| BackgroundLung cancer is the leading cause of mortality in oncology which have a rapid development, difficult early detection and diagnosis, poor treatment and prognosis. There are 222,520 cases of lung cancer in United States in 2010, accounting for 15% of new tumors; 157,300 cases of death, accounting for 28%; all these datas are the first in the world. Non small cell lung cancer (NSCLC) accounts for about 80-85% of the total number according to pathological types of lung cancer, whereas the remaining is small cell lung cancer. Among these newly diagnosed NSCLC cases, only 16% are early status, the majority of NSCLC patients are diagnosed advanced condition. Consequently, non-surgical treatment for these patients is the main therapeutic method. The research indicated that transmembrane protein tyrosine kinases widely expressed in a NSCLC cell surface:epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) which are closely related to the signaling pathways of amplification and regulation of tumor, survival, metastasis and angiogenesis. Based on this, gefitinib as a representative small molecule EGFR-TKI targeted drugs have emerged and became the principal treatment for patients with advanced NSCLC. For some patients with EGFR mutations, compared with conventional chemotherapy, the application of gefitinib can obtain better survival benefit. Especially in foreign citizen of Chinese origin, NSCLC patients with EGFR sensitive mutation rate up to 30%. Gefitinib seems to bring a great hope for lung cancer patients, but scientists found that all the current clinical application of small molecule EGFR TKI in the treatment period, patients are still resistant and recrudescent to the drugs along with long-time treatment. Once acquired drug resistance mutations emerged, the patient will lose the ultimate hope.The problem of solving EGFR TKI resistance generally has two ideas including development of new targeted therapy drugs or exploration of combined project for different drugs. The latter choice is more efficient and fast to implement. The anti-tumor effect of some flavonoids, polyphenols and low or non-toxic plant extracts have received much attention by scientists. Therefore, the combination with conventional anti-cancer drugs and plant compounds could reduce drug dosage effectively meanwhile reducing the toxic side effects of anti-cancer drugs and also increasing the biological activity of anti-cancer drugs for human. This will be especially urgent and indispensable to improve the quality of life of patients.Genistein is a natural tyrosine kinase inhibitor which anticancer effects have received much attention in recent years. Genistein can regulate multiple proteins of EGFR-mediated cell signaling pathways and inhibit tyrosine kinases in different levels. The TKI activities of its multi-targets have opened a door for designing a new combined project of targeted therapy. Genistein of certain doses not only induced tumor cells differentiation but also has the effect of apoptosis. However, genistein alone only produced weak effects on tumor cell differentiation and apoptosis. Consequently, the research on how to reduce the dose of anti-tumor drugs in order to reduce the toxic side effects and enhance effects of anti-tumor drugs is extremely significant.ObjectivesThe purpose of our study intends to compare the difference in cell proliferation, apoptosis and apoptosis related protein PARP,caspase-3 expression and phosphorylation level of pivotal protein such as EGFR,Ak,Erk1/2,mTOR in signaling pathways of human lung cacner cell lines H1975 among genistein single, gefitinib single and combination and to validate whether the combination possess enhanced anti-tumor effect compared to single agent in molecular level. The study also will explore the molecular mechanism of EGFR signaling pathway associated with drug resistance by nude mouse xenograft tumor experiment in vivo and in vitro. The results will help "dietary phytochemicals combined with targeted therapy or chemical prevention to inhibit acquired drug-resistance mutations in non-small cell lung cancer" to provide original theoretical evidence.Materials and methodsThe whole experiment is divided into two parts.Chapter One:Synergistic inhibitory effects by the combination of genistein and gefitinib on NSCLC with aquired drug-resistance in vitroThis study intend to select the EGFR mutant lung cancer cell lines with the purpose of comparing and researching the effect and mechanism of drug cytotoxicity among genistein single, gefitinib single and combination as well as helping "dietary phytochemicals combined with targeted therapy or chemical prevention to inhibit acquired drug-resistance mutations in non-small cell lung cancer" to provide theoretical evidence. Select EGFR resistant mutations lung cancer cell lines H1975, H1975 cells contain EGFR20 T790M mutation in exon and exon 21 L858R mutation as well as resistant to EGFR-TKI. Different doses of genistein single, gifitinib single and their combination treatment were applied in human lung cancer cell lines H1975. Growth condition of H1975 cells were observed dynamically, the inhibition of cells proliferation in each group were assayed by MTT; CI values (synergy index) were calculated by Chou-Talalay equation; apoptosis rates were detected by Flow cytometry; both protein expression of apoptosis-related gene such as PARP,caspase-3 and phosphorylation levels of EGFR,Erkl/2,Akt,mTOR were detected by western blotting, analyzing whether the combination with genistein and gefitinib could synergistically enhance anti-tumor effect on NSCLC with aquired drug-resistance.Chapter Two:Synergistic inhibitory effects and mechanism by the combination of genistein and gefitinib on NSCLC with aquired drug-resistance in vivoWe established the model on subcutaneously implanted tumor induced by human non small cell lung cancer cell line H1975 in nude mice. Female nude mice,6-8 weeks old were used. The cell line of human non small cell lung carcinoma named H1975 was cultured at 37℃,5%C02 in 1640 medium supplemented with 15% calf serum.Tumors were established by injection of about 4×106 tumor cells respectively into the back of each nude mouse. Once the tumors reached～100mm3 in diameter, the 40 mice were randomly divided into 4 groups of 10 each experiment:(1) Control group:5% Tween 80 solution; (2) Genistein group:150 mg/kg; (3) Gefitinib group: 50 mg/kg; (4) Combination group:Genistein 150 mg/kg+Gefitinib 50 mg/kg. At the end of five weeks, all surviving mice were killed after anesthesia and removed tumor tissues. Tumor volume was measured to calculate the value of synergy index "q" value and tumor inhibition rates. Tumor tissues were fised with 10% neutral formalin. Cell proliferation in situ was assayed to calculate the proliferation index by immunohistochemistry SP method; Cell apoptosis in situ was analyzed to calculate the apoptosis index by TUNEL method using. ResultsChapter One:Synergistic inhibitory effects by the combination of genistein and gefitinib on NSCLC with aquired drug-resistance in vitro1. Antiproliferative effects significantly differed between all groups of regimens in EGFR-TKI resistant NSCLC cell line with T790M.HI975 cells were treated with genistein single for 48 hours, gefitinib single for 48 hours, and combination with genistein and gefitinib co-treated for 48 hours, we observed that genistein single, gefitinib single and combination therapy of genistein and gefitinib produced a different antiproliferative effects. Every group along with drug concentration increased, the inhibitory effect gradually increased, combined group appeared the best inhibitory effect. Antiproliferative effects of combination group were stronger than any regimen alone on equal concentration or higher concentration, strengthen synergetic effect of anti-tumor, the number of cells reduced significantly under microscope.One-way ANOVA test analysis showed that obviously antiproliferative effects of combination group on cancer cells than any regimen alone, the inhibition rate increased significantly and appeared dose-dependment effect, the difference was significant (P=0.000, P=0.001, P=0.000, P=0.001, P=0.013, P=0.017). It indicated that co-treatment with genistein and gifitinib possesses synergetic antiproliferative effect in H1975.CI values of combined-drugs were calculated by Chou-Talalay software, the result showed that the more drug concentration increased (G1-G6), the more inhibitory effect gradually increased, CI values were less than 1 (CI<1, CI=1, CI> 1 represent synergistic, additional and antagonistic effect respectively), indicating that a combination of both genistein and gifitinib has a synergistic inhibition effect on cancer cells. It showed a strong synergistic effect among G1, G2, G3 three dose groups (CI<0.7). 2. Combination with gefitinib and genistein synergistically induces apoptosisMTT antiproliferative experiments show that compared to treatment of single drug, combination therapy with genistein and gefitinib can synergistically inhibit cell growth in H1975 which may be related to cell apoptosis and its mechanism, so the effect of tumor cell apoptosis of genistein alone, gefitinib alone and combination with genistein and gefitinib was detected by flow cytometry. The results of flow cytometry suggest that genistein or geifitinib treatment alone in H1975 cells for 48 hours can both promote apoptosis, apoptosis rates were (42.03±4.38)% and (35.95±2.32)% respectively. However, after combination treatment, apoptosis significantly increased in H1975 cells, was (68.70±7.13)% (combination vs. gefitinib, P=0.000), have significant difference.It indicated that co-treatment with genistein and gifitinib possesses synergistic effect of promoting apoptosis in H1975 cells.3. Gefitinib combined with genistein effect expression of apoptosis-related proteinOur study demonstrated that neither genistein nor gefitinib alone could activate these apoptosis-related molecules significantly, both expression of caspase-3 and PARP decreased, nevertheless in combination group, the cleaved form of caspase-3 and PARP could be obviously found. It suggesting that PARP and caspase-3 play an important role in gefitinib or/and genistein induced apoptosis in H1975 cells, PARP and caspase-3 might be two major regulatory genes.4. Treatment effects on expression of EGFR and its downstream signaling pathwayWe studied that EGFR and its downstream signaling pathways of two drugs in order to reveal the mechanism of action on genistein and gifitinib. After gefitinib or/ and genistein incubating H1975 cells for 12 hours, some related proteins were detected by western blotting. Compared with gefitinib group, combination group's phosphorylation levels of EGFR,Akt,mTOR were significantly lowered (P=0.000) while phosphorylation of Erkl/2 was not inhibited (P= 0.174).Chapter Two:Synergistic inhibitory effects and mechanism by the combination of genistein and gefitinib on NSCLC with aquired drug-resistence in vivo.1. The growth effect of implanted tumor by combination with gefitinib and genistein on nude mouse xenograft tumor modelAt the end of experiment, tumor volume was measured.The average tumor volumes of genistein, gefitinib and combination group were significantly lower than the control group.The tumor volume of control, genistein, gefitinib and combination group were respectively (2138.59±279.00) mm3, (1689.48±220.41) mm3, (1475.63±192.51) mm3 and (405.28±65.57) mm3, compared with combined group, the tumor volume of gefitinib group have significantly lowered (P=0.000); tumor inhibitory rates were 0.00%,21.00%,31.00% and 81.05%, combination group was the best. The "q" value of combined group is 1.78 according to formula, indicating that genistein combined gifitinib can synergistically inhibit tumor growth which consistent with the first part of experiments.2. The inhibitory effect of proliferation by combination with gefitinib and genistein on nude mouse xenograft tumor modelThe nuclear showed brown-yellow after Ki67 staining.The brown-yellow proliferating cells of genistein, gefitinib and the combination group significantly reduced compared to control group, cell proliferation rates were seperately (29.37±7.47)% (control), (21.64±6.30)%(genistein), (11.88±3.45)%(gefitinib) and (7.48±1.91)%(combination), which showed a lowered tendency. It indicate that combination have a co-suppression proliferation effect compared gefitinib alone (combination vs. gefitinib, P=0.000).3. The induced effect of apoptosis by combination with gefitinib and genistein on nude mouse xenograft tumor modelThe nuclear of tumor tissue showed uniform green fluorescence by TUNEL staining. The green fluorescence apoptosis cells of genistein, gefitinib and the combination group significantly increased compared to control group, cell apoptosis rates were seperately (9.31±1.63)%(control), (15.76±3.08)%(genistein), (32.63+ 6.24)%(Gefitinib) and (54.71±9.61)%(combination), which showed a gradual increased tendency. Combination vs. gefitinib (P=0.000), which indicated that combination have a co-induced apoptosis effect compared gefitinib alone.Conclusions1. Gefitinib combined with genistein has synergistic antiproliferation effect on H1975 human lung cancer cells. CI values of combined-drugs were calculated by Chou-Talalay software, CI<0.7, showed a strong synergistic effect.2. Gefitinib combined with genistein has synergistic promoting- apoptosis effects on H1975 human lung cancer cells which may associate with synergistically regulate apoptosis-related protein expression including PARP and caspase-3 protein.3. Gefitinib combined with genistein has significantly synergistic inhibitory effects on phosphorylation levels of EGFR,Akt,mTOR on H1975 human lung cancer cells which may inhibit invasion and metastasis of lung cancer cells.4. Gefitinib or genistein have inhibitory tumor growth effect on non small cell lung cancer with aquired drug-resistance by varying degrees and tumor growth inhibition effect of combination group is more obvious, "q" value is 1.78.5. Gefitinib combined genistein have synergistic inhibition of proliferation on non small cell lung cancer with aquired drug-resistance. 6. Gefitinib combined genistein have synergistic induction of apoptosis on non small cell lung cancer with aquired drug-resistance. |
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