BackgroundTetramethylpyrazine (TMP) is one of the active ingredients extracted from the Chinese herb Chuanxiong, which has been used to treat cerebrovascular and cardiovascular diseases, pulmonary diseases and cancer. However, the molecular mechanisms underlying the actions of TMP have not been fully elucidated. In a previous study we showed that TMP-mediated glioma suppression and neural protection involves the inhibition of CXCR4 expression. The SDF-1/CXCR4 axis plays a fundamental role in many physiological and pathological processes. In this study, we further investigated whether the regulation of the SDF-1/CXCR4 pathway is also involved in the TMP-mediated inhibition of neovascularization or fibrosis and improvement of microcirculation.Methodology/Principal FindingsUsing a scratch-wound assay, we demonstrated that TMP significantly suppressed the migration and tubule formation of the human umbilical vein endothelial cell line ECV304 in vitro. The expression of CXCR4 in ECV304 cells is notably down-regulated after TMP treatment. In addition, TMP significantly suppresses corneal neovascularization in a rat model of corneal alkali burn injury. The expression of CXCR4 on days 1, 3 and 7 post-injury was determined through RT-PCR analysis. Consistent with our hypotheses, the expression of CXCR4 in the rat cornea is significantly increased with alkali burn and dramatically down-regulated with TMP treatment. Moreover, TMP treatment significantly attenuates bleomycin-induced rat pulmonary fibrosis, while immunofluorescence shows a notably decreased amount of CXCR4-positive cells in the TMP-treated group. Furthermore, TMP significantly down-regulates the expression of CXCR4 in platelets, lymphocytes and red blood cells. Whole-blood viscosity and platelet aggregation in rats are significantly decreased by TMP treatment.ConclusionsThese results show that TMP exerts potent effects in inhibiting neovascularization, fibrosis and thrombosis under pathological conditions; thus, the underlying mechanism of TMP might partially contribute to the down-regulation of CXCR4.
Abstract. Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb Chuanxiong. Increasing numbers of studies have indicated that tetramethylpyrazine hydrochloride (TMPH) has anticancer effects. However, the molecular mechanisms underlying the actions of TMPH have not been fully elucidated. In this study, using real-time RT-PCR and western blot techniques, we demonstrate that TMPH significantly downregulates the expression of the chemokine receptor CXCR4 in C6 glioma cells. Consistent with a role for CXCR4 in cancer development, TMPH inhibits the migration, proliferation and colony formation of C6 glioma cells in vitro more effectively than the CXCR4 antagonist AMD3100. Interestingly, TMPH does not affect the cell cycle when the cells are grown to 50-80% confluency but induces S-phase arrest at 100% confluency, as indicated by a significant reduction in the G1 and G2 populations. These findings were also confirmed in vivo. Rats were implanted with C6 glioma cells and treated with 100 mg/kg TMPH for 20 days. Our data show that tumour growth was significantly inhibited in rats treated with TMPH (4.14±2.81 mm 3 ) compared with tumour growth in control rats (55.9±14.12 mm 3 ). Microcirculation in the implants was sparser in the TMPH-treated rats than that in the control rats, as measured by FITC-dextran staining. Consistent with the in vitro results, TMPH significantly downregulated the expression of CXCR4 in C6 glioma implantation compared with the control. This study provides new insights into the mechanisms of the TMPH anticancer effects.
Retinoblastoma is a childhood ocular tumor caused by the inactivation of both alleles of the retinoblastoma gene (Rb1). Without Rb1 gene function, chromosomal aberrations are observed in retinoblastoma cells. The instability of the genome is closely associated with the repair of DNA double-strand breaks (DSBs). However, the precise molecular mechanism of action of Rb1 in DNA DSB repair remains unclear. Thus, in this study, we aimed to investigate whether the Rb1 gene affects DNA stability by assaying DNA DSB repair and also whether it regulates the proliferation of retinoblastoma cells. Rb1 immunofluorescence and RT-PCR were performed, demonstrating that the Rb1 gene is silenced in SO-Rb50 retinoblastoma cells, and the karyotype analysis of SO-Rb50 cells indicated that the loss of Rb1 function led to genomic instability; both numerical and structural chromosomal aberrations were observed in our study. In addition, the DNA DSB repair efficiency of the SO-Rb50 cells was measured by γ-H2AX immunofluorescence, a commonly used in situ marker of DNA DSBs, following exposure to ionizing radiation (IR) (2.5 and 5.0 Gy). We found that the DNA repair efficiency was significantly increased following IR-induced damage (P<0.01). However, there was no significant difference in DNA repair efficiency between the cells expressing exogenous Rb1 and the control (P>0.05). The assay for the screening of the effect of Rb1 on the sub-pathway of DNA DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated that Rb1 did not affect NHEJ activity, although it significantly promoted the HR pathway (HR levels increased by 2.46-fold) compared with the control (P<0.01). Furthermore, we found that the cell viability of the SO-Rb50 cells transfected with exogenous Rb1 was significantly inhibited (P<0.01) and cell cycle assay indicated that exogenous Rb1 induced S phase arrest (P<0.001) which also inhibited the proliferation of retinoblastoma cells (SO-Rb50) in vitro. Therefore, this study provides new insight into the mechanisms of action of the Rb1 gene in regulating the proliferation of retinoblastoma cells.
Ku80 plays a critical role in DNA double strand breaks repair. However, Ku80 is silenced in mature neurocytes. In this study, the mechanism of Ku80 silencing and its role in DNA double strand break repair in retinal neurocytes was investigated. Our data show that Ku80 expression is activated in primary cultured retinal neurocytes after treatment with 5-azacytidine in vitro, whereas methylation of -179 bp in Ku80 promoter induces Ku80 silencing in retinal neurocytes. Ku80 reactivation in retinal neurocytes by 5-azacytidine enhances DNA integrity after treatment with H(2)O(2). Therefore, our data suggest Ku80 might be a target for reactivation to increase retinal neuronal DNA repair.
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