Correction: P62 regulates resveratrol-mediated Fas/Cav-1 complex formation and transition from autophagy to apoptosis.

[This corrects the article DOI: 10.18632/oncotarget.2733.].

Lasting DNA Damage and Aberrant DNA Repair Gene Expression Profile Are Associated with Post-Chronic Cadmium Exposure in Human Bronchial Epithelial Cells.

Cadmium (Cd) is a widespread environmental pollutant and carcinogen. Although the exact mechanisms of Cd-induced carcinogenesis remain unclear, previous acute/chronic Cd exposure studies have shown that Cd exerts its cytotoxic and carcinogenic effects through multiple mechanisms, including interference with the DNA repair system. However, the effects of post-chronic Cd exposure remain unknown. Here, we establish a unique post-chronic Cd-exposed human lung cell model (the "CR0" cells) and investigate the effects of post-chronic Cd exposure on the DNA repair system. We found that the CR0 cells retained Cd-resistant property even though it was grown in Cd-free culture medium for over a year. The CR0 cells had lasting DNA damage due to reduced DNA repair capacity and an aberrant DNA repair gene expression profile. A total of 12 DNA repair genes associated with post-chronic Cd exposure were identified, and they could be potential biomarkers for identifying post-chronic Cd exposure. Clinical database analysis suggests that some of the DNA repair genes play a role in lung cancer patients with different smoking histories. Generally, CR0 cells were more sensitive to chemotherapeutic (cisplatin, gemcitabine, and vinorelbine tartrate) and DNA damaging (H2O2) agents, which may represent a double-edged sword for cancer prevention and treatment. Overall, we demonstrated for the first time that the effects of post-chronic Cd exposure on human lung cells are long-lasting and different from that of acute and chronic exposures. Findings from our study unveiled a new perspective on Cd-induced carcinogenesis-the post-chronic exposure of Cd. This study encourages the field of post-exposure research which is crucial but has long been ignored.

DNA methylation of a non-CpG island promoter represses NQO1 expression in rat arsenic-transformed lung epithelial cells.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II flavoenzyme that catalyzes reduction reactions to protect cells against electrophiles and oxidants, is involved in tumorigenesis. Altered methylation of the NQO1 gene has been observed and is speculated to result in aberrant NQO1 expression in rat cells undergoing chemical carcinogenesis, although this has not been proven experimentally. In this study, we first investigated the potential epigenetic mechanisms underlying the phenomenon of NQO1 differential expression in individual subclones of rat arsenic-transformed lung epithelial cells (TLECs). NQO1 expression of TLEC subclones with or without 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis, and real-time PCR. Methylation status of the NQO1 promoter in TLEC subclones was analyzed by bisulfite sequencing. Transcriptional activity of NQO1 promoter in vitro methylated was determined by luciferase assay using a CpG-free luciferase reporter driven by the NQO1 promoter region (-435 to +229). We found that non-CpG island (non-CpGI) within the NQO1 promoter was hyper- or hypo-methylated in TLEC subclones and corresponded to low and high gene expressions, respectively. Following the treatment with 5-Aza-CdR, transcription of the NQO1 gene in the hypermethylated subclones was restored, accompanied by demethylation of the NQO1 promoter. In vitro promoter methylation almost completely silenced reporter activity in TLECs. These results indicate that DNA methylation of the non-CpGI promoter contributes to epigenetic silencing of NQO1 in rat TLECs.

Resveratrol induces autophagy-dependent apoptosis in HL-60 cells.

BACKGROUND: All known mechanisms of apoptosis induced by resveratrol act through cell cycle arrest and changes in mitochondrial membrane potential. It is currently unknown whether resveratrol-induced apoptosis is associated with other physiological processes, such as autophagy. METHODS: Apoptosis-related markers involved in the intrinsic and extrinsic apoptotic pathways, and autophagic markers were detected by using western blotting and immunofluorescence. Mitochondrial membrane potential was assayed by flow cytometry. Pharmaceutical or genetic inhibition of autophagy involved were carried by 3- methyladenine or knockdown of autophagy-related (Atg) genes by siRNA. Differences between two values were tested by Student's unpaired t test. RESULTS: We show that resveratrol-induced apoptosis occurs through both the intrinsic and extrinsic apoptotic pathways. Mitochondrial membrane potential and apoptosis-related markers, such as an increased Bax/Bcl-2 ratio, and cleaved forms of caspase-8 and caspase-3, arise following resveratrol addition. Moreover, we find that resveratrol increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, and further demonstrate that resveratrol-induced autophagy depends on the LKB1-AMPK-mTOR pathway. We next reveal that some apoptosis-related markers induced by resveratrol are further attenuated by the inhibition of autophagy with 3-methyladenine or knockdown of autophagy-related (Atg) genes by siRNA. CONCLUSIONS: These results suggest that resveratrol induced apoptotic cell death of HL-60 cells depends on the autophagy activated through both the LKB1-AMPK and PI3K/AKT-regulated mTOR signaling pathways.

Aberrant cytokine secretion and zinc uptake in chronic cadmium-exposed lung epithelial cells.

PURPOSE: Our previous results showed that cadmium (Cd)-adapted lung epithelial cells (LECs) developed resistance to apoptosis due to non-responsiveness of the c-Jun N-terminal kinase pathway and augmented expression of cytokeratin 8. Since cellular Cd entry is a prerequisite in order for Cd to elicit its cytotoxicity, therefore, we wonder if there are differential metal ion transport ability and also other phenotypic changes that occurred in these Cd-resistant LECs. EXPERIMENTAL DESIGN AND RESULTS: Here, we explored further and found that the zinc (Zn) importer Zip8 was stably abolished in these cells along with a marked decrease of Cd and Zn accumulation. Moreover, by cell migration assays and cytokine antibody array analysis, we found that Cd-adapted cells exhibit enhanced migratory ability possibly due to elevated secretions of vascular endothelial growth factor and macrophage inflammatory protein-3 alpha (MIP-3α). CONCLUSION AND CLINICAL RELEVANCE: Taken together, our results show that during chronic Cd exposure, lung cells antagonize excessive cellular Cd-influx by abolishing Zip8 expression to reduce Cd-toxicity; however, this also renders cells with a diminished Zn uptake. The imbalance of Zn homeostasis and elevation of angiogenic and epithelial-mesenchymal transition-promoting cytokines in Cd-adapted cells might thus likely promote Zn deficiency, angiogenesis, and cell invasion.

Upregulation of glycolysis and oxidative phosphorylation in benzo[α]pyrene and arsenic-induced rat lung epithelial transformed cells.

Arsenic and benzo[α]pyrene (B[a]P) are common contaminants in developing countries. Many studies have investigated the consequences of arsenic and/or B[a]P-induced cellular transformation, including altered metabolism. In the present study, we show that, in addition to elevated glycolysis, B[a]P/arsenic-induced transformation also stimulates oxidative phosphorylation (OXPHOS). Proteomic data and immunoblot studies demonstrated that enzymatic activities, involved in both glycolysis and OXPHOS, are upregulated in the primary transformed rat lung epithelial cell (TLEC) culture, as well as in subcloned TLEC cell lines (TMCs), indicating that OXPHOS was active and still contributed to energy production. LEC expression, of the glycolytic enzyme phosphoglycerate mutase (PGAM) and the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (OGDH), revealed an alternating cyclic pattern of glycolysis and OXPHOS during cell transformation. We also found that the expression levels of hypoxia-inducible factor-1a were consistent with the pattern of glycolysis during the course of transformation. Low doses of an ATP synthase inhibitor depleted endogenous ATP levels to a greater extent in TLECs, compared to parental LECs, indicating greater sensitivity of B[a]P/arsenic-transformed cells to ATP depletion. However, TLEC cells exhibited better survival under hypoxia, possibly due to further induction of anaerobic glycolysis. Collectively, our data indicate that B[a]P/arsenic-transformed cells can maintain energy production through upregulation of both glycolysis and OXPHOS. Selective inhibition of metabolic pathways may serve as a therapeutic option for cancer therapy.

Proteomics Based Identification of Proteins with Deregulated Expression in B Cell Lymphomas.

Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL.

Two-dimensional liquid chromatography with tandem mass spectrometry-based proteomic characterization of endometrial luminal epithelial surface proteins responsible for embryo implantation.

OBJECTIVE: To identify endometrial epithelial cell surface proteins essential for blastocysts implantation. DESIGN: Isolation of cell-surface labeled prereceptive (pregnancy day 1) and receptive (pregnancy day 4) mouse endometrial proteins coupled to two-dimensional liquid chromatography with tandem mass spectrometry. SETTING: University research laboratory. ANIMAL(S): Sexually mature female imprinting control region (ICR) mice. INTERVENTION(S): Labeling, purification, and identification of endometrial luminal surface proteins with differentially expressing proteins determined by significant analysis of a microarray algorithm and selected differentially expressed proteins verified by immunohistochemistry and functional assay. MAIN OUTCOME MEASURE(S): Investigation in endometrial luminal surface proteome of prereceptive and receptive endometria of the expression of four of the differentially expressed proteins and functional analysis of aminopeptidase N in a three-dimensional blastocyst-endometrial coculture model. RESULT(S): We identified 104 cell surface proteins from prereceptive and receptive pregnant mouse endometria and found that 27 were statistically significantly up-regulated and 18 were statistically significantly down-regulated in the receptive endometrium. Immunohistochemical analysis of four of the differentially expressed proteins in the endometrium showed concordant results. Functional assay showed that blastocyst attachment was statistically significantly reduced upon inhibition of aminopeptidase N. CONCLUSION(S): The luminal cell surface proteome of the prereceptive and receptive endometria differs, and aminopeptidase N is potentially involved in embryo attachment.

P62 regulates resveratrol-mediated Fas/Cav-1 complex formation and transition from autophagy to apoptosis.

Resveratrol is a potential polyphenol drug used in cancer treatment. We examined the relationship between autophagy and apoptosis in RSV-treated non-small lung adenocarcinoma A549 cells. Resveratrol treatment increased autophagy and autophagy-mediated degradation of P62. Immunocytochemistry revealed P62 co-localized with Fas/Cav-1 complexes, known to induce apoptosis. However, siRNA-mediated P62 downregulation enhanced formation of Fas/Cav-1 complexes, suggesting that P62 inhibited Fas/Cav-1 complex formation. Fas/Cav-1 complexes triggered caspase-8 activation and cleavage of Beclin-1, releasing a C-terminal Beclin-1 peptide that translocated to the mitochondria and initiate apoptosis. Inhibition of autophagy by siRNA-mediated repression of Beclin-1 also blocked RSV-induced apoptosis, showing a dependence of apoptosis on autophagy. P62 knockdown by siRNA accelerated the activation of caspase-8 and initiate apoptosis, while Cav-1 knockdown inhibited apoptosis, but increased autophagy. Inhibition of autophagy by 3-MA prevented both P62 degradation and induction of apoptosis, whereas inhibition of apoptosis by z-IETD-FMK or z-DEVD-FMK enhanced both P62 induction and autophagic cell death. In conclusion, P62 links resveratrol-induced autophagy to apoptosis. P62 blocks apoptosis by inhibiting Fas/Cav-1 complex formation, but RSV-induced autophagic degradation of P62 enables formation of Fas/Cav-1 complexes which then activate caspase-8-mediated Beclin-1 cleavage, resulting in translocation of the Beclin-1 C-terminal fragment to the mitochondria to initiate apoptosis.

Dioscin induced activation of p38 MAPK and JNK via mitochondrial pathway in HL-60 cell line.

Saponins have shown promise in cancer prevention and therapy; however, little is known about the detailed signaling pathways underlying their anticancer activities. In the present study, we examined the mechanisms of action of dioscin, a glucosides saponin isolated from Polygonatum zanlanscianense pump, in human myeloblast leukemia HL-60 cells. Dioscin suppressed HL-60 cell growth in a dose-dependent manner. This inhibition was due to the induction of apoptosis as revealed by the externalization of phosphatidylserine, and cleavages of lamin A/C and PARP-1. Treatment with dioscin induced apoptosis through activation of caspases 3, 7, 8, 9, and 10. Phosphorylation of p38 MAPK and JNK contributed to dioscin-induced apoptosis upstream of caspase activation. Using various inhibitors and antioxidant agents, we found that mitochondrial derived reactive oxygen species and depletion of mitochondrial transmembrane potential lead to the phosphorylation of p38 MAPK and JNK. Taken together, our results demonstrated that dioscin induces apoptosis by activation of p38 MAPK and JNK through the caspase-dependent mitochondrial death pathway. This work suggests that dioscin may be used as a drug lead for the treatment of myeloblast leukemia.

Resveratrol-induced apoptosis is enhanced by inhibition of autophagy in esophageal squamous cell carcinoma.

The anti-cancer activity of resveratrol in human esophageal squamous cell carcinoma (ESCC) was investigated focusing on the role of autophagy and its effects on apoptotic cell death. We demonstrated that resveratrol inhibits ESCC cell growth in a dose-dependent manner by inducing cell cycle arrest at the sub-G1 phase and resulting in subsequent apoptosis. Mechanistically, resveratrol-induced autophagy in the ESCC cells is AMPK/mTOR pathway independent. Since both pharmacological and genetic inhibition of autophagy enhanced the resveratrol-induced cytotoxicity to the ESCC cells, this provided a novel strategy in potentiating the anti-cancer effects of resveratrol and other chemotherapeutic reagents in ESCC cancer treatment.

An overview of esophageal squamous cell carcinoma proteomics.

Esophageal squamous cell carcinoma (ESCC) still remains the leading cancer-caused mortality in northern China, in particular in areas nearby Taihang Mountain. Late-stage diagnosis of ESCC increases the mortality and morbidity of ESCC. Therefore, it is imperative to identify biomarkers for early diagnosis, monitoring of tumor progression and identifying potential therapeutic targets of ESCC. Proteomics provides a functional translation of the genome and represents a richer source for the functional description of diseases and biomarkers implicated in cancer. In this review, we discuss the dysregulated proteins associated with ESCC identified by proteomic approaches and aim to enhance our understanding of molecular mechanisms implicated in ESCC development and progression from a proteomics perspective and discuss the potential biomarkers of ESCC as well.

Post-translational modification of human heat shock factors and their functions: a recent update by proteomic approach.

Heat shock factors (HSFs) are vital for modulating stress and heat shock-related gene expression in cells. The activity of HSFs is controlled largely by post-translational modifications (PTMs). For example, basal phosphorylation of HSF1 on three serine sites suppresses the heat shock response, and hyperphosphorylation of HSF1 on several other serine and threonine sites by stress-activated kinases results in its activation, while acetylation on K80 inhibits its DNA-binding ability. Sumoylation of HSF2 on K82 regulates its DNA-binding ability, whereas sumoylation of HSF4B on K293 represses its transcriptional activity. With the advancement of proteomic technology, novel PTM sites on various HSFs have been identified with the use of tandem mass spectrometry (MS/MS), but the functions of many of these PTMs are still unclear. Yet, it should be noted that the discovery of these novel PTM sites provided the necessary evidence for the existence of these PTM marks in vivo. Followed by subsequent functional analysis, this would ultimately lead to a better understanding of these PTM marks. MS/MS-based proteomic approach is becoming a gold standard in PTM validation in the field of life science. Here, the recent literature of all known PTMs reported on human HSFs and the resulting functions will be discussed.

Comparative proteomic analysis of differentiation of mouse F9 embryonic carcinoma cells induced by retinoic acid.

The multipotent mouse F9 embryonic carcinoma cell is an ideal model system to investigate the mechanism of retinoic acid (RA) in cell differentiation and cell growth control and the biochemical basis of early embryonic development. We reported here a proteomics approach to study protein expression changes during the differentiation of F9 cells into the visceral endoderm. F9 cells were incubated with or without RA at 0, 24, 48, and 72 h. Total proteins extracted were separated by two-dimensional electrophoresis (2-DE) and the protein patterns on the gels were comparatively analyzed by computer. Approximately 1,100 protein spots were detected in the F9 proteome, within the pH 3-10 range. Fourteen protein spots which the levels of expression were found to be altered dramatically during the F9 cells differentiating, and were identified by MALDI-TOF MS or ESI-MS/MS. These proteins included metabolism enzymes, HSP60s, RAN, hnRNP K, FUBP1, VDAC1, STI1, and prohibitin. These proteins are involved in cellar metabolism, gene expression regulation, stress response, and apoptosis, respectively. The data from proteomic analyze are consistent with the result obtained from Western blot analysis. This study increases our understanding of the proteomics changes during F9 cells differentiation induced by RA.

Multiple pathways were involved in tubeimoside-1-induced cytotoxicity of HeLa cells.

The Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae) is a Chinese herb with anticancer potential. Its main active component tubeimoside-1 (TBMS1), a triterpenoid saponin, was previously proved as a potent anticancer chemotherapeutic agent; however, the molecular basis for its activities is still elusive. In the present study, subcellular proteomic study in the cytoplasm and membrane protein fractions extracted from HeLa cells revealed that proteins act as mediators of ROS generation and Ca(2+) regulation were substantially altered in expression upon TBMS1 stimuli. We also found that TBMS1 induced cell cycle arrest at G2/M phase accompanied by a decrease in G0/G1 phase in HeLa cells. Further biochemical studies showed that TBMS1 inhibited the levels of cyclinB1, Cdc2 and Cdc25C, but enhanced Chk2 phosphorylation. In addition, the cytoplasm sequestration of Cdc25C, Cip1/p21 induction and tubulin dyspolymerization also contributed to the TBMS1-mediated cell cycle arrest on the G2/M phase.

Molecular changes during arsenic-induced cell transformation.

Arsenic and its derivatives are naturally occurring metalloid compounds widely distributed in the environment. Arsenics are known to cause cancers of the skin, liver, lung, kidney, and bladder. Although numerous carcinogenic pathways have been proposed, the exact molecular mechanisms remain to be delineated. To further characterize the role of oxidative stress in arsenite-induced cell transformation via the reactive oxygen species (ROS)-mediated Ras/Erk pathway, here we demonstrated arsenite-induced rat lung epithelial cell (LEC) transformation, epithelial-mesenchymal transition, stimulation of the extracellular signal-regulated kinase signaling pathway, and enhancement of cell proliferation. However, there was no evidence of activation of the phosphoinositide 3-kinase/protein kinase B pathway in arsenite-induced transformed LECs. Since ROS is involved in arsenite-induced LEC cell transformation, Redox-status regulatory proteins (Cu/Zn SOD and thioredoxin) and arsenite-induced LEC cell transformation were significantly inhibited by concurrent treatment with the antioxidants. Our experimental results clearly demonstrated that induction of p-ERK and cell proliferation by arsenite is mediated via oxidative stress, since antioxidants can inhibit arsenite-induced cell transformation.

Epidermal growth factor-induced epithelial-mesenchymal transition in human esophageal carcinoma cells--a model for the study of metastasis.

Deciphering the molecular basis of esophageal cancer metastasis requires adequate experimental models. Epithelial-mesenchymal transition (EMT) is the hallmark of tumor metastasis. As a promoter of the malignant progression of esophageal cancer, epidermal growth factor (EGF) has been shown to induce EMT in several cell lines. In this study we examined the effects of EGF on esophageal carcinoma EC109 cells. We found that EGF at high concentration induced the cells to undergo morphological change, exhibit higher invasive and metastatic potential, as well as change in the expression of lineage markers. This EMT model might facilitate mechanistic studies of esophageal cancer metastasis.

Down-regulation of HSP27 sensitizes TRAIL-resistant tumor cell to TRAIL-induced apoptosis.

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it preferentially induces apoptosis in human cancer over normal cells. Most tumor cells, including lung cancer cell line A549, unfortunately, are resistant to TRAIL treatment even at high dose. Recent studies indicated that TRAIL-resistant cancer cells could be sensitized to TRAIL by combination therapy. Stress and heat shock proteins such as HSP90, HSP70 and HSP27 are induced in response to a wide variety of physiological environmental insults including heat, reactive oxygen species or anticancer drugs. Their elevated expressions facilitate cells to survive in stress circumstances. The HSP27 expression is enhanced in many tumor cells, implying that it is involved in tumor progression and the development of treatment resistance in various tumors, including lung cancer. This fact suggests a novel strategy for the treatment of cancer via inhibiting the function of HSP27. In this study, we investigated the inhibitory effect of a small interfering (si) RNA on the expression of HSP27 gene in the TRAIL-resistant human lung adenocarcinoma cell line A549, and the effect of HSP27 siRNA on drug sensitization of A549 cells to TRAIL treatment. The results showed that treatment of A549 cells with HSP27 siRNA down-regulated HSP27 expression but did not induce significant apoptosis. However, combination of HSP27 siRNA with TRAIL-induced significant apoptosis in TRAIL-resistant A549 cells. In addition to inducing caspases activation and apoptosis, combined treatment with HSP27 siRNA and TRAIL also increased JNK and p53 expression and activity. Collectively, these findings provide a conclusion that siRNA targeting of the HSP27 gene specifically down-regulated HSP27 expression in A549 cells, and sensitized the cells to TRAIL-induced apoptosis.

Biomarkers of lung-related diseases: current knowledge by proteomic approaches.

The lung epithelial surface is one of the vital barriers or sensors in the body responding to the external atmosphere and thereby always subjecting to direct toxicological exposure, stress, stimulus, or infection. Due to its relatively higher sensitivity in response to toxicants, the use of lung epithelial cell culture and lung tissue from animal models or patients has facilitated our learning to lung physiopathology and toxicopharmacology. The recent advancement of proteomics has made it possible to investigate the cellular response at a global level. In this review, the potential applications of proteomic approach in studying lung-related diseases and biomarker discovery will be discussed.