calpain and Ovarian-Neoplasms

Aberrant expression of microRNAs (miRNAs) contributes to the development of high grade serous ovarian cancer (HGSOC). However, the molecular mechanism by which miRNA-585-3p mediates high-grade serous ovarian carcinogenesis is unclear. This study aims to investigate the specific mechanism of action of miR-585-3p in HGSOC.. Expression of miR-585-3p in HGSOC tissues and cell lines was detected by qRT-PCR. Cell viability and migration were detected using MTT and transwell system. The expression of target genes and target proteins of miR-585-3p was detected by dual luciferase reporter assay and western blot.. The expression of miR-585-3p was significantly lower in HGSOC tissues and cells than in normal ovarian tissues and cell lines. In HGSOC tissues, CAPN9 expression was inversely correlated with miR-585-3p expression. MiR-585-3p inhibited the proliferation and migration of HGSOC cells. MiR-585-3p bound to the 3'-untranslated region (UTR) of CAPN9 and inhibits CAPN9 expression. Overexpression of CAPN9 reduced the inhibitory effect of miR-585-3p in HGSOC cells.. miR-585-3p is significantly down-regulated in HGSOC tissues and cell lines. MiR-585-3p inhibits the proliferation and migration of HGSOC cells by targeting CAPN9.

Topics: Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms; Ovary

Long non-coding RNA (lncRNA) has been regarded as crucial regulator for cancer progression. Roles of KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) in cancers including osteosarcoma and colon cancer have been previously reported. However, its role in ovarian cancer (OC) remains unclear.. Expression level of KCNQ1OT1 on OC cells and normal cell was analyzed with quantitative real-time PCR. Gain and loss-of-function experiments were performed to analyze the biological roles of KCNQ1OT1 in OC. Moreover, whether KCNQ1OT1 functions its role via mediating MICRORNA-142-5p (MIR-142-5p)/calpain 10 (CAPN10) axis was analyzed. In addition, effects of KCNQ1OT1, MIR-142-5p, and CAPN10 on overall survival of OC patients were analyzed at Kaplan-Meier plotter website.. We showed KCNQ1OT1 was elevated expression in OC cells and indicated poorer overall survival of OC patients. Besides, we found KCNQ1OT1 could promote OC cell proliferation and migration in vitro. Moreover, MIR-142-5p was found reduced expression, while CAPN10 was found elevated expression in OC cells compared with normal cell. Kaplan-Meier curve analysis showed low MIR-142-5p or high CAPN10 expression were indicators for poorer overall survival of OC patients. At length, we showed KCNQ1OT1 could regulate OC development via MIR-142-5p/CAPN10 axis.. Taken together, KCNQ1OT1 upregulates CAPN10 expression via sponging MIR-142-5p, thus promoting the proliferation and migration of OC.

Topics: Calpain; Cell Line; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms; Potassium Channels, Voltage-Gated; Survival Analysis

Expression of members of the calpain system are associated with clinical outcome of patients with, amongst others, breast and ovarian cancers, with calpain-2 expression in ovarian cancer being implicated in chemo-resistance and survival. This study aimed, using a large patient cohort and in vitro models, to verify its importance and further investigate the role in ovarian cancer chemoresponse.. Calpain-1, calpain-2, calpain-4 and calpastatin expression were evaluated in primary ovarian carcinomas (n = 575) by immunohistochemistry. Protein expression was assessed, via western blotting, in five ovarian cancer cell lines with various sensitivities towards cisplatin/carboplatin. In vitro calpain activity was inhibited by calpeptin treatment to assess changes in platinum sensitivity by proliferation assay, with expression of genes associated with epithelial-mesenchymal transition being examined by RT. The current study confirmed previous data that high calpain-2 expression is associated with poor overall survival (P = 0.026) and that calpain-1 was not associated with overall survival or progression-free survival. Low expression of calpastatin (P = 0.010) and calpain-4 (P = 0.003) were also associated with adverse survival. Such prognostic associations do not seem to be linked with altered tumour sensitivity towards platinum-based chemotherapy. Interestingly, low calpain-1 expression was more frequent in patients with confined tumours (stage 1) (χ. The conventional calpains and calpastatin have been confirmed to play an important role in ovarian cancer; however, the precise mechanisms whereby they exert effects remain to be elucidated.

Topics: Adenocarcinoma, Clear Cell; Adenocarcinoma, Mucinous; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cohort Studies; Cystadenocarcinoma, Serous; Endometrial Neoplasms; Epithelial-Mesenchymal Transition; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Ovarian Neoplasms; Prognosis; Survival Rate; Tumor Cells, Cultured

We have previously reported on the prognostic importance of the calpain family of proteins in ovarian cancer, especially calpain-2. Spleen tyrosine kinase (Syk) phosphorylates a variety of cytoskeletal proteins with studies suggesting potential interactions between Syk and conventional calpains. Microtubule-associated protein 4 (MAP4) has been reported to be regulated by Syk.. The current study assessed Syk and MAP4 protein expression, by immunohistochemistry on a tissue microarray comprised of cores from primary ovarian carcinomas (n = 575), to evaluate associations with patient clinical outcomes and other clinicopathological factors and sought to determine whether there were any correlations between the expression of Syk, MAP4 and the calpain system.. MAP4 expression was significantly associated with ovarian cancer histological subtype (P < 0.001), stage (P = 0.001), grade (P < 0.001) and residual tumour (P = 0.005). Despite this finding, we found no significant association existing between MAP4 expression and overall survival. Syk expression was also found significantly associated with histological subtype (P < 0.001). Syk seems to play a contradictory role with respect to tumour progression: low cytoplasmic Syk expression was significantly associated with low stage (P = 0.013), and low nuclear Syk expression with chemo-resistance in patients treated with taxane-containing therapy (P = 0.006). Interestingly, despite the lack of association in the whole cohort, high nuclear Syk expression was significantly associated with better overall survival in certain subgroups (P = 0.001).. The current study indicates a lack of correlation between calpain-2 expression and Syk and MAP4. Syk, MAP4 and calpain-1 appeared to significantly correlate with each other in the whole cohort, with calpain-1 being more highly associated with MAP4 and Syk in mucinous carcinomas. Overall, the current results suggest that Syk, MAP4, and calpain-1 expression are correlated with each other and these proteins may be involved in early stages of tumour spread.

Topics: Calpain; Cytoplasm; Female; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Middle Aged; Neoplasm Grading; Ovarian Neoplasms; Syk Kinase; Tissue Array Analysis

Increasing evidence shows that the calpain regulatory subunit Capn4 can modulate the proliferation and metastasis of cancer cells, and plays an important role in the development of malignant tumors. However, there is no information on the clinical significance of Capn4 in epithelial ovarian carcinoma (EOC) or the molecular mechanisms by which Capn4 promotes the growth and metastasis of EOC. Therefore, the aim of this study was to clarify the role of Capn4 in EOC.. We evaluated Capn4 and osteopontin (OPN) expression in EOC cell lines and tissues from patients with ovarian cancer by western blotting and immunohistochemical analysis. We then created cell lines with downregulated and upregulated Capn4 expression, using Capn4-targeting small interfering RNA and a pcDNA3.1-Capn4 overexpression vector, respectively, to investigate its function in EOC in vitro. In addition, we investigated the potential mechanism underlying the function of Capn4 by examining the effect of modifying Capn4 expression on Wnt/β-catenin signaling pathway-related genes by western blotting.. Capn4 was overexpressed in clinical EOC tissues compared with that in normal ovarian epithelial tissue, and was associated with poor clinical outcomes. Upon silencing or overexpressing Capn4 in EOC cells, we concluded that Capn4 promotes cell proliferation and migration in vitro. Furthermore, Capn4 promoted EOC metastasis by interacting with the Wnt/β-catenin signaling pathway to upregulate OPN expression.. Our study indicates that Capn4 plays a critical role in the progression and metastasis of EOC, and could be a potential therapeutic target for EOC management.

Topics: beta Catenin; Calpain; Carcinoma, Ovarian Epithelial; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Osteopontin; Ovarian Neoplasms; Plasmids; RNA Interference; RNA, Small Interfering; Up-Regulation; Wnt Proteins; Wnt Signaling Pathway

Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for tumour spread of epithelial ovarian cancer (EOC), possibly determined by the intercellular connecting protein E-Cadherin (E-Cad) and its fragments.. Tumour tissue of 105 advanced EOC patients was evaluated for protein expression of E-Cad, β-Catenin and Calpain by western blotting and immunohistochemistry. Expression patterns were compared between tumours with solely intraperitoneal (pT3c, pN0; n=41) and tumours with retroperitoneal metastases (pT1a-3c, pN1; n=64). Lysates of the EOC cell line SKOV3 and tumour tissue from the intraperitoneal group were tested for E-Cad expression following Calpain treatment.. E-Cad full-length (E-Cad-FL, 120 kDa) and two major fragments at 85 kDa (E-Cad-85) and 23 kDa (E-Cad-23) were detected by western blotting. E-Cad-85 expression was significantly higher in tumours with solely intraperitoneal metastases and correlated strongly with E-Cad-23 and the protease Calpain. Calpain-mediated cleavage was identified as a potential mechanism to generate E-Cad-85 from E-Cad-FL by treating lysates from SKOV3 cells and tumour tissue with this enzyme. Increased cytoplasmic localisation of β-Catenin in tumours with high E-Cad-85 expression corroborates that E-Cad-85 loses the binding site for β-Catenin after fragmentation, enabling tumour cluster formation and peritoneal dissemination.. Calpain-mediated E-Cad fragmentation appears to promote intraperitoneal EOC progression. Understanding these mechanisms might eventually lead to new tailored subtype-specific diagnostic and therapeutic interventions.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Blotting, Western; Cadherins; Calpain; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Female; Humans; Immunohistochemistry; Lymph Nodes; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Peptide Fragments; Peritoneal Neoplasms; Retroperitoneal Neoplasms; Retroperitoneal Space; Retrospective Studies

Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1(+/+) and BRCA1(null) status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells.

Topics: 14-3-3 Proteins; Actin Cytoskeleton; BRCA1 Protein; Calpain; Cell Adhesion; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Microfilament Proteins; Neoplasms; Nuclear Proteins; Ovarian Neoplasms; Profilins; Protein Phosphatase 2; Proteomics; Spectrin

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA), and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways, and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA are unknown. Here, we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation, and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibition of calpain but not of caspases or the 26 S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca(2+)-dependent PARC down-regulation. The role of Ca(2+) in PARC down-regulation was further confirmed as ionomycin-induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicate the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca(2+)/calpain in PARC post-translational processing and chemosensitivity.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Calcium Ionophores; Calpain; Carrier Proteins; Caspase 3; Cell Line, Tumor; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Ionomycin; Ovarian Neoplasms; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; Transferases; Tumor Suppressor Protein p53

Ovarian cancer is routinely treated with surgery and platinum-based chemotherapy. Resistance is a major obstacle in the efficacy of this chemotherapy regimen and the ability to identify those patients at risk of developing resistance is of considerable clinical importance. The expression of calpain-1, calpain-2 and calpastatin were determined using standard immunohistochemistry on a tissue microarray of 154 primary ovarian carcinomas from patients subsequently treated with platinum-based adjuvant chemotherapy. High levels of calpain-2 expression was significantly associated with platinum resistant tumours (P = 0.031). Furthermore, high expression of calpain-2 was significantly associated with progression-free (P = 0.049) and overall survival (P = 0.006) in this cohort. The association between calpain-2 expression and overall survival remained significant in multivariate analysis accounting for tumour grade, stage, optimal debulking and platinum sensitivity (hazard ratio = 2.174; 95% confidence interval = 1.144-4.130; P = 0.018). The results suggest that determining calpain-2 expression in ovarian carcinomas may allow prognostic stratification of patients treated with surgery and platinum-based chemotherapy. The findings of this study warrant validation in a larger clinical cohort.

Topics: Adult; Aged; Aged, 80 and over; Calcium-Binding Proteins; Calpain; Chemotherapy, Adjuvant; Disease Progression; Female; Follow-Up Studies; Gene Expression Regulation; Humans; Immunohistochemistry; Middle Aged; Multivariate Analysis; Ovarian Neoplasms; Platinum Compounds; Prognosis; Proportional Hazards Models; Retrospective Studies

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

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Calcium; Calpain; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Dipeptides; DNA-Binding Proteins; Drug Resistance, Neoplasm; Female; Humans; Immunoprecipitation; Intracellular Space; Nuclear Proteins; Ovarian Neoplasms; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Tumor Protein p73; Tumor Suppressor Proteins

Calpains, also called calcium activated neutral proteases (CANP), are expressed ubiquitously. They are intracellular, non-lysosomal cytoplasmic cysteine endopeptidases. Calcium is required for their activation. Their endogenous specific inhibitor is calpastatin, which is expressed ubiquitously and coexists within cells besides calpain. When calcium is present, calpastatin and calpain attach to each other inhibiting the protease. The calpain system plays an important role in many processes including apoptosis, necrosis, ischemia formation and exocytosis. So far, many reports exist on studies about the influence of calpains in different tumors (skin, breast, renal cell and prostate cancers). The role of calpains in pathogenesis or further tumor progression has always been proved in related studies, but their exact function could not be demonstrated. So far, no studies on calpains being involved in the pathogenesis of ovarian cancer have been published. In our study we focused on the expression of the enzymes calpain 1, calpain 2 and their inhibitor calpastatin in normal and malign ovarian tissue. Therefore, we performed immunohistochemical stainings of paraffin slices and evaluated staining intensity (SI), percentage of positive cells (PP) and immunoreactive score (IRS). We evaluated the correlation between enzyme expression in malign and benign ovarian tissues. In malignant ovarian tissue, we found decreased expression, staining intensity and immunoreactive score of calpastatin. With higher grading of the ovarian carcinoma, staining intensity and immunoreactive score of calpain 1 decreased. Staining intensity of calpain 2 in ovarian carcinoma decreased with increasing lymph node status. We clearly demonstrated differences between enzyme expressions in malign and benign tissue. This study could not find any specific function of calpains. Only few studies in the literature have been found that deal with calpain evaluation of ovarian cancer. Additional studies including more patients are required to elucidate the functional role and impact of calpain in tumors in detail.

Topics: Calcium-Binding Proteins; Calpain; Female; Humans; Immunohistochemistry; Ovarian Neoplasms

ARHI is a maternally imprinted tumor suppressor gene that is downregulated in 60% of ovarian and breast cancers. Loss of ARHI expression is associated with tumor progression in breast cancer and decreased disease-free survival in ovarian cancer. ARHI encodes a 26-kDa protein with 55-62% homology to Ras and Rap. In contrast to Ras, ARHI inhibits growth, motility, and invasion. ARHI contains a unique 34 amino-acid extension at its N-terminus and differs from Ras in residues critical for GTPase activity and for its putative effector function. Deletion of ARHI's unique N-terminal extension markedly reduces its inhibitory effect on cell growth. The gene maps to chromosome 1p31 at a site of LOH in 40% of ovarian and breast cancers. Mutations have not been detected, but the remaining allele is silenced by methylation in approximately 10-15 % of cases. In the remaining cancers, ARHI is downregulated by transcriptional mechanisms that involve E2F1 and E2F4, as well as by the loss of RNA binding proteins that decrease the half-life of ARHI mRNA. Transgenic expression of human ARHI in mice produces small stature, induces ovarian atrophy, and prevents postpartum milk production. Reexpression of ARHI in cancer cells inhibits signaling through Ras/Map and PI3 kinase, upregulates P21(WAF1/CIP1), downregulates cyclin D1, induces JNK, and inhibits signaling through STAT3. Marked overexpression of ARHI with a dual adenoviral vector induces caspase-independent, calpain-dependent apoptosis. When ARHI is expressed from a doxycycline-inducible promoter at more physiological levels, autophagy is induced, rather than apoptosis. Growth of ovarian and breast cancer xenografts is reversibly suppressed by ARHI, but expression of the NTD mutant produced only a limited inhibitory effect on growth of xenografts.

Topics: Animals; Apoptosis; Breast Neoplasms; Calpain; Down-Regulation; Female; Genes, Tumor Suppressor; Genomic Imprinting; Humans; Mice; Mice, Transgenic; Ovarian Neoplasms; rho GTP-Binding Proteins; Signal Transduction

Se-methylselenocysteine (Se-MSC) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis, but its mechanism of action is still not well understood. The present study was designed to assess the mechanism of Se-MSC on the induction of apoptosis in SKOV-3 ovarian cancer cells. Se-MSC displayed strong inhibitory effects on cell proliferation and viability of SKOV-3 cells in dose and time dependent manners and induced apoptosis. Investigation of the mechanism of Se-MSC-induced apoptosis revealed that treatment with Se-MSC produced morphological features of apoptosis and DNA fragmentation. This was associated with caspase-3 activation and cleavage of poly(ADP-ribose) polymerase and phospholipase C-gamma1 proteins. However, SKOV-3 cells treated with Se-MSC did not demonstrate cytochrome c accumulation in the cytosol during apoptosis induction. Pretreatment of cells with the caspase inhibitors (z-VAD-fmk and DEVD-CHO) prevented Se-MSC-induced apoptosis. These results suggested that Se-MSC induces apoptosis through cytochrome c-independent caspase-3 activation in SKOV-3 cells. In late stage of apoptosis, p18kDa fragment of Bax was generated with the down-regulation of the expressions of survivin, X-linked inhibitor of apoptosis protein, and human inhibitor of apoptosis protein 1 following Se-MSC treatment, suggesting that the modulation of Bax and IAP (inhibitors of apoptosis) family proteins play some role in Se-MSC-mediated apoptosis. Pre-treatments of z-VAD-fmk and the calpain inhibitor, calpeptin inhibited Bax cleavage. These results suggested that Bax cleavage is mediated by calpain, and calpain activation may be a caspase-dependent one. Taken together, the chemopreventive effects of Se-MSC may be related in part to the caspase-3 activation, the down-regulation of IAP family proteins, and Bax cleavage mediated by caspase-dependent calpain activation.

Topics: Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Calpain; Caspases; Cysteine; Cytochrome c Group; Enzyme Activation; Female; Humans; Inhibitor of Apoptosis Proteins; Organoselenium Compounds; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Selenocysteine; Tumor Cells, Cultured

ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.

Topics: Adenoviridae; Animals; Apoptosis; Breast Neoplasms; Calpain; Caspase Inhibitors; Caspases; Cell Cycle; Cell Division; Female; Genes, Tumor Suppressor; Genetic Therapy; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; rho GTP-Binding Proteins; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays