Resveratrol improves the anticancer effects of doxorubicin in vitro and in vivo models: A mechanistic insight

Abstract

Background

Resveratrol (RSVL), a well known dietary compound and in combination with doxorubicin (DOX) has gained a global importance for cancer prevention. However, mechanism of action by this combination is not well understood till date.

Hypothesis

The synergistic combination of RSVL and DOX might be more effective in anti-cancer activity by modulating the diverse cancer signaling pathways as compared to their alone treatments.

Methods

The cytotoxicity of alone and combination doses of RSVL and DOX were analyzed by colorimetric MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) cell proliferation assay. The migration and colony forming abilities were evaluated by wound healing and clonogenic assays. Apoptosis was detected by Annexin V/PI and DAPI stainings. The cell cycle and intracellular reactive oxygen species (ROS) generation were measured by flow cytometry. The differential expression of genes and proteins were measured by qRT-PCR and western blotting analyses. Finally, in-vivo studies were performed in Ehrlich ascitic carcinoma (EAC) mouse model.

Results

The synergistic combination of DOX (IC20) and RSVL (IC30) was selected based on the combination index values in MCF-7 and MDA-MB-231 cell lines. This combination showed potent growth inhibition with ∼2.5 fold of dose advantage and also significantly decreased the wound healing and clonogenic potential of breast cancer cells. The combination treatment was also found to inhibit the inflammatory response (NF-kB, COX-2), autophagic flux (LC3, Beclin-1), redox regulation (Nrf2) and induces apoptosis (BAX: BCL-2 ratio and Caspase-9) in breast cancer cells. Further, combined dosages of DOX (5 mg/kg b.wt) and RSVL (10 mg/kg b.wt) inhibited tumor volume with increased life span (139%, p value < 0.05) in Ehrlich ascitic carcinoma (EAC) cells bearing mice.

Conclusion

In brief, our results suggested that resveratrol chemosensitizes doxorubicin in combination, through inhibiting breast cancer cells proliferation and invasion, and inducing apoptosis via suppression of chronic inflammation and autophagy.

Introduction

At present, cancer is the major global health concern with persisted high incidence and mortality rate (Ferlay et al. 2015), despite the availability of wide therapeutic options particularly in chemotherapeutic regimen. Majority of chemotherapeutic drugs have been synthesized based on recognized target genes or proteins associated with cancer cells survival or proliferation pathways (Galustian and Dalgleish 2010). However, most of these drugs are having severe side effects due to cytotoxicity on normal non-target cells. In addition, cancer cells also have capability to confer drug resistance due to increased expression of p-glycoprotein leading to increased efflux of these drugs (Lehne 2000). In the tumor initiation and progression, multiple signaling pathways are diverted to the oncogenic events by altering normal cellular homeostasis (Bianco et al. 2006). Continually studies on cancer depicted a complex network of signaling pathways assisting tumor progression by generating the favorable microenvironment for nourishment of cancer cells (Li et al., 2013, Wang et al., 2012). In the current scenario of chemotherapy, multiple combinations of anticancer drugs are being used for cancer patients, these combinations also evidenced to enhance survival rate. However, success of cancer chemotherapy is still limited due to persistence of several others reasons like drug resistance, target of non-cancer cells, metabolic stresses, which lead to severe side effects (Iwamoto 2013). Therefore, a novel combination of chemotherapeutic drugs is primarily required to solve all these issues (Kummar et al., 2010, Wang et al., 2010). From the last several years, combinations of chemotherapeutic drugs with phytochemicals have become a major area of research for all the oncologists. It is because; phytochemicals have reported to enhance chemo-sensitivity or therapeutic efficacy and limits high toxicity of chemotherapeutic drugs (Bolhassani 2015).

Doxorubicin (DOX) is widely used anticancer drug against different human malignancies; it has severe side effects in the form of typhlitis, cardiotoxicity, nephrotoxicity, hepatotoxicity and other toxicity (Pestalozzi et al., 1993, Rashid et al., 2013, Singal and Iliskovic, 1998). Doxorubicin causes a high cellular level of ROS to kill cancer cells; however, necrotic cell death is most predominant in killing of cancer cells, which generated inflammatory micro-environment, which also become a reason for undesired cellular toxicity (Thakur et al. 2008). Hence, doxorubicin alone is not preferable drug. A range of phytochemicals available showed a chemo-preventive effect and sensitize cancer cells toward doxorubicin (Vinod et al. 2013). Among diverse group of phytochemicals, resveratrol (RSVL) has proven their strong chemo-preventive effects in various types of human tumors (Delmas et al., 2014, Kim et al., 2014, Siddiqui et al., 2015), and shown to prevent initiation, promotion and progression of cancer (George et al., 2011, Roy et al., 2009). RSLV is a well known antioxidant and phenolic compound, and promoted growth-arresting ability of anticancer drugs with minimizing cellular toxicity generated by chemotherapeutic drugs (Delmas et al., 2011, Kim et al., 2014). These research outcomes suggested that RSVL governs redox homeostasis, inflammation, cell proliferation and death by modulating cancer associated signaling pathways (Colin et al., 2014, Tosetti et al., 2009). However, the mode of action of RSVL in combination with DOX has not been well understood in depth. Motivated by the present understanding of combination effect of drugs and phytochemicals, we made an effort to delineate the molecular pathways modulated by combined effect of DOX and RSV in breast cancer cell lines and Ehrlich ascitic carcinoma (EAC) mice model.