| Acrolein, crotonaldehyde and 4-HNE are highly reactiveα,β-unsaturated aldehyde produced endogenously during lipid peroxidation and naturally distributed pervasively in living environments, posing serious threats to human health. In this study, we report human aldose resductase-like-1 (ARL-1) as a novel enzyme that catalyzes the reduction of those aldehydes to their alcohol form and protects cells from their toxicity.Using purified ARL-1 protein, we determined its enzymatic activity in response to acrolein,crotonaldehyde and 4-HNE .Recombinant ARL-1 protein showed strong enzymatic activity to acrolein(Km:0.110±0.012mM,Vmax:3122.0±64.7nmol/mg/min),crotonaldehyde(Km:0.082±0.014mM,Vmax:2647.0±132.2 nmol /mg/min) and 4-HNE(Km :0.031±0.007mM,Vmax:3296±245.9nmol/mg/min).By introducing a functional EGFP/ARL-1 fusion protein into 293T cells, we demonstrated that plating efficiency in liquid culture and focus formation in soft agar increased by more than 60% (p<0.05), compared to the vector control cells. More significantly, at low dose of 5μM acrolein, EGFP/ARL-1 expression enhanced both plating efficiency and focus formation by more than 3 fold; and the foci (in soft agar) of 293T cells expressing EGFP/ARL-1 were significantly larger than those of the vector control cells. At high concentrations of acrolein (25 and 50μM), EGFP/ARL-1 protein prevented oncotic death of 293T cells, induced by acrolein, but did not affect cell death pathways. With two chemically synthesized small interfering RNAs (siRNA 1 and siRNA 2), ARL-1 in HCT 8 cells derived from colorectal cancer was downregulated by approximately 60% and 90%, resulting in significant inhibition of cell growth. Compared to control cells, ARL-1 knockdown in HCT 8 cells greatly decreased the plating efficiency and anchorage-independent cell growth, reflecting in reduced colony formation rates and diminished colony size. ARL-1 knockdown also resulted in dramatic cell death when exposed to acrolein (25μM).To further elucidate the intracellular function of ARL-1,I have established two stable cell models,ie,stable ARL-1 expression and stable ARL-1 silencing cell models.HCT8 cells with high endogenous ARL-1 were used for stable ARL-1 knockdown, while RKO cells(also a colorectal cancer cell line) with no ARL-1 expression were used for ARL-1 gene delivery. Totally, three HCT-8 stable clones with ARL-1 knockdown by 50– 90% and three RKO stable clones with ARL-1 activity increase by 5– 10 folds were established. Two vector control cell clones for each were also generated for controls. When exposed to acrolein (25μM), RKO cells with overexpression of ARL-1 showed exhibited higher viability by 19.5 - 34.8% than vector control cells (P<0.05), whereas HCT 8 cells with ARL-1 knockdown were more sensitive to acrolein by 31 - 39.1% than control cells (P<0.05). When exposed to 4-HNE(20μM),viability of RKO cells with expression of ARL-1 was increased by16.8-28.5% ,compared with control cells, whereas the sensitivity of HCT8 cells with ARL-1 knockdown to 4-HNE(25μM) was 24.9-52.4% higher than vector control cells(P<0.05). Similar results were observed in exposure to crotonaldehyde (50μM), with increase of cell viability by 12.5 - 23% in RKO cells (P<0.05) and decrease by 22.1 - 26.6% in HCT 8 cells (P<0.05).ARL-1 activity changes also altered clonogenic growth of cells. Plating efficiency of RKO cells with ARL-1 overexpression was enhanced by 4.4 - 50.8% in presence of acrolein (5μM, P<0.01), while HCT 8 cells with ARL-1 knockdown exhibited decreased efficiency by 4.2% - 24.1% (P<0.05), compared to control cells. ARL-1 knockdown also significantly affected anchorage-independent growth of HCT-8 cells in soft agar, exhibiting lower focus formation rates by 9.6 - 38.4% (P<0.01) and smaller colony size, compare to control cells. Genotoxicity induced by carbonyls was evaluated by comet assay and mutation rate. The results indicate that ARL-1 activity significantly correlated with DNA break and mutation induced by HNE and crotonaldehyde.Taken together, our data demonstrated that ARL-1 is a critical protein that protects cells from carbonyl toxicity including cytotoxicity and genotoxicity.Conclusion:1. ARl-1 expression can protect cells against cytotoxicity induced by reactive carbonyls.2. ARL-1 gene silencing results in growth inhibition of HCT8 cells.3. ARL-1 expression decreases reactive carbonyls induced gene mutation frequency.4. ARL-1 expression reduces cellular ROS generation.
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