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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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Related in: MedlinePlus

Wester blot analysis of AKT siRNA treated MDAH-2774 cells. A. Approximately 10 μg of protein extracts of control and treated samples of MDAH-2774 cells as indicated were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with Bcl-2. β actin was used as a loading control. B. SDS-PAGE analysis of ritonavir treated samples of MDAH-2774 cells.
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Figure 6: Wester blot analysis of AKT siRNA treated MDAH-2774 cells. A. Approximately 10 μg of protein extracts of control and treated samples of MDAH-2774 cells as indicated were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with Bcl-2. β actin was used as a loading control. B. SDS-PAGE analysis of ritonavir treated samples of MDAH-2774 cells.

Mentions: AKT, plays a critical role in controlling the balance between survival and apoptosis and plays a significant role in insulin stimulation of glucose transport[21]. Earlier work with protease inhibitors showed inhibition of AKT in breast and hematological malignancies [22-24]. We tested if ritonavir inhibited AKT signaling in ovarian cancer cell lines using synthetic siRNA as known inhibitor for AKT. We found that ritonavir was more efficacious in decreasing phosphoryated AKT than siRNA. Significant reduction in the expression of AKT was also observed transfected with siRNA, compared with scrambled siRNA transfected control cells. Furthermore ritonavir has synergistic effect on reducing AKT expression when treated together AKT siRNA. In addition, the suppression of AKT both ritonavir treatment and AKT siRNA decreased the expression of anti-apoptotic Bcl-2 expression (Fig. 6A). Further we observed a dose dependent decrease in the Hsp90 levels with ritonavir treatment. Heat shock protein 90 (Hsp90) binds to AKT and protects it from being inactivated by protein phosphatase 2A-mediated dephosphorylation (Fig. 6B).


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)

Wester blot analysis of AKT siRNA treated MDAH-2774 cells. A. Approximately 10 μg of protein extracts of control and treated samples of MDAH-2774 cells as indicated were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with Bcl-2. β actin was used as a loading control. B. SDS-PAGE analysis of ritonavir treated samples of MDAH-2774 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691728&req=5

Figure 6: Wester blot analysis of AKT siRNA treated MDAH-2774 cells. A. Approximately 10 μg of protein extracts of control and treated samples of MDAH-2774 cells as indicated were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with Bcl-2. β actin was used as a loading control. B. SDS-PAGE analysis of ritonavir treated samples of MDAH-2774 cells.
Mentions: AKT, plays a critical role in controlling the balance between survival and apoptosis and plays a significant role in insulin stimulation of glucose transport[21]. Earlier work with protease inhibitors showed inhibition of AKT in breast and hematological malignancies [22-24]. We tested if ritonavir inhibited AKT signaling in ovarian cancer cell lines using synthetic siRNA as known inhibitor for AKT. We found that ritonavir was more efficacious in decreasing phosphoryated AKT than siRNA. Significant reduction in the expression of AKT was also observed transfected with siRNA, compared with scrambled siRNA transfected control cells. Furthermore ritonavir has synergistic effect on reducing AKT expression when treated together AKT siRNA. In addition, the suppression of AKT both ritonavir treatment and AKT siRNA decreased the expression of anti-apoptotic Bcl-2 expression (Fig. 6A). Further we observed a dose dependent decrease in the Hsp90 levels with ritonavir treatment. Heat shock protein 90 (Hsp90) binds to AKT and protects it from being inactivated by protein phosphatase 2A-mediated dephosphorylation (Fig. 6B).

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