MicroRNA Profile of Tumorigenic Cells During Carcinogenesis of Lung Adenocarcinoma

To obtain microRNA (miRNA) profile and clarify their biological function in tumorigenic Sca‐1⁺CD34⁺ cells during carcinogenesis of lung adenocarcinoma. After intranasal infection with recombinant Adeno‐Cre viruses (AdV‐Cre), lung adenocarcinoma was identified pathologically in Lox‐stop‐lox Kras (LSL‐Kras) G12D mice. Sca‐1⁺CD34⁺ cells were sorted by flow cytometry and tested for tumor‐initiating ability, self‐renewal and tumorigenicity. MiRNA profiles were obtained using microarray and further confirmed by real‐time RT‐PCR (qRT‐PCR). MiRNA functions were predicted bioinformatically, and miR‐294 function was verified to explore its role in tumor migration and invasion. Lung adenocarcinoma was induced in LSL‐Kras G12D mice within 30 days. In vivo, the tumorigenicity of Sca‐1⁺CD34⁺ cells was 25 times stronger than Sca‐1^?CD34^? cells. During tumorigenesis of lung adenocarcinoma, the expression of 145 miRNAs in Sca‐1⁺CD34⁺ cells increased and 72 miRNAs decreased (P < 0.01). Four successively up‐regulated miRNAs (miR‐15a*, miR‐203, miR‐294 and miR‐295*) and three successively down‐regulated ones (miR‐19b, miR‐483 and miR‐615–5p) were identified. Among them, miR‐294 could constitutively bind to 3'‐UTR of matrix metalloproteinase 3 (MMP3), and down‐regulate MMP3 protein expression. MiR‐294 also significantly inhibited migration and invasion of Lewis lung cancer cells. MiRNAs are characteristically expressed in tumor‐initiating Sca‐1⁺CD34⁺ cells of lung adenocarcinoma, and may play important roles during the carcinogenesis of lung adenocarcinoma. J. Cell. Biochem. 116: 458–466, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

     

Lycopene Protects Keratinocytes Against UVB Radiation‐Induced Carcinogenesis via Negative Regulation of FOXO3a Through the mTORC2/AKT Signaling Pathway

Lycopene, one of the most potent anti‐oxidants, has been reported to exhibit potent anti‐proliferative properties in a wide range of cancer cells through modulation of the cell cycle and apoptosis. Forkhead box O3 (FOXO3a) plays a pivotal role in modulating the expression of genes involved in cell death. Herein, we investigated the role of FOXO3a signaling in the anti‐cancer effects of lycopene. Results showed that lycopene pretreatment attenuated UVB‐induced cell hyper‐proliferation and promoted apoptosis, accompanied by decreased cyclin‐dependent kinase 2 (CDK2) and CDK4 complex in both human keratinocytes and SKH‐1 hairless mice. FOXO3a is phosphorylated in response to UVB irradiation and sequestered in the cytoplasm, while lycopene pretreatment rescued this sensitization. Gene ablation of FOXO3a attenuated lycopene‐induced decrease in cell hyper‐proliferation, CDK2, and CDK4 complex, indicating a critical role of FOXO3a in the lycopene‐induced anti‐proliferative effect of keratinocytes during UVB irradiation. Transfection with FOXO3a siRNA inhibited the lycopene‐induced increase in cell apoptosis, BAX and cleaved PARP expression. Moreover, loss of AKT induced further accelerated lycopene‐induced FOXO3a dephosphorylation, while loss of mechanistic target of rapamycin complex 2 (mTORC2) by transfection with RICTOR siRNA induced levels of AKT phosphorylation comparable to those obtained with lycopene. In contrast, overexpression of AKT or mTORC2 decreased the effects of lycopene on the expression of FOXO3a as well as AKT phosphorylation, suggesting that lycopene depends on the negative modulation of mTORC2/AKT signaling. Taken together, our findings demonstrate that the mTORC2/AKT/FOXO3a axis plays a critical role in the anti‐proliferative and pro‐apoptotic effects of lycopene in UVB‐induced photocarcinogenesis. J. Cell. Biochem. 119: 366–377, 2018. © 2017 Wiley Periodicals, Inc.     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.