Jian-Ri Li

Docosahexaenoic acid reduces cellular inflammatory response following permanent focal cerebral ischemia in rats.

Cellular inflammatory response plays an important role in ischemic brain injury and anti-inflammatory treatments in stroke are beneficial. Dietary supplementation with docosahexaenoic acid (DHA) shows anti-inflammatory and neuroprotective effects against ischemic stroke. However, its effectiveness and its precise modes of neuroprotective action remain incompletely understood. This study provides evidence of an alternative target for DHA and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of 3 consecutive days of DHA preadministration on circulating and intracerebral cellular inflammatory responses in a rat model of permanent cerebral ischemia. DHA exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, edema and blood-brain barrier disruption. The results of enzymatic assay, Western blot, real-time reverse transcriptase polymerase chain reaction and flow cytometric analysis revealed that DHA reduced central macrophages/microglia activation, leukocyte infiltration and pro-inflammatory cytokine expression and peripheral leukocyte activation after cerebral ischemia. In parallel with these immunosuppressive phenomena, DHA attenuated post-stroke oxidative stress, c-Jun N-terminal kinase (JNK) phosphorylation, c-Jun phosphorylation and activating protein-1 (AP-1) activation but further elevated ischemia-induced NF-E2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) expression. DHA treatment also had an immunosuppressive effect in lipopolysaccharide/interferon-γ-stimulated glial cultures by attenuating JNK phosphorylation, c-Jun phosphorylation and AP-1 activation and augmenting Nrf2 and HO-1 expression. In summary, we have shown that DHA exhibited neuroprotective and anti-inflammatory effects against ischemic brain injury and these effects were accompanied by decreased oxidative stress and JNK/AP-1 signaling as well as enhanced Nrf2/HO-1 expression.

Infection of Pericytes In Vitro by Japanese Encephalitis Virus Disrupts the Integrity of the Endothelial Barrier

ABSTRACT Though the compromised blood-brain barrier (BBB) is a pathological hallmark of Japanese encephalitis-associated neurological sequelae, the underlying mechanisms and the specific cell types involved are not understood. BBB characteristics are induced and maintained by cross talk between brain microvascular endothelial cells and neighboring elements of the neurovascular unit. In this study, we show a potential mechanism of disruption of endothelial barrier integrity during the course of Japanese encephalitis virus (JEV) infection through the activation of neighboring pericytes. We found that cultured brain pericytes were susceptible to JEV infection but were without signs of remarkable cytotoxicity. JEV-infected pericytes were found to release biologically active molecules which activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1), and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. Infection of pericytes with JEV was found to elicit elevated production of interleukin-6 (IL-6), which contributed to the aforementioned endothelial changes. We further demonstrated that ubiquitin-protein ligase E3 component n-recognin-1 (Ubr 1) was a key upstream regulator which caused proteasomal degradation of ZO-1 downstream of IL-6 signaling. During JEV central nervous system trafficking, endothelial cells rather than pericytes are directly exposed to cell-free viruses in the peripheral bloodstream. Therefore, the results of this study suggest that subsequent to primary infection of endothelial cells, JEV infection of pericytes might contribute to the initiation and/or augmentation of Japanese encephalitis-associated BBB breakdown in concerted action with other unidentified barrier disrupting factors.

Dual inhibitor of phosphoinositide 3-kinase/mammalian target of rapamycin NVP-BEZ235 effectively inhibits cisplatin-resistant urothelial cancer cell growth through autophagic flux.

PURPOSE Therapeutically induced autophagic cell death has been proven to be effective in cases of solid tumors. The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 possesses antitumor activity against solid tumors. Inhibition of mTOR has been shown to elicit autophagy. In this study, we examined the antiproliferation and autophagic activities of NVP-BEZ235 in parental and cisplatin-resistant urothelial carcinoma (UC) cells. MATERIALS AND METHODS Two UC cell lines, NTUB1 and a cisplatin-resistant subline N/P(14), were applied to examine the cytotoxic effect of NVP-BEZ-235. The cell death mechanism was also evaluated. RESULTS NVP-BEZ235 was effective in inhibiting the growth of UC cells including parental and cisplatin-resistant cells on flow cytometry assay and Western blot. Although NVP-BEZ235 did not induce LC3-II conversion, it did elicit acidic vesicular organelle (AVO) development on flow cytometry. On Western blot, NVP-BEZ235 decreased p62 and phospho-Rb expressions in a concentration-dependent manner. GFP-LC3 conversion and the appearance of cleaved-GFP following NVP-BEZ235 treatment were demonstrated on Western blot. In addition, lysosomotropic inhibition of autophagy by chloroquine (CQ), an agent that is currently in clinical use and a known antagonist of autophagy, resulted in proliferation of UC cells. Thus, inhibition of autophagic flux by CQ appears to be a survival mechanism that counteracts the anticancer effects of NVP-BEZ235. CONCLUSIONS We demonstrated that NVP-BEZ235 inhibits UC cell proliferation by activating autophagic flux and cell cycle arrest, but does not induce apoptotic cell death. Our findings suggest that the anticancer efficacy of NVP-BEZ235 is due to autophagic flux and co-treatment with CQ counteracts the cytotoxic effect.

Disruption of in vitro endothelial barrier integrity by Japanese encephalitis virus‐Infected astrocytes

Blood–brain barrier (BBB) characteristics are induced and maintained by crosstalk between brain microvascular endothelial cells and neighboring cells. Using in vitro cell models, we previously found that a bystander effect was a cause for Japanese encephalitis‐associated endothelial barrier disruption. Brain astrocytes, which neighbor BBB endothelial cells, play roles in the maintenance of BBB integrity. By extending the scope of relevant studies, a potential mechanism has been shown that the activation of neighboring astrocytes could be a cause of disruption of endothelial barrier integrity during the course of Japanese encephalitis viral (JEV) infection. JEV‐infected astrocytes were found to release biologically active molecules that activated ubiquitin proteasome, degraded zonula occludens‐1 (ZO‐1) and claudin‐5, and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. JEV infection caused astrocytes to release vascular endothelial growth factor (VEGF), interleukin‐6 (IL‐6), and matrix metalloproteinases (MMP‐2/MMP‐9). Our data demonstrated that VEGF and IL‐6 released by JEV‐infected astrocytes were critical for the proteasomal degradation of ZO‐1 and the accompanying disruption of endothelial barrier integrity through the activation of Janus kinase‐2 (Jak2)/signal transducer and activator of transcription‐3 (STAT3) signaling as well as the induction of ubiquitin–protein ligase E3 component, n‐recognin‐1 (Ubr 1) in endothelial cells. MMP‐induced endothelial barrier disruption was accompanied by MMP‐mediated proteolytic degradation of claudin‐5 and ubiquitin proteasome‐mediated degradation of ZO‐1 via extracellular VEGF release. Collectively, these data suggest that JEV infection could activate astrocytes and cause release of VEGF, IL‐6, and MMP‐2/MMP‐9, thereby contributing, in a concerted action, to the induction of Japanese encephalitis‐associated BBB breakdown. GLIA 2015;63:1915–1932

Metallic stent in the treatment of ureteral obstruction: Experience of single institute

Background: The Resonance® metallic stent has been reported to be sufficient for the management of malignant extrinsic ureteral obstructions within a 12‐month time period. To determine the effectiveness in each specific patient group, we report our experience using the Resonance® stent in the treatment of ureteral obstructions. Methods: We retrospectively reviewed 20 patients (23 stents) who successfully received the Resonance® metallic stents and divided them into a patent group (n = 19) and an obstructive group (n = 4) according to the treatment results. Twenty‐one stents were inserted via cystoscopy or ureteroscopy in a retrograde fashion. The remaining two were inserted via percutaneous nephrostomy in an antegrade manner. Follow‐up serum creatinine measurements and sonography were performed. The overall ureteral patency rate and the risk of stent failure were evaluated. Results: The overall ureteral patency rate was 82.6% (19/23). Patients with previous radiotherapy had a 50% (4/8) patency rate which was significantly lower than non‐radiotherapy patients (100%, 15/15, p = 0.028). Malignant obstructions in those other than radiotherapy patients had a 100% patency rate (5/5). Benign obstructions in those other than radiotherapy patients had a 100% patency rate (10/10). In the radiotherapy patients, the mode of therapy did not dominate the stent outcome. Conclusion: Patients with ureteral obstructions can be treated sufficiently with the Resonance® metallic stent. Patients who had gynecological malignancies and received radiotherapy had a higher failure rate after Resonance® metallic stent insertion.

Luteolin sensitizes human 786-O renal cell carcinoma cells to TRAIL-induced apoptosis

AIMS Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be one of the most promising candidates in research on treatments for cancer, including renal cell carcinoma (RCC). However, many cells are resistant to TRAIL-induced apoptosis which limits the potential application of TRAIL in cancer therapy. Luteolin, a naturally occurring flavonoid, has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity. In this study, we investigated whether luteolin treatment could modulate TRAIL-induced apoptosis in RCC. MAIN METHODS The effect of luteolin on TRAIL sensitivity was assessed in human RCC 786-O, ACHN, and A498 cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. KEY FINDINGS We found that nontoxic concentration of luteolin alone had no effect on the level of apoptosis, but a combination treatment of TRAIL and luteolin caused significant extrinsic and intrinsic apoptosis. The sensitization was accompanied by Bid cleavage, Mcl-1 and FLIP down-regulation, DR4/DR5 protein expression and cell surface presentation, and Akt and signal transducer and activator of transcription-3 (STAT3) inactivation. Among these phenomena, changes in FLIP, Akt, and, STAT3 are more prone to the effects of luteolin treatment. Studies have further demonstrated that inactivation of Akt or STAT3 alone was sufficient to down-regulate FLIP expression and sensitized 786-O cells to TRAIL-induced apoptosis. SIGNIFICANCE Data from this study thus provide in vitro evidence supporting the notion that luteolin is a potential sensitizer of TRAIL in anticancer therapy against human RCC involving Akt and STAT3 inactivation.

Autophagy contributes to gefitinib-induced glioma cell growth inhibition.

Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation.

Rictor-dependent AKT activation and inhibition of urothelial carcinoma by rapamycin

OBJECTIVE We previously reported a very high cumulative incidence of urothelial carcinoma in Taiwanese kidney transplant recipients. Rapamycin, the inhibitor of mTOR Complex 1, provides alternative immunosuppressive therapy after kidney transplantation with less neoplastic potential. We examined the in vivo and in vitro effects of rapamycin on urothelial carcinoma. MATERIALS AND METHODS The rat model of urothelial carcinoma was induced by 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in Fischer F344 rats. The anti-tumor effect of rapamycin was assessed grossly, microscopically, and by Western blot analysis. The mechanism of rapamycins attenuation of urothelial carcinoma was also evaluated by T24 cells. RESULTS Rapamycin significantly reduced urinary bladder tumor growth in the rat model of 0.05% BBN-induced urothelial carcinoma (P < 0.001). The blood trough levels of rapamycin were correlated with the occurrence of urothelial carcinoma. In vitro, rapamycin also inhibited the cell proliferation, migration, and invasion, as well as the protein expression of vascular endothelial growth factor-A of T24 urothelial carcinoma cells, whereas rapamycin did not induce significant apoptosis in T24 cells. Rapamycin decreased the expression of phospho-mTOR, phospho-S6K, cyclin D1, and VEGF-A. Rapamycin also activated AKT in T24 cells in the rat model of urothelial carcinoma. The rapamycin-associated activation of AKT was inhibited by rictor siRNA, but not raptor siRNA. CONCLUSIONS This study provides in vitro and in vivo evidence that rapamycin may inhibit the development of urothelial carcinoma. The present findings also suggest rictor-dependent AKT activation as a consequence of mTORC1 inhibition.

Induction of apoptosis by luteolin involving akt inactivation in human 786-o renal cell carcinoma cells.

There is a growing interest in the health-promoting effects of natural substances obtained from plants. Although luteolin has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity, the molecular mechanisms have not been well elucidated. This study provides evidence of an alternative target for luteolin and sheds light on the mechanism of its physiological benefits. Treatment of 786-O renal cell carcinoma (RCC) cells (as well as A498 and ACHN) with luteolin caused cell apoptosis and death. This cytotoxicity was caused by the downregulation of Akt and resultant upregulation of apoptosis signal-regulating kinase-1 (Ask1), p38, and c-Jun N-terminal kinase (JNK) activities, probably via protein phosphatase 2A (PP2A) activation. In addition to being a concurrent substrate of caspases and event of cell death, heat shock protein-90 (HSP90) cleavage might also play a role in driving further cellular alterations and cell death, at least in part, involving an Akt-related mechanism. Due to the high expression of HSP90 and Akt-related molecules in RCC and other cancer cells, our findings suggest that PP2A activation might work in concert with HSP90 cleavage to inactivate Akt and lead to a vicious caspase-dependent apoptotic cycle in luteolin-treated 786-O cells.

A novel method of laparoscopy-assisted peritoneal dialysis catheter placement.

Peritoneal dialysis (PD) is a widely used renal replacement therapy for end-stage renal disease patients. We compared our novel laparoscopic-assisted method with conventional procedures in the catheter survival and complications. Seventy-three patients who underwent PD catheter placement were enrolled in this study. Our laparoscopic methods were characterized with smaller incision and additional fixation in the lower abdomen. Catheter migration developed in 4 patients in the conventional group compared with none in the laparoscopic group. The catheter dysfunction-free survival was significantly longer among the patients in the laparoscopic group than that in the conventional group (P=0.001). There were no significant differences between the 2 groups in peritonitis, exit-site hematoma, or exit-site infection. Laparoscopy-assisted PD catheter insertion with an intraperitoneal loop fixation is safe and provides good maintenance of catheter function (See the video, Supplemental Digital Content 1, http://links.lww.com/SLE/A28).