Function And Mechanisms Of Phospholipase Cγ1 In Cell Migration And Cell-matrix Adhesion In Colorectal Cancer Cells

Cancer progression is multi-step process.In order for a tumor to invade a tissue, it must first recognize and interact with the surrounding extracellular matrix (ECM). Secondly, tumor cells must degrade or remodel the surrounding ECM in order to migrate through the dissolved ECM to reach adjacent tissue. There are multiple cell properties, such as cell adhesion and migration, proteinases production, angiogenesis, and their controlling signaling pathways, affecting tumor behaviors and could be targeted for clinical therapeution.Colorectal carcinoma is one of the most common human tumors. Recently, the morbidity and the mortality of colorectal cancer increase worldwide. It is the second to the fourth most common cancer in industrialized countries. Nearly 50% of patients diagnosed with colorectal cancer develop metastasis within five years.Most deaths by colorectal cancer are due to invasion and distant metastasis. It is important to investigate the influence factors and the mechanisms of invasion and metastasis in colorectal cancer, which would contribute to identify the potential target for therapeutic intervention and anticancer drug development.Phospholipase C (PLC), a very important phosphoinositide-specific isozyme, can catalyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and results in the generation of two intracellular messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(l,4,5)P3]. These messengers then promote the activation of protein kinase C and the release of Ca~2+ from intracellular stores, respectively, and initiate a set of signaling cascades, which are involved in cell motility, development,cancer invasion and other processes.The PLC family comprises a diverse group of enzymes that differ in structure and tissue distribution. Until recently, 11 mammalian PLC isozymes had been identified and divided into four types: (3(1 - 4), y(l, 2), 8(1-4) and e(l). Two types of mammalian PLCy have been identified: PLCyl, which is ubiquitously expressed and PLCy2, whose pattern of expression, although widespread, is highest in cells of hematopoeitic origin.Phospholipase Cyl(PLCyl), is known to be activated in response to growth factor stimulation by a mechanism that relies on tyrosine phosphorylation and plays an important role in regulating cell behaviors. PLCyl was found increased in many cancer tissues and cancer cell lines and associated with tumor progression. It was reported that PLCyl may function as a key molecular switch in cancer motility.But, the signal transduction mechanisms of its role in cancer invasion and metastasis remains uncertain.Till now, the mechanisms that govern tumor cell invasion in colorectal cancer are incompletely understood. The relationship between cell-matrix adhension /cell motility with colorectal cancer invasion and metastasis is still to be elucidated. Also, little information is available concerning the role of PLCyl in colorectal cancer. From this point, colorectal cancer cell lines were used in this study to investigate function and mechanisms of phospholipase Cyl in cell-matrix adhesion and cell migration in colorectal cancer in order to provide the theoretical and experimental base for improving therapeutic approaches.In this study, firstly, with molecular clone technique, eukaryotic expression vectors for full length encoding region of human PLCyl cDNA and PLCz (dominant negative fragment of PLCyl) were constructed. After identification and packaging, the recombinant vectors were transfected into human colorectal cancer cell line LoVo. The expression of PLCyl and PLCz were studied at the RNA and protein level. Secondly, U73122, a specific inhibitor of PLC was used to study the effect of PLCyl on cell-matrix adhesion and cell migration in colorectal cancer cells. Finally, Western blot, gel electrophoresis mobility shift assay (EMSA), immunocytochemistry, zymography and RT-PCR were performed to invistigate the signal transduction mechanisms of PLCyl in colorectal cancer cells.The main results and conclusions are as follows:1. Recombinant eukaryotic expression plasmids pLNCX2/PLCyl and pLNCX2/PLCz were constructed successfully. The recombinant plasmids were indentified by PCR, restriction endonuclease maping and sequencing. Then retrovirus vectors were transducted into package cells PT67 for virus production using Lipofectamine? 2000 and then LoVo cells were infected with virus. RT-PCR and Western blot verfied that the PLCyl and PLCz can express in LoVo cells after transfected. This laid sound basis for the following research of PLCyl.2. PLCyl can regulate cell-matrix adhesion and cell migration in highly metastatic colorectal cancer cells. Cell-matrix adhesion assay showed that highly metastatic colorectal cancer cell LoVo displayed the dose-dependent decrease after treatment with different doses of U73122 (1 |J,M, 2.5 jxM, 5 |J,M, 10 fxM). Compared with control group,OD450 decreased from 1.102 ±0.135 to 0.773 + 0.154, 0.568±0.128, 0.504±0.063, 0.282+0.049, respectively (P < 0.05).But, in lowly metastatic colorectal cancer cell SW480, inhibition of PLCyl by U73122 had no significantly effect on cell-matrix adhesion, indicating that PLCyl plays different role in different cell lines. So, highly metastatic colorectal cancer cell line LoVo was chosen to be used in subsequent experiments.Similarly, pretreatment of LoVo cells with U73122(1 uM, 2.5 \iM, 5 jxM) decreased the cell migration significantly in a dose-dependent manner (from 100% to 88.49 + 2.68%, 69.91 +0.97%, 41.62+3.59%, respectively) (P < 0.05).3. EGF, as an upstream molecule, regulates cell migration and cell-matrix adhesion through PLCyl. It was shown that increased EGF receptor expression correlated with tumorigenic progression of colon cell lines in vitro and liver metastasis of colon tumors in v/vo.Here, in LoVo cells, EGF treatment resulted in increased phosphorylation of PLCyl in a dose-dependent manner. 20 nM EGF can result in the most significant increase in the level of phosphorylation of PLCyl. So, 20nM EGF was used to treat cells later. Additionally, it was noted that cell migration and cell-matrix adhesion were enhanced dramatically in the presence of EGF. And, the inhibition effect of U73122 could be reversed partially by EGF. The results indicated that the EGFR-PLCyl signaling pathway was active in the cells.4. NF-kB lies downstream of EGFR-PLCyl signaling pathway. Previous reportsshowed that a variety of human tumors and tumor cell lines exhibited enhanced NF-kB activity, including colorectal cancer tissues and highly metastatic colorectal tumour cell lines. Treated with various doses of PDTC (50 |iM, 100 uM, 150 |iM, 200 (AM), a NF-kB inhibitor, cell-matrix adhesion was decreased in a dose-dependent manner. Compared with control group,OD450 decreased from 2.148 + 0.094 to 1.587±0.328, 1.312 + 0.192, 1.115 + 0.177, 0.961+0.151, respectively (P < 0.05). Meanwhile, PDTC reduced cell migration while EGF reversed it partially. The results of EMSA and immunocytochemistry further verified that EGF-mediated NF-kB activity, at lease in part, was PLCyl-dependent.5. Hsp70 is one of the down intermediaries of EGFR-PLCyl-NF-kB signaling pathway and is regulated negatively by specific inhibitors. Some investigators demonstrated that high expression of Hsp70 was associated with invasion, metastasis and poor prognosis. More recently, mRNA microarray analysis showed that Hsp70 gene, lying downstream of PLCyl, was negatively associated with invasion in the human prostate carcinoma cells. Till now, the molecules and the signaling pathway correlated to Hsp70 in tumor are still to be elucidated.In this study, cells were exposed to various doses of U73122 (luM, 2.5|iM, 5 \nM) in the presence or absence of EGF (20 nM) or exposed to U73122 (2.5 jxM) or PDTC (100 |iM) for various time intervals(0, 0.5, 1, 4, 8, I2h).Treatment of U73122 or PDTC upregulated the expression of Hsp70 in LoVo cells. But the effects of the two inhibitors are different. The expression of Hsp70 was increased 30 min after U73122 treatment, keeping high till 12 h. However, the upregulation effect of PDTC last only about 4 hours, after which the expression of Hsp70 decreased to control level. Given that NF-kB is a family of transcription factors and regulated by a variety of molecules, we supposed that this difference may be caused by other signals, which augmented the inhibition of NF-kB by PDTC, resulting in the expression of Hsp70 come back to the basal level fast.6. The expression and activity of MMP-2 and TIMP-2 are not regulated by EGFR-PLCyl -NF-kB signaling pathway. The expression and activity of MMPs are increased in many human cancers, which correlate with advanced tumour stage, increased invasion and metastasis, and shortened survival. MMPs canpromote cancer progression by degrading structural components of the ECM, increasing cancer cell migration and angiogenesis. As an important member of MMPs family, MMP-2 plays important role in cancer progression. TIMP-2, an endogenous inhibitor, can specifically inhibit MMP-2. Thus, the expression and activity of MMP-2 and TIMP-2 were investigated. LoVo cells were subjected to different treatment with U73122, PDTC or/and EGF, then RNA was extracted. RT-PCR results showed that EGF, U73122 or PDTC had no significant effect on the expression of MMP-2, TIMP-2 at mRNA level. Meanwhile, RT-PCR using RNA extracted from LoVo cells transfected with pLNCX2/PLCyl or pLNCX2/PLCz also showed the same result. Furthermore, zymography indicated that the activity of MMP-2 did not change dramatically by EGF, U73122 or PDTC treatment.Taken together, all of the results indicated that PLCyl plays an important role in regulation of cell-matrix adhesion and cell migration in human highly metastatic colorectal cancer cells and further has an effect on tumor invasion and metastasis. EGF acts as upstream stimulus while NF-kB are the downstream intermediaries of PLCyl. Hsp70 lies downstream of EGFR-PLCyl-NF-kB signaling pathway and negative-regulated by the pathway. However, MMP-2 and TIMP-2 may not be regulated by EGFR-PLCyl -NF-kB signaling pathway. These consequences first demonstrate the relationship between PLCyl and colorectal cancer cells behaviors and indicate that EGFR-PLCyl -NF-kB -Hsp70 signaling pathway is operative in LoVo cells, which would not only provide the basis for the further study in the mechanisms of colorectal cancer invasion and metastasis but also suggest that EGFR-PLCyl-NF-kB-Hsp70 signaling pathway could be targeted for therapeutic intervention in colorectal cancer.