Studying The Effects Of Nordy And The Expression Of Functional CXCR4 On Rhabdomyosarcoma Cell Line PLA-802

BACKGROUND & PURPOSE:Rhabdomyosarcoma(RMS)is one of the most common malignant tumors in soft tissue.It can occur at any age and is high malignancy.With the application and improvement of chemotherapy,the therapy of RMS gets some improvement,but therapeutic effect is still dissatisfied.It is of great significance to develop new effective anti-RMS drugs.Nordy is a kind of compound of nordihydroguaiareticacid.It is reported that Nordy can apparently inhibit the growth of lung cancer in vitro and in vivo,and can inhibit the growth and induce the differentiation of many glioma cells. It is suggested that Nordy is a new kind of anti-cancer drugs.The chemokines and its receptors have many important and complex effects during the cancer development.It was reported that the chemokines receptor CXCR4 expressed in many RMS cell lines,but there is not known about the expression and function of CXCR4 protein.This research focuses on the effect of Nordy on RMS and the expression and function of CXCR4 protein. MATERIALS AND METHODS:24 clinical specimens and PLA-802 cells(derived from gland alveolar RMS)were investigated.Part 1:IC₅₀and the effects on the growth of PLA-802 cells were tested by cell counting;cell cycle and apoptosis were analyzed by FCM;morphological changes were observed by LM and EM; the expression of myosin and VEGF were tested by immunocytochemistry and ELISA.Part 2:PLA-802 cells were transplanted to subcutaneous tissue of the nude mice to establish a model.The mice were treated with Nordy through abdominal injection.The sizes of tumor were measured to certify the effects of Nordy on the growth of tumor cells;morphological changes were observed by LM and EM;the expression of MVD and VEGF were tested by immunocytochemistry and image analysis;the side effects of Nordy on nude mice at the concentration of 27mg/kg were tested.Part 3: The expression of CXCR4 in human RMS tissue and RMS cells in vitro were tested by immunocytochemistry.Part 4:The effects of CXCR4 on the proliferation of PLA-802 cells were tested by MTT;its effects on immigration and chemotaxis were tested by random immigration model in vitro,the effects on the intracellular concentration of Ca²⁺were analyzed by laser scanning confocal microscope,the expression of VEGF was tested by immunocyto-chemistry and ELISA.RESULTS:(1)The experiment in vitro: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 and this inhibit effect was dose and time dependent;3)The cell became longer,the nucleus and nucleolus became small after treated by Nordy;4) The cell mitochondrion became abnormal,and the endoplasmic reticulum(ER)became extension,the ribsome detached from the rough endoplasmic reticulum;5)The cells in G₀/G₁ cell cycle increased 15.1% and 8.7%respectively after treated by 100μmol/L Nordy for 12h and 24h. The cells in G₂/M phages in cell cycle increased 317.4%and 137.2%after treated by 100μmol/L Nordy for 48h and 72h.There was the significant difference comparing with the control(P＜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 the significant difference compared with the control(P＜0.05);7)The expression of myosin increased and that of VEGF decreased after treated with Nordy.There was the significant difference compared with the control(P＜0.05).(2)The experiment in vivo:1)The growth of tumor in the treated group 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 treated group; 3)There was not apparent tumor atypia and mitoschisis and blood vessel was decreased in the treated group;4)Myofilament and microfilament were increased in tumor cells and muscle fibril was formed under EM in the treated group;5)The expression of myosin increased and that of VEGF decrease in the treated group.There was the significant difference compared with the control(P＜0.05);6)A obvious apoptosis peak could be observed in PLA-802 cells,which accelerated as time went by after treated with 100μmol/L Nordy for 12h,there was the significant difference comparing with the control(P＜0.05);7)The expression of myosin increased and that of VEGF decreased after treated with Nordy.There was the significant difference compared with the control(P＜0.05)(3)Detection of CXCR4 protein:The expression of CXCR4 were presented in different human RMS tissue and RMS cells,and expression of CXCR4 in gland alveolar RMS was stronger than embryonal RMS(P＜0.05).(4)Detection of CXCR4 function:1)10,25ng/ml SDF-1αcan apparently stimulate proliferation of PLA-802 cell(P＜0.05),the optimal concentration is 10ng/ml SDF-1α;2)40ng/ml SDF-1αcan strenthen spontaneous movement of PLA-802 cell(P＜0.05);3)SDF-1αcan induce spontaneous movement of PLA-802 cell and it was dosage dependent in concentration of 10～100ng/ml,the highest concentration was 100ng/ml, CXCR4 antibody can inhibit SDF-1αinduced PLA-802 cell immigration, it was dosage dependent in concentration of 1～20ng/ml,30ng/ml SDF-1αinduced PLA-802 cell immigration could be completely inhibited by 20μg/ml;4)it was dosage dependent in concentration of 25～100ng/ml SDF-1α,which can cause elevation of Ca²⁺concentration in PLA-802 cells (P＜0.05);5)The expression of VEGF protein in PLA-802 cells is higher induced by SDF-1α.CONCLUSIONS:â‘ Nordy has the effect of inhibiting growth,promoting apoptosis, inducing differentiation on PLA-802 cell;â‘¡The expression of CXCR4 was present in PLA-802 cell and RMS tissue and there was strong expression in gland alveolar RMS.The CXCR4 can stimulate the RMS cell proliferation,invasion and the expression of VEGF.