| Backgrounds and objectivesHepatocellular carcinoma (HCC) is a kind of malignant tumor that threats the health of our people and researchers have got so many discoveries in the fields of early therapy, recurrence and metastasis of the cancer in recent decades. However, except the early small HCC, the 5 years survival rates is low and tumor recurrence and metastasis are thought to be the main cause. From this point of view, to seek a new potent target of intervention which point to the recurrence and metastasis of HCC would be a important mission in clinical and experimental research.In recent years, ezrin has been a hot spot in the researching fields of tumor metastasis. Ezrin is product of gene Vi112 which locates at 6q25, as a main molecular of the cell skeleton of the brush border in chicken’s small intestine epithelial has been founded for about 30 years. Studies discovered that Ezrin-Radixin-Moesin were a family of protein which had the similar structures and functions, so, they are called ERM family. Ezrin is abundantly located in the cell surface structures such as microvilli and membrane folds and can link the cell skeleton and the membrane. Ezrin is thought to be a organizer and linker between cell skeleton and cell membrane molecules such as CD44, ICAM-2, E-Cadherin, etc. Some studies had showed ezrin played an important role in the metastasis progression of children’s osteosarcoma and rhabdomyosarcoma. Nowadays, more and more clinical and experimental evidence indicating that ezrin and cell adherence molecules play a key role in tumor progression and cell adhension, however, little has been known about the function of ezrin in HCC. Finishend study of the author indicated that there was weak expression of ezrin in normal liver tissues and strong expression in metastatic focus in liver or in the portal vein thrombus. MHCC97-H is a human hepatocellular carcinoma cell line with high metastatic potential which constructed by the researchers of liver cancer institute of FuDan university. Some study indicated that there had high expression in MHCC97-H cells which cultured in vitro.Paclitaxel is a kind of inhibitor of caryomitosis, and it acts on the microtublin so as to block the mitosis of cancer cells. At the same time, paclitaxel can inhibit cancer cell growth by inducing apoptosis.Although it has been used in the therapy of lung cancers, breast carcinomas, pancreas carcinomas and so on, little has been mentioned in the use of liver cancer treatment. In recent years there were sporadic reports on the effects of paclitaxel inhibiting the growth and invasion ability of HCC.In consideration of the above, we have a tentative idea as blow:As we know microtubule is a part of cell skeleton, and paclitaxel can inhibit the tumor cell mitosis by acting on the microtubule, however, ezrin has been improved as a linker between cell skeleton and cell membrane molecules. The question is:What the ezrin expression level would be if paclitaxel acted on the HCC cells? What effects would be shown on the HCC cells’ motility and invasion ability when the ezrin expression level changed?Methods1. Immunohistochemistry and Western blot to test ezrin expression in human liver cancer tissues and their metastatic focus.2. Electronicmicroscopy, MTT assay, Flow cytometry were used to survey the effect of paclitaxel on the morphorgh, grouth circle and grouth curve. And immunocellularhistology, RT-PCR, and Western blot were used to test the expression of ezrin.3. Constructing the over-expressing vector of ezrin and transinfection was done to the liver cancer cells. Detecting the cancer cells motility and invasion ability in vivo and in vitro with or without paclitaxel by the way of Wound healing assay, Transwell assay, clone formation assay, and the nude mouse tail vein injection with 4 groups of tumor cells.Results:1. The metastatic lesions had higher ezrin expression than in the primary liver cancers. The high level of ezrin expression had relationships with larger size/lower TNM stage/lower differentiation and metastatic leision tumors.2. The results of cell circles and morphology after MHCC97-H incubated with different concentration of paclitaxel.2.1 The proportion of G2-M phase cells in group A was 15.8% vs cells control 15.4%. While phase S cells proportion was 57.2% vs 29.6%, and apoptosis ratio was 4.4% vs 2.9%.2.2 The proportion of G2-M phase cells in group B was 40.2% vs cells control 15.4%.2.3 The proportion of G2-M phase cells in group c was 46.3% vs cells control 15.4%.2.4 There was increment of apoptosis in MHCC97-H cells when incubated with higher concentration of paclitaxel.2.5 After incubated with 20mg/L of paclitaxel for 24 hours, the cell was swell, decreased pseudopods, swelling cell organelle.2.6 After incubated with 40mg/L of paclitaxel for 24 hours, cell organelle was more swelling, nucleus membrane shrinking, chromatin was concentrated at the margin of nucleus membrane, and apoptosis body was seen around the tumor cell.2.7 After incubated with 80mg/L of paclitaxel for 24 hours, we could not see the pseudopods on the cell surface at some cancer cells, the nucleus was even bigger than before, with little neoplasm,.2.8 After incubated with different concentration of paclitaxel (20/40/80mg/L) and for different duration (12/24/48h), MTT assay to detect the tumor cells growth inhibition. For the same duration, with the increment of drug concentration, the value of IC50 increased (P<0.05).. For the same concentration, IC50 value increased with the duration time (P<0.05).3. ezrin expression after incubated with different concentration of paclitaxel.3.1 Immunocytochemistry. MHCC97-H cells(cells control) have high level expression of ezrin after 48 hours culture, and the strong positive expression cells proportion is over 50%. In contrast to cells control, we got decreased ezrin expression in experimental groups. In the same duration, the higher concentration of paclitaxel, the lower expression of ezrin.3.2 Western blot:We repeated experiments 5 times, and got the grey scale value of each band. The data are presented as mean±SD. In this study, we set the grey scale value of GAPDH as 10.00, then we got the comparative ezrin expression value as blow:cells control 4.05±0.36(n=5), group A3.65±0.27(n=5), groupB 2.38±0.25(n=5), groupC1.66±0.19(n=5). Statistic results show that:there is no difference between group A and cells control. However, there is significance when control VS group B (P<0.05), and control VS group C (P< 0.05), respectively. 3.3 RT-PCR:The gel images were scanned and analysed by the VDS500 image analysis system. We repeated experiments 5 times, and got the optical density (OD) value of each band. The data are presented as mean±SD. In this study, we set the OD value of GAPDH as 10.00, then we got the comparative ezrin mRNA expression value as blow:cells control 8.16±0.58(n=5), group A7.12±0.32(n=5), groupB 3.26±0.17(n=5), groupC 2.99±0.21(n=5). One-way ANOVA results show that:concerning about ezrin m-RNA expression, there is no difference between group A and control. However, there is significant difference when group BVS control (P<0.05), and group C VS control (P<0.05), respectively.4. We construct wild type ezrin vector pcDNA-WT-ezrin and transinfection was done successfully. Cells motility and invasion ability in vivo and in vitro after MHCC97-H incubated with different concentration of paclitaxel.4.1 Wound Scratch Assay: Transinfected cells without paclitaxel could heal the 5mm wound within 16 hours. Nontransinfented cells without paclitaxel could almost heal the 4mm wound after 24hours.While the transinfected cancer cells which dealed with 40mg/L paclitaxel depressed their ability of wound healing.4.2 Transwell assay:The average numbers of tumor cells which could pass through the artificial membrane are shown as blow. Transinfected cells without paclitaxel could penetrate the membrane in cells control were counted as 42.5±3.8(cells/pore), and the other three groups was 35.2±3.2, 27.9±2.4,12.4±1.1, respectively (n=9).4.3 Tumor cells clone formation assay:Transinfected cells without paclitaxel formatted the most clones numbers, while the uninfected cells with 40mg/L paclitaxel formatted the least number of tumor clones.4.4 Nude mouse tail vein injection assay:We counted the metastatic nodules in lung under microscope, and the average lung lesions were: Transinfected cells without paclitaxel 12.2±2.6 (n=10), nontransinfected cells without paclitaxel 10.7±2.2(n=10), Transinfected cells with 40mg/L paclitaxel 6.5±1.9(n=10), nontransinfected cells with 40mg/L paclitaxel 3.1±1.2(n=10).Conclusions:1. Hepatocellular carcinoma tissues had positive expression of ezrin, while the metastatic focus had over-expression of ezrin. The over-expression of ezrin had relationship to the clinic-pathology characters such as the tumor size, differentiation, TNM stages and the metastasis. Ezrin expression level in the dissected liver cancer tissues can be an independent predictive factor.2. Under a certain concentration, with the increment of drug concentration, both the proportion of G2-M cells and apoptosis increased. Paclitaxel led to the morphology change of MHCC97-H cells.3. Within a certain concentration, paclitaxel down-regulated ezrin expression in the tumor cells. The higher concentration of paclitaxel, the lower expression of ezrin.4. Paclitaxel can inhibit the wound scrath healing ability/ penetrating the artificial membrane ability/clones formation ability in vitro and can decrease the numbers of metastatic tumors in the lung of nude mice by down-regulating the expression of ezrin.5. The tumor cell’s clones formation ability in vitro and nude mice lung metastatic lesion formation ability in vivo can be strengthened by up-regulating the expression of ezrin.6. Paclitaxel may depress the reproduction/motility/ transformation and invasion ability of MHCC97-H cells by inhibiting the expression of ezrin, indicating ezrin is a critical molecular target of HCC metastasis progression. Paclitaxel is an effective drug on depressing the reproductive/invasive /metastatic ability of liver cancer cells. |
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