Synergistic Effect And Its Mechanism Of Thymoquinone And Cisplatin On Gastric Cancer Both In Vitro And In Vivo

Background and objectiveGastric cancer(gastric cancer,GC),remains one of the most common gastrointestinal malignant tumors,ranking fifth in global morbidity and third in mortality.China is one of the high incidence regions of GC,the number of new cases each year is about 400,000,and the overall mortality rate is increasing year by year.GC are usually atypical and non-specific at early stage.Therefore,majority of GC were diagnosed at advanced stages,with poor clinic treatment effects and low 5-year survival rates,which seriously endangers the health of Chinese people.Chemotherapy is a common treatment for GC at present,but its clinic application is limited due to its serious side effects,adverse reactions and drug resistance.It is very important to develop new chemotherapeutic drugs and seek effective combination treatment in order to provide an improved therapy for advanced GC.Recently,the clinical application of black grass seeds has attracted a lot of attention due to its multiple medicinal value.The various active substances extracted from black grass seeds have been reported that they could induce oxidization,inflammation,immunologic function,anti-bacterial,anti-tumor or other biological effects.Thymoquinone(TQ),is an active monomer extracted and purified from black grass seeds.Precious studies have demonstrated that TQ can exert its anti-tumor effects on various types of cancers,including colorectal cancer,breast cancer,lung cancer,prostate cancer,and so on.It can significantly inhibit the proliferation,invasion and migration of cancer cells,and induce apoptosis of cancer cells.In addition,TQ can enhance the apoptosis of colon cancer cells induced by5-fluorouracil(5-Fu)both in vitro and in vivo,and strengthen the anti-tumor effect of gemcitabine,a conventional chemotherapeutic drug,on pancreatic cancer.However,the specific mechanism of action is unclear.Cisplatin is the basic drug for the treatment of GC.By interfering with DNA repair,cisplatin can induce DNA damage and then induce apoptosis of cancer cells.However,cisplatin treatment alone is not ideal at present for GC,and the drug resistance rate of relapsed tumor patients after cisplatin therapy is quite high.Several studies have found that combined therapy(based on cisplatin)is superior to cisplatin alone,including ovarian cancer,gastric cancer,esophageal cancer,lung cancer,pancreatic cancer,and so on.Therefore,more and more attention has been paid to the clinical application of cisplatin combined therapy strategy in order to improve the current situation of tumor treatment.PI3K/AKT signaling pathway is one of the important pathways involved in the regulation of cell proliferation.It is often activated abnormally in the carcinogenesis and development of tumors,which were recognized as a key"tumor driver".There are many subunits and kinases in this pathway,thus providing many potential targets for cancer therapy.Tumor suppressor gene(PTEN(phosphatase and tension homlog,PTEN)was first discovered in 1997.Its expressed product has dual activities of protein phosphatase and lipid phosphatase,which can dephosphorylate PIP3 into inactive PIP2,to block the activation of AKT,inhibiting activation of PI3K/AKT signaling pathway.PTEN gene mutations,deletions or promoter hypermethylation occur widely in many human malignant tumors.However,few research was conducted on the role of PTEN gene in the carcinogenesis and development of GC.It has been shown that abnormal activation of Wnt/?-catenin signaling pathway is closely related to tumor invasion and migration ability.It has been reported that,as an important negative regulatory factor of Wnt/?-catenin signaling pathway,GSK-3?can be closely regulated by PI3K/AKT signaling pathway.Several studies have confirmed that the change of PI3K/AKT pathway activity plays an important role in the anti-tumor process of TQ.TQ can improve the sensitivity of tumor-resistant cells to doxorubicin and docetaxel by targeting PI3K/AKT signaling pathway.Our previous research has confirmed TQ can exert anti-tumor effects on GC both in vitro and in vivo.We hypothesized that TQ might inhibit the activity of PI3K/AKT signaling pathway by up-regulating the PTEN gene,thus enhancing the sensitivity of gastric cancer cells to cisplatin,and that PTEN gene may play an important role in the invasion and migration of gastric cancer.Therefore,on the basis of the previous study,we further studied the effect of TQ combined with cisplatin on the biological behaviors of GC cells,and explored the mechanism of PTEN gene regulating the carcinogenesis and development of gastric cancer.The anti-tumor effects of TQ and cisplatin on gastric cancer were also confirmed in vivo.In the first part,human gastric cancer cell lines(HGC-27,SGC-7901,MGC-803)were treated with TQ and/or cisplatin.The effects of thymoquinone and/or cisplatin on the proliferation and apoptosis of GC cells were detected by CCK-8,clone formation,Hoechst 33258 staining,flow cytometry and western blot.We also construct stable transfected gastric cancer cell lines to explore the effect of TQ on the sensitivity of gastric cancer cells to cisplatin and its mechanism.In the second part,the mechanism of PTEN gene in regulating the invasion and migration of gastric cancer cells was further discussed.In the third part,a subcutaneous xenograft tumor model of GC cells was conducted to explore the effect of TQ and cisplatin therapy on the biological behaviors of gastric cancer cells in nude mice.Materials and methods1.The human gastric cancer cells including HGC-27,SGC-7901 and MGC-803,were treated with different concentrations TQ(0,5,10,20,40,80?M)and/or cisplatin(0,0.25,0.5,1,2,4?g/m L),respectively.The inhibitory rates of TQ and/or cisplatin on the proliferation of GC cells were evaluated by CCK-8,and the IC50 of TQ and/or cisplatin was calculated by using the Grah Pad software.The combined effects of TQ and cisplatin on GC cell viability were analysis by Calcinyn 2.0software calculating combination index(CI).By means of CCK-8,clone formation,flow cytometry,and Hochest 33258 staining,the effects of TQ and cisplatin on the proliferation and apoptosis of gastric cancer cells were detected.The expression of PTEN,p-AKT,AKT,P-gp,Bax,Bcl-2,Cyt C,AIF,procaspase-3,clear caspase-3,procaspase 7,clear caspase 7,pro-caspase-9 and clear caspase-9 were measured by western blot.2.Construct stable transfected GC cell lines with down-regulation of PTEN.After stimulation of the cells with TQ and/or cisplatin,CCK-8,clone formation,Hoechst33258 apoptosis staining and western blot assay were used to verify whether TQ enhanced the sensitivity of gastric cancer cells to cisplatin by modulating PTEN gene.3.Gastric cancer tissue chip were purchased from Wuhan Iwill Biotechnology Company(IWLT-N-96G43,China).The cancer tissues and adjacent normal tissues from a total of 48 GC patients,were contained in this tissue chip.Immunocytochemistry,a wound healing assay,a Matrigel invasion assay,an immunofluorescence staining were performed to detect expression of PTEN and?-catenin in gastric cancer and adjacent normal tissues.The effects of of PTEN knockdown on GC cells migration and invasion were accessed.Further,MMP-2 and MMP-9 activities were analyzed by zymography assay.The changes in related proteins were further quantified by western blotting.4.The SGC-7901 cells were selected and resuspended by PBS to adjust the cell concentration 4×10~7/m L.The cells were then subcutaneously inoculated into the right back of the nude mice(each nude mouse was successfully inoculated with 100?L in volume).After 7 days,we successfully constructed subcutaneous transplanted tumor models.The nude mice were then randomly divided into four groups and injected intraperitoneally with physiological saline,cisplatin,TQ,and the combination of TQ and cisplatin,respectively.The state of nude mice and the growth of tumor were observed and recorded.The drug was given at intervals of 2 days.During the treatment,tumor size was measured in two dimensions using vernier caliper by two researchers(2-3times/week),and the tumor volume(TV)was calculated using the formula:TV(mm~3)=0.5×d~2×D,where d and D are the shortest and longest diameters,respectively.After 10 times of administration,the blood was obtained from the eyeball.The tumors were harvested,weighed,and then were analyzed by HE staining and TUNEL assay.Liver and renal function were measured through detection of the levels of ALT,AST,Urea and Cr.Results1.GC cells including SGC-7901,HGC-27,MGC-803,were incubated with TQ(0,5,10,20,40,80?M)and cisplatin(0,0.25,0.5,1,2,4,8?g/ml)at different concentrations for 24h respectively.TQ and cisplatin exhibited inhibition of cell growth by CCK-8 assay,respectively,in a concentration-dependent manner.In addition,it was observed SGC-7901 was the most sensitive to TQ.TQ,at a concentration up to 5?M,was of no obvious cytotoxicity with approximately 90%of cells viability in all GC cell lines tested.GC cells pretreated with TQ(5?M),were more sensitively to cisplatin(0,0.25,0.5,1,2,4?g/m L),the IC50 values of cisplatin in combination with TQ(5?M)in GC cells were much lower than of cisplatin alone(P<0.05).The analysis of CI using Calcusyn 2.0 softwore indicated that TQ and cisplatin have synergistic inhibitory effects on the proliferation of gastric cancer cells.In the colony formation,the numbers of HGC-27 cells colony in control,cisplatin(2?g/ml),TQ(5?M),and TQ pretreatment following cisplatin were169.3±10.6,126.3±11.6,120.0±3.6,49±6.9,respectively.Hoechst 33258 staining showed that significant differences were found in the number of apoptotic cells,the percentage of apoptotic cells induced by TQ and/or cisplatin was significant higher than that of the untreated control group(*P<0.05 versus untreated cells),meanwhile,the apoptotic cells of combined treatment was higher than those of cisplatin alone.The Annexin PE/7-AAD apoptosis staining showed SGC-7901 cells treated with a combination(5?M TQ+2ug/ml cisplatin)contained more early and late apoptotic cells(19.15%and 63.5%)than control(1.77%and 5.23%),TQ(3.47%and 9.58%),cisplatin(11.24%and 22.15%).Western blot was further conducted and we found TQ led to an obvious increase in PTEN proteins,and a clear decrease in p-AKT and P-gp proteins in a concentration-dependent manner;combined group led to an increased in PTEN proteins level,but a decreased in p-AKT and P-gp proteins level;the apoptotic protein Bax,Cyt-C,AIF,cleaved caspase-3,cleaved caspase-7,cleaved caspase-9 was increased significantly in the combined group,but the level of anti-apoptotic protein Bcl-2,procaspase-3,procaspase-7 and procaspase-9 was decreased significantly(P<0.05,respectively).2.GC cell lines were transfected with PTEN-sh RNA in order to down-regulate the PTEN expression in GC cells,and blank control were also be conducted.In CCK-8 assay,cells were treated with cisplatin alone or TQ+cisplatin,the sensitivity of gastric cancer cells with down-regulation of PTEN was lower than that of the NC group.Colony formation assay showed the combined HGC-27/PTEN sh RNA has higher clonoy counts than in NC.We also found that the apoptosis rate of GC cells in cisplatin or combined group,was decreased after interfering with PTEN expression(P<0.05).SGC-7901/NC,and SGC-7901/PTEN-sh RNA were incubated with 2?g/ml cisplatin alone,and a combination of 5?M TQ+2?g/ml cisplatin,respectively.TQ and cisplatin led to a clear increase in PTEN protein,an obvious decrease in p-AKT,P-gp proteins,and a clearly increase in apoptotic proteins Bax,cleaved-caspase-9,and cleaved-caspase-3 protein than cisplatin alone in NC cells(P<0.05).These results above suggest TQ can enhance the sensitivity of gastric cancer cells to cisplatin,while down-regulation of PTEN contributes to weakening the apoptosis induced by cisplatin alone and a combination of TQ and cisplatin,respectively.3.Low expression of PTEN was found in majority of gastric cancer tissues,which showed significant associations with differentiation grade in gastric cancer patients.Further,a negative correlation was revealed between PTEN and?-catenin protein expression in gastric cancer tissues(r=-0.546,P<0.01).Additionally,PTEN knockdown promoted the migration and invasion of cells and caused an obvious increase in p-AKT,p-GSK-3?,?-catenin,E-cadherin,MMP-7,MMP-2,and MMP-9in gastric cancer cells.Gelatin zymogram showed that the activity of MMP-2 and MMP-9 in human gastric cancer cells were increased by interfering with the expression of PTEN.4.In vivo experiment of gastric cancer cells in nude mice,24 nude mice were successfully established subcutaneous transplantation tumor model.They were randomly divided into four groups:control group,cisplatin,TQ combined with cisplatin group.The transplanted tumor grew gradually.At the end of experiment,tumors were harvested,weighed,and we measured the length and short diameter of the tumors.Tumors grew progressively and reached approximately 1214.92±207.99mm~3 in control group.However,tumor growth was suppressed by TQ and cisplatin in vivo,respectively(582.49±75.43mm~3 and 845.01±53.09mm~3)(*P<0.05 versus control).Furthermore,the combination of TQ and cisplatin significantly inhibited tumor growth in vivo(345.99±54.83 mm~3)compared with cisplatin(*#P<0.05 versus cisplatin alone).The weight of the tumors in control group was heavier than that of the tumors in cisplatin group,TQ and combination group,furthermore,tumors growth were significantly inhibited in combination group than in cisplatin alone.Further TUNEL analysis revealed the combination of TQ and cisplatin resulted in an apparent increase in cell apoptosis in the tumor mass than that in other group(P<0.05).IHC staining showed the expression level of PTEN protein in the combined group was higher than that in either treatment alone(P<0.05).The results showed that TQ could enhance the antitumor effect of cisplatin by up-regulating the expression of PTEN gene in vivo.ConclusionTQ significantly augments cisplatin-induced anti-tumor effects on gastric cancer both in vitro and in vivo by up-regulating PTEN gene.Meanwhile,PTEN Gene induces cell invasion and migration via regulating AKT/GSK-3?/?-catenin signaling pathway in human gastric cancer.TQ might be as a promising candidate as a cancer chemopreventive or chemotherapeutic agent for antineoplastic combination therapy and merits further clinical investigation.