Jinsong Kang

Oxidative Stress Induces Parallel Autophagy and Mitochondria Dysfunction in Human Glioma U251 Cells

Accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) is an oxidative stress response, which induced various defense mechanisms or programmed cell death (PCD). As one of the major types of PCD, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. To date, however, the exact mechanism by which ROS regulates autophagy is still poorly understood. Thus, the purposes of this study were to elucidate how H(2)O(2) exerts its cytotoxic effects on malignant glioma U251 cells and to uncover the molecular mechanism that might be involved. Here, we show that H(2)O(2)-induced autophagy and apoptosis in U251 cells are mediated through the Beclin 1 and Akt/mTOR pathways. Accumulation of ROS leads to changes in mitochondrial permeability with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics at a transcriptional level of fission and fusion. Overexpression of cellular Bcl-2 partially inhibited autophagy through both the Beclin 1 and the Akt/mTOR pathways and led to recovery of mitochondrial dynamics. When autophagy was prevented at an early stage by 3-methyladenine, apoptosis significantly increased. Our data provide the first evidence that H(2)O(2) induces autophagy through interference with the Beclin 1 and Akt/mTOR signaling pathways and is regulated by the anti-apoptotic gene Bcl-2 in glioma U251 cells.

Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells.

The function of autophagy in cisplatin-treated cancer cells is not fully understood. Cisplatin treatment induced degradation of ubiquitinated proteins by autophagy, which reduced apoptosis induced by endoplasmic reticulum (ER) stress and downregulated the mitochondrial pathway of apoptosis. Inhibition of autophagy using 3-methyladenine (3-MA) or chloroquine (CQ) increased the levels of intracellular misfolded proteins, which enhanced cellular apoptosis. We found that tunicamycin, an ER stress inducer, augmented cisplatin cytotoxicity by upregulating ER stress-mediated apoptosis. Our data indicates that autophagy plays an important role in preventing cisplatin-induced apoptosis in HeLa cells, thus inhibition of autophagy may improve cisplatin chemotherapy.

p62/SQSTM1 involved in cisplatin resistance in human ovarian cancer cells by clearing ubiquitinated proteins

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we show a critical role for the ubiquitin-binding protein p62/SQSTM1 in cisplatin resistance in human ovarian cancer cells (HOCCs). Specifically, we found that cisplatin-resistant SKOV3/DDP cells express much higher levels of p62 than do cisplatin-sensitive SKOV3 cells. The protein p62 binds ubiquitinated proteins for transport to autophagic degradation, reducing apoptosis induced by endoplasmic reticulum (ER) stress in SKOV3/DDP cells. Knockdown of p62 or inhibition of autophagy using 3-methyladenine resensitises SKOV3/DDP cells to cisplatin. Collectively, our data indicate that p62 acts as a receptor or adaptor for autophagic degradation of ubiquitinated proteins, and plays an important role in preventing ER stress-induced apoptosis, leading to cisplatin resistance in HOCCs.

Inhibition of CLIC4 Enhances Autophagy and Triggers Mitochondrial and ER Stress-Induced Apoptosis in Human Glioma U251 Cells under Starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.

Suppression of Chloride Channel 3 Expression Facilitates Sensitivity of Human Glioma U251 Cells to Cisplatin Through Concomitant Inhibition of Akt and Autophagy

Cisplatin resistance is a difficult problem in clinical chemotherapy, and the mechanisms involved in cisplatin resistance require further study. In this study, we investigated the role of chloride channel‐3 (ClC‐3) in cisplatin resistance. Autophagy was demonstrated by accumulation of LC3‐II, beclin 1 and Atg12‐Atg5. The ultrastructure changes were observed under electron microscope. Chemical staining with acridine orange or MDC was used to detect acidic vesicular organelles. Quantification of apoptosis was detected by PI and Annexin V staining. The mechanisms involved in the Akt pathway and autophagy were studied by western blot analysis. Our results showed that Akt phosphorylation and autophagy were induced by cisplatin in human glioma U251 cells. Specific inhibition of ClC‐3 by ClC‐3 siRNA sensitized the apoptosis‐resistant U251 cells to cisplatin‐mediated cell death and downregulated phosphorylated Akt. Interestingly, ClC‐3 suppression also inhibited induction of autophagy by cisplatin although the Akt/mTOR pathway was deregulated. Counteracting the autophagic process by 3‐methylademine enhanced cytotoxicity of cisplatin, revealing that autophagy plays a key role in chemoresistance. Suppressing the Akt/mTOR pathway by the NADPH oxidase inhibitor diphenyl iodonium (DPI) indicated that cisplatin‐induced activation of Akt/mTOR pathway requires generation of reactive oxygen species (ROS) through NADPH oxidase. Collectively, our results suggest that ClC‐3 suppression causes the inhibition of Akt and autophagy, which can enhance the therapeutic benefit of cisplatin in U251 cells. Anat Rec, 296:595–603, 2013.

Lysosome Dysfunction Enhances Oxidative Stress-Induced Apoptosis Through Ubiquitinated Protein Accumulation in Hela Cells

The role of lysosomal system in oxidative stress‐induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH4Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH4Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione‐induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress. Anat Rec, 2013.

Hyperbaric oxygen enlarges the area of brain damage in MCAO rats by blocking autophagy via ERK1/2 activation

Hyperbaric oxygen (HBO) is emerging as a therapy for brain ischemia, although its benefits are still debated. The present study aimed to investigate the effect of HBO on brain damage in a rat model of transient focal cerebral ischemia and its underlying mechanism of action. Male Wistar rats, which had suffered 1.5h of transient middle cerebral artery occlusion (tMCAO) and had a Longas neuron score of 3, were given pure oxygen at 3.0 atm absolute, for 60 min after the third hour of reperfusion. After 24h of reperfusion, rat brains were removed and studied. 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin staining revealed that the infarct ratio in the HBO group increased remarkably when compared with the MCAO group. Up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation was detected in the HBO group because of reactive oxygen species (ROS) generation. Autophagy appeared to be obstructed in the HBO group. Administration of the ERK1/2 inhibitor U0126 decreased the infarct ratio and improved protein clearance by autophagy in the HBO group. Collectively, these results suggest that HBO enlarges the area of brain damage via reactive oxygen species-induced activation of ERK1/2, which interrupts autophagy flux.

5-Nitro-2-(3-phenylpropylamino) benzoic acid induced drug resistance to cisplatin in human erythroleukemia cell lines.

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we showed a critical role for the chloride channel‐3 (ClC‐3) in cisplatin resistance in human erythroleukemia K562 and RK562 cells. We found that a chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) could protect cells from cisplatin‐induced apoptosis. NPPB treatment decreased the mRNA and the protein expression of Bax/Bcl‐2, decreased the protein expressions of cytochrome C and caspase‐3, and increased the mRNA expressions of cyclin D1 and ClC‐3 in cells treated with cisplatin. The caspase‐3 activity was decreased significantly and the rate of cell apoptosis was decreased. NPPB treatment increased CIC‐3 expression, which could increase acidification of intracellular compartments, and increased sequestration of cisplatin, inducing decreased effective drug concentrations, and subsequently cell death. Collectively, our data indicate that NPPB can induce drug resistance to cisplatin by upregulating the expression of CIC‐3. NPPB‐induced CIC‐3 expression facilitates acidification of sequestrated cisplatin, and plays an important role in preventing cisplatin‐induced apoptosis in human erythroleukemia K562 and RK562 cells. Anat Rec,, 2011.

SIRT3 participates in glucose metabolism interruption and apoptosis induced by BH3 mimetic S1 in ovarian cancer cells.

The Bcl-2 antiapoptotic proteins are important cancer therapy targets; however, their role in cancer cell metabolism remains unclear. We found that the BH3-only protein mimetic S1, a novel pan Bcl-2 inhibitor, simultaneously interrupted glucose metabolism and induced apoptosis in human SKOV3 ovarian cancer cells, which was related to the activation of SIRT3, a stress-responsive deacetylase. S1 interrupted the cellular glucose metabolism mainly through causing damage to mitochondrial respiration and inhibiting glycolysis. Moreover, S1 upregulated the gene and protein expression of SIRT3, and induced the translocation of SIRT3 from the nucleus to mitochondria. SIRT3 silencing reversed the effects of S1 on glucose metabolism and apoptosis through increasing the level of HK-II localized to the mitochondria, while a combination of the glycolysis inhibitor 2-DG and S1 intensified the cytotoxicity through further upregulation of SIRT3 expression. This study underscores an essential role of SIRT3 in the antitumor effect of Bcl-2 inhibitors in human ovarian cancer through regulating both metabolism and apoptosis. The manipulation of Bcl-2 inhibitors combined with the use of classic glycolysis inhibitors may be rational strategies to improve ovarian cancer therapy.

p62 participates in the inhibition of NF-κB signaling and apoptosis induced by sulfasalazine in human glioma U251 cells

Nuclear factor-κB (NF-κB) is constitutively activated in most malignant gliomas and is involved in cancer progression and drug resistance to chemotherapy. Sulfasalazine (SAS) is a classic inhibitor of NF-κB. Apoptosis and autophagy were induced by SAS accompanied by inhibition of NF-κB signaling in U251 cells. Inhibition of autophagy by 3-MA suppressed the effects of SAS on NF-κB signaling and apoptosis in U251 cells. Multifunctional scaffold protein p62 is well known as an autophagy marker protein and provides crosstalk for important signaling pathways, including NF-κB signaling. SAS-induced decrease in the p62 protein levels may be the result of degradation through autophagy. SAS induced the inhibition of NF-κB signaling and apoptosis at least partly via a p62-dependent effect in U251 cells. Collectively, our data shed light on the link between p62 and the NF-κB signaling pathway, particularly in glioma cells. The results may facilitate the design of more effective targeted therapies for the treatment of tumors in which NF-κB signaling is altered.