November 2021
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13 Reads
Free Radical Biology and Medicine
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Publications (403)
January 2021
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23 Reads
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27 Citations
Toxicology
Cadmium (Cd) is recognized as a highly toxic heavy metal for humans in part because it is a multi-organ carcinogen. To clarify the mechanism of Cd carcinogenicity, we have established an experimental system using rat liver TRL1215 cells exposed to 2.5 μM Cd for 10 weeks and then cultured in Cd-free medium for an additional 4 weeks (total 14 weeks). Recently, we demonstrated, by using this experimental system, that 1) Cd stimulates cell invasion by suppression of apolipoprotein E (ApoE) expression, and 2) Cd induces DNA hypermethylation of the regulatory region of the ApoE gene. However, the underlying mechanism(s) as well as other potential genetic participants in the Cd-stimulated invasion are undefined. In the present work, we found that concurrent with enhanced invasion, Cd induced oxidative stress, coupled with the production of oxidative stress-sensitive metallothionein 2A (MT2A), which lead to down-modulation of ten-eleven translocation methylcytosine dioxygenase 1 (TET1: DNA demethylation) in addition to ApoE, without impacting DNA methyltransferases (DNMTs: DNA methylation) levels. Furthermore, the expression of tissue inhibitor of metalloproteinase 2 and 3 (TIMP2 and TIMP3) that are positively regulated by TET1, were decreased by Cd. The genes (ApoE/TET1/TIMP2/TIMP3) suppressed by Cd were further suppressed by hydroquinone (HQ; a reactive oxygen species [ROS] producer), whereas N-acetyl-l-cysteine (NAC; a ROS scavenger) prevented the suppression of their expression by HQ. In addition, NAC reversed their expression suppressed by Cd. Cd-stimulated cell invasion was clearly dampened by NAC in a concentration-dependent manner. Overall these findings suggest that 1) altered TET1 expression and activity together with ApoE are likely involved in the enhanced invasiveness due to Cd exposure, and 2) Cd down-regulation of TET1 likely evokes a reduction in ApoE expression (possible by DNA hypermethylation), and 3) anti-oxidants are effective in abrogation of the enhanced invasiveness that occurs concurrently with Cd-induced malignant transformation.
June 2020
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40 Reads
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8 Citations
Toxicology and Applied Pharmacology
CAsE-PE cells are an arsenic-transformed, human prostate epithelial line containing oncogenic mutations in KRAS compared to immortalized, normal KRAS parent cells, RWPE-1. We previously reported increased copy number of mutated KRAS in CAsE-PE cells, suggesting gene amplification. Here, KRAS flanking genomic and transcriptomic regions were sequenced in CAsE-PE cells for insight into KRAS amplification. Comparison of DNA-Seq and RNA-Seq showed increased reads from background aligning to all KRAS exons in CAsE-PE cells, while a uniform DNA-Seq read distribution occurred in RWPE-1 cells with normal transcript expression. We searched for KRAS fusions in DNA and RNA sequencing data finding a portion of reads aligning to KRAS and viral sequence. After generation of cDNA from total RNA, short and long KRAS probes were generated to hybridize cDNA and KRAS enriched fragments were PacBio sequenced. More KRAS reads were captured from CAsE-PE cDNA versus RWPE-1 by each probe set. Only CAsE-PE cDNA showed KRAS viral fusion transcripts, primarily mapping to LTR and endogenous retrovirus sequences on either 5′- or 3′-ends of KRAS. Most KRAS viral fusion transcripts contained 4 to 6 exons but some PacBio sequences were in unusual orientations, suggesting viral insertions within the gene body. Additionally, conditioned media was extracted for potential retroviral particles. RNA-Seq of culture media isolates identified KRAS retroviral fusion transcripts in CAsE-PE media only. Truncated KRAS transcripts suggested multiple retroviral integration sites occurred within the KRAS gene producing KRAS retroviral fusions of various lengths. Findings suggest activation of endogenous retroviruses in arsenic carcinogenesis should be explored.
April 2019
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13 Reads
PCR amplification of wt and mutant cDNA KRAS in RWPE-1, CAsE-PE and normal human prostate cells. (PPTX)
April 2019
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250 Reads
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20 Citations
Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5μM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q<0.05 by RNA-seq. Notably, KRAS expression was highly elevated in CAsE-PE cells, with pathway analysis supporting increased cell proliferation, cell motility, survival and cancer pathways. Targeted DNA sequencing of KRAS revealed a mutant specific allelic imbalance, ‘MASI’, frequently found in primary clinical tumors. We found high expression of a mutated KRAS transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by lower expression of a wild-type allele. Parallel cultures of RWPE-1 cells retained a wild-type KRAS genotype. Copy number analysis and sequencing showed amplification of the mutant KRAS allele. KRAS is expressed as two splice variants, KRAS4a and KRAS4b, where variant 4b is more prevalent in normal cells compared to greater levels of variant 4a seen in tumor cells. 454 Roche sequencing measured KRAS variants in each cell type. We found KRAS4a as the predominant transcript variant in CAsE-PE cells compared to KRAS4b, the variant expressed primarily in RWPE-1 cells and in normal prostate, early passage, primary epithelial cells. Overall, gene expression data were consistent with KRAS-driven proliferation pathways found in spontaneous tumors and malignantly transformed cell lines. Arsenite is recognized as an important environmental carcinogen, but it is not a direct mutagen. Further investigations into this in vitro transformation model will focus on genomic events that cause arsenite-mediated mutation and overexpression of KRAS in CAsE-PE cells.
April 2019
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11 Reads
April 2019
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10 Reads
RNA-seq total reads and alignment summary. (XLSX)
April 2019
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9 Reads
KRASP1 and KRAS alignment. (DOCX)
April 2019
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13 Reads
qPCR of CNVs for KRAS exons 4a and exon 3, intron 5 and intron 3. (PPTX)
April 2019
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12 Reads
Structure of KRAS variants, KRAS4a and KRAS4b. (DOCX)
Citations (72)
... The aging of the brain or the development of diseases is associated with the deregulation of the management of metal ions [10]. Particularly, recent evidence indicates that if different types of metalloproteins interact in a certain salt solution, the potential galvanic erosion may dissolute the compound surface and result in inducing tumor formation [12,13]. ...
- Citing Chapter
July 2015
... Inorganic As has been shown to enhance the expression of MMP genes, which play a role in degrading the ECM 12 , thereby influencing As-related cancers. In rat hepatocytes, Cd was found to decrease expressions of TIMP2 and TIMP3, which are positively regulated by ten-eleven translocation methylcytosine dioxygenase 1 (TET1), which participates in DNA demethylation 13 . Moreover, urinary levels of As and Cd exhibited a positive and significant correlation with TIMP1, a marker associated with renal damage 14 . ...
- Citing Article
January 2021
Toxicology
... On the other hand, some other elements such as lead (Pb), cadmium (Cd), and mercury (Hg) are not vital to the body and are toxic even at low concentrations (Nyambura et al. 2020;Han et al. 2020;Engwa et al. 2019). Previous studies demonstrate that the adverse effects of human health concerning the exposure to heavy metals vary even at low concentrations and are not limited only to neurological and cancerous complications (e.g., ATSDR 2005ATSDR , 2008 Castro-González and Méndez-Armenta 2008; Jomova and Valko 2011;Tokar et al. 2011). Noorpoor and Sadri (2014) evaluated the health risk of heavy metals and found that people were not exposed to non-cancerous diseases by inhaling these metals and the total number of lifetime cancers was caused by inhaling metals such as chromium (Cr), arsenic (As), Cd, and Ni 5. Additionally, Ali-Taleshi et al. (2020) investigated several heavy metals (e.g., Al Fe, Zn, Pb, Cr, and Cd) in suspended particles in Tehran. ...
- Citing Chapter
January 2010
Metal Ions in Life Sciences
... To date, several processes associated with proliferation and apoptosis regulation, such as miR-143, the RAS/ERK pathway, and hypermethylation-mediated silencing of MLH1 (Ngalame et al., 2016;Treas et al., 2013), have been observed. Additionally, mitochondrial dysfunction (Singh et al., 2016), hypomethylated genomic DNA and activated K-ras (Benbrahim-Tallaa et al., 2005), gained independence of androgen (Merrick et al., 2020), as well as activated p62-Nrf2 axis (Xie et al., 2020) have also been confirmed with arsenic carcinogenesis. Importantly, with respect to arsenic carcinogenesis, accumulating evidence suggests that the phenotypic alternation shift from epithelial to mesenchymal transition (EMT) is a prerequisite step in the development of the malignant phenotype (Li et al., 2022;Xu et al., 2014). ...
- Citing Article
June 2020
Toxicology and Applied Pharmacology
... For RNA isolation (Qiagen; RNeasy Mini, Cat No 74004), cells were washed with PBS prior to addition of lysis buffer and spin columns with on-column DNAase-1 digestion followed by washes, elution, and storage at −80 • C. RNA integrity was measured on an Agilent Model 5300 Fragment Analyzer (Agilent, Santa Clara, CA, USA) with RQN values of 9-10. RNA samples were prepared for RNA-seq by rRNA depletion, fragmentation (Covaris Inc., Woburn, MA, USA), bar coding, and library construction as previously described [102]. Briefly, pooled libraries were analyzed for cluster generation of 150 bp paired end fragments to produce a~30X to 40X coverage of the mouse transcriptome using an Illumina NovaSeq instrument. ...
- Citing Article
- Full-text available
April 2019
... For instance, apoE is an essential composition of mature HCV particle engaging in the infection process and able to protect the virus from immune clearance (Fukuhara et al. 2014;Hueging et al. 2014;Lavie and Dubuisson 2017). In addition, low apoE expression is associated with malignant transformation of hepatocytes (Hirao-Suzuki et al. 2018), and decreased apoE levels in peripheral blood was found to be related to the metastasis of HCC (Fu et al. 2009;Yokoyama et al. 2006). These studies suggest that apoE and its associated biomolecules play multiple roles in the pathogenesis of various liver others are in the supplementary materials (supplementary Fig. 3). ...
- Citing Article
September 2018
The Journal of Toxicological Sciences
... MSCs also support haemopoiesis in the first trimester of pregnancy [11]. Increasing evidence indicates that arsenic exposure targets stem cells and causes disruption of stem cell differentiation [12] thereby promoting the development of cancer and other diseases. Such changes alter stem cell adaptability and induce normal stem cells to acquire a cancer stem cell (CSC) phenotype, which is linked to both arsenic-associated cancer and prenatal arsenic exposure-associated CSC emergence [12]. ...
- Citing Chapter
October 2015
... The prenatal exposure levels and the MEG3 CpG hypermethylation are significant geographic co-clustering in the whites and African Americans in a NEST cohort study [65]. In studies about specific DNA hypermethylation and downstream target genes, Cd decreased the cell invasion inhibitor, called apolipoprotein E (ApoE), by hypermethylating the ApoE promoter [61], and Cd interfered with the liver X receptor (LXR) which controls ApoE transcription [147]. Cd upregulates the oxidative stress-sensitive metallothionein 2A (MT2A) and downregulates the TET1-regulated metalloproteinases 2 and 3 (TIMP2/3) by the ROS, but it is independent of DNA methyltransferase (DNMT). ...
- Citing Article
March 2017
Toxicology
... The PI3K/Akt array analysis revealed that Cd regulates the PI3K/Akt pathway on both transcriptional and translational levels, proposing that the PI3K/Akt signaling pathway plays a crucial role in the transformation of normal prostate cells into a malignant form due to Cd exposure, offering insights into potential therapeutic strategies for cadmium-induced prostate cancer . Another study shows that KRAS silencing abrogates Cdmediated carcinogenic transformation of prostate epithelial cells RWPE1 and downregulation of the PI3K/Akt signaling pathway, which further highlights the importance of this pathway in the transformation process (Ngalame et al., 2016). ...
- Citing Article
- Full-text available
August 2016
Chemical Research in Toxicology
... Several in vitro studies have demonstrated that low-level exposure to As in human prostate stem cells induces transformation in cancer stem-like cells characterized by an increased release of matrix metalloproteinase-9 (MMP-9), inhibition of apoptosis, DNA methylation, and activation of K-RAS oncogene [39][40][41]. An increased expression of anti-apoptotic genes BCL2 and BCL-XL, which induced resistance to cell death, has also been observed, while the expression of MMP-9 supported the metastatic process [42]. Furthermore, during the development of PCa, normal cells undergo a process where tumor suppressor genes are silenced due to promoter methylation. ...
- Citing Article
- Full-text available
December 2015
Toxicology and Applied Pharmacology
