Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1)

Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted.     

Effect of Ovarian Cancer Ascites on Cell Migration and Gene Expression in an Epithelial Ovarian Cancer In Vitro Model

A third of patients with epithelial ovarian cancer (EOC) present ascites. The cellular fraction of ascites often consists of EOC cells, lymphocytes, and mesothelial cells, whereas the acellular fraction contains cytokines and angiogenic factors. Clinically, the presence of ascites correlates with intraperitoneal and retroperitoneal tumor spread. We have used OV-90, a tumorigenic EOC cell line derived from the malignant ascites of a chemonaive ovarian cancer patient, as a model to assess the effect of ascites on migration potential using an in vitro wound-healing assay. A recent report of an invasion assay described the effect of ascites on the invasion potential of the OV-90 cell line. Ascites sampled from 31 ovarian cancer patients were tested and compared with either 5% fetal bovine serum or no serum for their nonstimulatory or stimulatory effect on the migration potential of the OV-90 cell line. A supervised analysis of data generated by the Affymetrix HG-U133A GeneChip identified differentially expressed genes from OV-90 cells exposed to ascites that had either a nonstimulatory or a stimulatory effect on migration. Ten genes (IRS2, CTSD, NRAS, MLXIP, HMGCR, LAMP1, ETS2, NID1, SMARCD1, and CD44) were upregulated in OV-90 cells exposed to ascites, allowing a nonstimulatory effect on cell migration. These findings were validated by quantitative polymerase chain reaction. In addition, the gene expression of IRS2 and MLXIP each correlated with prognosis when their expression was assessed in an independent set of primary cultures established from ovarian ascites. This study revealed novel candidates that may play a role in ovarian cancer cell migration.     

The Role of Heat Shock Protein 90B1 in Patients with Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is a heterogenetic disorder in women that is characterized by arrested follicular growth and anovulatory infertility. The altered protein expression levels in the ovarian tissues reflect the molecular defects in folliculogenesis. To identify aberrant protein expression in PCOS, we analyzed protein expression profiles in the ovarian tissues of patients with PCOS. We identified a total of 18 protein spots that were differentially expressed in PCOS compared with healthy ovarian samples. A total of 13 proteins were upregulated and 5 proteins were downregulated. The expression levels of heat shock protein 90B1 (HSP90B1) and calcium signaling activator calmodulin 1 (CALM1) were increased by at least two-fold. The expression levels of HSP90B1 and CALM1 were positively associated with ovarian cell survival and negatively associated with caspase-3 activation and apoptosis. Knock-down of HSP90B1 with siRNA attenuated ovarian cell survival and increased apoptosis. In contrast, ovarian cell survival was improved and cell apoptosis was decreased in cells over-expressing HSP90B1. These results demonstrated the pivotal role of HSP90B1 in the proliferation and survival of ovarian cells, suggesting a critical role for HSP90B1 in the pathogenesis of PCOS. We also observed a downregulation of anti-inflammatory activity-related annexin A6 (ANXA6) and tropomyosin 2 (TPM2) compared with the normal controls, which could affect cell division and folliculogenesis in PCOS. This is the first study to identify novel altered gene expression in the ovarian tissues of patients with PCOS. These findings may have significant implications for future diagnostic and treatment strategies for PCOS using molecular interventions.     

B19, a Novel Monocarbonyl Analogue of Curcumin,Induces Human Ovarian Cancer Cell Apoptosis via Activation of Endoplasmic Reticulum Stress and the Autophagy Signaling Pathway

Background: The unfolded protein response, autophagy and endoplasmic reticulum (ER) stress-induced apoptosis regulate tumor cell fate and have become novel signaling targets for the development of cancer therapeutic drugs. Curcumin has been used to treat several different cancers, including ovarian cancer, in clinical trials and research; however, the role of ER stress and autophagy in the therapeutic effects of curcumin and new curcumin analogues remains unclear.Methods: Cell viability was determined using the MTT assay. Apoptosis was detected using flow cytometry with PI/Annexin V-FITC staining. The expression levels of ER stress- and autophagy-related proteins were analyzed by western blotting. The activation of autophagy was detected using immunofluorescence staining.Results: We demonstrated that B19 induced HO8910 cell apoptosis in a dose-responsive manner. We also determined and that this effect was associated with corresponding increases in a series of key components in the UPR and ER stress-mediated apoptosis pathways, followed by caspase 3 cleavage and activation. We also observed that B19 treatment induced autophagy in HO8910 cells. The inhibition of autophagy using 3-methyladenine (3-MA) increased levels of intracellular misfolded proteins, which enhanced ovarian cancer apoptosis.Conclusions: Our data indicate that ER stress and autophagy may play a role in the apoptosis that is induced by the curcumin analogue B19 in an epithelial ovarian cancer cell line and that autophagy inhibition can increase curcumin analogue-induced apoptosis by inducing severe ER stress.     

Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)     

Salinomycin inhibits Akt/NF-κB and induces apoptosis in cisplatin resistant ovarian cancer cells

BackgroundDespite advances in treatment, ovarian cancer is the most lethal gynecologic malignancy. Therefore significant efforts are being made to develop novel strategies for the treatment of ovarian cancer. Salinomycin has been shown to be highly effective in the elimination of cancer stem cells both in vitro and in vivo. The present study focused on investigating important cell signaling molecules such as Akt and NF-κB during salinomycin-induced apoptosis in cisplatin resistant ovarian cancer cells (A2780cis).MethodsMTT assay was performed to determine cell viability. Flow cytometry and DNA fragmentation assay were performed to analyze the effect on cell cycle and apoptosis. The expression of apoptosis related proteins was evaluated by Western blot analysis.ResultsThe cell viability was significantly reduced by salinomycin treatment in a dose dependent manner. The flow cytometry result showed an increase in sub-G1 phase. Salinomycin inhibited the nuclear transportation of NF-κB, and downregulated Akt expression. Declined Bcl-2, activation of caspase-3 and increased PARP cleavage triggered apoptosis. Moreover, DNA fragmentation assay also revealed apoptotic induction.ConclusionThe result suggested that salinomycin-induced apoptosis in A2780cis was associated with inhibition of Akt/NF-κB. It may become a potential chemotherapeutic agent for the cisplatin resistant ovarian cancer therapy.     

Elaiophylin,a novel autophagy inhibitor,exerts antitumor activity as a single agent in ovarian cancer cells

Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. Here, we screened the North China Pharmaceutical Group Corporation''s pure compound library of microbial origin using GFP-LC3B-SKOV3 cells and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin promotes autophagosome accumulation but blocks autophagic flux by attenuating lysosomal cathepsin activity, resulting in the accumulation of SQSTM1/p62 in various cell lines. Moreover, elaiophylin destabilizes lysosomes as indicated by LysoTracker Red staining and CTSB/cathepsin B and CTSD/ cathepsin D release from lysosomes into the cytoplasm. Elaiophylin eventually decreases cell viability, especially in combination with cisplatin or under hypoxic conditions. Furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis; however, high doses (8 mg/kg) of elaiophylin lead to dysfunction of Paneth cells, which resembles the intestinal phenotype of ATG16L1-deficient mice. Together, these results provide a safe therapeutic window for potential clinical applications of this compound. Our results demonstrate, for the first time, that elaiophylin is a novel autophagy inhibitor, with significant antitumor efficacy as a single agent or in combination in human ovarian cancer cells, establishing the potential treatment of ovarian cancer by this compound.     

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade.

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.     

Combination of AT-101/cisplatin overcomes chemoresistance by inducing apoptosis and modulating epigenetics in human ovarian cancer cells

We investigated the effects of AT-101/cisplatin combination treatment on the expression levels of apoptotic proteins and epigenetic events such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme activities in OVCAR-3 and MDAH-2774 ovarian cancer cells. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were performed. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. DNMT and HDAC activities were evaluated by ELISA assay and mRNA levels of DNMT1 and HDAC1 genes were quantified by qRT-PCR. Combination of AT-101/cisplatin resulted in strong synergistic cytotoxicity and apoptosis in human ovarian cancer cells. Combination treatment reduced some pivotal anti-apoptotic proteins such as Bcl-2, HIF-1A, cIAP-1, XIAP in OVCAR-3 cells, whereas p21, Bcl-2, cIAP-1, HSP27, Clusterin and XIAP in MDAH-2774 cells. Among the pro-apoptotic proteins, Bad, Bax, Fas, phospho-p53 (S46), Cleaved caspase-3, SMAC/Diablo, TNFR1 and Cytochrome c were induced in OVCAR-3 cells, whereas, Bax, TRAILR2, FADD, p27, phospho-p53 (S46), Cleaved caspase-3, Cytochrome c, SMAC/Diablo and TNFR1 were induced in MDAH-2774 cells. Combination treatment also inhibited both DNMT and HDAC activities and also mRNA levels in both ovarian cancer cells. AT-101 exhibits great potential in sensitization of human ovarian cancer cells to cisplatin treatment in vitro, suggesting that the combination of AT-101 with cisplatin may hold great promise for development as a novel chemotherapeutic approach to overcome platinum-resistance in human ovarian cancer.     

Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas

Background

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.

Methodology/Principal Findings

In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.

Conclusions/Significance

In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.     

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

Tubocapsenolide A, a novel withanolide, inhibits proliferation and induces apoptosis in MDA-MB-231 cells by thiol oxidation of heat shock proteins

Tubocapsenolide A (TA), a novel withanolide-type steroid, exhibits potent cytotoxicity against several human cancer cell lines. In the present study, we observed that treatment of human breast cancer MDA-MB-231 cells with TA led to cell cycle arrest at G(1) phase and apoptosis. The actions of TA were correlated with proteasome-dependent degradation of Cdk4, cyclin D1, Raf-1, Akt, and mutant p53, which are heat shock protein 90 (Hsp90) client proteins. TA treatment induced a transient increase in reactive oxygen species and a decrease in the intracellular glutathione contents. Nonreducing SDS-PAGE revealed that TA rapidly and selectively induced thiol oxidation and aggregation of Hsp90 and Hsp70, both in intact cells and in cell-free systems using purified recombinant proteins. Furthermore, TA inhibited the chaperone activity of Hsp90-Hsp70 complex in the luciferase refolding assay. N-Acetylcysteine, a thiol antioxidant, prevented all of the TA-induced effects, including oxidation of heat shock proteins, degradation of Hsp90 client proteins, and apoptosis. In contrast, non-thiol antioxidants (trolox and vitamin C) were ineffective to prevent Hsp90 inhibition and cell death. Taken together, our results demonstrate that the TA inhibits the activity of Hsp90-Hsp70 chaperone complex, at least in part, by a direct thiol oxidation, which in turn leads to the destabilization and depletion of Hsp90 client proteins and thus causes cell cycle arrest and apoptosis in MDA-MB-231 cells. Therefore, TA can be considered as a new type of inhibitor of Hsp90-Hsp70 chaperone complex, which has the potential to be developed as a novel strategy for cancer treatment.     

Cathepsins as effector proteases in hepatocyte apoptosis

Conclusion Cathepsins B and D appear to act as part of the effector protease cascade in hepatocyte apoptosis, both in bile salt-induced apoptosis and CPT-induced apoptosis of hepatocellular cancer cell lines. It is important to note that these proteases do not appear to participate in many models of apoptosis studied to date; in fact, cathepsin inhibitors have been used as negative controls to show that enzymes other than caspases are not involved in apoptosis. In particular, it has been shown that cathepsin B inhibitors do not prevent many models of apoptosis in lymphocytes (43). Further, our experiments have shown that not all models of hepatocyte apoptosis are mediated by cathepsins. For example, staurosporine-induced apoptosis is not inhibited by cathepsin B inhibitors in primary hepatocytes or in cell lines stably transfected with the cathepsin B antisense construct. Although the signaling pathways leading to activation of cathepsins B and D in hepatocyte apoptosis are not completely understood, we hypothesize that a caspase 8-like protein may be involved proximal to cathepsins D and B (Fig. 6). The precise mechanism by which cathepsin B is translocated from lysosomes to “apoptotic targets” is currently under investigation in our laboratory. Because of the relative promiscuity of cathepsin B as protease, it is likely that it is involved in nonspecific protein degradation in apoptotic bodies; however, cathepsin B has also been shown to degrade certain specific proteins, such as histones, which may be directly relevant to the apoptotic process. Further evaluation of the role of cathepsins B and D in apoptosis should include the determination of specific proteolytic targets that result in the biochemical and morphologic manifestations of apoptosis.     

Recognition of cytoplasmic RNA results in cathepsin-dependent inflammasome activation and apoptosis in human macrophages

dsRNA is an important pathogen-associated molecular pattern that is primarily recognized by cytosolic pattern-recognition receptors of the innate-immune system during virus infection. This recognition results in the activation of inflammasome-associated caspase-1 and apoptosis of infected cells. In this study, we used high-throughput proteomics to identify secretome, the global pattern of secreted proteins, in human primary macrophages that had been activated through the cytoplasmic dsRNA-recognition pathway. The secretome analysis revealed cytoplasmic dsRNA-recognition pathway-induced secretion of several exosome-associated proteins, as well as basal and dsRNA-activated secretion of lysosomal protease cathepsins and cysteine protease inhibitors (cystatins). Inflammasome activation was almost completely abolished by cathepsin inhibitors in response to dsRNA stimulation, as well as encephalomyocarditis virus and vesicular stomatitis virus infections. Interestingly, Western blot analysis showed that the mature form of cathepsin D, but not cathepsin B, was secreted simultaneously with IL-18 and inflammasome components ASC and caspase-1 in cytoplasmic dsRNA-stimulated cells. Furthermore, small interfering RNA-mediated silencing experiments confirmed that cathepsin D has a role in inflammasome activation. Caspase-1 activation was followed by proteolytic processing of caspase-3, indicating that inflammasome activation precedes apoptosis in macrophages that had recognized cytoplasmic RNA. Like inflammasome activation, apoptosis triggered by dsRNA stimulation and virus infection was effectively blocked by cathepsin inhibition. In conclusion, our results emphasize the importance of cathepsins in the innate immune response to virus infection.     

Specific human antibodies do not inhibit Trypanosoma cruzi oligopeptidase B and cathepsin B, and immunoglobulin G enhances the activity of trypomastigote-secreted oligopeptidase B

Trypanosoma cruzi expresses oligopeptidase B and cathepsin B that have important functions in the interaction with mammalian host cells. In this study, we demonstrated that sera from both chagasic rabbits and humans have specific antibodies to highly purified native oligopeptidase B and cathepsin B. Levels of antibodies to cathepsin B were higher than those observed to oligopeptidase B by absorbance values recorded upon ELISA. We next showed that 90% and 30% of sera from individuals with mucocutaneous leishmaniasis have antibodies that recognize oligopeptidase B and cathepsin B as antigens, respectively. In addition, 55% and 40% of sera from kala-azar patients have antibodies to oligopeptidase B and cathepsin B, respectively. Sera from malaria patients did not recognize the proteases as antigens. Despite high levels of specific antibodies, sera from T. cruzi-infected patients did not inhibit the activities of either oligopeptidase B or cathepsin B. Furthermore, sera or IgG purified from either infected or non-infected individuals enhanced the enzymatic activity of the secreted oligopeptidase B. Oligopeptidase B secreted by trypomastigotes and cathepsin B released upon parasite lysis retain their enzymatic activities and may be associated with Chagas' disease pathogenesis by hydrolyzing host proteins and inducing host immune responses.     

Anti-proliferative and metastatic protease inhibitory activities of protoberberines: An in silico and in vitro approaches

In metastasis, tumour cells interact with numerous factors and one of them is extracellular matrix (ECM). Earlier studies have emphasised the on role of matrix metalloproteases (MMPs) and cathepsin B in ECM degradation and cancer metastasis. In silico docking approaches are a boon in exploring the physicochemical characteristics like angiogenesis, growth and repair of cancerous cells. The binding affinities of berberrubine, jatrorrhizine and thalifendine with caspase 3, MMP-9, cathepsin B and telomeric DNA were performed using hex 6v and iGEMDOCK v2.1 software tools. The present investigation on berberrubine, jatrorrhizine and thalifendine revealed the formation of potentially stable complexes with caspase 3, cathepsin B and telomeric DNA similar to doxorubicin, a well-known anticancer drug. Further, in vitro approaches were made to study anti-proliferative activity against colon, lymphoma and ovarian cancer cell lines and enzyme inhibition activity against MMP-9 and cathepsin B. The results obtained reveal that these protoberberines alkaloids have potential inhibitory effect on cancer cell proliferation as well as metastatic proteases.     

Geraniin suppresses ovarian cancer growth through inhibition of NF‐κB activation and downregulation of Mcl‐1 expression

This study investigated the anticancer effects of geraniin on ovarian cancer cells and the signaling pathways involved. Ovarian cancer cells were treated with different concentrations of geraniin for 48 h and examined for viability, apoptosis, mitochondrial membrane depolarization, and gene expression. Xenograft tumor studies were performed to determine the anticancer activity of geraniin in vivo. Geraniin significantly decreased cancer cell viability in a concentration‐dependent fashion. Geraniin significantly triggered apoptosis, which was accompanied by loss of mitochondrial membrane potential and increased cytochrome c release and caspsase‐3 activity. Mechanistically, geraniin significantly downregulated Mcl‐1 and impaired NF‐κB p65 binding to the mcl‐1 promoter. Overexpression of Mcl‐1 significantly reversed geraniin‐induced apoptosis in OVCAR3 cells. In addition, geraniin retarded ovarian cancer growth and reduced expression of phospho‐p65 and Mcl‐1. Collectively, geraniin elicits growth suppression in ovarian cancer through inhibition of NF‐κB and Mcl‐1 and may provide therapeutic benefits for this malignancy.     

Genetic down-regulation of phosphoinositide 3-kinase by bikunin correlates with suppression of invasion and metastasis in human ovarian cancer HRA cells

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.