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1.  Sirt3-mediated mitophagy protects tumor cells against apoptosis under hypoxia 
Oncotarget  2016;7(28):43390-43400.
Sirt3, a mitochondrial deacetylase, participates in the regulation of multiple cellular processes through its effect on protein acetylation. The objective of this study was to explore the role of Sirt3 in the mitochondrial autophagy (mitophagy), a process of the specific autophagic elimination of damaged mitochondria. We found that silencing of Sirt3 expression in human glioma cells by RNA interference blunted the hypoxia-induced the localization of LC3 on the mitochondria, and the degradation of mitochondria. These results suggest an important involvement of this protein deacetylase in the induction of mitophagy in cancer cells subjected to hypoxia. Further, we demonstrated that Sirt3 activated the hypoxia-induced mitophagy by increasing the interaction of VDAC1 with Parkin. In the cells subjected to hypoxia, inhibition of Sirt3-mediated mitophagy further decreased the mitochondrial membrane potential, and increased the accumulation of ROS that triggers the degradation of anti-apoptotic proteins Mcl-1 and survivin through the proteasomal pathway. Silencing of Sirt3 expression also promoted apoptosis, and enhanced the sensitivity of cancer cells to hypoxia. The regulatory role of Sirt3 in autophagy and apoptosis was also observed in human breast cancer cells. The results of the current study reveal Sirt3 as a novel regulator coupling mitophagy and apoptosis, two important cellular processes that determine cellular survival and death.
PMCID: PMC5190031  PMID: 27270321
Sirt3; mitophagy; hypoxia; apoptosis; cancer cells
2.  Survival and death of endoplasmic-reticulum-stressed cells: Role of autophagy 
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) results in ER stress, which subsequently activates the unfolded protein response that induces a transcriptional program to alleviate the stress. Another cellular process that is activated during ER stress is autophagy, a mechanism of enclosing intracellular components in a double-membrane autophagosome, and then delivering it to the lysosome for degradation. Here, we discuss the role of autophagy in cellular response to ER stress, the signaling pathways linking ER stress to autophagy, and the possible implication of modulating autophagy in treatment of diseases such as cancer.
PMCID: PMC3202126  PMID: 22031845
Endoplasmic reticulum stress; Autophagy; Apoptosis; Cell survival; Cell death
3.  Zinc transporters and dysregulated channels in cancers 
As a nutritionally essential metal ion, zinc (Zn) not only constitutes a structural element for more than 3000 proteins but also plays important regulatory functions in cellular signal transduction. Zn homeostasis is tightly controlled by regulating the flux of Zn across cell membranes through specific transporters, i.e. ZnT and ZIP family proteins. Zn deficiency and malfunction of Zn transporters have been associated with many chronic diseases including cancer. However, the mechanisms underlying Zn regulatory functions in cellular signaling and their impact on the pathogenesis and progression of cancers remain largely unknown. In addition to these acknowledged multifunctions, Zn modulates a wide range of ion channels that in turn may also play an important role in cancer biology. The goal of this review is to propose how zinc deficiency, through modified Zn homeostasis, transporter activity and the putative regulatory function of Zn can influence ion channel activity, and thereby contribute to carcinogenesis and tumorigenesis. This review intends to stimulate interest in, and support for research into the understanding of Zn-modulated channels in cancers, and to search for novel biomarkers facilitating effective clinical stratification of high risk cancer patients as well as improved prevention and therapy in this emerging field.
PMCID: PMC5199720  PMID: 27814637
Zn Homeostasis; ZnT; ZIP; TRP Channels; Store-operated Ca2+ Entry; Orai Channels; STIM; Prostate Cancer; Breast Cancer; Esophageal Cancer; Review
4.  Endotoxins enhance hepatocarcinogenesis induced by oral intake of thioacetamide in rats 
AIM: To clarify whether endotoxin is of pathogenic importance for hepatocarcinogenesis,or the increased cancer risk results solely from thecirrhotic process.
METHODS: The rat model of hepatoma was treated by the intake of 0.03% thioacetamide in drinking water for six months. During induction of hepatoma, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The liver nuclear DNA index and proliferation index were quantitatively analyzed by flow cytometry. Hepatic histology was examined with light and electron microscopes. Plasmic endotoxin concentration and γ-glutamyl transpeptidase activity were measured, and hepatoma incidence was recorded.
RESULTS: Thioacetamide induced cirrhosis and hepatoma in Wistar rats with histology or regenerative nodule, fibrosis and neoplastic foci were quite similar to the pathogenic process of human cirrhosis leading to hepatoma. In comparison with TAA controls (DNA index: 1.15 ± 0.21), exo-endotoxin increased the DNA index by 7.8% (1.24 ± 0.25, P < 0.02) and hepatoma rate by 16.7. Splenectomy-induced enteric endotoxemia increased the DNA index by 25% (1.44 ± 0.15, P < 0.01) and hepatoma rate by 33%. A summation of the effects of these two factors increased the DNA index by 36% (P£¼0.01)and hepatoma incidence by 50%, moreover, the level of endotoxemia showed a close relation with DNA index (r = 0.96, P < 0.01), as well as with the occurrence rate of hepatoma (r = 0.00, P < 0.01). Histological findings further verified such alterations.
CONCLUSION: Lipopolysaccharide administration and/or splenectomy-induced enterogenic endotoxemia may enhance rat hepatocarcinogenesis induced by oral intake of thioacetamide.
PMCID: PMC4688633  PMID: 11819255
liver neoplasms; carcinoma, hepatocellular; endotoxins; thioacetamide; glutamyl transpeptidase/metabolism; flow cytometry; DNA, neoplasm; rats, Wistar
5.  Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats 
AIM: To evaluate the relationship between expression of ras, p53 and bcl-2 gene products and hepatocarcinogenesis since the endotoxemia produced from lipopolysaccharide administration and/or the hypophagocytic state of splenectomy significantly accelerated hepatocarcinogenesis induced by thioacetamide.
METHODS: The hepatocarcinoma model was induced by 6-mo oral intake of 0.03% thioacetamide. During the hepatocarcinoma modeling process, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The techniques of flow cytometry, immunohistochemistry and immunoelectronmicroscopy were applied for quantitative analysis of the expression of oncogene proteins.
RESULTS: In this model system, overexpression of ras p21 protein mainly occurred in the precancerous cell population or in cells in the early stage of hepatocyte transformation. The levels of ras p21 declined when nuclear DNA aneuploidy increased. Expression of bcl-2 protein slowly and steadily rose, with more hepatocytes staying in S + G2M phases, as the hepatocarcinoma became more malignant. p53 was moderately expressed during hepatocarcinogenesis. There was no statistical correlation between endotoxemia levels and the changes in levels of ras, p53 and bcl-2 gene products.
CONCLUSION: Overexpression of oncogene ras p21 was considered likely to be a precursor of premalignant hepatocytes and possibly as responsible for the initiation of hepatocarcinogenesis. Bcl-2 protein expression is proportional to the severity of malignancy in hepatocarcinogenesis. p53 may be involved in a key pathway underlying the transformation and development processes of hepatocarcinoma. This study confirmed the hypothesis that there are multiple genes and multiple steps involved in hepatocarcinogenesis. Expression of oncogene proteins reflects the properties of the premalignant and malignant cells, but is not directly related to endotoxemia statistically.
PMCID: PMC4806235  PMID: 27053867
Genes, ras; Genes, p53; Oncogene proteins; Gene expression; Liver neoplasms; Thioacetamide
6.  Induction of autophagy contributes to crizotinib resistance in ALK-positive lung cancer 
Cancer Biology & Therapy  2014;15(5):570-577.
Use of the inhibitor of ALK fusion onco-protein, crizotinib (PF02341066), has achieved impressive clinical efficacy in patients with ALK-positive non-small cell lung cancer. Nevertheless, acquired resistance to this drug occurs inevitably in approximately a year, limiting the therapeutic benefits of this novel targeted therapy. In this study, we found that autophagy was induced in crizonitib-resistant lung cancer cells and contributed to drug resistance. We observed that ALK was downregulated in the crizotinib-resistant lung cancer cell line, H3122CR-1, and this was causally associated with autophagy induction. The degree of crizotinib resistance correlated with autophagic activity. Activation of autophagy in crizotinib-resistant H3122CR-1 cells involved alteration of the Akt/mTOR signaling pathway. Furthermore, we demonstrated that chloroquine, an inhibitor of autophagy, could restore sensitivity of H3122CR-1 to crizotinib and enhance its efficacy against drug-resistant lung cancer. Thus, modulating autophagy may be worth exploring as a new strategy to overcome acquired crizonitib resistance in ALK-positive lung cancer.
PMCID: PMC4026079  PMID: 24556908
crizotinib; ALK; autophagy; drug resistance; lung cancer
7.  The NFκB inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype 
Cancer Biology & Therapy  2014;15(5):602-611.
The malignant phenotype of glioblastoma multiforme (GBM) is believed to be largely driven by glioma stem-like cells (GSCs), and targeting GSCs is now considered a promising new approach to treatment of this devastating disease. Here, we show that SN50, a cell-permeable peptide inhibitor of NFκB, induced robust differentiation of human GSCs, causing loss of their oncogenic potential. We observed that following treatment of GSCs with SN50, their differentiated progeny cells showed significant decreases in their capability to form neuro-spheres and to invade in vitro and a reduction in their tumorigenicity in mouse xenograft models, but had increased sensitivity to the chemotherapeutic drug temozolomide and to radiation treatment. These results suggest that blocking the NFκB pathway may be explored as a useful mean to induce differentiation of GSCs, and provide another supportive evidence for the promise of differentiation therapy in treatment of malignant brain tumors.
PMCID: PMC4026083  PMID: 24557012
NFκB inhibitor; SN50; brain tumor; differentiation; glioma stem cells
8.  Integrated regulation of autophagy and apoptosis by EEF2K controls cellular fate and modulates the efficacy of curcumin and velcade against tumor cells 
Autophagy  2013;9(2):208-219.
Endoplasmic reticulum (ER) stress induces both autophagy and apoptosis yet the molecular mechanisms and pathways underlying the regulation of these two cellular processes in cells undergoing ER stress remain less clear. We report here that eukaryotic elongation factor-2 kinase (EEF2K) is a critical controller of the ER stress-induced autophagy and apoptosis in tumor cells. DDIT4, a stress-induced protein, was required for transducing the signal for activation of EEF2K under ER stress. We further showed that phosphorylation of EEF2K at Ser398 was essential for induction of autophagy, while phosphorylation of the kinase at Ser366 and Ser78 exerted an inhibitory effect on autophagy. Suppression of the ER stress-activated autophagy via silencing of EEF2K aggravated ER stress and promoted apoptotic cell death in tumor cells. Moreover, inhibiting EEF2K by either RNAi or NH125, a small molecule inhibitor of the enzyme, rendered tumor cells more sensitive to curcumin and velcade, two anticancer agents that possess ER stress-inducing action. Our study indicated that the DDIT4-EEF2K pathway was essential for inducing autophagy and for determining the fate of tumor cells under ER stress, and suggested that inhibiting the EEF2K-mediated autophagy can deteriorate ER stress and lead to a greater apoptotic response, thereby potentiating the efficacy of the ER stress-inducing agents against cancer.
PMCID: PMC3552884  PMID: 23182879
EEF2K; ER stress; autophagy; apoptosis; tumor cells
9.  Altered energy metabolism in cancer 
Cancer Biology & Therapy  2013;14(2):81-89.
The early observations by Dr Otto Warburg revealed that fundamentally metabolic differences exist between malignant tumor cells and adjacent normal cells. Many studies have further reported the relationship between altered cellular metabolism and therapeutic outcomes. These observations suggest that targeting the peculiar metabolic pathways in cancer might be an effective strategy for cancer therapy. In recent years, investigations have accelerated into how altered cellular metabolism promotes tumor survival and growth. This review highlights the current concepts of altered metabolism in cancer and the molecular targets involved in glycolysis, mitochondria and glutamine metabolism and discusses future perspective of cellular metabolism-based cancer treatment.
PMCID: PMC3572003  PMID: 23192270
cancer metabolism; cancer therapy; cell survival; cell death
10.  The Impact of Cellular Senescence in Cancer Therapy: Is it True or Not? 
Acta pharmacologica Sinica  2011;32(10):1199-1207.
Cellular senescence is defined as the physiological program of terminal growth arrest, which can be triggered by various endogenous or exogenous stress signals. Cellular senescence can be induced in response to oncogenic activation, acting as a barrier to tumorigenesis. Tumor cells can undergo senescence when exposed to chemotherapeutic agents. In addition to suppressing tumorigenesis, senescent cells remain metabolically active and may contribute to tumor formation and to therapy resistance. In the current review, we discuss the molecular regulation of cellular senescence, the potential implications of senescence in human cancer, and the possibility of exploiting cellular senescence as a therapeutic intervention in the treatment of cancer.
PMCID: PMC3760423  PMID: 21909124
senescence; oncogenesis; cancer therapy; cell survival; cell death
11.  Anticancer strategies based on the metabolic profile of tumor cells: therapeutic targeting of the Warburg effect 
Acta Pharmacologica Sinica  2016;37(8):1013-1019.
Tumor cells rely mainly on glycolysis for energy production even in the presence of sufficient oxygen, a phenomenon termed the Warburg effect, which is the most outstanding characteristic of energy metabolism in cancer cells. This metabolic adaptation is believed to be critical for tumor cell growth and proliferation, and a number of onco-proteins and tumor suppressors, including the PI3K/Akt/mTOR signaling pathway, Myc, hypoxia-inducible factor and p53, are involved in the regulation of this metabolic adaptation. Moreover, glycolytic cancer cells are often invasive and impervious to therapeutic intervention. Thus, altered energy metabolism is now appreciated as a hallmark of cancer and a promising target for cancer treatment. A better understanding of the biology and the regulatory mechanisms of aerobic glycolysis has the potential to facilitate the development of glycolysis-based therapeutic interventions for cancer. In addition, glycolysis inhibition combined with DNA damaging drugs or chemotherapeutic agents may be effective anticancer strategies through weakening cell damage repair capacity and enhancing drug cytotoxicity.
PMCID: PMC4973382  PMID: 27374491
glycolysis cancer cells; cancer cells metabolism; Warburg effect; anticancer strategy
12.  Dysfunction of Nucleus Accumbens-1 Activates Cellular Senescence and Inhibits Tumor Cell Proliferation and Oncogenesis 
Cancer research  2012;72(16):4262-4275.
Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, has emerging roles in cancer. We report here that NAC1 acts as a negative regulator of cellular senescence in transformed and non-transformed cells, and dysfunction of NAC1 induces senescence and inhibits its oncogenic potential. We show that NAC1 deficiency markedly activates senescence and inhibits proliferation in tumor cells treated with sub-lethal doses of γ-irradiation. In mouse embryonic fibroblasts (MEFs) from NAC1 knockout mice, following infection with a Ras virus, NAC1−/− cells undergo significantly more senescence and are either non- or less transformed in vitro and less tumorigenic in vivo when compared with NAC1+/+ cells. Furthermore, we show that the NAC1-caused senescence blunting is mediated by ΔNp63, which exerts its effect on senescence through p21, and that NAC1 activates transcription of ΔNp63 under stressful conditions. Our results not only reveal a previously unrecognized function of NAC1, the molecular pathway involved and its impact on pathogenesis of tumor initiation and development, but also identify a novel senescence regulator that may be exploited as a potential target for cancer prevention and treatment.
PMCID: PMC3614094  PMID: 22665267
NAC1; senescence; ΔNp63; oncogene; tumorigenesis
13.  Autophagic pathways as new targets for cancer drug development 
Acta pharmacologica Sinica  2010;31(9):1154-1164.
Autophagy is an evolutionarily conserved lysosomal self-digestion process involved in degradation of long-lived proteins and damaged organelles. In recent years, increasing evidence indicates that autophagy is associated with a number of pathological processes, including cancer. In this review, we focus on the recent studies of the evolutionarily conserved autophagy-related genes (ATGs) that are implicated in autophagosome formation and the pathways involved. We discuss several key autophagic mediators (eg, Beclin-1, UVRAG, Bcl-2, Class III and I PI3K, mTOR, and p53) that play pivotal roles in autophagic signaling networks in cancer. We discuss the Janus roles of autophagy in cancer and highlighted their relationship to tumor suppression and tumor progression. We also present some examples of targeting ATGs and several protein kinases as anticancer strategy, and discuss some autophagy-modulating agents as antitumor agents. A better understanding of the relationship between autophagy and cancer would ultimately allow us to harness autophagic pathways as new targets for drug discovery in cancer therapeutics.
PMCID: PMC3587337  PMID: 20694022
autophagy; cancer; autophagy-related gene (ATG); Beclin-1; Bcl-2; Class III and I PI3K; mTOR; p53
14.  Expression of elongation factor-2 kinase contributes to anoikis resistance and invasion of human glioma cells 
Acta pharmacologica Sinica  2011;32(3):361-367.
To determine whether elongation factor-2 kinase (eEF-2 kinase) contributes to the malignant phenotype of glioblastoma multiforme by promoting the migration and invasion of glioma cells. The mechanism involved was also explored.
Human glioma cell lines T98G and LN-229 were used. The expression of eEF-2 kinase was silenced using siRNA, and the invasive potential of tumor cells was assessed using a wound-healing assay and a Matrigel invasion assay. Apoptosis was determined using propidium iodide (PI) staining and western blot analysis of cleaved caspase-3.
Silencing the expression of eEF-2 kinase by siRNA significantly suppressed both the migration and invasion of human glioma cells. Silencing eEF-2 kinase expression also sensitized glioma cells to anoikis, thereby decreasing tumor cell viability in the absence of attachment. Treatment of tumor cells with the caspase inhibitor z-VAD-fmk down-regulated Bim accumulation and abolished glioma cell sensitivity to anoikis.
The results suggest that the expression of eEF-2 kinase contributes to migration and invasion of human glioma cells by protecting them from anoikis. eEF-2 kinase expression may serve as a prognostic marker and a novel target for cancer therapy.
PMCID: PMC3586188  PMID: 21278783
eEF-2 kinase; migration; invasion; anoikis; glioma
15.  Autophagy and apoptosis: rivals or mates? 
Chinese Journal of Cancer  2013;32(3):103-105.
Autophagy, a cellular process of “self-eating” by which intracellular components are degraded within the lysosome, is an evolutionarily conserved response to various stresses. Autophagy is associated with numerous patho-physiological conditions, and dysregulation of autophagy contributes to the pathogenesis of a variety of human diseases including cancer. Depending on context, activation of autophagy may promote either cell survival or death, two major events that determine pathological process of many illnesses. Importantly, the activity of autophagy is often associated with apoptosis, another critical cellular process determining cellular fate. A better understanding of biology of autophagy and its implication in human health and disorder, as well as the relationship between autophagy and apoptosis, has the potential of facilitating the development of autophagy-based therapeutic interventions for human diseases such as cancer.
PMCID: PMC3845593  PMID: 23419194
Autophagy; apoptosis; cancer; molecular regulation
16.  MK-2206, a novel allosteric inhibitor of Akt, synergizes with gefitinib against malignant glioma via modulating both autophagy and apoptosis* 
Molecular Cancer Therapeutics  2011;11(1):154-164.
Gefitinib, a small molecule inhibitor of the epidermal growth factor receptor tyrosine kinase, has been shown to induce autophagy as well as apoptosis in tumor cells. Yet, how to exploit autophagy and apoptosis to improve therapeutic efficacy of this drug against cancer remains to be explored. We reported here that MK-2206, a potent allosteric Akt inhibitor currently in Phase I trials in patients with solid tumors, could reinforce the cytocidal effect of gefitinib against glioma. We found that co-treatment with gefitinib and MK-2206 increased the cytotoxicity of this growth factor receptor inhibitor in the glioma cells, and the Compusyn synergism/antagonism analysis showed that MK-2206 acted synergistically with gefitinib. The benefit of the combinatorial treatment was also demonstrated in an intracranial glioma mouse model. In the presence of MK-2206, there was a significant increase in apoptosis in glioma cells treated with gefitinib. MK-2206 also augmented the autophagy-inducing effect of gefitinib, as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by silencing of the key autophagy gene, beclin 1 or 3-MA, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agents plays a cytoprotective role. Notably, at 48 hours following the combinatorial treatment, the level of LC3-II began to decrease but Bim was significantly elevated, suggesting a switch from autophagy to apoptosis. Based on the synergistic effect of MK-2206 on gefitinib observed in this study, the combination of these two drugs may be utilized as a new therapeutic regimen for malignant glioma.
PMCID: PMC3302182  PMID: 22057914
MK-2206; gefitinib; apoptosis; autophagy; glioblastoma
17.  NAC1 and HMGB1 enter a partnership for manipulating autophagy 
Autophagy  2011;7(12):1557-1558.
Our recent study revealed a new role of nucleus accumbens-1 (NAC1), a transcription factor belonging to the BTB/POZ gene family, in regulating autophagy. Moreover, we found that the high-mobility group box 1 (HMGB1), a chromatin-associated nuclear protein acting as an extracellular damage associated molecular pattern molecule (DAMP), is the downstream executor of NAC1 in modulating autophagy. In response to stress such as therapeutic insults, NAC1 increases the expression, cytosolic translocation and release of HMGB1; elevated level of the cytoplasmic HMGB1 leads to activation of autophagy. The NAC1-HMGB1 partnership may represent a previously unrecognized pathway that regulates autophagy in response to various stresses such as chemotherapy.
PMCID: PMC3327620  PMID: 22024751
Apoptosis; autophagy; cisplatin; HMGB1; NAC1
18.  Inhibition of eEF-2 kinase Sensitizes Human Glioma Cells to TRAIL and Down-regulates Bcl-xL Expression 
Elongation factor-2 kinase (eEF-2 kinase, also known as calmodulin-dependent protein kinase III), is a unique calcium/calmodulin-dependent enzyme that inhibits protein synthesis by phosphorylating and inactivating elongation factor-2 (eEF-2). We previously reported that expression/activity of eEF-2 kinase was up-regulated in several types of malignancies including Gliomas, and was associated with response of tumor cells to certain therapeutic stress. In the current study, we sought to determine whether eEF-2 kinase expression affected sensitivity of glioma cells to treatment with tumor the necrosis factor-related apoptosis-inducing ligand (TRAIL), a targeted therapy able to induce apoptosis in cancer cells but causes no toxicity in most normal cells. We found that inhibition of eEF-2 kinase by RNA interference (RNAi) or by a pharmacological inhibitor (NH125) enhanced TRAIL-induced apoptosis in the human glioma cells, as evidenced by an increase in apoptosis in the tumor cells treated with eEF-2 kinase siRNA or the eEF-2 kinase inhibitor. We further demonstrated that sensitization of tumor cells to TRAIL was accompanied by a down-regulation of the anti-apoptotic protein, Bcl-xL, and that overexpression of Bcl-xL could abrogate the sensitizing effect of inhibiting eEF-2 kinase on TRAIL. The results of this study may help devise a new therapeutic strategy for enhancing the efficacy of TRAIL against malignant glioma by targeting eEF-2 kinase.
PMCID: PMC3210449  PMID: 21945617
eEF-2 kinase; TRAIL; Bcl-xl; apoptosis; glioblastoma
19.  NAC1 Modulates Sensitivity of Ovarian Cancer Cells to Cisplatin via Altering the HMGB1-Mediated Autophagic Response* 
Oncogene  2011;31(8):1055-1064.
Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to play important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We reported here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3, and SKOV3. We further demonstrated that knockdown of NAC1 by RNAi or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors, 3-MA and chloroquine, and siRNAs targeting beclin 1 or Atg5, on the cytotoxicity of cisplatin. Treatment with 3-MA, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated via high mobility group box1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity, but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1- mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.
PMCID: PMC3275651  PMID: 21743489
NAC1; autophagy; apoptosis; HMGB1; cisplatin; ovarian cancer
20.  eEF-2 kinase 
Autophagy  2011;7(6):660-661.
Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.
PMCID: PMC3127050  PMID: 21460616
eEF-2 kinase; Akt; autophagy; apoptosis; MK-2206; cancer treatment
21.  eEF-2 Kinase Dictates Crosstalk between Autophagy and Apoptosis Induced by Akt Inhibition, thereby Modulating Cytotoxicity of Novel Akt Inhibitor MK-2206* 
Cancer research  2011;71(7):2654-2663.
Inhibition of the survival kinase Akt can trigger apoptosis but also has been found to activate autophagy, which may confound tumor attack. In this study, we investigated regulatory mechanisms through which apoptosis and autophagy were modulated in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt to enter clinical development. In human glioma cells, Akt inhibition by MK-2206 or siRNA-mediated attenuation strongly activated autophagy, whereas silencing of eukaryotic elongation factor-2 (eEF-2) kinase, a protein synthesis regulator, blunted this autophagic response. Suppression of MK-2206-induced autophagy by eEF-2 silencing was accompanied by a promotion of apoptotic cell death. Similarly, siRNA-mediated inhibition of eEF-2 kinase potentiated the efficacy of MK-2206 against glioma cells. Together, these results demonstrated that blunting autophagy and augmenting apoptosis by inhibition of eEF-2 kinase could modulate the sensitivity of glioma cells to Akt inhibition. Our findings suggest that targeting eEF-2 kinase may reinforce the anti-tumor efficacy of Akt inhibitors such as MK-2206.
PMCID: PMC3210447  PMID: 21307130
Elongation factor-2 kinase; Akt; Autophagy; Apoptosis; MK-2206; Glioblastoma
22.  Interaction of Beclin 1 with Survivin Regulates Sensitivity of Human Glioma Cells to TRAIL-induced Apoptosis 
FEBS letters  2010;584(16):3519-3524.
We reported a novel interaction between Beclin 1, a key regulator of autophagy, and survivin, a member of the IAP family. We found that knock-down of Beclin 1 down-regulated survivin protein, and the turnover rate of survivin was increased when Beclin 1 expression was silenced. Knock-down of Beclin 1 sensitized glioma cells to TRAIL-induced apoptosis, and introduction of survivin antagonized the sensitizing effect, suggesting that down-regulation of survivin mediates the enhanced sensitivity to TRAIL-induced apoptosis. These results demonstrate a novel interaction between Beclin 1 and survivin, and may provide a potential mechanism underlying the cross-talk between autophagy and apoptosis.
PMCID: PMC3210451  PMID: 20638385
apoptosis; autophagy; Beclin 1; survivin; TRAIL
23.  Expression of elongation factor-2 kinase contributes to anoikis resistance and invasion of human glioma cells 
Acta Pharmacologica Sinica  2011;32(3):361-367.
To determine whether elongation factor-2 kinase (eEF-2 kinase) contributes to the malignant phenotype of glioblastoma multiforme by promoting the migration and invasion of glioma cells. The mechanism involved was also explored.
Human glioma cell lines T98G and LN-229 were used. The expression of eEF-2 kinase was silenced using siRNA, and the invasive potential of tumor cells was assessed using a wound-healing assay and a Matrigel invasion assay. Apoptosis was determined using propidium iodide (PI) staining and Western blot analysis of cleaved caspase-3.
Silencing the expression of eEF-2 kinase by siRNA significantly suppressed both the migration and invasion of human glioma cells. Silencing eEF-2 kinase expression also sensitized glioma cells to anoikis, thereby decreasing tumor cell viability in the absence of attachment. Treatment of tumor cells with the caspase inhibitor z-VAD-fmk down-regulated Bim accumulation and abolished glioma cell sensitivity to anoikis.
The results suggest that the expression of eEF-2 kinase contributes to migration and invasion of human glioma cells by protecting them from anoikis. eEF-2 kinase expression may serve as a prognostic marker and a novel target for cancer therapy.
PMCID: PMC3586188  PMID: 21278783
eEF-2 kinase; migration; invasion; anoikis; glioma
24.  A role for p53 in the regulation of extracellular matrix metalloproteinase inducer in human cancer cells 
Cancer Biology & Therapy  2009;8(18):1722-1728.
EMMPRIN, a transmembrane glycoprotein known to pro-mote survival, invasion and metastasis of tumor cells through multiple pathways and mechanisms, has been found to be overexpressed in various types of cancer cells. Here we report that loss of the function of p53, a tumor suppressor protein that is mutated in approximately 50% of human cancers, contributes to the upregulation of EMMPRIN protein. We observed an inverse association between the activity of p53 and the level of EMMPRIN protein in several cancer cell lines. We further demonstrated that p53 is able to negatively regulate EMMPRIN protein, but downregulation of EMMPRIN by p53 is independent of repression of the EMMPRIN transcription. Furthermore, downregulation of EMMPRIN by p53 can be rescued by chloroquine, a lysosome inhibitor, but not by MG132, a proteasome inhibitor, suggesting an involvement of the lysosomal pathway in the p53-regulated degradation of EMMPRIN. Downregulation of EMMPRIN by p53 leads to a decrease in the activity of MMP-9 and an inhibition of tumor cell invasion. Our study suggests that the upregulation of EMMPRIN seen in many cancers can be attributed to, at least in part, the dysfunction of p53 and thus provides new evidence for the roles of p53 in tumor development and progression.
PMCID: PMC3630173  PMID: 19597352
p53; extracellular matrix metalloproteinase inducer; matrix metalloproteinase; tumor progression
25.  Regulation of autophagy by a beclin 1-targeted microRNA, miR-30a, in cancer cells 
Autophagy  2009;5(6):816-823.
beclin 1, the mammalian homologue of the yeast Atg6, is a key autophagy-promoting gene that plays a critical role in the regulation of cell death and survival of various types of cells. However, recent studies have observed that the expression of beclin 1 is altered in certain diseases including cancers. The causes underlying the aberrant expression of beclin 1 remain largely unknown. We report here that microRNAs (miRNAs), a class of endogenous, 22–24 nucleotide noncoding RNA molecules able to affect stability and translation of mRNA, may represent a previously unrecognized mechanism for regulating beclin 1 expression and autophagy. We demonstrated that beclin 1 is a potential target for miRNA miR-30a, and this miRNA could negatively regulate beclin 1 expression resulting in decreased autophagic activity. Treatment of tumor cells with the miR-30a mimic decreased, and with the antagomir increased, the expression of beclin 1 mRNA and protein. Dual luciferase reporter assay confirmed that the miR-30a binding sequences in the 3′-UTR of beclin 1 contribute to the modulation of beclin 1 expression by miR-30a. Furthermore, inhibition of beclin 1 expression by the miR-30a mimic blunted activation of autophagy induced by rapamycin. Our study of the role of miR-30a in regulating beclin 1 expression and autophagy reveals a novel function for miRNA in a critical cellular event with significant impacts in cancer development, progression and treatment, and in other diseases.
PMCID: PMC3669137  PMID: 19535919
beclin 1; autophagy; microRNA; miR-30a; gene expression

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