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Ritonavir blocks AKT signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells.

Kumar S, Bryant CS, Chamala S, Qazi A, Seward S, Pal J, Steffes CP, Weaver DW, Morris R, Malone JM, Shammas MA, Prasad M, Batchu RB - Mol. Cancer (2009)

Bottom Line: Over a 3 day period with 20 muM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line.These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, MI, USA. skumar@med.wayne.edu

ABSTRACT

Background: Ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stages. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the 5-year survival rate. Chemotherapy with paclitaxel after surgery increases median survival only by 2 to 3 years in stage IV disease highlights the need for more effective drugs. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening of immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail treatment known as HAART (Highly Active Anti-Retroviral Therapy).

Results: Here we show that ritonavir, a HIV protease inhibitor effectively induced cell cycle arrest and apoptosis in ovarian cell lines MDH-2774 and SKOV-3 in a dose dependent manner. Over a 3 day period with 20 muM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line. Ritonavir caused G1 cell cycle arrest of the ovarian cancer cells, mediated by down modulating levels of RB phosphorylation and depleting the G1 cyclins, cyclin-dependent kinase and increasing their inhibitors as determined by gene profile analysis. Interestingly, the treatment of ritonavir decreased the amount of phosphorylated AKT in a dose-dependent manner. Furthermore, inhibition of AKT by specific siRNA synergistically increased the efficacy of the ritonavir-induced apoptosis. These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.

Conclusion: Our results demonstrate a potential use of ritonavir for ovarian cancer with additive effects in conjunction with conventional chemotherapeutic regimens. Since ritonavir is clinically approved for human use for HIV, drug repositioning for ovarian cancer could accelerate the process of traditional drug development. This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.

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Analysis of cell cycle regulatory proteins. A. Approximately 10 μg of protein extracts of control and 5 μM and 20 μM ritonavir treated MDAH-2774 cells for 48 h were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with RB and E2F-1. β actin was used as a loading control. RB-p: hyper phosphorylated retinoblastoma protein; RB: Under phosphorylated retinoblastoma protein. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and total RNA was isolated to generate cRNA and was hybridized to Whole Human Genome (G4112A) arrays (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. B. Analysis of RB family of pocket proteins. C. Analysis of E2F family of transcription factors. D, E and F represent the gene profile analysis of the expression of CDKs and Cyclins and CDKIs respectively that are involved in G0 to G1 phase transition.
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Figure 5: Analysis of cell cycle regulatory proteins. A. Approximately 10 μg of protein extracts of control and 5 μM and 20 μM ritonavir treated MDAH-2774 cells for 48 h were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with RB and E2F-1. β actin was used as a loading control. RB-p: hyper phosphorylated retinoblastoma protein; RB: Under phosphorylated retinoblastoma protein. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and total RNA was isolated to generate cRNA and was hybridized to Whole Human Genome (G4112A) arrays (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. B. Analysis of RB family of pocket proteins. C. Analysis of E2F family of transcription factors. D, E and F represent the gene profile analysis of the expression of CDKs and Cyclins and CDKIs respectively that are involved in G0 to G1 phase transition.

Mentions: Since we observed a significant increase in the population of G0/G1 phase of the cells with the treatment of ritonavir, we evaluated genes that control the cell cycle progression at G0/G1 phase. The cell cycle progression at G0/G1 phase is inhibited by active under-phosphorylated form retinoblastoma protein (RB) which sequesters growth promoting E2F-1 transcription factor. To evaluate if the observed block of S phase entry is due to the activation of RB protein, we first analyzed the levels of phosphorylation of RB and expression of E2F-1 by western blot analysis. We observed inhibition of phosphoryation status of the RB in response to ritonavir in dose dependent manner compared with control along with decrease in the E2F-1 protein levels (Fig. 5A). Gene expression analysis of RB and its related tumor suppressor proteins revealed an increase of 1.44 folds in the RB expression and 1.30 folds of p107 expression but there is decrease in the expression of p130 by 1.1 folds (Fig. 5B). Further we analyzed expression levels of three of the E2F family of proteins, E2F-1, 2 and 3 which interact with RB and as expected we observed 1.53 fold, 3.05 folds and 1.05 folds reduction in the expression levels of E2F-1, 2 and 3 respectively (Fig. 5C). Cyclins and cyclin dependent kinases (CDKs) exhibit distinct expression patterns which contribute to the temporal coordination of each event in cell cycle progression. G1 phase of cell cycle is regulated mainly by cyclin D, E and CDK2, 4 and 6. Ritonavir treatment resulted in the decreased expression levels of G1 phase cyclins and CDKs corroborating inactivation of RB proteins (Fig. 5D and 5E). Further we observed increased expression cyclin dependent kinase inhibitors (CDKIs) which bind and inhibit the activity of cyclin/Cdk complexes and negatively regulate cell cycle progression (Fig. 5F).


Ritonavir blocks AKT signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells.

Kumar S, Bryant CS, Chamala S, Qazi A, Seward S, Pal J, Steffes CP, Weaver DW, Morris R, Malone JM, Shammas MA, Prasad M, Batchu RB - Mol. Cancer (2009)

Analysis of cell cycle regulatory proteins. A. Approximately 10 μg of protein extracts of control and 5 μM and 20 μM ritonavir treated MDAH-2774 cells for 48 h were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with RB and E2F-1. β actin was used as a loading control. RB-p: hyper phosphorylated retinoblastoma protein; RB: Under phosphorylated retinoblastoma protein. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and total RNA was isolated to generate cRNA and was hybridized to Whole Human Genome (G4112A) arrays (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. B. Analysis of RB family of pocket proteins. C. Analysis of E2F family of transcription factors. D, E and F represent the gene profile analysis of the expression of CDKs and Cyclins and CDKIs respectively that are involved in G0 to G1 phase transition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Analysis of cell cycle regulatory proteins. A. Approximately 10 μg of protein extracts of control and 5 μM and 20 μM ritonavir treated MDAH-2774 cells for 48 h were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with RB and E2F-1. β actin was used as a loading control. RB-p: hyper phosphorylated retinoblastoma protein; RB: Under phosphorylated retinoblastoma protein. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and total RNA was isolated to generate cRNA and was hybridized to Whole Human Genome (G4112A) arrays (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. B. Analysis of RB family of pocket proteins. C. Analysis of E2F family of transcription factors. D, E and F represent the gene profile analysis of the expression of CDKs and Cyclins and CDKIs respectively that are involved in G0 to G1 phase transition.
Mentions: Since we observed a significant increase in the population of G0/G1 phase of the cells with the treatment of ritonavir, we evaluated genes that control the cell cycle progression at G0/G1 phase. The cell cycle progression at G0/G1 phase is inhibited by active under-phosphorylated form retinoblastoma protein (RB) which sequesters growth promoting E2F-1 transcription factor. To evaluate if the observed block of S phase entry is due to the activation of RB protein, we first analyzed the levels of phosphorylation of RB and expression of E2F-1 by western blot analysis. We observed inhibition of phosphoryation status of the RB in response to ritonavir in dose dependent manner compared with control along with decrease in the E2F-1 protein levels (Fig. 5A). Gene expression analysis of RB and its related tumor suppressor proteins revealed an increase of 1.44 folds in the RB expression and 1.30 folds of p107 expression but there is decrease in the expression of p130 by 1.1 folds (Fig. 5B). Further we analyzed expression levels of three of the E2F family of proteins, E2F-1, 2 and 3 which interact with RB and as expected we observed 1.53 fold, 3.05 folds and 1.05 folds reduction in the expression levels of E2F-1, 2 and 3 respectively (Fig. 5C). Cyclins and cyclin dependent kinases (CDKs) exhibit distinct expression patterns which contribute to the temporal coordination of each event in cell cycle progression. G1 phase of cell cycle is regulated mainly by cyclin D, E and CDK2, 4 and 6. Ritonavir treatment resulted in the decreased expression levels of G1 phase cyclins and CDKs corroborating inactivation of RB proteins (Fig. 5D and 5E). Further we observed increased expression cyclin dependent kinase inhibitors (CDKIs) which bind and inhibit the activity of cyclin/Cdk complexes and negatively regulate cell cycle progression (Fig. 5F).

Bottom Line: Over a 3 day period with 20 muM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line.These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, MI, USA. skumar@med.wayne.edu

ABSTRACT

Background: Ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stages. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the 5-year survival rate. Chemotherapy with paclitaxel after surgery increases median survival only by 2 to 3 years in stage IV disease highlights the need for more effective drugs. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening of immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail treatment known as HAART (Highly Active Anti-Retroviral Therapy).

Results: Here we show that ritonavir, a HIV protease inhibitor effectively induced cell cycle arrest and apoptosis in ovarian cell lines MDH-2774 and SKOV-3 in a dose dependent manner. Over a 3 day period with 20 muM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line. Ritonavir caused G1 cell cycle arrest of the ovarian cancer cells, mediated by down modulating levels of RB phosphorylation and depleting the G1 cyclins, cyclin-dependent kinase and increasing their inhibitors as determined by gene profile analysis. Interestingly, the treatment of ritonavir decreased the amount of phosphorylated AKT in a dose-dependent manner. Furthermore, inhibition of AKT by specific siRNA synergistically increased the efficacy of the ritonavir-induced apoptosis. These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.

Conclusion: Our results demonstrate a potential use of ritonavir for ovarian cancer with additive effects in conjunction with conventional chemotherapeutic regimens. Since ritonavir is clinically approved for human use for HIV, drug repositioning for ovarian cancer could accelerate the process of traditional drug development. This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.

Show MeSH
Related in: MedlinePlus