Role of Kir4.1 Channels in Aminoglycoside-Induced Ototoxicity of Hair Cells.

The Kir4.1 channel, an inwardly rectifying potassium ion (K+) channel, is located in the hair cells of the organ of Corti as well as the intermediate cells of the stria vascularis. The Kir4.1 channel has a crucial role in the generation of endolymphatic potential and maintenance of the resting membrane potential. However, the role and functions of the Kir4.1 channel in the progenitor remain undescribed. To observe the role of Kir4.1 in the progenitor treated with the one-shot ototoxic drugs (kanamycin and furosemide), we set the proper condition in culturing Immortomouse-derived HEI-OC1 cells to express the potassium-related channels well. And also, that was reproduced in mice experiments to show the important role of Kir4.1 in the survival of hair cells after treating the ototoxicity drugs. In our results, when kanamycin and furosemide drugs were cotreated with HEI-OC1 cells, the Kir4.1 channel did not change, but the expression levels of the NKCC1 cotransporter and KCNQ4 channel are decreased. This shows that inward and outward channels were blocked by the two drugs (kanamycin and furosemide). However, noteworthy here is that the expression level of Kir4.1 channel increased when kanamycin was treated alone. This shows that Kir4.1, an inwardly rectifying potassium channel, acts as an outward channel in place of the corresponding channel when the KCNQ4 channel, an outward channel, is blocked. These results suggest that the Kir4.1 channel has a role in maintaining K+ homeostasis in supporting cells, with K+ concentration compensator when the NKCC1 cotransporter and Kv7.4 (KCNQ4) channels are deficient.

Potentiation of TRAIL­induced cell death by nonsteroidal anti­inflammatory drug in human hepatocellular carcinoma cells through the ER stress­dependent autophagy pathway.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed primary liver malignancy. The limited success with relapse of the disease in HCC therapy is frequently associated with the acquired resistance to anticancer drugs. To develop a strategy and design for overcoming the resistance of HCC cells to TNF­related apoptosis inducing ligand (TRAIL)­induced cell death, we evaluated the efficacy of a non­steroidal anti­inflammatory drug (NSAID) in combination with TRAIL against TRAIL­resistant HCC cells expressing a high level of CD44. We revealed by MTT and western blotting, respectively, that celecoxib (CCB), an NSAID, and 2,5­dimethyl celecoxib (DMC), a non­cyclooxygenase (COX)­2 inhibitor analog of CCB, were able to sensitize TRAIL­resistant HCC cells to TRAIL, implicating a COX­independent mechanism. CCB dose­dependently enhanced LC3­II and reduced p62 levels through AMPK activation and inhibition of the Akt/mTOR pathway and upregulated expression of ATF4/CHOP, leading to activation of endoplasmic reticulum (ER) stress­dependent autophagy. The TRAIL sensitization capacity of CCB in TRAIL­resistant HCC cells was abrogated by an ER stress inhibitor. In addition, we also revealed by flow cytometry and western blotting, respectively, that accelerated downregulation of TRAIL­mediated c­FLIP expression, DR5 activation and CD44 degradation/downregulation by NSAID resulted in activation of caspases and poly(ADP­ribose) polymerase (PARP), leading to the sensitization of TRAIL­resistant HCC cells to TRAIL and thereby reversal of TRAIL resistance. From these results, we propose that NSAID in combination with TRAIL may improve the antitumor activity of TRAIL in TRAIL­resistant HCC, and this approach may serve as a novel strategy that maximizes the therapeutic efficacy of TRAIL for clinical application.

Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation.

Recently, novel therapeutic strategies have been designed with the aim of killing cancer stem-like cells (CSCs), and considerable interest has been generated in the development of specific therapies that target stemness-related marker of CSCs. In this study, nonsteroidal anti-inflammatory drugs (NSAIDs) significantly potentiated Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-mediated cytotoxicity through apoptotic and autophagic cell death induction, but COX-2-inhibitory function was not required for NSAID-induced autophagy in CD44-overexpressing human chronic myeloid leukemia K562 (CD44highK562) cells. Importantly, we found that treatment with NSAIDs resulted in a dose-dependent increase in LC3-II level and decrease in p62 level and simultaneous reduction in multiple stemness-related markers including CD44, Oct4, c-Myc, and mutant p53 (mutp53) in CD44highK562 cells, suggesting that NSAIDs could induce autophagy, which might mediate degradation of stemness-related marker proteins. Activation of AMPK and inhibition of Akt/mTOR/p70S6K/4EBP1 participated in NSAID-induced autophagy in CD44highK562 cells. In addition, treatment of CD44highK562 cells with NSAIDs inhibited expression of HSF1/Hsps, which resulted in suppression of 17-AAG-induced activation of Hsp70, leading to reversal of 17-AAG resistance and sensitization of CD44highK562 cells to 17-AAG by NSAIDs. In conclusion, combining NSAIDs with Hsp90 inhibitor may offer one of the most promising strategies for eradication of CD44-overexpressing CSCs.

Sensitization of multidrug-resistant cancer cells to Hsp90 inhibitors by NSAIDs-induced apoptotic and autophagic cell death.

NSAIDs (non-steroidal anti-inflammatory drugs) have potential use as anticancer agents, either alone or in combination with other cancer therapies. We found that NSAIDs including celecoxib (CCB) and ibuprofen (IBU) significantly potentiated the cytotoxicity of Hsp90 inhibitors in human multidrug-resistant (MDR) cells expressing high levels of mutant p53 (mutp53) protein and P-glycoprotein (P-gp), and reversed Hsp90 inhibitor resistance caused by activation of heat shock factor 1 (HSF1) and by up-regulation of heat shock proteins (Hsps) and P-gp. Inhibition of Akt/mTOR and STAT3 pathways by CCB induced autophagy, which promoted the degradation of mutp53, one of Hsp90 client proteins, and subsequently down-regulated HSF1/Hsps and P-gp. Inhibition of autophagy prevented mutp53 degradation and CCB-induced apoptosis, and inhibition of caspase-3-mediated apoptotic pathway by Z-DEVD-FMK did not completely block CCB-induced cell death in MDR cells, suggesting that autophagic and apoptotic cell death may contribute to CCB-induced cytotoxicity in MDR cells. Furthermore, CCB and IBU suppressed Hsp90 inhibitor-induced HSF1/Hsp70/P-gp activity and mutp53 expression in MDR cells. Our results suggest that NSAIDs can be used as potential Hsp90 inhibitor chemosensitizers and reverse resistance of MDR cells to Hsp90 inhibitors via induction of apoptosis and autophagy. These results might enable the use of lower, less toxic doses of Hsp90 inhibitors and facilitate the design of practically applicable, novel combination therapy for the treatment of MDR cancer.

The impact of a dose of the angiotensin receptor blocker valsartan on post-myocardial infarction ventricular remodelling.

AIMS: Although clinical guidelines advocate the use of the highest tolerated dose of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers after acute myocardial infarction (MI), the optimal dosing or the risk-benefit profile of different doses have not been fully identified. METHODS AND RESULTS: In this multicentre trial, 495 Korean patients with acute ST segment elevation MI and subnormal left ventricular (LV) ejection fraction (<50%) were randomly allocated (2:1) to receive maximal tolerated dose of valsartan (titrated up to 320 mg/day, n = 333) or low-dose valsartan (80 mg/day, n = 162) treatment. The primary objective was to assess the changes in echocardiographic parameters of LV remodelling from baseline to 12 months after discharge. After treatment, end-diastolic LV volume (LVEDV) decreased significantly in the low-dose group, but the difference in LVEDV changes was insignificant between the maximal-tolerated-dose and low-dose groups. End-systolic LV volume decreased significantly in both groups, to a similar degree between groups. LV ejection fraction rose significantly in both study groups, to a similar degree. Changes in plasma levels of neurohormones were also comparable between the two groups. Drug-related adverse effects occurred more frequently in the maximal-tolerated-dose group than in the low-dose group (7.96 vs. 0.69%, P < 0.001). CONCLUSIONS: In the present study, treatment with the maximal tolerated dose of valsartan did not exhibit a superior effect on post-MI LV remodelling compared with low-dose treatment and was associated with a greater frequency of adverse effect in Korean patients. Further study with a sufficient number of cases and statistical power is warranted to verify the findings of the present study.

Potential Role of CD133 Expression in the Susceptibility of Human Liver Cancer Stem-Like Cells to TRAIL.

Hepatocellular carcinoma (HCC) is one of the most common malignancies, with a poor prognosis and high recurrence rate. In the present study, we identified CD133, one of the markers of cancer stem cells, as a novel molecular target of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In four human HCC cell lines established from primary HCC tumors, we found that CD133-high human liver cancer stem-like cells (CD133hi) derived from the SNU-475 cell line were highly susceptible to TRAIL compared to other HCC cell lines with a small population of CD133. CD133hi SNU-475 cells showed upregulation of TRAIL receptor DR5 and stemness-related genes such as c-Myc and ABC transporters compared to their CD133-low (CD133lo) cells. Hypersensitivity of CD133hi cells to TRAIL was associated with c-Myc-mediated upregulation of DR5 and downregulation of c-FLIPL in the cells. Knockdown of CD133 expression in CD133hi cells resulted in the downregulation of c-Myc, and depletion of c-Myc caused a decrease in the cell surface expression of DR5 and an increase in the expression of c-FLIPL and, consequently, attenuated TRAIL-induced cytotoxicity and apoptosis of CD133hi cells. These results suggest that TRAIL may provide a new strategy for CD133hi CSCs of HCC-targeted therapies and, potentially, for therapies of other CD133-expressing types of cancer.

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1.

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp.

Sensitization of chemo-resistant human chronic myeloid leukemia stem-like cells to Hsp90 inhibitor by SIRT1 inhibition.

Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44(high) K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, ß-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44(high) K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44(high) cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.

Amurensin G enhances the susceptibility to tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity of cancer stem-like cells of HCT-15 cells.

Cancer stem cells (CSCs) are resistant to radiotherapy and chemotherapy and play a significant role in cancer recurrence. Design of better treatment strategies that can eliminate or otherwise control CSC populations in tumors is necessary. In this study, the sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity and the effect of amurensin G, a novel sirtuin 1 (SIRT1) inhibitor, were examined using the CSC-enriched fraction of HCT-15 human colon cancer cells. Cancer stem cell-enriched HCT-15 colony cells were paradoxically less sensitive to doxorubicin, and more sensitive to TRAIL-induced cytotoxicity, than their parental cells. Also, CD44(+) HCT-15 cells were more susceptible to TRAIL-mediated cytotoxicity than CD44(-) HCT-15 cells, possibly due to increased levels of death receptors DR4 and DR5 as well as c-Myc, and decreased levels of c-FLIPL /S in CD44(+) cells compared with CD44(-) HCT-15 cells. The combination effect of amurensin G on TRAIL-mediated cytotoxicity was much more apparent in CD44(+) cells than in CD44(-) HCT-15 cells, and this was associated with more prominent downregulation of c-FLIP(L/S) in CD44(+) cells than in CD44(-) HCT-15 cells. These results indicate that HCT-15 colony or CD44(+) cells, which may have CSC properties, are more sensitive to TRAIL than parental or CD44(-) HCT-15 cells. Amurensin G may be effective in eliminating colon CSCs and be applicable to potentiate the sensitivity of colon CSCs to TRAIL.

Ku70 acetylation and modulation of c-Myc/ATF4/CHOP signaling axis by SIRT1 inhibition lead to sensitization of HepG2 cells to TRAIL through induction of DR5 and down-regulation of c-FLIP.

In this study, we investigated the role of c-Myc/ATF4/CHOP signaling pathway in sensitization of human hepatoma HepG2 cells to TRAIL. Knockdown of SIRT1 or treatment with SIRT1 inhibitor caused the up-regulation of DR5 and down-regulation of c-FLIP through modulation of c-Myc/ATF4/CHOP pathway, and subsequently enhanced the cytotoxic and apoptotic effects of TRAIL on HepG2 cells. Interestingly, SIRT1 interacted directly with c-FLIP(L) and Ku70, and treatment with SIRT1 inhibitor enhanced acetylation of Ku70 and subsequently decreased its binding to c-FLIP. And this was followed by degradation of c-FLIP. Moreover, Ku70(-/-) MEF and Ku70-knockdown HepG2 cells showed the increased levels of c-Myc, ATF4, CHOP, and DR5 and decreased level of c-FLIP. These results were followed by increased sensitivity of Ku70(-/-) MEF cells and Ku70-knockdown HepG2 cells to TRAIL compared with their control cells. These findings reveal for the first time that SIRT1 inhibition increases Ku70 acetylation, and the acetylated Ku70 with a decreased function mediates the induction of DR5 and the down-regulation of c-FLIP by up-regulating c-Myc/ATF4/CHOP pathway, and consequently promotes the TRAIL-induced apoptosis of HepG2 cells. This study provides important mechanistic insight of the synergism exhibited by SIRT1 inhibition and TRAIL.

Amurensin G, a novel SIRT1 inhibitor, sensitizes TRAIL-resistant human leukemic K562 cells to TRAIL-induced apoptosis.

Many types of cancer cells remain resistant towards TRAIL-induced cytotoxicity by the blockade of apoptotic signaling cascades. Thus, sensitizers are needed to enhance the effect of TRAIL-based cancer therapies. Although synergistic tumor cell death has been reported when various HDAC inhibitors were administered with TRAIL in a variety of human cancers, the effect of inhibitors of Class III HDAC such as SIRT1 have not been reported. We reported here for the first time that inhibition of SIRT1 augmented the cytotoxic and apoptotic effects of TRAIL on human leukemic K562 cells. Knockdown of SIRT1 or treatment with amurensin G, a potent new SIRT1 inhibitor, up-regulated the levels of DR5 and c-Myc and down-regulated the level of c-FLIP(L/S). Furthermore, knockdown of SIRT1 or treatment with amurensin G augmented the molecular responses to TRAIL, including activation of caspase-8, -9 and -3, PARP cleavage, up-regulation of Bax, and down-regulation of Bcl-2. Amurensin G-enhanced TRAIL-induced apoptosis was abrogated by caspase inhibitor Z-VAD-FMK. These findings suggest that the suppression of SIRT1 with siRNA or amurensin G sensitize the TRAIL-resistant K562 cell to TRAIL-induced apoptosis, possibly by the up-regulation of c-Myc and DR5 surface expression and the down-regulations of c-FLIP and Mcl-1. In addition, amurensin G, a potent new SIRT1 inhibitor, would be used as a sensitizer of TRAIL in TRAIL-resistant leukemic cells.