The Effect Of Lapatinib And DPP On Cervical Adenocarcinoma Cells And Its Mechanism

Background and purposeCervical cancer is one of the most common gynecoloigic malignant tumor in China, and It ranks the third killer among all gynecoloigic malignant tumor death.Every year 30,000 people die of cervical cancer (CC) in China.In recent 20 years, with An increase in the incidence of CC in China,There is a tendency of patients with CC more younger. The percentage of patients with adenocarcinoma of the uterine cervix (AUC) who were associated with poor prognosis compared to squamous cell cervical cancer (SCC). Because AUC grow inward and apt to infiltrating through the stroma and the clearance of vascular and lymph vessels, which is the cause of AUC patients with poor prognosis.Currently the comprehendsive treatment of surgery, radiotherapy, chemotherapy could cure most of the patients with CC in early and middle stage, but the patients with advanced or recurrent CC did not achieve satisfactory results.Palliative chemotherapy is the main way for the patients with advanced CC.Cisplatin is considered the most effective chemotherapy drugs to patients with recurrence or metastasis CC, recommended for first-line chemotherapy, but tumor cells can easily produce their resistance to it.Therefore, it's very important to further study of cervical cancer and seek new treatments and drugs. Molecular targeted therapy is a new biological treatment model, blocking the malignant behavior of the tumor from the molecular level,inhibiting tumor cell growth and metastasis, and even deleting the tumor. Epidermal growth factor receptor (EGFR/HER) family consists of four glycoproteins, comprising ErbB1 (EGFR), ErbB2 (HER-2), ErbB3 (HER-3), ErbB4 (HER-4). EGFR is associated with tumor cell proliferation, angiogenesis, tumor invasion, metastasis and the inhibition of apoptosis. Studies have shown that EGFR is higher expression in some patients with CC,and its expression indicates poor prognosis.Lapatinib (lapatinib, Tykerb) is a quinazoline amine compounds, is an oral small molecule EGFR / HER-2 double-receptor tyrosine kinase inhibitors.Its chemical name is lapatinib ditosyla temonohydrate. It can inhibit the EGFR (ErbB-1) and HER-2 (ErbB-2) of the ATP sites, prevent the phosphorylation and activation of tumor cells and block EGFR (ErbB-1) and HER-2 (ErbB-2) downstream signal path-way, thereby inhibiting tumor growth. Lapatinib was launched by laxoSmithKline R & D in British, in May 2007 FDA has approved lapatinib and capecitabine for patients with advanced or metastatic breast cancer patients and over-expression ErbB-2,who had received anthracycline, paclitaxel, trastuzumab (Herceptin) therapy. Lapatinib has been some research in cervical cancer.ether a go-go potassium channel (Eag1) is a member of the voltage-gated potassium channel Eag family, that is including Eag(including Eag1 and Eag2), Erg (including 3 members) and Elk (including 2 Members) channels.Human Eagl gene is located on chromosome 1q 32.1-2 .Eagl is a new independent genetic loci which was found in 1969 by Kaplan when he detect mutation in drosophila melanogaster caused leg tremors after ether anesthesia .Eagl is a voltage-gated potassium channels, activated by depolarization and showing permeability to both K+ and Ca2+ . Eagl channel express abundantly in the brain,but the expression in peripheral tissues is very limited.Some study found that its expression could be detected in myoblasts the placenta . Eagl could specifically expressed highly in some primary tumor cells and tumor cell lines, closely associated with malignant cell transformation, tumor proliferation, metastasis and prognosis.Therefore, we chosen lapatinib and cisplatin acting on AUC Hela cells, and investigated the inhibition of the two drugs on CC in vitro. Also we reseached the role of lapatinib to cell cycle, cell apoptosis and Eagl potassium channels to Explore the possible mechanism and provided theoretical basis for comprehensive treatment and improving efficacy of CC.Part I:Coadministration of lapatinib with DDP inhibits cell proliferation in AUC Hela cells, lapatinib acting on the Hela cell cycle and apoptosisMaterial and Methods1. AUC Hela cells were cultured in RPMI 1640 containing 10% fetal calf serum and subcultured every 3 to 5 days. 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 on10 consecutive days. The growth curve drew using Excel.3. EGFR and HER-2 gene expression in AUC Hela cells:We collected the cells after its covered flask 80% area, and extracted total RNA by Trizol reagent. Total RNA was reversed transcription into cDNA which was amplified by polymerase chain reaction (PCR) kit ,then ran agarose gels electrophoresis with the products of PCR amplification and observed EGFR, HER-2gene expression in the UV light.4. The cells were divided into 4 groups:lapatinib-treated group, DDP-treated group, lapatinib combined DDP group and control group. The inhibition rates of cells was detected when the concentration of lapatinib group were 0.5,1,2,4,8,10,20μmol/L,DDP were 0.75,1.5,3,6,12,24,48μg/ml for 24h,48h,72h respectively. The concentration of lapatinib combined DDP group were 1μmol/L + 0.75μg/ml,2μmol/L+1.5μg/ml,4μmol/L + 3μg/ml for 72h.Inverted microscope observed, cell cycle and apoptosis analysis include cells in the concentration of 1μmol/L,2μmol/L, 4μmol/L.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 lapatinib and DDP was assessed using the q value, when q>1.15,0.85≤q≤1.15, and q<0.85 indicated 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 SPSS 13.0 statistical 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 T3 multiplecomparison 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 to 7 days,and more faster growth was from 4 to 7 days, then the growth gradually slowed down.2. EGFR and HER-2 gene expression in AUC Hela cells:The result showed that EGFR gene was expressed in Hela cells By RT-PCR,but HER-2 gene was not.3. Combination of lapatinib and DDP inhibit proliferation of Hela cells:By MTT assay, lapatinib and DDP alone both inhibited proliferation of Hela cells in different concentrations and different time points,and the inhibition rate was significantly higher (P<0.05)with the concentration and time increased. Inhibitory rate of Lapatinib and DDP combination group(1μmol/L+0.75μg/ml and 4μmol/L+3μg/ml) was significantly higher than lapatinib alone group(P<0.05),but there was no difference,compared with DDP alone group. Combination group show additive effect in 4μmol/L+3μg/ml concentrations for 72 hours (q= 0.99), while displayed antagonism in 1μmol/L +0.75μg/ml and 2μmol/L + 1.5μg/ml concentrations (q<0.85).4. Morphology of Hela cells observed under inverted microscope in lapatinib group:Control cells adhered in good condition, showing polygonal cells,larger and plump cytoplasm, cell borders clearly. Experimental cells have different levels of morphological changes, becaming round and size reduced, but the cell membrane structural integrity. Adherent cells were shrunken, rounded off. The cells occurred morphological changes increased in the concentrations of 2μmol/L,and with high doses abnormal cells became more common, the total numbers of cells significantly reduced.5. Lapatinib 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 G0/G1 phase was increase as lapatinib concentration increased, while the proportion of cells in S and G2/M phase was decreased.6. Lapatinib affected to cell apoptosis of Hela cells:Hela cells were detected by flow cytometry after lapatinib-treated 72h.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<0.01).The apoptosis rate increased with the dose increasing.Part II:Lapatinib effected to the expression of Eag-1 potassium channel in AUC Hela cells.Material and Methods1. AUC Hela cells were cultured in RPMI 1640 containing 10% fetal calf serum and subcultured every 3 to 5 days. Exponentially growing cells were chosen for experient.2. The cells were divided into 2 groups:control group and lapatinib-treated group (lμmol/L,2μmol/L,4μmol/L). extracted RNA and the whole protein after using the drug 72h.3. Detected the changes of Eagl gene expression among the groups by RT real-time PCR and Western-blot.4. Statistical analysis:All datas were analysised by SPSS 13.0 statistical 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.Results Compared with the control group, Eagl gene expression (1,2 umol/L) was significantly down-regulated in lapatinib-treated group (P<0.01), and there was no Eagl gene expression in lapatinib 4 umol/L treated group.ConclusionEGFR gene expressed in CC Hela cells,and it could be used as cancer therapeutic target. lapatinib and DDP alone both inhibited proliferation of CC Hela cells time-and dose-dependently. Combination group showed additive effect in high dose group.The cell rate of G0/G1 phase was significantly increased, while the proportion of cells in S and G2/M phase was Obviously decreased. lapatinib could induce Hela cells'apoptosis,and The apoptosis rate increased with the dose increasing. Eagl gene expression was significantly down-regulated,therefore we consider that lapatinib may be regulating cell cycle, inhibition of tumor cell proliferation by preventing Eagl potassium channel,that is worthy of further study.