The Experimental Study Of Proliferation Inhibititon, Inducing Apoptosis, Inhibiting Angiogenesis, Downregulating LPS Induced TLR4Expression And NF-kappa B Modulation By Celastrol On LP-1Human Multiple Myeloma Cell Line

Objective:To investigate the effect of celastrol on cell proliferation, cell cycle, apoptosis, angiogenesis, modulation of NF-kappa B signaling pathway in LP-1human multiple myeloma cell line. Methods:The effect of celastrol on cell growth curve was investigated by trypan-blue staining. The activity of celastrol on LP-1cell proliferation were detected by CKK-8assay for IC50value. Celastrol-induced cell cycle arrest was analyzed by flow cytometry after propidium iodide (PI) staining. Celastrol induced LP-1cell apoptosis was observed by fluorescence microscope after Annexin V-EGFP staining and further quantitatively analyzed by flow cytometry. Procaspase-3activation by celastrol was measured by colorimetric pNA-based caspase-3assay monitored by a spectrophotometer at405nm. Cell surface TLR4expression was analyzed by flow cytometry. The Cyclin D1, P21wap1/cip1, P27kip1, Bcl-2, Bax, survivin, NFkappa B p65, IKKa, IκBα protein expression was analyzed by Western blot. NF-κB nuclear translocation was observed by fluorescence microscope. Celastrol-induced angiogenesis inhibition on human umbilical vascular endothelial cells (HUVECs) was determined by in vitro wound-healing assay. Invasion of HUVECs was assayed using a Transwell chamber. Angiogenic factor VEGF level was quantification by LUMINEX. Results:Celastrol significantly inhibited the proliferation of MM cell lines LP-1in a time and dose dependent manner, with the IC50value0.8817μM.Cell cycle analysis indicated that the G1phase cells were increased while S phase cells were decreased after treatment with celastrol for24hrs (**p<0.01). The Cyclin D1and P27kip1expression was significantly downregulated in the process. LP-1cell apoptosis induced by celastrol was also in a dose dependent manner. The percentage of apoptotic cell was71.2%after treatment with1μM celastrol for48h accompanied by obvious procaspase-3activation in the apoptosis process. Celastrol also inhibited the wound-healing of HUVECs in dose dependent manner. At the concentration of1μM, Celastrol significantly inhibited wound-healing of HUVECs after24hrs treatment, while cell cytoxicity effect was observed after48hrs treatment. Celastrol inhibited LPS-stimulated LP-1human multiple myeloma cell induced HUVECs cell migration and invasion in a concentration-dependent manner. The wound diameter increased by72.9%,165.4%and246.2%at0.025,0.05and0.1μM, respectively, compared with the LPS treatment. A45%-74%inhibition of LPS-dependent cell invasion in the presence of0.025-0.1μM celastrol was observed. Celastrol significantly downregulated LPS induced TLR4expression on LP-1human multiple myeloma cells. Celastrol significantly inhibited LPS induced VEGF secretion in LP-1human multiple myeloma cells. The VEGF level was decreased by64.8%,84.4%and92.9%at0.025,0.05and0.1μM, respectively, compared with the LPS treatment. Celastrol down-modulates antiapoptotic proteins including Bcl-2and survivin expression. Celastrol inhibited_the IKK/NF-kB pathway induced by LPS. The protein levels of NF-κB p65, IKKa and IκB-α were decreased in a dose-dependent manner after co-treatment with celastrol. Celastrol effectively blocked nuclear translocation of the p65subunit. Conclusion: Celastrol inhibited cell proliferation of human multiple myeloma LP-1cells and inhibited angiogenesis of HUVECs in vitro. Celastrol induced cell cycle arrest of LP-1cells on G1phrase, which could inhibit further DNA synthesis and cell mitosis. Celastrol induces human multiple myeloma cell cycle arrest and apoptosis by p27upregulation and NF-kappaB modulation. Celastrol could inhibit lipopolysaccharide induced angiogenesis by suppressing TLR4triggered NF-kappa B activation. Celastrol inhibited LPS-stimulated LP-1human multiple myeloma cell induced HUVECs cell migration and invasion in a concentration-dependent manner. Celastrol significantly downregulated LPS induced TLR4expression on LP-1human multiple myeloma cells. Celastrol significantly inhibited LPS induced VEGF secretion in LP-1human multiple myeloma cells. Celastrol inhibited the IKK/NF-kB pathway induced by LPS. Celastrol effectively blocked nuclear translocation of the p65subunit. Celastrol may be used as a NFkappa B inhibitor to inhibit myeloma cell proliferation. Celastrol could inhibit lipopolysaccharide induced angiogenesis by suppressing TLR4triggered NF-kappa B activation.