Determination of intermediate biomarker expression levels by quantitative reverse transcription-polymerase chain reaction in oral mucosa of cancer patients treated with liarozole

Liarozole is a 1-substituted imidazole derivative that inhibits cytochrome P450 activity and increases endogenous plasma concentrations of retinoid ac id (RA). We have previously demonstrated that RA down-modulates transformin g growth factor (TGP)-alpha and epidermal growth factor receptor (EGFR) lev els in head and neck squamous cell carcinoma by decreasing the transcriptio n rate of these two genes. Previous reports suggest that RA receptor (RAR)- beta levels are down-modulated in head and neck cancer and are restored by RA therapy. Cellular RA-binding protein (CRABP)-II is up-regulated by RA an d appears to modulate intracellular RA metabolism. In conjunction with a Ph ase I clinical trial, total intact RNA was extracted from oral cavity mucos a biopsied from 17 patients with advanced malignancies, before and after tr eatment with a 4-week course of liarozole, To analyze these limited quantit ies of total RNA (as little as 0.6 mu g/sample), a quantitative reverse tra nscription-PCR assay,vas developed using delayed dropping of the 5' p-actin primer to amplify the highly abundant p-actin gene as an internal control. We used this method to determine the expression levels of TGF-or, EGFR, RA R-beta, and CRABP-II before and after treatment. There was a trend toward e levation of RAR-beta levels in oral mucosa after liarozole therapy (P = 0.1 07), whereas TGF-alpha, EGFR, and CRABP-II were not modulated by systemic l iarozole treatment. Those results suggest that liarozole may up-regulate RA R-beta in tissues from cancer patients and that expression levels of potent ial intermediate biomarkers may be determined in small tissue biopsies usin g a quantitative reverse transcription-PCR assay.