Cheng Yang

Central Arkansas Veterans Healthcare System, Washington, Washington, D.C., United States

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Publications (6)32.59 Total impact

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    ABSTRACT: Cisplatin injury to renal tubular epithelial cells (RTEC) is accompanied by autophagy and caspase activation. However, autophagy gradually decreases during the course of cisplatin injury. The role of autophagy and the mechanism of its decrease during cisplatin injury are not well understood. This study demonstrated that autophagy proteins beclin-1, Atg5, and Atg12 were cleaved and degraded during the course of cisplatin injury in RTEC and the kidney. zVAD-fmk, a widely used pancaspase inhibitor, blocked cleavage of autophagy proteins suggesting that zVAD-fmk would promote the autophagy pathway. Unexpectedly, zVAD-fmk blocked clearance of the autophagosomal cargo, indicating lysosomal dysfunction. zVAD-fmk markedly inhibited cisplatin-induced lysosomal cathepsin B and calpain activities and therefore impaired autophagic flux. In a mouse model of cisplatin nephrotoxicity, zVAD-fmk impaired autophagic flux by blocking autophagosomal clearance as revealed by accumulation of key autophagic substrates p62 and LC3-II. Furthermore, zVAD-fmk worsened cisplatin-induced renal dysfunction. Chloroquine, a lysomotropic agent that is known to impair autophagic flux, also exacerbated cisplatin-induced decline in renal function. These findings demonstrate that impaired autophagic flux induced by zVAD-fmk or a lysomotropic agent worsened renal function in cisplatin acute kidney injury (AKI) and support a protective role of autophagy in AKI. These studies also highlight that the widely used antiapoptotic agent zVAD-fmk may be contraindicated as a therapeutic agent for preserving renal function in AKI.
    AJP Renal Physiology 08/2012; 303(8):F1239-50. · 4.42 Impact Factor
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    ABSTRACT: We demonstrate the effect of proteasome inhibitors in mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-exposed renal tubular epithelial cells (LLC-PK(1) cells) and in a model of cisplatin nephrotoxicity. Immunofluorescence and subcellular fractionation studies revealed cisplatin-induced translocation of AIF from the mitochondria to nucleus. Mcl-1, a pro-survival member of the Bcl-2 family, is rapidly eliminated on exposure of renal cells to cisplatin. Proteasome inhibitors PS-341 and MG-132 blocked cisplatin-induced Mcl-1 depletion and markedly prevented mitochondrial release of AIF. PS-341 and MG132 also blocked cisplatin-induced activation of executioner caspases and apoptosis. These studies suggest that proteasome inhibitors prevent cisplatin-induced caspase-dependent and -independent pathways. Overexpression of Mcl-1 was effective in blocking cisplatin-induced cytochrome c and AIF release from the mitochondria. Downregulation of Mcl-1 by small interfering RNA promoted Bax activation and cytochrome c and AIF release, suggesting that cisplatin-induced Mcl-1 depletion and associated Bax activation are involved in the release of AIF. Expression of AIF protein in the mouse was highest in the kidney compared to the heart, brain, intestine, liver, lung, muscle, and spleen. In an in vivo model of cisplatin nephrotoxicity, proteasome inhibitor MG-132 prevented mitochondrial release of AIF and markedly attenuated acute kidney injury as assessed by renal function and histology. These studies provide evidence for the first time that the proteasome inhibitors prevent cisplatin-induced mitochondrial release of AIF, provide cellular protection, and markedly ameliorate cisplatin-induced acute kidney injury. Thus, AIF is an important therapeutic target in cisplatin nephrotoxicity and cisplatin-induced depletion of Mcl-1 is an important pathway involved in AIF release.
    Biochemical pharmacology 09/2009; 79(2):137-46. · 4.25 Impact Factor
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    ABSTRACT: Autophagy has emerged as another major "programmed" mechanism to control life and death much like "programmed cell death" is for apoptosis in eukaryotes. We examined the expression of autophagic proteins and formation of autophagosomes during progression of cisplatin injury to renal tubular epithelial cells (RTEC). Autophagy was detected as early as 2-4 h after cisplatin exposure as indicated by induction of LC3-I, conversion of LC3-I to LC3-II protein, and upregulation of Beclin 1 and Atg5, essential markers of autophagy. The appearance of cisplatin-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in RTEC provided further evidence for autophagy. The autophagy inhibitor 3-methyladenine blocked punctated staining of autophagosomes. The staining of normal cells with acridine orange displayed green fluorescence with cytoplasmic and nuclear components in normal cells but displayed considerable red fluorescence in cisplatin-treated cells, suggesting formation of numerous acidic autophagolysosomal vacuoles. Autophagy inhibitors LY294002 or 3-methyladenine or wortmannin inhibited the formation of autophagosomes but induced apoptosis after 2-4 h of cisplatin treatment as indicated by caspase-3/7 and -6 activation, nuclear fragmentation, and cell death. This switch from autophagy to apoptosis by autophagic inhibitors further suggests that the preapoptotic lag phase after treatment with cisplatin is mediated by autophagy. At later stages of cisplatin injury, apoptosis was also found to be associated with autophagy, as autophagic inhibitors and inactivation of autophagy proteins Beclin 1 and Atg5 enhanced activation of caspases and apoptosis. Our results demonstrate that induction of autophagy mounts an adaptive response, suppresses cisplatin-induced apoptosis, and prolongs survival of RTEC.
    American journal of physiology. Renal physiology 05/2008; 294(4):F777-87. · 3.61 Impact Factor
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    ABSTRACT: One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.
    Autophagy 01/2008; 4(5):710-2. · 12.04 Impact Factor
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    ABSTRACT: Mcl-1 is an antiapoptotic member of the Bcl-2 family that plays an important role in cell survival. We demonstrate that proteasome-dependent regulation of Mcl-1 plays a critical role in renal tubular epithelial cell injury from cisplatin. Protein levels of Mcl-1 rapidly declined in a time-dependent manner following cisplatin treatment of LLC-PK(1) cells. However, mRNA levels of Mcl-1 were not altered following cisplatin treatment. Expression of other antiapoptotic members of the Bcl-2 family such as Bcl-2 and BclxL was not affected by cisplatin treatment. Cisplatin-induced loss of Mcl-1 occurs at the same time as the mitochondrial release of cytochrome c, activation of caspase-3, and initiation of apoptosis. Treatment of cells with cycloheximide, a protein synthesis inhibitor, revealed rapid turnover of Mcl-1. In addition, treatment with cycloheximide in the presence or absence of cisplatin demonstrated that cisplatin-induced loss of Mcl-1 results from posttranslational degradation rather than transcriptional inhibition. Overexpression of Mcl-1 protected cells from cisplatin-induced caspase-3 activation and apoptosis. Preincubating cells with the proteasome inhibitor MG-132 or lactacystin not only restored cisplatin-induced loss of Mcl-1 but also resulted in an accumulation of Mcl-1 that exceeded basal levels; however, Bcl-2 and BclxL levels did not change in response to MG-132 or lactacystin. The proteasome inhibitors effectively blocked cisplatin-induced mitochondrial release of cytochrome c, caspase-3 activation, and apoptosis. These studies suggest that proteasome regulation of Mcl-1 is crucial in the cisplatin-induced apoptosis via the mitochondrial apoptotic pathway and that Mcl-1 is an important therapeutic target in cisplatin injury to renal tubular epithelial cells.
    American journal of physiology. Renal physiology 07/2007; 292(6):F1710-7. · 3.61 Impact Factor
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    ABSTRACT: We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-alpha markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-alpha or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.
    Journal of Biological Chemistry 10/2005; 280(35):31230-9. · 4.65 Impact Factor

Publication Stats

211 Citations
32.59 Total Impact Points

Institutions

  • 2005–2012
    • Central Arkansas Veterans Healthcare System
      Washington, Washington, D.C., United States
  • 2007–2009
    • University of Arkansas at Little Rock
      Little Rock, Arkansas, United States