The Study Of B7-H3 Molecular In Drug-Resistance In Colorectal Cancer Cells

Colorectal cancer(CRC)is one of the most common types of cancer.Each year 1.2 million new cases are diagnosed,and more than 600,000 patients die from CRC.An important reason for the high mortality rate of CRC is due to the emergence of chemotherapeutic drug resistance.Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may help identify novel targets and drugs that will,in turn,improve therapy.B7-H3,first recognized as a co-stimulatory molecule,has been associated with poor prognosis.Our lab found that B7-H3 could promote colorectal cancer cells migration and invasion.Since metastasis is closely related to chemoresistance,in present work we have investigated the role of B7-H3 in drug-resistance in colorectal cancer(CRC).We evaluated the expression of co-stimulatory protein B7-H3 in HCT-116、HCT-8、HT29、SW480、SW620、RKO、LOVO cells.As revealed by Western blotting,a higher expression of B7-H3 was found in HCT-8、HT29、HCT-116 cells with respect to SW480、SW620、RKO、LOVO cells.We found that HCT-8/5-Fu and HCT-116/L-OHP cells have more B7-H3 expression than in their wild type(HCT-8/WT,HCT-116/WT).In order to evaluate the possible role of B7-H3 in affecting the drug sensitivity of CRC cells,we silenced the expression of B7-H3 in HCT-116 and HCT-8 cells by using specific siRNAs.The results of Cell viability assay showed that HCT-116/RNAi and HCT-8/RNAi cells were much more sensitive to Oxaliplatin than HCT-116/Scrambled and HCT-8/Scrambled cells.To corroborate this conclusion,we obtained two stable B7-H3 overexpressing cell models(i.e.SW480-B7H3 and SW620-B7H3)with their respective controls(i.e.SW480-NC and SW620-NC).As the results showed,SW480-B7H3 and SW620-B7H3 cells were more resistant to oxaliplatin compared with their respective negative control.And then we also evaluated the effect of B7-H3 on apoptosis and DNA damage induced by oxaliplatin treatment of CRC cells.Results suggested that CRC cells with low B7-H3 expression(i.e.HCT-8/RNAi and SW480-NC)were more sensitive to oxaliplatininduced apoptosis and DNA damage.In our experiments,we found that the expression of XRCC1,an important scaffold protein for DNA damage repair,in CRC cells was related to B7-H3.The results of real-time PCR,western blot analysis and immunofluorescence showed that the expression of XRCC1 at the mRNA level and protein level was consistent with that of B7-H3,such that B7-H3-overexpressing cells(SW480-B7H3 and HCT-8/Scrambled)demonstrated higher XRCC1 expression compared to low B7-H3 expression cells(SW480-NC)or B7-H3 knocked down cells(HCT-8/RNAi).Immunofluorescent staining showed the DNA damage increased in SW480-B7H3 cells silenced for XRCC1 upon treatment with oxaliplatin.These data suggested B7-H3 upregulated XRCC1 expression so that the oxaliplatin-induced DNA damage can be repaired.The phenomenon that B7-H3 enhanced drug resistance and DNA damage repair ability through up-regulation of XRCC1 was observed,and next aimed to identify the signaling pathway involved.It has been reported that the XRCC1 expression is regulated by PI3K/AKT and MAPK-ERK1/2 pathway.In the subsequent experiments,we performed western blotting to detect the expression of AKT,ERK1/2 and their phosphorylated forms in B7-H3 overexpressing cells(HCT-8/Scrambled,SW480-B7H3)and their negative control group(HCT-8/RNAi,SW480-NC).The phosphorylation level of AKT(p-AKT)was higher in SW480-B7H3 and HCT-8/Scrambled cells,whereas phosphorylated ERK 1/2(p-ERK1/2)did not significant change.We therefore inferred that PI3K-AKT might be a major pathway by which B7-H3 regulated the expression of XRCC1.To confirm this hypothesis,we analyzed pAKT in SW480-NC and SW480-B7H3 cells that were treated with or without LY294002(a specific inhibitor of PI3 K,100μM).The level of phosphorylated AKT was significantly increased in the SW480-B7-H3 cells compared with the SW480-NC cells.But when the SW480-B7-H3 cells were treated with LY294002,B7-H3-induced increased in AKT phosphorylation and XRCC1 expression were blocked by LY294002.These results confirmed that PI3K-AKT pathway is strictly involved in the B7-H3-mediated regulation of XRCC1.Moreover,SW480-B7H3 cells were more sensitive to oxaliplatin-induced DNA damage and cell death after the inhibition of PI3K-AKT pathway exerted by LY294002.In summary,our study demonstrated that B7-H3 decreases oxaliplatin-induced DNA damage by promoting the expression of XRCC1 via the PI3K/AKT signaling pathway.These findings provide new insight into the role of B7-H3 in cancer treatment and may have significant implications in the development of targeted therapeutics for overcoming drug resistance.