Increased aldehyde reductase expression mediates acquired radioresistance of laryngeal cancer cells via modulating p53

The main obstacle to cure tumors by radiotherapy has been ascribed to tumor radioresistance. To determine the mechanisms underlying resistance to irradiation, it is essential to compare proteins differentially expressed from radiotherapy-sensitive and -resistant cancer cells. Aldehyde reductase (AKR1A1) was recently identified as increased in radioresistant laryngeal cancer cells by comparative proteomics approach. Here, we provide the mechanism of AKR1A1-mediated radioresistance via p53 regulation in laryngeal cancer cells. AKR1A1 induction was correlated with the radioresistant phenotype of laryngeal cancer HEp-2 cells. AKR1A1 depletion with siRNA significantly enhanced radiation sensitivity of radioresistant HEp-2 cells by promoting radiation-induced cell death and accelerated radiation-mediated inhibition of cell proliferation, without affecting either the PI3K-Akt or MAPK-ERK pathways. Intriguingly, AKR1A1 depletion induced phosphorylation of p53 at serine 15 and G 2/M transition in response to irradiation. We further found that AKR1A1 interacted with p53 and this interaction was dramatically increased in the irradiated radioresistant cells compared with the control cells. AKR1A1 expression also regulated p53 stability in response to irradiation. Furthermore, AKR1A1 depletion only sensitized HCT116 cells expressing p53 to irradiation and not p53-deficient cells. Therefore, our data suggest that radiation-inducible AKR1A1 contributes to acquired radioresistance of laryngeal cancer cells by suppressing p53 activation through inhibitory interaction.     

Hypoxia increases resistance of human pancreatic cancer cells to apoptosis induced by gemcitabine.

PURPOSE: Hypoxia, frequently found in the center of solid tumor, is associated with resistance to chemotherapy by activation of signaling pathways that regulate cell pro-liferation, angiogenesis, and apoptosis. We determined whether hypoxia can increase the resistance of human pancreatic carcinoma cells to gemcitabine-induced apoptosis by activation of phosphatidylinositol 3'-kinase (PI3K)/Akt, MEK/mitogen-activated protein kinase (extracellular signal-regulated kinase) [MAPK(Erk) kinase (MEK)], and nuclear factor kappa B (NF-kappa B) signaling pathways. EXPERIMENTAL DESIGN: We evaluated the phosphorylation of Akt and MAPK(Erk), DNA binding activity of NF-kappa B, and apoptosis induced by gemcitabine in L3.6pl human pancreatic cancer cells under normoxic and hypoxic conditions. We then examined the effects of the PI3K inhibitor LY294002, MEK inhibitor U0126, and the epidermal growth factor receptor tyrosine kinase inhibitor PKI 166 on these signaling pathways and induction of apoptosis. RESULTS: Hypoxic conditions increased phosphorylation of Akt and MAPK(Erk) and NF-kappa B DNA binding activity in L3.6pl cells. The activation of Akt and NF-kappa B was prevented by LY294002, whereas the activity of MAPK(Erk), but not NF-kappa B, was inhibited by U0126. The increased activation of Akt, NF-kappa B, and MAPK(Erk) was inhibited by PKI 166. Under hypoxic conditions, L3.6pl cells were resistant to apoptosis induced by gemcitabine. The addition of LY294002 or PKI 166 abrogated cell resistance to gemcitabine, whereas U0126 only partially decreased this resistance. CONCLUSIONS: These data demonstrate that hypoxia can induce resistance of pancreatic cancer cells to gemcitabine mainly through the PI3K/Akt/NF-kappa B pathways and partially through the MAPK(Erk) signaling pathway. Because PKI 166 prevented the activation of PI3K/Akt/NF-kappa B and MAPK(Erk) pathways, the combination of this tyrosine kinase inhibitor with gemcitabine should be an effective therapy for pancreatic cancer.     

INPP4B overexpression enhances the antitumor efficacy of PARP inhibitor AG014699 in MDA-MB-231 triple-negative breast cancer cells

Although preclinical and clinical studies on poly-(adenosine diphosphate ribose) polymerase (PARP) inhibitor alone or in combination with DNA-damaging agents have shown promising results, further research to improve and broaden the application scope of this therapeutic approach is needed. The main aim of this study was to evaluate whether overexpressing inositol polyphosphate 4-phosphatase type II (INPP4B) gene, a novel tumor suppressor gene negatively regulating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, could enhance the antitumor efficacy of PARP inhibitor AG014699 used in the treatment of triple-negative breast cancer (TNBC). Here in this report, we used a TNBC cell line MDA-MB-231 without expression of INPP4B as the study model and a lentiviral system to stably overexpress INPP4B gene in MDA-MB-231 cells. We detected that the overexpression of INPP4B could significantly suppress cell proliferation and block cell cycle progression in G1 phase via decreasing the protein level of phosphorylated AKT. It is further revealed that PARP inhibitor AG014699 induced DNA damage conferring a G2/M arrest and decreased cell viability, which is paralleled by the induction of apoptosis. However, PARP inhibitor AG014699 could activate the PI3K/AKT signaling pathway activity and partially offset its therapeutic efficacy. In our study, a significant enhancement of proliferation inhibition was observed when INPP4B overexpression was combined with PARP inhibitor AG014699 in comparison with either single treatment. The suppression of PI3K/AKT pathway caused by the overexpression of INPP4B contributed to the enhanced antitumor efficacy of the combined therapy. Our in vitro results indicated that this experimental therapeutic strategy combining INPP4B overexpression and PARP inhibitor AG014699 might be of potential therapeutic value as a new strategy for the treatment of patients with TNBC and is worthy of further study.     

Loss of INPP4B causes a DNA repair defect through loss of BRCA1, ATM and ATR and can be targeted with PARP inhibitor treatment

Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.     

Loss of caspase-8 activation pathway is a possible mechanism for CDDP resistance in human laryngeal squamous cell carcinoma, HEp-2 cells

Cisplatin (CDDP) is among the most widely used and most effective chemotherapeutic agent for many types of human cancer. Because killing cancer cells by chemotherapy is principally executed by apoptosis, a defective apoptotic program might acquire drug resistance. Flow cytometric Annexin V assay demonstrated that HEp-2 cells (human laryngeal cancer) were persistently resistant to CDDP as compared to HeLa cells (human uterine cervical cancer), despite the same histological type and wild-type p53 status. CDDP treatment caused steady induction of p53 protein in both cancer cell types, although it was more dramatic in CDDP-resistant HEp-2 cells, which was correlated well with p53 Ser15 phosphorylation, but not with the expression level of HPV type 18 E6 oncoprotein in these cells. Importantly, CDDP differently activated caspase cascades between HEp-2 and HeLa cells. CDDP activated the caspase-8 pathway through TNFR superfamily receptors such as Fas, but not caspase-9 in HeLa cells. On the other hand, the caspase-9 pathway was significantly activated in HEp-2 cells, although the activation of caspase-8 by CDDP was deficient. This different response to CDDP in caspase-8 activation was not related with the expression level of either Fas or FasL in these cells. We concluded from these results that loss of the caspase-8 activation pathway in HEp-2 cells was a possible mechanism for its resistance to CDDP-induced apoptosis. The caspase-8 pathway might play an important role in CDDP-induced apoptosis in HPV-positive human squamous cell carcinomas.     

Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells

Activated Ras utilises several downstream pathways, including the mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway and the phosphoinositide 3-kinase (PI-3k)/Akt pathway, to promote cell proliferation and to inhibit apoptosis. To investigate which pathway plays a major role in Ras-induced drug resistance to chemotherapeutic agents in breast cancer cells, we transfected MCF7 breast cancer cells with a constitutively active H-RasG12V and examined the toxicities of three commonly used breast cancer chemotherapeutic agents, paclitaxel, doxorubicin, and 5-fluorouracil in these cells under the conditions that PI-3K or MEK were selectively inhibited by their respective specific inhibitors or dominant negative expression vectors. We found that Ras-mediated drug resistance is well correlated with resistance to apoptosis induced by anticancer agents in MCF7 breast cancer cells. Although inhibition of MEK/MAPK or PI-3K/Akt can each enhance the cytotoxicity of paclitaxel, doxorubicin, or 5-fluorouracil, inhibition of the PI-3K/Akt pathway seems to have a greater effect than inhibition of the MEK/MAPK pathway in reversing Ras-mediated drug resistance. Our results indicate that the PI-3K pathway may play a more important role in receptor tyrosine kinase-mediated resistance to chemotherapy and suggest that PI-3K/Akt might be a critical target molecule for anticancer intervention in breast cancer.     

Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy

Autophagy, or programmed cell death type II, is one of the responses of cancer cells to various therapies, including ionizing radiation. Recently, we have shown that radiation induces autophagy, but not apoptosis, in various malignant glioma cell lines. Autophagy is mainly regulated by the mammalian target of rapamycin (mTOR) pathway. The Akt/mTOR pathway also mediates oncogenesis and radioresistance. Thus, we hypothesized that inhibiting this pathway has both an anticancer and radiosensitizing effect by activating autophagy. The purpose of our study was therefore to determine whether and by which mechanisms an Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, had anticancer and radiosensitizing effects on malignant glioma U87-MG and radioresistant U87-MG cells with a consistitutively active form of epidermal growth factor receptor (U87-MGDeltaEGFR). Treatment with the Akt inhibitor successfully inhibited Akt activity and reduced cell viability in both cell lines. In terms of the mechanism, the Akt inhibitor decreased phosphorylated p70S6 kinase, a downstream target of Akt, and induced autophagy, but not apoptosis. Furthermore, the Akt inhibitor radiosensitized both U87-MG and U87-MGDeltaEGFR cells by enhancing autophagy. Specific inhibition of Akt using the dominant-negative Akt plasmid also resulted in enhanced radiation-induced autophagy. In conclusion, an Akt inhibitor showed anticancer and radiosensitizing effect on U87-MG and U87-MGDeltaEGFR cells by inducing autophagy. Thus, Akt inhibitors may represent a promising new therapy as a single treatment or used in combination with radiation for malignant gliomas, including radioresistant ones that express DeltaEGFR.     

Down-regulation of p21Waf-1 protein facilitates IR- and UV-induced apoptosis in human squamous carcinoma cells

DNA damage via radiation exposure and administration of chemotherapeutic agents induce apoptosis, which is a basic mechanism for non-surgical anti-cancer treatment. We analyzed ionizing radiation (IR)- or ultraviolet (UV)-induced apoptosis in human laryngeal carcinoma (HEp-2) and uterine cervical carcinoma (HeLa) cells, and found that HeLa cells were significantly more sensitive to both IR- and UV-induced apoptosis compared to HEp-2 cells, in spite of the same histological type and p53 status. The cyclin-dependent kinase (Cdk) inhibitor, p21Waf-1 was modified differently between the two cancer cell types, whereas p53 protein was induced in a similar manner after IR or UV treatment. IR steadily induced p21Waf-1 protein in HEp-2 cells, but not in HeLa cells. Additionally, p21Waf-1 protein recovered close to the basal level only in HEp-2 cells, although UV caused rapid, dramatic and caspase-independent reduction of p21Waf-1 protein in both cancer cells. Furthermore, overexpression of p21Waf-1 protein in these cells by transient transfection and in stable cell lines in a tetracycline-regulated system, rescued IR- and UV-induced apoptosis. Finally, suppression of p21Waf-1 protein using antisense oligodeoxynucleotide transfection facilitated UV-induced apoptosis in both HEp-2 and HeLa cells. We concluded that p21Waf-1 protein is modified independently of p53 and functions as an inhibitor of IR- as well as UV-induced apoptosis in squamous carcinoma cells.     

Role of phosphatidylinositol-3 kinase/Akt pathway in bladder cancer cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand

TRAIL/Apo2L is a pro-apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Among various molecular strategies by which cancer cells evade apoptosis, PI3K/Akt signaling represents a dominant survival pathway. In this report, we investigated the role of PI3K/Akt pathway in TRAIL-induced apoptotic death in human bladder cancer cells. We observed that RT4 cells had very low level of constitutively active Akt and were sensitive to TRAIL, whereas UM-UC-3 and T24 cells had higher levels of constitutively active Akt and were resistant to TRAIL. Downregulation of constitutively active Akt by PI3K inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. However, transfecting constitutively active Akt into RT4 cells increased Akt activity and inhibited TRAIL-induced apoptosis. These results suggest that elevated Akt activity protects UM-UC-3 and T24 cells from TRAIL-induced apoptosis, and the PI3K/Akt signaling might inhibit apoptotic signals. Thus, the modulation of Akt activity by combining pharmacological drugs or genetic alterations of the Akt expression could induce cellular responsiveness to TRAIL and PI3K/Akt signaling pathway could serve as a novel target for therapeutic intervention in bladder cancer.     

AKT activation and response to interferon-beta in human cancer cells

Significant growth inhibition and induction of apoptosis by IFN-beta in cancer cells including colorectal cancer cells have been observed. We and others have previously reported the Stat 1 induction of TRAIL is a crucial step in the IFN-beta induced apoptosis pathway. However, when evaluating the sensitivity of a panel of colorectal cancer cell lines, we found no clear correlation between activation of the Jak/Stat signaling pathway and response to interferon. In the present study, we have evaluated the interaction of the PI3k/Akt pathway and IFN-beta induced apoptosis in human colorectal cancer cells. The results demonstrate a correlation between Akt activity, phosphorylation of Bad and resistance to interferon-induced apoptosis in these cells. The association of activation of Akt, phosphorylation of Bad and resistance to IFN-beta-induced apoptosis was further supported by the observation that disruption of the pathway in a more resistant cell line led to sensitization, and expression of an activated Akt in a more sensitive cell line led to increased resistance. Taken together, this data indicates that the PI3/Akt kinase pathway may be an important contributor to IFN-beta sensitivity and resistance in colorectal cancer cells. This data demonstrates a potential pathway by which cells may develop resistance to IFN, and further elucidation of this process may allow us to better target IFN therapy.     

INPP4B is upregulated and functions as an oncogenic driver through SGK3 in a subset of melanomas

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates PI3K/Akt signalling and has a tumour suppressive role in some types of cancers. However, we have found that it is upregulated in a subset of melanomas. Here we report that INPP4B can function as an oncogenic driver through activation of serum- and glucocorticoid-regulated kinase 3 (SGK3) in melanoma. While INPP4B knockdown inhibited melanoma cell proliferation and retarded melanoma xenograft growth, overexpression of INPP4B enhanced melanoma cell and melanocyte proliferation and triggered anchorage-independent growth of melanocytes. Noticeably, INPP4B-mediated melanoma cell proliferation was not related to activation of Akt, but was mediated by SGK3. Upregulation of INPP4B in melanoma cells was associated with loss of miRNA (miR)-494 and/or miR-599 due to gene copy number reduction. Indeed, overexpression of miR-494 or miR-599 downregulated INPP4B, reduced SGK3 activation, and inhibited melanoma cell proliferation, whereas introduction of anti-miR-494 or anti-miR-599 upregulated INPP4B, enhanced SGK3 activation, and promoted melanoma cell proliferation. Collectively, these results identify upregulation of INPP4B as an oncogenic mechanism through activation of SGK3 in a subset of melanomas, with implications for targeting INPP4B and restoring miR-494 and miR-599 as novel approaches in the treatment of melanomas with high INPP4B expression.     

Overcoming mTOR inhibition-induced paradoxical activation of survival signaling pathways enhances mTOR inhibitors' anticancer efficacy

The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. Several mTOR inhibitors are currently being tested in cancer clinical trials. Both PI3K/Akt and MEK/ERK signaling regulate mTOR axis. However, inhibition of mTOR activates Akt survival signaling, which in turn attenuates mTOR inhibitors' anticancer efficacy. We are interested in developing strategies for enhancing mTOR-targeted cancer therapy. In this study, we report that mTOR inhibition also induced activations of the MEK/ERK signaling pathway in some cancer cell lines after a prolonged treatment. The combination of rapamycin with the MEK inhibitor U0126 significantly enhanced growth inhibitory effects of cancer cells, suggesting that MEK/ERK activation may counteract mTOR inhibitors' anticancer efficacy. Similarly, the combination of an mTOR inhibitor with the EGF receptor inhibitor erlotinib synergistically inhibited the growth of both human cancer cells in cell cultures and xenografts in nude mice. Moreover, the presence of erlotinib suppressed rapamycin-induced phosphorylation of Akt, ERK and eIF4E as well, implying that erlotinib can suppress mTOR inhibition-induced feedback activation of several survival signaling pathways including Akt, ERK and eIF4E. Thus, we suggest a therapeutic strategy for enhancing mTOR-targeted cancer therapy by preventing mTOR inhibition-induced feedback activation of several survival mechanisms.     

Adenovirus-mediated PTEN treatment combined with caffeine produces a synergistic therapeutic effect in colorectal cancer cells

The tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene is a negative regulator of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt/PKB) signaling pathway. Overexpression of PTEN in cancer cells results in cell-cycle arrest and cell death through inhibition of PI3K. Caffeine, a xanthine analogue, is well known to enhance the cytocidal and growth-inhibitory effects of DNA-damaging agents such as radiation, UV light, and anticancer agents on tumor cells by abrogating DNA-damage checkpoints through inhibition of ataxia-telangiectasia-mutated (ATM), and ATM and Rad3-related (ATR) kinase activity. In this study, we demonstrate that treatment with a combination of adenovirus-mediated transfer of PTEN (Ad-PTEN) and caffeine synergistically suppressed cell growth and induced apoptosis in colorectal cancer cells but not in normal colorectal fibroblast cells. This synergistic effect was induced through abrogation of G(2)/M arrest, downregulation of the Akt pathway, and modulation of the p44/42MAPK pathway. Thus, combined treatment with Ad-PTEN and caffeine is a potential therapy for colorectal cancer.     

Semaphorin 3B inhibits the phosphatidylinositol 3-kinase/Akt pathway through neuropilin-1 in lung and breast cancer cells

Semaphorin 3B (SEMA3B), located at 3p21.3, is a secreted member of the semaphorin family important in axonal guidance. SEMA3B undergoes allele and expression loss in lung and breast cancer and can function as a tumor suppressor. Previously, we found that SEMA3B induces apoptosis in tumor cells either by reexpression or when applied as a soluble ligand. SEMA3B-induced apoptosis was mediated, in part, by blocking vascular endothelial growth factor autocrine activity in tumor cells. In the current study, treatment of lung and breast cancer cells with picomolar concentrations of soluble SEMA3B inhibited their growth; induced apoptosis; and was associated with decreased Akt phosphorylation, increase in cytochrome c release and caspase-3 cleavage, as well as increased phosphorylation of several proapoptotic proteins, including glycogen synthase kinase-3beta, FKHR, and MDM-2. Lung and breast cancer lines resistant to SEMA3B did not show these signaling changes and a tumor-derived missense SEMA3B mutant was inactive in this regard, providing specificity. SEMA3B-mediated inhibition of proliferation and induction of apoptosis in cancer cells were blocked by expressing a constitutively active Akt mutant and are linked to tumor cell expression of neuropilin-1 (Np-1). SEMA3B-insensitive Np-1-negative tumor cells acquired sensitivity to SEMA3B after forced expression of Np-1, whereas SEMA3B-sensitive Np-1-positive tumor cells lost sensitivity to SEMA3B after knockdown of Np-1 by small interfering RNA. We conclude that SEMA3B is a potential tumor suppressor that induces apoptosis in SEMA3B-inactivated tumor cells through the Np-1 receptor by inactivating the Akt signaling pathway. CA118384     

Antiapoptotic signaling in LNCaP prostate cancer cells: a survival signaling pathway independent of phosphatidylinositol 3'-kinase and Akt/protein kinase B

Constitutive activation of the phosphatidylinositol 3'-kinase (PI3 kinase)-Akt/protein kinase B (PKB) "survival signaling" pathway is a likely mechanism by which many cancers become refractory to cytotoxic therapy. In LNCaP prostate cancer cells, the PTEN phosphoinositide phosphatase is inactivated, leading to constitutive activation of Akt/PKB and resistance to apoptosis. However, apoptosis and inactivation of Akt/PKB can be induced in these cells by treatment with PI3 kinase inhibitors. Surprisingly, androgen, epidermal growth factor, or serum can protect these cells from apoptosis, even in the presence of PI3 kinase inhibitors and without activation of Akt/PKB, indicating the activity of a novel, Akt/PKB-independent survival pathway. This pathway blocks apoptosis at a level prior to caspase 3 activation and release of cytochrome c from mitochondria.     

Interactions of the integrin subunit beta1A with protein kinase B/Akt, p130Cas and paxillin contribute to regulation of radiation survival.

BACKGROUND AND PURPOSE: Cell adhesion-mediated radioresistance is a common phenomenon particularly relevant in tumor cells, which might hamper anticancer therapies. To analyze the role of adhesion-mediating beta1-integrins, stably transfected functional beta1A-integrin-expressing GD25beta1A and GD25beta1B cells, which express mutant beta1B-integrins, were compared in terms of radiation survival and beta1-integrin signaling. MATERIALS AND METHODS: Cells grown on fibronectin, collagen-III, laminin, vitronectin, anti-beta1-integrin-IgG (beta1-IgG) or poly-l-lysine were irradiated with 0-6Gy in presence or absence of growth factors or inhibitors for phosphatidylinositol-3 kinase (PI3K), i.e. Ly294002 and wortmannin. In addition to colony formation, protein kinase B/Akt (PKB/Akt) kinase activity, focal adhesion kinase (FAK), p130Cas, paxillin and c-Jun N2-terminal kinase (JNK) expression and phosphorylation were analyzed by Western blot technique. RESULTS: Adhesion of GD25beta1A cells to extracellular matrix proteins or beta1-IgG resulted in growth factor-independent radiation survival. In contrast, serum starved GD25beta1B cells showed a significant (P<0.01) reduction in radiation survival on all substrates. PI3K inhibition moderately or strongly radiosensitized GD25beta1A or GD25beta1B cells, respectively. The pro-survival effects detected in serum starved GD25beta1A cells were due to direct, PI3K-mediated stimulation of PKB/Akt activity by beta1-integrins and induced p130Cas and paxillin phosphorylation. Phosphorylated p130Cas and paxillin subsequently prevented activation of cell death-regulating JNK. CONCLUSIONS: The data show that beta1-integrin-mediated signaling through the cytoplasmic integrin domains is critical for efficient pro-survival regulation after irradiation. Profound knowledge of the underlying mechanisms of integrin-mediated cellular radioresistance could foster the design of new molecular-targeted anticancer therapies.     

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.