Experimental Study On Reversion Of Human Nasopharyngeal Carcinoma Drug-Resistance By Bevacizumab

BACKGUOUND & OBJECTIVES:Nasopharyngeal Carcinoma (NPC) is one of the most common malignant tumorin south China and Southeast Asia. Most patients have been in advanced stage whendetected, as data shows, that stageⅢ～Ⅳare about 85% of all. A great quantityclinical research shows that combination of radiation therapy and chemotherapy mayget higher cure rate, even the chemotherapy alone are effective in late patients,especially with the use of new drugs such as gemcitabine. Gemcitabine(2', 2-difluorodeoxycytide) is a new analog of arabinosylcytosin, belonging toanti-metabolism anticancer drugs, and it is catalyzed into active diphosphate dioxygencytidine and triphosphare dioxygen cytidine by nucleoside monophosphate kinase incell, the latter can interfere DNA synthesis by restraining DNA pclymerase, theninhibit the growth of tumor cells. Gemcitabine is the only drug that was approved in1996 by American Food and Drug administration for treatment of pancreatic cancer.Further studies indicate that gemcitabine are effective to head and neck tumors too,especially to those advanced stage patients. However the whole therapeutic efficacyof NPC is not satisfacted, because most patients died from abortive chemotherapy,which is greatly incriminated to multidrug resistance (MDR) of cancer cell. TumorMDR is defined as the cross tolerance to many antitumor drugs after chemotherapy,which is untouched, structure independent, and mechanism various. Establishing celllines is the basis of studying MDR in virto. Anti angiogenesis becomes a new methodto treat tumor recently, because of the generation, development and metastasis of tumor depend on angiogenesis. Vascular endothelial growth factor (VEGF) is the keyfactor in tumor angiogenesis, which is the most active and authoritative angiogenesisfactor. Bevacizumab, the recombinant humanized monoclonal antibody to vascularendothelial growth factor, is a new anticancer therapy that was approved in Februaryof this year by the Food and Drug administration for the use of first line treatment ofadvanced colorectal cancer. VEGF and its receptors expresses in many tumor, as isreported that there are 60 to 90 percent of NPC patients expresses VEGF, and whichis related to bad clinical features and prognosis of NPC. Bevacizumab has showedgood effect in variousâ…¡toâ…¢phase clinical trials including NPC. We found thatbiochemotherapy mode, which is made by combining bevacizumab and chemicmedicine, can gain not only more effective power than chemotherapy or bevacizumabalone, but also exceed the algebraic sum of the two. Some inefficient drugs regainsensitivity after using the molecular targeted drugs such as bevacizumab inrecrudescent or metastatic circumstance. It seems that bevacizumab treat tumor notonly by inhibiting neovascularization but also by increasing sensitivity of tumor cellsto chemic medicine. The latter is most probably the cause why bevacizumab canreverse MDR.We designed this study to: 1. Establish a gemcitabine-induced humannasopharyngeal carcinoma (NPC) drug-resistant cell line for the first time and studyits characteristics. 2. Investigate the effect of bevacizumab on drug fast tumor cell,research whether bevacizumab can reverse MDR.Materials & METHODS:1. After investigating NPC cell lines 5-8F, C666-1, CNE2 by RT-PCR, we foundVEGF and VEGFR-2 are expressed in CNE2 cells. Human NPC poorly differentiatedcell line CNE2 was cultivated in high glucose DMEM medium, supplemented with10% calf serum, in 37℃, 5%CO₂ saturated humidity incubate box. The drug-resistantcell line CNE2/Gem was established by a procedure of treating the human NPC cellsCNE2 with gemcitabine by pulse drug selection and continuous stepwise selection.The IC₅₀ and resistance index (RI) to several commonly used anti-tumor drugs weretested by MTT assay. Fluorescence activated cell analysis (FACS) was used for determining the cell cycle and concentration of fluorescence dye rhodamine123within the cells. Cell growth curve, doubling time, and cell morphology weremeasured and observed.2. CNE2 cells, be of exponential phase of growth, were inoculated in 96-wellplates at 10⁴/well in 100ul culture volum. There were a control group and 7experimental groups which contained gemcitabine 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 thekill rate of drugs. As is shown in formula: kill rate=(1—OD value of eachexperimental group/average OD value of control group)×100%. One-Way Anova inSPSS10.0 was used to analyze the experimental data, and we defined statisticalsignificance as P＜0.05.3. CNE2 and CNE2/Gem cells were conventionally cultivated in 50ml-cultureflasks, grew in adherence observed by microscope, then add gemcitabine andbevacizumab of certain density to the cells respectively: a flask of CNE2 and a flaskof CNE2/Gem with 20ug/ml gemcitabine, a flask of CNE2/Gem with both 20ug/mlgemcitabine and 10ug/ml bevacizumab, another flask of CNE2 and CNE2/Gem werecontrol. All cells were put into 37℃5%CO₂ saturated humidity incubate box.Collected the cells and dyed them with Annexinâ…¤and PI, then detected the apoptosisrate with flow cytometry. This procedure repeated 3 times, and we analyzed theexperimental data with one-Way Anova in SPSS 10.0.4. Some CNE2/Gem cells were. cultivated in culture fluid supplemented with10ug/ml bevacizumab, went down to a new generation once per four days, 4generations later, the cells were called CNE2/Gem/Bev. Detected the mRNA ofMDR1, VEGF, Bcl-2 and Bax expressed in CNE2, CNE2/Gem and CNE2/Gem/Bevcells, and computed the relative express quantity.RESULTS:1. Double time of CNE2 and CNE2/Gem were 17.13h and 23.13h respectively,as evaluated by the growth curve. The IC₅₀ to gemcitabine increased from 2.12ug/ml in CNE2 to 37.04ug/ml in CNE2/Gem as tested by MTT assay at 48～72h exposure,the RI was 17.62. The RI to cisplatin (DDP), 5-fluoroutacil (5-FU), and vincristine(VCR) were 15, 6.98, and 12.8 respectively, indicating its multidrug resistant prorerty.FACS analysis showed that the concentration of rhodamine123 was much lower inCNE2/DDP cells than in CNE2 cells (3.56 vs. 220.35). The CNE2/Gem cellsappeared more round in various size under light microscopy, with more granulation incytoplasm. CNE2/Gem cells could go down to the future generation efficiently, withstable drug resistance.2. The kill rate of 10ug/ml bevacizumab group was similar to that of controlgroup, without statistical significant difference (P=0.140); moreover higher drugdensity did not mean higher kill rate, there was no statistical significant differencebetween high and low density group (P=0.531). However when combined with20ug/ml or 40ug/ml gemcitabine, the kill rate of 10ug/ml bevacizumab was higherthan that of using gemcitabine alone with statistical significant difference (P=0.026,P=0.001). Likewise, with the same density of gemcitabine, double bevacizumab didnot bring out higher kill rate (P=0.591).3. Compared with control cells, early apoptosis rate was higher (23.21% vs.5.2%, 8.66% vs. 4.71%, 39.46% vs. 4.71%), late apoptosis and dead rate was muchhigher (50.48% vs. 3.1%, 48.21% vs. 2.5, 42.68% vs. 2.5%), after being treated with20ug/ml gemcitabine or combined with 10ug/ml bevacizumab. Early apoptosis rate ofCNE2/Gem was much lower than that of CNE2 (8.87±1.06% vs. 23.50±4.21%,P=0.005), late apoptosis rate was lower too (41.70±9.78% vs. 60.52±9.94%,P=0.036). To CNE2/Gem, the early apoptosis rate, which was caused by combined20ug/ml gemcitabine and 10ug/ml bevacizumab, was much higher than that ofgemcitabine alone (39.87±5.85% vs. 8.87±1.06% , P＜0.001); And the lateapoptosis rate differed little (37.64±5.11% vs. 41.70±9.78%, P=0.582).4. As was shown in half-quantitative RT-PCR assay, the relative express quantityof the four mRNA in CNE2, CNE2/Gem and CNE2/Gem/Bev was: MDR1 0.623,1.364 and 1.165, VEGF 0.241, 1.652 and 1.547, Bcl-2 0.613, 0.952 and 0.135, Bax0.665, 0.387 and 1.751. CONCLUSION:1. CNE2/Gem, the gemcitabine resistant phenotype, which showed prominentdrug resistant, was established successfully. It is a good model for exploring themechanism of resistance of NPC to gemcitabine.2. Bevacizumab could greatly increase the sensitivity of CNE2/Gem cells togemcitabine. The lethal effect was weak when bevacizumab was used only, but theeffect was great when combined with gemcitabine.3. The early apoptosis cells decreased obviously in CNE2 when treated withgemcitabine, and increased again when treated with bevacizumab. So we might drawa conclusion that bevacizumab could improve early apoptosis in MDR cells.4. Bevacizumab had effect not on MDR related genes but on apoptosis relatedgenes, and promoted MDR cells to apoptosis. To CNE2 cells, when induced them toMDR with gemcitabine, then treated them with bevacizumab, their expression ofMDR1 increased greatly, then decreased slightly; VEGF increased greatly, then keptstable; Bcl-2(apoptosis inhibiting gene) increased, then decreased greatly;Bax(apoptosis enhancing gene) decreased, then increased greatly.In short, bevacizumab could treat cancer through reversing MDR, in other words,increasing the sensitivity of MDR cells to chemotherapy.