The Effect And Mechanism Of Nimotuzumab And DPP On Cervical Adenocarcinoma Hela Cell

Background and purposeIn the world,every year has500000new cervical cancer cases,and the death cases is200000or so;in our country,the new cases overtake130000,and the death cases is about200000.what is more? The rate of death is the first in gynecological malignancies.It is known that the main cause is humanpapilloma virus infection.Cervical adenocarcinoma accounts for10-34%.Although the proportion is less,the trend is increasing and younger. Cervical adenocarcinoma rises from cervical endometium and invades growth inwardly.Lack of obvious clinical symptom and early matastasis,poor prognosis,the combinded treatments of surgery and radiotherapy,chemotherapy are difficult to achieve good outcomes. Cisplatinum is the most effective drug and the first-line chemotherapy for cervical cancer,Besides,the tumour cells are easy to resistance to cisplatium,and the result is in vain.Because of biology anf treatment response is different from squamous cell carcinoma,it is very important to seek for new treatments and new drugs.Tumor molecular targeted therapy is a mew biological treatment modalities with the most specificity in the treatment.To carcinogenesis and proliferation, metastasis,blocking tumor bad behavior,and inhibiting tumor cell grouth and metastasis and even tumor regression.In recent years,with tumor molecular targetd therapy development the drugs are endless. And this is good for patients with Cervical adenocarcinoma.The drugs are foolowing:①Epidermal growth factor receptor inhibitors:EGFR monoclonal antibody, Small molecule tyrosine kinase inhibitorsthe;②anti angiogenesis inhibitors;③the multiple target inhibitors;④the mTOR pathway inhibitors.Part I:Nimotuzumab with DDP inhibits cell proliferation in Cervical adenocarcinoma Hela cells, nimotuzumab acting on the Hela cell cycle and apoptosisMaterial and Methods1. Hela cells were cultured in RPMI1640containing10%fetal calf serum and subcultured every3to5days. Exponentially growing cells were chosen for experient.2. Growth curves of Hela cells detected by MTT assay:Setting the normal group and blank control group, which were detected after seeded plates overnight, once a day on seven consecutive days. The growth curve drew using Excel.3. EGFR gene expression in Hela cells:We collected the cells after its covered flask80%area, and extracted total RNA by Trizol reagent. Total RNA was reversed transcription into cDNA which was amplified by RT-PCR.4. The cells were divided into4groups:nimotuzumab-treated group, DDP-treated group, nimotuzumab combined DDP group and control group. The inhibition rates of cells was detected when the concentration of nimotuzumab group were12.5,25,50,75,100ug/ml,DDP were0.75,1.5,3,6,12μg/ml for24h,48h,72h respectively. The concentration of nimotuzumab combined DDP group were50ug/ml+0.75μg/ml,50ug/ml+1.5μg/ml,50ug/ml+3μg/ml for72h.I cell cycle and apoptosis analysis include cells in the concentration of25ug/ml,50ug/ml,100ug/ml.5. Taking the above group, the inhibitory of Hela cells were detected by MTT assay after treatment. Calculate the inhibitory rates of cells according OD value.6. Interaction between nimotuzumab and DDP was assessed using the q value, when q>1.15,0.85≤q≤1.15, and q<0.85indicated synergistic, additive, and antagonistic effects respectively.7. Taking the above group, collected cells and analyzed cell cycle and apoptosis by flow cytometry.8. Statistical analysis:All datas were analysised by SPSS13.0statistical softwire. The results of experiment were expressed by the way of mean±SD. The Inhibitory rate was analyzed by the way of General Linear Models of Univariate followed by LSD or Dunnett’s T3multiplecomparison test. Data of cell cycle and apoptosis were analyzed by One-way ANOVA followed by LSD multiple comparison test. P Values were considered to be significant at≤0.05.Results1. Growth curves of Hela cells:Observed from the growth curve, species Hela cells showed exponential growth froml to7days,and more faster growth was from4to7days, then the growth gradually slowed down.2. EGFR gene expression in Hela cells:The result showed that EGFR gene was expressed in Hela cells By RT-PCR,.3. Combination of nimotuzumab and DDP inhibit proliferation of Hela cells:By MTT assay, nimotuzumab and DDP alone both inhibited proliferation of Hela cells in different concentrations and different time points,and the inhibition rate was significantly higher (P<0.001)with the concentration and time increased.4. nimotuzumab affected to cell cycle of Hela cells:The result of cell cycle analysis indicated that there were significantly different in the cell rate of G0/G1, S and G2/M phase between the control group and the experiment group (all P<0.05). The cell rate of GO/G1phase was increase as nimotuzumab concentration increased, while the proportion of cells in S and G2/M phase was decreased.5. nimotuzumab affected to cell apoptosis of Hela cells:Hela cells were detected by flow cytometry after lapatinib-treated72h.The result showed that lapatinib could significantly induce apoptosis in Hela cells. The rate of cell apoptosis in experim-ental group were significantly different from control group (P<0.05).The apoptosis rate increased with the dose increasing. Part II:nimotuzumab effected to the expression of AREG gene Hela cells.Material and Methods1. Hela cells were cultured in RPMI1640containing10%fetal calf serum and subcultured every3to5days. Exponentially growing cells were chosen for experient.2. The cells were divided into2groups:control group and nimotuzumab-treated group (25ug/ml,50ug/ml,100ug/ml). extracted RNA after using the drug72h.3. Statistical analysis:All datas were analysised by SPSS13.0statistical softwire. The results of experiment were expressed by the way of mean±SD. The dates was analyzed by One-way ANOVA followed by LSD multiple comparison test. P Values were considered to be significant at≤0.05.ResultsCompared with the control group, AREG gene expression (25ug/ml,50ug/ml,100ug/ml) was significantly UP-regulated in nimotuzumab-treated group (P<0.05).ConclusionEGFR gene expressed in Hela cells,and it could be used as cancer therapeutic target. nimotuzumab and DDP alone both inhibited proliferation of Hela cells time-and dose-dependently. Combination group showed additive effect in high dose group.The cell rate of G0/G1phase was significantly increased, while the proportion of cells in S and G2/M phase was Obviously decreased. nimotuzumab could induce Hela cells’apoptosis,and The apoptosis rate increased with the dose increasing. AREG gene expression was significantly up-regulated.therefore we consider that AREG may be a treatment and predict targets,it is worth further study.