| BACKGROUND & PURPOSE:Rhabdomyosarcoma(RMS)is one of the most common malignancies arising from soft tissue. It can occur at any age with high malignancy. With the application and improvement of chemotherapy, the prognosis of RMS has been improved a lot but remains to be improved. It is of great importance to discover its tumorigenesis and develop new effective anti-RMS drugs. Nordy is a kind of compound of nordihydroguaiaretic acid. It has been reported that Nordy can apparently inhibit the growth of lung cancer cell line A549 and many kinds of glioma cell lines both in vitro and in vivo,and even holds many other biological activities, such as inducing differentiation, against angiogenesis, etc. Our study was conducted to investigate the effect and mechanism of Nordy on human RMS cell line PLA-802 and its xenograft, so as to search the feasibility of using Nordy in treating RMS.MATERIALS AND METHODS:24 clinical RMS specimens and PLA-802 cell line were investigated. PLA-802 cell line and its xenograft were treated with Nordy. CXCR4 expression in RMS specimens were tested by immunohistochemistry. IC50 and the effects of Nordy on the growth of PLA-802 cells were tested by cell counting;Cell cycle and apoptosis were analyzed by FCM; Morphological and ultrastructural changes of PLA-802 cell and its xenograft were observed by light microscope and electron microscope; The mRNA and protein expressions of myosin, VEGF and CXCR4,and Microvascular density (MVD) in PLA-802 cell line and its xenograft were tested by real time RT-PCR, western blot, ELISA, immunohistochemistry and image analysis. The chemotaxis and calcium mobilization of CXCR4 with or without Nordy treatment on PLA-802 cell line was tested by chemotaxis analysis and laser scanning confocal microscopy (LSCM).RESULTS:Part1: (1) IC50 of Nordy on PLA-802 cell line was 69.23μmol/L; (2) 50～200μmol/L Nordy had inhibitory effect on PLA-802 cell growth and this effect was dose- and time-dependent; (3) The cell became longer, the nucleus and nucleolus became small with nucleus/plasma ratio decreased after treated with 100μmol/L Nordy; (4) The ridges of cell mitochondrion developed abnormal derangement, breakage, cavitated or even formed myelin sheath-like structure, and the endoplasmic reticulum(ER) became extended, the ribsomes detached from the rough endoplasmic reticulum at the time point of 24 hours after treated with Nordy; At the time point of 72 hours, the cells presented with ellipse-like or fusiform shape with extended mitochondrion relieved, less cavitated while with more myelin sheath-like structure, and various amout of actin microfilament would be easily seen in plasma; (5) The cells in G0/G1 phase in cell cycle increased by 15.1% and 8.7% respectively after treated with 100μmol/L Nordy for 12h and 24h. The cells in G2/M phase in cell cycle increased 317.4% and 137.2% after treated with 100μmol/L Nordy for 48h and 72h. There was a significant difference as compared with the contro(lP<0.05); (6) An obvious apoptosis peak could be observed, which accelerated as time went by after treated with 100μmol/L Nordy for 12h. There was a significant difference as compared with the control ( P<0.05 ) ; (7) The expression of myosin ( mRNA and protein )increased and that of VEGF decreased after treated with Nordy. There was a significant difference from the control(P<0.05).Part 2: (1) Detection of CXCR4 protein: The expressions of CXCR4 were presented in different types of human RMS tissues, and expression of CXCR4 in alveolar RMS was stronger than that in embryonal RMS (p<0.05); (2) CXCR4 function in RMS cell line PLA-802: chemotaxis could be induced by 10～100ng/ml SDF-1αand this effect was dose-dependent, with the greatest immigration at the concentration of 100ng/ml; 25～100ng/ml SDF-1αcould increase the concentration of plasma calcium in PLA-802 cells, and the increase in the calcium usually persists a little while and then decrease later. It was an identical reaction in condition with or without extracellular calcium; (3) The effect of Nordy upon the expression and function of CXCR4 in PLA-802 cells: 1) CXCR4 mRNA and protein expressions in PLA-802 cells could be reduced with 50～100μmol/L Nordy treatment; 2) 50～100μmol/L Nordy had inhibitative effect on chemotaxis induced by SDF-1α(50ng/ml); 3) The increase in plasma calcium concentration induced by SDF-1α(50ng/ml)could be inhibited with 50～150μmol/L Nordy, and such inhibitative effect was dose-dependent irrespective of extracellular calcium.Part 3: (1) The growth of tumor in the treated groups was hampered obviously after treated with 13.5mg/kg and 27mg/kg Nordy(P< 0.05),the former inhibition rate was 47.43%,and the latter was 60.02%; (2) No apparent side effects were observed in the Nordy treated groups; (3) There were no apparent tumor atypia and mitoschisis, and microvascular density(MVD) was decreased in the Nordy treated groups; (4) The mRNA and protein expressions of myosin were increased, of VEGF and CXCR4 were decreased in the Nordy treated groups, There was a significant difference as compared with the control(P<0.05)CONCLUSIONS:Nordy has effect of inhibiting growth on PLA-802 cell line and its xenograft; The mechanism maybe relate to promoting apoptosis, suppressing cell-cycle, inducing differentiation, inhibiting functional expression of CXCR4 and decreasing angiogenesis, etc.
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