| Gastric cancer(GC)is the fifth most common malignancy but the third leading cause of cancer-related deaths worldwide.Surgery represents the main approach for this disease,but the advances in surgical techniques achieves a minimal improvement in terms of overall survival for the patients within later stage of GC.Chemotherapy,therefore,remains as the main therapeutic approach in clinical treatment of gastric carcinoma.Studies have shown that cisplatin(DDP)in combination with fluorouracil and its derivant are the elemental chemotherapeutics in gastric carcinoma.However,high resistance to chemotherapy drug becamde a crucial bottleneck.Despite the development of new drugs has significantly improved the effectiveness of chemotherapy,the prognosis of patients with unresectable or metastatic gastric adenocarcinoma remains poor due to drug resistance.Drug resistance frequently occurs as a primary(inherent)or secondary(acquired following chemotherapeutic treatment)tumor defense mechanism against multiple cytotoxic drugs,which severs as a major hindrance to curative cancer therapy.Thus,there is a substantial need for other therapeutic agents that may offer superior efficacy for patients suffering from gastric cancer.Tumors exploit various pathways to recruit blood vessel for tumor growth and metastasis.Angiogenesis,the development of new blood vessels from the existing vasculature,has become the most widely investigated mode of new vessel formation in gastric cancer.Thus,inhibition of angiogenesis remarkably ameliorates gastric cancer,which suggesting the potential role of anti-angiogenesis strategy in the treatment of gastric cancer.Anti-angiogenicdrugs,such as Endostar,bevacizumab,Sutent and Sorafenib came out in succession since then.Back in 2004,bevacizumab has been approved by FDA in America.Bevacizumab is a monoclonal antibody targeting VEGF-A that showed activity in several solid tumors such as colorectal,breast,ovarian and non-small cell lung cancer.It binds to VEGF,preventing its interaction to VEGFR-1 and VEGFR-2 on the surface of endothelial cells.The biological activity of VEGF interferes with the formation of new tumor blood vessels thus preventing tumor growth.However,whether combination DDP with bevacizumab could offer superior efficacy for patients suffering from gastric cancer is unknown.Thus,further studies remain to be carried out on advantages of whether and how applying them in combination to overcome drug-resistance in GC.We applied MGC 803,higher VEGF expression gastric carcinoma cell line,to explore the inhibitory effect of bevacizumab in combination with DDP on MGC 803 in different time series.We also explored the therapeutic effects of bevacizumab in combination with DDP on carcinoma-bearing mice.Flow cytometry,Western blot and IHC were applied for observing the VEGF/AKT pathway as well as apoptosis-related protein Bcl-2 and BAX expression in gastric carcinoma cells after treatment in vivo and in vitro.Part 1 Investigating the mechanism and inhibitory effect of bevacizumab in combination with the FP on MGC 803 in different time courses.Objective: Exploring the inhibitory effect of bevacizumab in combination with FP on MGC 803 in different time series.,and try to find out the optimum combination timing sequence of anti-agiogenic drug bevacizumab and FP.Method: Human gastric carcinoma cells MGC 803 were cultured with bevacizumab in combination with FP treatment following different series.Cells growth inhibition was measured by MTT method,andcell cycle phase and apoptosis rate were detected using flow cytometry(PI staining).1 There were statistic significancein the inhibition rate of MGC 803 cells treated with different concentrations of single drugs(P<0.05).Further comparison between each group revealed that there were statistic significance(P<0.05)in the inhibition rate under the situation of different concentration of single drug treatment.2 The drug administration in different time series has statistic significance(P<0.001)on the inhibition rate of MGC-803 cells.The highest inhibition rate(68.63 %)occurred on the cells treated with bevacizumab plus24 h FP chemotherapy treatment before,which was followed by 48 h(63.46 %)and 72 h(62.89 %)before FP chemotherapy treatment.The inhibition rate of combining FP chemotherapy and bevacizumab at the same time accounts for60.96%,a little higher than that following treatment of combining bevacizumab 24 h after FP chemotherapy,59.64%.Then the rate of cells treated with bevacizumab 48 h and 72 h after FP chemotherapy occupied57.53% and 56.63% separately.The inhibition rate of FP group was the lowest,52.33%.The results showed that the combination of the first dose of bevacizumab combinated with FP chemotherapy could inhibit the growth of gastric cancer cells.3 There were no statistic significance(P > 0.05)in G0/G1 phase and G2/M phase in cell cycle of MGC 803 gastric carcinoma cell lines.There was,however,statistic significance(P<0.05)in S phase.The results showed,in S phase,the highest inhibition rate was found in cells treated with bevacizumab24 h before chemotherapy treatment,48.13%,which exceeded the rate of all other groups(P<0.05).The lowest inhibition rate was found in the FP plan group,23.50%,which was lower than the rate of all other groups(P<0.05).There were no statistic significance(P>0.05)between the rest of the groups.The results showed that the FP combined withmonoclonal antibody for 24 hours before chemotherapy significantly induced the cell cycle arrest in S phase in gastric cancer cells.4 There were statistic significance(P<0.001)in apoptosis rate of gastric carcinoma cells treated with different dosing timing.Processing cells with Results:bevacizumab 24 h before FP chemotherapy occupied the highest apoptosis rate(4.76%),which was higher than that in other groups.The apoptosis rate of combining FP chemotherapy and bevacizumab at the same time accounts for2.99%,which is a little higher than that in the cells treated with combining bevacizumab 24 h after FP chemotherapy,2.90%.The results showed that the combined FP treatment with monoclonal antibody for 24 hours before chemotherapy significantly promoted the apoptosis of gastric cancer cells.Conclusions:1 Different dosing timing results in different inhibition rate of gastric carcinoma cells.The highest inhibition rate occurred in the group treated with bevacizumab 24 h before FP chemotherapycombination.2 Different dosing timing inhibited the S phase of cell cycle of gastric carcinoma cells in different extent.The highest rate of S phase is the cells treated with bevacizumab 24 h before FP chemotherapy combination.3 Different dosing timing results in different apoptosis rate of gastric carcinoma cell lines MGC 803.The highest apoptosis rate occurred in the group treated with bevacizumab 24 h before FP chemotherapy combination..Part 2 Applying animal imaging technology in vivo to analysis the curative effects of bevacizumab in combination with chemotherapy treatment on gastric carcinoma-bearing mice in different timing sequence.Objective: Comparing the difference of curative effect of bevacizumab in combination with chemotherapy treatment on tumour development in different timing sequence by observing the growth changes of fluorescent-labelled tumour cells inside gastric carcinoma-bearing nude mice.Method: After bevacizumab in combination with FP in different timing sequence treatment gastric carcinoma-bearing nude mice model,Theon tumour growth was measured by calculating the inhibition rate via tumour volume,and observing the changing value of fluorescent signals.Group A Bevacizumab +FP Simultaneous administration;Group B FP after 24 hr Bevacizumab;Group C Bevacizumab after 24 hr FP;Group D Bevacizumab(only);GroupE Control,normal saline was injected in the abdominal cavity?Results:1 There were no statistic significance in wight changes among different groups(P = 0.219).There were statistic significance(P < 0.001)in body weight changes at different time points after treatment.There was no statistic significance in weight changes at different time point in group A(P = 0.773)by contrast to the group B,C,D and E where had found an increasing trend of weight changes with time changes(P<0.05).2 According to the calculation based on the tumour volume,the anti-tumour rate in group A,B,C and D were 40.95%,37.57%,68.42%,19.84% respectively.It demonstrated that there were no statistic significance in the changes of tumour volume among different groups(P=0.166).There were,however,statistic significance(P=0.006)in the changes of tumour volume at different time points after treatment.There were,however,statistic significance at the second and the third week after medication(P<0.05).The tumour volume of group C was obviously less than that of group D and E(P<0.05).There were no statistic significance among group A,B and D in the volume changes of nude mice at different time point(P>0.05).The tumor volume changes of group C presented an decreasing trend with time changing(P<0.05),while that of group E appeared an increasing tendency with time changing(P<0.05).3 There were statistic significances in the changing value of fluorescent signals of tumour among nude mice in different groups(P < 0.001).There were statistic significances(P<0.001)in the changing value of fluorescent signals of tumour at different time points after the treatment.Interactions exits between time and groups(P<0.001).There was no statistic significance in the changing value of fluorescent signals of tumour before medication(P =0.074).There were,however,statistic significances at the first,the second and the third week after the medication(P<0.05).In the first and the second week,the changing value of fluorescent signals in group E distinctly exceeded that in group A,B,C and D.The changing value of fluorescent signals before medication in group C was obviously weaker than that after medication(P<0.05).The changing value of fluorescent signals in group E appeared an increasing tendency with time changing(P<0.05).Conclusions:1 The combined the bevacizumab treatment with chemotherapy showed an interaction effect,and achieved the most prominent anti-tumour effects compared with FP chemotherapy treatment.2 The study in vivo found tumor volume no longer reduces,the fluorescence signal to decay,namely drugs continue to play an anti-tumor effect,used dynamic small animal imaging system to obtain the major axis,minor axis and signal value of fluorescence.Part 3 The molecular mechanism of anti-tumor effect of bevacizumab combined with FP chemotherapy in different dosing timing on gastric tumour cellObjective: To explore the molecular mechanism of anti-tumor effect of bevacizumab combined with FP treatment by observing the VEGF/AKT pathway as well as apoptosis-related protein Bcl-2 and BAX expression in gastric carcinoma cells after treatment in vivo and in vitro.Method: Western blot and immunohistochemistry were used for detecting the expression of AKT,Bax,Bcl-2,Cyclin D1,P21,PI3 K,MVD,VEGFR in tumour tissues of tumour-bearing mice and the the human gastric cell line MGC 803 at different timing sequence after the treatment of bevacizumab in combination with chemotherapy.Results:1 The effect ofcombined administration of drugs in different timing sequence on the expression of relevant proteins in gastric carcinoma cell line MGC 803.After the combined administration in different timing sequence on gastric carcinoma cell line MGC 803,we have found that there were statisticsignificance in OD of PIK3,AKT and P21 between groups that were treated with combined drugs and those were only treated with chemotherapy(P <0.05).Pairwise comparison between groups indicated that the highest expression among each group was the FP chemotherapy group,PIK3(1.11±0.03),AKT(1.27±0.0),P21(1.51±0.04).The group treated with bevacizumab 72 h after FP chemotherapy treatment showed PIK3(0.97±0.04),AKT(1.13±0.10),P21(1.38±0.05),which the group treated with bevacizumab48 h after FP chemotherapy treatment showed PI3K(0.90±0.05),AKT(1.04±0.12),P21(1.35±0.05).The lowest expression among each group was those treated with bevacizumab 24 h before FP chemotherapy treatment,PI3K(0.07±0.03),AKT(0.02±0.01),P21(0.13±0.02).The significant therapeutic effects may be mediated by inhibition of PI3 K and AKT.Apart from the group that was treated with bevacizumab 48 h before FP chemotherapy treatment,the OD of the expression of Bcl-2 protein in other groups compared with that of the group that only treated with chemotherapy,presented a statistic significance(P<0.05).Pairwise comparison between groups indicated that the highest expression was found in group which was only treated by chemotherapy,at 1.12±0.04,which was followed by the group treated with with bevacizumab 72 h and 24 h after FP chemotherapy treatment,at 1.00±0.06 and 0.74±0.04 respectively.The lowest expression among each group was those treated with bevacizumab 24 h before FP chemotherapy treatment,0.05±0.04..OD of the expression of Bax protein in other groups was compared with that of the group that only treated with chemotherapy,presenting a statistic significance(P<0.05).Pairwise comparison between groups provided that the highest expression among each group was the groups treated with bevacizumab 72 h before FP chemotherapy,1.02±0.04.Then came the groups which were treated with bevacizumab 48 h and 24 h before FP chemotherapy treatment,at 0.99±0.05 and 0.95±0.04 respectively.The lowest expression found in the group only treated with FP chemotherapy,0.07±0.01.OD of the expression of Cyclin D1 protein in other groups compared with that of the group that only treated with chemotherapy,revealed a statistic significance(P<0.05).Pairwise comparison between groups provided that the lowest expression was found in the group only treated with FP chemotherapy,0.04±0.03.Then came the groups which were treated with bevacizumab 72 h after FP chemotherapy and 48 h before FP chemotherapy,at0.13±0.05 and 0.28±0.04 respectively.The highest expression among each group was the groups treated with bevacizumab 24 h before FP chemotherapy,0.71±0.05.2 Combined administration in different timing sequence has an impact on the expression of relevant proteins in tumour tissues from carcinoma-bearing nude mice.After the combined administration in different timing sequence on carcinoma-bearing nude mice(gastric cell line MGC 803),we have found that there were statistic significance in OD of PIK3,AKT and Bcl-2 between groups treated with combined and control group(P < 0.05).Pairwise comparison between groups showed that the highest expression was found in control group,PIK3(0.73±0.04),AKT(1.13±0.05),Bcl-2(1.40±0.04).Then follows the group treated with bevacizumab 24 h after FP chemotherapy treatment,PIK3(0.44±0.03),AKT(0.46±0.02),Bcl-2(1.14±0.04),and the group that only treated with bevacizumab,PI3K(0.14±0.03),AKT(0.35±0.03),Bcl-2(0.74±0.04).The lowest expression was the group treated with bevacizumab 24 h before FP chemotherapy,PI3K(1.13±0.05),AKT(0.06±0.03),Bcl-2(0.19±0.02).OD of the expression of P21 protein in other groups compared with that of control group,presenting a statistic significance(P < 0.05).Pairwise comparison between groups provided that the highest expression among each group was the groups treated with bevacizumab 24 h before FP chemotherapy,0.85±0.02.Then came the group which were only treated with bevacizumab,0.61±0.02,and the group dosed with two treatments at the same time,0.48±0.04.The lowest expression was found in the group treated with bevacizumab 24 h after FP chemotherapy,0.43±0.01,and the control group,0.43±0.03.OD of the expression of Bax protein in othergroups compared with the control group,presenting a statistic significance(P < 0.05).Pairwise comparison between groups provided that the highest expression among each group was the groups treated with bevacizumab 72 h before FP chemotherapy,1.02±0.04.The lowest expression found in the group only treated with FP chemotherapy,0.07±0.01.OD of the expression of Cyclin D1 protein in other groups compared with control group,revealing a statistic significance(P < 0.05).Pairwise comparison between groups provided that the lowest expression was found in control group,0.31±0.04.The highest expression among each group was the group treated with bevacizumab 24 h before FP chemotherapy,0.92±0.01.Conclusions:1 Bevacizumab up-regulates the expression of Bax and Cyclin D1,and down-regulates the expression of Bcl-2 and P21 to inhibit the proliferation of tumour cells by regulating the PI3K/AKT signal pathway in MGC 803 cell line.2 FP before chemotherapy plus bevacizumab upregulates the expression of Bax and Cyclin D1,and down-regulates the expression of Bcl-2 and P21 protein in tumor tissues.The carcinoma-bearing nude mice treated with bevacizumab 24 h before FP chemotherapy showed the most predominant effect on down-regulating the PI3K/AKT signal pathway compared with the mice only treated with bevacizumab. |
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