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Helicobacter pylori infection of the human stomach causes chronic gastritis that can lead to gastric cancer. Because activated lymphocytes persist in the gastric mucosa, and because a high multiplicity of infection (MOI) of H. pylori is needed to induce apoptosis in vitro, we speculated that resistance of lymphocytes to apoptosis is an important feature of the immune response to H. pylori. Freshly isolated mouse splenocytes underwent substantial spontaneous apoptosis and displayed a biphasic response to H. pylori, in which low MOI (1-10) markedly inhibited apoptosis, whereas high MOI (> or =75) potentiated apoptosis. Low MOI reduced mitochondrial membrane depolarization, caspase-3 and caspase-9 activation, and cytochrome c release and increased Bcl-2 levels. Low MOI also induced cellular proliferation. When cells were subjected to fluorescence-activated cell sorting after coculture with H. pylori, CD19+ B cells were found to be protected from apoptosis and undergoing proliferation at low MOI, whereas CD3+ T cells did not exhibit this pattern. The protective effect of low MOI on apoptosis persisted even when B cells were isolated before activation. Immunophenotyping showed that all B-cell subsets examined were protected from apoptosis at low MOI. Additionally, gastric infection with H. pylori resulted in protection of splenic B cells from spontaneous apoptosis. Our results suggest that the low levels of H. pylori infection that occur in vivo are associated with B-cell survival and proliferation, consistent with their potential to evolve into mucosa-associated lymphoid tissue lymphoma.

2-Methoxyestradiol is a physiologic metabolite of 17beta-estradiol. This orally active compound can inhibit tumor growth or metastasis in tumor models without inducing any clinical sign of toxicity. Our previous studies indicated that 2-methoxyestradiol-mediated apoptosis involves the disappearance of intact 21-kDa Bid protein, cytochrome c release, and predominant procaspase-3 cleavage. Here, using MIA PaCa-2 cells as a model, we investigated whether this estrogen metabolite induces apoptosis by converging two major pathways: the death receptor-mediated extrinsic and the mitochondrial intrinsic pathway. Exogenous expression of dominant-negative caspase-8 or dominant-negative FADD reverts the effect of 2-methoxyestradiol-mediated cell death. In parallel with this observation, Z-IETD-FMK, a cell permeable irreversible inhibitor of caspase-8, can render significant protection against 2-methoxyestradiol-induced apoptosis. RNase protection assay and cell surface receptor analysis by flow cytometry show the up-regulation of members of death receptor family in 2-methoxyestradiol-exposed pancreatic cancer cells. Our mechanistic studies also implicate that oxidative stress precedes 2-methoxyestradiol-mediated c-Jun NH2-terminal kinase activation, leading to elevated Fas level. Because 2-methoxyestradiol is able to trigger death receptor signaling, we were interested in examining the effects of 2-methoxyestradiol and Fas ligand (FasL)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) together on pancreatic cancer cell death. Interestingly, the endogenous angiogenesis inhibitor 2-methoxyestradiol augments FasL/TRAIL-induced apoptosis in these cells. Moreover, the combination of 2-methoxyestradiol and TRAIL reduces the tumor burden in vivo in MIA PaCa-2 tumor xenograft model by caspase-3 activation.

Evidence suggests that an inflammatory profile of cytokines and chemokines persisting at a particular site would lead to the development of a chronic disease. Recent studies implicate bacterial infection as one possible link between inflammation and carcinogenesis; however, the crucial molecular pathways involved remain unknown. We hypothesized that one possible upstream signaling pathway leading to inflammation in carcinogenesis may be mediated by Toll-like receptors (TLR). We describe for the first time an adaptive mechanism acquired by ovarian cancer cells that allows them to promote a proinflammatory environment and develop chemoresistance. We propose that the TLR-4-MyD88 signaling pathway may be a risk factor for developing cancer and may represent a novel target for the development of biomodulators. Our work explains how bacterial products, such as lipopolysaccharide, can promote, directly from the tumor, the production of proinflammatory cytokines and the enhancement of tumor survival. In addition, we provide new evidence that links TLR-4 signaling, inflammation, and chemoresistance in ovarian cancer cells.

4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a recently identified metabolite of fenretinide (4-HPR). We explored the effectiveness of 4-oxo-4-HPR in inducing cell growth inhibition in ovarian, breast, and neuroblastoma tumor cell lines; moreover, we investigated the molecular events mediating this effect in two ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/HPR) to 4-HPR. 4-oxo-4-HPR was two to four times more effective than 4-HPR in most cell lines, was effective in both 4-HPR-sensitive and 4-HPR-resistant cells, and, in combination with 4-HPR, caused a synergistic effect. The tumor growth-inhibitory effects of 4-oxo-4-HPR seem to be independent of nuclear retinoid receptors (RAR), as indicated by the failure of RAR antagonists to inhibit its effects and by its poor ability to bind and transactivate RARs. Unlike 4-HPR, which only slightly affected the G(1) phase of the cell cycle, 4-oxo-4-HPR caused a marked accumulation of cells in G(2)-M. This effect was associated with a reduction in the expression of regulatory proteins of G(2)-M (cyclin-dependent kinase 1 and cdc25c) and S (cyclin A) phases, and with an increase in the expression of apoptosis-related proteins, such as p53 and p21. Apoptosis was induced by 4-oxo-4-HPR in both 4-HPR-sensitive and 4-HPR-resistant cells and involved activation of caspase-3 and caspase-9 but not caspase-8. We also showed that 4-oxo-4-HPR, similarly to 4-HPR, increased reactive oxygen species generation and ceramide levels by de novo synthesis. In conclusion, 4-oxo-4-HPR is an effective 4-HPR metabolite that might act as therapeutic agent per se and, when combined with 4-HPR, might improve 4-HPR activity or overcome 4-HPR resistance.

Apoptotic signaling defects both promote tumorigenesis and confound chemotherapy. Typically, chemotherapeutics stimulate cytochrome c release to the cytoplasm, thereby activating the apoptosome. Although cancer cells can be refractory to cytochrome c release, many malignant cells also exhibit defects in cytochrome c-induced apoptosome activation, further promoting chemotherapeutic resistance. We have found that breast cancer cells display an unusual sensitivity to cytochrome c-induced apoptosis when compared with their normal counterparts. This sensitivity, not observed in other cancers, resulted from enhanced recruitment of caspase-9 to the Apaf-1 caspase recruitment domain. Augmented caspase activation was mediated by PHAPI, which is overexpressed in breast cancers. Furthermore, cytochrome c microinjection into mammary epithelial cells preferentially killed malignant cells, suggesting that this phenomenon might be exploited for chemotherapeutic purposes.

Apoptosis is implemented by death machinery that involves the caspase family of proteins, under the condition of a variety of stresses. Previous studies have shown that mouse embryonic stem cells deficient for caspase-3 are resistant to UV-induced apoptosis and that the active form of caspase-3 translocates to the nucleus. It has also been shown that the breast cancer tumor suppressor, BRCA1, is phosphorylated on Ser1423 and Ser1524 by the ataxia telangiectasia mutated-related kinase (ATR) after UV damage. Here, we show that activation of caspase-3 by UV is abrogated in BRCA1-mutated SNU251 and HCC1937 cells but was restored by reexpressing wild-type (wt) BRCA1, but not phosphorylation-deficient BRCA1Ser1423Ala/Ser1524Ala (BRCA1(S1423/1524A)). In SNU251 cells expressing BRCA1(S1423/1524A), the inhibitory interaction between X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 remains intact after UV, compared with cells expressing wt BRCA1. We determined that XIAP and BRCA1 interact and upon phosphorylation this complex is disrupted. Nuclear translocation of active caspase-3 is detected only when wt BRCA1 is reexpressed. Consistent with this, Rad21, a known substrate of caspase-3, is cleaved only when wt BRCA1 is expressed in vivo. These results propose a model that inhibition of BRCA1 phosphorylation leads to the abrogation of a specific form of apoptosis that is mediated by caspase-3.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA-mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16-induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.

Caspase-1 plays a key role in the processing of cytokines and in the apoptosis of neurons and macrophages. Whether it also causes apoptosis of cancer cells has been unclear. In this study, we screened an array of apoptosis-related proteins in ovarian carcinoma cell lines and their tissue of origin, ovarian surface epithelium (OSE). Caspase-1alpha protein was abundant in OSE and in nontumorigenic OSE with extended but limited life spans (immortalized OSE), but was reduced in the cancer lines A2780 and OVCAR10. By Western blot and immunofluorescence, caspase-1alpha levels were greatly reduced in six of eight ovarian carcinoma lines compared with OSE. By real-time reverse transcription-PCR, steady-state transcripts of the CASP1 gene were proportional to protein levels. Caspase-1alpha overexpression caused significant apoptosis, but overexpression of a caspase-1alpha mutant without catalytic activity did not, confirming that the effect was caspase-1alpha-specific. Immunofluorescence of caspase-1alpha and terminal nucleotidyl transferase-mediated dUTP-X nick end labeling colocalization clearly established a link between apoptosis and caspase-1alpha expression. Caspase-9 and caspase-3 were activated in caspase-1alpha overexpressing A2780 cells, suggesting involvement of an intrinsic apoptotic pathway. Caspase-1alpha overexpression did not change the apoptotic effect of cisplatin in A2780 and OVCAR10 cells, suggesting that this agent activates a different pathway. Immunohistochemically, caspase-1 was lower in ovarian serous carcinomas than in OSE. Our study indicates, for the first time, that caspase-1alpha is proapoptotic in ovarian cancer cells, and raises the possibility that its down-regulation is one of the mechanisms which increase resistance to apoptosis in cancer cells.

Retinoic acid (RA) displays pronounced anticarcinogenic activities in several types of cancer. Whereas the mechanisms that underlie this activity remain incompletely understood, tumor suppression by RA is believed to emanate primarily from its ability to regulate transcription of multiple target genes. Here, we investigated molecular events through which RA inhibits the growth of MCF-7 mammary carcinoma cells, focusing on the involvement of the two proteins that mediate transcriptional activation by RA, the nuclear hormone receptor retinoic acid receptor (RAR) and the cellular retinoic acid-binding protein (CRABP) II, in this process. RA treatment of MCF-7 cells did not affect cell cycle distribution but triggered pronounced apoptosis. Accordingly, expression array analyses revealed that RA induces the expression of several proapoptotic genes, including caspase 7 and caspase 9. Whereas caspase 7 is an indirect responder to RA signaling, caspase 9 is a novel direct target for RAR, and it harbors a functional retinoic acid response element in its second intron. In agreement with the known role of CRABP-II in enhancing the transcriptional activity of RAR, the binding protein augmented RA-induced up-regulation of caspase 9, cooperated with RA in activating both caspase 7 and 9, and amplified the ability of RA to trigger apoptosis. Surprisingly, the data indicate that CRABP-II also displays proapoptotic activities on its own. Specifically, overexpression of CRABP-II, in the absence of RA, up-regulated the expression of Apaf1 and triggered caspase 7 and caspase 9 cleavage. These observations suggest that, in addition to its known role in direct delivery of RA to RAR, CRABP-II may have an additional, RA-independent, function.

The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides -1,194 and -1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl-transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl-transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.

We are currently conducting clinical trials of E1A gene therapy for patients with ovarian cancer. The adenovirus type 5 E1A gene suppresses growth of ovarian cancer cells that overexpress HER-2/neu (HER2) and growth of some--but not all--that express low HER2. In HER2-overexpressing cells, suppression by E1A is predominantly by down-regulation of HER2, but the mechanism in low HER2-expressing cells is not fully understood. The adenoviral E1B protein has sequential and functional homology to Bcl-2 and prolongs the viability of adenovirus host cells by inhibiting E1A-induced apoptosis. Bcl-2 is overexpressed in ovarian cancer and participates in chemoresistance; we hypothesized that Bcl-2 inhibits E1A-induced apoptosis leading to resistance to E1A gene therapy. E1A suppressed colony formation of ovarian cancer cells that express low levels of Bcl-2 and HER2 (OVCAR-3 and OVCA 433), but enhanced colony formation in low HER2-, high Bcl-2-expressing ovarian cancer cells (2774 and HEY). Treating 2774 or HEY cells with antisense oligonucleotide Bcl-2 (Bcl-2-ASO) did not reduce cell viability. E1A combined with Bcl-2-ASO led to significant decreases in cell viability resulting from increased apoptosis relative to cells treated with E1A alone (P < 0.05). The increase in apoptosis was partly due to cytochrome c release and subsequently caspase-9 activation by Bcl-2-ASO. Finally, in an ovarian cancer xenograft model, treatment with Bcl-2-ASO did not prolong survival, but E1A plus Bcl-2-ASO did (P < 0.001). In conclusion, ovarian tumors overexpressing Bcl-2 may not respond well to E1A gene therapy, but treatment with a combination of E1A and Bcl-2-ASO may overcome this resistance.

The antiapoptotic molecule galectin-3 was previously shown to regulate CD95, a member of the tumor necrosis factor (TNF) family of proteins in the apoptotic signaling pathway. Here, we question the generality of the phenomenon by studying a different member of this family of proteins [e.g., TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in a wide variety of cancer cells]. Overexpression of galectin-3 in J82 human bladder carcinoma cells rendered them resistant to TRAIL-induced apoptosis, whereas phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY-294002) blocked the galectin-3 protecting effect. Because Akt is a major downstream PI3K target reported to play a role in TRAIL-induced apoptosis, we questioned the possible relationship between galectin-3 and Akt. Parental J82 and the control vector-transfected J82 cells (barely detectable galectin-3) exhibit low level of constitutively active Akt, resulting in sensitivity to TRAIL. On the other hand, J82 cells overexpressing galectin-3 cells expressed a high level of constitutively active Akt and were resistant to TRAIL. Moreover, the blockage of TRAIL-induced apoptosis in J82 cells seemed to be mediated by Akt through the inhibition of BID cleavage. These results suggest that galectin-3 involves Akt as a modulator molecule in protecting bladder carcinoma cells from TRAIL-induced apoptosis.

Toward the goal of developing effective treatments for prostate cancers, we examined the effects of cyclin-dependent kinase inhibitors on the survival of prostate cancer cells. We show that roscovitine, R-roscovitine, and CGP74514A (collectively referred to as CKIs) induce the apoptosis of LNCaP and LNCaP-Rf cells, both of which express wild-type p53. Apoptosis required caspase-9 and caspase-3 activity, and cytochrome c accumulated in the cytosol of CKI-treated cells. Amounts of p53 increased substantially in CKI-treated cells, whereas amounts of the endogenous caspase inhibitor XIAP decreased. CKIs did not appreciably induce the apoptosis of LNCaP cells treated with pifithrin-alpha, which prevents p53 accumulation, or of prostate cancer cells that lack p53 function (PC3 and DU145). Ectopic expression of p53 in PC3 cells for 44 hours did not reduce XIAP abundance or induce apoptosis. However, p53-expressing PC3 cells readily apoptosed when exposed to CKIs or when depleted of XIAP by RNA interference. These findings show that CKIs induce the mitochondria-mediated apoptosis of prostate cancer cells by a dual mechanism: p53 accumulation and XIAP depletion. They suggest that these events in combination may prove useful in the treatment of advanced prostate cancers.

Nuclear factor-kappaB (NF-kappaB) plays an important role during liver neoplastic development through transcriptional regulation of prosurvival genes, which then counteract the death-inducing signals elicited by the host immune response. The c-Myc proto-oncogene is frequently deregulated in liver tumors. Furthermore, enforced expression of c-Myc in the liver promotes the development of hepatocellular carcinomas, a process that is accelerated by coexpression with transforming growth factor-alpha (TGF-alpha). TGF-alpha/c-Myc-derived hepatocellular carcinomas display reduced apoptotic levels compared with those of single c-Myc transgenic hepatocellular carcinomas, suggesting that TGF-alpha provides a survival advantage to c-Myc-transformed hepatocytes. Given that TGF-alpha/c-Myc hepatocellular carcinomas display constitutive NF-kappaB activity, here, we have tested the hypothesis that enforced expression of TGF-alpha results in constitutive NF-kappaB activation and enhanced cell survival using TGF-alpha/c-Myc-derived hepatocellular carcinoma cell lines. We show that TGF-alpha induces NF-kappaB through the phosphatidylinositol 3-kinase/Akt axis in these bitransgenic hepatocellular carcinomas. Furthermore, we found that adenovirus-mediated inhibition of NF-kappaB activity impairs the ability of TGF-alpha/c-Myc-derived tumor cells to grow in an anchorage-independent fashion due to sensitization to c-Myc-induced apoptosis. Lastly, we show that NF-kappaB inhibits c-Myc-induced activation of caspase-9 and caspase-3 through up-regulation of the antiapoptotic target genes Bcl-X(L) and X-linked inhibitor of apoptosis (XIAP). Overall, these results underscore a crucial role of NF-kappaB in disabling apoptotic pathways initiated by oncogenic transformation.

Consumption of the traditional kava preparation was reported to correlate with low and uncustomary gender ratios (more cancer in women than men) of cancer incidences in three kava-drinking countries: Fiji, Vanuatu, and Western Samoa. We have identified flavokawain A, B, and C but not the major kavalactone, kawain, in kava extracts as causing strong antiproliferative and apoptotic effect in human bladder cancer cells. Flavokawain A results in a significant loss of mitochondrial membrane potential and release of cytochrome c into the cytosol in an invasive bladder cancer cell line T24. These effects of flavokawain A are accompanied by a time-dependent decrease in Bcl-x(L), a decrease in the association of Bcl-x(L) to Bax, and an increase in the active form of Bax protein. Using the primary mouse embryo fibroblasts Bax knockout and wild-type cells as well as a Bax inhibitor peptide derived from the Bax-binding domain of Ku70, we showed that Bax protein was, at least in part, required for the apoptotic effect of flavokawain A. In addition, flavokawain A down-regulates the expression of X-linked inhibitor of apoptosis and survivin. Because both X-linked inhibitor of apoptosis and survivin are main factors for apoptosis resistance and are overexpressed in bladder tumors, our data suggest that flavokawain A may have a dual efficacy in induction of apoptosis preferentially in bladder tumors. Finally, the anticarcinogenic effect of flavokawain A was evident in its inhibitory growth of bladder tumor cells in a nude mice model (57% of inhibition) and in soft agar.

CD8(+) cells expressing high numbers of TCR per cell (TCR(hi)) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CD8(+) cells expressing high levels of E75-TCR (TCR(hi)) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-gamma(+) Perf(-) and to TCR(hi) IFN-gamma(+) Perf(+) cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCR(hi) Perf(hi) cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCR(hi) Perf(+) CTL required an agonist of optimal CH(2) side chain length, which in this study was equal to two CH(2) groups appended to E75 at the Gly(4) position. Side chains one CH(2) shorter or longer than optimal were either less stimulatory or induced death of TCR(hi) Perf(+) cells. Differentiation of TCR(hi) CD8(+) cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.

Although a number of p53 target genes have been identified, the mechanisms of p53-dependent activities that determine cellular survival or death are still not fully understood. Here we report isolation of a novel p53 target gene, designated p53-inducible cell-survival factor (p53CSV). p53CSV contains a p53-binding site within its second exon and the reduction of expression by small interfering RNA enhanced apoptosis, whereas overexpression protected cells from apoptosis caused by DNA damage. p53CSV is induced significantly when cells have a low level of genotoxic stresses, but not when DNA damage is severe. p53CSV can modulate apoptotic pathways through interaction with Hsp70 that probably inhibits activity of apoptosis protease activating factor-1. Our results imply that under specific conditions of stress, p53 regulates transcription of p53CSV and that p53CSV is one of the important players in the p53-mediated cell survival.

Cancer cells frequently possess defects in the genetic and biochemical pathways of apoptosis. Members of the Bcl-2 family play pivotal roles in regulating apoptosis and possess at least one of four Bcl-2 homology (BH) domains, designated BH1 to BH4. The BH3 domain is the only one conserved in proapoptotic BH3-only proteins and plays an important role in protein-protein interactions in apoptosis by regulating homodimerization and heterodimerization of the Bcl-2 family members. To date, 10 BH3-only proapoptotic proteins have been identified and characterized in the human genome. The completion of the Human Genome Project and the availability of various public databases and sequence analysis algorithms allowed us to use the bioinformatic database-mining approach to identify one novel BH3-only protein, apolipoprotein L6 (ApoL6). The full-length cDNA of ApoL6 was identified, cloned, and functionally expressed in p53-null colorectal cancer cells (DLD-1). We found that overexpression of wild-type ApoL6 induced mitochondria-mediated apoptosis in DLD-1 cells characterized by release of cytochrome c and Smac/DIABLO from mitochondria and activation of caspase-9, whereas ApoL6 BH3 domain deletion allele did not. In addition, overexpression of ApoL6 also induced activation of caspase-8. Furthermore, we showed that adenovirus harboring the full-length cDNA of ApoL6 induced marked apoptosis in a variety of cancer cell types, and ApoL6 recruited and interacted with lipid/fatty acid components during the induction of apoptosis. To our knowledge, this is the first example that intracellular overproduction of an apolipoprotein induces marked apoptosis.

Insulin significantly reduced tumor necrosis factor (TNF)-alpha-induced cleavage of procaspase-8, -9, and -3 and poly(ADP-ribose) polymerase when observed for up to 24 hours in a dose-dependent manner. Signaling pathways responsible for the inhibitory effects of insulin were investigated by using protein kinase inhibitors. Both phosphatidylinositol 3'-kinase (PI3K) and mitogen-activated protein kinase kinase pathways mediate the ability of insulin to decrease the TNF-alpha-induced cleavage of procaspase-8. In contrast, only the PI3K inhibitor reversed the effect of insulin on the TNF-alpha-induced cleavage of procaspase-9. Moreover, insulin decreased the apoptotic level induced by TNF-alpha, whereas the PI3K inhibitor enhanced it. The protein level of Apaf-1, an activator of procaspase-9, remained constant with the application of agents affecting the cleavage of procaspase-9. In examining another regulator of cleaved caspase-9, X chromosome-linked inhibitor of apoptosis protein (XIAP), we observed that TNF-alpha treatment induced fragmentation of XIAP, which was also enhanced by the PI3K inhibitor. In addition, XIAP was coimmunoprecipitated with procaspase-9. The treatment with TNF-alpha reduced the level of XIAP precipitated with procaspase-9, whereas insulin reversed this effect. Moreover, PI3K and Akt inhibitors, but not mammalian target of rapamycin inhibitor, inhibited the effect of insulin on the coprecipitation of procaspase-9 and XIAP. Our data suggest that insulin decreases the TNF-alpha-induced cleavage of procaspase-9 and subsequent apoptosis by regulating XIAP via the PI3K/Akt pathway.

2-(8-Hydroxy-6-methoxy-1-oxo-1Eta-2-benzopyran-3-yl)propionic acid (NM-3) is a small molecule isocoumarin derivative that has recently entered clinical trials as an orally bioavailable anticancer agent. NM-3 induces lethality of human carcinoma cells by both apoptotic and nonapoptotic mechanisms and potentiates the effects of cytotoxic chemotherapeutic agents. The present studies have evaluated the effects of NM-3 on human multiple myeloma (MM) cells. The results demonstrate that NM-3 potentiates dexamethasone-induced killing of both dexamethasone-sensitive MM1.S and dexamethasone-resistant RPMI8226 and U266 MM cells. We show that NM-3 enhances dexamethasone-induced release of the mitochondrial apoptogenic factors cytochrome c and Smac/DIABLO. The results also demonstrate that NM-3 enhances dexamethasone-induced activation of the intrinsic caspase-9->caspase-3 apoptotic pathway. In concert with these results, NM-3 potentiates dexamethasone-induced apoptosis of MM1.S cells. Moreover, NM-3 acts synergistically with dexamethasone in inducing apoptosis of the dexamethasone-resistant RPMI8226 and U266 MM cells. These findings indicate that NM-3 may be effective in combination with dexamethasone in the treatment of MM.

Many molecules are inducibly localized in lipid rafts, and their alteration inhibits early activation events, supporting a critical role for these domains in signaling. Using confocal microscopy and cellular fractionation, we have shown that the pool of Bad, attached to lipid rafts in proliferating cells, is released when cells undergo apoptosis. Kinetic studies indicate that rafts alteration is a consequence of an intracellular signal triggered by interleukin-4 deprivation. Growth factor deprivation in turn induces PP1alpha phosphatase activation, responsible for cytoplasmic Bad dephosphorylation as well as caspase-9 and caspase-3 activation. Caspases translocate to rafts and induce their modification followed by translocation of Bad from rafts to mitochondria, which correlates with apoptosis. Taken together, our results suggest that alteration of lipid rafts is an early event in the apoptotic cascade indirectly induced by interleukin-4 deprivation via PP1alpha activation, dephosphorylation of cytoplasmic Bad, and caspase activation.

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1-dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1(-) cells) nor up-regulated (in APAF-1(+) cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1(+) and APAF-1(-) tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 -specific inhibitors in both APAF-1(+) and APAF-1(-) tumor cells. In response to 1 to 100 micromol/L of cisplatin, camptothecin, or celecoxib, APAF-1(+) melanomas (n = 12) did not show significantly increased levels of apoptosis compared with APAF-1(-) tumors (n = 7), with the exception of enhanced apoptosis in response to a very high dose (100 micromol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.

We have shown previously that ionizing radiation (IR) induces a persistent G(2)-M arrest but not cell death in MCF-7 breast carcinoma cells that harbor functional p53 but lack caspase-3. In the present study, we investigated the mechanisms of apoptosis resistance and the roles of p53, caspase-3, and cell cycle arrest in IR-induced apoptosis. The methylxanthine caffeine and the staurosporine analog UCN-01, which can inhibit ATM and Chk kinases, efficiently abrogated the IR-induced G(2)-M arrest and induced mitochondrial activation as judged by the loss of the mitochondrial membrane potential and the release of cytochrome c and Smac/Diablo. However, despite these proapoptotic alterations, cell death and activation of the initiator caspase-9 were not induced in MCF-7 cells but were interestingly only observed after reexpression of caspase-3. Sensitization to IR-induced apoptosis by caffeine or UCN-01 was abrogated neither by cycloheximide nor by pifithrin-alpha, an inhibitor of the transcriptional activity of p53. Furthermore, suppression of p53 by RNA interference could not prevent caffeine- and IR-induced mitochondrial alterations and apoptosis but resulted in an even more pronounced G(2)-M arrest. Collectively, our results clearly show that the resistance of MCF-7 cells to IR-induced apoptosis is caused by two independent events; one of them is a caffeine- or UCN-01-inhibitable event that does not depend on p53 or a release of the G(2)-M arrest. The second event is the loss of caspase-3 that surprisingly seems essential for a fully functional caspase-9 pathway, even despite the previous release of mitochondrial proapoptotic proteins.

E7389, a macrocyclic ketone analog of the marine natural product halichondrin B, currently is undergoing clinical trials for cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via tubulin-based antimitotic mechanisms, which are similar or identical to those of parental halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G2-M phase of the cell cycle, beginning as early as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on acridine orange/ethidium bromide staining, and cell surface annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria, proteolytic activation of caspase-3 and -9, and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). In LNCaP human prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

Although a recent study (Zhang et al. Cell Death Differ 2002; 9; 790-800) presented that acetylcholinesterase (AChE) might be an important common component in leading to various types of apoptosis, the molecular mechanism, by which AChE functions, had remained elusive before that study. We explored the role of AChE in apoptosis by silencing the AChE gene. Silencing of the AChE gene abolished the expression of AChE and prevented caspase-9 activation, decrease of cell viability, nuclear condensation and poly(adenosine diphosphate-ribose) polymerase cleavage but not mitochondrial events. Importantly, silencing of the AChE gene blocked the interaction between apoptotic protease-activating factor-1 and cytochrome c. Here we propose that AChE plays a pivotal role in the formation of apoptosome.

Present studies demonstrate that treatment with the histone deacetylases inhibitor LAQ824, a cinnamic acid hydroxamate, increased the acetylation of histones H3 and H4, as well as induced p21(WAF1) in the human T-cell acute leukemia Jurkat, B lymphoblast SKW 6.4, and acute myelogenous leukemia HL-60 cells. This was associated with increased accumulation of the cells in the G(1) phase of the cell cycle, as well as accompanied by the processing and activity of caspase-9 and -3, and apoptosis. Exposure to LAQ824 increased the mRNA and protein expressions of the death receptors DR5 and/or DR4, but reduced the mRNA and protein levels of cellular FLICE-inhibitory protein (c-FLIP). As compared with treatment with Apo-2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or LAQ824 alone, pretreatment with LAQ824 increased the assembly of Fas-associated death domain and caspase-8, but not of c-FLIP, into the Apo-2L/TRAIL-induced death-inducing signaling complex. This increased the processing of caspase-8 and Bcl-2 interacting domain (BID), augmented cytosolic accumulation of the prodeath molecules cytochrome-c, Smac and Omi, as well as led to increased activity of caspase-3 and apoptosis. Treatment with LAQ824 also down-regulated the levels of Bcl-2, Bcl-x(L), XIAP, and survivin. Partial inhibition of apoptosis due to LAQ824 or Apo-2L/TRAIL exerted by Bcl-2 overexpression was reversed by cotreatment with LAQ824 and Apo-2L/TRAIL. Significantly, cotreatment with LAQ824 increased Apo-2L/TRAIL-induced apoptosis of primary acute myelogenous leukemia blast samples isolated from 10 patients with acute myelogenous leukemia. Taken together, these findings indicate that LAQ824 may have promising activity in augmenting Apo-2L/TRAIL-induced death-inducing signaling complex and apoptosis of human acute leukemia cells.

Maspin, a serine protease inhibitor (serpin), can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. This may occur through maspin-mediated inhibition of pericellular proteolysis. In a recent report, we provided evidence that maspin may also suppress tumor progression by enhancing cellular sensitivity to apoptotic stimuli. To our knowledge, maspin is the only proapoptotic serpin among all of the serpins implicated thus far in apoptosis regulation. The goal of the present study is to identify the specific target molecule(s), the modification of which by maspin renders tumor cells sensitive to chemotherapeutic agents. Our cellular, molecular, and biochemical studies demonstrate an essential role of Bax in the proapoptotic effect of maspin. First, Bax was up-regulated in maspin-transfected prostate and breast tumor cells, whereas the levels of other Bcl-2 family members including Bcl-2, Bcl-xl, and Bak remained unchanged. Second, on apoptosis induction, a greater amount of Bax was translocated from cytosol to mitochondria in maspin-transfected cells. After treatment with a Bax-silencing small interfering RNA, maspin-transfected cells became significantly more resistant to drug-induced apoptosis. Consistently, the release of cytochrome c and Smac/DIABLO from mitochondria was more responsive to apoptosis stimuli in maspin-transfected cells than in the mock-transfected cells. Third, the apoptosis induction of maspin-transfected cells was associated with increased activation of both caspase-8 and caspase-9. However, a caspase-9-specific inhibitor blocked the sensitization effect of maspin in a dose-dependent and time-dependent manner, demonstrating a rate-limiting role for caspase-9. In line with the central role of the Bax-mediated mitochondrial apoptotic pathway, maspin sensitized the apoptotic response of breast and prostate carcinoma cells to various drugs, ranging from death ligands to endoplasmic reticulum stress. The link between maspin and Bax up-regulation explains the loss of maspin-expressing tumor cells in invasive breast and prostate carcinomas. Our data reveal a novel mechanism for tumor suppressive maspin and suggest that maspin may be used as a modifier for apoptosis-based cancer therapy.

Cephalostatin 1 is a bis-steroidal marine natural product with a unique cytotoxicity profile in the in vitro screen system of the National Cancer Institute, suggesting that it may affect novel molecular target(s). Here we show that cephalostatin 1 induces a novel pathway of receptor-independent apoptosis that selectively uses Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) as a mitochondrial signaling molecule. At nanomolar concentrations, cephalostatin 1 triggers dose- and time-dependent DNA fragmentation in leukemia Jurkat T cells. Apoptosis was found to be dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone blocks cephalostatin 1-mediated DNA fragmentation. The CD95 death receptor as well as other caspase-8-requiring death receptors were not involved because Jurkat T cells lacking the CD95 receptor or caspase-8 and control cells responded equally to cephalostatin 1. Although cephalostatin 1 affects mitochondria by dissipating the mitochondrial membrane potential, neither cytochrome c nor apoptosis-inducing factor is released, as shown by Western blot analysis. Interestingly, cephalostatin 1 selectively triggers the mitochondrial release of the inhibitor of apoptosis antagonist Smac/DIABLO. Overexpression of the antiapoptotic protein Bcl-x(L) delayed both Smac/DIABLO release and onset of apoptosis, suggesting that Smac/DIABLO is required for cephalostatin 1-induced apoptosis. This new mitochondrial pathway is accompanied by marked structural changes of mitochondria as shown by transmission electron microscopy.

Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.

The phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival during oxidative stress. The role of the PI3K/Akt pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was phosphorylated rapidly in response to radiation in primary culture human umbilical vein endothelial cells in the absence of serum or growth factors. Akt phosphorylation occurred after doses as low as 1 Gy. PI3K consists of p85 and p110 subunits, which play a central role in Akt activation in response to exogenous stimuli. A mutation within the Src homology region 2 domain of mutant p85 (Deltap85) prevented radiation-induced Akt phosphorylation, when overexpressed in endothelial cells. Vascular endothelial cells transduced with control vector were resistant to radiation-induced apoptosis, whereas endothelial cell transduction with adenovirus encoding the mutated p85 (Ad.Deltap85) reversed this resistance to apoptosis after treatment with intermediate radiation doses (2-6 Gy). Deltap85 overexpression alone had no effect on the viability or apoptosis of endothelial cells. However, irradiated endothelial cells overexpressing Deltap85 released cytochrome c into the cytosol fraction and activated proteolytic cleavage of caspases 3 and 9, thereby inducing the apoptotic response. Inhibition of caspase 3 blocked endothelial apoptosis induced by overexpression of Deltap85 and radiation. These findings suggest that growth factor-independent activation of Akt contributes, in part, to the inherent resistance of irradiated vascular endothelium to the activation of apoptotic response.

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein pVHL is the cause of the familial VHL disease and the majority of sporadic renal clear cell carcinomas (RCCs). RCCs pose a significant problem for conventional cancer treatment protocols because of their highly recalcitrant characteristics to radio- and/or chemotherapies. In fact, the leading cause of morbidity and mortality of VHL patients is RCC. Recently, global gene profiling of RCC cells has revealed that sensitivity to tumor necrosis factor (TNF)-alpha-mediated cytotoxicity is pVHL dependent. Here, we report that although RCC cells devoid of functional pVHL (RC3) were resistant to the cytotoxic effects of TNF-alpha, reconstitution of these RCC cells with wild-type pVHL (WT8) restored their sensitivity to TNF-alpha cytotoxicity. The major TNF-alpha-inducible transcription factor nuclear factor (NF)-kappaB in the nuclear fraction capable of binding NF-kappaB-binding motifs was significantly increased in RC3 cells. Concordantly, the expression of NF-kappaB-target antiapoptotic genes c-FLIP, Survivin, c-IAP-1, and cIAP-2, which block the activities of caspases 8 and 3, were dramatically elevated in RC3 cells. Indeed, RC3 cells showed low caspases 8 and 3 activities. These results demonstrate that pVHL facilitates TNF-alpha-induced cytotoxicity in RCC cells, at least in part, through the down-regulation of NF-kappaB activity and subsequent attenuation of antiapoptotic proteins c-FLIP, Survivin, c-IAP-1, and c-IAP-2.

We have previously shown a critical role for IFN regulatory factor 5 (IRF-5) in the innate immune response to virus infection. For the first time, we now show that although IRF-5 is a direct target of p53, its cell cycle regulatory and proapoptotic effects are p53 independent. IRF-5 inhibits both in vitro and in vivo B-cell lymphoma tumor growth in the absence of wild-type p53. The molecular mechanism(s) of IRF-5-mediated growth inhibition is associated with a G(2)-M cell cycle arrest and modulation of growth regulatory and proapoptotic genes, including p21, Bak, DAP kinase 2, and Bax. Taken together, these data indicate that although IRF-5 is a downstream target of p53, its growth inhibitory and proapoptotic effects are independent of p53.

The novel oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) induces apoptosis of human leukemia cells by activation of the extrinsic caspase-8 pathway. The mechanisms responsible for the proapoptotic effects of CDDO are unknown. The present studies demonstrate that CDDO activates the c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase in U-937 leukemia cells. The results also show that CDDO activates stress kinases by increasing levels of reactive oxygen species and decreasing intracellular glutathione (GSH) concentrations. Similar findings were obtained with the C-28 methyl ester (CDDO-Me) and C-28 imidazolide ester (CDDO-Im) derivatives. The results also demonstrate that CDDO-induced: (a) stimulation of Jun NH(2)-terminal kinase; (b) activation of caspase-8; (c) loss of mitochondrial transmembrane potential; (d) release of cytochrome c; and (e) cleavage of caspase-3 are blocked by pretreatment with the antioxidant N-acetyl-L-cysteine and GSH but not with cysteine. In concert with these results, CDDO-induced apoptosis is also abrogated by N-acetyl-L-cysteine and GSH. These findings demonstrate that CDDO and its derivatives disrupt intracellular redox balance and thereby induce apoptosis.

Angiostatin, a proteolytic cleavage product of plasminogen, acts via a selective, yet poorly understood mechanism to potently inhibit angiogenesis (M. S. O'Reilly et al., Cell, 79: 315-328, 1994). Vascular endothelial cell proliferation assays revealed that angiostatin(4.5), a naturally occurring human isoform consisting of plasminogen kringle domains 1-4 and most of kringle domain 5 (G. A. Soff, Cancer Metastasis Rev., 19: 97-107, 2000), dose dependently reduces cell number despite the presence of a potent stimulus of proliferation. Flow cytometry using the vital dyes Hoechst 33342 and Pyronin Y revealed that approximately 40% of both control and angiostatin(4.5)-treated cells were in the proliferative phase, indicating that cell cycle progression is not impaired by exposure to angiostatin(4.5). Both bovine aortic endothelial cells and human umbilical endothelial cells were shown to undergo apoptosis in response to angiostatin(4.5). Caspases-3, -8, and -9 activation, specified by cleavage of fluorophore-conjugated specific peptide substrates, revealed a cascade of caspase activation that peaks at 36 h of angiostatin(4.5) treatment. Angiostatin(4.5) exposure induced release of cytochrome c from mitochondria in a caspase-dependent manner, but a pan-caspase inhibitor, zVAD-fmk, blocked cytochrome c release. Overall, these data indicate that human angiostatin(4.5) may function in vivo to block blood vessel formation by specifically inducing vascular endothelial cells to apoptose in a process likely involving both the intrinsic and extrinsic apoptosis pathways.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Glucocorticoids (GCs) are frequently used as cotreatment because they may have potent proapoptotic properties and reduce nausea, hyperemesis, and acute toxicity on normal tissue. In contrast to the proapoptotic effect of GCs in lymphoid cells, resistance toward cancer therapy-mediated apoptosis was induced in solid tumors of human cervix and lung carcinomas. Filter hybridization, expression data, as well as functional assays identified multiple core apoptosis molecules, which are regulated by GCs in a pro- or antiapoptotic manner. Both antiapoptotic genes such as FLIP and members of the Bcl-2 and IAP family as well as proapoptotic elements of the death receptor and mitochondrial apoptosis pathways were down-regulated in carcinomas resulting in a decreased activity of caspase-8, caspase-9, and caspase-3. In contrast, death receptor and mitochondrial apoptosis signaling as well as caspase activity was enhanced by dexamethasone in lymphoid cells. To restore apoptosis sensitivity in dexamethasone-treated carcinomas, caspase-8 and caspase-9 were transfected. This resensitized tumor cells in vitro and xenografts in vivo to cisplatin induced cell death. These data therefore raise concern about the widespread combined use of GCs with antineoplastic drugs or agents in the clinical management of cancer patients.

Several chaperone-binding drugs based on geldanamycin (GA) have been synthesized, and one of them, 17-allylamino-17-demethoxygeldanamycin (17-AAG), is being developed in the clinic. Interest in the use of 17-AAG in combination with cytotoxic drugs led us to study both GA and 17-AAG with cisplatin (DDP) in the human colon adenocarcinoma cell lines HT29 and HCT116. We performed isobologram analysis of combinations of DDP with GA or 17-AAG in these cell lines using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay to evaluate cell survival. In HCT116, the effects of GA and 17-AAG with DDP were additive and schedule dependent. In HT29 both GA and 17-AAG antagonized DDP effects resulting in cytotoxicity less than expected. We hypothesized that the antagonism in HT29 cells might be a consequence of altered p53 function in this cell line. Accordingly, we tested GA/17-AAG and DDP in combination in the HCTp5.2 cell line, which expresses a dominant-negative form of p53. In these cells too, the GA analogues antagonized DDP, suggesting a role for p53 in the observed effects. Investigation of the DDP-induced signaling pathways revealed that ansamycins block the activation of mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase pathways and c-Jun expression in HT29 cells while exerting incomplete inhibitory effects in HCT116 and HCTp5.2 cell lines. Therefore, effects on signaling are thought not to underlay the antagonism in the latter model. The ansamycins inhibited DDP-induced activation of caspases 8 and 3 in HT29 and HCTp5.2 but not in HCT116 cells, which we postulate to be the basis for higher survival of p53-deficient cells when treated with combinations of the two drugs.

Bax is a crucial mediator of the mitochondrial pathway for apoptosis, and loss of this proapoptotic Bcl-2 family protein contributes to drug resistance in human cancers. We report here that the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (THG) induces apoptosis of human colon cancer HCT116 cells through a Bax-dependent signaling pathway controlling the cytosolic release of mitochondrial apoptogenic molecules. Treating HCT116 cells with THG results in caspase-8 activation; Bid cleavage; Bax conformational change and mitochondrial translocation; the release of cytochrome c, Smac/Diablo, and Omi/HtrA2 into the cytosol; caspase-3 activation; and apoptosis. In contrast, knockout of Bax completely abrogates the full processing/activation of caspase-3 but has no effect on the processing of caspase-8 and the initial cleavage of caspase-3 to p24 fragment after THG treatment. The caspase-8-specific inhibitor z-IETD-fmk, as well as pan-caspase inhibitor z-VAD-fmk, but not the calpain inhibitor E-64d, prevents Bid cleavage, Bax conformational change, and subsequent caspase-3 processing and apoptosis. Caspase-8 processing is dependent on de novo protein synthesis; DR5 expression is strongly up-regulated by THG treatment. Moreover, the absence of Bax blocks THG-induced Omi and Smac release from mitochondria, and expression of cytosolic Omi (GFP-IETD-Omi) or Smac (GFP-IETD-Smac) restores the sensitivity of Bax-knockout HCT116 cells to apoptosis in response to THG treatment. Taken together, our results indicate that Bax-dependent Smac and Omi release plays an essential role in caspase-3 activation and apoptosis induced by THG in human colon cancer HCT116 cells.

The p53 mutant 143Ala is a human temperature-sensitive mutant with two conformational states. To definitively determine whether the Fas signal transduction pathway and the function of the pathway are dependent on p53 status, we have established stable transfectants of p53 mutant 143Ala in two human cancer cell lines: H1299 (lung cancer line) and PC-3 (prostate cancer line), the native state of which contains null p53 status and can grow at 37 degrees C and 32.5 degrees C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis showed inhibition of the growth of cells overexpressing p53 mutant 143Ala in the wild-type p53 form at 32.5 degrees C because of induction of G0/G1 arrest. Transfected cells had increased protein expression of p21, Fas, and MDM2 at the wild-type p53 conformation at 32.5 degrees C, but not in the mutant p53 form at 37 degrees C. However, there was no change in protein expression of FADD, FAP-1, Bcl-2, or Bax at 32.5 or 37 degrees C. Assays for apoptosis demonstrated that anti-Fas antibody CH-11 and FasL induced apoptosis only in cells that overexpress p53 mutant 143Ala at 32.5 degrees C with the wild-type p53 form. Both caspase-3 and caspase-8 activities were increased by anti-Fas antibody CH-11 only in cells at 32.5 degrees C with wild-type p53. Our results demonstrated that Fas-mediated apoptosis in H1299 and PC-3 cells expressing p53 mutant 143Ala occurred only with the wild-type p53 phenotype. These results support the hypothesis that Fas-mediated apoptosis is dependent, at least partially, on the presence of a functional wild-type p53 state. This model may be a useful tool for dissecting the specific interactions between wild-type p53 and the Fas signal transduction pathway in human cancer cells.

Chlorophyllin (CHL), an antimutagenic and anticarcinogenic water-soluble derivative of chlorophyll, was recently found to be highly effective as a chemopreventive agent in a high-risk population exposed unavoidably to aflatoxin B(1) in the diet (P. A. Egner et al., Proc. Natl. Acad. Sci. USA, 98: 14601-14606, 2001). The current study examined the response of HCT116 human colon cancer cells to CHL treatment. Cells exposed to concentrations in the range 0.0625-0.5 mM CHL underwent growth arrest and apoptosis after 24 h, with the formation of a sub-G(1) peak in the attached cell population and nuclear condensation in the floating cell population. There was a concentration-dependent attenuation of mitochondrial membrane potential (deltapsi(m)) without the release of cytochrome c or activation of the caspase-9/caspase-3/poly(ADP-ribose) polymerase pathway. However, apoptosis-inducing factor was released from mitochondria into the cytosol and translocated to the nucleus, leading to concentration-dependent cleavage of nuclear lamins. The upstream mediators of this CHL-induced apoptosis pathway were identified as caspase-8/caspase-6 and truncated Bid, acting in conjunction with other proapoptotic members of the Bcl-2 family, such as Bak. These findings suggest that CHL might trigger apoptosis via interaction with putative "death receptors" in the plasma membrane of cancer cells, leading to initial cleavage of procaspase-8 and activation of subsequent downstream events, resulting in the destruction of nuclear lamins. Importantly, E-cadherin and alkaline phosphatase, which are indicators of cell differentiation, were strongly induced at all concentrations of CHL. Thus, in addition to being an effective blocking agent during the initiation phase, these findings support a role for CHL as a suppressing agent and as a possible novel therapeutic strategy directed toward aberrant cell proliferation in the colon.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

The resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been attributed to a lack of caspase 8 expression. Here we demonstrate a clinically applicable molecular targeting strategy that not only increases caspase 8 expression ex vivo in NB cell lines but also in the tumor tissues of NB patients receiving IFN-gamma treatment. We identify the functional caspase 8 promoter, which is different from the methylated region reported previously, and show promoter activity is up-regulated by IFN-gamma through a IFN-gamma activation site-containing region. IFN-gamma also induces TRAIL expression in NB cell lines. However, the IFN-gamma restoration of caspase 8 in some NB cells revealed persistent TRAIL resistance in most NB cell lines examined. This additional lesion in the TRAIL path is because of a loss of cell membrane TRAIL receptors (TR1/TR2) not only in cell lines but in most of the NB tumor tissues evaluated. Restoration of TR2 expression by transfection enhances IFN-gamma-induced TRAIL sensitivity. Furthermore, we have found that we can improve TRAIL sensitivity in NB by reconstituting caspase 8 with IFN-gamma and TR2 with chemotherapeutic agents.

Apo-2L/TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis through engagement of the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). The transcription factor nuclear factor (NF)-kappa B regulates the expression of genes involved in cancer cell invasion, metastasis, and resistance to chemotherapy. In normal unstimulated cells, NF-kappa B is maintained in the cytoplasm with its inhibitor protein I kappa B, whereas in cancer cells, NF-kappa B is in the nucleus and constitutively activates target genes. To understand the function of NF-kappa B in TRAIL-induced apoptosis, we have analyzed the specific roles of NF-kappa B subunits. Overexpression of a transdominant-negative mutant of the inhibitory protein I kappa B alpha results in down-regulation of constitutively active NF-kappa B, induction of DR5, and tumor necrosis factor receptor (TNFR) 1-associated death domain expression and enhancement of TRAIL sensitivity. Overexpression of RelA or a transcriptional-deficient mutant of c-Rel inhibits TRAIL-induced apoptosis. Depletion of RelA in mouse embryonic fibroblasts increases cytokine-induced apoptosis, whereas depletion of c-Rel blocks this process. Overexpression of RelA subunit inhibits caspase-8 and DR4 and DR5 expression and enhances expression of cIAP1 and c-IAP2 after TRAIL treatment. By comparison, overexpression of c-Rel enhances DR4, DR5, and Bcl-X(s) and inhibits cIAP1, cIAP2, and survivin after TRAIL treatment. These results suggest that the RelA subunit acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2. The dual function of NF-kappa B, as an inhibitor or activator of apoptosis, depends on the relative levels of RelA and c-Rel subunits. Thus, NF-kappa B activity may play an important role in tumor progression, and down-regulation of RelA or up-regulation of c-Rel represents a possible therapeutic target for the treatment of cancer.

The inhibitor of apoptosis proteins (IAPs) plays a central role in repressing caspase-mediated cell death. However, little is known about the actual role of endogenously expressed IAPs in cancer cells. We found that the cytochrome c/apoptotic protease-activating factor-1 (apoptosome)-dependent caspase activation is deficient in human non-small cell lung cancer (NSCLC) NCI-H460 cells. This dysfunctional apoptosome activity was not correlated with any decrease of apoptosome component factors, but it was linked to an increased X-linked inhibitor of apoptosis protein (XIAP). In H460 cells, the overexpressed XIAP, but not c-IAP1, bound to the processed form of caspase-9 and suppressed the activation of downstream effector caspases. Moreover, the defect in apoptosome activity in H460 cells was dramatically restored by the IAP-targeting SmacN7 peptide, which disrupted XIAP-caspase-9 binding, indicating an essential role of the IAP in the apoptosome inhibition. However, the SmacN7 did not show any striking effect on the apoptosome activity of normal lung fibroblast cells, although these cells also expressed modest amounts of IAP. To explore the therapeutic approach, we additionally developed SmacN7(R)8, a newly designed cell permeable peptide. The SmacN7(R)8 selectively reversed the apoptosis resistance of H460 cells, and when in combination with chemotherapy, regressed the tumor growth in vivo with little toxicity to the mice. Our results indicate that IAP-dependent suppression of apoptosome predominantly occurs in IAP-overexpressing tumor, and the IAP-targeting Smac peptide is an effective molecule to increase tumor cell death induced by chemotherapy in vitro and in vivo.

Testicular germ cell cancer is one of the very few cancers that are highly sensitive to and curable by cisplatin-based chemotherapy even in an advanced stage. However, in a few cases resistance to cisplatin occurs and patients subsequently die from progressive disease. The molecular basis for this resistance remains to be determined. Using two cisplatin-sensitive (2102EP and H12.1) and one cisplatin-resistant human testicular germ cell cancer cell line (1411HP), we investigated molecular mechanisms in the induction of apoptosis after cisplatin-treatment focusing on the cleavage and activation of caspase-2, caspase-3, caspase-7, caspase-8, and caspase-9. The cell line 1411HP showed a 3.3-fold cisplatin resistance when compared with the sensitive cell lines 2102EP and H12.1 by IC(90)s, which was treatment schedule independent (2- or 24-h incubation). Cisplatin resistance was associated with substantially decreased apoptosis in vitro and in derived nude mice xenografts as determined by Apo 2.7 detection, DNA-laddering, immunohistochemistry of active caspase-3, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Total DNA platination as assessed by ELISA after cisplatin treatment in equimolar doses did not differ between cisplatin-resistant or -sensitive cells. In separate analysis of cells of early and late apoptotic stages, initiation of cisplatin-induced apoptosis appeared to be rather mediated by caspase-9 than by caspase-8. Resistant 1411HP cells failed to activate caspase-9 during the induction of apoptosis after cisplatin treatment at the IC(90) dose. Interestingly, inhibition of caspase-9 in sensitive H12.1 almost completely blocked apoptosis and induced cisplatin resistance to the same extent as in 1411HP so that apoptosis could only be induced by 3.3-fold higher cisplatin doses. Furthermore, in caspase-9 blocked cells, initiation of apoptosis occurred in a caspase-9 independent manner accompanied by activation of caspase-2 and caspase-3, which are intrinsic characteristics of resistant 1411HP cells. Failure of caspase-9 activation and cisplatin resistance was independent of the expression of p53, Bcl-2 family proteins, Fas receptor, and Fas ligand. In conclusion, failure of activation of the caspase-9 pathway induces a higher cellular threshold for cisplatin-mediated induction of apoptosis in testicular cancer cells. However, this higher threshold can be overcome by higher cisplatin doses, conceivably by using an alternate, caspase-9-independent apoptotic pathway. This supports the current clinical strategy of high-dose chemotherapy in patients with chemorefractory germ cell tumors. However, additional defining and eventually targeting the exact molecular mechanism blocking caspase-9 activation might lead to more selective therapeutic approaches to overcome cisplatin resistance in germ cell cancer.

Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%). Methylation frequencies were low in Wilms' tumors (19%) and absent in hepatoblastomas, acute leukemias, osteosarcomas, Ewing's sarcomas, and ganglioneuromas and in normal tissues. Methylation of CASP8 and the tumor suppressor gene RASSF1A were highly significantly correlated in all tumor types by both the chi(2) and the Fisher's exact tests (P < 0.0001 for both tests). Because the region of the gene examined by us and others is not located in the promoter region and lacks features of a CpG island, we explored the relationship between methylation and gene silencing in detail using 23 pediatric tumor cell lines. Studies included relating the methylation of the region to gene expression at mRNA and protein levels, enzymatic assays of gene function, clonal analysis of PCR amplicons of the region, and exposure to a demethylating agent. These studies indicated that methylation correlated with the loss of gene function in most cases; however, other mechanisms of gene inactivation were present in some cases. Posttranscriptional inactivation of the closely related gene caspase 10 was present in many cell lines. Our results suggest that deregulation of the death receptor pathway to apoptosis is frequent in many types of pediatric tumors and their cell lines.