| Objective: The mortality of lung cancer ranks first in all malignant tumors, and the morbidity is still increasing. Platinum-based chemotherapy has a major role in the treatment of lung cancer, but was limited by its adverse effects. Clinical practices show that Kanglaite(KLT) can cause synergistic antitumor effects, reduces adverse events and improve quality of life when combined with chemotherapy. Tumor microenvironment is becoming a intense study recently. Tumor associated macrophages(TAM) are the most inflammatory cells in tumor immune microenvironment and hypoxia inducible factor-1 alpha(HIF-1α) is one of the key factor in tumor hypoxia microenvironment. The aim of the research is to study the impact of KLT coupled with cisplatin on the growth of tumor, immunity, TAM and HIF-1α, and the relationship between TAM and HIF-1α in tumor tissues by establishing Kunming mice with Lewis lung carcinoma,in order to investigate the mechanism of synergistic effects, reducd toxicity and developed immunity, and the interaction between immunity and hypoxia in tumor microenvironment.Methods: 1 Lewis lung carcinoma cells were cultured in vitro and were subcutaneously injected in the left axilla of each mouse when they reached senescence at passages 3-4, in order to establish mouse models with Lewis lung carcinoma. General condition of the mice was observed and recorded. 24 Lewis lung carcinoma mice with similar tumor sizes were selected and randomly divided into four groups of 6 animals in each: the control group(control), KLT group(KLT), cisplatin group(DDP) and combination-therapy group(DDP+KLT) when the maximum diameter of the tumors reached 0.8-1cm. Then all of the mice were injected peritoneally with corresponding drugs for 14 day: saline(12.5 m L/kg NS) and NS(12.5 m L/kg NS), KLT(12.5 m L/kg KLT) and NS(12.5m L/kg NS), NS(12.5m L/kg NS) and DDP(1mg/Kg DDP), KLT(12.5 m L/kg KLT) and DDP(1mg/Kg DDP), with 12 h intervals between two injections every day. 2 The mice were then sacrificed in the 15 th day(12 hours after the last injection) and the tumors were harvested. The tumor, the spleen and the body weight were measured and the tumor growth inhibition rate and the spleen index of each group were calculated. 3 The percentage of TAM in tumor tissues of every mouse was determined by flow cytometry(FCM). 4 The expression of HIF-1α m RNA in tumor tissues of every mouse was detected by realtime-PCR. 5 Statistical calculations were performed using SPSS 13.0 for Windows software. All values are expressed as means±SD. Single-factor analysis of variance was performed to evaluate the differences within groups and LSD test for multiple comparisons between groups. Bivariate Pearson correlation was employed to explore the association between TAM and the expression of HIF-1α m RNA. A P-value<0.05 indicates a significant difference.Results:1 General condition of the mice: Lewis lung carcinoma models were established 6-7 days after subcutaneous injection of Lewis lung carcinoma cells to Kunming mice. The maximum diameter of the tumors reached 0.8-1.0cm 10 days after the injection. Reduced diet and activity, lustreless hair were observed in the mice, which is the most obvious in the control group. 2 Antitumor effects of each group: The mean tumor weight of the control group, KLT group, DDP group and DDP+KLT group was(2.1517±0.29936) g,(1.1217±0.22798) g,(0.8867±0.18694) g and(0.7050±0.23227) g. The tumor weight of the control group was higher than the other three groups, with statistical significance(P<0.01). There was no significant difference between the tumor weight of KLT group, DDP+KLT group and that of DDP group(P=0.105, 0.205). 3 Spleen index in the groups: Spleen index was calculated as splenic weight divided by body weight. The spleen index in the control group, KLT group, DDP group and DDP+KLT group was 7.31±1.50, 9.70±1.53, 4.58±1.29 and 6.54±1.68. The spleen index was higher in the control group, KLT group and DDP+KLT group than in the DDP group, with statistical significance(P=0.005, 0.000, 0.036<0.05). There’s no significant difference between the spleen index of the control group and that of DDP+KLT group(P=0.385), which was higher than the DDP group and lower than the KLT group, with statistical significance(P<0.05). 4 Percentage of CD11b+F4/80+ cells in tumor tissues of each group: Compared with the percentage of CD11b+F4/80+ cells in tumor tissues of the control group, that of the KLT group and DDP+KLT group was lower, both with statistical significance(P=0.020, 0.013<0.05), and that of the DDP group was higher, without statistical significance(P=0.898). The prcentage of CD11b+F4/80+ cells in tumor tissues of the DDP+KLT group was significantly lower than that of the DDP group(P=0.009<0.01). 5 Expression of HIF-1α m RNA in mouse tumor tissues of each group: The expressions of HIF-1α m RNA in tumor tissues of the KLT, DDP and DDP+KLT group were lower than that of the control group, and the difference was statistically significant(P=0.002, 0.011, 0.000<0.05). HIF-1α m RNA expression in tumor tissues of the DDP+KLT group was lower than the KLT group and DDP group, with statistical significance(P=0.039, 0.009). There was no difference in expression of HIF-1α m RNA between the DDP group and KLT group(P=0.518). 6 The correlation between TAM and the expression of HIF-1α m RNA in mouse tumor tissues: There was no bivariate correlation between TAM and the expression of HIF-1α m RNA in all of the four groups(P=0.545,0.332,0.751,0.776>0.05).Conclusions:1 KLT itself can inhibit the tumor growth, enhance the immune function in mice with Lewis lung carcinoma. KLT can also enhance the antitumor effect and reduce the immune suppression of chemotherapy when combined with DDP. 2 DDP inhibit the tumor growth of mice with Lewis lung carcinoma effectively, but it also decreases body immune function meanwhile. KLT relieve the immune suppression caused by chemotherapy, which may be related to the inhibition of TAM. 3 KLT, DDP can improve the hypoxic status of mice with Lewis lung, and the effect of combined application is superior to either drug alone. Its antitumor and synergistic effects on chemotherapy may be accomplished by downregulating the HIF-1α m RNA level and improving hypoxia status. High expression of HIF-1α is correlated with tumor development. |
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