The Effects Of TGF-β/Smad Signal On The Growth And Apoptosis Of Human Rhabdomyosarcoma Cell Line RD

Transforming growth factor-β(TGF-β) belongs to a large family of multifunctional polypeptides that are involved in the regulation of cell growth, apoptosis, adhesion, and differentiation. TGF-β binding to TβR-n then results in transphosphorylation and activation of TβR-I by TβR-Π. The activated TβR-I then propagates the signal through phosphorylation of receptor-restricted Smad2 and Smad3. Phosphorylated Smad2 and Smad3 associate with Smad4 and translocate to the nucleus and modulate transcription of TGF-β target genes. The regulation of TGF-β to different type cells take on different specificities. Loss of responsiveness to TGF-β/Smad signal frequently accompanies the procession of malignancy and may contribute to the development of many types of human cancers. Previous studies mostly focused on leukemia and many kind of carcinoma. The effects of TGF-β on soft tissue sarcoma were rarely reported.Rhabdomyosarcoma (RMS) is a common childhood malignant tumor with a 10~20% morbidity in soft tissue tumors. RMS has the characteristics of speedy growth and early hematogenous metastasis. Despite aggressive approaches incorporating surgery, dose-intensive combination chemotherapy, and radiation therapy, the outcome for patients with metastatic diseaseremains poor. It has been shown that various autocrine growth factor loops can be expressed by RMS and are likely to be involved in the growth of cancer cells in vitro. Better understanding of how these growth factors regulate proliferation and apoptosis of RMS cells may ultimately have therapeutic implications.In our previous study, we have identified that both alveolus rhabdomyosarcoma and embryonal rhabdomyosarcoma expressed TGF-/31, T/3R-I, TpR-FI, and down-stream molecules-Smads. Human embryonal rhabdomyosarcoma cell line RD also autocrines TGF-/31 and expresses T/3Rs and Smads. It has been identified that TGF-/3/Smad signaling exists and may play a role in RD. hi the study, we first use RNA interference to mediate Samd4 "gene silencing", so interrupting the transduction of endogenous TGF-j3/Smad signal pathway in RD. This strategy helps us to understand the effects of endogenous TGF-/3/Smad signal on RD cell. At the same time, the regulation of exogenous TGF-j31 to RD also was examined. Then the molecular mechanisms of cells growth by exogenous TGF-/31 in RD were also examined. The results were summarized as follows:1. According to definite proportion, biologically synthesized shRNA were transfected into RD by cation liposome vector. Through RT-PCR and Western blot methods, the optimal interference-efficacy shRNA was demonstrated and the time-course of shRNA interference also was identified. The shRNA Yl can effectively operate from 12h to 96h after transfection. Immunofluorescence staining showed that TGF-/31 -treatment lead Smad2/3 to enter into nucleus in RD, while suppression of Smad4 expression by shRNA Yl can block the translation of Smad2/3 by TGF-/31 signal. It issuggested that suppression of Smad4 by RNAi can efficiently interrupt the endogenous TGF-/3/Smad signaling.2. [3H]thymidine incorporation assay and MTT assay were used to measure the proliferative ability of RD. The viability of RD was obviously reduced in a time-dependent manner in shRNA-group, shRNA+TGF-/31-group, and TGF-/31 -group, respectively. To monitor TGF-/?/Smad-derived changes in cell cycle of RD, cell cycle profiles with time following shRNA or/and TGF-01 treatment were analysed by flow cytometry. Either obstruction of endogenous TGF-/3/Smad signaling or treatment of exogenous TGF-/31 could increase Gl population in RD, and prevent most cells from re-entering the S phase. A number of apoptosis bodies were observed by both Dapi staining and electron microscopy examination in ShRNA-group and shRNA + TGF-/31 -group. Flow cytometric analysis revealed that apoptosis index greatly increased in ShRNA-group and shRNA + TGF-/31-group, in a time-dependent manner. In DNA fragmentation assay, DNA ladder were detected in ShRNA-interference groups, other than control-group and TGF-jSl-group. All of above results suggested that obstruction of endogenous TFG-/3/Smad signaling by ShRNA interference can inhibit growth of RD and induce apoptosis of RD. And exogenous TGF-/31 can also powerfully suppress growth of RD, but has no obvious effect on apoptosis of RD. Physiologically TGF-/?/Smad signal is indispensable to growth of RD.3. To assess the mechanisms of RD growth inhibition induced by exogenous TGF-/31, the expression of various cell cycle regulators were examined by RT-PCR and Western blot analysis. Remarkable stimulation of P27 was observed in RD treated with TGF-/31. P21 also increased in responseto TGF-/31. But at both mRNA and protein levels, no obvious changes of PI 5, CDK2, and CDK4 were detected. TGF-/31 also decreased the expression of C-myc. Immunoprecipitation with CDK2 followed by Western blot analysis indicated that TGF-/31 obviously increased CDK2-bound P27, but marginally influenced CDK2-bound P21. In addition, kinase assay showed that CDK2 kinase activity is dramatically suppressed within 12h following TGF-/31 administration, whereas kinase activity of CDK4 has not much changes after TGF-/31 treatment. Through immunofluorescence staining, an interesting phenomenon was observed that TGF-/31 induced P27 congregated around the nucleus, and P21 diffused from nucleus to both nucleus and cytoplasm.In conclusion, physiological TGF-/3/Smad signal is indispensable to growth of RD. Interruption of endogenous TGF-|8/Smad signal transduction not only inhibits the growth of RD but also induces apoptosis of RD. Exogenous TGF-/31 up-regulates P27 to suppress CDK2 kinase activity, which process induces RD Gl-arrest of cell cycle and ultimately inhibits growth of RD. Exogenous TGF-/31 can't induce apoptosis of RD. The shRNA interference in Smad4 expression representing an extremely powerful tool may provide a novel basis for the development of rational intervention strategies in RMS therapy. Our experiments supplied a new insight for how TGF-/3/Smad signal regulates the growth and apoptosis of RMS cell line RD, and an experimental base for anticipative RMS gene therapy.