| Cancer is one of the most horrible and complex disease that taken lives away from people consistently, and is so called'wound that never heal'. After so many years'fighting against cancer, few progress of the anticancer therapy do people make. However, hundreds and thousands researches have got to expose the subtle network of tumorigenesis, and the relationship with vascularization. The formation of the new functional microvascular derived from the preexisting blood vessel is called angiogenesis, which is induced by the tumor, and subsequently satisfied all the nutrients and oxygen that tumor needs for growth and metastasis. Both the endothelial cells and the tumor cells are responsible for this pathological process, in which lots of pro-angiogenic factors and the endogenous angiogenesis inhibitors secreted by them are playing important roles. The two sides of scale were once balance with one side was the pro-angiogenic factors and the other side was the endogenous angiogenesis inhibitors. Once the scale loses its balance, the tumor swells. Different pro-angiogenic factors are overexpressed in different types of tumors. Vascular endothelial growth factor (VEGF) is one of the most well studied pro-angiogenic factors, found to be overexpressed in many types of cancer. VEGF binds to its major receptor tyrosine kinase, VEGFR2, which is primarily expressed on vascular endothelial cells (ECs). The binding of the two resulted in the activating of a cascade signal pathway which consequently mediate the proliferation and migration of vascular endothelial cells, facilitate the formation of microvascular. The epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), play critical roles in the progression of cancer through their effects on cellular proliferation, apoptosis, angiogenesis, and metastasis. EGFR provides an essential survival signal to tumor cells by activating the antiapoptotic Akt pathway and was found to be overexpressed in many tumors of epithelial and glial origin. It is also a kind of pro-angiogenic factor, blocking of EGFR of the tumor cells demonstrated direct or indirect anti-angiogenesis and tumor inhibition effect which was found to work by blocking the VEGF receptor. The non small cell lung cancer, is one of the most malignant cancer, found upregulation of the EGFR and VEGFR2, both of which are members of the pro-angiogenic factors.Small interfering RNA (siRNA) is a small molecular with infinite power that can solidly silence any gene involved in any pathophysiologic processes. It cost less than a decade for siRNA therapeutics to be available in clinical application from the RNAi first being discovered by Tuschl et al.. As the first tumor targeted siRNA therapeutics has been going through the evaluation of phase I clinical trial, more and more siRNA drugs are being explored. In this study, siRNA targeted the murine VEGFR2 gene was designed, validated and found to be robust in silencing in vitro. In the A549 NSCLC tumor xenograft mouse model, siRNAs targeted VEGR2 and EGFR respectively were delivered in combination with cisplatin, to investigate the anti-tumor effect compared with siRNA monotherapy, siNRA combination therapy, or chemotherapy alone. The inhibition of tumor vascularization by blocking both VEGFR2 and EGFR signaling pathways, combined with cytotoxic chemotherapy, resulted in profound antitumor effects and prolonged survival. These results inspire us of a new anti-cancer therapy that would potentially benefit a large population of cancer patients.Objectives:1. To design the most effectively silencing sequence that target to the murine VEGFR2 mRNA, and validate the silencing founction in the MS1 cells in vitro. 2. To investigate the anti-tumor effect of siRNAs targeted VEGR2 and EGFR respectively delivered together in combination with cisplatin, and compared with that of siRNA monotherapy, siNRA combination therapy, or chemotherapy alone, in the A549 NSCLC tumor xenograft mouse model.3. To investigate the anti-tumor mechanism of the therapies by determination of mRNA and protein expression level of both VEGFR2 and EGFR in the tumors.Methods:1. Based on the design principles of siRNA made by Tuschl, three pairs of VEGFR2/Flk-1 siRNA sequences were designed according to mouse mRNA (NM010612) published by GenBank. The sequence of VEGFR2 siRNA4 were from reference article[1]. A scrambled siRNA was used as an control. siRNAs were transfected into MS1 cells using LipofectamineTM 2000 (Invitrogen). Cells were cultured under standard conditions for a further 48 h before being examined by semi-quantitative RT-PCR or quantitative RT-PCR. For western blot cells were cultured for a further 72 h. The statistical significances were measured by ANOVA.2. 5×106 A549 cells in 0.2 ml of buffered saline were injected s.c. into the flank region of the athymic nude mice (BALB/C, nu/nu,4-6 weeks) to allowed the tumors to establish. tumor volume was measured with a caliper every three days and calculated using the formula:Volume=1/2×length×width2, where length represented the largest tumor diameter and width represented the smallest tumor diameter[5]. Animals were randomized in different groups for therapy when tumor volume reached 30-100 mm3. Each treatment group consisted of four to five animals. Animals were injected with (1) saline (intratumorally, twice/week), (2) PEI alone (intratumorally, twice/week), (3) 0.5nmol siVEGFR2 (intratumorally, twice/week), (4) 0.5nmol siEGFR (intratumorally, twice/week), (5) 0.5nmol siControl (intratumorally, twice/week), (6) 0.25nmol siVEGFR2+0.25nmol siEGFR (intratumorally, twice/week), (7) 5mg/kg cisplatin (intraperitoneally, once/week), (8) 0.25nmol siVEGFR2+0.25nmol siEGFR+ 3mg/kg cisplatin (siRNA:intratumoral injection, twice/week, cisplatin: intraperitoneal injection, once a week on the day after the first delivery of siRNA). siRNA was complexed with PEI before injection as described above. The animals were all sacrificed when the mice became moribund or experiment finished, and the tumors were carefully dissected and snap frozen for RNA, and protein analysis. The statistical significances of tumor volumes and mice body weight of each group were measured by factor analysis. The survival time of tumor bearting mice was measured by Kplan-Meier survival analysis.3. Total RNA was isolated from tumor tissues and the mRNA expression levels of VEGFR2 and EGFR in vivo were determined by two-steps quantitative real time reverse transcription PCR. Proteins were seperated from tumor tissues and analyzed by western blot for the detection of VEGFR2 and EGFR protein expression level. The statistical significances were measured by ANOVA.Results:1. VEGFR2 siRNA sequence screening in vitro.1.1.48hours after transfection, the VEGFR2 mRNA expression level was determined by both semi-quantitative reverse transcription PCR and quantitative real time PCR. The result revealed a profound approximately 60%-75% reduction of VEGFR2 mRNA led by siRNA 2, compared to that of 50%-60% by siRNA 1 or 3, and about 40% by siRNA 4, with all the VEGFR2 mRNA expression levels compared with that of control siRNA-treated cells.1.2. Quantitative real time PCR was used for further validation on the silencing of VEGFR2 mRNA expression (Figure 1C). The result turns to be up to 98%-99% silencing of VEGFR2 by siRNA 2, compared to that of 96%-98% by siRNA 1 or 4, and 85% by siRNA 3.1.3.VEGFR2 protein levels were determined by western blot after 72hours of transfection. The cell transfected with siRNA 2 significantly decreased the protein expression by 85%, compared to that conducted by control siRNA. The reduction of VEGFR2 protein expressed in MS1 cells caused by siRNA 1 and 3 was 55% and 42% respectively. No significant decline was observed in the VEGFR2 protein expression led by siRNA 4. 2. Tumor inhibitor effect of therapies conducted on the human NSCLC tumor xenograft mice model.2.1 Tumor volume and mice body weight were monitered every three days. The mice body weight of the control siRNA treated group was significantly lower than the PEI, saline, and EGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group (all P<0.01). Besides, the body weight of EGFR2 siRNA+EGFR siRNA group was also significantly lower than the saline group(P<0.05). There was significant difference of tumor volumes between the groups(F=57.301,P=0.000). The tumor volume of VGFR2 siRNA group(P<0.01),5mg/kg cisplatin group(P<0.01),EGFR siRNA group (P<0.01),EGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group (P<0.01) were significantly reduced.2.2 The survival time of tumor bearting mice was measured by Kplan-Meier survival analysis. The Log-Rank(Mantel-Cox) (Chi-Square=0.135,P=0.713), Breslow(Generalized Wilcoxon) (Chi-Square=0.400,P=0.527),Tarone-Ware (Chi-Square=0.034,P=0.853) methods were used, and the difference of mean survival time of each group was not significant.3. Determination of the protein expression level of VEGFR2 and EGFR. The VEGFR2 protein expression level of VEGFR2 siRNA treated group showed downregulation, but it is just parallel with that of control siRNA group. However, the EGFR protein expression of this group decreased markedly, compared with that of PEI group, sailine group or control siRNA group (P<0.05). Both the VEGFR2 and EGFR protein expression levels of EGFR siRNA treated group were reduced significantly (P<0.05 and<0.01 respectively), but had no significant difference with that of the VEGFR2 siRNA group. The VEGFR2 and EGFR protein expression of the siVEGFR2+siEGFR treated group were downregulated remarkably, and the levels were significantly lower than that in the VEGFR2 group (both VEGFR2 and EGFR protein, P<0.05) or the EGFR group (P<0.01 for just EGFR protein). Both the two kinds of protein levels of 5mg/kg cisplatin treated group were lowered, and were significantly lower than that in the VEGFR2 siRNA treated group or EGFR siRNA group. No significant difference of the VEGFR2 and EGFR protein expression was observed in both of the 5mg/kg cisplatin group and the siVEGFR2+siEGFR treated group. The VEGFR2 protein expression level of the siVEGFR2+siEGFR+3mg/kg cisplatin treated group is significant reduced compared with PEI or saline group, but fairly even with the control siRNA group. However, the level of VEGFR2 protein of this group was much higher than the 5mg/kg cisplatin group and the siVEGFR2+siEGFR group (P<0.01), and is no significant difference between this group with the VEGFR2 siRNA group or the EGFR siRNA group. The EGFR protein is almost completely knocked down, which could be easily shown in the western blot, and reduced significantly when compared to that in the PEI or saline group, and is lower than the EGFR siRNA group (P<0.01) and the siVEGFR2+siEGFR group (P<0.05). There is no significant difference between this group and the VEGFR2 siRNA treated group or the 5mg/kg cisplatin treated group.4. Determination of the mRNA expression level of VEGFR2 and EGFR. The VEGFR2 mRNA expression levels of VEGFR2 siRNA treated group (P<0.05), EGFR siRNA treated group (P=0.000),5mg/kg cisplatin treated group (P<0.05), VEGFR2 siRNA+EGFR siRNA treated group (P=0.000), VEGFR2 siRNA+EGFR siRNA+3mg/kg cisplatine treated group (P=0.000) were significantly higher than the PEI treated group. The VEGFR2 mRNA expression levels of EGFR siRNA treated group was significantly higher than VEGFR2 siRNA group,5mg/kg cisplatin group,VEGFR2 siRNA+EGFR siRNA group,VEGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group (P=0.000). The VEGFR2 mRNA expression levels of VEGFR2 siRNA+EGFR siRNA group was significantly higher than VEGFR2 siRNAgroup (P＜0.05) and 5mg/kg cisplatin group (P＜0.05). The VEGFR2 mRNA expression levels of VEGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group was significantly higher than VEGFR2 siRNA group (P<0.01) and 5mg/kg cisplatin group(P＜0.01). There was no significant difference between the VEGFR2 siRNA group and 5mg/kg cisplatin group, or between the VEGFR2 siRNA+EGFR siRNA group and VEGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group. The EGFR mRNA expression level of EGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group was significantly lower than the saline group(P<0.05),VGFR2 siRNA group (P<0.05),5mg/kg cisplatin group (P<0.05).Conclusion:1. The reduction of VEGFR2 protein expressed in the MS1 cells as a consequence of gene silencing, to some extent directly reflected the powerfulness of siRNA, with a sequence-dependent manner. Based on the results above, with the robust capability in decreasing both mRNA and protein expression level of VEGFR2 in the MS1 cells, siRNA 2 was chosen for the subsequent experiments of tumor inhibition in vivo.2. There was no significant difference beteen the treatment groups. However, the tumor growth was significantly inhibited by VEGFR2 group,5mg/kg cisplatin group,EGFR siRNA group,EGFR2 siRNA+EGFR siRNA+3mg/kg cisplatin group, and the tumor volume was significantly reduced. The was a significantly reduction in mice body weight of EGFR2 siRNA+EGFR siRNA group and control siRNA group. No significant difference of survival time was observed in the 8 treatment groups.3. The determination of both of the VEGFR2 and EGFR expression level on the mRNA and protein levels revealed that the tumor growth inhibition was mediated by the reduction of VEGFR2 and EGFR protein expression directly or indirectly.4. The combination therapy of VEGFR2 siRNA and EGFR siRNA enhanced the tumor inhibition effct of cisplatin with decreased side effcts and toxicity, and extended the survival time of tumor bearing mice. |
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