| Background and Objective:As one of the most common malignant tumors, lung cancer posc a serious thrcatto the public health and life. Its incidence and mortality are still on the riseworldwide. About 70-80％preclinical-diagnosis patients belonging to intermediate oradvance stage have lost surgery chance.Adjuvant chemotherapy has been recognizedfor its definite effect in prevention and treatment of tumor recurrence andmetastasis.Unfortunately, despite of the development of new chemotherapyregimens,the curative effect for advanced inoperable lung cancer remains poor, and itsefficacy is just 20％-40％, five-year survival rate is only 15％. Especially in NSCLCpatients, tumors are not sensitive to chemotherapeutic agents, one of reasons is theinternal and(or) acquired multidrug resistance(MDR) generated from tumorcells. Chemoresistance is major obstacle for successful treatment of cancer, and it isclear that new therapeutic options are necessary for lung cancer clinically. Recent study found that vascular endothelial growth factor(VEGF) is up-regulated in themajority of human tumors,which can stimulate tumor's angiogenesis, specific forendothelial cells, and this has effect on the tumors'growth, metastasis andprognosis. Because angiogenesis is a essential condition in tumor growth, invasionand metastasis, destruction or inhibition of tumor angiogenesis is the target of anti-angiogenesis therapy. Bevacizumab, a humanized recombinant monoclonalantibody, possess high affinity for binding to VEGF and execute its anti-tumor effectby blocking the angiogenetic process. Preclinical studies found that single-agenttherapy with Bevacizumab resulted in tumor growth inhibition of 20 different humantumor cell lines(13 tumor types) implanted into nude mice irrespective of the route ofadministration or tumor location. As the first FDA approved antiangiogenic agent forcancer therapy, Bevacizumab has previously shown good clinical activity in reducingtumors' angiogenesis when either used alone or in combination with otherchemotherapeutic drugs. Several of these studies also observed significant inhibitionof tumor metastases. Bevacizumab is currently applied in phaseⅡ/Ⅲtrials in a widerange of tumor types,and inspiriting results have been abtained inNSCLC, colorectal cancer and renal cancer. We aimed to investigate the effect ofanti-VEGF treatment on growth of A549/DDP(cisplatin resistance phenotype)xenografts model of human lung adenocarcinoma, offer useful preclinical data fornovel cancer treatments, and provide the basis for establishing a newbiochemotherapeutic approach for lung cancer. Specific contents are as follows:1. To appraise the biological characteristics and mechanism of multidrugresistance about A549/DDP cell line, and to establish models in which subcutaneousxenografts were induced in nude mice using the human lung adenocarcinoma cell lineA549/DDP.2. To explore the effects of Bevacizumab, a monoclonal antibody developed against VEGF, with or without cisplatin(DDP) on the growth of lung adenocarcinomaA549/DDP cell xenografts in mice.Methods:1. Serial subcultivations of three kinds of cells(A549, A549/DDP, SKOV-3) wereprepared for experimentation. The cell growth curve was drawn by MTT assay. Thecell cycle of A549 and A549/DDP cell line were compared. Both cell lines induced byDDP 24 hours later were double stained with AnnexinVand PI(propidiumiodide), then average apoptosis rates were analyzed with the flowcytometry(FCM). The IC50 and resistance index(RI) of A549/DDP cell line to DDPwere tested by MTT assay. The cross-resistance profile of A549/DDP cell line wastested though other five common chemotherapeuticdrugs(carboplatin, vincristine, epirubicin, gemzar and paclitaxel). The mRNAexpression of VEGF,apoptotic-associated gene(bcl-2) and multidrug resistancegenes(MDR1,LRP,GST-p)was assessed with semi-quantitative reversetranscription-polymerase chain reaction(RT-PCR) analyses.2. A model of human lung cancer was established by subcutaneous transplantationof A549/DDP cells in nude mice. After tumor engraftment(21days), 25 mice wererandomly divided into 5 groups(only animals with established tumors of at least 5ramin diameter were enrolled). Treatments was designed that:the mice in control group(Agroup) received sterile saline; the mice in Bevacizumab group(B group) receivedBevacizumab 5mg/kg; the mice in DDP group(C group) received DDP 4mg/kg;combination routine treatment group(D group) received Bevacizumab 5mg/kg+DDP4mg/kg and combination semi-treatment group(E group) received Bevacizumab5mg/kg+DDP 2mg/kg. Among the total, mice of C,D,E groups receivedtropisetron(3mg/kg) s.c. 30min before DDP to ameliorate gastrointestinal dysfunction. Treatment was given twice(0.5ml/boluse/time) per week byintraperitoneally and continued for 4 weeks, then all mice were killed by anestheticoverdose, the tumors were obtained from xenografts. At the same time, tumors wereweighed, and inhibition rates were evaluated, tumor microvessel density(MVD) weremeasured determined by immunohistochemistry, the mRNA expression ofapoptosis-associated gene(bcl-2) and multidrug resistance genes(LRP, GST-p) wereassessed by reverse transcription-polymerase chain reaction(RT-PCR) analyses.3. The results of experiment were expressed by the way of "Mean±SD", and all thedata were analyzed statistically by using SPSS13.0 software. The cell cycle of twokind of cells(A549,A549/DDP) was employed Independent-Samples T Test, and theanalysis of mRNA expression about four genes(bcl-2,MDR1,LRP and GST-π) wasin the same way. The comparison of average apoptosis rate between A549 andA549/DDP cell induced by DDP was applied to Paired-Samples T Test andIndependent-Samples T Test. Paired-Samples T Test was also adopted in data aboutsensitivity and cross resistance of A549 and A549/DDP cells to various cytotoxicdrugs. One-way ANOVA(Least-significant-Difference, LSD test) was introduced intoanalysis of the different data,as fllows: VEGFmRNA expression in three kind ofcells(A549, A549/DDP and SKOV-3) was determined by Dunnett test(SKOV-3 cellas positive control); inhibition rate and MVD of tumor growth in five groups; mRNAexpression about three genes(bcl-2,LRP and GST-π)of each group. P≤0.05 isdefined statistical significance.Results:1. Compared to parental cell, A549/DDP cell exhibited a lot of difference inbiological characteristics evaluated by the growth curve,for example the proliferationof A549/DDP cells accelerated, whose exponential growth stage was early to the parental cells.Flow cytometry was used in cell cycle and cell apoptosis analysis.Thenumber of A549/DDP cells in S phase increased(51.73±0.44)％, P＜0.001, 95％confidence interval(CI) of the difference was[36.07,37.94]) while in G1 phasedecreased ((37.25±0.21)％, P＜0.001, 95％CI was[43.30,44.18]), compared withparent cell line, suggesting that the DNA synthesis was increased in A549/DDP cellline. Average apoptosis rate was increased from(5.32±0.19)％to (34.5±2.24)％,(P=0.002), 95％CI was[24.15,34.36] in A549 cell line after being treated by DDP(10g/ml) for 24 hours. However, the change of average apoptosis rate in A549/DDP cellline was no difference(0.46±0.25)％, (P=0.086). We could found that remarkableapoptotic changebetween A549 cell line and A549/DDP cell line(P＜0.001). The IC50increased from (5.35±0.37)μM/L in A549 cell line to (92.88±4.10)μM/L inA549/DDP cell line as tested by MTT assay at 48h exposure of DDP, the RI was17.36(P=0.001). A549/DDP cell line was cross-resistant tocarboplatin(RI=6.23), epirubicin(RI=4.64), gemzar(RI=20.80), (P=0.002), but notresistant to vincristine(RI=1.39) and paclitaxel(RI=1.09). It was validated thatapoptotic gene(bcl-2) and multidrug resistance genes(LRP,GST-p) were up-regulatedin A549/DDP cell line by semi-quantitative RT-PCR(P＜0.001), and MDRlmRNAwas not detected. In contrast with SKOV-3 cell line, both A549/DDP cell line andA549 cell line showed higher expression in VEGFmRNA.2. At the end of the experimental period,the body weight of DDP routine treatmentgroups(C,D) were lighter than any other groups(P≤0.001). In singleBevacizumab(B) and both combination(D,E) groups, tumor growth was suppressedsignificantly, with growth inhibition rates of 20.96％, 51.67％and 50.95％respectively(P＜0.001), and more effect in both combination groups. Between DDPgroup and control group no statistically difference were seen.The expression of MVDin of those three groups above-mentioned were 18.6±1.14, 13.6±1.14, 14.4±0.55 respectively(P＜0.001). Compared to control group, RT-PCR analyses revealed adecrease in expression of bcl-2 genes in A549/r)DP tumors by anti-VEGF treatmentgroups(B,D,E groups), (P＜0.001), and no statistically difference in these threegroups. Either group showed no statistically difference in LRP and GST-pmRNAexpression.Conclusion:1. The changes of biological characters in A549/DDP cells were related with themultidrug resistance(MDR). A549/DDP cell line possessed the typical and stablemultidrug resistant phenotype by identification and showed higher VEGFexpression(target for Bevacizumab-treatment), which was suitable for next research ofDDP resistance in lung cancer.2. The A549/DDP cells can resistant the apoptosis which was caused by DDP. Itsmechanism might be closely related with the overexpression of bcl-2 and multidrugresistant genes(LRP,GST-p).3. Bevacizumab has synergetic cytostatic activity with DDP on growth of lungadenocarcinoma A549/DDP cell xenografts in mice through inhibitingangiogenesis, and may enhance the sensitivity of A549/DDP cells to DDP by inducingcell apoptosis.4. It is strong supported that tumor angiogenesis inhibitor in combination withtraditional cytotoxic agents would be a new innovative treatment regimen fornonsquamous advanced non-small-cell lung cancer.
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