Metformin reduces the cellular DNA repair capacity and enhances the effect of curcumin on the induction of apoptosis in AGS gastric cancer cells

Cancer results from the accumulation of mutations in critical genes, such as DNA repair genes. But these genes are a double-edged sword, because the basis of current cancer treatment is DNA damage from chemotherapy and radiation, and the repair system can slow the healing process by repairing the induced damage. Therefore, any substance that can reduce the DNA repair capacity of cancer cells can make the cells more sensitive to treatment. Metformin and curcumin, as low-complication compounds, can play this role well. In the present work, changes in the expression of CASP3, BAX, and BCL2L1 apoptotic genes, and nine genes involved in DNA repair pathways (XRCC1, XRCC2, XRCC3, XRCC4, XRCC5, XRCC6, XRCC7, BRCA1 and BRAC2) were measured comparatively by real-time PCR in AGS gastric cancer cell line under single and co-treatments with metformin and curcumin. Our findings showed that co-treatment of metformin and curcumin induced decreasing the expression of anti-apoptotic BCL2L1 and increasing expression of proapoptotic CASP3 and BAX. Metformin decreased the expression levels of seven genes, while curcumin did not alter the expression levels. The co-treatment of metformin and curcumin showed that although the XRCC2, XRCC3, XRCC4, XRCC5, XRCC6, XRCC7, BRCA1, and BRCA2 were down-regulated, there was no difference between metformin and co-treatment for mRNA levels. Our results suggest that metformin increases the sensitivity of cancer cells to anticancer drugs by suppressing several DNA repair pathways and that curcumin may induce apoptosis.


Introduction
Alterations in cellular DNA repair capacity play a critical role in the onset, progression, and even response to cancer treatment [1].Because anti-cancer therapies, including ionizing radiation and chemotherapy, are based on DNA damage, regulating the DNA damage response may lead to tumor susceptibility to treatment or resistance to genotoxic agents.Therefore, targeting DNA repair pathways may be a potential therapeutic approach to cancer treatment [2].
Metformin is the first-line treatment for type 2 diabetes mellitus and the most widely used drug for this disease worldwide.Numerous epidemiologic studies have shown a significant association between the use of metformin in people with type 2 diabetes mellitus and a reduction in the risk of many types of cancer [3,4].Metformin, in addition to its protective role in cancer, induces apoptosis and inhibits the proliferation of many cancer cells, tumorigenesis, tumor progression in vivo, metastasis, and angiogenesis [5,6].On the other hand, metformin improves the response to chemotherapy and radiotherapy, reduces the anticancer drug resistance in diabetic patients with breast cancer, and increases the sensitivity of cancer cells to drugs [7].Also, metformin protects the skin against UVB damage by reducing its DNA repair capacity [8].
Curcumin is the main active ingredient of turmeric (Curcuma longa) and has antioxidant, antiseptic, antiseptic, antiseptic effects [9].Epidemiological studies attribute the reduction in cancer incidence to the curcumin-rich diets [10].The results of treating cells with curcumin show that it inhibits cell proliferation and induces apoptosis [11].Also, studies proved the induction of DNA damage and the reduction of the expression of DNA repair genes by curcumin in different cancer cell lines [12].Although the anticancer effects of curcumin have been confirmed in population-based studies regardless of its molecular pathways, there are inconsistencies in the performance of curcumin in various laboratory studies.Studies indicate its protective role against DNA damage as well as reduced DNA repair capacity, especially in the expression of genes involved in doublestrand break (DSB) repair pathways [13].
X-ray cross-complementing (XRCC) and breast cancer (BRCA) genes play a role in various pathways of DNA repair pathways [14,15].XRCC1 is involved in the base excision repair (BER) pathway; in addition, XRCC2, XRCC3, BRCA1, and BRCA2 are involved in the homologous recombination (HR) pathway, and XRCC4, XRCC5, XRCC6, and XRCC7 are active in non-homologous end joining (NHEJ).BRCA1 works jointly in NHEJ repair, the nucleotide excision repair (NER), HR pathways [14]; however, BRCA2 has a more specific role in DNA repair [15].Damaged eukaryotic cells are not repaired, but are removed by apoptosis.Increasing the expression of proapoptoses, especially caspase-3 (CASP3) as a "point of no return", and/or decreasing anti-apoptotic proteins in the Bcl-2 protein family, such as BCL2L1, can shift the balance in favor of cell death after DNA damage treatment [16].We have previously observed the dose-dependent cytotoxic effect of metformin and curcumin on AGS gastric cancer cell line.Also, based on Chou-Talalay method, it was found that metformin and curcumin with a ratio of 1:625 in 72 h could have a strong synergistic interaction.Furthermore, the results of our previous study showed that metformin and curcumin could inhibit the EMT mechanisms by impeding cell migration and invasion, as well as colony formation; these changes were significantly higher in combination treatments.Furthermore, the combination of metformin and curcumin significantly and selectively increased the cytotoxic effects of chemotherapy drugs on the cancer cell line compared to the normal cell line [17].According to these results and as the changes in DNA repair capacity and cell apoptosis potential are affected by EMT changes and cancer progression, changes in the expression of genes involved in these pathways were investigated.
Due to the contradictions in the studies and the lack of reports on the effect of curcumin and metformin combination on DNA repair genes in a gastric cancer cell line, our present study aimed at investigating this area.

Reagents
Metformin powder (Santa Cruz, USA) was dissolved in phosphate buffer solution (PBS) (Merck, Germany), and curcumin powder (Merck, Germany) was dissolved in DMSO (Shellmax, China).To test cytotoxicity, Cell Proliferation Kit (MTT) was prepared from Roche Company.Human AGS gastric cancer cell line was prepared from National Cell Bank of Iran and cultivated in RPMI 1640 culture medium (Bio-idea, Iran) enriched with (10% v/v) heat-inactivated fetal bovine serum (FBS, Gibco) and 100 units/mL penicillin and 100 μg/mL streptomycin (Bio-idea, Iran) under standard conditions of a humidified 5% CO 2 incubator at 37 °C.RNX-Plus Solution for total RNA isolation was purchased from Cinagen (Iran) and cDNA synthesis kit from Takara (Japan).RealQ Plus Master Mix Green was provided by Ampliqon (Denmark).

RNA extraction, cDNA synthesis, and gene expression analysis
The concentrations used for metformin (0.625 mM) and curcumin (1 μM) were selected based on the Chou synergistic relationship with a combined index <1 (CI = 0.3), so that the viability of the cells under single treatments was more than 97%, while it decreased to about 60% in combined treatments.Based on previous observations, these values were used to examine the gene expression changes [17].AGS gastric cancer cells (5 × 10 5 ) were treated with metformin, curcumin, and their combination for 72 h.Total RNA was extracted and cDNA was synthesized.Altered gene expression patterns were detected by real-time quantitative polymerase chain reaction (qPCR) in a Rotor-Gene 6000 instrument (Corbett, Australia) with the thermal profile as follows one step of 95 °C for 15 min, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. XRCC1, XRCC2, XRCC3, XRCC4, XRCC5, XRCC6, XRCC7, BRCA1, and BRAC2 genes as well as CASP3, BAX and BCL2L1 genes were studied in our study.Primer sequences designed with the aid of AllelID software version 7.0 are summarized in Table 1.GAPDH gene was considered as the reference gene and alterations in gene expression were determined by 2 −ΔΔCt method [18].

Statistical analysis
All experiments were done in triplicate.The data are expressed as the mean ± standard deviation (SD).Statistical analyses were carried out via one-way analysis of variance (ANOVA) and Duncan's post hoc test using SPSS 16.0 (SPSS, Inc., USA) with a significance level of less than 0.05 (p < 0.05).

Combination of metformin and curcumin alters the expression of apoptotic genes in favor of apoptosis
Our results demonstrated that treatment with metformin and curcumin induced decreasing the expression of anti-apoptotic BCL2L1 gene and increasing expression of proapoptotic CASP3 and BAX genes.Although these expressional alterations were observed in single treatments, co-treatment with metformin and curcumin significantly changed the expression patterns towards apoptosis (Fig. 1).In summary, metformin treatment did not cause a significant change in the expression of apoptotic genes; however, curcumin treatment increased the expression of BAX gene by 8.6-fold and decreased the expression of BCL2L1 anti-apoptotic gene by 0.57-fold compared to the untreated control cell.However, their combination treatment showed a significant increase in proapoptotic CASP3 (2.8-fold) and BAX (15-fold) genes and a decrease in BCL2L1 anti-apoptotic expression (0.76-fold).The BAX/BCL2L1 ratio in curcumin (20fold) and co-treatment (86-fold) increased significantly (Fig. 1).

Metformin and curcumin reduce the DNA repair capacity in AGS cancer cells
In the present study, changes in the mRNA levels of nine genes involved in DNA repair pathways were examined Table 1 The primer sequences used in this study in AGS cells treated with metformin and curcumin (Fig. 2).Single treatment with metformin during 72 h showed that the expression levels of XRCC2, XRCC3, XRCC5, XRCC6, XRCC7, BRCA1, and BRCA2 significantly decreased.In single treatment with curcumin, the alteration of mRNA levels was not statistically significant.The combined treatment of metformin and curcumin also demonstrated a significant decrease in the expression of XRCC2, XRCC3, XRCC5, XRCC6, XRCC7, BRCA1, and BRCA2 genes.There was no significant difference between the studied mRNA levels of metformin and co-treatment of metformin and curcumin, indicating that curcumin has no effect on the expression levels of the examined DNA repair genes.

Discussion
In the present report, we provided evidence of the effect of metformin and curcumin on the expression levels of three genes involved in apoptosis and nine genes involved in DNA repair pathways.In this study, the selected DNA repair genes show a wider range of activity in DNA damage response.
Combination therapy and interests in low-complication and natural compounds are new topics in cancer Fig. 1 Diagram of changes in CASP3, BAX and BCL2-L1 gene expression as well as the BAX/BCL2-L1 ratio expression after treatment with 0.625 mM metformin, 1 μM curcumin and their combination over a 72 h time interval.Gene expression was normalized with GAPDH as internal control.The results (mean ± SEM) were obtained from 3 independent experiments.All experiments were performed independently in triplicate.Statistical analysis was performed using one-way ANOVA and Duncan's post-hoc test.In each panel, similar letters indicate no statistically significant difference Fig. 2 Combination of 0.625 mM metformin and 1 μM curcumin inhibits DNA repair genes expression in AGS cell line.Alterations in the expression of genes involved in DNA repair were measured using real-time qPCR after 72-h treatments with metformin, curcumin, and their combination.Graphs represent mean ± SEM for three independent experiments conducted in triplicates.Statistical analysis was performed using one-way analysis of variance (ANOVA) and Duncan's post hoc test.In each panel, similar letters indicate no statistically significant difference.All experiments were performed independently in triplicate research and treatment, and the study of the simultaneous effect of curcumin and metformin in recent years has received special attention.Our previous results showed that metformin and curcumin could synergistically reduce the survival of gastric cancer cells and significantly inhibit migration, invasion, and colony formation.Therefore, changes in the expression of genes involved in DNA repair and apoptosis were expected [17].
Examination of apoptotic genes expression patterns showed that single treatment with curcumin induced apoptosis due to up-regulation of BAX and downregulation of BCL2L genes.In co-treatment of metformin and curcumin, CASP3 and BAX genes were up-regulated and BCL2L1 gene was down-regulated.The BAX/BCL2L1 ratio, an index of apoptosis induction [16], increased more than 4 times compared to single-curcumin treatment and up to 82 times compared to the untreated control cells (Fig. 1).Therefore, co-treatment may increase apoptosis in the AGS cells.This is in accordance with the results of the cytotoxicity test in MTT assay and explains those results.In a similar study, it has been reported that the combination of metformin and curcumin induced apoptosis in LNCaP [19].
In the present study, investigation of changes in the expression of DNA repair genes shows that although metformin alone and in low non-cytotoxic doses can reduce the repair capacity, curcumin and its combination with metformin did not significantly alter the expression of DNA repair genes.Consistent with our results, other studies show that curcumin has no effect at concentrations below 14 μM [20] and the effects of curcumin are dose-and time-dependent [21], as reflected in our MTT results.
Our results showed that curcumin alters the expression of apoptotic genes, while having no effect on the expression of DNA repair genes.In contrast, metformin only reduced the expression of DNA repair genes.Inhibiting the DNA repair pathway and inducing apoptosis are ways to make cancer treatment more effective.Based on the link between DNA repair pathways and cancer progression, a new treatment strategy is being considered to increase the effect of DNA-damaging agents through DNA repair inhibitors.Our findings suggested that metformin increased the sensitivity of cancer cells to anticancer drugs by suppressing several DNA repair pathways, and that curcumin could induce apoptosis and accelerate cancer cell death.As demonstrated in our previous study, metformin and curcumin enhanced the effectiveness of chemotherapy drugs [17].This could be the basis for further studies to investigate the anti-cancer effects of metformin and curcumin.

Conclusions
Our current results show that curcumin may increase the expression of proapoptotic genes and by downregulation of the anti-apoptotic gene, it can sensitize AGS cells to apoptosis.Metformin potentiated this effect of curcumin.Metformin also reduced DNA repair capacity by reducing the expression of genes involved in DSB repair.Reducing the repair capacity and facilitating the onset of apoptosis can provide insights into cancer treatment.