Study On Anti-ovarian Cancer Of Amomum Tsaoko By Inhibiting Angiogenesis

Objective:Ovarian Cancer（OC）is one of the most common malignant tumors of the female reproductive system.According to statistics,there are239,000 new cases and 152,000 new deaths of Ovarian Cancer worldwide every year,and 52,000 new cases and 22,500 deaths of Ovarian Cancer in China every year.Hidden,the biggest characteristic is ovarian cancer disease diagnosed early diagnosis for its difficulty,about two-thirds of patients have been found in the middle-late stage of cancer.The ovarian cancer cells when the peritoneal spread fast,wide scope,drugs prone to resistance,although the detection methods and treatment for ovarian cancer,there is a big development.But the five-year relative survival rate for ovarian cancer has remained low at 30 to 40 percent.At present,surgical treatment and chemotherapy are usually used in clinical comprehensive treatment,but with the extension of the treatment process,the toxic and side effects of chemotherapy drugs and drug resistance in the body become more and more serious.Traditional Chinese medicine in the treatment of ovarian cancer,especially advanced ovarian cancer after surgery or non-surgery patients shows obvious advantages.TCM treatment of ovarian cancer can not only improve the immunity of patients,reduce the side effects of radiotherapy and chemotherapy,but also prolong the life of patients and improve their quality of life.Zingiberaceaae plants have been proved to have a good anticancer effect on a variety of tumors.The fruit is the mature and dry fruit of Zingiberaceaae Amomum tsaoko fruit,which is characterized by temperature,goes to the spleen and stomach meridian,and has the effect of dampness and dampness,preventing malaria and removing phlegm.Used for the treatment of cold and dampness,abdominal distension and pain,spleen full vomiting,malaria cold and fever,plague and fever and other syndromes.As a traditional medicinal and food homologous plant,Amomum tsaoko is widely used in food and spice industry in China.Existing studies mainly focus on the analysis,extraction and planting of volatile components.Pharmacological experimental studies have shown that the biological activities of Amomum tsaoko are also diverse:anti-tumor,anti-oxidation,anti-inflammatory,hypidemia,hypoglycemia and other activities are involved.The antitumor study showed that the essential oil could induce the apoptosis of nasopharyngeal carcinoma 6-10B cells and inhibit the growth of tumor cells in H22 tumor bearing mice.It was recorded in ancient medicine that the herb and fruit prescription could be used for various diseases of gynecology.Modern clinical practice found that the decoction of Amomum tsaoko could be used to treat abdominal distension and pain of gynecology abdominal surgery.In the preliminary test of screening the anti-ovarian cancer activity of Zingiberaceae herbs,the anti-ovarian cancer activity of the ethanol extract of Amomum tsaoko（At-EE）is the best,so this paper takes At-EE as the anti-ovarian cancer research object.Angiogenesis is one of the key factors in tumor growth,and targeting angiogenesis has been one of the methods for clinical treatment of cancer.In this study,we found the relationship between the anti-ovarian cancer activity of At-EE and its inhibition of angiogenesis,and identified the cytokines and potential pathways related to the inhibition of tumor angiogenesis of At-EE,which provided favorable evidence for the isolation and screening of anti-ovarian cancer drugs from Amomum tsaoko.Methods:1.Rutin and gallic acid were used as standards to determine the contents of flavonoids and phenolic compounds in At-EE.To prepare stable and reliable Amomum tsaoko extract.2.a)Establish a luciferase labeled mouse model of in-situ transplanted SKOV3 tumor,and investigate the distribution and diffusion of ovarian tumor in mice after At-EE administration;b)Ovarian tumor bearing mouse model was established by subcutaneous transplantation of SKOV3 tumor cells on the back of mice,and the inhibitory effect of At-EE on ovarian tumor was investigated;c)The expression of CD31 was detected by immunohistochemical analysis after staining of the tumor tissue sections,and the mean MVD of microvessels in tumor cells was counted.3.a)In vitro inhibitory effects of At-EE on proliferation of ovarion cancer SKOV3 cells,human umbilical vein endothelial cells HUVEC and human immortalized epidermal cells Ha Ca T were investigated by MTT assay.b)Annexin V-FITC double staining was used to investigate the effect of At-EE on apoptosis of SKOV3 cells,HUVEC cells and Ha Ca T cells.c)Transwell chamber was used to establish the co-culture model of SKOV3cells and HUVEC cells.Scratch experiment,invasion experiment and in vitro angiogenesis experiment were performed on normal HUVEC cells and HUVEC cells co-cultured with SKOV3 cells by At-EE.To study the effects of At-EE on migration,invasion and in vitro angiogenesis of HUVEC cells and SKOV3 cells.4.a)The m RNA levels of secretion-related cytokines in SKOV3 cells were detected by RT-PCR,and IL-6 and VEGF were determined to be the key cytokines affecting the migration,invasion and angiogenesis of co-cultured cells.b)ELISA was used to verify that At-EE could inhibit the expression levels of VEGF and IL-6 of SKOV3 cytokines in ovarian cancer;c)The involvement of VEGF and IL-6 in At-EE inhibition of HUVEC cells migration was verified by scratch test,and the involvement of VEGF and IL-6 in At-EE inhibition of endothelial cell HUVEC tubulogenesis was verified by in vitro angiogenesis test.5.Western Blot was used to analyze the expression of STAT3 and NF-κB in At-EE treated SKOV3 cells,and the interaction between NF-κB and P-STAT3,IL-6 and VEGF.6.The expression of GRP78 and CHOP in At-EE SKOV3 cells was detected by WB.The expression of NF-κB/P-STAT3 was detected by WB after silencing CHOP gene in SKOV3 cells.The expression of IL-6/VEGF in SKOV3 cells was detected by ELISA after SKOV3 CHOP gene was silenced.The expressions of NF-κB,P-STAT3,CHOP,VEGF and IL-6were detected by immunohistochemical staining.Results:1.For 1kg of Amomum tsaoko ethanol reflux extraction 198.6 g powder after drying.The average content of total flavonoids was 32.14mg/g and RSD was 1.134%.The average content of total phenol was112.23 mg/g with RSD of 0.532%.2.a)Fluorescent spot area and brightness in the lower abdomen of mice in the blank control group were higher than those in the At-EE group.The number of SKOV3 cells in each tumor-bearing mouse 4 weeks later was calculated according to the fluorescent spot intensity area.The number of SKOV3 cells in At-EE group 3.5×10~6 was significantly lower than that in blank group 6×10~6（P<0.01）.b)The weight of the stripped tumor in the blank control group（1.18±0.31g）was significantly heavier than that in the administration group（0.78±0.12g）,and the tumor volume also showed that the tumor volume in the blank control group increased rapidly over time,indicating that At-EE had a good inhibitory effect on the growth of SKOV3 solid tumor.c)A large number of patchy dark brown particles were observed in the cytoplasm of blank control group with strong positive expression,while a small number of scattered brown particles were observed in the cytoplasm of At-EE administration group with weak positive expression.The value of MVD-CD31 in tumor tissues of blank control group was all higher than that of At-EE group,and there was significant difference between the mean value of MVD-CD31 between the two groups（P<0.05）.3.a)SKOV3,HUVEC and Ha Ca T cells were treated with 0,20,40,60,80 and 100μg/m L At-EE,respectively,and the relative cell viability（%）was measured and calculated by MTT.The results showed that SKOV3cell viability decreased gradually with the increase of At-EE concentration.The cell viability of HUVEC and Ha Ca T were not affected.b)SKOV3cells were treated with 0,10,20,40,60,80 and 100μg/m L At-EE,respectively,and the apoptosis rate was detected by Annexinv-FITC/PI assay.With the increase of At-EE concentration,the apoptosis rate of SKOV3 cells was significantly increased.The same method was used to detect the apoptosis rates of HUVEC and Ha Ca T cells treated with different concentrations of At-EE.There was no significant difference in the apoptosis rates of HUVEC and Ha Ca T cells in the range of 0-100μg/m L At-EE concentration.c)For HUVEC cells,co-cultured HUVEC cells,co-cultured HUVEC cells treated with 5μg/m L At-EE,co-cultured HUVEC cells treated with 10μg/m L At-EE,scratch test,invasion test and in vitro angiogenesis test indicated that At-EE did not directly affect HUVEC cells.However,At-EE can significantly inhibit the improvement of HUVEC migration,invasion and in vitro angiogenesis of endothelial cells after co-culture,and this ability is enhanced with the increase of At-EE drug concentration in a concentration-dependent manner.4.a)m RNA expressions of VEGF,IL-6,IL-8,FGF,Mc P-1 and OPN in SKOV3 cells and SKOV3 cells treated with 10μg/m L At-EE were detected by RT-PCR assay.The results showed that the m RNA expression levels of VEGF（P<0.05）and IL-6（P<0.01）in At-EE treatment group were significantly decreased compared with control group.b)Protein concentration of IL-6 detected by ELISA:IL-6 concentration in blank control group was 14.87±1.31 pg/m L,while IL-6 concentration in 5μg/m L and 10μg/m L At-EE treated SKOV3 cell culture medium was 8.75±0.73pg/m L and 6.97±0.87 pg/m L,respectively.There were significant differences between the two groups and the control group.Protein concentration of VEGF detected by ELISA:VEGF concentration in blank control group was 21.14±1.56 pg/m L,while VEGF concentration in 5μg/m L and 10μg/m L At-EE group was 14.39±0.62 pg/m L and 12.21±0.87pg/m L,respectively,which were significantly different from Ctrl group.c)At-EE can significantly inhibit the migration of HUVEC in endothelial cells.When exogenous IL-6 or VEGF is added,this inhibition effect is reversed,indicating that IL-6 and VEGF secreted by SKOV3 are key molecules of At-EE inhibiting the migration ability of HUVEC cells.In vitro experiments on angiogenesis also showed similar results.5.WB analysis showed that p-STAT3 and NF-κB expression were down-regulated in At-EE.The interaction between NF-κB and p-STAT3was verified by WB experiments with Stattic and PDTC inhibitors.After NF-κB and p-STAT3 were down-regulated by Stattic and PDTC inhibitors,IL-6 and VEGF expression were inhibited in SKOV3 cells.WB analysis showed that the addition of exogenous IL-6 and VEGF could reverse the down-regulation of NF-κB and P-STAT3 expression in SKOV3 cells by At-EE.6.WB analysis showed that At-EE could significantly up-regulate the expression levels of GRP78 and CHOP proteins in a concentration-dependent manner,which was direct evidence of ER stress induced by At-EE.The down-regulation of NF-κB/p-STAT3 by At-EE was reversed by WB silencing CHOP gene in SKOV3 cells.ELISA showed that silencing of CHOP gene in SKOV3 cells could reverse the down-regulation of IL-6/VEGF by At-EE.Immunohistochemical staining confirmed that At-EE down-regulated NF-κB,p-STAT3,VEGF,IL-6,and up-regulated CHOP expression.Conclusion:Through the experiments in vivo and in vitro confirmed the At-EE has a resistance to the activity of ovarian cancer,the activity related to anti-tumor angiogenesis.At-EE does not directly affect normal vascular endothelial cells,but down-regulates ovarian cancer cell secretion of IL-6and VEGF by inhibiting the activation of p-STAT3 and NF-κB,resulting in inhibition of tumor angiogenesis.In addition,we demonstrated that p-STAT3 and NF-κB interregulate each other,and that IL-6 and VEGF also mediate p-STAT3 and NF-κB activation,thus forming a cycle.In addition,it has been confirmed that At-EE can block the p-STAT3/NF-κB/IL-6 and VEGF circuits by inducing ER stress.This study laid a certain foundation for the anti-ovarian cancer effect of Amomum tsaoko,and provided a possible research direction for screening and isolating anti-ovarian cancer drugs from Amomum tsaoko extracts.