DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in many human diseases, including cancer. The tobacco-specific carcinogen NNK also induces DNA methylation. However, the role of DNMT1-mediated methylation in tobacco carcinogenesis remains unclear. Here we used human and mouse lung cancer samples and cell lines to determine a mechanism whereby NNK induced DNMT1 expression and activity. We determined that in a human lung cell line, glycogen synthase kinase 3β (GSK3β) phosphorylated DNMT1 to recruit β-transducin repeat-containing protein (βTrCP), resulting in DNMT1 degradation, and that NNK activated AKT, inhibiting GSK3β function and thereby attenuating DNMT1 degradation. NNK also induced βTrCP translocation to the cytoplasm via the heterogeneous nuclear ribonucleoprotein U (hnRNP-U) shuttling protein, resulting in DNMT1 nuclear accumulation and hypermethylation of the promoters of tumor suppressor genes. Fluorescence immunohistochemistry (IHC) of lung adenomas from NNK-treated mice and tumors from lung cancer patients that were smokers were characterized by disruption of the DNMT1/βTrCP interaction and DNMT1 nuclear accumulation. Importantly, DNMT1 overexpression in lung cancer patients who smoked continuously correlated with poor prognosis. We believe that the NNK-induced DNMT1 accumulation and subsequent hypermethylation of the promoter of tumor suppressor genes may lead to tumorigenesis and poor prognosis and provide an important link between tobacco smoking and lung cancer. Furthermore, this mechanism may also be involved in other smoking-related human diseases.

Purpose: CYP1B1and CYP1A1expression is up-regulated by activation of the aryl hydrocarbon receptor (AhR) through binding of ligands such as cigarette smoke components. We examined the association between AhR, CYP1B1, and CYP1A1expression in noninvasive bronchioloalveolar carcinomas (BAC) and lung adenocarcinomas and investigated the effects of AhR overexpression on cell physiology. Experimental Design: AhR, CYP1B1, and CYP1A1 expression was examined in 107 lung adenocarcinomas and 57 BAC by immunohistochemistry. AhR expression in lung adenocarcinoma H1355 cells was stably reduced by RNA interference (RNAi). AhR, CYP1B1, and CYP1A1 expression was examined using real-time reverse transcription-PCR. Cell physiology was evaluated by measuring anchorage-independent growth and intracellular reactive oxygen species. Results: Expression of AhR and CYP1A1 was associated in smoking adenocarcinoma patients, whereas expression of AhR and CYP1B1was associated regardless of smoking status. The level of CYP1B1, but not CYP1A1, was positively associated with AhR overexpression in BAC. 2,3,7,8-Tetrachlorobenzo-p-dioxin^induced CYP1A1/1B1 expression was reduced in AhR RNAi clones. In the absence of 2,3,7,8-tetrachlorobenzo-p-dioxin, CYP1B1 mRNA levels were reduced in AhR RNAi clones, whereas CYP1A1 mRNA levels were barely detectable. Furthermore, anchorageindependent growth and intracellular oxidative stress were significantly reduced in AhR RNAi cells. Conclusions: In the absence of exogenous AhR ligands (such as cigarette smoke components), AhR overexpression up-regulated the expression of CYP1B1in the early stage of lung adenocarcinoma. Elevated AhR expression in lung adenocarcinoma cells could increase intracellular oxidative stress and promote cell growth, implying that disrupting AhR expression might prevent the early development of lung adenocarcinomas.

Quantum dots (QDs) are one of most utilized nanomaterials in nanocrystalline semiconductors. QDs emit near-infrared fluorescence and can be applied as probes for detecting vasculature and imaging in biological systems. Since QDs have potential in clinical application, the toxicity of QDs needs to be carefully evaluated. In our present study, we elucidate the cytotoxic mechanisms of QDs using a mouse renal adenocarcinoma (RAG) cell line. QDs in RAG cells increased intracellular reactive oxygen species (ROS) levels and induced autophagy at 6 h, leading to subsequent apoptosis at 24 h. QDs entered the cells and were located within the endoplasmic reticulum (ER), endosome, and lysosome at 6 h and endosome, lysosome, and mitochondria at 24 h. However, QDs only affected mitochondrial function and did not induce ER stress. N-Acetylcysteine, an antioxidant agent, reduced intracellular ROS levels and decreased QD-induced autophagy but enhanced QD-induced cell death. Moreover, 3-methylamphetamine (an autophagy inhibitor) also reduced the cell viability in QD-treated cells. These findings suggest that ROS plays an essential role in the regulation of QD-induced autophagy, which subsequently enhances cell survival. Taken together, these results suggest that oxidative stress-induced autophagy is a defense/survival mechanism against the cytotoxicity of QD.

ABSTRACT

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Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.

The objective of this study was to investigate whether quantum dot 705 (QD705) disrupts the cellular antioxidant systems leading to hepatotoxicity in mice. Mice were intravenously injected with QD705 and then sacrificed at week 12 or 16. Homeostasis of antioxidant-related metals, antioxidant activities, induction of oxidative stress, and toxicity in the liver were investigated. Although no histopathological change was observed, a time- and dose-dependent increase in metallothionein expression and reduction in liver function was noticed. Increased copper, zinc, and selenium levels and enhancements of the trace metal-corresponding transporters were noted at week 12. At week 16, a decline of selenium from its elevated level at week 12 was observed, which was accompanied by changes in glutathione peroxidase activity as well as in redox status. A significant reduction in superoxide dismutase activity was observed at 16 weeks. Furthermore, a corresponding elevation of heme oxygenase-1 expression, 8-oxo-7,8-dihydro-2'-deoxyguanosine, interleukin-6 and tumor necrosis factor-alpha suggested the presence of oxidative stress, oxidative DNA damage and inflammation.

QD705 is a cadmium/selenium/tellurium (Cd/Se/Te)-based quantum dot with good potential for biomedical applications. Although the biological fate of QD705 is established, its chemical fate in the biological system is still unknown. Since the chemical nature of Cd in QD705 (either stays as bounded Cd or becomes free Cd) is closely related to the toxicity of this nanocrystal, information on its chemical fate is critically needed. In this study we investigated the chemical fate of QD705 in the kidneys of mice. We used the molar ratio of Cd and Te (increased Cd/Te ratio signifies increased Cd release from QD705) and the induction of tissue metallothionein (MT) as markers for elevated free Cd in tissues. Our study indicated that 100% of QD705 (measured as Cd) was still retained in the body 16 weeks after exposure, with significant time redistribution to the kidneys. Furthermore, there were an elevation in both the molar Cd/Te ratio and MT-1 expression in the kidneys, suggesting that free Cd was released from QD705. Thus QD705 is not as stable or biologically inert as many may have once believed. Our study demonstrated that free Cd indeed can be released from QD705 in the kidneys and increases the risk of renal toxicity.