curcumin strongly suggest that it could be used as a cancer chemopreventive agent.     

Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia.

We have evaluated the role of caspases and the mitochondrial apoptosis inducing-factor (AIF) in apoptosis induced by cladribine (2CdA), in vitro, in cells from patients of B-CLL and in peripheral blood lymphocytes from normal donors. In sensitive B-CLL cells, apoptosis was characterized by cell shrinking, loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, activation of caspases 3, 7, 8 and 9, reduction of Mcl-1 levels, translocation of AIF from mitochondria to nucleus and chromatin condensation. No significant variations in the levels of Bcl-2, Bax and Bak proteins were noticed upon treatment with 2CdA. Co-treatment of cells with the pan-caspase inhibitor Z-VAD-fmk attenuated some morphological and biochemical characteristics of apoptosis and delayed 2CdA-induced DeltaPsi(m) loss, but did not prevent cell death. Z-VAD-fmk did not prevent 2CdA-induced AIF translocation but in this case apoptotic cells displayed only peripheral chromatin condensation, characteristic of AIF action. Reduced or negligible caspase 3 expression did not prevent 2CdA toxicity in cells from four patients. Cells from three patients that responded poorly to 2CdA lacked expression of caspases 9 or 3. Cells from another patient resistant to 2CdA expressed caspases 3, 7, 8 and 9 but they were not activated by treatment. These results indicate that execution of apoptosis is carried out independently by AIF and caspases, which are responsible for the development of apoptotic phenotype in response to 2CdA. Although caspases can also collaborate in DeltaPsi(m) loss, proapoptotic proteins from the Bcl-2 superfamily may be the key inducers of DeltaPsi(m) loss and apoptosis in B-CLL cells sensitive to 2CdA.     

Justicidin A decreases the level of cytosolic Ku70 leading to apoptosis in human colorectal cancer cells

The natural product justicidin A, an arylnaphthalide lignan isolated from Justicia procumbens, significantly inhibited the growth of human colorectal cancer cells HT-29 and HCT 116 at day 6 post-treatment. Further study revealed that justicidin A-treated HT-29 and HCT 116 colorectal cancer cells died of apoptosis. Justicidin A treatment caused DNA fragmentation and an increase in phosphatidylserine exposure of the cells. The number of cells in the sub-G1 phase was also increased upon justicidin A treatment. Caspase-9 but not caspase-8 was activated, suggesting that justicidin A treatment damaged mitochondria. The mitochondrial membrane potential was altered and cytochrome c and Smac were released from mitochondria to the cytoplasm upon justicidin A treatment. The level of Ku70 in the cytoplasm was decreased, but that of Bax in mitochondria was increased by justicidin A. Since Ku70 normally binds and sequesters Bax, these results suggest that justicidin A decreases the level of Ku70 leading to translocation of Bax from the cytosol to mitochondria to induce apoptosis. Oral administration of justicidin A was shown to suppress the growth of HT-29 cells transplanted into NOD-SCID mice, suggesting chemotherapeutic potential of justicidin A on colorectal cancer cells.     

Expression and activation of apoptosis-related molecules involved in interferon-alpha-mediated apoptosis in human liver cancer cells

Interferon (IFN)-alpha directly inhibits proliferation of liver cancer cells by inducing apoptosis, but the molecular mechanisms by which IFN-alpha induces apoptosis in these cells are not fully understood. We examined the effect of broad spectrum caspase inhibitor, Z-VAD-fmk, and the caspase activation in IFN-alpha-mediated apoptosis by using 4 liver cancer cell lines that were sensitive or resistant to IFN-alpha-mediated apoptosis. Involvement of apoptosis-related mitochondrial proteins and Bcl-2 family proteins in IFN-alpha-mediated apoptosis was further examined in 1 sensitive cell line (KIM-1). The Z-VAD-fmk completely or moderately inhibited IFN-alpha-mediated apoptosis in the sensitive cells. IFN-alpha induced time-dependent activation of caspase-3 in the sensitive cells, while the resistant cells showed mild or no activation. Activation of caspase-9, caspase-8, and caspase-7, and the cleavage of poly(ADP-ribose)polymerase were identified in either or both of the sensitive cell lines, but not in the resistant cells. In KIM-1 cells, the release of cytochrome c and Smac/DIABLO from mitochondria to cytosole was confirmed. Meanwhile, Bcl-xL was upregulated, and Bid activation or translocation, or conformational changes of Bax were not identified. In conclusion, our results suggest IFN-alpha-mediated apoptosis in liver cancer cells involves the mitochondrial apoptotic pathway and is induced by activating various caspases.     

Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-x(L) in a strictly caspase-3-dependent manner in human carcinoma cells

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.     

Smac is required for cytochrome c-induced apoptosis in prostate cancer LNCaP cells.

Release of cytochrome c from mitochondria to cytosol has been identified as one of the central events of apoptosis. Direct injection of cytochrome c induces apoptosis in some but not in all cell types. We observed that LNCaP prostate cancer cells failed to undergo apoptosis induced by cytochrome c microinjections. Microinjection of cytochrome c with another mitochondrial protein, Smac, was sufficient to activate caspases, however. Smac is believed to function as a neutralizer of caspase inhibitors, and mass spectrometry analysis identified XIAP as a predominant Smac binding protein in LNCaP cells. These findings are consistent with a requirement for a release of Smac from mitochondria to enable caspase activation in prostate cells. Indeed, translocation of Smac from mitochondria to cytosol was observed in LNCaP cells that undergo apoptosis and was inhibited by epidermal growth factor, which is a survival factor for these cells. These results further emphasize the central role of mitochondria in the regulation of apoptosis in prostate cancer cells.     

Transfection of Smac sensitizes tumor cells to etoposide-induced apoptosis and eradicates established human hepatoma in vivo

A major concern in clinical treatment of cancers is resistance of tumors such as hepatocellular carcinoma (HCC) and osteosarcoma to current chemotherapy protocols. Here, we reported that overexpression of second mitochondria-derived activator of caspase (Smac) sensitized osteosarcoma cells and HCC cells in vitro to chemotherapeutic drugs-induced apoptosis. Constitutive expression of Smac resulted in enhanced Bax accumulation on mitochondria upon etoposide stimulation and inhibited Bcl-2-induced antiapoptosis activity. Thus, Smac would sensitize tumor cells to chemotherapeutic drugs in part through promoting Bax translocation to mitochondria and bypassing Bcl-2 block. Moreover, we demonstrated that blockade of Smac expression by antisense smac did not impair etoposide-induced apoptosis; however, p53-induced apoptosis was impaired in smac deficient Saos-2 cell. This suggested Smac might be required in p53-induced apoptosis. Most importantly, complete eradication of HepG2 xenografts in vivo was achieved upon combined therapy with Ad-Smac and 5-Fu. Thus, overexpression of Smac in tumor cells might be a potent strategy for cancer treatment by sensitization of tumor cells to chemotherapeutic drugs.     

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells

Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.     

Bisindolylmaleimide IX is a potent inducer of apoptosis in chronic lymphocytic leukaemic cells and activates cleavage of Mcl-1.

New agents are required for the treatment of chronic lymphocytic leukaemia (CLL). We show here that a protein kinase C inhibitor, bisindolylmaleimide IX, is a potent inducer of apoptosis in CLL cells, and investigate the mechanisms by which this is induced. Bisindolylmaleimide IX induced a conformational change and subcellular redistribution of Bax from the cytosol to the mitochondria, resulting in the release of the proapoptotic mediators cytochrome c, Smac and Omi/HtrA2 from the mitochondrial inner membrane space. This was followed by the activation of caspase-9 as the apical caspase and subsequent activation of effector caspases. CLL cells undergoing apoptosis showed a rapid caspase-mediated cleavage of Mcl-1, an antiapoptotic member of the Bcl-2 family implicated in CLL survival and poor prognosis. This cleavage was mediated primarily by caspase-3. Cleavage of Mcl-1 may provide a feed-forward amplification loop, resulting in the rapid induction of apoptosis. Bisindolylmaleimide IX or a related derivative may be of clinical use in the treatment of CLL.     

Sensitization for gamma-irradiation-induced apoptosis by second mitochondria-derived activator of caspase

Resistance to current treatment regimens, such as radiation therapy, remains a major concern in oncology and may be caused by defects in apoptosis programs. Because inhibitor of apoptosis proteins (IAPs), which are expressed at high levels in many tumors, block apoptosis at the core of the apoptotic machinery by inhibiting caspases, therapeutic modulation of IAPs could target a key control point in resistance. Here, we report for the first time that full-length or mature second mitochondria-derived activator of caspase (Smac), an inhibitor of IAPs, significantly enhanced gamma-irradiation-induced apoptosis and reduced clonogenic survival in neuroblastoma, glioblastoma, or pancreatic carcinoma cells. Notably, Smac had no effect on DNA damage/DNA repair, activation of nuclear factor-kappaB, up-regulation of p53 and p21 proteins, or cell cycle arrest following gamma-irradiation, indicating that Smac did not alter the initial damage and/or cellular stress response. Smac enhanced activation of caspase-2, caspase-3, caspase-8, and caspase-9, loss of mitochondrial membrane potential, and cytochrome c release on gamma-irradiation. Inhibition of caspases also blocked gamma-irradiation-induced mitochondrial perturbations, indicating that Smac facilitated caspase activation, which in turn triggered a mitochondrial amplification loop. Interestingly, mitochondrial perturbations were completely blocked by the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or the relatively selective caspase-2 inhibitor N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone, whereas caspase-8 or caspase-3 inhibitors only inhibited the increased drop of mitochondrial membrane potential provided by Smac, suggesting that caspase-2 was acting upstream of mitochondria after gamma-irradiation. In conclusion, our findings provide evidence that targeting IAPs (e.g., by Smac agonists) is a promising strategy to enhance radiosensitivity in human cancers.     

Apoptotic death in curcumin-treated NPC-TW 076 human nasopharyngeal carcinoma cells is mediated through the ROS, mitochondrial depolarization and caspase-3-dependent signaling responses

Curcumin, a potent candidate anticancer agent, is a dietary pigment (phenolic compound) derived from the food flavoring spice turmeric (Curcuma longa), and it has been shown to have inhibitory effects on tumor cells through anti-proliferative and proapoptotic activities. However, there is no report showing curcumin-induced apoptotic cell death in human nasopharyngeal carcinoma cells in vitro. Thus, this study was performed to elucidate whether mitochondria and caspase cascades are involved in the modulation of apoptosis and cell cycle arrest in curcumin-treated NPC-TW 076 human nasopharyngeal carcinoma cells. The effects of curcumin on cell cycle arrest and apoptosis were measured by flow cytometry, and caspase-3 activity, apoptosis-associated protein levels and its regulated molecules were studied by flow cytometric assay and immunoblots. The results indicated that curcumin-induced G2/M phase arrest was associated with a marked decrease in the protein expression of cyclin A, cyclin B and cyclin-dependent kinase 1 (Cdk1). Curcumin-induced apoptosis was accompanied with upregulation of the protein expression of Bax and downregulation of the protein levels of Bcl-2, resulting in dysfunction of mitochondria and subsequently led to cytochrome c release and sequential activation of caspase-9 and caspase-3 in NPC-TW 076 cells in a time-dependent manner. These findings revealed that mitochondria, AIF caspase-3- dependent pathways play a vital role in curcumin-induced G2/M phase arrest and apoptosis of NPC-TW 076 cells in vitro.     

A novel cyano derivative of 11-keto-β-boswellic acid causes apoptotic death by disrupting PI3K/AKT/Hsp-90 cascade, mitochondrial integrity, and other cell survival signaling events in HL-60 cells

Intervention of apoptosis is a promising strategy for discovery of novel anti‐cancer therapeutics. In this study, we examined the ability of a novel cyano derivative of 11‐keto‐β ‐ boswellic acid, that is, butyl 2‐cyano‐3,11‐dioxours‐1,12‐dien‐24‐oate (BCDD) to induce apoptosis in cancer cells. BCDD inhibited cell proliferation with 48 h IC₅₀ of 0.67 µM in HL‐60, 1 µM in Molt4, and 1.5 µM in THP1 cells. The mechanism of cell death was investigated in HL‐60 cells where it caused apoptosis by acting against several potential apoptosis suppressive targets. It inhibited phosphatidylinositol‐3‐kinase (PI3K)/AKT activity, NF‐κB, Hsp‐90, and survivin which may enhance the sensitivity of cells to apoptosis. Also, BCDD decreased the activity of Bid and Bax in cytosol, caused ΔΨ_mt loss, releasing pro‐apoptotic cytochrome c, SMAC/DIABLO leading to caspase‐9‐mediated down stream activation of caspase‐3, ICAD, and PARP1 cleavage. Translocation of apoptotis‐inducing factor (AIF) from mitochondria to the nucleus indicated some caspases‐independent apoptosis. Though it upregulated DR‐5 and caspase‐8, the caspase inhibitor yet had no effect on apoptosis as against 75% inhibition by caspase‐9 inhibitor. Attempts were made to examine any acclaimed role of AIF in the activation of caspase‐8 using siRNA where it had no effect on caspase‐8 activity while the Bax‐siRNA inhibited caspase‐3 activation suggesting predominance of intrinsic signaling. Our studies thus demonstrated that BCDD exerts multi‐focal action in cancer cells while it required 10‐fold higher the concentration to produce cytotoxicity in normal human PBMC and gingival cell line, and therefore, may find usefulness in the management of human leukemia. © 2011 Wiley Periodicals, Inc.     

Fas-mediated apoptosis in neuroblastoma requires mitochondrial activation and is inhibited by FLICE inhibitor protein and Bcl-2.

Fas-mediated apoptosis proceeds though mitochondria-dependent or -independent pathways and is deficient in drug-resistant cells. Neuroblastoma, a common pediatric malignancy, often develops drug-resistance and has a silenced caspase 8 (FLICE) gene, which has been associated with Fas- and drug-resistance. We report that besides caspase 8, which was absent in approximately one-third of 26 neuroblastoma cases in this study, other proteins such as bcl-2 and FLICE-inhibitory protein (FLIP), are equally important in conferring Fas-resistance to neuroblastoma cells. Both bcl-2 and FLIP were frequently expressed in neuroblastoma tissues. Our in vitro studies showed that FLIP was recruited to the death-inducing signaling complex and interfered with the recruitment of caspase 8 in neuroblastoma cells. bcl-2 inhibited the activation of the mitochondria; but it also lowered the free cytoplasmic levels of caspase 8 by binding and sequestering it, thus acting through a novel antiapoptotic mechanism upstream of the mitochondria. In vitro down-regulation of bcl-2 with antisense oligonucleotides allowed the release of cytochrome c from mitochondria and the activation of caspases 8 and 3 upon Fas activation as well as sensitized neuroblastoma cells to Fas-mediated apoptosis. Down-regulation of FLIP had only a modest apoptotic effect because of the coexistent mitochondrial block. However, combined treatment with bcl-2 and FLIP antisense oligonucleotides had a statistically significant synergistic effect reversing Fas-resistance in neuroblastoma cells in vitro. These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation. Thus, gene-targeting therapies for bcl-2 and FLIP may reverse Fas-resistance and prove useful in the treatment of drug-resistant neuroblastomas.     

Mcl-1 is required for Akata6 B-lymphoma cell survival and is converted to a cell death molecule by efficient caspase-mediated cleavage

Enforced expression of the antiapoptotic Bcl-2 family protein Mcl-1 promotes lymphomagenesis in the mouse; however, the functional role of Mcl-1 in human B-cell lymphoma remains unclear. We demonstrate that Mcl-1 is widely expressed in malignant B-cells, and high-level expression of Mcl-1 is required for B-lymphoma cell survival, since transfection of Mcl-1-specific antisense oligodeoxynucleotides was sufficient to promote apoptosis in Akata6 lymphoma cells. Mcl-1 was efficiently cleaved by caspases at evolutionarily conserved aspartic acid residues in vitro, and during cisplatin-induced apoptosis in B-lymphoma cell lines and spontaneous apoptosis of primary malignant B-cells. Overexpression of the Mcl-1 cleavage product that accumulated during apoptosis was sufficient to kill cells. Therefore, Mcl-1 is an essential survival molecule for B-lymphoma cells and is cleaved by caspases to a death-promoting molecule during apoptosis. In contrast to Mcl-1, Bcl-2 and Bcl-XL were relatively resistant to caspase cleavage in vitro and in intact cells. Interfering with Mcl-1 function appears to be an effective means of inducing apoptosis in Mcl-1-positive B-cell lymphoma, and the unique sensitivity of Mcl-1 to caspase-mediated cleavage suggests an attractive strategy for converting it to a proapoptotic molecule.