Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.

Effect of Spindle Checkpoint on Akt2-mediated Paclitaxel-resistance in A2780 Ovarian Cancer Cells

Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel （PTX）-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint. In the present study, we investigated the role of the Akt2/Bubl cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel （PTX）. Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000, and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting. Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX. Moreover, we compared the expression level of Bubl in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G2/M arrest, and inhibited Bubl expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.

A STUDY OF DECREASING DRUG-RESISTANCE OF HUMAN OVARIAN CANCER CELLS IN VITRO

A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition to ovarian cancer were investigated. A 5- fold increase of cisplatin density converted the tumors which were initially resistance to standard-dose cisplatin Into drug-sensitive ones. Vera-pamil was found to be able to overcome vincristine-resistance of some tumors in vitro. These results suggest that using high dose cisplatin therapy or increasing local drug concentration by using other administration way, we could expect some ovarian cancers that had failed to standard dose cisplatin therapy to be effective. Combination of vincristine with verapamll may be helpful in treating some vincristineresistant cases.

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3(GSK-3)might act as a tumor suppressor since its inhibition is expected tomimic the activation of Wnt-signaling pathway,GSK-3β may contribute to NF-κ3 activation in cancer cells leading toincreased cancer cell proliferation and survival.Here we report that GSK-3β activity was involved in the proliferationof human ovarian cancer cell both in vitro and in vivo.Inhibition of GSK-3 activity by pharmacological inhibitors sup-pressed proliferation of the ovarian cancer cells.Overexpressing constitutively active form of GSK-3β induced entryinto the S phase,increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells.Furthermore,GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovariancancer cells.Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells,implicating this kinase as a potential therapeutic target in ovarian cancer.

Changes of TIZ expression in epithelial ovarian cancer cells

Objective:To study the change of TIZ expression in epithelial ovarian cancer cells.Methods:HO8910 cells were transinfected with siRNA to inhibit the expression of TIZ.pcDNA3.1-TIZ vectors were combined to increase the TIZ expression level.The cell viabilily,colony forming efficiency and cycle distribution of HO8910,HO8910/NC,HO8910/pcDNA3.1-NC,HO8910/ TIZ-573 and HO8910/pcDNA3.l-TIZ were compared,and the invasion rate,migration rate and adhesion rate between 5 groups of cells were compared.Results:Compared with those of HO8910,HO8910/NC and HO8910/pcDNA3.1-NC,the cell viability,colony forming efficiency and cell cycle distribution of HO8910/ TIZ-573 were increased,while the indexes of HO8910/pcDNA3.1-NC were decreased with statistical significant difference(P<0.05).There was no statistical significant difference in the invasion rate,migration rate and adhesion rate between 5 groups of cells(P>0.05).Conclusions:The expression of TIZ can inhibit the proliferation of epithelial ovarian cancer cells.

N-myc downstream-regulated gene 2 promotes proliferation of HO-8910 ovarian cancer cells

Objective To investigate N-myc downstream-regulated gene 2(NDRG2) expression in ovarian cancer cells and its potential usefulness as a diagnostic marker and/or target for therapeutic intervention.Methods Human NDRG2 L/S gene was obtained by revers-transcription polymerase chain reaction(RT-PCR). Sequence analysis confirmed the identity of NDRG2 L/S gene, which was then inserted into a eukaryotic vector p LNCX2, which was in turn transfected into NDRG2 gene-negative HO-8910 cells. Flow cytometry(FCM) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay were conducted to determine the proliferation rate of HO-8910 cells. Cisplatin resistance of HO-8910 cells transfected with p LNCX2-NDRG2 L/S was evaluated by FCM. Tumors were generated in female nude mice by subcutaneous injection of HO-8910 cells.Results NDRG2 gene was isolated and its expression vector was successfully constructed. NDRG2 expression positively correlated with the proliferation of HO-8910 cells. NDRG2 L/S promoted tumorigenicity in HO-8910 cells.Conclusion The present study identified a novel function of NDRG2 L/S gene and demonstrated its involvement in the promotion of ovarian cancer cell proliferation and enhancement of cisplatin resistance in HO-8910 cells. Future studies are warranted to determine the relationship between NDRG2 upregulation and ovarian cancer progression.

Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

Summary： To investigate the expression of cyclooxygenase-2 （COX-2） in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a Basis for the chemoprevention of ovarian cancer.

Expression of cyclooxygenase-2 mRNA in drug-sensitive cell and drug-resistant strains of ovarian cancer cell lines

Objective： To investigate the expression of cyclooxygenase-2 （COX-2） mRNA in drug-sensitive cell and drugresistant clones of ovarian cancer cell lines. Methods： RT-PCR and immunocytochemistry were used to investigate the expression of cyclooxygenase-2 in 3 clones drug-sensitive and 5 clones drug-resistant ovarian cancer cell. Results： Strong COX-2 mRNA expressions were detected in 3 clones of drug-sensitive cell and weak expressions were detected in 5 clones of drug-resistant cell. The protein expression of COX-2 in drug-sensitive cell was strongly positive reaction in immunocytochemistry stain and there was a weak positive reaction in 5 clones of drug-resistant cell. Conclusion： The expression of COX-2 mRNA in drug-sensitive cell strains is much higher than that in drugresistant strains of ovarian cancer cell lines, providing a basis of the chemoprevention for ovarian cancer.

Effects of HLEC on the secreted proteins of epithelial ovarian cancer cells prone to metastasize to lymph nodes

Objective:To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer(EOC)cells(SKOV3-PM4)with directional highly lymphatic metastasis.Methods:Supernatants of four groups of cultured cells,namely,SKOV3(A),SKOV3+HLEC(B),SKOV3-PM4(C),SKOV3-PM4+HLEC(D),were collected,and their proteins were detected by antibody arrays and iTRAQ-2D-LC-MALDITOF/TOF/MS.Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA.Results:Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C(compared with group A),whereas IGFBP7 and SPARC were downregulated in group D(compared with group C).Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other.Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers,benign tumors,and control groups.Two proteins were upegulated in the first group.VEGFA in the control group was downregulated.For IGFBP,upregulation in the control group and down-regulation in the first group were also observed.Conclusion:The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors.The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.

Cinobufotalin as a Novel Agent to Inhibit in Vitro Epithelial Ovarian Cancer Cell Proliferation, Migration and Invasion

Objective: Cinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of giant toads and is utilized in traditional Chinese medicine (Chan Su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Recently, CINO has been shown to inhibit lung cancer cell function and has been implicated in several other disease processes. In this study, we pursued the potential anticancer application of CINO using the ovarian tumor cell line SK-OV-3. Study Design: We evaluated the effect of CINO on cultures of SK-OV-3. Cells were treated with 0.1, 0.5, 1, 5, and 10 μM CINO. Cell proliferation, migration, invasion, and viability were measured using commercially available kits. Cell cycle progression was evaluated by a Cell Cycle Phase Determination Kit. Apoptosis was evaluated by an Apoptotic Blebs Assay Kit;cell cycle arrest and apoptotic signaling were determined by fluorescence-activated cell sorting (FACS) analysis. Results: CINO at ≥ 0.5 μM inhibited SKOV-3 cell proliferation, migration, and invasion (p < 0.05). There was a higher (p < 0.05) percentage of S phase cells in groups treated with CINO at 0.5 μM. CINO at ≥ 0.5 μM down regulated expression of Proliferating Cell Nuclear Antigen (PCNA) and caused cell death. Conclusion: This data demonstrates that CINO impairs SK-OV-3 cell function via cell cycle arrest and apoptotic signaling, suggesting CINO be further investigated as a novel anti-ovarian cancer agent.

Overexpression of OGFr Downregulates Ovarian Cancer Cell Proliferation <i>In Vitro</i>and Inhibits Tumorigenesis

The opioid growth factor (OGF) and its receptor (OGFr) regulate human ovarian cancer cell proliferation through a tonically active inhibitory axis. We investigated the effect of OGFr overexpression on ovarian tumorigenesis. Clonal cell lines of SKOV-3 human ovarian cancer were established to stably overexpress OGFr. shRNA constructs were evaluated for antitumor activity in vitro, as well as in vivo using mouse models of s.c. and i.p. tumor transplantation. The 5 clonal cell lines were characterized by increases in OGFr protein (62% to 245%) and binding capacity (51% - 154%), and decreases (36% - 185%) in cell number, relative to untransfected wild-type (WT) cells and empty vector (EV) transfected clones. Nude mice receiving s.c. injection of 2 overexpressing OGFr cell lines (OGFr-3 and OGFr-22) had reduced tumor incidence, delayed tumor appearance (up to 12 days), and decreased tumor volume (up to 87%) relative to WT and EV controls. Mice injected i.p. with these clonal lines displayed reduced formation of tumor nodules (up to 95%), and depressed tumor weights (up to 99%) compared to WT and EV groups. DNA synthesis, but not cell survival, was depressed in cells and s.c. tumors overexpressing OGFr in comparison to the WT and EV groups. Angiogenesis was reduced up to 86% in clonal tumors compared to WT and EV groups. This preclinical evidence demonstrates that OGFr expression is a molecular determinant of ovarian cancer progression, and has important relevance to understanding the pathogenesis and treatment of this deadly disease.

Dysregulation of the TGF-β Postreceptor Signaling Pathway in Cell Lines Derived from Primary or Metastatic Ovarian Cancer

Transforming growth factor beta (TGF β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF β. Mechanisms of resistance to TGF β caused by modulation of cell cycle regulators and/or inactivation of components of the TGF β signaling transduction pathway such as C myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF β and expression status of transforming growth factor receptor Ⅱ (TβRⅡ), Smad4, CDC25A and C myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi quantitative RT PCR. Normal ovarian surface tissues were used as controls. The expression of TβRⅡ was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88 % (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20 % (1/5) of non tumorigenic cell lines ( P <0.05). C myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF β of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF β induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF β is not associated with a lack of TβRⅡ.

Ovarian cancer stem cells: Can targeted therapy lead to improved progression-free survival?

Despite significant effort and research funds, epithelial ovarian cancer remains a very deadly disease. There are no effective screening methods that discover early stage disease; the majority of patients are diagnosed with advanced disease. Treatment modalities consist primarily of radical debulking surgery followed by taxane and platinum-based chemotherapy. Newer therapies including limited targeted agents and intraperitoneal delivery of chemotherapeutic drugs have improved disease-free intervals, but failed to yield longlasting cures in most patients. Chemotherapeutic resistance, particularly in the recurrent setting, plagues the disease. Targeting the pathways and mechanisms behind the development of chemoresistance in ovarian cancer could lead to significant improvement in patient outcomes. In many malignancies, including blood and other solid tumors, there is a subgroup of tumor cells, separate from the bulk population, called cancer stem cells(CSCs). These CSCs are thought to be the cause of metastasis, recurrence and resistance. However, todate, ovarian CSCs have been difficult to identify, isolate, and target. It is felt by many investigators that finding a putative ovarian CSC and a chemotherapeutic agent to target it could be the key to a cure for this deadly disease. This review will focus on recent advances in this arena and discuss some of the controversies surrounding the concept.

Aiming to immune elimination of ovarian cancer stem cells

Ovarian cancer accounts for only 3% of all cancers in women, but it causes more deaths than any other gynecologic cancer. Treatment with chemotherapy and cytoreductive surgery shows a good response to the therapy. However, in a large proportion of the patients the tumor grows back within a few years. Cancer stem cells, that are less responsive to these treatments, are blamed for this recurrence of disease. Immune therapy either cellular or humoral is a novel concept to treat cancer. It is based on the notice that immune cells invade the tumor. However, the tumor invest heavily to escape from immune elimination by recruiting several immune suppressive mechanisms. These processes are normally in place to limit excessive immune activation and prevent autoimmune phenomena. Here, we discuss current knowledge about the immune(suppressive)status in ovarian cancer. Moreover, we discuss the immunological targets of ovarian cancer stem cells.

γ-Secretase Inhibitor, DAPT Inhibits Self-renewal and Stemness Maintenance of Ovarian Cancer Stem-like Cells In Vitro

Objective: The Notch signaling pathway plays an important role in the stem cell signaling network and contributes to tumorigenesis. However, the functions of Notch signaling in ovarian cancer stem cells (OCSCs) are not well understood. We aimed to investigate the effects of Notch blockade on self-renewal and stemness maintenance of OCSCs. Methods: Ovarian cancer stem-like cells were enriched from ovarian cancer cell lines in serum-free medium. A γ-secretase inhibitor, (DAPT), was used to block Notch signaling. MTT assays were performed to assess self-renewal and proliferation inhibition, flow cytometry was performed to analyze cell surface marker and immunofluorescence, Western Blot and Real-time RT-PCR assays were performed to detect Oct4 and Sox2 protein and mRNA expression of the Ovarian cancer stem-like cells treated with DAPT. Results: Notch blockade markedly inhibits self-renewal and proliferation of ovarian cancer stem-like cells, significantly downregulates the expression of OCSCs-specific surface markers, and reduces protein and mRNA expression of Oct4 and Sox2 in OCSC-like cells. Conclusion: Our results suggest that Notch signaling is not only critical for the self-renewal and proliferation of OCSCs, but also for the stemness maintenance of OCSCs. The γ-secretase inhibitor is a promising treatment targeting OCSCs.

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma

Cancer stem cells (CSCs) or tumor initiating cells are rare cells that are able to establish a tumor or metastasis. Identification of those CSCs is, however, cumbersome even in established cell lines. Several cancer stem cell markers were reported to be expressed by ovarian cancer. Those cancer stem cells are gifted with lower vulnerability to irradiation and cytostatic drugs explaining the high incidence of recurrence after treatment. A variety of different cancer stem cell markers were described for epithelial tumors. Also, cancer cell lines were assessed for stem cell markers with no common denominator. The expression of CD24, CD44, CD117, CD133, ABCG2, ALDH was determined for cells from 22 patients. Ovarian cancer cells were collected from ascites. Part of the tumor cells were analyzed immediately and stained for the above mentioned cancer stem cell markers. The remainder of the cells was cultured for several weeks using standard stem cell culture conditions. We observed a large variety in expression of putative stem cell markers for primary tumors. After two weeks of culture spheres were seen in several cultures, indicative for cancer stem cells, though not all patients’ cells were able to form spheres. Our data show for the first time the heterogeneity in marker display in primary tumors. Also for the cultured cells stem cell markers were determined. None of the stem cell markers was expressed by all patients’ cells. No correlation with tumor type was demonstrated. The complexity of expression challenges the isolation of cancer stem

Presence and role of stem cells in ovarian cancer

Ovarian cancer is the deadliest gynecological malignancy.It is typically diagnosed at advanced stages of the disease,with metastatic sites disseminated widely within the abdominal cavity.Ovarian cancer treatment is challenging due to high disease recurrence and further complicated pursuant to acquired chemoresistance.Cancer stem cell(CSC)theory proposes that both tumor development and progression are driven by undifferentiated stem cells capable of self-renewal and tumor-initiation.The most recent evidence revealed that CSCs in terms of ovarian cancer are not only responsible for primary tumor growth,metastasis and relapse of disease,but also for the development of chemoresistance.As the elimination of this cell population is critical for increasing treatment success,a deeper understanding of ovarian CSCs pathobiology,including epithelial-mesenchymal transition,signaling pathways and tumor microenvironment,is needed.Finally,before introducing new therapeutic agents for ovarian cancer,targeting CSCs,accurate identification of different ovarian stem cell subpopulations,including the very small embryoniclike stem cells suggested as progenitors,is necessary.To these ends,reliable markers of ovarian CSCs should be identified.In this review,we present the current knowledge and a critical discussion concerning ovarian CSCs and their clinical role.

Regulatory Effect of E2,IL-6 and IL-8 on the Growth of Epithelial Ovarian Cancer Cells

To determine the regulatory effects of estrogen and cytokine IL-6 and IL-8 on the growth of epithelial ovarian cancer （OVCA）, we first examined the status of estrogen receptors （ERα and ERβ）, IL-6 receptor （IL-6Rα and gp130）, and IL-8 receptor （IL-8RA and IL-8RB） on five epithelial OVCA cell lines by semiquantitative RT-PCR and Western blot analysis. Results showed that the expressions of these receptors were variable on the five cells. Those OVCA ceUs expressing the receptors were selected to study related molecular mechanism. MTT assay was performed to observe the effects of 17β-estradiol （E2）, IL-6 and IL-8 on cell proliferation. We discovered that E2 markedly promoted the proliferation of CAOV-3 and OVCAR-3 cell in a time- and dose-dependent manner. Tamoxifen （Txf）, an ER inhibitor, completely blocked the proliferation of the E2-induced cells, and IL-6- or/and IL-8-neutralizing antibody only showed partially blocking activity. IL-6 and IL-8 were able to significantly stimulate CAOV-3 and OVCAR-3 cell proliferation in a time- and dose-dependent manner, which had a potential synergistic effect on CAOV-3 cells but not on OVCAR-3 cells. The cell proliferation induced by these two cytokines was abolished completely by their specific neutralizing antibodies, partially by Txf, but not by unrelated goat IgG. Taken together, our results suggested that estrogen, IL-6 and IL-8 could modulate OVCA growth by forming a reciprocal cascade with amplifying effect.

EFFECTS AND MECHANISMS OF ENDOSTATIN ON THE GROWTH OF OVARIAN CANCER SKOV_3 CELLS IN VITRO AND IN VIVO

Objective： To evaluate the inhibitory effect of Endostatin on ovarian cancer cell line SKOV3 and to investigate the possible mechanism of the inhibition. Methods： Using MTT, transmission electron microscope （TEM） and immunocytochemistry, the effects of Endostatin on the proliferation of SKOV3 cells were studied. Nude mice were subcutaneously implanted with SKOV3 cells. The cell apoptosis of implanted tumor was detected by TUNEL and TEM. The expressions of bcl-2 and bax in implanted tumor tissues were measured by RT-PCR and immunohistochemistry. Results： Endostatin significantly inhibited the proliferation of SKOV3 cells in vitro （P〈0.01） and induced cell apoptosis, whereas the expressions of bcl-2 and bax were not changed obviously in SKOV3 cell treated with Endostatin. The mean tumor weight of Endostatin treated group was markedly lower than that of PBS control group （P〈0.05）. The expression of bcl-2 was down-regulated in Endostatin treated group, but bax was not influenced. Conclusions： The results demonstrated that Endostatin might have anti-tumor effect on ovarian carcinoma. One of the important mechanisms of Endostatin effect of anti-angiogenic and anti-tumor activities might involve regulating the bcl-2/bax expression and inducing apoptosis.

Dietary Conjugated Linoleic Acids Arrest Cell Cycle Progression and Prevent Ovarian Cancer Xenografts Growth Suggesting a Trans-10 Cis-12 Isoform Specific Activity

Therapies for treating ovarian cancer (OvCa) successfully are largely inadequate. Alternative therapies and diet(s) with preventive potential to debilitated onset, and reduced OvCa tumor burden in situ, have not been systematically studied. Preventive role of conjugated linoleic acids (CLAs) has been reported in many other cancers. We report the first systematic in vitro and in vivo study modeling potential preventive mechanism(s) of CLA, an octadecadienolic fatty acid in clear cell OvCa cell line TOV-21G. We demonstrate that a dose and time-dependent down-regulation of cyclin E and A proteins (p 0.05) by CLA (t10,c12) was concomitant with cell cycle arrest of TOV-21G cell lines in S phase. To understand the molecular mechanism underlying CLA (t10,c12) induced S phase arrest, levels of cell cycle regulatory proteins were determined by western blot analyses. Exposure to CLA (t10,c12) increased p21(CIP1/WAF1), and p27(KIP1) protein levels in a time and dose-dependent manner. Interestingly CLA (t10,c12) did not significantly affect protein levels of cyclin-dependent kinase (cdk) 2, and p53, however, hyperphosphorylated form of pRb (p 0.05) was abrogated. Exposure to CLA (c9,t11) indicated a modest increase in p21(CIP1/WAF1) and p27(KIP1) levels, but changes in cyclin A and E levels were statistically insignificant. These results indicate that CLA (t10,c12) mediated p27(KIP1) upregulation and inhibition of hyperphosphorylation of ppRb may be the possible mechanism for the S phase arrest in TOV-21G cell line. Our in vivo data showed that CLA reduced the progression of TOV-21G xenografts by >50%. Together our results provide evidence of CLA exerted preventive effect on OvCa cell and tumor growth. Tumor growth arrest may be resultant from CLA (t10,c12) mediated modulation of cell cycle arrest.