caveolae与肿瘤细胞的多药耐药性

 caveolae是特化的细胞膜微结构域，它由其特异性的被覆蛋白caveolin及多种脂类分子和膜蛋白组成，在细胞外分子的内化、信号的跨膜转导和胆固醇的转运过程中起着重要的作用。新近的研究表明，caveolae及其某些组成成分在肿瘤多药耐药细胞中表达上调，并有可能参与了肿瘤细胞多药耐药性的形成。对caveolae与肿瘤细胞多药耐药性的研究进展进行了综述。     

Immunohistochemical study on caveolin-1α in regenerating process of tubular cells in gentamicin-induced acute tubular injury in rats

Caveolin-1, a principal component of caveolae, modulates growth signaling, endocytosis, and intracellular transport. We examined the expression of caveolin-1α and its relation to cell cycle and caveolin-interacting growth factor receptors in regenerating proximal tubules (PTs) after gentamicin-induced acute renal failure in rats. Caveolin-1α appeared in regenerating PTs as early as day 4 after last gentamicin, peaked at days 6 to 8, and showed cytoplasmic pattern after day 8. Immunoelectron microscopy revealed caveolin-1α-positive caveolae on the cell membrane and in cytoplasms in regenerating PTs at days 4 to 8 and caveolin-positivity confined to cytoplasms after day 10. The number of PT cells with proliferation markers peaked at day 6 and decreased afterwards as expression of cyclin-dependent kinase inhibitors increased. Platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) were colocalized with caveolin-1α in proliferating PTs as early as day 4. Phosphorylated EGFR increased at day 8 and afterwards when caveolins dissociated from EGFR or decreased. In case of PDGFR-β, phosphorylation seemed to be associated with the increase and association of caveolins to the receptors. Our results suggest that transient expression of caveolin-1α in early regenerating PTs might contribute to the regenerating process of PTs through modulating growth factor receptors.     

Caveolae/Caveolin-1在血管生成和平滑肌细胞增殖中的作用

目的 小凹(cavcolae)是细胞膜上的特异性凹陷,小凹蛋白(caveolin)是构成Caveolae的标志性蛋白,起到维持Caveolae结构的作用并参与胞吞和胞内运输作用、胆固醇运输、信号传导等过程.Caveolin-1富含于多种类型的细胞内,其中内皮细胞和平滑肌细胞是含量最丰富的细胞类型,对血管生成和平滑肌细胞增殖起重要作用.     

Mechanisms of the increase in the permeability of the blood–tumor barrier obtained by combining low-frequency ultrasound irradiation with small-dose bradykinin

The research was conducted to study the characteristics of the noninvasive, reversible, targeted opening of the blood–brain barrier (BBB) by use of low-frequency ultrasound (LFU) irradiation and the selective opening of the blood–tumor barrier (BTB) by intracarotid infusion of bradykinin (BK) in small-dose, with the objective of exploring maximum opening of the BTB by combining LFU irradiation with BK infusion. Thus, it provides new therapeutic strategies for targeted transport of macromolecular or granular drugs to the brain. By using the rat C6 glioma model it was shown that extravasation of Evans blue (EB) through the BTB was significantly increased by combining LFU irradiation (frequency = 1.0 MHz, power = 12 mW, duration = 20 s) with intracarotid small-dose BK infusion, compared with utilizing the two methods separately. By transmission electron microscopy (TEM) we observed that this combination significantly increased the number of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB. An even more significant increase was observed by using RT-PCR, western blot, immunohistochemistry, and immunofluorescence to detect mRNA and changes of expression of the caveolae structure proteins caveolin-1 and caveolin-2 of BMECs. In summary, this research concludes that LFU irradiation and small-dose BK together selectively enhance the permeability of the BTB and increase the number of pinocytic vesicles of BMECs to a maximum. Significant up-regulation of the level of expression of caveolae structure proteins caveolin-1 and caveolin-2 might be the molecular mechanism of the co-enhanced endocytotic transport by BMECs. Thus, this research provides new therapeutic strategies for targeted transport of macromolecular drugs and the design of drugs. Chun-yi Xia and Zhen Zhang contributed equally to this work.     

Enhanced caveolae-mediated endocytosis by diagnostic ultrasound in vitro

The modulation of cellular endothelial permeability is a desirable goal for targeted delivery of labels and therapeutic macromolecules; the underlying mechanisms, however, remains poorly understood. Here, we hypothesize that a higher endothelial permeability may result as an outcome of selective enhancement of caveolar endocytosis by ultrasound (US), in the frequency and intensity range of current clinical diagnostic use. To assess the role of free radicals in this phenomenon, we exposed confluent human endothelial cells to pulsed diagnostic US for 30 min, with a mechanical index (MI) of 0.5 and 1.2, using a 1.6-MHz cardiac US scan, and endothelial cells not exposed to US were used as control. Here we show that pulsed diagnostic US with a MI of 1.2 (high mechanical index ultrasound [HMIUS]) were able to selectively enhance endothelial caveolar internalization of recombinant glutathione-S-transferase (GST)-Tat11-EGFP fusion protein (26 +/- 1 vs. 11.6 +/- 1 A.U, p < 0.001 vs. control), without disruption of plasma membrane integrity. Moreover, pulsed diagnostic US with a MI of 0.5 (low mechanical index ultrasound) did not increase caveolar endocytosis compared with control (11.4 +/- 1.2 vs. 11.6 +/- 1). Free-radical generation inhibitors, such as catalase and superoxide dismutase, reduced the HMIUS-induced caveolar internalization by a 49.29% factor; finally, HMIUS-induced caveolar endocytosis was found to be associated with a significant increase in the phosphorylation of tyr-14-caveolin1, ser1177-eNOS and Thr202/Tyr204-ERK(1/2) compared with control. These findings show how HMIUS irradiation of human endothelial cells cause a selective enhancement of caveolar-dependent permeability, partially mediated by free radicals generation, inducing a marked increase of phosphorylation of caveolar-related proteins. Thus, the use of diagnostic US could potentially be used as an adjuvant to drive caveolar traffic of extracellular peptides by using a higher level of US energy.     

Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy

Field emission scanning electron microscopy (FE SEM) was used to visualize the distribution of virus-associated components, the virus-attachment (G) protein, and the host-cell-derived lipid, GM1, in respiratory syncytial virus (RSV) filaments. RSV-infected cells were labeled in situ with a G protein antibody (MAb30) whose presence was detected using a second antibody conjugated to colloidal gold. No bound MAb30 was detected in mock-infected cells, whereas significant quantities bound to viral filaments revealing G protein clusters throughout the filaments. GM1 was detected using cholera toxin B subunit conjugated to colloidal gold. Mock-infected cells revealed numerous GM1 clusters on the cell surface. In RSV-infected cells, these gold clusters were detected on the filaments in low, but significant, amounts, indicating the incorporation of GM1 within the viral envelope. This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus.     

Drug transport to the brain: key roles for the efflux pump P-glycoprotein in the blood-brain barrier

1. The blood-brain barrier (BBB) contributes to brain homeostastis and fulfills a protective function by controlling the access of solutes and toxic substances to the central nervous system (CNS). The efflux transporter P-glycoprotein (P-gp) is a key element of the molecular machinery that confers special permeability properties to the BBB. 2. P-gp, which was initially recognized for its ability to expel anticancer drugs from multidrug-resistant cancer cells, is strongly expressed in brain capillaries. Its expression in the BBB limits the accumulation of many hydrophobic molecules and potentially toxic substances in the brain. 3. The purpose of this review is to summarize the current state of knowledge about the expression of P-gp, its cellular localization as well as its possible functions in the BBB.     

Identification of caveolae and caveolin in C6 glioma cells

Caveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identification of CAV and the known caveolin isoforms (1alpha, 1beta, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immunofluorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely fluorescent puncta throughout the cytoplasm and diffuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two different methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin-2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5-40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain.     

Caveolin-1在内皮素-1诱导血管平滑肌细胞增殖中的作用

目的: 探讨caveolin-1蛋白在VSMC的分布、在ET-1诱导的VSMC增殖中表达的变化情况。方法: 在培养的VSMC上,使用 TdR掺入法观察ET-1和BQ123对其DNA合成的影响;用荧光免疫组化方法观察caveolin-1蛋白在VSMC的分布,ET-1对caveolin-1蛋白表达的影响;用Western blot检测ET-1和BQ123对caveolin-1蛋白表达的影响。结果: ET-1可以呈浓度梯度地刺激VSMC的增殖,ETAR特异性阻断剂BQ123可以抑制ET-1的作用;免疫荧光证实了在VSMC上,caveolin-1分布于细胞表面,在ET-1刺激增殖过程中,VSMC上caveolin-1蛋白荧光强度下降;Western blot证实ET-1可以抑制caveolin-1蛋白的表达。结论: 在VSMC上,caveolin-1主要分布于细胞表面,在ET-1刺激增殖过程中,caveolin-1蛋白表达发生了变化,ET-1可通过ETAR抑制caveolin-1蛋白的表达。