The Mechanism Of Lithium-induced Growth Inhibition,Migration Suppression, Apoptosis Promotion In Tumor Cells And FAK’s Influence On Growth, Migration In B16F10Cells

Lithium is a drug for mental illness and used in clinics for many years, clinical investigations show that it can reduce the risk of malignant tumors. Recent studies reveal that lithium inhibits growth of tumor cells by regulating their cell cycle, lithium induced G2/M transition block in various tumor cells including colon cancer cells (Caco2), thyroid cancer, leukemia cells (HL-60), hepatoma cells (SMMC-7721), esophageal cells (Eca109), glioma cells, and the embryonal carcinoma cells (P19), while inducing S phase arrest in prostate cancer cells and non-small cell lung cancer cells. In addition, lithium can inhibit the migration of glioma, promote apoptosis of HL-60and pancreatic cancer cells. Moreover, studies have shown that lithium plus TNFa synergistically enhanced apoptosis of fibrosarcoma (L929) and rhabdomyosarcoma (KYM). Lithium influences the proliferation, migration and apoptosis of various tumor cells, but the mechanisms underlying its actions remain unclear. In the present study, we try to elucidate the influence of cell growth and migration by LiCl in B16F10cells, and the synergetic apoptotic effect of LiCl combination with TRAIL on A549cells.First, LiCl inhibited the growth of B16F10cells both in vitro and in vivo. Cell cycle profiles by Flow cytometry showed that LiCl induced G2/M arrest in B16F10cells; we further studied the underlying mechanisms. Results showed that LiCl inhibited the activity of cyclinB1/cdc2complex through increasing the protein expression of p-cdc2(Tyrl5), p53, p21; upregulation the transcriptional expression of several G2phase-related genes (p53, p21, gadd45and14-3-3); and reducing the expression of cdc25c both in translational and transcriptional level. In addition, data showed that LiCl activated ERK, and inhibited JNK. Furthermore, the ERK inhibitor U0126can moderately reverse the G2/M arrest caused by LiCl, and the JNK inhibitor SP600125can induce G2/M arrest as LiCl does, the results revealed that ERK and JNK at least partly involved in the G2/M arrest induced by LiCl. Further studies showed that LiCl caused DNA damage, activated DNA damage repair system by upregulation expression of several key genes (MRE11, RAD50, Ku70, Ku80and MLH1), and the inhibitors of ATM/ATR-Chkl pathway can abrogate G2/M arrest effect of LiCl. On the other hand, GSK3β and the IMPase are two important intracellular targets of lithium. Although LiCl can inhibit the activity of GSK30effectively, another GSK30inhibitor SB216763and GSK30gene silencing failed to mimic the action of LiCl. It is reported that lithium can decrease the intracellular inositol level by inhibiting IMPase, but in our research, inositol supplementation does not influence the cell cycle distribution. In addition, LiCl also activated P38, and increased the reactive oxygen in B16F10cells, but the P38inhibitor SB203580and ROS antagonist GSH didn’t affect the cycle arrest caused by LiCl. Therefore, GSK30, IMPase, ROS and P38MAPK were not involved in LiCl-induced G2/M arrest.Next, wound healing assay and Transwell cell migration assay showed that LiCl inhibited the migration of B16F10cells in vitro; LiCl administration significantly suppressed pulmonary metastasis of B16F10cells in the experimental passive metastasis model in vivo. Furthermore, our preliminary results showed that LiCl decreased the expression of matrix metalloproteinase MMP2, and increased the expression of several important metastasis suppressor genes (nm23, KAI1, Kissl, Timp3, PTEN) related to melanoma at transcriptional level. In addition, LiCl can also downregulate the metastasis promoter miR-21and upregulate the metastasis inhibitors miR-31and miR-34a.Finally, TRAIL is considered as a new anticancer drug with high efficiency and low toxicity in recent years, however, many tumor cells are resistant to TRAIL, so the combination therapy has become a new strategy. We found LiCl combination with TRAIL exhibited a striking synergic anti-tumor effect both in vitro and in vivo. We next studied the mechanism of their synergic effect. Results showed that LiCl increased the expression of death receptors DR4and DR5, but decreased the transcription of anti-apoptotic genes FLIPs, Bcl-2, Bcl-xl and Surivin. Combined treatment with TRAIL and LiCl enhanced apoptosis by activated Caspase-3, Caspase-8, Caspase-9and cleavage of PARP, Bid. Moreover, LiCl induced G2/M arrest of A549cells through inhibition of JNK and activation of ATM/ATR-Chkl pathway, which increased the sensitivity of TRAIL on A549cells. However, although LiCl can activate p38and inhibit GSK30in A549cells, our results ruled out the participation of ERK, p38, GSK3P and IMPase in apoptosis induced by co-treatment with TRAIL and LiCl.In summary, the mechanisms of the influence on tumor cells proliferation, migration and apoptosis by LiCl is as follows:(1) LiCl increased expression of p53、p21、gadd45、14-3-3and decreased the expression of cdc25c through activation of ERK、ATM/ATR-Chk1pathway, and inhibition of JNK pathway. As a result, they together inhibited the activity of cyclinB1/cdc2complex, induced G2/M arrest, and inhibited the growth of B16F10cells.(2) LiCl inhibited migration of B16F10cells possibly through downregulation of MMP2, upregulation of several metastasis suppressor genes and influence on expression of microRNA.(3) LiCl combination with TRAIL synergistically enhanced apoptosis of A549cells via upregulation of death receptors DR4and DR5, downregulation of the anti-apoptotic protein genes transcription, and activation of the caspases cascades. In addition, LiCl induced G2/M arrest by inhibition of JNK and activation of Chkl, and then increased the sensitivity of TRAIL on A549cells. In short, it will have important theoretical significance and potential applications values to clarify the anti-tumor effects and mechanisms of LiCl.Focal-adhesion kinase (FAK) is a non-receptor protein tyrosine kinase which is localized to the cytoplasmic side of focal adhesion and plays an important role in survival, proliferation, migration of tumor cells. More and more evidences indicated that overexpression of FAK is common in a variety of malignant tumors, such as that the expression of the FAK increase along the malignant degree of melanoma. The other part of the study focused on how the stable interference of FAK can influence the survival and migration of B16F10cells. Results indicated that the stable interference of FAK slowed the growth of B16F10cells and reduced its migration ability both in vitro and in vivo. Further study found that the stable interference of FAK can inhibit the activation of ERK, reduce transcription of NCOA3and upregulate the mRNA level of CCDC80. Besides, overexpression of CCDC80can inhibit the migration of B16F10cells and NCI-H446cells. These results suggest that FAK may influence expression of NCOA3through ERK pathway, which in turn affect expression of CCDC80, and finally regulate the survival and migration of B16F10cells.