| Research background and objective Gastric cancer,one of the most common cancer.In recent years,the prognosis of gastric cancer has improved owing to the great progress of surgical techniques,chemotherapies,radiation therapy and so on.Nonetheless,the 5-year survival rate remains low.Thus,new strategies to overcome gastric cancer are urgently required.Compared with normal cells,cancer cells more preferentially depend on glycolysis to produce adenosine triphosphate(ATP)for growth and proliferation.This phenotype,referred to as “aerobic glycolysis”,was first observed 80-years ago by Otto Warburg.In addition,this glycolytic metabolic character in cancer cells also suggest that target glycometabolism may be an effective way to selectively inhibit cancer cells Hexokinase(HK),phosphofructokinase 1(PFK-1),and pyruvate kinase(PK)are crucial enzymes that regulate the rate of glycolysis.Therefore,if suppression of glycolysis can induce cancer cell growth inhibition and/or apoptosis,then HK,PFK-1 and PK may be potential targets for developing novel anti-cancer agents.3-Bromopyruvate(3-Br PA)is an alkylating agent,Our group and other scholar's researchs have shown 3-Br PA can inhibit Hexokinase(HK)activity by binding with its inhibitory sites.Sodium citrate(SCT),a common organic compound which widely used in food additives and stabilizers,Our previous studies have shown that SCT can bind and suppress the 6-Phosphofructokinase-l(PFK-1)activity.Thus,we suspect 3-Br PA and SCT can suppress the rate of glycolysis by inhibiting key enzymes,cause malignant cells energy depletion and kill them.Moreover,researchs had confirmed that DNA will be persistently degradation when energy exhaust in tumor cells,and cause intensive permeability of mitochondrion,then lead to the release cytochrome C(Cyt-C)and activatie caspases to induce cell apoptosis.It is generally known that potential anticancer ability of a new therapeutic approach are often evaluated by activation of tumor cells apoptosis.In our previous researchs,we had elucidated that 3-Br PA and SCT could inhibit malignant pleural mesothelioma and gastric cancer cells growth.However,the antitumor effect and its mechanism of intra-abdominal application of 3-Br PA and SCT has not reported at home or abroad,further study is unravel the specific molecular mechanism what not entirely clear.In the present experiment,we employed human gastric cancer cell line SGC-7901 and built an orthotopic transplantated tumor model in nude mice.We aimed to study the effects of 3-Br PA and SCT in vitro and vivo,and to explore its specific inhibitory mechanisms,which will provide some research basiss for the development of gastric cancer therapy.Part One Experimental study in vitro of effects on gastric cancer cell by 3-Br PA and SCTMethods Cultured human gastric cancer cell line SGC-7901,and assessed the cell viability by MTT after treated with 3-Br PA and SCT.Then,we made eight groups in vitro study: blank control group,5-FU positive control group,3-Br PA low,medium and high concentration groups,SCT low,medium and high concentration groups.Gastric cancer cell growth and morphological changes were observed under Inverted microscope.Cell apoptosis and cell cycle changes were detected by using flow cytometry and Hoechst 33258 staining.Reactive oxygen species(ROS)production was detected by fluorescent probe and Laser confocal microscope.HK,PFK-1,PK activity were detected by using ultraviolet colorimetry.ATP and Lactic acid content were measured by spectrophotometry assay.The expression of protein related to apoptosis was detected respectively by using WB.Results Cell viability reduced in a time-and dose-dependent manner after exposured to 3-Br PA or SCT.The IC50 values of 3-Br PA against SGC-7901 cells after being treated for 24 h or 48 h were 9.88 ± 1.07 g/ml and 5.97 ± 0.95 g/ml,respectively;however,the IC50 values of SCT were 6.47 ± 0.87 mg/ml and 3.84± 0.59 mg/ml.The gastric cancer cells in 5-FU,3-Br PA,or SCT treated groups became round or inflated,with fewer cellular contacts.However,untreated cells attached closely to one another,and were polygonal in shape.Cell apoptosis and cell cycle were detected by FCM.The percentage of apoptosis was increased from 15.60 ± 1.83% after 24 h to 84.45 ± 3.97% after48 h in the 3-Br PA treated groups,and increased from 19.31 ± 1.89% to 88.34 ± 5.22% in the SCT groups.As a positive control,the percentage was increased from 61.57 ± 4.30% to 92.64 ± 4.15% after treated with 5-FU.Moreover,after exposure to 3-Br PA,SCT or 5-FU for 24 h,the cell population in the G2/M phases significantly increased(P < 0.05).The cell population in the S phase of5-FU group also obviously increased(P < 0.05).The apoptotic cells,characterized by chromatin condensation and fragmentation,were observed after exposed to 3-Br PA and SCT.Percentage of apoptotic cells increased along with the concentrations of 3-Br PA and SCT.The results strongly indicated that 3-Br PA and SCT could induced SGC-7901 cells apoptosis in vitro.ROS production was significantly increased upon treatment with 3-Br PA,SCT and 5-FU compared with control(P < 0.05).Furthermore,the fluorescence intensity detected with laser scanning confocal microscope was increased along with the dose of 3-Br PA and SCT.3-Br PA significantly reduced HK activity in a time-and dose-dependent manner(P < 0.05).The HK activity observed in the 5-FU group was also less than control group(P < 0.05).HK activity in the SCT groups had no difference compared with control group(P > 0.05).PFK-1 activity decreased in a timeand dose-dependent manner in the SCT groups(b P < 0.05).However,PFK-1activity was not inhibited by 3-Br PA or 5-FU(P > 0.05).There are no significant difference of PK activity in 5-FU,3-Br PA,and SCT groups compared with the control group(a P > 0.05).Both the 3-Br PA and SCT groups exhibited a significant time-and dose-dependent decrease in cellular ATP levels and lactate production(P < 0.05).Moreover,the cellular ATP levels in the 5-FU group after treated for 8 h and lactate content after treated for 48 h were both obviously reduced compared with the control group(P < 0.05).Compared with the control group,the protein expression of Bax,Cyt-C and cleaved caspase-3 were increased,while the protein expression of Bcl-2 and Survivin were decreased after exposure to 3-Br PA and SCT(P < 0.05).Furthermore,we found that 3-Br PA or SCT produced these effects in a dose-dependent manner.5-FU can also increase protein expression of Bax,Cyt-C,cleaved caspase-3 and decrease the expression of Bcl-2 and Survivin,but its ability was weaker than 3-Br PA-M and SCT-M(P < 0.05).Conclusion The present in vitro study identified that 3-Br PA and SCT could obviously inhibit gastric cancer cell SGC-7901 proliferation,arrest cell-cycle,suppress the activities of glycolytic enzymes,increase the generation of ROS to activate mitochondrial-mediated apoptosis,downregulate the expression of Survivin to induce gastric cancer cell apoptosis.Part Two Built human gastric orthotopic transplanted tumor model in nude mice and Micro-PET/CT imagingMethods Six female BALB/C nude mice were randomly selected,and the back of axillary region of each mouse was subcutaneously injected with 200 ul gastri cancer cell suspension.When the subcutaneous tumor became palpable(i.e.,8~10mm),the tumor tissue was excised from the nude mouse,and then subcutaneous transplanted between nude mice for six generations.Randomly selected 150 female BALB/C nude mice,then orthotopic transplanted the tumor tissue inside the seromuscular layer of greater curvature and used 1-2 drops of medical OB glue to bind the serosal layer incision.The tumor was allowed to grow for 2 weeks before treatment was initiated.Then,mice were randomized into 8 groups(18 mice per group).All mice were intraperitoneally injected with relevant drug or PBS once per day for 4 weeks.Any changes of the nude mice,such as body weight,behavior,eating and excretion were monitored.After4-week treatment,6 nude mice in each group were imaged with micro-PET/CT by using 18F-FDG as radioactive tracer.Results The gastric orthotopic transplanted tumor model in nude mice were successfully built and the tumor formation rate was about 95%.The behavior,eating and body weight of nude mice have no significant abnormality were seen by intraperitoneally injected 3-Br PA and SCT with experiment concentrations.And,the radiotracer accumulation(%ID/g)in the orthotopic transplanted tumor were as follow: 3-Br PA-L group 2.703±0.214;3-Br PA-M group 2.047±0.286;3-Br PA-H group 1.348 ± 0.142;SCT-L group 2.527 ± 0.279;SCT-M group1.975± 0.245;SCT-H group 1.228 ± 0.136;5-FU group 2.187 ±0.276;PBS group 3.285 ±0.389.The 18F-FDG uptake were significantly decreased along with the increased concentrations of 3-Br PA and SCT(P < 0.05).The radioactivity uptake was also reduced in 5-FU group when compared with PBS group(P < 0.05).Conclusion The human gastric orthotopic transplanted tumor model in nude mice could be successfully built with medical OB glue paste method,and intraperitoneally injected of 3-Br PA and SCT was a safe and effective way to suppress tumor metabolism.Part Three Experimental study in vivo of effects on gastric gastric orthotopic transplanted tumor in nude mice by 3-Br PA and SCTMethods Successfully built human gastric orthotopic transplanted tumor model and intraperitoneally injected of 3-Br PA and SCT.After 4-week treatment,6 nude mice in each group were randomly selected.Blood routine and hepatic and renal function of nude mice were detected.Orthotopic transplanted tumor,liver and kidney tissue were observed by HE staining.HK,PFK-1,PK activity in tumor were detected by using ultraviolet colorimetry.ATP and Lactic acid content in tumor were measured by spectrophotometry assay.Cell apoptosis of orthotopic transplanted tumor were detected by using TUNEL and TEM.The expression of apoptosis related proteins were measured by Immunohistochemical method(IHC).The expression of gene m RNA related to apoptosis was detected by using RT-q PCR.Results Compared with PBS group,the WBC,PLT,ALT,ALB,TBil,BUN and Cr levels in 3? Br PA and SCT groups had no significantly difference(P>0.05).The WBC,PLT and ALB levels in 5? FU group were significantly decreased when compared with PBS group(P<0.05).The serum ALT,BUN,TBil,BUN and Cr levels in the 5? FU-treated mice were obvious higher than those in PBS-treated group(P<0.05).However,the HGB levels have no obvious differences in each groups(P>0.05).The mice in 3-Br PA and SCT groups exhibited a tumor growth inhibition compared with the control group(P<0.05).Moreover,the life prolonging rate increased in 3-Br PA and SCT groups((P<0.05)).HK activity were significantly reduced in dose-dependent manner in3-Br PA treated groups(P<0.05).However,no differences were observed on the HK activity in SCT treated groups compared with control group(P>0.05). Interestingly,HK activity was also inhibited by 5? FU(P<0.05).The PFK-1activity was considerably decreased in SCT treated groups with dose-dependent manner(P<0.05).3-Br PA and 5-FU had no distinct effect on the PFK-1 activity in tumor(data not shown).In addition,the ATP and lactate production were dose-dependent prominently reduced in 3-Br PA and SCT groups(P<0.05),indicated their ability to induce apoptosis by blocking energy metabolism.5-FU also exhibited a moderate ability to decrease the production of ATP and lactate(P<0.05).From the results of liver and kidney HE staining,histological examination did not reveal any apparently abnormality,indicated the safety of 3-Br PA and SCT doses used in our study.However,slight vacuolar changes and spotty necrosis in hepatocytes,hydropic changes and leukocyte infiltration in kidney cells were observed in 5-FU treated mice.The orthotopic transplanted tumor tissue HE staining further proved that the model was successfully established.Results from TUNEL staining showed that the average apoptotic index(AI)was increased along with the doses of 3-Br PA and SCT.Futhermore,intraperitoneal injected of 5-FU could also induce tumor cells apoptosis in vivo(P < 0.05).And,this treatment-dependent changes was further supported by the TEM assay,where the 3-Br PA,SCT and 5-FU treated tumors showed mitochondria swollen with disorganized cristae,apoptotic bodies formation,nuclear fragmentation or condensation,and margination of chromatin,which presented some typical morphological features of cell apoptosis.Otherwises,no typical manifestation of tumor cell apoptosis was observed in the PBS groups.From the IHC analysis,the tumors from mice treated with 3-Br PA and SCT exhibited increased levels of cleaved caspase-9 and-3 with a dose-dependent manner(P < 0.05).Similarly,a significant increase expression of cleaved casapase-9 and-3 was also found in tumors with 5-FU treatment.With the RT-q PCR assay,the 3-Br PA and SCT treated groups exhibited an increased expression of Bax and Cyt-C,and a decreased expression of Bcl-2 and Survivin,in a concentration-dependent manner(P < 0.05).Besides,with 5-FU treatment,Bax and Cyt-C were also upregulated while Bcl-2 and Survivin were downregulated(P < 0.05).Conclusion Intraperitoneal injection of 3-Br PA and SCT could inhibit orthotopic transplanted tumor growth,extent survival time and induce apoptosis in vivo.Research conclusion Our study have taken the in vitro and in vivo experiments together to study the influence of 3-Br PA and SCT on gastric cancer SGC-7901 cells and explore its antitumor mechanisms.(1)The in vitro experiments showed that 3-Br PA and SCT can block the cell cycle,inhibit activities of key enzymes HK and PFK-1 to inhibit the rate of glycolysis in gastric cancer cell,decrease the production of ATP and lactic acid,lead to energy depletion of cell.And for the first time found3-Br PA and SCT can promote the accumulation of ROS in gastric cancer cells,increase Bax protein expression and down-regulate the Bcl-2 protein expression in mitochondrial apoptotic pathways,thus cause the release of Cyt C,activate caspase 3,and inhibit the expression of antiapoptotic proteins Survivin to promote gastric cancer cell apoptosis.(2)We establish gastric orthotopic transplantation tumor model in nude mice,and for the first time in domestic and foreign by intraperitoneal injection of 3-Br PA and SCT,then employ 18F-FDG Micro-PET/CT to evaluate their antitumor effect.The results confirmed that Application of 3-Br PA and SCT intraperitoneal injected can also inhibit glycolysis enzymes activities to reduce glycolysis levels in stomach orthotopic transplantation tumor tissue,then lead to cell energy depletion,acidic environment destruction and cause DNA degradation,promote m RNA expression of Bax and Cyt C,suppress m RNA expression of Bcl-2 and Survivin,then upregulate the proteins expression of activated caspase-9 and caspase-3 to play its antitumor function.Taken together,our findings highlight the potential of 3-Br PA and SCT as a safe,novel and attractive strategy in gastric cancer treatment,worth further investigation. |
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