Molecular Mechanism Studies Of Neferine-induced G1Cell Cycle Arrest In Human Osteosarcoma Cells

Osteosarcoma is a type of malignancy derived from mesenchymal stem cells or osteoblasts. It is the most common form of primary bone cancers. Clinically, the tumor is characterized by early age of onset, high malignancy, and frequent metastases to lung. Osteosarcoma is conventionally treated by the combination of chemotherapy and surgery. Given the multidrug resistance and high toxicity of chemotherapy, great efforts have been devoted to searching for new cancer chemopreventive agents from natural products.Benzylisoquinoline alkaloid (BA) is the structural backbone of many alkaloids. Many benzylisoquinoline alkaloids have been reported to show therapeutic properties. Neferine, a bis-benzylisoquinoline alkaloid isolated from the green seed embryos of lotus, has been shown to attenuate bleomycin-induced pulmonary fibrosis, to inhibit the proliferation of vascular smooth muscle cells, and to reduce platelet aggregation. Recently, neferine was shown to reverse drug resistance in human hepatocarcinoma cells HepG2and human gastric carcinoma cells SGC7901/VCR. It is still unknown whether neferine possesses direct anti-tumor effect.In our previous study, the growth-inhibitory effects of nerferine were evaluated on Hela, MCF-7, Saos-2and U20S cells. Neferine significantly inhibited the proliferation of those cell lines. Neferine indued G1-phase cell cycle arrest in human osteosarcoma cells with down regulation of cyclin E and upregulation of p21. However, the mechanism by which p21is upregulated remains to be determined. Since neferine does not cause DNA damage in U20S cells, it probably does not act as a genotoxicant.To understand the molecular mechanism underlying the neferine-mediated G1cell cycle arrest, we characterized the signal pathways that might be involved in the regulation of Gl/S progression.· Neferine-induced cell cycle arrest is caused by up-regulation of p211. To determine the role of p21in the neferine-induced U20S cell growth inhibition, siRNA was used to knock down p21expression in U20S cells. BrdU incorporation assay was used to determine the S phase population in the presence or absence of neferine. Down-regulation of p21by RNAi markedly attenuated the reduction of S phase cell population caused by neferine, indicating that the up-regulation of p21was responsible for G1cell cycle arrest induced by neferine.2. Neferine did not affect the transcription of p21in U20S cells. The up regulation of p21at protein level by neferine is probably mediated by post-transcriptional mechanism. To determine whether neferine upregulates p21through transcriptional activation, real-time PCR was used to measure mRNA level in U20S cells treated with neferine. The result showed that there was no obvious changes of p21mRNA level in U20S cells treated with neferine compared to DMSO treated cells. The result suggested up regulation of p21by neferine was not due to transcriptional activation. To further clarify the mechanism by which neferine up regulated p21, the half-life p21protein in U20S cells was determined. After blocking translation with cycloheximide (CHX), the half-life of p21was analyzed by Western blotting. The half-life of p21was significantly prolonged in cells treated with neferine when compared with control. These data indicated that neferine increased p21abundance by prolonging the half-life of the p21protein in U20S cells.·Neferine activates p38MAPK and JNK pathways but not the AKT and MEK/ERK pathways. A number of studies have demonstrated that MAPK (Mitogen-activated protein kinase), MEK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal kinase) and AKT (also known as protein kinase B) cascade are involved in p21accumulation and G1cell cycle arrest. We next determined which signaling pathway mediated the upregulation of p21at protein level.1. We treated the U20S cells with neferine and examined the activation status of the MEK/ERK, AKT, p38MAPK and JNK1/2pathways using phospho-specific antibodies. There were no obvious changes in the levels of phosphorylated AKT and ERK in U20S cells treated with neferine. However, the levels of phosphorylated p38MAPK and JNK were significantly increased. From these results, we concluded that AKT and ERK pathways were not involved in the upregulation of p21at protein level.2. JNK is not involved in neferine-induced p21accumulation in U20S cells.(1) To test whether JNK1/2pathway is involved in neferine-induced p21accumulation in U20S cells, we used SP600125to block JNK activity. SP600125treatment itself led to a significant elevation in the level of p21accumulation, indicating that the increase in p21abundance could not be caused by JNK1/2activation.(2) siRNAs were used to knock down JNK1and JNK2expression in U20S cells. Consistent with the upregulation of p21as a result of pharmacological inhibition of JNK1/2, p21was up regulated by knockdown of JNK1/2. These results suggested that the activaton of JNK is not responsible for the upregulation of p21.3. p38MAPK is required for neferine-induced p21accumulation. To determine the role of p38MAPK in the neferine-induced U20S cell G1arrest, the following experiments were performed:(1) SB203580(a p38MAPK inhibitor) was used to block p38MAPK activity. Neferine treatment alone significantly increased the level of p21in both U20S and Saos-2cells; SB203580significantly attenuated the increase of p21protein.(2) When p38a and p were knocked down in U20S cells, the up-regulation of p21and the reduction of S phase cell population caused by neferine were both attenuated.4. p38MAPK up-regulated p21protein throuth phosphorylation of p21at Ser130.(1) We compared the half-life of p21protein in U20S cells treated with neferine alone or in combination with SB203580. SB203580attenuated the prolonged p21half-life caused by neferine. These data indicated that neferine increased p21abundance by stabilizing the p21protein through the activation of p38MAPK.(2) The phosphorylated p21(p-p21Ser130), together with the total p21, was found to be upregulated by neferine. In SB203580treated cells and p38MAPK knockdown cells, upregulation of phosphorylated p21by neferine was attenuated compared to control cells. These results indicated that neferine exhibited a potent growth-inhibitory effect on human osteosarcoma cells, primarily by inducing cell cycle arrest at G1stages. The G1arrest was partially due to an up-regulation of p21. The up-regulation of p21by neferine was not caused by transcriptional activation. p38MAPK is required for neferine-induced p21accumulation. p38MAPK phosphorylated p21at Ser130and prolonged its half-life.