Effects Of Bevacizumab On Apoptosis In Drug-Resistant Nasopharyngeal Carcinoma Cells

BACKGROUND & OBJECTIVES:Nasopharyngeal Carcinoma(NPC) is one of the most common malignant tumors in south China and Southeast Asia.Most patients have been in advanced stage once detected,and researches showed that stageⅢ～Ⅳcases had a proportion of about 85%.The combination of radiation therapy and chemotherapy, as main therapy to NPC,still can not get good therapeutic efficacy yet,because most patients died from the failure of chemotherapy,which is greatly incriminated to multidrug resistance(MDR) of cancer cells.Tumor MDR is the cross tolerance to many antitumor drugs after chemotherapy,which is untouched,structure independent,and mechanism various.Some latest viewpoints show that it is the apoptosis down regulation caused by various kinds of factors that lead to MDR, and cancer cells escape from being killed by resisting apoptosis.All researchers tried to find ways to enhance apoptosis of drug-resistant cancer cells.Recent studies found that vascular endothelial growth factor(VEGF) is up-regulated in the majority of human tumors,which can stimulate tumor's angiogenesis,specific for endothelial cells,and this has effect on the tumors'growth,metastasis and prognosis.Bevacizumab,the recombinant humanized monoclonal antibody to vascular endothelial growth factor(VEGF),is a new anticancer therapy that was approved by the Food and Drug administration for the use of first line treatment of advanced colorectal cancer in 2004 and advanced head and neck cancer in 2007.We found in clinic it difficult to get obvious antitumor effect by using bevacizumab only,whereas the biochemotherapy mode,which is made by combining bevacizumab and chemic medicines,is effective to some drug resistant recurrent and metastatic patients.It seems that bevacizumab treat tumor patients not only by inhibiting neovascularization but also by increasing sensitivity of tumor cells to chemic medicine,and the latter is most probably caused by promoting apoptosis.We designed this study to:1.Study the bionomics of human nasopharyngeal carcinoma(NPC) DDP-resistant cell line CNE2/DDP.2.Establish models in which subcutaneous xenografts were induced in nude mice using CNE2/DDP.3. Investigate the effects of bevacizumab on apoptosis of CNE2/DDP in cell and animal level.MATERIALS & METHODS:1.Human NPC poorly differentiated cell line CNE2 and its DDP-resistant subseries CNE2/DDP were cultivated in high glucose DMEM medium, supplemented with 10%calf serum,in 37℃,5%CO₂ saturated humidity incubate box.Fluorescence activated cell analysis(FACS) was used for determining the cell cycle and concentration of fluorescence dye rhodamine123 within the cells. Cell growth curve,doubling time,and cell morphology were measured and observed.The IC₅₀ and resistance index(RI) to DDP,gemcitabine, 5-fluorouracilseveral and vincristine were tested by MTT assay.2.CNE2/DDP cells,in exponential phase of growth,were inoculated in 96-well plates at 10⁴/well in 100μl culture volum.There were one control group and 5 experimental groups which contained DDP and bevacizumab in certain density, with 6 repeated units in each group.The plates were put into incubate box for 72h, then detected each group of cells' survival rate with MTT assay,then worked out the kill rate of drugs.As is showed in formula:kill rate =(1-OD value of each experimental group/average OD value of control group)×100%.One-Way Anova in SPSS10.0 was used to analyze the experimental data,and we defined statistical significance as P＜0.05. 3.CNE2 and CNE2/DDP cells were conventionally cultivated in 50ml-culture flasks,grew in adherence observed by microscope,then 0.1μg/ml DDP and 0.1μg/ml DDP + 10μg/ml bevacizumab were added to the cells respectively, CNE2/DDP without drugs was blank.All cells were put into 37℃5%CO₂ saturated humidity incubate box.Collected the cells and dyed them with Annexin V and PI,then detected the apoptosis rate with flow cytometry.This procedure repeated 3 times,and we analyzed the experimental data with Kruskal-Wallis H rank test in SPSS10.0.4.Some CNE2/DDP cells were cultivated in culture fluid supplemented with 10μg/ml bevacizumab,went down to a new generation once per 4 days,4 generations later,the cells were called CNE2/DDP/Bev.Detected the mRNA of MDR1,Bcl-2 and Bax expressed in CNE2,CNE2/DDP and CNE2/DDP/Bev cells by reverse transcription-polymerase chain reaction(RT-PCR) analyses,and computed the relative express quantity.5.A model of human NPC was established by subcutaneous transplantation of CNE2/DDP cells in nude mice.After tumor engraftment(21days),25 mice were randomly divided into 5 groups(only animals with established tumors of at least 5mm in diameter were enrolled).Treatments were designed as:the mice in control group(A group) received sterile saline;the mice in bevacizumab group(B group) received bevacizumab 5mg/kg;the mice in DDP group(C group) received DDP 4mg/kg;combination routine treatment group(D group) received bevacizumab 5mg/kg+DDP 4mg/kg and combination semi-treatment group(E group) received bevacizumab 5mg/kg+DDP 2mg/kg.The mice of C,D,E groups received tropisetron(3mg/kg) s.c.30min before DDP to ameliorate gastrointestinal dysfunction.Treatments were given twice(0.5ml/boluse/time) per week by intraperitoneally and continued for 4 weeks,then all mice were killed by anesthetic overdose,the tumors were obtained from xenografts.At the same time, tumors were weighed,and inhibition rates were evaluated,tumor microvessel density(MVD) were measured determined by immunohistochemistry,the mRNA expression of multidrug resistance genes(MDR1) and apoptosis-associated gene (Bcl-2,Bax) were assessed by RT-PCR.One-Way Anova and Least-significant-Difference analysis in SPSS10.0 was used to analyze the experimental data,and we defined statistical significance as P＜0.05.RESULTS:1.Double time of CNE2 and CNE2/DDP were 17.13h and 23.13h respectively,as evaluated by the growth curve.The IC₅₀ to DDP increased from 0.03μg/ml in CNE2 to 0.45μg/ml in CNE2/DDP as tested by MTT assay at 48～72h exposure, the RI was 14.94.The RI to gemcitabine,5-fluoroutacil(5-FU),and vincristine (VCR) were 17.62,6.60,and 14.17 respectively,indicating its multidrug resistant prorerty.FACS analysis showed that the concentration of rhodamine123 was much lower in CNE2/DDP cells than in CNE2 cells(3.56 vs.220.35).The CNE2/DDP cells appeared more round in various size under light microscopy, with more granulation in cytoplasm.2.The killing rate of 10μg/ml bevacizumab group was similar to that of blank group(5.2%±4.3%vs.0.0%±4.1%,P=0.180),while the rates of combination of 10μg/ml bevacizumab and DDP(0.1μg/ml and 0.2μg/ml separately) were higher than that of using DDP only(42.3%±6.5%vs.34.4%±5.4%,P=0.041; 62.6%±5.5%vs.50.0%±5.9%,P=0.009).3.The early apoptosis rate of DDP group and DDP+bevacizumab group were higher than that of control group(8.87%±1.06%vs.3.25%±0.75%, 38.20%±3.51%vs.3.25%±0.75%),the late apoptosis and dead rate of DDP group and DDP+bevacizumab group were higher than that of control group (41.71%±9.78%vs.5.70%±0.43%,48.96%±6.90%vs.5.70%±0.43%) too.The apoptosis rates of control group and experimental groups were significantly different(P=0.027),the rate of combining 0.1μg/ml DDP and 10μg/ml bevacizumab group was higher than that of DDP group(87.29%±3.38%vs. 50.58%±8.83%).4.As was shown in half-quantitative RT-PCR assay,the relative express quantity of the four mRNA in CNE2,CNE2/DDP and CNE2/DDP/Bev was:MDR1 0.623, 1.364 and 1.165,Bcl-2 0.613,0.952 and 0.135,Bax 0.665,0.387 and 1.751. 5.At the end of the experimental period,the body weight of DDP routine treatment groups(C,D) were lighter than any other groups(P＜0.001).Compared with control group,tumor growth of both combination groups(D,E) were suppressed significantly,with growth inhibition rates of 59.37%±3.29%and 63.17%±3.05%respectively(P＜0.001).The tumor growth inhibition rates of bevacizumab group and DDP group were 6.03%±4.68%and 5.71%±8.30% respectively,with no statistically difference compared with control group (P=0.067,P=0.081).The expression of MVD in both bevacizumab group and combination groups(B,D,E) were 13.60±1.11,12.80±0.90 and 13.22±1.02 respectively(P＜0.001).Compared with control group,RT-PCR analysis revealed a decrease in expression of Bcl-2 gene and increase in Bax gene in CNE2/DDP tumors of both bevacizumab group and combination groups(B,D,E),(P＜0.001), and no statistically difference in these three groups.Each group showed no statistically difference in MDR1 mRNA expression(P=0.918).CONCLUSION:1.Bevacizumab could greatly increase the sensitivity of CNE2/DDP cells to DDP. The lethal effect was strongest when combining bevacizumab and DDP,but was weak when using bevacizumab only,and the lethal effect did not increase but the effect was great when combined with gemcitabine.2.Bevacizumab could promote apoptosis of human NPC DDP-resistant cell CNE2/DDP.The apoptosis rate of combining bevacizumab and DDP was much higher than that of using DDP only.3.Bevacizumab had effects not on MDR related genes but on apoptosis related genes,and promoted MDR cells to apoptosis.To CNE2 cells,when induced them to MDR with DDP,then treated them with bevacizumab,their expression of MDR1 increased greatly,then decreased slightly;Bcl-2(apoptosis inhibiting gene) increased,then decreased greatly;Bax(apoptosis enhancing gene) decreased,then increased greatly.4.Bevacizumab has synergetic cytostatic activity with DDP on growth of NPC CNE2/DDP cell xenografts in mice through inhibiting angiogenesis,and may enhance the sensitivity of CNE2/DDP cells to DDP by inducing cell apoptosis.In short,bevacizumab could heal cancer through promoting NPC cells apoptosis.