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Tacrolimus Enhances CXCR4/SDF-1α Expression and Promotes Hepatocellular Carcinoma Metastasis

Author: ZhuHuaZuo
Tutor: ZhouJian；TanChangJun；DaiZhi
School: Fudan University
Course: Surgery
Keywords: Hepatocellular carcinoma Tacrolimus Tumor metastasis FK506 CXCR4 SDF-1α ACI MH3924a Rat hepatoma model
CLC: R735.7
Type: Master's thesis
Year: 2009
Downloads: 77
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Abstract

Background primary liver cancer (HCC) is one of the most common malignancy in the world, the mortality rate of the total cancer mortality, global cancer mortality third. With the proposal of the concept of liver cancer early diagnosis, early treatment, the 5-year survival rate of patients with liver cancer than in the past, have been greatly improved. Currently, surgery is still the preferred method of treatment is long-term survival of patients with liver cancer, but liver cancer patients is often associated with cirrhosis of the liver and liver dysfunction, only 20% of patients until the time when a clear diagnosis often meet hepatectomy surgical indications, In this case, the liver transplant as both resection of liver cancer and hardening of the liver, but also to improve the means of a treatment for end-stage liver disease, began to be gradually accepted. After transplantation, however, is still unable to avoid the recurrence and metastasis of liver tumors, the immunosuppressant is one of the major risk factors. After liver transplantation is widely recognized conventional immunosuppressant treatment regimens triple FK506/CsA plus MMF and hormone medication, some transplant centers try rapamycin alternative FK506 or CsA. FK506 and CsA are calcineurin inhibitors, by inhibiting the activity of nerve calmodulin phosphatase, further inhibiting the synthesis and release of IL-2, inhibition of cell proliferation and activation of T, B, the clinical immunosuppressive effects. Part of the cell experiment results show no significant effect of CsA and FK506 on the growth of liver cancer cells, but more animal experiments reported clinical retrospective research tips, calcineurin inhibitors indeed lead to liver cancer recurrence and metastasis after liver transplantation The increased rate of risk factors. The immunosuppressant calcineurin enzymes indeed contribute to the postoperative tumor recurrence and metastasis, there is still controversy. Chemokines are a class of small molecule secreted proteins through the chemokine receptors involved in the body pathophysiological regulation. Was originally thought that chemokines mainly involved in chemotaxis and activation of some inflammatory cells and some immune inflammatory reaction. However, in recent years, reported chemotactic factor more and more involved in tumor growth and metastasis. The most notable is CXCR4/SDF-1α biological axis. The biological axis that broad participation in the process of directed chemotaxis of tumor cells and transfer. CXCR4 is a variety of tumor cells expressing the chemokine receptor. SDF-1α secreted by the stromal cells, persistent high expression in the bone marrow, lymph nodes, lungs and liver, these organs are often the target organ metastasis. This prompted the CXCR4/SDF-1α of likely has played an important role in a variety of tumor target organ directional transfer in. A number of experimental studies have shown that, CXCR4/SDF-1α biological axis balance change may lead to changes in tumor invasion and metastasis, if FK506 can promote liver transplant postoperative tumor recurrence and metastasis, whether it is because the regulation of hepatic tumor cells and surrounding stromal cells the biology of CXCR4/SDF-1α axis balance? corresponding chemotactic effect between CXCR4/SDF-1α blocked by the CXCR4 antagonist AMD3100, could weaken role of FK506 promoting tumor growth and metastasis? This is The significance of this study. Research purposes by in vitro experiments and experimental study of the rat immunosuppressant FK506 whether explicitly promote the growth and metastasis of liver cancer in rats, as well as CXCR4/SDF-1α altered expression and its clinical significance in the process. Research methods. Vitro: 1.1 tumor cell proliferation assay - thiazolyl blue colorimetry (MTT) the rat hepatoma MH3924a cells grouped culture, alone or in combination with different concentrations of FK506 and CXCR4 antagonist of AMD3100 MTT assay. proliferation. 1.2 Scanning Electron Microscopy MH3924a 5 × 10 ~ 3 was plated on coverslips (8 × 8mm ~ 2) and replaced with complete medium containing different immune inhibitor concentration in the incubation 1d after culture was continued, and by scanning electron microscopy specimen preparation procedures processing After observation. 1.3 Immunohistochemical staining MH3924a8 × 10 ~ 3 passages coverslip (8 × 8mm to 2), incubated 1d replaced with complete medium containing different concentrations of immunosuppressants continue to foster. Immunohistochemical operation flow processing line CXCR4 antibody staining, IPP software analysis of protein expression. 1.4 cell invasion assay the MH3924a cell groups, plus the negative control of the upper chamber, FK506 intervention, intervention of AMD3100, the lower chamber and with different concentrations of SDF-1α, follow the standard process of cell migration assay and invasion assay, analysis of the changes in cell invasion force . Vivo experiments 2.1 2mm ~ 3MH3924a liver cancer tumor mass surgically implanted ACI rats left liver, rat liver cancer model. 2.2 postoperative day 5 ACI tumor-bearing rats were randomly divided into two groups, one group was given saline injected subcutaneously 3mg/kg/d ~ * 14d; given a set of FK506 subcutaneous injection 0.3mg/kg/d to * 14d. 2.3 observed 40 days later put to death, records tumor growth in experimental animals, the number of metastatic lung nodules and other organ metastasis. 2.4 collect rat liver tumor specimens, adjacent tissues and liver normal tissue, Immunohistochemical detection of tumor CXCR4 paracancerous of SDF-1α expression. Lungs filling 15% of India ink dye, rinse, and count the number of metastatic nodules. Differences between the Mann-Whitney rank sum test was used to compare the groups. Results. Vitro, FK506 in low concentrations (10ug / L), of MH3924a hepatoma cells proliferation had no effect (P = 0.135); while in high concentrations (100μg/L-1000μg/L), MH3924a hepatoma cell proliferation in vitro a significant role (P <0.01); FK506 constant concentration (100μg / L), grouped with different concentrations of AMD3100 (10ng/ml, 50ng/ml, 100ng/ml) can not change FK506 role of liver cancer cells in vitro in MH3924a proliferation (P <0.01). Vitro, of FK506 (100μg / L) intervention training, CXCR4 hepatoma cells in MH3924a expression compared to the negative control group has increased, but no significant difference (P> 0.05); via AMD3100 (50ng/ml) intervention cultured hepatoma cells in MH3924a, CXCR4 expression was decreased (P <0.05). Electron microscopy experiments: MH3924a cells by the FK506 (100ug / L) after the treatment, the surface rich microvilli, richer morphology invasive. After combined treatment FK506 and AMD3100 microvilli did not significantly reduce the performance continued to show high invasive phenotype. Tumor cell invasion assay in hepatoma cells of FK506 (100ug / L) after the intervention of MH3924a number of plastic than the negative control group was significantly increased, the difference was statistically significant (P <0.01); via SDF-1α (10ng/ml the control group was significantly increased (P <0.01) the induced MH3924a hepatoma cells wear glue quantity than the negative control group increased significantly, the difference was statistically significant (P <0.01); cells of of the joint training MH3924a liver cancer after FK506 AMD3100 wearing plastic than negative ), compared with FK506 intervention group showed decreasing trends, but the difference was not statistically significant (P = 0.607), and significantly reduced compared with the SDF-1α induced group (P = 0.507). 5. Vivo animal experiment results show that: the number of metastatic nodules was 1.39 ± 1.25 in the saline group rat lung FK506 group, metastatic lung nodules was 6.50 ± 4.63 (P <0.05). 100% of the the FK506 group of rats occurred multiple organ other than the lung metastases and liver spread outside the transfer, including abdominal muscles, lymph nodes; this case only 20% of the saline group rats. 6. Vivo experimental animal tissues by immunohistochemistry results showed that: compared to the negative control group, FK506 intervention group rat liver tumor CXCR4 upregulation, the difference was statistically significant (P = 0.048); cancer tissue and normal liver tissue SDF-1α significantly increased, and the difference was statistically significant (P = 0.026). Conclusion 1. Vitro, FK506 can significantly promote liver cancer cell invasiveness (10 ug/L-1000 ug / L) and proliferative capacity (100 ug/L-1000 ug / L); vivo experiments, FK506 significantly promote large rat liver cancer growth and metastasis. 2.FK506 promotion of rat liver tumors in vivo CXCR4 and SDF-1α expression increased cancer tissue. 3 blocking the tumor associated CXCR4/SDF-1α biological axis can enhance tumor invasion force of some weakened FK506 lead.

Tacrolimus Enhances CXCR4/SDF-1α Expression and Promotes Hepatocellular Carcinoma Metastasis

Abstract

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