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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

Analysis apoptosis related proteins with the treatment of ritonavir. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and cell lysate was prepared. Approximately 10 μg of total cell extracts of control and ritonavir treated MDAH-2774 cells were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with PARP, Bcl-2, Bak and p53. β actin was used as a loading control. Native and cleaved PARP are indicated by arrows.
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Figure 3: Analysis apoptosis related proteins with the treatment of ritonavir. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and cell lysate was prepared. Approximately 10 μg of total cell extracts of control and ritonavir treated MDAH-2774 cells were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with PARP, Bcl-2, Bak and p53. β actin was used as a loading control. Native and cleaved PARP are indicated by arrows.

Mentions: The balance between pro and anti-apoptotic signals decides the fate of the cells and therefore we examined the expression levels of genes that are involved in the apoptotic pathway by western blot analysis. Approximately 10 fold increase in the expression of p53 was observed by western blot analysis in the same samples (Fig. 3). Primary cellular function of Poly (ADP-ribose) polymerase-1 (PARP) is not entirely certain, never the less it plays an important role in apoptosis as a cellular response to chemotherapeutic and DNA damaging agents [20]. PARP cleavage is an indirect way of semi-quantitative measurement of the activated caspase-3 and a direct measurement of the extent of the apoptosis. Western blot analysis revealed dose dependent activation of the caspase-3 as determined by its cleavage of PARP. Treatment for 48 h resulted in a dose-dependent cleavage of the 117-kDa PARP to the smaller 85-kDa product. Cleavage was first seen at 15 μM of ritonavir, with further increases in the levels of the 85-kDa cleavage product seen with 25 μM. A dose-dependent increase in the expression of the pro-apoptotic protein Bak with concomitant inhibition of anti-apoptotic protein Bcl-2 was also observed (Fig 3).


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 apoptosis related proteins with the treatment of ritonavir. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and cell lysate was prepared. Approximately 10 μg of total cell extracts of control and ritonavir treated MDAH-2774 cells were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with PARP, Bcl-2, Bak and p53. β actin was used as a loading control. Native and cleaved PARP are indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Analysis apoptosis related proteins with the treatment of ritonavir. MDAH-2774 cells treated with 15 μM ritonavir for 48 h were harvested and cell lysate was prepared. Approximately 10 μg of total cell extracts of control and ritonavir treated MDAH-2774 cells were resolved by SDS-PAGE, transferred to nitrocellulose membrane; and probed with PARP, Bcl-2, Bak and p53. β actin was used as a loading control. Native and cleaved PARP are indicated by arrows.
Mentions: The balance between pro and anti-apoptotic signals decides the fate of the cells and therefore we examined the expression levels of genes that are involved in the apoptotic pathway by western blot analysis. Approximately 10 fold increase in the expression of p53 was observed by western blot analysis in the same samples (Fig. 3). Primary cellular function of Poly (ADP-ribose) polymerase-1 (PARP) is not entirely certain, never the less it plays an important role in apoptosis as a cellular response to chemotherapeutic and DNA damaging agents [20]. PARP cleavage is an indirect way of semi-quantitative measurement of the activated caspase-3 and a direct measurement of the extent of the apoptosis. Western blot analysis revealed dose dependent activation of the caspase-3 as determined by its cleavage of PARP. Treatment for 48 h resulted in a dose-dependent cleavage of the 117-kDa PARP to the smaller 85-kDa product. Cleavage was first seen at 15 μM of ritonavir, with further increases in the levels of the 85-kDa cleavage product seen with 25 μM. A dose-dependent increase in the expression of the pro-apoptotic protein Bak with concomitant inhibition of anti-apoptotic protein Bcl-2 was also observed (Fig 3).

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