Glycosylation Of CACNA2D1 Plays A Key Role In Multidrug Resistance Of Gastric Cancer

Gastric cancer is one of the most common malignant cancers in the world. According to the literature, An estimated 951,600 new stomach cancer cases and 723,100 deaths occurred in 2012.With advances in medical research the methods for the treatment of gastric cancer have been continuously improved, but multi-drug resistance is still a major problem which hampered the clinical efficacy of chemotherapy.Scholars have done a lot of researches to elucidate the mechanism of multidrug resistance in gastric cancer. We have done a lot of researches. In our previous work, we successfully screened and identified a number of genes and molecules related with MDR through glycoproteomics identification and lnc RNA array. Due to the complexity of resistance mechanisms of gastric cancer the molecules related with MDR could not clarify the nature of the multidrug resistance of gastric cancer.Correct glycosylation is the basis for the maintenance of normal biological activity of proteins, glycosylation error will result in a reduction of the synthesis of glycoproteins or loss of function. Many articles have been reported that glycosylation plays an important role in a variety of tumors during the occurrence of multi-drug resistance. Recently, more and more researchers are focusing on the impact of the glycoprotein on tumor.By subjecting drug resistant cell lines and the parental cell line SGC7901 to a modified cell surface capturing strategy to examine the differential expression of cell surface N-glycoproteins, 11 N-glycoproteins were identified. These glycoproteins are quite possible to be biomarkers for predicting MDR or key regulators for targeted therapy We focused on CACNA2D1 which was one of the significantly changed proteins in both drug resistant cell lines compared with parental cell line. Previously reporting that CACNA2D1 plays an important role in nerve signaling aspects, but the role of CACNA2D1 in gastric cancer and the mechanisms of multidrug resistance in gastric cancer has not been reported. It is significant that we investigate the relationship between the glycosylation of CACNA2D1 and the MDR of gastric cancer.Objectives To identify the relationship between the expression levels of CACNA2D1 in human GC specimens and the clinical prognosis; We evaluated the role of glycosylation of CACNA2D1 in GC multidrug resistance; We primarily investigated the possible mechamisms of glycosylaiton of CACNA2D1 in GC multidrug resistance.Methods 1. Immunohistochemistry was performed to detect the expression of CACNA2D1 in the tissues of gastric cancer, then analyze the correlation between the expression of CACN2D1 and prognosis of patients with gastric cancer. 2. q RT-PCR and Western-blot were carried out to evaluate the expression of CACNA2D1 in drug resistant cell lines and the parental cell line. 3. We establish stably transfected cell lines with amplifiedd CACNA2D1 sense and sh RNA vectors. MTT assay, flow cytometry and drug bump were preformed to evaluate the effects of CACNA2D1 on MDR 4. The vectors expressing glycosylation-site mutated CACNA2D1 were constructed and transfected to establish stably transfected cell lines.MTT assay, flow cytometry and drug bump were preformed to evaluate the effects of glycosylation of CACNA2D1 on MDRResults 1、The expression of CACNA2D1 is closely related to the prognosis of patients with gastric cancerq RT-PCR to detect the 16 pairs of fresh gastric cancer tissues and matched normal tissues CACNA2D1 found expression in gastric carcinoma was significantly higher than the adjacent tissues.We detected the expression of CACNA2D1 in 89 cases of patients with gastric cancer by using Immunohistochemistry.43 samples came out with positive of CACNA2D1 and 46 with negative of CACNA2D1. The positive rate of gastric cancer48.3%(43/89) is significant higher than that of Adjacent tissues13.4%(12/89). the expression of CACNA2D1 is closely related to the prognosis of patients with gastric cancer by univariate statistical analysis2. The expression of CACNA2D1 in drug resistant cell lines were increasedq RT-PCR and Western-blot were carried out to evaluate the expression of CACNA2D1 in drug resistant cell lines and the parental cell line. The expression of CACNA2D1 in drug resistant cell lines were increased compared with the parental cell line.3. CACNA2D1 can promote gastric cancer cells multidrug resistanceThe results showed that knock down of CACNA2D1 resulted in decreased sensitivity to ADR, 5-fu and CDDP; over expression of CACNA2D1 can reduce the sensitivity of gastric cancer cells to chemotherapeutic drugs.Apoptosis was measured by flow cytometry results showed that knock down of CACNA2D1 can promote sensitivity to 5-fu sensitivity. Over expression of CACNA2D1 can inhibit the sensitivity of gastric cancer cells to chemotherapeutic drugs.Drug pump out experiments showed that knock down of CACNA2D1 can inhibit the ability of drug resistant cell lines pumping drug out. Over expression of CACNA2D1 can promote the ability of gastric cancer cells pumping drug out.4. The glycosylation of CACNA2D1 can promote gastric cancer multidrug resistanceIn order to verify the role of glycosylation of CACNA2D1 in multidrug resistance in gastric cancer, we constructed glycosylation site mutations vector of CACNA2D1 by site-directed mutagenesis method and transfected in gastric cancer cells, The MTT results showed that the lost of glycosylation of CACNA2D1 resulted in decreased sensitivity to chemotherapeutic drug; Drug pump out experiments showed that the lost of glycosylation of CACNA2D1 can inhibit the ability of pumping drug out.Conclusions 1. In the gastric cancer tissues, CACNA2D1 expression levels in gastric cancer tissues was significantly higher than the adjacent tissues; the expression of CACNA2D1 is closely related to the prognosis of patients with gastric cancer by Statistical analysis 2. CACNA2D1 with high glycosylated form in gastric resistant cell lines was significantly higher than the parental cells. 3. Overexpression of CACNA2D1 in gastric cancer cells can change the ability of drug pumping out which significantly facilitate the generation of drug-resistant phenotype 4. Compared with wild-type CACNA2D1, reducing their level of glycosylation can inhibit drug resistance in gastric cancer cells, suggesting that glycosylation plays a key role in the regulation of gastric drug CACNA2D1 process.