L. S. CHATURVEDI1,2,3, D. VYAS1,3 1SAN JOAQUIN GENERAL HOSPITAL,SURGERY,FRENCH CAMP, CALIFORNIA, USA 2CALIFORNIA NORTHSTATE UNIVERSITY,PHARMACEUTICAL SCIENCES AND BIOMEDICAL SCIENCES,ELK GROVE, CALIFORNIA, USA 3CALIFORNIA NORTHSTATE UNIVERSITY,COLLEGE OF MEDICINE,ELK GROVE, CALIFORNIA, USA
Introduction:
It is estimated that one million cases of breast cancer are diagnosed annually worldwide. Of these, approximately 12-20% are of the triple-negative breast cancer (TNBC) that do not express receptors for estrogen (ER), progesterone (PR) or human epidermal growth factor receptor 2 (HER2). TNBC is typically treated with a combination of other therapies such as chemotherapy, radiation, and surgery. Therefore, there is urgent need of new therapy for TNBC patients. Carfilzomib (CFZ) is a selective irreversible second-generation proteasome inhibitor being used for the treatment of relapsed and refractory multiple myeloma as the anticancer therapy. We have previously reported antiproliferative and apoptotic effects of CFZ alone or in combination with Doxorubicin (DOX) on human TNBC-MDA-MB-231 cancer cells. Overexpression of cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1 and an elevated phosphorylation of stress-activated c-Jun NH2-terminal kinase has been reported in basal-types and TNBCs with poor prognosis. However, the role of CFZ in the regulation of CDKN1A/p21Waf1/Cip1 protein expression and c-Jun NH2-terminal kinase activation has not been determined in human TNBC-MDA-MB-231 cancer cells. Herein, we investigated the role of CFZ in the modulation of CDKN1A/p21Waf1/Cip1 and c-Jun NH2-terminal kinase activation in human TNBC-MDA-MB-231 breast cancer cells.
Methods:
Human TNBC-MDA-MB-231 cell-line, a model for TNBC was treated by various concentrations of CFZ alone, in a combination with DOX or vehicle control dimethyl sulfoxide (DMSO). Cell Counting Kit-8 (CCK-8) using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) was used to detect cell viability. Annexin V-FITC apoptosis detection kit and flow cytometry were used to analyze the cell cycle. Western blot was used to detect the expression of cyclin-dependent kinase inhibitor CDKN1A/p21Waf1/Cip1 protein and phosphospecific c-Jun NH2-terminal kinase antibodies
Results:
We confirmed the antiproliferative and apoptotic effects of CFZ alone or in combination with DOX using CCK-8 viability and Annexin V-FITC apoptosis detection Kit respectively. The immunoblot analysis revealed that CFZ alone significantly inhibited CDKN1A/p21Waf1/Cip1 in a dose-dependent manner and as well as in a combination with DOX in comparison to vehicle control DMSO. Furthermore, CFZ alone and in a combination with DOX also significantly inhibited the phosphorylation of stress-activated c-Jun NH2-terminal kinase in human TNBC- MDA-MB-231 cancer cells.
Conclusion:
Our data suggest that irreversible proteasome inhibitor CFZ alone and in combination with DOX may induce apoptosis and inhibit proliferation by modulating CDKN1A/p21Waf1/Cip1 protein expression and phosphorylation of stress-activated c-Jun NH2-terminal kinase in human MDA-MB-231 breast cancer cells. Further investigation will encourage the potential use of CFZ alone and in the combination of DOX against tumors harboring drug-resistant phenotypes.