Mechanisms Of The Effect Of AKR1B10 On Colon Carcinoma And Lung Cancer Cells Growth

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine, but overexpressed in liver and lung cancer. AKR1B10 efficiently catalyzes the reduction of aldehydes and ketones to form corresponding alcohols. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-a (ACCA). ACCA is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers. The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. This study discussed the effect of AKR1B10 on colon tumor cells and the related mechanisms, and then we further studied the effect of AKR1B10 on non-small cell lung cancer growth by in vivo and in vitro experiments to test its universal effectiveness on cancer cell growth.In view of the regulation of AKR1B10 on ACCA activity, in this study, we firstly studied the effect of ACCA on lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15 by using TOFA(5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, and ACCA siRNA. TOFA is cytotoxic to NCI-H460, HCT-8 and HCT-15, with an IC50 at approximately 5.0,5.0, and 4.5μg/ml, respectively. TOFA at 1.0-20.0μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Consistent with this, ACCA siRNA also induced cell apoptosis. Supplementing simultaneously the cells with palmitic acids (100μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA.Thereafter, we investigated the effect of AKR1B10 silencing on cell growth and survival/death. Our results showed that in human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA (siRNA)-induced AKR1B10 silencing resulted in caspase-3 mediated apoptosis. In these cells, the total and sub-species of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2-to 3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux and caspase-3 cleavage. AKR1B10 silencing also disturbed the homeostasis ofα,β-unsaturated carbonyls, leading to 2-to 3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 and NCI-H460 cells with palmitic acids (80μM), the end-products of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival via mediating lipid metabolism and eliminating cellular carbonyls.Studies on lung cancer were expanded to evaluate the AKR1B10 as a potential target for lung cancer intervention. We silenced AKR1B10 in human lung carcinoma cells (NCI-H460 and A549), using AKR1B10 siRNA and evaluated the cell and tumor growth. Results exhibited that AKR1B10 silencing prevented cells growth and significantly reduced plating efficiency in liquid culture and focus formation by more than 40% in semisolid culture. Phosphorylation of H2AX, a marker of DNA damage, and alkaline comet assays showed that AKR1B10 downregulation increased DNA damage by almost 2 folds versus control. Increased genotoxic stress in turn led to p53 activation and increase of ratio of proapoptotic protein Bax/antiapoptotic protein Bcl-2, activating caspase 3 mediated poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis. More importantly, in nude mice experiment, AKR1B10 downregulation delayed formation and progression of subcutaneous lung tumor xenografts. In NCI-H460 lung cancer animal models, tumors did not appear until the 17th day after injection of tumor cells when AKR1B10 was silenced, while in the control group, tumors occurred on the 9th day. The growth rate of tumors in AKR1B10 silencing group decreased 50% compared with control group. Meanwhile, in the tumor models of A549 cells, we also observed similar results.To further investigate the effect of AKR1B10 on lung cancer development and interference, We applied the technology of tetracycline-inducible gene expression to establish the cell mode of regulating AKR1B10 expression by tetracycline. Firstly, the regulatory TetR expression construct (pcDNA6/TR) was tansfected into NCI-H460 cells, and Transfectants were selected with 10μg/ml Blasticidin. The positive clones were selected using PCR and RT-PCR. Secondary, inducible AKR1B10 siRNA expression construct (pENTRTM H1/TO/ siRNA) was transfected into the cell clones with high TetR expression. Stable clones selected with 400μg/ml Zeocin were subjected to 3μg/ml doxycycline induction tests. The clones with doxycycline-inducible AKR1B10 knockdown were selected and propagated for future studies.In summary, this study demonstrated that AKR1B10 plays a critical role in lung and colon cancer cell growth by modulating lipid synthesis, mitochondrial function and oxidative status, as well as carbonyl levels. AKR1B10 also affects the formation and progression of lung cancer in animal tumor models. These data provides important experiment evidences and theoretical basis for targeting AKR1B10 for lung cancer therapy. This study also generated a doxycycline-inducible AKR1B10 silencing cell model, a convenient and economic experiment tool for the study of lung cancer intervention and drug development.