Biomimetic hypoxia-triggered RNAi nanomedicine for synergistically mediating chemo/radiotherapy of glioblastoma.

Although RNA interference (RNAi) therapy has emerged as a potential tool in cancer therapeutics, the application of RNAi to glioblastoma (GBM) remains a hurdle. Herein, to improve the therapeutic effect of RNAi on GBM, a cancer cell membrane (CCM)-disguised hypoxia-triggered RNAi nanomedicine was developed for short interfering RNA (siRNA) delivery to sensitize cells to chemotherapy and radiotherapy. Our synthesized CCM-disguised RNAi nanomedicine showed prolonged blood circulation, high BBB transcytosis and specific accumulation in GBM sites via homotypic recognition. Disruption and effective anti-GBM agents were triggered in the hypoxic region, leading to efficient tumor suppression by using phosphoglycerate kinase 1 (PGK1) silencing to enhance paclitaxel-induced chemotherapy and sensitize hypoxic GBM cells to ionizing radiation. In summary, a biomimetic intelligent RNAi nanomedicine has been developed for siRNA delivery to synergistically mediate a combined chemo/radiotherapy that presents immune-free and hypoxia-triggered properties with high survival rates for orthotopic GBM treatment.

Inhibition of the Rac1-WAVE2-Arp2/3 signaling pathway promotes radiosensitivity via downregulation of cofilin-1 in U251 human glioma cells.

The Ras-related C3 botulinum toxin substrate 1 (Rac1)-WASP-family verprolin-homologous protein-2 (WAVE2)-actin-related protein 2/3 (Arp2/3) signaling pathway has been identified to be involved in cell migration and invasion in various types of cancer cell. Cofilin­1 (CFL­1), which is regulated by the Rac1­WAVE2­Arp2/3 signaling pathway, may promote radioresistance in glioma. Therefore, the present study aimed to investigate the potential role of the Rac1­WAVE2­Arp2/3 signaling pathway in radioresistance in U251 human glioma cells and elucidate its affect on CFL­1 expression. Western blot analysis was performed to evaluate the protein expression of CFL­1. In the present study, Rac1 was inhibited by NSC 23766, WAVE2 was inhibited by transfection with short hairpin (sh)RNA­WAVE2 using Lipofectamine™ 2000 and Arp2/3 was inhibited by CK­666. Cell viability was measured using the 3­(4,5­dimethylthiazol­2­yl)-2,5­diphenyltetrazolium bromide assay, the cell migration ability was examined by a wound­healing assay, and the cell invasion ability was assessed using a Transwell culture chamber system. The results showed that inhibition of the Rac1­WAVE2­Arp2/3 signaling pathway using NSC 23766, shRNA­WAVE2 or CK­666 reduced the cell viability, migration and invasion abilities in U251 human glioma cells, concordant with a reduced expression of CFL­1. Furthermore, the expression of CFL­1 was significantly increased in radioresistant U251 glioma cells when compared with normal U251 human glioma cells. These findings indicate that inhibition of the Rac1­WAVE2­Arp2/3 signaling pathway may promote radiosensitivity, which may partially result from the downregulation of CFL­1 in U251 human glioma cells.

Phosphoglycerate kinase 1 promotes radioresistance in U251 human glioma cells.

Phosphoglycerate kinase 1 (PGK1) has been found to be increased in radioresistant astrocytomas. The present study was designed to investigate the potential role of PGK1 in the radioresistance in U251 human cells. Quantitative PCR and western blot analysis were performed to evaluate PGK1 expression for mRNA levels and protein levels, respectively. The short hairpin RNA (shRNA)-PGK1 and the high expression plasmids were transfected to radioresistant U251 cells (RR-U251 cells) or normal U251 cells using lipofectamine™ 2000. The cell viability was determined by MTT assay. The wound-healing assay (WHA) was used to evaluate cell migration ability. Cell invasion abilities were examined using a Transwell culture chamber system. Our results showed that the expression of PGK1 was significantly increased in RR-U251 cells compared to normal U251 cells. Following irradiation, the cell viability as well as the migration and invasion ability were significantly higher in RR-U251 cells compared with normal U251 cells. Downregulating PGK1 using shRNA induced a significantly downregulated cell viability and decreased migration and invasion ability, and overexpression of PGK1 contributed to upregulated cell viability and increased migration and invasion ability, both in RR-U251 cells and normal U251 cells. These findings suggest that PGK1 could promote radioresistance in U251 human cells.

Gossypin induces G2/M arrest in human malignant glioma U251 cells by the activation of Chk1/Cdc25C pathway.

Gossypin is a flavone that was originally isolated from Hibiscus vitifolius and has traditionally been used for the treatment of diabetes, jaundice, and inflammation. Recently, gossypin was found to have potent anticancer properties; however, its effect on human gliomas still remain unknown. To investigate the potential anticancer effects of gossypin on malignant gliomas and analyze the associated molecular mechanisms, we treated human glioma U251 cells with gossypin. Our study showed that the treatment of U251 cells with gossypin inhibited cell proliferation in a dose- and time-dependent manner and was observed to be minimally toxic to normal human astrocytes. Gossypin's effect on cell cycle distribution was observed, and we found that it induced G2/M-phase arrest in U251 cells. An analysis of cell-cycle regulatory proteins indicated that the arresting effect of gossypin on the cell cycle at G2/M phase was involved in the phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinase 1 (Chk1). These findings indicate that gossypin is a potential treatment of gliomas because of gossypin's potential to regulate the proliferation of U251 cells via the cell-cycle regulatory proteins Chk1 and Cdc25C.

Icariin attenuates cardiac remodelling through down-regulating myocardial apoptosis and matrix metalloproteinase activity in rats with congestive heart failure.

OBJECTIVES: In this study, the anti-heart failure effect of icariin, a natural flavonol glycoside, and the underlying mechanisms were investigated. METHODS: Heart failure was induced by isoproterenol in male Sprague-Dawley rats. Matrix metalloproteinase activity was determined by gelatin zymography assay. The mRNA expression was determined by real-time PCR. The protein expression was determined by Western bolt. Mitochondria structure was examined by transmission electron microscopy. KEY FINDINGS: Isoproterenol administration resulted in a severe heart failure, as shown by the increased levels of left ventricular weight index, heart rate, left ventricular end diastolic pressure, maximal rate of left ventricular pressure decline (dp/dt(min) ), decreased levels of left ventricular systolic pressure and maximal rate of left ventricular pressure rise (dp/dt(max) ). Against these, icariin dose-dependently reversed the changes of these cardiac morphometric and haemodynamic parameters. In addition, icariin significantly inhibited serum levels of tumour necrosis factor-α, noradrenaline, angiotensin II and brain natriuretic peptide in rats with congestive hear failure and improved the histological changes, including cardiocyte hypertrophy, cardiocyte degeneration, inflammatory infiltration and cardiac desmoplasia. Furthermore, the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, which regulate collagen production, were also blocked by icariin. Moreover, myocardial apoptosis was remarkably attenuated by icariin through regulating Bcl-2/Bax axle. CONCLUSIONS: Icariin ameliorates left ventricular dysfunction and cardiac remodelling through down-regulating matrix metalloproteinase-2 and 9 activity and myocardial apoptosis in rats with congestive heart failure.

[Effect of bone morphogenetic protein 4 on glioma stem cell proliferation and apoptosis in vitro].

OBJECTIVE: To investigate the role of bone morphogenetic protein 4 (BMP4) on the proliferation and apoptosis in glioma stem cells. METHODS: Stem cells were isolated from a human glioma cell line U87 by using vincristine and characterized by immunofluorescence assay. Proliferation and apoptosis were determined by soft agar colony assay and flow cytometry; Cyclin D1, Bcl-2 and Bax were detected by Western blot analysis. RESULTS: BMP4 inhibited cell proliferation and promoted apoptosis in U87 glioma stem cells. Moreover, Bcl-2 and Cyclin D1 expression were decreased by BMP4, while Bax level was elevated. CONCLUSION: BMP4 can inhibit U87 glioma stem cells proliferation through downregulating Cyclin D1 level, and promote apoptosis through induction of Bax expression and inhibition of Bcl-2 level. It suggests that BMP4 plays an important role in human glioma stem cell biology.

[Effect of osteopontin silencing by lentivirus-mediated delivery of siRNA on glioma cell invasion and apoptosis].

OBJECTIVE: To investigate the effect of osteopontin silencing on the invasion and apoptosis of U251 cells. METHODS: The invasion, apoptosis and levels of uPA, MMP-2 and MMP-9 were determined by invasion assay, flow cytometry, Western blot and real-time fluorescence quantitative PCR respectively. RESULTS: Osteopontin small interference RNA (siRNA) inhibited osteopontin expression and cell invasion, promoted apoptosis in U251 cells. In addition, the expression of Bcl-2, uPA, MMP-2 and MMP-9 was decreased, while Bax level was elevated. CONCLUSION: Osteopontin siRNA can inhibit U251 cells invasion via the down-regulation of uPA, MMP-2 and MMP-9 levels, and promote apoptosis through induction of Bax expression and inhibition of Bcl 2 level. It suggests that osteopontin plays an important role in human glioma progression.

Notch1 induces enhanced expression of Delta-like-1 in the U251MG glioma cell line.

The Notch signaling pathway takes part in coordinated regulation of cell growth, survival and differentiation. Previous findings have shown that Notch1 and Delta-like-1 (DLL1) are overexpressed in many glioma cell lines and primary human gliomas. Down-regulation of DLL1 by RNA interference inhibits proliferation and induces apoptosis in multiple glioma cell lines. Our studies showed that Notch1 expression plasmid induced more expression of DLL1 in the U251MG glioma cell line. Adversely, blocking Notch1 receptors down-regulated the expression of DLL1. Both down-regulating DLL1 and blocking Notch1 receptors induced U251MG cell apoptosis and proliferation inhibition, and combining the two treatments produced stronger effects than the sum of a single treatment. These findings suggest a positive feedback loop between Notch1 and DLL1, which may become an effective combined therapeutic target.