An Initial Approach To The Effects Of Inhibiting Phospholipase C Gammal Signaling Pathway On The Colorectal Cancer Cell's Proliferation And Apoptosis And Its Mechanism

Colorectal cancer is a usual type of malignant neoplasm with a high mortality rate ranked fourth or fifth in all the malignant neoplasms. About half of the patients failed to be cured due to the unsatisfied treatment efficacy of colorectal cancer. Thus, it is urgent to seek efficient methods to interfere and block the colorectal cancer's genesis and development and the main work is to seek the pathogenesis and target. It shows that the dysfunction of proteins and coherent signaling pathways has a certain role in colorectal cancer genesis. The signaling pathways related to cell growth, mitogenesis and proliferation are at an abnomal activated state, while those related to apoptosis are always blocked. The studies of signaling pathways on colorectal cancer genesis not only broaden the views on studying colorectal cancer's pathogenesis but also provide more targets for the signal transduction depressant drug therapy for colorectal cancer.Phospholipase C gamma 1 (PLC-γ1), a member of phospholipase C family, is a vital middle signal molecule in transmembrane signaling. When activated by acceptors or some other molecules such as hormones, neurotransmitters and growth factors, it hydrolyzes phosphatidylinositol-4, 5-bisphosphate (PIP2), generating two second messenger molecules: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which regulates intracellular levels of Ca~2+ and activates protein kinase C(PKC) respectively, and then triggers a downstream kinase cascade, which plays an important role in a series of cell's physiological function such as secretion, proliferation, growth and differentiation. There are two completely different cognitions of the roles of PLC-γl in cell proliferation. In some experimental models PLC-γl is found to be a vital molecule in the regulation of cell mitogenesis and proliferation. It can promote mitogenesis and proliferation through activating PKC and ERK cascade depending on its phospholipase activity or through activating intranuclear PI3K signaling pathway by nuclear translocation. While in other models, it is found that PLC-γl has little effect on cell mitogenesis and proliferation. PLC-γl is also an important molecule negatively regulating apoptosis and necessary to cell's survival in many conditions. PLC-γl is also highly related to colorectal cancer, the expression level of which is much higher in malignant tissues than in normal tissues with higher enzymatic activity. PLC-γl is also involved in cell transformation, the genesis and invasion of tumor. Heat shock protein (HSP70), a member of heat shock protein family and an important molecular chaperon, plays an equal important role in regulating cell proliferation and apoptosis, which also shows increased expression in colorectal cancer. Does the high expression of PLC-γl in colorectal cnacer cell have any effect on the cell's over-proliferation and deactivated apoptosis? Is it possible to inhibit colorectal cancer cell's over-proliferation and induce apoptosis through blocking the PLC-yl signaling pathway? Is there any relationship between HSP70 and PLC-yl signaling pathway? What effect does HSP70 have on the colorectal cancer cell's proliferation and apoptosis? There have been no researches done to solve these problems so far.We selected colorectal cancer cell line LOVO cell to be our experimental model and blocked PLC-yl signaling pathway to observe the effect on its proliferation and apoptosis and probe into the possible signal mechanism. We mean to provide cell biology and molecular biology data for the study of colorectal cancer's pathogenesis and the finding of new medicine targeted to the key regulating molecule. For this purpose, this study consists of the following two parts.Part one, to block PLC-yl signaling pathway and observe its effect on colorectal cancer cell's proliferation and apoptosis and approach the possible signaling mechanism.Methods: To select colorectal cancer cell line LOVO cell to be the study model, block PLC-yl signaling pathway in it using chemical blocking agent U73122 and study the effects on LOVO cell from the following aspects which include: 1 .the effect on cell proliferation: to make routine cell growth curve and determine cell proliferation inhibition ratio by MTT method; 2.the effect on cell cycle progress: to detect and analyze the proportion of cells in each cell cycle phase using flow cytometry assay; 3.the effect on LOVO cell's growth characteristic: to observe the cells' morphology under light microscope and scanning electron microscope and the colony formation; 4.the effect on the expression of heat shock protein70(HSP70): to detect the expression of HSP70 by Western blot.Results: l.LOVO cell's adherence ability decreased notably when PLC-yl signaling pathway was blocked. We observed more round cells and less spindle cells with good adherence ability under light microscope and shorter ecphymas of cells and fewer microvillis under scanning electron microscope; 2. LOVO cell's proliferation got slower after blocking PLC-yl signaling pathway and the effect depended on the concentration of U73122. The inhibition ratio reached 35% and 45% with the treatment of 10|xmol/L U73122 for 24h and 48h respectively; 3. LOVO cell's colony formation ability decreased after blocking PLC-yl signaling pathway. The number of LOVO cell's colony formation decreased about half with the treatment of l|.imol/L U73122 while hardly a colony was observed with the treatment of lOjxmol/L U73122; 4. The proportion of LOVO cell in Gl phase increased while the one in S phase decreased after blocking PLC-yl signaling pathway, which resulted in the inhibition of cell cycle progress; 5.The expression of HSP70 in LOVO cell increased significantly after blocking PLC-yl signaling pathway.Discussion: In this study we found that LOVO cell's over-proliferation was inhibited notably after blocking PLC-yl signaling pathway, indicating that PLC-yl is an important signal molecule regulating LOVO cell' proliferation and supporting the important roles of PLC-yl in controlling cell proliferation. The effects of inhibiting PLC-yl signaling pathway should have something done with its over-expressed PLC-yl and we induce that PLC-yl might have the same important roles in controlling proliferation of other cancer cell which also over-expresses PLC-yl. The proportion of LOVO cell in Gl phase increased while the one in S phase decreasedafter blocking PLC-yl signaling pathway, indicating that one of the mechanisms of cell proliferation inhibition was cell cycle progress inhibition, ie, LOVO cell was detented in Gl phase and the transition from Gl phase to S phase slowed down; The expression of HSP70 increased after blocking PLC-yl signaling pathway, hinting that cell cycle progress inhibition may be due to the chaperone role of HSP70 which binds to molecules regulating cell cycle progress, stabilizating its conformation, and makes the molecules activated or deactivated. It is hopeful to treat colorectal cancer by the method of blocking abnormally activated signaling pathways, and we confer that PLC-yl will be a new target choice for colorectal cancer therapy.Part two, to block PLC-yl signaling pathway and observe the effect on LOVO cell's apoptosis.Methods: To choose colorectal cancer cell line LOVO cell to be the study model, block PLC-yl signaling pathway in it using chemical blocking agent U73122, and study the effect on LOVO cell from the following aspects: 1.observation of cells' morphologic change by light microscope; 2.detection by agarose gel electrophoresis; 3.detection by flow cytometry assay with PI simple staining; 4.detection of apoptosis-executing protein Capspase-3 :(Western blot).Results: After blocking PLC-yl signaling pathway, we found that, 1.Although most LOVO cells became round, no apoptosis specific shrinkage of cells was found by light microscope. The number of dead cells was not found increased and the cell viability was not changed by the trypan blue dye exclusion test; 2. No apoptosis specific DNA ladder was found by agarose gel electrophoresis; 3.No apoptosis specific hypodiploid peeks occurred; 4.No activated Caspase-3 was detected by Western blot.Discussion: No apoptosis specific conformational and biochemical changes were found and no activated Caspase-3 was detected after blocking PLC-yl signaling pathway, indicating that blocking PLC-yl signaling pathway can't induce LOVO cell apoptosis and PLC-yl is not the key regulator of LOVO cell's apoptosis. We infer that no apoptosis after blocking PLC-yl signaling pathway might be due to the compensated activating of other signaling pathways, such as the increased expression of HSP70 and its anti-apoptosis effect. Blocking PLC-yl signaling pathway probably can reduce colorectal cancer cell's anti-apoptosis ability.Conclusions:First, to block PLC yl signaling pathway can inhibit LOVO cell's over-proliferation, change their growth characteristic. One of the mechanisms of cell proliferation inhibition is that LOVO cell is detented at Gl phase and that cell's transition from Gl to S phase is prolonged, ie, cell cycle progess is inhibited; to block PLC yl signaling pathway can upregulate the expression of HSP70. LOVO cell's cell cycle inhibition might be due to the chaperone role of HSP70; PLC-yl and HSP70 are both vital molecules regulating LOVO cell's proliferation and cell cycle progress and the latter is a possible downstream signal molecule in PLC-yl signaling pathway.Second, blocking PLC-yl signaling pathway can't induce LOVO cell's apoptosis. PLC-yl is not a key regulator of LOVO cell's apoptosis. Increased expression of HSP70 may take a certain effect on maintaining LOVO cell at deactivated apoptosis state.The genesis and development of colorectal cancer involve many abnormal signaling pathways, and the loss control of colorectal cancer cell proliferation and apoptosis is the consequence of crosstalk of many signaling pathways. How is the PLC-yl signaling pathway related to these dysfunctional pathways? What causes the abnormality of these signaling pathways? To solve these problems might be the key means to probe into the pathogeny mechanism and therapy measures for colorectal cancer, which would be a very interesting research direction. It will be an effective avenue for us to solve these problems through studying the functions of the different-expression proteins in colorectal cancer by means of functional proteomics.