Liankun Sun

THE LATE AND PERSISTENT PATHOGENIC EFFECTS OF CADMIUM AT VERY LOW LEVELS ON THE KIDNEY OF RATS

Cadmium (Cd) is an important nephrotoxic pollutant. To examine late effects on the kidney of individuals previously exposed to chronic Cd at very low levels, male Wistar rats were given 20 nmol/kg i.p. injections of Cd every other day for 4 weeks. At the 20th, 28th, 36th, 44th and 52nd week of the study, renal metal accumulation, morphology and function were examined. Immunochemical staining was performed to detect renal 3-nitrotyrosine (3-NT) accumulation, metallothionein (MT) expression, cell proliferation and global DNA methylation. Results showed that renal Cd concentration and MT expression along with 3-NT accumulation were significantly higher in the Cd group than that in the control. Histopathologically renal tubule damage at the early stage and hyperplasia at the late stage were observed in the Cd group. Renal fibrosis in glomeruli was evident in the Cd group, particularly at the late stage of the study. Immunoreactivity of global DNA methylation was markedly diminished in the Cd group at both 20th and 52nd weeks. These results suggest that previous exposure to chronic Cd at very low level induced persistent damaging effects on the kidney along with increases in cell proliferation and global DNA hypomethylation.

Cytoprotective Effect of the UCP2-SIRT3 Signaling Pathway by Decreasing Mitochondrial Oxidative Stress on Cerebral Ischemia–Reperfusion Injury

Recovered blood supply after cerebral ischemia for a certain period of time fails to restore brain function, with more severe dysfunctional problems developing, called cerebral ischemia–reperfusion injury (CIR). CIR involves several extremely complex pathophysiological processes in which the interactions between key factors at various stages have not been fully elucidated. Mitochondrial dysfunction is one of the most important mechanisms of CIR. The mitochondrial deacetylase, sirtuin 3 (SIRT3), can inhibit mitochondrial oxidative stress by deacetylation, to maintain mitochondrial stability. Uncoupling protein 2 (UCP2) regulates ATP (Adenosine triphosphate) and reactive oxygen species production by affecting the mitochondrial respiratory chain, which may play a protective role in CIR. Finally, we propose that UCP2 regulates the activity of SIRT3 through sensing the energy level and, in turn, maintaining the mitochondrial steady state, which demonstrates a cytoprotective effect on CIR.

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.

Intracellular pH sensing using polymeric micelle containing tetraphenylethylene-oxazolidine

A novel amphiphilic copolymer consisting of tetraphenylethylene-oxazolidine (TPE-OX) as a pH-sensitive chromophore and poly(ethylene glycol) methyl ether methacrylate (PEGMA) as a water-soluble chain has been well designed and synthesized. A self-assembled polymeric micelle based on the copolymer exhibited clear dual-emission switching between cyan and red with a decrease of pH value in Tris-HCl buffer solution due to the extended conjugation and emerging intramolecular charge transfer effect when opening the spiro-ring of OX moiety. Fluorescence imaging of HepG2 cells stained by the polymeric micelle shows switched luminescence from cyan to red with high selectivity and contrast, indicating that the polymeric micelle was an effective probe for intracellular pH detection. Additionally, the red emission of the polymeric micelle in lysosomes can reversibly switch back to the original cyan emission in response to the two lysosomal activity inhibitors chloroquine and bafilomycin, which demonstrates that the polymeric micelle has potential applications in detecting the activity of lysosomal and further autophagy in cancer cells.

p62/SQSTM1 as an oncotarget mediates cisplatin resistance through activating RIP1-NF-κB pathway in human ovarian cancer cells

Platinum‐based therapeutic strategies have been widely used in ovarian cancer treatment. However, drug resistance has greatly limited therapeutic efficacy. Recently, tolerance to cisplatin has been attributed to other factors unrelated to DNA. p62 (also known as SQSTM1) functions as a multifunctional hub participating in tumorigenesis and may be a therapeutic target. Our previous study showed that p62 was overexpressed in drug‐resistant ovarian epithelial carcinoma and its inhibition increased the sensitivity to cisplatin. In this study, we demonstrate that the activity of the NF‐κB signaling pathway and K63‐linked ubiquitination of RIP1 was higher in cisplatin‐resistant ovarian (SKOV3/DDP) cells compared with parental cells. In addition, cisplatin resistance could be reversed by inhibiting the expression of p62 using siRNA. Furthermore, deletion of the ZZ domain of p62 that interacts with RIP1 in SKOV3 cells markedly decreased K63‐linked ubiquitination of RIP1 and inhibited the activation of the NF‐κB signaling pathway. Moreover, loss of the ZZ domain from p62 led to poor proliferative capacity and high levels of apoptosis in SKOV3 cells and made them more sensitive to cisplatin treatment. Collectively, we provide evidence that p62 is implicated in the activation of NF‐κB signaling that is partly dependent on RIP1. p62 promotes cell proliferation and inhibits apoptosis thus mediating drug resistance in ovarian cancer cells.

SIRT3 aggravates metformin-induced energy stress and apoptosis in ovarian cancer cells

ABSTRACT Increasing evidence suggests that mitochondrial respiratory chain complex I participates in carcinogenesis and cancer progression by providing energy and maintaining mitochondrial function. However, the role of complex I in ovarian cancer is largely unknown. In this study we showed that metformin, considered to be an inhibitor of complex I, simultaneously inhibited cell growth and induced mitochondrial‐related apoptosis in human ovarian cancer cells. Metformin interrupted cellular energy metabolism mainly by causing damage to complex I that impacted mitochondrial function. Additionally, treatment with metformin increased the activation of sirtuin 3 (SIRT3), a mitochondrial deacetylase. We demonstrated that SIRT3 overexpression aggravated metformin‐induced apoptosis, energy stress and mitochondrial dysfunction. Moreover, treatment with metformin or SIRT3 overexpression increased activation of AMP‐activated protein kinase (AMPK), a major sensor of cellular energy status. AMPK compensated for energy loss by increasing glycolysis. The impact of this was assessed by reducing glucose levels in the media or by using inhibitors (2‐deoxyglucose, Compound C) of glycolysis and AMPK. The combination of these factors with metformin intensified cytotoxicity through further downregulation of ATP. Our study outlines an important role for SIRT3 in the antitumor effect of mitochondrial complex I inhibitors in human ovarian cancer cells. This effect appears to be mediated by induction of energy stress and apoptosis. Strategies that target the mitochondria could be enhanced by modulating glycolysis to further aggravate energy stress that may increase the antitumor effect. HIGHLIGHTSMetformin induces mitochondrial‐apoptosis by targeting respiratory chain complex 1.The expression and activity of sirtuin 3 (SIRT3) are increased by metformin.SIRT3 aggravates metformin‐induced energy stress and apoptosis.Inhibition of the AMPK / glucose pathway enhances the antitumor effect of metformin.

TAT‑fused IP3R‑derived peptide enhances cisplatin sensitivity of ovarian cancer cells by increasing ER Ca2+ release

Ovarian cancer is the most common gynecological malignancy. At present, cisplatin is used to treat ovarian cancer; however, the development of cisplatin resistance during therapy is a common obstacle to achieving favorable outcomes. Recently, the B-cell lymphoma 2 (Bcl-2) BH4 domain has been reported to mediate the prosurvival activity of Bcl-2 in cancer; however, the involvement of the BH4 domain of Bcl-2 in the cisplatin resistance of ovarian carcinoma cells is not entirely clear. In this study, we observed the cytoplasmic and mitochondrial levels of Ca2+ by confocal laser microscopy. We also detected cell apoptosis using western blot analysis and flow cytometry. The present study demonstrated that TAT-fused inositol 1,4,5-trisphosphate receptor-derived peptide (TAT-IDPS), which targets the BH4 domain of Bcl-2, increased cisplatin-induced Ca2+ flux from the endoplasmic reticulum (ER) into the cytosol and mitochondria. In addition, TAT-IDPS increased cisplatin-induced expression of mitochondrial apoptosis-associated proteins and ER stress-associated proteins. These results indicated that TAT-IDPS may enhance the cytotoxicity of cisplatin toward ovarian carcinoma cells by increasing ER Ca2+ release.

Autophagy eliminates ER membrane reorganization induced by Bcl-2 inhibitor in HeLa cells

The endoplasmic reticulum (ER) is a membranous network within cells that is important for several cellular functions including translation and folding of secretory and membrane proteins, lipid biogenesis and sequestration of Ca2+. Disruption of ER structure might affect the normal physiology of the cells. In yeast, expansion of the ER is observed under unfolded protein response (UPR) and subsequently induces autophagy initiated from the ER. In this study, we demonstrated a drastic and specific ER membrane reorganization (EMR), characterized by the clustering of the ER membrane into large and compact aggregates and occurring independent of UPR in HeLa cells treated with S1 combined with ABT-737. Subsequently, combined with S1 and ABT-737 triggered autophagy. Herein, we report a key step for removal of damaged and superfluous cellular constituents, by a mechanistic link between ER aggregation and autophagic activation. Our study is the first time to show that autophagy may be a way to remove the ER membrane reorganization induced by Bcl-2 inhibitors ABT-737 and S1 and it may help us to analyze autophagy in certain diseases.

Zinc enhances chemosensitivity to paclitaxel in PC‑3 prostate cancer cells

Paclitaxel‑based chemotherapy is a promising approach for prostate cancer treatment. However, single‑drug chemotherapy is associated with an increased risk of drug resistance. Therefore, novel combination chemotherapy regimens are a popular topic of research. Zinc participates in the regulation of apoptosis, for example in the form of Zn2+ and via zinc‑dependent enzymes. Zinc can either induce or suppress apoptosis, and its effect depends primarily on its concentration. Previous research has demonstrated that physiological concentrations of zinc can directly induce apoptosis of PC‑3 prostate cancer cells via the mitochondrial pathway. In prostate cancer tissues, zinc concentrations have been demonstrated to be reduced compared with non-cancerous tissues. Furthermore, the concentration of zinc has been demonstrated to decrease further with cancer progression. In the present study, it was investigated whether exposure of PC‑3 cells to zinc improved their sensitivity to the chemotherapeutic agent, paclitaxel. MTT assays, cell clone formation assays, Hoechst staining and flow cytometry revealed that zinc enhanced PC‑3‑cell chemosensitivity to paclitaxel. Western blotting and reverse transcription‑polymerase chain reaction were used to determine that the mitochondria‑mediated apoptosis signaling pathway is involved with zinc/paclitaxel‑induced cell death. The present study provides a foundation for the development of novel tumor combination therapy.

Autophagy inhibitor chloroquine induces apoptosis of cholangiocarcinoma cells via endoplasmic reticulum stress

Poor prognosis and chemotherapy tolerance are the main obstacles encountered in the treatment of cholangiocarcinoma. Chloroquine (CQ), an antimalarial agent, is able to induce sustained endoplasmic reticulum (ER) stress by functioning as an autophagy inhibitor. The present study indicated that CQ had the ability to induce apoptosis in QBC939 cholangiocarcinoma cells. Furthermore, using western blotting, Hoechst staining and flow cytometry, it was demonstrated that CQ induced the apoptosis of QBC939 cholangiocarcinoma cells. Analysis by a polymerase chain reaction (PCR) array and confirmation via quantitative PCR technology indicated that the expression levels of growth arrest and DNA damage 153 [C/EBP homologous protein (CHOP)], a key molecule involved in ER stress-induced apoptosis, and its downstream death receptors were increased following CQ stimulation. It was considered that the upregulation of CHOP may mediate CQ-induced extrinsic pathways and autophagy-dependent apoptosis; therefore, the role of autophagy in cholangiocarcinoma treatment was elucidated based on the data demonstrating that CQ regulates the ER-autophagy network in tumor cells. Furthermore, it was considered that CQ may become a novel and effective strategy for the treatment of cholangiocarcinoma.