VCPA,A Novel Synthetic Derivative Of ?-tocopheryl Succinate,Sensitizes Human Gastric Cancer To Doxorubicin-induced Apoptosis Via ROS-dependent Mitochondrial Dysfunction

Gastric carcinoma is a common malignant disease worldwide and has a dismal prognosis.Doxorubicin(DOX),one of the most widely used chemotherapeutic agents,has limited use because of its side effects and the development of tumor-cell resistance.Combinations of doxorubicin and non-cross-resistant agents have been required for adjuvant chemotherapy of gastric cancer.Here,we report that VCPA,a novel synthetic derivative of a-Tocopheryl Succinate,induced apoptosis via production of reactive oxygen species(ROS).When used in combination with doxorubicin,lower doses of VCPA sensitized human gastric cancer cells to DOX-induced apoptosis.The DOX/VCPA combination treatment caused an imbalance in the ratio of Bcl-2 to Bax and induced a lethal mitochondrial dysfunction.MAPKs were also activated in response to the DOX/VCPA treatment but played a protective role in DOX-induced cell death.In vivo studies further confirmed the sensitizing effect of VCPA.Combining DOX with VCPA markedly inhibited tumor growth in a tumor xenograft model of human gastric cancer.Taken together,our study revealed that VCPA,through increased ROS production,could synergize with DOX and circumvent DOX resistance in human gastric cancer cells.This study was divided into two parts,as the following:Part 1.VCPA sensitized doxorubicin induced apoptosis on human gastric cancer cells in vitro.Objective:Gastric cancer remains the second leading cause of cancer-related deaths in the world.Almost two-thirds of new diagnoses occur in developing countries,with 42%in China alone.Although surgery is the main treatment for gastric cancer,chemotherapy is also necessary for tumor metastasis.VCPA is a novel derivative of a-tocopheryl succinate that has been shown to increase the efficacy of numerous chemotherapeutic drugs.The aim of our study was to explored the effects and mechanisms of VCPA combined with DOX-induced apoptosis in human gastric cancer cells.Methods:The detection of cell viability and cell growth were performed with a cholecystokinin-8(CCK-8)assay and the IC50 values were also calculated.Colony formation assay was used to detect the capacity of cells proliferation.Cell cycles and cell apoptosis were analysised by flow cytometry.Intracellular reactive oxygen species(ROS)was detected by reactive oxygen species assay kit.Mitochondrial membrane potential was detected by mitochondrial membrane potential assay kit with JC-1.The proteins levels of mitochondrial related apoptotic pathway and MAPK signaling pathway were measured by western blot.Resluts:VCPA inhibited the growth of SGC-7901 and MGC-803 cells in a concentration-dependent manner.The IC50 values of VCPA against SGC-7901 and MGC-803 were 14.25 and 16.91 mM,respectively.The inhibitory effect was also noted in a clonogenic survival assay of SGC-7901 and MGC-803 cells treated with VCPA.VCPA induced ROS-dependent apoptosis and potentiated doxorubicin-induced apoptosis in human gastric cancer cells.In addition,treatment of cells with the combination of 10 mM of VCPA and DOX significantly reduced the IC50 of DOX in SGC-7901 and MGC-803 cells(6.68-and 9.68-fold reductions,respectively)compared with DOX-only treatment.VCPA enhanced DOX-induced intracellular ROS accumulation and mitochondrial dysfunction.Mitochondrial pathways played mian role in DOX/VCPA-induced apoptosis and MAPK signaling might induce resistance rather than sensitization to DOX/VCPA-induced apoptosis.Conclusion:VCPA,a novel synthetic derivative of a-tocopheryl succinate,sensitizes human gastric cancer to doxorubicin-induced apoptosis via ROS-dependent mitochondrial dysfunctionPart 2.The anti-tumor effects of VCPA combined with doxorubicin in human gastric carcinoma MGC-803 cells tumor xenografts in nude mice.Objective:To investigate the effects of VCPA combined with DOX on inhibiting the growth and inducing apoptosis of human gastric carcinoma MGC-803 cells tumor xenografts in nude mice.Methods:Six-week-old athymic BALB/cA nu/nu female mice were purchased from Vital River Laboratory Animal Technology Company and maintained in an Animal Biosafety Level 3 Laboratory at the Animal Experimental Center of Wuhan University.All animal experiments were performed according to the Wuhan University Animal Care Facility and National Institutes of Health guidelines.Approximately 5 × 106 MGC-803 cells were harvested and suspended in 200 ml of PBS and Matrigel(1:1)and injected subcutaneously into the right flank of each mouse.Two weeks after the xenotransplantation,mice were randomized into four groups and treated as follows:VCPA(10 mg/kg i.p.every other day for 3 weeks),DOX(1 mg/kg i.p.every other day for 3 weeks),a combination of both,or saline as an untreated vehicle.The size of subcutaneous tumors and weight of the mice were recorded every two days.Tumor volume(V)was calculated according to the formula V = 0.5 ×1 × w2,where 1 is the greatest diameter and w is the diameter at the point perpendicular to 1.At the end of treatment,mice were sacrificed,and the tumors were removed and used for immunohistochemical staining.Histopathological abnormalities were observed in the vital organs such as heart,liver,and kidney by HE staining.Ki67 and CD31 were examined by immunohisto-chemistry in the tumor sections.The level of tumor tissue apoptosis in vivo was determined using TUNEL assay.Resluts:VCPA alone failed to inhibit the growth of the xenograft tumor.Although DOX alone significantly inhibited tumor growth,the combination of VCPA and DOX was much more effective in reducing tumor burden.The tumor volume and weight in the combinational group were significantly lower than DOX alone.Additionally,none of the mice showed obvious body weight loss or abnormal symptoms in drug treatments.Furthermore,no evident histopathological abnormalities were observed in the vital organs such as heart,liver,and kidney by HE staining.Although DOX alone down-regulated the expression of Ki67,the combination was more effective.The expression of CD31 was also decreased in the DOX/VCPA group compared with the control.TUNEL analyses revealed that the DOXA/VCPA combination treatment effectively promoted apoptosis in xenograft tumors.Conclusion:VCPA amplifies the therapeutic effect of DOX in a xenograft gastric cancer nude mouse model.