Effect of PI3K/Akt pathway inhibition-mediated G1 arrest on chemosensitization in ovarian cancer cells.

BACKGROUND: Pharmacological inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway prevents G(1) cell cycle progression into S, resulting in G(1) accumulation. The hypothesis that this arrest might negatively impact on chemotherapeutic agents primarily effective in S, G(2) or M-phase was investigated. MATERIALS AND METHODS: Inhibition of PI3K/Akt pathway signaling via LY294002 and Akti-1/2 was demonstrated by immunoblotting. Cell cycle progression was determined by flow cytometric analysis. Cell proliferation was assayed using the XTT cell viability assay. The Chou and Talalay median effect principal was used to evaluate drug interaction. RESULTS: In SKOV3 and IGROV1 human ovarian cancer cells, LY294002 and Akti-1/2 increased the percentage of cells in G(1) and reversed the cell cycle effects of cisplatin, paclitaxel, gemcitabine and topotecan. Pathway blockade synergistically enhanced the cytotoxicity of cisplatin and paclitaxel, but antagonized gemcitabine and topotecan effects. CONCLUSION: Pharmacological PI3K/Akt inhibition antagonizes the efficacy of chemotherapeutic agents primarily effective in the S or G(2)-phase of the cell cycle.

Dual targeting of phosphoinositide 3-kinase and mammalian target of rapamycin using NVP-BEZ235 as a novel therapeutic approach in human ovarian carcinoma.

PURPOSE: This study evaluates the effect of dual PI3K and mTOR inhibition using NVP-BEZ235 in preclinical models of ovarian cancer as a potential novel therapeutic strategy. EXPERIMENTAL DESIGN: Inhibition of PI3K/Akt/mTOR signaling by NVP-BEZ235 was demonstrated by immunoblotting. The effect on cell proliferation was assessed in 18 ovarian cancer cell lines, including four pairs of syngeneic cisplatin-sensitive and cisplatin-resistant cell lines. The in vivo effects of NVP-BEZ235 on established tumor growth were evaluated using an immunocompetent, transgenic murine ovarian cancer model (LSL-K-ras(G12D/+)Pten(loxP/loxP)). RESULTS: NVP-BEZ235 decreased cell proliferation in all ovarian cancer cell lines assayed and sensitized cisplatin-resistant cells to the cytotoxic effects of cisplatin. Cell lines with PI3K-activating mutations or Pten deletions were significantly more sensitive to the effect of NVP-BEZ235 than cell lines without these mutations (P < 0.05). A statistically significant correlation was found between relative levels of p4E-BP1 and the IC(50) for NVP-BEZ235. In LSL-K-ras(G12D/+)Pten(loxP/loxP) mice with established intraperitoneal tumor disease, oral administration of NVP-BEZ235 decreased pAkt, p4E-BP1 and Ki67 in tumor tissue, and resulted in significantly longer survival compared to control animals (P < 0.05). NVP-BEZ235 also induced cell cycle arrest, caspase 3 activity, and reduced cell migration. CONCLUSIONS: Targeting PI3K and mTOR simultaneously using NVP-BEZ235 effectively inhibits ovarian cancer cell growth even in the presence of platinum resistance and prolongs survival of mice with intra-abdominal ovarian tumor disease. We propose that dual PI3K and mTOR inhibition using NVP-BEZ235 may be an effective novel therapeutic approach in patients with ovarian cancer.

Cisplatin and PI3kinase inhibition decrease invasion and migration of human ovarian carcinoma cells and regulate matrix-metalloproteinase expression.

Targeting of the PI3K (phosphoinositide3-kinase)/Akt/mTOR pathway in human ovarian cancer cells is a promising novel therapeutic strategy. We investigated the effects of cisplatin and the PI3K inhibitor LY294002 on invasion, migration and the expression of essential matrix metalloproteinases (MMPs) in ovarian cancer cells. SKOV3, OVCAR5 and IGROV1 human ovarian cancer cell lines were treated with cisplatin, LY294002 and a combination of both drugs. Invasion and migration of treated cells was assessed using Matrigel and uncoated PET membrane assays. Expression levels of pro-MMP2, MMP2, TIMP1, TIMP2 and MT1-MMP were determined using Western Blotting. Gel zymography was used to quantitate the functional levels of active MMP2. All three cell lines showed significantly reduced invasion and migration after treatment with cisplatin, LY294002, and the combination of both drugs compared to untreated controls. In SKOV3 cells, cisplatin alone and in combination with LY294002 resulted in a 6.3 and 7.1-fold reduction in the total amount of activated MMP2. TIMP1 expression decreased by 5.0, 6.6 and 28.4-fold with cisplatin, LY294002 and the combination respectively (P < 0.05). In contrast, only cisplatin and the combination of both drugs resulted in a significant, 3.7 and 5.1-fold reduction in the level of TIMP2. Expression levels of MT1-MMP remained unchanged. These observations were corroborated in IGROV1 cell lines that showed similar changes of activated MMP2 and TIMP2 expression, but no significant decrease in TIMP1 levels. Our data suggests that inhibition of ovarian cancer cell motility is mediated via down-regulation of activated MMP2, TIMP1 and TIMP2 expression under these treatment conditions.

Anthracyclines, proteasome activity and multi-drug-resistance.

BACKGROUND: P-glycoprotein is responsible for the ATP-dependent export of certain structurally unrelated compounds including many chemotherapeutic drugs. Amplification of P-glycoprotein activity can result in multi-drug resistance and is a common cause of chemotherapy treatment failure. Therefore, there is an ongoing search for inhibitors of P-glycoprotein. Observations that cyclosporin A, and certain other substances, inhibit both the proteasome and P-glycoprotein led us to investigate whether anthracyclines, well known substrates of P-gp, also inhibit the function of the proteasome. METHODS: Proteasome function was measured in cell lysates from ECV304 cells incubated with different doses of verapamil, doxorubicin, daunorubicin, idarubicin, epirubicin, topotecan, mitomycin C, and gemcitabine using a fluorogenic peptide assay. Proteasome function in living cells was monitored using ECV304 cells stably transfected with the gene for an ubiquitin/green fluorescent protein fusion protein. The ability of the proteasome inhibitor MG-132 to affect P-glycoprotein function was monitored by fluorescence due to accumulation of daunorubicin in P-glycoprotein overexpressing KB 8-5 cells. RESULTS: Verapamil, daunorubicin, doxorubicin, idarubicin, and epirubicin inhibited 26S chymotrypsin-like function in ECV304 extracts in a dose-dependent fashion. With the exception of daunorubicin, 20S proteasome function was also suppressed. The proteasome inhibitor MG-132 caused a dose-dependent accumulation of daunorubicin in KB 8-5 cells that overexpress P-glycoprotein, suggesting that it blocked P-glycoprotein function. CONCLUSION: Our data indicate that anthracyclines inhibit the 26S proteasome as well as P-glycoprotein. Use of inhibitors of either pathway in cancer therapy should take this into consideration and perhaps use it to advantage, for example during chemosensitization by proteasome inhibitors.