Mechanism Studying Of The Effects Of Ni2+on Human Lung Cancer Cell Growth And Invasion

Nickle compounds are the human carcinogens according to the report from the International Cancer Research Center published in2009. Recently, both a lot of epidemiological and animal studies suggest a significant correlation among exposure to Ni2+, increased risk of lung cancer and tumorigenesis. However, the effect of nickle compounds on lung cancer development and lung cancer cell invasion remains unclear. Therefore, study on the effects of nickle compounds on the tumorigenesis of lung cancer will be of great importance in prevention and treatment of lung cancer.Toll-like receptor4(TLR4) is becoming a hot topic in the field of cancer research. It has been reported that TLR4is involved in tumor invasion, metastasis, relapse, drug resistance, and immune escape. Recent studies have demonstrated the increased expression of TLR4at both mRNA and protein levels in lung cancer tissues compared with normal tissues. In addition, TLR4expression level is positively correlated with the malignancy degree of lung cancer. A recent report suggested that nickle compounds triggers inflammatory response of cells through directly activating TLR4. Inflammation may induce tumorigenesis. But it is still unknown whether nickle compounds can promote lung cancer growth and invasion.In this project, using water nickel chloride (NiCl2) as the research factors, using human non-small cell lung cancer cell lines A549and H1299as the research object, we discuss the lung cancer cell invasion and its related mechanisms by two valent nickel (Ni2+).First, we treated the human non-small cell lung cancer cell lines A549and H1299with Ni2+, checked the number of invasive cells by invasion assays in vitro, and tested of the effect of Ni2+on lung cancer cell growth and invasion. Second, by using ELISA, we examined the levels of cell secreted interleukin-8(IL-8) and tumor growth factor beta (TGF-beta) which may be induced by Ni2+. Third, after48h's treatment of A549and H1299cells with Ni2+, we tested if Ni2+is able to affect the TLR4pathway by examining the protein levels of the TLR4pathway components, such as matrix metalloproteinase-2(MMP-2), matrix-mefalloproteinase-9(MMP-9), myeloid differe ntiation factor88(MyD88), Toll-like receptor interferon activation of the expression changes of TRIF, interleukin-1receptor-associated kinase4(IRAK4), tumor necrosis factor receptor associated factor6(TRAF6), nuclear factor kappa B (NF-kappa B) and activation protein1(AP-1). Fourth, we tested the cancer cell invasion ability after knocking down TLR4and/or MyD88in A549and H1299cells. We also investigated the roles of p38MAPK and NF-κB in Ni2+-induced non-small cell lung cancer invasion by treating the A549and H1299cells with p38MAPK-specific inhibitor SB203580, NF-κB inhibitor PDTC and JNK inhibitor1, respectively.This study includes the following four parts.Part Ⅰ Effects of Ni2+on human lung cancer invasionIn this section, we used A549and H1299as the cell model to study the Ni2+induced lung cancer invasion. We tested cancer cell invasion ability using Transwell assays after treating the cells with different dose of Ni2+. On the other hand, we tested the levels of cell secreted IL-8and TGF-β through ELISA assays after treating the cells with different dose of Ni2+. It was reported that the level of matrix metalloproteinases is related to cancer invasion. So the protein and mRNA levels of MMP-2and MMP-9were examined in A549and H1299cells before and after treatment with different dose of Ni2+. Through these experiments, we clarified the function of Ni2+-induced non-small cell lung cancer invasionThe results showed that following Ni2+treatment, the number of A549and H1299cells which went through the artificial matrix membrane was significantly increased. The levels of cancer cell secreted IL-8and TGF-β were also significantly increased after treatment with Ni2+. So did the expression levels of MMP-2and MMP-9. The study of Ni2+affecting human lung cancer cell proliferation indicated that continuous treatment of the A549and H1299cells with2mM of Ni2+for48h had the greatest impact on the ability of cell proliferation.Our findings demonstrated that Ni2+treatment of lung cancer cells is able to promote cell invasion, induce cellular secretion of IL-8and TGF-β, and increase the expression of MMP-2and MMP-9. All these factors can help cancer cells to digest the artificial matrix membrane, which suggested Ni2+may induce lung cancer invasion by regulating the secretion of IL-8, TGF-(3and expression of MMP-2and MMP-9.Part II Ni2+targeting TLR4signaling pathway in lung cancerIn this section, we tested if Ni2+can target TLR4signaling pathway in human lung cancer. First, through flow cytometry analysis, we examined expression induction of TLR4by Ni2+in A549and H1299cells. Furthermore, we examined the TLR4mRNA level to further verify the results by using quantitative RT-PCR. TRIF and MyD88are two most important downstream factors of TLR4which control two different signal transduction pathways.Through Western Blot and quantitative RT-PCR assays, we examined the protein and mRNA levels of TRIF and MyD88following Ni2+treatment. In addition, we checked the downstream genes of MyD88, such as IRAK4, TRAF6, NF-κB and AP-1.The results showed that in lung cancer cell lines A549and H1299, Ni2+treatment did not affect the expression of TLR4, but significantly increased the transcriptional level of MyD88. These results suggest that Ni2+may specifically activate MyD88-dependent signaling pathway but independent of TRIF. The analysis on the dowmstream gene of MyD88, such as IRAK4, TRAF6, NF-κB and AP-1, gave consistent results. These results suggest in A549and H1299cells, both the mRNA and protein levels of TLR4are not changed after Ni2+treatment, so do the gene TRIF, a downstream factor of TLR4. Interestingly, MyD88, another downstream gene of TLR4, and its downstream factors including IRAK4, TRAF6, NF-κB and AP-1were significantly increased following Ni2+treatment. These findings further confirmed our hypothesis that Ni2+can upregulate TLR4-MyD88pathway in non-small cell lung cancer.Part Ⅲ Ni2+promotes lung cancer invasion through TLR4/MyD88pathwayIn this section, we focused on the relationship between TLR4/MyD88pathway and cancer invasion. First, we knocked down TLR4and MyD88in lung cancer cells using siRNAs, and tested the knockdown efficiency by quantitative RT-PCR. Following the knocking-down, we treated the cells with Ni2+and tested the cell invasion. The results suggested that the knockdown efficiency of TLR4and MyD88in cells were very high. The Transwell results showed the Ni2+-induced cancer cell invasion was significantly decreased in TLR4or MyD88knockdown cells. It indicated that Ni2+induces non-small cell lung cancer invasion through regulating TLR4/MyD88pathway. In cells without TLR4and MyD88expression, Ni2+can not induce cancer invasion, suggesting TLR4/MyD88pathway is required for lung cancer invasion induced by Ni2+.Part Ⅳ p38MAPK pathway and NF-κB pathway are necessary for Ni2+induced lung cancer invasionThe first three sections have demonstrated that Ni2+is able to enhance lung cancer invasion, which is dependent on TLR4/MyD88signaling pathway. In this section, we studied the downstream genes of MyD88and tried to find any specific target. NF-κB and MAPK are two important downstream pathways of MyD88. We treated the lung cancer cell with specific inhibitors targetng these two pathway and checked effects of Ni2+on cancer cell invasion. After treating cells with specific inhibtors to p38MAPK and NF-κB, the Ni2+-induced cancer cell invasion was significantly decreased. However, JNK inhibitor didn't block Ni2+-induced cancer cell invasion. The results suggested that Ni2+induces lung cancer invasion through TRL4/MyD88/p38MAPK/NF-κB pathway.