Introduction
Breast cancer is the leading cause of death in women worldwide and it is the most common cancer in the Arab world. It affects women at an early age compared with women in western countries[
1]. Doxorubicin (DOX), an anthracycline antibiotic is among the most effective anticancer agents used to treat breast cancer[
2]. It exerts its cytotoxic effect by intercalating between DNA base pairs on the double helix and inhibiting topoisomerase II (TOPO-II), the enzyme responsible for DNA helix conformation and stability. Unfortunately, chronic cardiotoxicity including development of a cardiomyopathy is a major limiting factor of the chemotherapeutic use of doxorubicin[
3]. In an attempt to minimize DOX effective chemotherapeutic dose and thereby its side effects, a variety of approaches have been Investigated. One of them is the search for natural compounds with chemopreventive or anticancer properties that can be used in combination with doxorubicin. Resveratrol (RSVL) (trans – 3, 5, 4 – trihydroxystilbene) is a naturaly occurring poly-phenolic compound found primarily in root extracts of the oriental plant Polygonum cuspidatum and many other plant species[
4]. It is highly abundant in skins of red grapes and moderately abundant in peanuts and blueberries[
4]. It has recently been discovered that it has many beneficial effects in different biological systems, which include anti-inflammatory, antioxidant, anti-neoplastic, anti-carcinogenic, anti-tumorigenic, cardioprotective, neuroprotective, anti-aging and antiviral effects[
4]. Its potential chemopreventive and chemotherapeutic activities have been demonstrated in all three stages of carcinogenesis (initiation, promotion, and progression)[
5]. Resveratrol exhibits anticancer properties in a wide variety of tumor cells, including breast cancer cells[
6]. The growth-inhibitory effect of RSVL is mediated through different mechanisms[
7]. Therefore this study was aimed to explore whether the natural product resveratrol could enhance the cytotoxic effect of DOX against the growth of human breast cancer cell line (MCF-7 cell line). We investigated the possible mechanisms of interaction between DOX and RSVL regarding DOX cytotoxicity, apoptosis induction, cellular uptake and cell cycle progression of breast cancer cells in presence and absence of RSVL.
Discussion
Doxorubicin is the most widely used drug in the treatment of a variety of human neoplasms, However, with the increasing use of DOX, acute as well as chronic cumulative dose-dependent cardiomyopathy has been recognized as the major limiting factor for DOX chemotherapy[
12,
13]. Therefore, in this study we investigated the modulatory effect of the natural polyphenolic compound, RSVL on DOX cytotoxicity in MCF-7 human breast cancer cell line.
Treatment of MCF-7 cells with different DOX doses alone was observed to be cytotoxic to the cells. The cytotoxicity of DOX has been confirmed by the results of induction of apoptosis and cell cycle progression, where 0.25 μg/ml DOX induced 49 –fold increase in early apoptotis and 2-fold increase in arrest of the cells in S phase in comparison with control cells.
Similar results was obtained following single treatment of DOX in MCF-7 cells[
14]. In support of the importance of cell-cycle arrest to DOX cytotoxicity, it has been found that P388 leukemia cells synchronized in S and G
2/M phases were more sensitive to DOX than cells in G
1 phase[
15]. Our results, have further confirmed the fact that anthracyclines are mostly active on proliferating cells in S and G
2/M phases due to the maximal expression of their target enzyme TOPO II at these phases[
16,
17].
Resveratrol is known to have both cardioprotective and antitumor activities[
7,
18] and it can attenuate DOX-induced early cellular damage in cancer patients[
19]. Thus RSVL is a perfect candidate to be used as a sensitizing agent to modulate the cytotoxic effect of DOX against the growth of breast cancer cells. We also observed that, MCF-7 cells treated with RSVL alone showed high increase in early apoptosis, S-phase and in G
0 phase (Figures
2 and
3). Resveratrol has previously been shown to induce dose-dependent cell cycle arrest, growth inhibition or apoptosis in several human cancer cell lines[
20]. Resveratrol apoptosis induction effect in tumor cell line from different origins was shown to be through a lot of different regulatory mechanisms[
21,
22]. Previous studies on the effects of RSVL on the cell cycle of many cell lines including MCF-7 cells, demonstrated the ability of RSVL to block the S–G
2 transition resulting in a concentration-dependent accumulation of cells in S or G
1 phase which may be due to inhibition of the enzymes used for DNA replication such as ribonucleotide reductase[
20,
23‐
25]. Other mechanisms that could explain RSVL-induced S phase arrest is the increase expression of p53, a tumor suppressor protein[
26], the increase expression of positive G
1/S regulators, such as cyclin D1 and cyclin E which are responsible for S phase entry[
27], depletion of survivin, an inhibitor of apoptosis protein[
7]. Resveratrol-induced S phase arrest would eventually lead to apoptotic death as indicated by the very high increase in G
0 phase arrest (Figure
3).
Treatment with 15 μg/ml RSVL supplied simultaneously with different DOX concentrations enhanced the cytotoxic effect of DOX significantly. There was a 7.4-fold decrease in IC50 in cells treated with DOX and RSVL simultaneously as compared with DOX treated cells (Table
2). To gain further insight into the interaction mechanisms between DOX and RSVL, apoptosis assay, flow cytometric DNA analysis and DOX cellular uptake assay were performed. Apoptosis assay showed a small increase of the early apoptotic cell percentages in the simultaneous treated cells as compared with DOX treated group. The smaller DOX dose used simultaneously with RSVL showed a stronger increase in apoptosis as compared with DOX treated group (Figure
2). Furthermore, flow cytometric analysis revealed that simultaneous treatment of DOX with RSVL induced preferential cell arrest at G
0, there were 41-fold increase in percentages of G
0 phase arrest for treated cells (Figure
3). Several studies have reported that RSVL molecular mechanisms of sensitization for drug induced apoptosis involved cell cycle arrest in S phase[
27,
28], which has been used as a strategy to increase drug incorporation into cells. Thus, the cooperative effect of RSVL and the cell cycle-dependent drug DOX may result from RSVL-induced cell cycle arrest in S phase, thereby exposing a higher proportion of tumor cell population to DOX, therefore, more cells will undergo apoptosis and leave the cycle to enter the apoptotic G
0 phase.
These findings have been further confirmed by the observed increased in DOX cellular uptake after the simultaneous treatment with RSVL, which was in a dose dependent manner. There were an increase in DOX accumulation ratios for cells treated with DOX and RSVL, (Figure
4). This implies that, RSVL not only exposed higher proportion of MCF-7 cells to DOX by inducing cell cycle arrest in S phase but it also increased the DOX concentration available inside the cells. The increase in DOX cellular uptake inside the MCF-7 cells may be explained based on the inhibition of P-glycoprotein and multidrug resistance (MDR)[
29] that plays very important role in the absorption, distribution, and elimination of DOX, and thus determines its efficacy and toxicity[
29,
30]. Surprisingly our results showed that when RSVL was given prior to DOX, although it was more cytotoxic against the growth of MCF-7 cells, we noticed slight inhibition of DOX-induced apoptosis, less percentage of cells arrest in G
0 and decreased DOX cellular uptake into the cells compared with simultaneous treatment with DOX and RSVL.
The decrease of DOX cellular uptake in MCF-7 cells and the arrest of cells in S phase suggest that the enhanced growth inhibitory effects observed after the sequential RSVL and DOX treatment may not be caused by the synergism between DOX and RSVL or by the increased DOX cellular uptake, but this may be caused by the cytotoxic activity of RSVL itself[
20,
27].
Recently (2012), RSVL was found to reduce the intracellular accumulation of rhodamine 123 in colon cancer cell line suggesting that RSVL enhances the activity of P-glycoprotein[
31]. These conflicting findings could be explained on the following basis: MDR can be acquired after initial exposure to the anticancer drugs[
32]. In addition several studies have found that some of the well known P-glycoprotein antagonists such as verapamil and cyclosporine A can induce P-glycoprotein expression in colon carcinoma cells[
33]. It is important to note that the time needed for expression and inhibition of P-glycoprotein by their antagonists is controversial. Therefore, based on our results we can say that RSVL antagonizes or inhibits P-glycoprotein when it is given simultaneously with DOX thereby causing an increase in DOX cellular uptake[
29]. However, when it is given 24 h before DOX it enhances the P-glycoprotein expression. The 24 h period between RSVL and DOX is considered as an intial exposure that will enhance the expression of P glycoprotein and thereby MDR that will lead to the decrease in DOX cellular uptake. Further studies are needed to investigate how different sequence of treatment of RSVL and DOX could affect the P-glycoprotein activity and hence by the DOX intracellular accumulation in MCF-7 cells.
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
Abdel-Moneim, Zohir, sameer and Hadeel sharing in experimental work and writing the manuscript Mohamed Elshal did the flow cytometric analysis. All authors read and approved the final manuscript.