The Effect And Mechanism Of Cucurbitacin D On Gastric Cancer Cell Survival

BackgroundGastric cancer（GC）,which is the fourth most commonly cancer in the world,is a major public health problem that needs to be solved on a global scale.According to the epidemiological statistics,about 990,000 people are diagnosed as gastric cancer and 738,000 people die due to the gastric cancer every year on the world.Therefore,it is also the second major deadly cancer.The incidence rate of gastric cancer varies according to age,sex,and region.The age of onset is over 50 years old.The incidence rate of gastric cancer among men is 2 to 3 times more than that of women.Worldwidely,the incidence rate is the highest in Eastern Asia（including China）,Eastern Europe,and South America,rather the morbidity of the most region of North America and Africa is the lowest.With the rapid pace of modern people’s life,the change of diet structure,and the increase of learning and work pressure,the age of onset has younger trend and the number of the incidence is increasing year by year.According to the epidemiological survey,the number of cases of gastric cancer and the number of deaths in China accounted for 42.6%and 45%of total number worldwide.Respectively,among the 183 countries in the world,the incidence rate of China ranks fifth and the mortality rate of China ranks sixth.In 2012,there were about 424,000 new cases of gastric cancer in China,of which 298,000 were males and 126,000 were females.The gastric cancer’s incidence rates of national total population and male which only lower than lung cancer ranks second in the same period of malignant tumor.The incidence of female gastric cancer is lower than that of breast cancer,lung cancer and colorectal cancer,ranking the fourth.In 2012,there were about 298,000 deaths from gastric cancer in China,of which about 206,000were males and 92,000 females.The death rate of gastric cancer in China is 22.04/10million.The death rate of the total population and male gastric cancer are only lower than that of lung cancer and liver cancer,ranking third in the same period.The mortality rate of female gastric cancer is only lower than that of lung cancer,taking the second place.China is a country with high incidence rate of gastric cancer,and gastric cancer is still one of the major diseases that seriously threaten the health of Chinese residents.Reducing the incidence rate and mortality of gastric cancer in China is becoming a major public health problem that we must face directly.At present,most of the patients with gastric cancer are diagnosed with advanced gastric cancer,and the 65%⁷5%of patients with radical desire were faced with the recurrence and the outcome of the systemic disease after the treatment.In recent years,with the development of science and technology,the diagnosis and treatment technology of gastric cancer have been improved,especially the progress of comprehensive treatment of tumor,such as surgery,endoscopy,radiotherapy,chemotherapy,and targeting therapy.The treatment effect of gastric cancer has been improved,but the prognosis of comprehensive treatment is still not ideal for advanced and metastatic gastric cancer patients.Chemotherapy is an important clinical treatment for patients diagnosed with carcinoma in situ which is not suitable for operation or metastatic cancer.The commonly used drugs are oxaliplatin,5-fluorouracil（5-FU）,docetaxel and irinotecan.However,the drug resistance rate is high and the cytotoxic effect of the drug is increasing due to the long term application.So chemotherapy can only bring the median survival rate of 12 months.Therefore,searching for new chemical drugs with low toxicity and high efficiency is of great significance in the treatment of gastric cancer.Cucurbitacins are a group of natural three terpene compounds extracted from cucurbit and cruciferous plants.Modern studies have reported that Cucurbitacins play anti-inflammatory and anti-tumor effects in various tumor diseases,which can inhibit cell growth and promote apoptosis of various tumor cell lines.Cucurbitacin B,D,E,I,F,O,P and Q can inhibit the proliferation and differentiation of tumor cells by inhibiting the phosphorylation of STAT3.It has been reported that cucurbitacin D extracted from Trichosanthes can induce apoptosis of various tumor cells,and can affect various signal transduction pathways such as Stat3 and NF-kappa B.Chemotherapy-induced apoptosis of tumor cells is an important means of clinical treatment of tumor diseases,cucurbitacin D as a potential anti-tumor drug has research value.However,the role of cucurbitacin D in gastric cancer cells and its specific molecular mechanism of inducing apoptosis are still unclear,and need to be further studied.At the cellular level,the effect of cucurbitacin D on the proliferation and apoptosis of gastric cancer cells（AGS,SNU1,Hs746T）was studied in the multi concentration gradient and time gradient.Inducing the activation of Akt with SC79and inhibiting the expression of iNOS by L-canavanine to discuss the potential mechanismes of cucurbitacin D in gastric cancer.At the organizational level,the transplanted tumor model of nude mice was constructed to research the effect of cucurbitacin D on the proliferation and apoptosis of gastric cancer in vivo and its molecular mechanism.Method1.The effects of Cucurbitacin D with different concentration gradient and time gradient on the proliferation of gastric cancer cells（AGS,SUN1 and Hs746T）were detected by CCK-8.The effects of Cucurbitacin D on the levels of ROS in gastric cancer cells（AGS,SUN1 and Hs746T）were detected by DCFH-DA fluorescence probe.The effects of Cucurbitacin D on the apoptosis of gastric cancer cells（AGS,SUN1 and Hs746T）were detected by TUNEL.The level of Ca2+,ATP and NO in gastric cancer cells（AGS,SUN1 and Hs746T）were also detected.2.Western blot was used to detect the effect of Cucurbitacin D on the apoptosis related signaling pathway of iNOS,Bax,and Akt signaling pathway in gastric cancer AGS cells.L-canavanine was used to interfere the expression of iNOS in the AGS cells,and the SC79 was used to activate the Akt.The effect of L-canavanine and SC79 on the inhibitory of the proliferation in gastric cancer cells induced by Cucurbitacin D in vitro was detected by CCK-8,and the effect of L-canavanine and SC79 on the increasing apoptosis of AGS cells induced by Cucurbitacin D was detected by TUNEL.The BALB/c-nu/nu mice were injected with 1 x 10⁶ AGS cells per mouse to construct a subcutaneous tumor model（Intraperitoneal injection）.And the growth of subcutaneous xenografts in nude mice was observed and recorded carefully.The length and diameter of the tumor were recorded and the volume was calculated.When the volume of the tumor reached 200mm³,the BALB/c-nu/nu mice were injected with Cucurbitacin D（1 mg/kg）and SC79（20 mg/kg）,both were intraperitoneal injection.After the experiment,the growth curve of subcutaneous transplanted tumor in nude mice was drawn to detect the effects of Cucurbitacin D and SC79 on the growth of subcutaneous transplanted tumor,and TUNEL method was used to detect the effects of Cucurbitacin D and SC79 on the cell apoptosis in subcutaneous transplanted tumor.Result1.The result of CCK-8 test showed that the inhibitory effect of Cucurbitacin D on the proliferation of gastric cancer cells（AGS,SUN1 and Hs746T）in vitro in a concentration and time dependent manner.That is,the inhibition effect gradually increased with the increasing of drug concentration and the prolongation of action time.The results of ROS detection showed that the intracellular ROS content of gastric cancer cells（AGS,SUN1 and Hs746T）cells increased obviously after the treatment of Cucurbitacin D（P<0.05）,and the effect in a dose dependent matte.The concentration of ROS increased gradually with the increase of drug concentration（P<0.05）.The result of TUNEL staining showed that the apoptosis rate of the gastric cancer cells（AGS,SUN1 and Hs746T）cells increased gradually with the increase of Cucurbitacin D concentration（P<0.05）.The Ca²⁺concentration,and the contents of NO in the gastric cancer cells（AGS,SUN1 and Hs746T）cells gradually increased with the increase of Cucurbitacin D concentration and the prolongation of the action time（P<0.05）.2.The result of Western blot showed that the expression of iNOS,Bax,Cleaved caspase-9 and cytochrome proteins in gastric carcinoma AGS cells increased significantly with the increase of Cucurbitacin D concentration,and the expression of Bcl-2 protein decreased significantly（P<0.05）,the level of phosphorylation among the Akt,mTOR,and rs6 proteins were significantly reduced as well（P<0.05）.In order to further explore the potential mechanism of Cucurbitacin D in inhibiting the proliferation and promoting apoptosis of gastric cancer cells,the L-canavanine and SC79 were used to inhibit the expression of iNOS and the activation of Akt in gastric cancer AGS cells respectively,and set up six groups:the control group,Cucurbitacin D group,SC79 group,Cucurbitacin D+SC79 group,L-canavanine group,and Cucurbitacin D+L-canavanin group.The results of CCK-8 and TUNEL staining in vitro showed that Akt agonist SC79 significantly eliminated the inhibition of proliferation and increasing of apoptosis on gastric cancer AGS cells induced by Cucurbitacin D,compared with the normal control group and Cucurbitacin D group（P<0.05）.Similarly,iNOS inhibitor L-canavanin also could antagonize the effects of Cucurbitacin D on gastric cancer AGS cells in vitro.The model of subcutaneous transplantation of gastric cancer in nude mice was constructed,and randomly divided into the control group,the Cucurbitacin D group,the SC79 group and the Cucurbitacin D+SC79 group.The long diameter（L）and the short diameter（W）of the tumor were observed and recorded,and the volume of the tumor was calculated by formula V（mm3）=LW² x 0.5236 and the growth curve of the nude mouse subcutaneous transplanted tumor was plotted.The results showed that Cucurbitacin D significantly inhibited the growth of tumor compared with the control group（P<0.05）,and SC79 antagonized the effect.The tumor tissue was embedded and stained with TUNEL.The results showed that,compared with the negative control group,the brown yellow apoptotic cells of the Cucurbitacin D treatment group were much more,the percentage of apoptotic cells was higher（P<0.05）,and the SC79 treatment blocked the apoptosis induced by Cucurbitacin D（P<0.05）.ConclusionCucurbitacin D has an anti-tumor effect on gastric cancer cells both in vitro and in vivo;Cucurbitacin D interferes with the expression of iNOS and the activation of Akt to promote the accumulation of ROS and Ca²⁺in cells,increases the content of NO in cells.Cucurbitacin D plays an important role in gastric cancer by affecting iNOS/NO signaling pathway and Akt signal pathway.