Colorectal cancer is the third deadliest cancer in the world and many people die every year in the world due to this diease. Chemotherapy takes an important place among colon cancer treatment options and 5-FU is widely used as a chemotherapeutic agent . In the treatment of colorectal cancer, it is important to develop drug combinations that will increase the effectiveness of chemotherapy and to determine the molecular targets of the drugs . A decrease in the response rate can be encountered in case of prolonged use of 5-FU . For these reasons, combined therapies that can increase the sensitivity of cells against 5-FU and the molecular pathways involved in this process are important in the treatment of the disease. Our results show that HCT-116 p53 +/+ cells are more sensitive to 5-FU (IC50 = 2.703 ±0.2) to HCT-116 p53-/- cells (IC50=5.806±0.34) (Figure 1G and figure 2G). These results are consistent with the studies done before with 5-FU . We demonstrated first time that chemotherapeutic agent 5-FU decrease SIRT5 and FOXO3a protein expressions significantly and p53 indepedently (p<0.05) (Fig 3A, 3B, 4A, 4B). SIRT5 is a member of the sirtuin family whose role in cancer has been least studied. It has dual roles in cancer as tumor supressor or tumor promoter depending cell conditions, tumor stage and tissue of origin [12,19]. Some studies indicate that possible tumor suppressor function of SIRT5. SIRT5 has been shown to be significantly downregulated in head and neck and hepatocellular carcinoma tissues [20,21]. In addition, there are many studies showing that it is a tumor promoter. SIRT5 overexpression and its role in facilitating drug resistance has been indicated in human non-small cell lung cancer cells by reducing the expression of Nrf2 and downstream targets . SIRT5 overexpression was reported in Waldenstrom's macroglobulinemia (WM) CD19+ cells and colon cancer cell lines (HCT-116 and Lovo) and colorectal cancer tissues when compared to normal tissues. siRNA silencing of SIRT5 has been shown to inhibit cancer cell proliferation [23-25]. Upregulation of SIRT5 was observed in hepatocellular carcinoma cell lines and cancerous tissues and association between poor survival and increase in SIRT5 expression was shown . In another study, regarding the role of SIRT5 in colon cancer, it was shown that the combination of SIRT5 inhibitors with chemotherapeutic agents (cetuximab) exhibited a therapeutic approach in wild type Kras colorectal patients . SIRT5-mediated Lactate Dehydrogenase B (LDHB) deacetylation has been shown to trigger autophagy and tumorigenesis in colorectal cancer . In the literature, there is very few study about the relationship between SIRT5 and FOXO3a. SIRT5 has an effect on the deacetylation of FOXO3a in lung epithelial cells induced by cigarette smoke extract and deacetylation of FOXO3a by SIRT5 leads to prevention of lung epitelial cell apoptosis. FOXO3a is a transcription factor that induces gene expression involved in antioxidant defense and translocates to the nucleus upon deacetylation by SIRT5 . Some studies indicated the relationship between FOXO3a and metastasis/bad prognosis in cancer [29,30]. It was shown that FOXO3a and β-catenin was accumulated in nucleus and this is correlated with metastasis of colon cancer . In acute myeloid leukemia patients which have somatic mutations, there is a linkage between lower overall survival and high FOXO3 expression . Also, in glioblastoma and pancreatic ductal adenocarcinoma progression, it was detected the high FOXO3a expression [33,34]. But besides that there are many studies that indicate the tumor suppressor roles of FOXO3a proteins [35,36]. Acetylation of FOXO has apoptosis promoting effect via activating the proapoptotic genes (Bim, p21, FASL6 etc) and as a result of deacetylation of FOXO3a by SIRT1 leads to cell survival and inhibition of FOXO3a mediated apoptosis [37,38]. According to our results, due to the reducing effect of 5-FU on SIRT5 expression, deacetylation of FOXO3a is alleviated and FOXO3a-dependent apoptotic pathways are induced. This idea is consistent with the stimulating effect of 5-FU on Bim protein. SIRT5 is known to deacetylate FOXO3a  and FOXO3a leads to the induction of apoptosis via Bim . We examined the effects of SIRT5 and p53 modulators when used alone or in combination with 5-FU on proliferation of colon cancer cells p53 dependent and independently and showed that these modulators have significant effects on 5-FU sensitivity of colon cancer cells. While we determined that RSV and Nutlin3a are more effective in p53+/+ cells than p53-/- cells (Fig. 1D and 1F and fig. 2D and 2F), suramin is more effective in p53 -/- cells when compared to p53 +/+ cells (Fig. 1E and 2E). In our study, in order to explain why the increase in the sensitivity of cells to 5-FU, we investigated the effects of these modulators and 5-FU on SIRT5 and p53 protein expressions as well as FOXO3a and Bim expressions. In this study, we examined the effect of 5-FU on the SIRT5-FOXO3a-Bim axis p53 dependent and independently. Nutlin3a has been shown that to induce p53 expression .For this purpose, while activation of the p53 pathway was provided by nutlin3a in HCT-116 p53 +/+ cells, HCT-116 p53 -/- cells took part in the study as counterpart cells that did not express p53. There are limited studies in the literature on the relationship between SIRT5 and p53 protein. It has been shown that p53 cannot be deacetylated by sirtuins except SIRT1 . Solomon et al., showed that SIRT5 can deacetylate cytochrome c but not p53 . Lieber et al. showed that the decrease in SIRT1 and SIRT5 expressions caused posttranslational modification or hyperacetylation of PGC1-α and p53 . We found that SIRT5 modulator RSV increased the sensitivity of HCT-116 p53 +/+ cells to 5-FU much more than HCT-116 p53 -/- cells (Figure 1D and figure 2D). Our results of western blot experiments indicate that RSV hasn’t any significant effect on the SIRT5 protein expression in both cell lines (Fig. 3A and fig. 3B) We found that RSV increase SIRT5 protein expression 1.34 fold (0.634 ± 0.08 to 0.851± 0.180) in HCT 116 p53+/+ cells and increase 1.17 fold (1.00± 0.2 to 1.179± 0.38) HCT 116 p53-/- cells but it is not statistically significant. According to the previous studies, it has been found that deacetylase activity of SIRT5 is activated and desuccinylase activity is inhibited by resveratrol  and SIRT5 enzyme activity is decrased by suramin and increased by resveratrol . Besides these studies, it has been shown that SIRT5 protein expression is not altered by resveratol in H295R human adrenal cell lines . We also found that RSV decreased FOXO3a protein expression in HCT-116 p53 -/- cells significantly (Fig. 3B), but it didn’t have any significant effect on the FOXO3a protein in HCT-116 p53 +/+ cells (Fig. 4B). So this difference may be due to p53 and FOXO3a protein expression difference. RSV in both cells increase the sensitivity of colon cancer cells to 5-FU. But increase in the sensitivity is much more in HCT-116 p53 +/+ cells than HCT-116 p53 -/- cells. According to Yu et al, when HCT-116 cells were treated with 15µM RSV and 0.5µM 5-FU, cells was blocked at S phase, enhanced DNA damage and apoptosis was observed. Mohapatra et al, reported that 5-FU can increase the chemotherapy efficiacy of RSV which is used in colon cancer treatment . The difference of our study from these studies is to be performed all analysis both p53 +/+ and p53 -/- cells to compare the effects due to differences in p53 expression and cells were treated with 5-FU at increasing concentration (0.01-20 µM) and 50 µM RSV. In our study and we found that HCT-116 p53 +/+ (IC50 = 48.51± 2.25) cells are more sensitive to resveratrol than HCT-116 p53 -/- cells (IC50= 53.52 ±.7.162). Dermani et al reported that apoptosis and cell cycle arrest is induced by resveratrol . In 3DC, resvaratrol induce PDE4 activity and luminal cavity formation and induced apoptosis . Resveratrol inhibited colon cancer cell invasion and metastasis by Wnt/β-catenin signal pathway [48-53]. In this study, we aimed to investigate the effect of SIRT5 modulators on the sensitivity of HCT-116 colon cancer cells to 5-FU. We found that RSV (50µM) decrease the IC50 of HCT-116 p53 -/- cells from 5.80 ± 0.34 to 2.58 ± 0.512 and decrease the IC50 of HCT-116 p53 +/+ cells from 2.703± 0.2 to 0.024 ± 0.002. There are no studies in the literature investigating the effects of SIRT5 and p53 modulators on the sensitivity of colon cancer cells to 5-FU and examining the effect of 5-FU, SIRT5 and p53 modulators on the SIRT5, p53, FOXO3a and Bim protein expressions in both p53 +/+ and p53 -/- colon cancer cells. This is the first study to examine changes in SIRT5 protein expression as a result of p53 activation by nutlin3a and to be used p53 -/- cells as a counterpart cell line. For this purpose, both cells were incubated with nutlin3a for 72h. According to our results, nutlin-3a increased p53 protein expression in p53 +/+ cells compared to the control group (Nulin-3a free cells) (Fig. 4C). When the effect of nutlin-3a on the expression of SIRT5, FOXO, Bim proteins was investigated, it was found that nutlin-3a significantly increase Bim protein in only p53 +/+ cells (Fig. 4D) and it has not been detected any significant effect on on the SIRT5 or FOXO3a expression in p53 +/+ and p53 -/- colon cancer cells. Similar to our results, the increase in nutlin-3a-mediated Bim protein expression found p53 dependent . Renault et al compared FOXO3a protein levels in p53 +/+ and p53 -/- mouse embryonic fibroblasts (in the presence and absence of doxorubucin) that doxorubicin and nutlin-3a increases FOXO3a in p53 +/+ cells but it didn’t change in p53 -/- cells (Renault et al., 2011). RSV and Suramin were used as SIRT5 modulators in this study. And both cells were incubated with RSV(50µM) and Suramin (100µM) for 72h it was shown that both suramin and RSV increased p53 protein expressions in HCT p53 +/+ cells (Figure 4C). In the previous studies, suramin has been shown increases p53 protein levels, but fails to increase p21 mRNA levels or to activate the G~ checkpoint. These data suggest that suramin induces growth arrest in NIH-3T3 cells by a mechanism that is independent of cellular p53 status . According to our results, RSV decreased FOXO3a protein expression significantly in HCT-116 p53-/- cells, but it didn’t any significant effect in HCT-116 p53 +/+ cells (0.54±0.05 to 0.35 ± 0.12) (p<0.01) (Fig. 3B and fig. 4B). And RSV increase p53 and Bim protein expression significantly in HCT-116 p53+/+ cells (Fig. 4 C and 4D) and increse Bim expression in HCT-116 p53-/- cells significantly (Fig. 3D). In previous studies, RSV (20uM) has been shown that reversed SIRT1-7 activity reduced by TNF-α in HUVECs . It has been reported that RSV promote nuclear translocation of FOXO3a and cause apoptosis in HeLa cells . RSV inhibited PI3K-AKT signal transduction pathway, decrease Foxo protein expression and induce apoptosis in H2O2 treated FLS cells . Resveratrol exhibits apoptotic effects via FOXO proteins . It was reported that RSV inhibits orthotopic pancreatic tumor growing via FOXO activation . Resveratrol activates FOXO in prostate cancer xenografts factor [59,60]. In the literature, there is no study that investigates the effect of suramin on the proliferation of HCT-116 p53 +/+ and p53 -/-colon cancer cells. Suramin has been shown that inhibited SIRT5 NAD(+)-dependent deacetylase activity [61,12]. We found that while suramin hasn’t any statistically significant effect on SIRT5 expression in HCT-116 p53 +/+ cells. We found that suramin decrases SIRT5 protein expression from 0.63 ± 0.08 to 0.47± 0.14) but this decrease couldn’t be found statistically significant (Fig. 4A), and decreases SIRT5 protein expression significantly in HCT-116 p53 -/- cells (p<0.05) (Fig. 3 A). Suramin decreases FOXO3a in both cells but it is not significant (Fig. 3B and fig. 4B). Suramin increases p53 and Bim protein expressions in HCT-116 p53 +/+ cells (Fig. 4D) and exhibits any significant effect on Bim protein expressions in HCT-116 p53 -/- cells (Fig. 3D). Falcone et al. showed that suramin made colorectal cancer cells sensitive to 5-FU + LV .
In conclusion, this study showed that 5-FU decreased SIRT5 and FOXO3a protein expressions in p53 independent manner for the first time. And SIRT5 and p53 modulators increase the sensitivity of colon cancer cell lines to 5-FU. The role of SIRT5 protein expression on the cell proliferation of colon cancer cell lines was explained. SIRT5 inhibition by suramin leads the increase in the sensitivity of colon cancer cells to 5- FU in p53 -/- cells. Thus, it is important in terms of being a guide for new approaches for colon cancer treatment and new drugs that are planned to be developed.