The Effect And Mechanism Of Kanglaite Injection In Combination With Gefitinib On Angiogenesis In Mice With Lewis Lung Cancer

Objective:Overall, Lung cancer has the highest morbidity and mortality rates in human malignant tumors. It seems to pose a major threat to the health of all mankind. Lung cancer, especially in non-small cell lung cancer (NSCLC), the treatment is still less than satisfactory. In recent years, with the new molecular targeted drug-gefitinib (Iressa) put into clinical, it brings the dawn for the treatment of NSCLC. As a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), gefitinib has become the first choice in the first-line treatment of advanced NSCLC with EGFR mutation. Nevertheless, Clinical studies has shown that the total efficiency in the treatment of NSCLC is only10%to20%. And the emergence of drug resistance also can not be ignored. Therefore, looking for ways to increase the efficacy of EGFR-TKI, improve or even reverse the resistance of EGFR-TKI drugs for clinical treatment is necessary. KLT injection is a traditional Chinese medicine, made of Yiyiren oil extracted from the Chinese medicine Yiyiren, studies have shown that it has strong killing and inhibition effects to various tumor cells. Several studies have shown that KLT combined with chemotherapy can improve the treatment efficacy and chemotherapy sensitivity of advanced NSCLC significantly; it also can inhibit tumor angiogenesis significantly. Studies have found that the tumor-induced angiogenesis may be a major reason for limiting the efficacy of EGFR-TKI. In this study, According to build the Lewis lung carcer animal models, we apply a series of experimental methods,weigh the average tumor weight of each group, calculate the inhibitory rate and tumor microvascular density (MVD), measure the mRNA and protein expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2(VEGFR-2/ kinase-insert domain-containing receptor, KDR), to explore the effect and mechanism of KLT in combination with Gefitinib on angiogenesis in mice with Lewis lung cancer.Methods:1Animals and treatment:Cultured Lewis lung carcer (LLC) cells in vitro, selected the logarithmic growth phase cells.40heathy male C57BL/6mice were randomized into4groups:control group, KLT treatment group (1.67g/kg), gefitinib treatment group (45mg/kg), KLT (1.67g/kg)+gefitinib (45mg/kg) treatment group. The cells were inoculated into the right upper limb axillary of each mouse by Subcutaneous way to establish model.2Evaluation of tumor inhibiting effects:Then examined the growth of each tumor ever other day. The7th day after inoculation, tumor could be touthed. When in the12th day, the tumors became to about1.0cm×1.0cm×1.0cm on average, the drugs started to be used in animals. KLT treatment group was injected with KLT of1.67g/kg/d (0.3ml/20g) by tail vein for two weeks; gefitinib treatment group was given gefitinib of45mg/kg/d (0.1ml/10g) by intragastric administration for two weeks; KLT+gefitinib treatment group was administered as described above; model group was given normal saline and5%glucose, the dose and methods was like KLT+gefitinib treatment group. All mice were sacrificed24hours later, subcutaneous tumors were processed for tumor weight and tumor inhibition rate of each group were calculated.3Immunohistochemistry (IHC)image analysis was performed for detecting vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2(VEGFR-2/kinase-insert domain-containing receptor, KDR) and microvessel density (MVD).4Semi-quantitative RT-PCR was used to detect the expression of VEGF and KDR mRNA in the transplanted tumor.Results:1The average tumor weight and tumor inhibition rate:The average weight in KLT treatment group and gefitinib treatment group were (1.20±0.15) g and (1.25±0.11) g, lower than control group (2.13±0.17) g (P<0.01). KLT+gefitinib treatment group were (0.48±0.07) g, much lower than control group too (P<0.01). The average weight in KLT+gefitinib treatment group was lower than KLT treatment group and gefitinib treatment group, the difference had statistically significant (P<0.01). But between the two single drug groups, there was no significant difference(P>0.05). Compared with the control group, KLT treatment group, gefitinib treatment group, and KLT+gefitinib treatment group can inhibit transplanted tumor, especially the combination group, the inhibition rates in the three group were43.77%,41.53%,77.69%respectively. The q value is equal to1.16, it indicates that the two drugs have a synergistic effect.2Immunohistochemical results:The MVD positive labeling index (LI) in control treatment group, KLT treatment group, gefitinib treatment group and the combination group were:24.345±1.06,18.247±1.36,20.089±1.46,14.459±0.98. The two single drug group was lower than control treatment group significantly, the difference had statistically significant (P<0.01); combination therapy group was lower than the two single drug group, the difference also had statistically significant (P<0.01); But between the two single drug groups, there was no significant difference (P>0.05). The expression level of VEGF and KDR protein in KLT treatment group and gefitinib treatment group were lower than the control group (P<0.05); combination therapy group was lower than the two single drug group, the difference also had statistically significant (P <0.05);3RT-PCR results:The expression level of VEGF and KDR mRNA in KLT treatment group and gefitinib treatment group were lower than the control group (P<0.05); The expression level of VEGF and KDR mRNA in KLT+gefitinib treatment group was more lowered than the control group (P <0.05); combination therapy group was lower than the two single drug group, the difference also had statistically significant (P<0.05).Conclusions:1KLT and gefitinib have inhibitory effect on mice with Lewis lung tissue, and KLT combined with gefitinib can enhance this inhibitory effect.2KLT and gefitinib can inhibit tumor angiogenesis, the tumor angiogenesis inhibition effect in combination group is more pronounced.3The down-regulation of the expression of VEGF and KDR may be one of the mechanisms for the combination of two drugs inhibit tumor angiogenesis.