Study On Mechanism Of Lung Cancer Metastasis And Lung Cancer In Preventing Lung Metastasis By Lung Cancer

Background:Tumor metastasis is the main cause which induces most of cancer patients’ curative failure and death. Some researches indicate that tumor microenvironment can change the potential of tumor metastasis. In tumor prevention and treatment, microcosmic integral regulation is attached importance to. Tumor microenvironment refers to the tumor and its external environment, which is a complex dynamic network. On the basis of tumor entity, tumor microenvironment can be divided into tumor core microenvironment, tumor invasion microenvironment, and tumor metastasis microenvironment. In the past, Chinese medical researches always focused on tumor itself (tumor core microenvironment), but ignored non-cancer components (tumor metastatic microenvironment). Theoretically, integral regulation of tumor microenvironment needs to be broad-spectrum, bidirectional, low-toxic, and highly-effective. Traditional Chinese medicine satisfies these principles, which is in single or combined use for tumor prevention and treatment. Clinical practice and researches show that long-term use of Chinese medicine has the advantage of tumor metastasis prevention. According to recent studies, drug combination therapy is preferred due to increase in the efficacy. Anti-inflammatory drugs are also used for prevention and combination therapy, because they have fewer side effects and spend less than targeting drugs and chemotherapeutic drugs. Nevertheless, it is rarely reported that the combined use of Chinese medicine and anti-inflammatory drugs applies to tumor metastasis prevention in experimental researches.On the basis, this study researched molecular mechanisms of Chinese medical effects on lung metastasis microenvironment of tumor bearing mice (tumor bearing mice’s lung tissue). Fei-liu-ping ointment (FLP), Celecoxib (CLB), and cyclophosphamide all have clear anti-tumor effects, which were chose in the experiment.Purpose:Experiment in vivo of FLP and combination of FLP and different types of drugs (chemotherapy drugs, anti-inflammatory drugs) is proceeded, especially the molecular mechanisms of tumor metastasis microenvironment regulation by FLP combined with anti-inflammatory drugs.Methods:(1) Transplanted tumor model of Lewis lung cancer in C57BL/6 mice was established. Small animal in vivo imaging technology and regular sampling methods were used, to observe the effects of FLP and combination of FLP with different types of drugs on transplanted tumor, together with time and dose dependent lung metastasis.(2) Through gene chip technology, key genes that FLP effects on in tumor metastasis microenvironment were screened, and thus the gene signal transduction network was constructed.(3) The key genes screened from step (2) in tumor metastasis microenvironment were verified by Western blot and immunohistochemistry methods. The effects of FLP combined with different types of drugs on key molecular expression of protein in different stages (14d,21d,28d) were studied, including the mediated downstream genes and metastasis-associated molecules.(4) According to the result of step (3), through Western blot and immunohistochemistry methods, molecular mechanisms of lung metastasis prevention by FLP combined with CLB in the COX-2 mediated tumor metastasis microenvironment were studied.Results:(1) Small animal in vivo imaging showed that the tumors of mice p/sec/cm2/sr were inhibited significantly in FLP-L group and FLP-M group (P<0.05), in addition, FLP-M group having the most significant anti-tumor effect compared with other groups. The inhibitory effect on tumors of mice p/sec/cm2/sr was not significant in FLP-H group (P>0.05). When FLP was combined with different types of drugs, in vivo imaging on the 7d,14d,21d,28d showed a synergistic or additive inhibitory effect on tumors of mice p/sec/cm2/sr in FLP+CTX group and FLP+CLB group (P<0.05). On the 7d, there was no significant difference in inhibition of tumors of mice p/sec/cm2/sr between FLP+CLB group and CLB group (P>0.05).(2) Tumor inhibition rate:mice were regularly sacrificed on the 14d,21d,28d, and the tumors were sampled and weighed for tumor inhibition rate calculation. On the 14d, 21d,28d, tumor inhibition rates of FLP-L group, FLP-L group, FLP-M group and FLP-H group were:4.9%, 12.7%, 6.9%; 28.1%, 37.9%, 24.6%; and 14.7%, 24.6%, 13.5% respectively. On the 14d, 21d, 28d, the tumor inhibition rates of FLP+CTX group and FLP+CLB group were:42.2%,62.3%; 79.6%, 58.7%; and 67.7%,29.8%.(3) Lung metastasis:the result suggested that lung metastasis did not occur in each group on the 14d. On the 21d, micrometastases began to form, accompanied by a large number of inflammatory cells infiltration. On the 28d, metastases became larger, and the internal turned to be consolidation with a large amount of inflammatory cells infiltration. On the 21d and 28d, the incidence of lung metastasis in Control group, FLP-M group,FLP+CTX group, and FLP+CLB group were 50%,30%; 20%,20%; 80%,50%; and 20%, 20%. Lung metastases total in Control group, FLP-M group, FLP+CTX group, and FLP+CLB group were 23,15; 7,8; 36,19; 11,8. The inhibition rates of lung metastasis were 34.7%,69.6%,65.2%; 47.2%,69.4%,69.4%. From the analysis of the inhibition rate of lung metastasis, FLP-M group achieved the greatest improvement among all groups. FLP-M group:CTX group:CLB group=12.5%:9.4%:11.3%.(4) 1068 differential genes were screened by gene expression profile chip, including 713 down-regulated genes,355 up-regulated genes. There were 67 genes different more than 2 times among down-regulated genes, while no gene differed by over 2 times was screened among up-regulated genes. The significant accurate signal pathways were found through differential genes, among which NF-kappaB signaling pathway and PI3K-Akt signaling pathway were concerned in this experiment. From study on the signal transduction network constructed by 104 key genes, we could see genes Prkca, Mapk10, Pik3r2, Plcdl, and Nfkb1 had strong intermediary ability, which held a key node position in the whole Signal-Net.(5) The expression of key regulatory factor PI3K/AKT/NF-kB was studied in the tumor metastasis microenvironment. Immunohistochemistry results showed that in lung metastasis microenvironment, PI3K, AKT, and NF-kB expression increased within time in Control group, but in contrast, the PI3K, AKT, and NF-kB expression decreased in treatment group. Western blot results showed that on the 14d (before metastasis microenvironment), the expression of PI3K, AKT, NF-I3B was inhibited in FLP group, but the difference was not significant (P>0.05). On the other hand, the expression of AKT, NF-kB could be significantly inhibited in FLP+CLB group (P<0.05). On the 21d (metastasis microenvironment), the expression of PI3K protein was inhibited significantly in FLP group (P<0.05), and moreover, the expression of PI3K, AKT, NF-and NF-kB protein was inhibited in FLP+CLB group with a significant difference (P<0.05). On the 28d (metastasis microenvironment), the inhibitory effect on PI3K, AKT, NF-kB expression was not found in FLP group (P>0.05), while the inhibition of PI3K and AKT protein expression was significant in FLP+CLB group (P<0.05).(6) We studied regulatory effect of FLP and FLP combined with CLB on lung metastasis microenvironment mediated by COX-2 to prevent lung metastasis. Western blot results showed that in lung metastasis microenvironment, the expression of COX-2 could be inhibited significantly in FLP+CLB group on the 14d (P<0.05). On the 21d and 28d, the expression of COX-2 could be inhibited in both FLP group and FLP+CLB group with significant difference (P<0.05). On the 28d, inhibitory effect on the VEGF and PDGFR-βexpression was showed in FLP group, and the E-Cadherin protein expression was up-regulated in FLP group, but there was no significant difference (P>0.05). N-Cadherin, Vimentin, and MMP-9 protein could be inhibited significantly in FLP group (P<0.05). In terms of E-Cadherin expression up-regulation, and inhibition of VEGF, PDGFR-β, N-Cadherin, Vimentin, MMP-2, MMP-9 expression, FLP+CLB group was superior to FLP single group and CLB single group with significant difference (P<0.05).Conclusions:(1) The middle dose of FLP is optimal for tumor inhibition rate, and the inhibition rate of lung metastasis by FLP improves with the time. In addition, combined use of FLP and CLB has the advantage of inhibiting lung metastasis.(2) FLP involved in the regulation of down-regulated genes in lung metastasis microenvironment has advantages, which down-regulates Prkca, Mapk10, Pik3r2, Plcdl, and Nfkbl as the core, in order to regulate the balance of the whole gene network.(3) The combination of FLP and CLB can markedly inhibit PI3K-AKT-NF-kB-COX-2 molecules in lung metastasis microenvironment, and it may be in a time dependent manner.(4) The combination of FLP and CLB can regulate EMT, angiogenesis, ECM, and metastasis-associated molecules coordinately, which has the advantage of metastasis inhibition.