Cd-exposed Cells Research Articles

M6A-methylated Lonp1 drives mitochondrial proteostasis stress to induce testicular pyroptosis upon environmental cadmium exposure

Cadmium (Cd) is a widely distributed typical environmental pollutant and one of the most toxic heavy metals. It is well-known that environmental Cd causes testicular damage by inducing classic types of cell death such as cell apoptosis and necrosis. However, as a new type of cell death, the role and mechanism of pyroptosis in Cd-induced testicular injury remain unclear. In the current study, we used environmental Cd to generate a murine model with testicular injury and AIM2-dependent pyroptosis. Based on the model, we found that increased cytoplasmic mitochondrial DNA (mtDNA), activated mitochondrial proteostasis stress occurred in Cd-exposed testes. We used ethidium bromide to generate mtDNA-deficient testicular germ cells and further confirmed that increased cytoplasmic mtDNA promoted AIM2-dependent pyroptosis in Cd-exposed cells. Uracil-DNA glycosylase UNG1 overexpression indicated that environmental Cd blocked UNG-dependent repairment of damaged mtDNA to drive the process in which mtDNA releases to cytoplasm in the cells. Interestingly, we found that environmental Cd activated mitochondrial proteostasis stress by up-regulating protein expression of LONP1 in testes. Testicular specific LONP1-knockdown significantly reversed Cd-induced UNG1 protein degradation and AIM2-dependent pyroptosis in mouse testes. In addition, environmental Cd significantly enhanced the m6A modification of Lonp1 mRNA and its stability in testicular germ cells. Knockdown of IGF2BP1, a reader of m6A modification, reversed Cd-induced upregulation of LONP1 protein expression and pyroptosis activation in testicular germ cells. Collectively, environmental Cd induces m6A modification of Lonp1 mRNA to activate mitochondrial proteostasis stress, increase cytoplasmic mtDNA content, and trigger AIM2-dependent pyroptosis in mouse testes. These findings suggest that mitochondrial proteostasis stress is a potential target for the prevention of testicular injury.

Cadmium Activates EGFR/STAT5 Signaling to Overcome Calcium Chelation and Promote Epithelial to Mesenchymal Transition.

Cadmium (Cd) is a heavy metal found in cigarette smoke, as well as in air and drinking water due to agricultural and industrial activities, and it poses a health risk to the general population. Prolonged low-dose Cd exposure via inhalation or ingestion causes lung and kidney cancers in humans and in animal models. While high doses of Cd exposure are correlated with the occupational setting and are cytotoxic, low doses of Cd are mainly correlated with exposure in the general population and induce carcinogenesis. The mechanism by which Cd-exposed cells overcome calcium chelation and induce malignant transformation remains unclear. This study examines how cells exposed to low doses of Cd survive loss of E-cadherin cell-cell adhesion via activation of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 5 (STAT5), which work to upregulate genes associated with survival and proliferation. To demonstrate the role of Cd in EGFR/STAT5 activation, we exposed two epithelial cell lines, BEAS-2B and HEK293, to two different doses (0.4 µM and 1.6 µM) of Cadmium chloride hemipentahydrate (CdCl2·2.5H2O) that are environmentally relevant to levels of Cd found in food and cigarettes for 24 h (hours) and 9 weeks (wks). When comparing cells treated with Cd with control cells, the Cd treated cells exhibited faster proliferation; therefore, we studied activation of EGFR via the STAT5 pathway using immunofluorescence (IF) for protein expression and localization and, in addition, RT-qPCR to examine changes in EGFR/STAT5 inducible genes. Our results showed an increase in EGFR and phosphorylated EGFR (p-EGFR) protein, with 1.6 µM of Cadmium having the highest expression at both 24-hour (hr) and 9-week (wk) exposures. Moreover, the IF analysis also demonstrated an increase of STAT5 and phosphorylated STAT5 (pSTAT5) in both short-term and long-term exposure, with 0.4 µM having the highest expression at 24 h. Finally, via Western blot analysis, we showed that there was a dose-dependent decrease in E-cadherin protein expression and increased N-cadherin in cells treated with low doses of Cd. These data demonstrate that epithelial cells can overcome Cd-mediated toxicity via activation of EGFR pathway to induce cell proliferation and survival and promote epithelial to mesenchymal transition.

Molybdenum and cadmium co-exposure induces CaMKKβ/AMPK/mTOR pathway mediated-autophagy by subcellular calcium redistribution in duck renal tubular epithelial cells

Excessive molybdenum (Mo) and cadmium (Cd) are toxic environmental pollutants. Our previous research confirmed excessive Mo and Cd co-induced calcium homeostasis disorder and autophagy in duck kidneys, but how calcium ion (Ca2+) regulates autophagy is unclear. The results revealed that the Mo- and/or Cd-induced cytosolic Ca2+ concentration ([Ca2+]c) increase mainly came from intracellular calcium stores. Mo and/or Cd caused mitochondrial Ca2+ content ([Ca2+]mit) and [Ca2+]c increase with endoplasmic reticulum (ER) Ca2+ content ([Ca2+]ER) decrease and upregulated calcium homeostasis-related factor expression levels, but 2-Aminoethoxydiphenyl borate (2-APB) reversed subcellular Ca2+ redistribution. Increased Phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) activities and inositol 1,4,5-trisphosphate receptor (IP3R) expression level were observed in Mo- and/or Cd-treated cells, which was reversed by the PLC inhibitor U-73122. 2-APB and 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) addition mitigated [Ca2+]c and autophagy (variations in microtubule-associated protein light chain 3 (LC3), LC3B-II/LC3B-I, autophagy related 5 (ATG5), sequestosome-1(P62), programmed cell death-1 (Beclin-1) and Dynein expression levels, LC3 puncta, autophagosomes and acid vesicle organelles) under Mo and/or Cd treatment, respectively, while thapsigargin (TG) had the opposite impacts. Additionally, the calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor STO-609 reversed the increased CaMKKβ, adenosine 5′-monophosphate-activated protein kinase (AMPK), Beclin-1, and LC3B-II/LC3B-I protein expression levels and reduced mammalian target of rapamycin (mTOR) and P62 protein expression levels in Mo- and/or Cd-exposed cells. Collectively, the results confirmed that [Ca2+]c overload resulted from PLC/IP3/IP3R pathway-mediated ER Ca2+ release, and then activated autophagy by the CaMKKβ/AMPK/mTOR pathway in Mo- and/or Cd-treated duck renal tubular epithelial cells.

Proteomic Perspective of Cadmium Tolerance in Providencia rettgeri Strain KDM3 and Its In-situ Bioremediation Potential in Rice Ecosystem.

In this study, a multi-metal-tolerant natural bacterial isolate Providencia rettgeri strain KDM3 from an industrial effluent in Mumbai, India, showed high cadmium (Cd) tolerance. Providencia rettgeri grew in the presence of more than 100 ppm (880 μM) Cd (LD50 = 100 ppm) and accumulated Cd intracellularly. Following Cd exposure, a comparative proteome analysis revealed molecular mechanisms underlying Cd tolerance. Among a total of 69 differentially expressed proteins (DEPs) in Cd-exposed cells, de novo induction of ahpCF operon proteins and L-cysteine/L-cystine shuttle protein FliY was observed, while Dps and superoxide dismutase proteins were overexpressed, indicating upregulation of a robust oxidative stress defense. ENTRA1, a membrane transporter showing homology to heavy metal transporter, was also induced de novo. In addition, the protein disaggregation chaperone ClpB, trigger factor, and protease HslU were also overexpressed. Notably, 46 proteins from the major functional category of energy metabolism were found to be downregulated. Furthermore, the addition of P. rettgeri to Cd-spiked soil resulted in a significant reduction in the Cd content [roots (11%), shoot (50%), and grains (46%)] of the rice plants. Cd bioaccumulation of P. rettgeri improved plant growth and grain yield. We conclude that P. rettgeri, a highly Cd-tolerant bacterium, is an ideal candidate for in-situ bioremediation of Cd-contaminated agricultural soils.

Down-regulation of lncRNA MEG3 promotes chronic low dose cadmium exposure-induced cell transformation and cancer stem cell-like property

Cadmium (Cd) is a toxic heavy metal and one of carcinogens that cause lung cancer. However, the exact mechanism of Cd carcinogenesis remains unclear. To investigate the mechanism of Cd carcinogenesis, we exposed human bronchial epithelial cells (BEAS-2B) to a low dose of Cd (2.5 μM, CdCl2) for 9 months, which caused cell malignant transformation and generated cancer stem cell (CSC)-like cells. The goal of this study is to investigate the underlying mechanism. The long non-coding RNA (lncRNA) microarray analysis showed that the expression level of a tumor suppressive lncRNA maternally expressed 3 (MEG3) is significantly down-regulated in Cd-transformed cells, which is confirmed by further q-PCR analysis. Mechanistically, it was found that chronic Cd exposure up-regulates the levels of DNA methyltransferases (DNMTs), which increases the methylation of the differentially methylated region (DMR) 1.5 kb upstream of MEG3 transcription start site to reduce MEG3 expression. Functional studies showed that stably overexpressing MEG3 in Cd-transformed cells significantly reduces their transformed phenotypes. Moreover, stably overexpressing MEG3 in parental non-transformed human bronchial epithelial cells significantly impaired the capability of chronic Cd exposure to induce cell transformation and CSC-like property. Further mechanistic studies revealed that the cell cycle inhibitor p21 level is reduced and retinoblastoma protein (Rb) phosphorylation is increased in Cd-transformed cells to promote cell cycle progression. In addition, Cd-transformed cells also expressed higher levels of Bcl-xL and displayed apoptosis resistance. In contrast, stably overexpressing MEG3 increased p21 levels and reduced Rb phosphorylation and Bcl-xL levels in Cd-exposed cells and reduced their cell cycle progression and apoptosis resistance. Together, these findings suggest that MEG3 down-regulation may play important roles in Cd-induced cell transformation and CSC-like property by promoting cell cycle progression and apoptosis resistance.

LncRNA DUXAP10 Upregulation and the Hedgehog Pathway Activation Are Critically Involved in Chronic Cadmium Exposure-Induced Cancer Stem Cell-Like Property.

Cadmium (Cd) is a well-known lung carcinogen. However, the mechanism of Cd carcinogenesis remains to be clearly defined. Cd has been shown to act as a weak mutagen, suggesting that it may exert tumorigenic effect through nongenotoxic ways, such as epigenetic mechanisms. Long noncoding RNAs (lncRNAs) refer to RNA molecules that are longer than 200 nucleotides in length but lack protein-coding capacities. Regulation of gene expressions by lncRNAs is considered as one of important epigenetic mechanisms. The goal of this study is to investigate the mechanism of Cd carcinogenesis focusing on the role of lncRNA dysregulations. Cd-induced malignant transformation of human bronchial epithelia BEAS-2B cells was accomplished by a 9-month low-dose Cd (CdCl2, 2.5 µM) exposure. The Cd-exposed cells formed significantly more colonies in soft agar, displayed cancer stem cell (CSC)-like property, and formed tumors in nude mice. Mechanistically, chronic low-dose Cd exposure did not cause significant genotoxic effects but dysregulated lncRNA expressions. Further Q-PCR analysis confirmed the significant upregulation of the oncogenic lncRNA DUXAP10 in Cd-transformed cells. DUXAP10 knockdown in Cd-transformed cells significantly reduced their CSC-like property. Further mechanistic studies showed that the Hedgehog pathway is activated in Cd-transformed cells and inhibition of this pathway reduces Cd-induced CSC-like property. DUXAP10 knockdown caused the Hedgehog pathway inactivation in Cd-transformed cells. Furthermore, Pax6 expression was upregulated in Cd-transformed cells and Pax6 knockdown significantly reduced their DUXAP10 levels and CSC-like property. In summary, these findings suggest that the lncRNA DUXAP10 upregulation may play an important role in Cd carcinogenesis.

Puerarin Restores Autophagosome-Lysosome Fusion to Alleviate Cadmium-Induced Autophagy Blockade via Restoring the Expression of Rab7 in Hepatocytes.

Autophagic dysfunction is one of the main mechanisms by which the environmental pollutant cadmium (Cd) induces cell injury. Puerarin (Pue, a monomeric Chinese herbal medicine extract) has been reported to alleviate Cd-induced cell injury by regulating autophagy pathways; however, its detailed mechanisms are unclear. In the present study, to investigate the detailed mechanisms by which Pue targets autophagy to alleviate Cd hepatotoxicity, alpha mouse liver 12 (AML12) cells were used to construct a model of Cd-induced hepatocyte injury in vitro. First, the protective effect of Pue on Cd-induced cell injury was confirmed by changes in cell proliferation, cell morphology, and cell ultrastructure. Next, we found that Pue activated autophagy and mitigated Cd-induced autophagy blockade. In this process, the lysosome was further activated and the lysosomal degradation capacity was strengthened. We also found that Pue restored the autophagosome-lysosome fusion and the expression of Rab7 in Cd-exposed hepatocytes. However, the fusion of autophagosomes with lysosomes and autophagic flux were inhibited after knocking down Rab7, and were further inhibited after combined treatment with Cd. In addition, after knocking down Rab7, the protective effects of Pue on restoring autophagosome-lysosome fusion and alleviating autophagy blockade in Cd-exposed cells were inhibited. In conclusion, Pue-mediated alleviation of Cd-induced hepatocyte injury was related to the activation of autophagy and the alleviation of autophagy blockade. Pue also restored the fusion of autophagosomes and lysosomes by restoring the protein expression of Rab7, thereby alleviating Cd-induced autophagy blockade in hepatocytes.

\u201cMetalloestrogenic\u201d effects of cadmium downstream of G protein-coupled estrogen receptor and mitogen-activated protein kinase pathways in human uterine fibroid cells

Cadmium (Cd) is a toxic metal reported to act as an estrogen “mimic” in the rat uterus and in vitro. We have reported that Cd stimulates proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM; fibroid) cells through nongenomic signaling involving the G protein-coupled estrogen receptor (GPER), with activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (pMAPK44/42). In this study, we explored Cd-induced mechanisms downstream of MAPK and whether Cd could stimulate phosphorylation of Histone H3 at serine 10 (H3Ser10ph) through activated Aurora B kinase (pAurora B), a kinase important in activation of histone H3 at serine 10 during mitosis, and if this occurs via Fork head box M1 (FOXM1) and cyclin D1 immediately downstream of MAPK. We found that Cd increased proliferating cell nuclear antigen (PCNA) and H3Ser10ph expression by immunofluorescence, and that H3ser10ph and pAurora B were coexpressed along the metaphase plate in ht-UtLM cells. In addition, Cd-exposed cells showed higher expression of pMAPK44/42, FOXM1, pAurora B, H3ser10ph, and Cyclin D1 by western blotting. Immunoprecipitation and proximity ligation assays further indicated an association between FOXM1 and Cyclin D1 in Cd-exposed cells. These effects were attenuated by MAPK kinase (MEK1/2) inhibitor. In summary, Cd-induced proliferation of ht-UtLM cells occurred through activation of Histone H3 and Aurora B via FOXM1/Cyclin D1 interactions downstream of MAPK. This provides a molecular mechanism of how Cd acts as an “estrogen mimic” resulting in mitosis in hormonally responsive cells.

Human placental cell line HTR-8/SVneo accumulates cadmium by divalent metal transporters DMT1 and ZIP14

Cadmium (Cd) is a global pollutant that accumulates in the placenta and can cause placental dysfunction. Although iron transporters have been suggested to participate in placental Cd uptake, it is still unknown which transporters are actually involved in this process. We specifically aimed to study the role of three iron transporters in the uptake of Cd into the placental cell line HTR-8/SVneo. For this purpose, Divalent Metal Transporter (DMT)1 and ZRT/IRT like protein (ZIP)8 and ZIP14 were downregulated and changes in cellular Cd levels analysed in relation to controls. As clearly shown by the reduction of the Cd content by ∼60% in DMT1- and ZIP14-downregulated cells, the two proteins are essential for Cd accumulation in HTR-8/SVneo cells. Using a validated antibody, we show DMT1 to be localised in situ in trophoblast and stromal cells. We further wanted to investigate how placental cells cope with Cd loading and which metallothionein (MT) isoforms they express. Cd-exposed cells accumulate Cd in a dose-dependent manner and upregulate MT2A accordingly (up to 15-fold induction upon 5 μM CdCl2 treatment for 72 h). 5 μM Cd exposure for 72 h decreased cell number to 60%, an effect that was aggravated by MT2A depletion (cell number reduced to 30%) indicating additive effects. In conclusion, our data suggest that DMT1 and ZIP14 are required for Cd uptake into human placental cells that upregulate MT2A to store and detoxify the metal. Cd storage in the placenta reduces Cd transport to the fetus, which, however, could impair placental functions and fetal development.

Role of the PI3K/Akt pathway in cadmium induced malignant transformation of normal prostate epithelial cells

This study investigated the role of the PI3K/Akt pathway in cadmium (Cd) induced malignant transformation of normal prostate epithelial (PWR1E and RWPE1) cells. Both PWR1E and RWPE1 cells were exposed to 10 μM Cd for one year and designated as Cd-PWR1E and Cd-RWPE1. Cd-RWPE1 cells robustly formed tumors in athymic nude mice. Functionally, Cd-exposure induced tumorigenic attributes indicated by increased wound healing, migration and invasion capabilities in both cell lines. RT2-array analysis revealed many oncogenes including P110α, Akt, mTOR, NFKB1 and RAF were induced whereas tumor suppressor (TS) genes were attenuated in Cd-RWPE1. This was validated by individual quantitative-real-time-PCR at transcriptional and by immunoblot at translational levels. These results were consistent in Cd-PWR1E vs parental PWR1E cells. Gene Set Enrichment Analysis revealed that five prostate cancer (PCa) related pathways were enriched in Cd-exposed cells compared to their normal controls. These pathways include the KEGG- Pathways in cancer, Prostate Cancer Pathway, ERBB, Apoptosis and MAPK pathways. We selected up- and down-regulated genes randomly from the PI3K/Akt pathway array and profiled these in the TCGA/GDC prostate-adenocarcinoma (PRAD) patient cohort. An upregulation of oncogenes and downregulation of TS genes was observed in PCa compared to their normal controls. Taken together, our study reveals that the PI3K/Akt signaling is one of the main molecular pathways involved in Cd-driven transformation of normal prostate epithelial cells to malignant form. Understanding the molecular mechanisms involved in the Cd-driven malignant transformation of normal prostate cells will provide a significant insight to develop better therapeutic strategies for Cd-induced prostate cancer.

Cadmium inhibits apoptosis of human esophageal epithelial cells by upregulating CDK6

Cadmium (Cd) exposure in environment is associated with development of esophageal cancer. However, the mechanisms of Cd-induced carcinogenesis are still not been fully cleared, and the present study aimed to explore the possible etiological mechanism of Cd-induced esophageal cancer. Human esophageal epithelial cell lines (HET-1A and KYSE450) were treated with CdCl2 at 0.05 mg/l for 12, 24 h, and the then the apoptosis were detected using flow cytometry with annexin-V-FITC/PI staining. Results showed that apoptosis of treatment groups was significantly inhibited, and decreased reactive oxygen species (ROS) production played a key role in the inhibitory effects by upregulating Bcl-2 and downregulating Caspase-3/9. The relief of oxidative stress during Cd exposure was actively promoted by the increased nicotinamide adenine dinucleotide phosphoric acid and glutathione levels. To investigate the causes of enhanced intracellular antioxidant capacity, the activity of pyruvate kinase (PK), a key enzyme of glycolysis, was detected. Our results showed that PK activity was inhibited, suggesting that glycolysis process was blocked which promoted more intermediate metabolites of glycolysis to be used for reduced nicotinamide adenine dinucleotide phosphoric acid (NADPH) or other antioxidants synthesis. PK activity was closely correlated with phosphorylation of pyruvate kinase M2 (PKM2), and a highly negative correlation (correlation coefficients: −0.835, p < 0.05) between them was found. Western blotting showed the overphosphorylation of PKM2 in Cd-exposed cells, resulting from increased expression of cyclin-dependent kinases 6 (CDK6). These results suggested a possible mechanism of carcinogenic: Cd-induced upregulation of CDK6 in esophageal cell lines caused PKM2 overphosphorylation inhibiting PK activity, thereby shunting glucose-derived carbon into the pentose phosphate pathway and promoting the production of NADPH and reduced glutathione (GSH) to neutralize ROS, which finally results in the inhibited apoptosis.

Chronic exposure to cadmium induces a malignant transformation of benign prostate epithelial cells

Epidemiological evidence suggests that cadmium (Cd) is one of the causative factors of prostate cancer, but the effect of Cd on benign prostatic hyperplasia (BPH) remains unclear. This study aimed to determine whether Cd exposure could malignantly transform BPH1 cells and, if so, to dissect the mechanism of action. We deciphered the molecular signaling responsible for BPH1 transformation via RNA-sequencing and determined that Cd induced the expression of zinc finger of the cerebellum 2 (ZIC2) in BPH1 cells. We noted Cd exposure increased ZIC2 expression in the Cd-transformed BPH1 cells that in turn promoted anchorage-independent spheroids and increased expression of stem cell drivers, indicating their role in stem cell renewal. Subsequent silencing of ZIC2 expression in transformed cells inhibited spheroid formation, stem cell marker expression, and tumor growth in nude mice. At the molecular level, ZIC2 interacts with the glioma-associated oncogene family (GLI) zinc finger 1 (GLI1), which activates prosurvival factors (nuclear factor NFκB, B-cell lymphoma-2 (Bcl2), as well as an X-linked inhibitor of apoptosis protein (XIAP)) signaling in Cd-exposed BPH1 cells. Conversely, overexpression of ZIC2 in BPH1 cells caused spheroid formation confirming the oncogenic function of ZIC2. ZIC2 activation and GLI1 signaling induction by Cd exposure in primary BPH cells confirmed the clinical significance of this oncogenic function. Finally, human BPH specimens had increased ZIC2 versus adjacent healthy tissues. Thus, we report direct evidence that Cd exposure induces malignant transformation of BPH via activation of ZIC2 and GLI1 signaling.

Abstract 4277: Inhibition of autophagy (Plac8) prevents malignant transformation of cadmium induced prostate carcinogenesis

Abstract Introduction: Cadmium (Cd) exposure is associated with an increased risk of prostate cancer (CaP). Previously we reported that Cd induces Plac8 expression may be responsible for defective autophagy which facilitates the transformation of prostate epithelial cells. The goal of this study is to decipher the molecular signaling responsible for defective autophagy by plac8 and whether inhibition of plac8 facilitates pro-apoptotic machinery in chronically exposed -Cd in prostate epithelial cells. Methods: Overexpression or silencing Plac8 in chronically exposed Cd in RWPE-1 cells or cadmium, transformed prostate epithelial (CTPE) cells were subjected to cell viability, apoptosis, autophagy functional studies, Western blot and in vivo analyses. For statistical analysis, data were analyzed using Student’s‘t’ test with a p-value less than 0.05 considered significant. Results: Our results suggest that during cellular transformation, Cd exposure induced Plac8 expression, which caused defective-autophagy that resulted in the proliferation of damaged cells. Inhibition of Plac8 resulted in suppression of pro-survival machinery and concomitantly induced severe endoplasmic reticulum (ER) stress that resulted in caspase-mediated cell death in Cd-exposed cells. More specifically, inhibition of Plac8 by shRNA resulted in induction of ER stress markers (PREK/ATF4) which facilitate caspase-mediated cell death in Cd- transformed prostate epithelial cells. Activation of NFKB is one of the downstream events of Plac8 that transcriptionally regulates NFKB activation in Cd-transformed cells. We identified, Plac8 binding sites in NFKB promoter. Hence inhibition of plac8 resulted in the complete shutdown of NFKB mediated pro-survival signaling in Cd-transformed prostate epithelial cells. While analyzing autophagy signaling network, we found induction of Atg family proteins, however, in the absence of Plac8, the induction of autophagy fails to protect the Cd-transformed cells. Similar results were observed in plac8 silenced cd-transformed cells in xenotransplanted mice, where Cd-transformed cells induced aggressive tumors, and Plac8 knockdown significantly inhibited the tumor growth. While ATF4, PLac8, NFKB were not expressed in normal human prostate tissues, their expression was significantly increased in BPH followed by CaP specimens; normal prostate &amp;lt; BPH &amp;lt;&amp;lt; Gleason 6-7 CaP &amp;lt; Gleason 8-10 CaP. Cd levels were much higher in tumor tissues (21.43 μg/g) of CaP patients than in healthy controls (1.12 μg/g). The levels also correlated with Plac8 expression. These results demonstrate the clinical significance of Cd levels and of Plac8 expression as plausible drivers of Cd-induced transformation in prostate carcinogenesis. Conclusions: These results suggest that Plac8 may be an essential component in the Cd-induced transformation of normal prostate epithelial cells to a cancerous state. Citation Format: Venkatesh Kolluru, Ashish Tyagi, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Inhibition of autophagy (Plac8) prevents malignant transformation of cadmium induced prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4277.

Sulforaphane Protect Against Cadmium-Induced Oxidative Damage in mouse Leydigs Cells by Activating Nrf2/ARE Signaling Pathway.

Cadmium (Cd) is harmful for humans and animals, especially for the reproductive system. However, the mechanism of its toxicity has not been elucidated, and how to alleviate its toxicity is very important. This study aimed to explore the role and mechanism of action of sulforaphane (SFN) in protecting mouse Leydigs (TM3) cells from cadmium (Cd)-induced damage. The half-maximal inhibitory concentration (IC50) of Cd and the safe doses of SFN were determined using a methyl thiazolyl tetrazolium (MTT) assay. The testosterone secretion from TM3 cells was measured using the enzyme-linked immunosorbent assay. The intracellular oxidative stress was evaluated using corresponding kits. The cell apoptosis was detected using flow cytometry. The mRNA expression of genes associated with NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling was detected using reverse transcription–polymerase chain reaction, including Nrf2, heme oxygenase I (HO-1), glutathione peroxidase (GSH-Px), NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS). The protein expression of Nrf2, GSH-Px, HO-1, γ-GCS, and NQO1 was detected using Western blot analysis. The results showed that the IC50 of Cd to TM3 cells was 51.4 µmol/L. SFN reduced the release of lactate dehydrogenase from Cd-exposed cells. Cd + SFN 2.5 treatment significantly elevated testosterone concentration compared with the Cd group (p < 0.05). SFN significantly increased total superoxide dismutase (T-SOD) and GSH-Px activity and GSH content in Cd-treated cells (p < 0.05; p < 0.01), inhibited the production of malondialdehyde or reactive oxygen species caused by Cd (p < 0.05; p < 0.01), and reduced the apoptotic rate of Cd-induced TM3 cells (p < 0.01). SFN upregulated the mRNA expression of Nrf2, GSH-Px, HO-1, NQO1, and γ-GCS in Cd-treated cells, indicating the protective effect of SFN against Cd-induced oxidative stress or cell apoptosis by activating the Nrf2/ARE signaling pathway.

Assessment of sulforaphane-induced protective mechanisms against cadmium toxicity in human mesenchymal stem cells.

Cd is a hazardous substance and carcinogen that is present in the environment; it is known to cause toxic effects in living organisms. Sulforaphane is a naturally available phytochemical with antioxidant, anti-inflammatory, and anticarcinogenic properties. However, the effects of sulforaphane on Cd toxicity in human mesenchymal stem cells (hMSCs) are unknown. In the present study, we investigated the molecular mechanisms of the effects of sulforaphane on Cd toxicity in hMSCs by using MTT assays, acridine orange/ethidium bromide staining, Hoechst staining, LysoRed staining, assessment of mitochondrial membrane potential, and gene expression analysis. Cd decreased hMSC viability in a dose-dependent manner with an IC50 value of 56.5μM. However, sulforaphane did not induce any significant reduction in cell viability. Nuclear morphological analysis revealed that Cd induced necrotic cell death. Additionally, Cd caused mitochondrial membrane potential loss in hMSCs. The treatment of Cd-exposed cells with sulforaphane (Cd-sulforaphane co-treatment) resulted in a significant recovery of the cell viability and nuclear morphological changes compared with that of cells treated with Cd only. The gene expression pattern of cells co-treated with Cd-sulforaphane was markedly different from that of Cd-treated cells, owing to the reduction in Cd toxicity. Our results clearly indicated that sulforaphane reduced Cd-induced toxic effects in hMSCs. Overall, the results of our study suggested that sulforaphane-rich vegetables and fruits can help to improve human health through amelioration of the molecular effects of Cd poisoning.

Role of subcellular calcium redistribution in regulating apoptosis and autophagy in cadmium-exposed primary rat proximal tubular cells

Ca2+ signaling plays a vital role in regulating apoptosis and autophagy. We previously proved that cytosolic Ca2+ overload is involved in cadmium (Cd)-induced apoptosis in rat proximal tubular (rPT) cells, but the source of elevated cytosolic Ca2+ concentration ([Ca2+]c) and the effect of potential subcellular Ca2+ redistribution on apoptosis and autophagy remain to be elucidated. Firstly, data showed that Cd-induced elevation of [Ca2+]c was primarily generated intracellularly. Moreover, elevations of [Ca2+]c and mitochondrial Ca2+ concentration ([Ca2+]mit) with depletion of endoplasmic reticulum (ER) Ca2+ levels ([Ca2+]ER) were revealed in Cd-treated rPT cells, but this subcellular Ca2+ redistribution was significantly suppressed by 2-Aminoethoxydiphenyl borate (2-APB). Elevated inositol 1,4,5-trisphosphate (IP3) levels with up-regulated IP3 receptor (IP3R) protein levels were shown in Cd-exposed cells, confirming that IP3R-mediated ER Ca2+ release results in the elevation of [Ca2+]c. Up-regulated sequestosome 1 (p62) protein levels and autophagic flux assay demonstrated that Cd impaired autophagic degradation, while N-acetylcysteine (NAC) markedly attenuated Cd-induced p62 and microtubule-associated protein 1 light chain 3-II (LC3-II) accumulation, implying that the inhibition of autophagic flux was due to oxidative stress. Furthermore, pharmacological modulation of [Ca2+]c with 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and 2-APB alleviated Cd-mediated apoptosis, inhibition of autophagic degradation and subsequent cytotoxicity, while thapsigargin (TG) had the opposite regulatory effect on them. In summary, cytosolic calcium overload originated from IP3R-mediated ER Ca2+ release has a negative impact on Cd nephrotoxicity through its promotion of apoptosis and inhibition of autophagic flux.

Salidroside Protects against Cadmium-Induced Hepatotoxicity in Rats via GJIC and MAPK Pathways.

It is known that cadmium (Cd) induces cytotoxicity in hepatocytes; however, the underlying mechanism is unclear. Here, we studied the molecular mechanisms of Cd-induced hepatotoxicity in rat liver cells (BRL 3A) and in vivo. We observed that Cd treatment was associated with a time- and concentration-dependent decrease in the cell index (CI) of BRL 3A cells and cellular organelle ultrastructure injury in the rat liver. Meanwhile, Cd treatment resulted in the inhibition of gap junction intercellular communication (GJIC) and activation of mitogen-activated protein kinase (MAPK) pathways. Gap junction blocker 18-β-glycyrrhetinic acid (GA), administered in combination with Cd, exacerbated cytotoxic injury in BRL 3A cells; however, GA had a protective effect on healthy cells co-cultured with Cd-exposed cells in a co-culture system. Cd-induced cytotoxic injury could be attenuated by co-treatment with an extracellular signal-regulated kinase (ERK) inhibitor (U0126) and a p38 inhibitor (SB202190) but was not affected by co-treatment with a c-Jun N-terminal kinase (JNK) inhibitor (SP600125). These results indicate that ERK and p38 play critical roles in Cd-induced hepatotoxicity and mediate the function of gap junctions. Moreover, MAPKs induce changes in GJIC by controlling connexin gene expression, while GJIC has little effect on the Cd-induced activation of MAPK pathways. Collectively, our study has identified a possible mechanistic pathway of Cd-induced hepatotoxicity in vitro and in vivo, and identified the participation of GJIC and MAPK-mediated pathways in Cd-induced hepatotoxicity. Furthermore, we have shown that salidroside may be a functional chemopreventative agent that ameliorates the negative effects of Cd via GJIC and MAPK pathways.

Alpha-lipoic acid protects against cadmium-induced hepatotoxicity via calcium signalling and gap junctional intercellular communication in rat hepatocytes.

This study investigated the protective effect of alpha-lipoic acid (LA) on cadmium (Cd)-induced hepatotoxicity in BRL 3A rat liver cells. We demonstrated that LA ameliorated Cd-induced cellular injury in cell viability and nuclear fragmentation in BRL 3A cells. Furthermore, LA markedly ameliorated Cd-induced gap junctional intercellular communication (GJIC) inhibition and Cx43 mRNA expression decrease, as well as disassembly of gap junctions. The gap junction blocker carbenoxolone disodium (CBX) as well as LA protected healthy cells from Cd-exposed cells in Transwell co-culture system. LA also protected BRL 3A cells against Cd-induced elevation of the intracellular concentration of free calcium ([Ca(2+)]i). Pretreatment with a chelater of intracellular Ca(2+) BAPTA-AM or chelater of extracellular Ca(2+) EGTA attenuated Cd-induced cytotoxicity and GJIC inhibition. CBX exacerbated the decrease in cell viability and further elevated the increase in [Ca(2+)]i induced by Cd, whereas BAPTA-AM partly attenuated these phenomena, while EGTA had little effects. These results suggested that Cd-induced hepatotoxicity via GJIC inhibition and [Ca(2+)]i elevation, which originates mainly from intracellular stores. GJIC inhibition has dual effects: (i) it restricts release of Ca(2+) from the cell, which exacerbates the [Ca(2+)]i elevation and cytotoxicity induced by Cd; and (ii) it protects healthy cells from their dangerous neighbors by blocking intercellular communication. Above all, our results indicated that LA partly prevented Cd-induced cytotoxicity via GJIC and calcium signaling in BRL 3A rat liver cells.

Abstract 5131: Cadmium induces cytotoxicity in human bronchial epithelial cell in a p53-independent, nuclear factor NF-kappaB-dependent manner

Abstract Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well known carcinogens. Here, we report that in CdCl2-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 μM) of Cd can up-regulate the protein levels of eukaryotic translation initiation factor 5A-1 (eIF-5A-1) and redox-sensitive transcription factor NF-κB p65 in a time- and dose-dependent manner. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF-5A-1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF-5A-1 in Cd cytotoxicity. As eIF-5A-1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF-5A-1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF-5A-1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF-5A-1 promoter region of Cd-treated cells. Taken together, our findings report for the first time a regulatory mechanism for the pro-apoptotic protein eIF-5A-1 in which its level is controlled in an NF-κB-dependent manner. Citation Format: Ji-Ying Du, De-Ju Chen, Yan-Ming Xu, Andy T. Y. Lau. Cadmium induces cytotoxicity in human bronchial epithelial cell in a p53-independent, nuclear factor NF-kappaB-dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5131. doi:10.1158/1538-7445.AM2014-5131

Oxidative stress and Ca(2+) signals involved on cadmium-induced apoptosis in rat hepatocyte.

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5∼10μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5∼10μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5h at 5μM Cd. Significant (P < 0.01) reactive oxygen species (ROS) production at 5μM Cd and 0.75h occurred prior to the decrease of the mitochondrial membrane potential, suggesting the involvement of ROS in mitochondrial membrane damage. Glutathione (GSH) level significantly decreased after cell treatment with 5 and 10μM Cd after 12h (P < 0.01). Meanwhile, the intracellular free Ca(2+) concentration ([Ca(2+)] i ) of Cd-exposed cells significantly increased (P < 0.01) at 1.5h, and pretreatment with the calcium chelator Bapta-AM partially blocked Cd-induced apoptosis. This finding indicated that the elevation of [Ca(2+)] i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca(2+) signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.