Furthermore, the clonogenic assay performed with a sustained treatment of U251 cells with -asarone for two weeks also indicated that 60 and 240 M of -asarone reduced 21.83% and 50.09% of colony formation rate compared with that of the untreated control, respectively (Figure 1C). Open in a separate window Figure 1 -asarone inhibited the growth of human glioma U251 cells. cells may be related to the suppression of hnRNPA2/B1-mediated signaling pathway. gene, is among the most abundant Keratin 18 antibody hnRNP proteins . Accumulating evidence has demonstrated that hnRNP A2/B1 is oncogenic and overexpressed in various tumor cells, including breast , pancreas , liver , gastric , and lung carcinoma cells . Moreover, hnRNP A2/B1 overexpression has also been observed in human glioma tissue specimens and is closely correlated with advanced glioma grades [5,11]. It is becoming increasingly clear that deregulation of alternative splicing involved in processing pre-mRNAs of diverse signaling proteins plays a direct role in cancer development and progression . Recently, hnRNP A2/B1 has been described in the regulation of alternative splicing of several tumor suppressors and oncogenes, such as Bcl-x [13,14,15], which is an anti-apoptotic protein belonging to the well-known Bcl-2 family. Moreover, accumulating evidence also revealed that suppression of hnRNP A2/B1 induced cell cycle arrest at G1 phase in cervical cancer cells , lung cancer cells [17,18], and human embryonic stem cells , which renders it a potential novel target for tumor therapy. -asarone is the main component in the volatile oil of Rhizoma, a Chinese herbal medicine proved to possess anti-glioma activity in our recent study . It has been described that -asarone exhibited anti-tumor activities on colorectal cancer cells [21,22] and gastric cancer cells . Recently, we found that -asarone obviously inhibited the growth of glioma cells , which was further confirmed by another group . Moreover, -asarone has been shown to not only directly cross the bloodCbrain barrier (BBB), but also to improve the permeability of the BBB and inhibit the function of P-glycoprotein [26,27,28]. A two-dimensional gel electrophoresis-based proteomics has been recently employed by our group to comprehensively investigate the cellular targets of -asarone. HnRNP A2/B1 was successfully Thymol identified as one of the key protein targets regulated by -asarone . Most recently, we found that -asarone inhibited invasion and the epithelialCmesenchymal transition (EMT) in U251 cells by suppressing HnRNP A2/B1 . Thus, it is interesting for us to further explore the potential role of hnRNP A2/B1-mediated signaling pathway in the anti-glioma effect of -asarone. In the current study, we further characterized the inhibitory effect of -asarone on the growth of U251 cells. Then, the induction of apoptosis and cell cycle arrest by -asarone was determined. Furthermore, we also sought to identify the underlying Thymol role of hnRNP A2/B1 and its relevant mechanisms during these processes. Finally, the anti-glioma effect and the underlying mechanisms were further confirmed in nude mice bearing U251 tumor xenografts. 2. Results 2.1. -Asarone Inhibited the Growth of U251 Cells To determine the influence of -asarone on the growth of human glioma cells, we first evaluated the inhibitory effect of -asarone on the cell viability of human glioma U251 cells by sulforhodamine B (SRB) assay. Figure 1A demonstrated that -asarone obviously inhibited the cell viability of U251 cells in a concentration-dependent manner (IC50 = 361 M). Then, the trypan blue exclusion assay was performed to determine the cell proliferation. Our results showed that -asarone suppressed the proliferation of U251 cells in a concentration- and time-dependent manner (Figure 1B). Furthermore, the clonogenic assay performed with a sustained treatment of U251 cells with -asarone for two weeks also indicated that 60 and 240 M of -asarone reduced 21.83% and 50.09% of colony formation rate compared with that of the untreated control, respectively (Figure Thymol 1C). Open in a separate window Figure 1 -asarone inhibited the growth of human glioma U251 cells. (A) Cells were treated with -asarone as indicated for 72 h and the cell viability was determined by SRB assay. (B) Cells were treated with -asarone as indicated for 96 h and cell proliferation was measured by trypan blue exclusion assay. (C) Colony formation ability was determined in the cells treated with -asarone (60 and 240 M) for two weeks and then stained with 0.005% crystal violet. (D) Quantification.