Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx.

BACKGROUND: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

Differential Effects of Nano TiO2 and CeO2 on Normal Human Lung Epithelial Cells In Vitro.

Nano-TiO2 and nano-CeO2 are among the most widely used engineered nanoparticles (NPs). We investigated a variety of endpoints to assess the toxicity of eight of these NPs to induce potentially adverse health effects in an In Vitro human respiratory epithelial cell model. These endpoints include cytotoxicity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) production, 8-hydroxy-2_-deoxyguanosine (8-oxo-dG), endogenous DNA adducts, Apurinic/apyrimidinic (AP) sites, 4-Hrdoxynonenal (4-HNE) protein adducts, Malondialdehyde (MDA) protein adducts, and genomics analysis on altered signaling pathways. Our results indicated that cytotoxicity assays are relatively insensitive, and we detected changes in other endpoints at concentrations much lower than those inducing cytotoxicity. Among the ROS-related endpoints, 8-oxo-dG is relatively more sensitive than other assays, and nano-TiO2 induced more 8-oxo-dG formation than nano-CeO2. Finally, there are many signaling pathways changes at concentrations at which no cytotoxicity was observed. These alterations in signaling pathways correlated well with In Vitro toxicity that was observed at higher concentrations, and with in vivo adverse outcome pathways caused by nano-TiO2 and nano-CeO2 in experimental animals.

Signaling Pathways and MicroRNA Changes in Nano-TiO2 Treated Human Lung Epithelial (BEAS-2B) Cells.

The effect of titanium dioxide nanoparticles (nano-TiO2 Degussa p25) treatment of human lung epithelial cells (BEAS-2B) was examined by analyzing changes in messenger [mRNA] and microRNA [miRNA]. BEAS-2B cells were treated with 0, 3, 10, 30 or 100 µg/ml nano-TiO2 for 1 day (for mRNA analysis) or 3 days (for miRNA analysis). Differentially expressed mRNA and miRNA were analyzed using Affymetrix microarrays and Affymetrix miRNA microarrays, respectively. Although, the tested doses were not cytotoxic, there were alterations in both mRNA and miRNA expression. The expression of mRNA/miRNA changes were examined in MetaCore (GeneGo) and IPA (Ingenuity Pathway Analysis) to delineate associated canonical/signaling pathways. Canonical/signaling pathways altered by nano-TiO2 treatments included: cell cycle regulation, apoptosis, calcium signaling, translation, NRF2-mediated oxidative response, IGF1 signaling, RAS signaling, PI3K/AKT signaling, cytoskeleton remodeling, cell adhesion, BMP signaling, and inflammatory response. Many of the genes in these pathways are known to be regulated by the miRNAs whose expressions were altered by the nano-TiO2 treatment. The miRNA 17-92 cluster and let-7 miRNA family that are involved in lung cancer formation were altered by nano-TiO2 treatment. The miR-17-92 cluster, an oncogenic microRNA cluster, is induced while the tumor suppressor microRNA, let-7 family, is suppressed. The changes of let-7/KRAS signaling pathway was observed in all the doses treated. The observed changes in miRNA expression introduces an additional mechanistic dimension that supports the significance of the observed mRNA expression changes, and demonstrated that the nano-TiO2 in vitro treatment in human lung cells can cause diverse but coordinated pathway alterations associated with changes in in vivo response to tumorigenes.

Cellular interactions and biological responses to titanium dioxide nanoparticles in HepG2 and BEAS-2B cells: role of cell culture media.

We showed previously that exposure of human lung cells (BEAS-2B) to TiO2 nanoparticles (nano-TiO2 ) produced micronuclei (MN) only when the final concentration of protein in the cell-culture medium was at least 1%. Nanoparticles localize in the liver; thus, we exposed human liver cells (HepG2) to nano-TiO2 and found the same requirement for MN induction. Nano-TiO2 also formed small agglomerates in medium containing as little as 1% protein and caused cellular interaction as measured by side scatter by flow cytometry and DNA damage (comet assay) in HepG2 cells. Nano-TiO2 also increased the activity of the inflammatory factor NFkB but not of AP1 in a reporter-gene HepG2 cell line. Suspension of nano-TiO2 in medium containing 0.1% protein was sufficient for induction of MN by the nanoparticles in either BEAS-2B or HepG2 cells as long the final concentration of protein in the cell-culture medium was at least 1%.

Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: effect of size, surface coating, and intracellular uptake.

Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key stress-responsive pathways activated by Nrf-2/ARE, NFκB, and AP1 during exposure to Ag NP of two distinct sizes (10 and 75 nm) and coatings (citrate and polyvinylpyrrolidone), as well as silver nitrate (AgNO3), and CeO2 nanoparticles. The in vitro assays assessed the cellular response in a battery of stable luciferase-reporter HepG2 cell lines. We further assessed the impact of Ag NP and AgNO3 exposure on cellular redox status by measuring glutathione depletion. Lastly, we determined intracellular Ag concentration by inductively coupled plasma mass spectroscopy (ICP-MS) and re-analyzed reporter-gene data using these values to estimate the relative potencies of the Ag NPs and AgNO3. Our results show activation of all three stress response pathways, with Nrf-2/ARE displaying the strongest response elicited by each Ag NP and AgNO3 evaluated here. The smaller (10-nm) Ag NPs were more potent than the larger (75-nm) Ag NPs in each stress-response pathway, and citrate-coated Ag NPs had higher intracellular silver concentrations compared with both PVP-coated Ag NP and AgNO3. The cellular stress response profiles after Ag NP exposure were similar to that of AgNO3, suggesting that the oxidative stress and inflammatory effects of Ag NP are likely due to the cytotoxicity of silver ions.

Chloral hydrate decreases gap junction communication in rat liver epithelial cells.

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1­5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell­cell contact was apparent in CH (0.5­5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.

A potential microRNA signature for tumorigenic conazoles in mouse liver.

Triadimefon, propiconazole, and myclobutanil are conazoles, an important class of agricultural fungicides. Triadimefon and propiconazole are mouse liver tumorigens, while myclobutanil is not. As part of a coordinated study to understand the molecular determinants of conazole tumorigenicity, we analyzed the microRNA expression levels in control and conazole-treated mice after 90 d of administration in feed. MicroRNAs (miRNAs) are small noncoding RNAs composed of approximately 19-24 nucleotides in length, and have been shown to interact with mRNA (usually 3' UTR) to suppress its expression. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Groups of mice were fed either control diet or diet containing 1800 ppm triadimefon, 2500 ppm propiconazole, or 2000 ppm myclobutanil. MicroRNA was isolated from livers and analyzed using Superarray whole mouse genome miRNA PCR arrays from SABioscience. Data were analyzed using the significance analysis of microarrays (SAM) procedure. We identified those miRNAs whose expression was either increased or decreased relative to untreated controls with q < or = 0.01. The tumorigenic conazoles induced many more changes in miRNA expression than the nontumorigenic conazole. A group of 19 miRNAs was identified whose expression was significantly altered in both triadimefon- and propiconazole-treated animals but not in myclobutanil-treated animals. All but one of the altered miRNAs were downregulated compared to controls. This pattern of altered miRNA expression may represent a signature for tumorigenic conazole exposure in mouse liver after 90 d of treatment.