Effects of Curcumin in Combination with Doxorubicin in Human Colorectal Cancer Cell Line

Colorectal cancer (CRC) is one of the main leading causes of cancer related mortality worldwide [1]. Although the patients survival has recently increased due to improved surgery, local radiotherapy and chemotherapy, but the high rate of metastatic patients prognosis are not good [1-2]. Indeed, one of the main goals in cancer therapy is to predict response to chemotherapeutic drugs by drug response assays [3]. Anthracycline drugs including doxorubicin Abstract


Introduction
Colorectal cancer (CRC) is one of the main leading causes of cancer related mortality worldwide [1]. Although the patients survival has recently increased due to improved surgery, local radiotherapy and chemotherapy, but the high rate of metastatic patients prognosis are not good [1][2]. Indeed, one of the main goals in cancer therapy is to predict response to chemotherapeutic drugs by drug response assays [3]. Anthracycline drugs including doxorubicin (DOX) considered as furthermost active wide-spectrum and Inexpensive drugs in cancer treatment. However, CRC cells are characteristically resistant to anthracyclines [4]. Indeed cancer cells shows resistance toward anti-cancer agents in major cases [1][2]. On the other hands, numerous routine chemotherapeutics have adverse side effects; dietary manipulations might have an important role in the prevention of numerous human cancers. Curcumin the yellow pigment in turmeric has been extensively utilized in the Asian countries [5]. Previous data has been revealed that curcumin prevent the growing of transformed cells and colon carcinogenesis at the beginning and progression phases in carcinogen-induced models [6][7]. We aimed to evaluate the possible sensitizing effects of curcumin in HCT-116 CRC cells treated with DOX.

Cell lines and Cell Cultures
Human HCT-116 cells were purchased from the Pasteur Institute (Tehran, Iran). Cells were maintained in growth medium consisting of Dulbecco's modified Eagle's medium [DMEM (BioWest, France)] supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic (BioWest, France) at 37°C and 5% CO 2 . Cells were passaged utilizing trypsin/EDTA. Curcumin was purchased from Sigma Chemical Co. (USA), dissolved in 100% ethanol, and then diluted with the DMEM. Doxorubicin hydrochloride purchased from sigma-Aldrich (USA) and solubilized in pure water.
The E group (control) considered as control which include the untreated cells had any exposure to DOX and curcumin.
At the end of the 48 h incubation period, the reaction was terminated by adding 10 µl of WST-1 stock to each well. The reaction was allowed to proceed for 3-4 hours at 37°C. Cell viability was assayed according to the cleavage of the tetrazolium salt, water-soluble tetrazolium-1 (WST-1), to dark red formazan. Absorbance was read at 420 nm with a reference wavelength > 650 using the enzyme-linked immunosorbent assay microplate reader. All values were compared to the related controls. All assays were performed with triple replicates.

Statistical analysis
Data shown as the mean ± SD. The differences among more than two means were evaluated by one-way analysis of variance (ANOVA) with SPSS software, V. 10.0 (SPSS Inc, Chicago, Illinois). P < 0.05 considered as significance level.

Effects of DOX on cell viability
The cytotoxic effects of the single treatments of DOX and curcumin after 48 h of exposure in CRC cell line plotted as the percentage of viable cells versus control cells ( Figure 1). As presented in Figure 1, the exposure of HCT-116 cells to various concentrations of single agents (DOX and curcumin) decreased the cellular viability in a dose-dependent pattern.

Cytotoxic effects of DOX in combination with curcumin on HCT-116 cells
Different concentrations of DOX in combination with curcumin were selected according to the initial tests mentioned in the methods to evaluate the cytotoxic effects of drug combinations on the HCT-116 cell line.   to induce the immuno-, hepato-, and cardioprotective characteristics of DOX [18] which is similar to our data. Also, Meiyanto et al utilized curcumin in combination with DOX that increased the sensitivity of resistant MCF-7 cells to DOX, which was comparable to our results [19].
Other study showed that two drug (curcumin+DOX) loaded in a single nanoparticle formulation decrease the drug resistance by effects on gradual mRNA expression of MDR1 and Bcl-2 in CML blast-like K562 cells [20].
In addition, Huang et al reported that curcumin could increase the impacts of irinotecan on CRC cells by effect on cell viability and apoptosis, which is similar to our results [21].
In conclusion , it could be deduced that curcumin as natural agents can affect main carcinogenic pathways without demonstrating noticeable adverse impacts, which might be considered as perfect chemosensitizer agents in combination with DOX. In this study, we confirmed the cytotoxic effects of dietary agent's curcumin for chemosensitizing of DOX.
WST-1 results from double combination cases are presented in Figure 2.
The DOX in combination with curcumin showed increased cytotoxicity compared to higher concentrations (the doses, which led to 50% of cell killed in single treatments) of each single agents. The D group had higher cytotoxic effects in compared to A,B,C and E groups (p<0.05). In addition, the C and B group showed increased toxicity in compared to A and E groups (p<0.05). Indeed, in combination cases in the low concentrations of DOX and curcumin, the cellular viability were decreased in compared to each single drug treatments in the concentration which led to 50% of cell toxicity [C and D treatments had significant elevated toxicity versus to each single treatments (p<0.05)].

Discussion
Amongst diverse ranges of disorders, cancer has always considered as the furthermost important condition, which affected many individuals word width. In this regards, different cancer therapeutics applied to overcome cancer cell growth [3][4][5][6][7][8]16]. CRC considered as one of the commonest cancers all over the worlds. In the face of innovative treatment strategies, CRC is infrequently cured because of recurrence. Numerous studies have utilized curcumin, as anti-cancer agents in numerous types of cancer, such as CRC [17]. Since, Anthracycline drugs such as DOX remain some of the most active wide-spectrum and cost-effective drugs in cancer therapy and CRC cells are resistant to anthracyclines, we evaluated the effects of curcumin in combination with DOX in CRC cells.
Based on our data DOX and curcumin in the dose dependent manner reduced the cellular viability. In combination cases in the lower concentrations of DOX and curcumin, the cellular viability were decreased in compared to each single drug treatments in the concentration which led to 50% of cell killed.
Our study assess the curcumin effects in combination with DOX, to evaluate how curcumin increase the cytotoxic effects of DOX in CRC cells. For this elevated cytotoxic effects in combination cases, it could be proposed with that fact which described by Chauhan et al. in this study , they indicated that curcumin should be considered as a harmless, non-toxic chemotherapeutic drug [7] which impedes lipooxygenase activity , impeding the cytochrome P-450 enzyme activity and promotion/progression stages of carcinogenesis. The anti-cancer properties of curcumin has been associated with arrest of cancer cells in S, G2/M cell cycle phase and apoptosis activation [7].
Recently researchers indicated that curcumin in combination with DOX was effective in sensitizing cancer cells that are resistant to DOX and in decreasing cancer cells viability. Curcumin has also been shown