Abstract
Application of doxorubicin (Dox) for the treatment of cancer is restricted due to its severe side effects. We used combination strategy by combining doxorubicin (Dox) with withaferin A (WFA) to minimize the ill effects of Dox. Treatment of various epithelial ovarian cancer cell lines (A2780, A2780/CP70 and CaOV3) with combination of WFA and Dox (WFA/DOX) showed a time- and dose-dependent synergistic effect on inhibition of cell proliferation and induction of cell death, thus reducing the dosage requirement of Dox. Combination treatment resulted in a significant enhancement of ROS production resulting in immense DNA damage, induction of autophagy analyzed by transmission electron microscope and increase in expression of autophagy marker LC3B, and culminated in cell death analyzed by cleaved caspase 3. We validated combination therapy on tumor growth using an in vitro 3Dimension (3D) tumor model and the more classic in vivo xenograft model of ovarian cancer. Both tumor models showed a 70 to 80% reduction in tumor growth compared to control or animals treated with WFA or Dox alone. Immunohistochemical analysis of the tumor tissues from animals treated with WFA/Dox combination showed a significant reduction in cell proliferation and formation of microvessels accompanied by increased in LC3B level, cleaved caspase 3, and DNA damage. Taken together, our data suggest that combining WFA with Dox decreases the dosage requirement of Dox, therefore, minimizing/eliminating the severe side effects associated with high doses of DOX, suggesting the application of this combination strategy for the treatment of ovarian and other cancers with no or minimum side effects.
Highlights
Ovarian cancer is the most lethal malignancy of the female reproductive tract [1]
We examined the effect of combination treatment in vivo on tumor growth, proliferation, angiogenesis, autophagy, cell death, and DNA damage using xenograft tumors produced by injecting A2780 cells in nude mice
We demonstrated that a low dose of Dox (1 mg/kg) combined with a suboptimal dose of withaferin A (WFA) (2 mg/kg) was highly effective in suppressing tumor progression (Fig. 9) by reducing proliferation and angiogenesis while increasing autophagy, DNA damage, and apoptosis (Fig. 10), indicating that combining WFA with Dox reduces the dosage requirement of Dox to suppress tumor growth, and could minimize or eliminate the side effects including myocardial toxicity associated with high doses of Dox used to treat various solid cancers including ovarian cancer
Summary
Ovarian cancer is the most lethal malignancy of the female reproductive tract [1]. Due to lack of symptoms at an early stage of the disease, the five-year survival rate is only 27.2% [1]. The mainline treatment of ovarian cancer is cytoreductive surgery followed by platinum-based chemotherapy [2]. Ovarian cancer responds positively in 70 to 80% of the cases [3]. Within 18 to 24 months after initial treatment, tumor relapse occurs, which (for approximately 70% of patients) is attributed to the carcinomas having become platinum-resistant [3] This poor survival rate for women with platinum-resistant ovarian carcinomas points to an urgent need for an alternative treatment strategy. Doxorubicin (Dox) is a broad-spectrum anthracylin isolated from Streptomyces peucetius that has been used for the treatment of several cancers, including ovarian, breast, and prostate [4]. Pegylated liposomal doxorubicin (PLD) (DOXIL) is regarded as one of the standard treatment options in recurrent ovarian cancers (ROC) [6]. Despite comparatively lower side effects, Doxil has very low response rate (,20%) [6]
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