Aldo-keto reductase family 1 B10 gene silencing results in growth inhibition of colorectal cancer cells: Implication for cancer intervention.

Aldo-keto reductase family 1 B10 (AKR1B10), a member of aldo-keto reductase superfamily, is overexpressed in human hepatocellular carcinoma, lung squamous cell carcinoma and lung adenocarcinoma. Our previous study had demonstrated that the ectopic expression of AKR1B10 in 293T cells promotes cell proliferation. To evaluate its potential as a target for cancer intervention, in the current study we knocked down AKR1B10 expression in HCT-8 cells derived from a colorectal carcinoma, using chemically synthesized small interfering RNA (siRNA). The siRNA 1, targeted to encoding region, downregulated AKR1B10 expression by more than 60%, and siRNA 2, targeted to 3' untranslational region, reduced AKR1B10 expression by more than 95%. AKR1B10 silencing resulted in approximately a 50% decrease in cell growth rate and nearly 40% suppression of DNA synthesis. More importantly, AKR1B10 downregulation significantly reduced focus formation rate and colony size in semisolid culture, indicating the critical role of AKR1B10 in HCT-8 cell proliferation. Recombinant AKR1B10 protein showed strong enzymatic activity to acrolein and crotonaldehyde, with K(m) = 110.1 +/- 12.2 microM and V(max) = 3,122.0 +/- 64.7 nmol/mg protein/min for acrolein and K(m) = 86.7 +/- 14.3 microM and V(max) = 2,647.5 +/- 132.2 nmol/mg protein/min for crotonaldehyde. AKR1B10 downregulation enhanced the susceptibility of HCT-8 cells to acrolein (25 microM) and crotonaldehyde (50 microM), resulting in rapid oncotic cell death characterized with lactate dehydrogenase efflux and annexin-V staining. These results suggest that AKR1B10 may regulate cell proliferation and cellular response to additional carbonyl stress, thus being a potential target for cancer intervention.

Molecular-cytogenetic analysis of HER-2/neu gene in BRCA1-associated breast cancers.

The BRCA1 tumor suppressor gene and the HER-2/neu oncogene are located in close proximity on the long arm of chromosome 17 (17q11-21). Absence of BRCA1 or functional overexpression of the HER-2/neu gene presumably contributes to the somatic phenotype of breast cancer in premenopausal women, characterized by unfavorable prognostic features such as high tumor grade, hormone receptor negativity, and high proliferation rate. To examine whether amplification of HER-2/neu contributes to the aggressive biology of BRCA1-associated tumors, we have performed fluorescence in situ hybridization on formalin-fixed paraffin-embedded breast tumor tissue sections from 53 BRCA1 mutation carriers and 41 randomly selected, age-matched sporadic breast cancer cases. Although BRCA1-associated and sporadic tumors were equally likely (19% versus 22%) to exhibit HER-2/neu amplification [defined as a ratio of HER-2/neu copies to chromosome 17 centromere (CEP17) copies > or = 2], 6 (15%) of the sporadic tumors were highly amplified (defined as a ratio greater-than-or-equal 5) versus none of the BRCA1-associated tumors (P = 0.048). HER-2 protein overexpression as measured by immunohistochemical analysis was not observed among the BRCA1-associated cases (P = 0.042). Four of 21 (19%) sporadic tumors exhibited strong membranous staining of HER-2 (intensity level of 3+) as compared with 0 of 39 BRCA1-associated tumors. Our data suggest that a germ-line mutation in the BRCA1 tumor suppressor gene is associated with a significantly lower level of HER-2/neu amplification. Thus, it is possible that BRCA1-associated and HER-2/neu-highly amplified tumors progress through distinct molecular pathways, and the aggressive pathological features of BRCA1-associated tumors appear unrelated to amplification of the adjacent HER-2/neu oncogene.