Si-Ho Park

Efficacy validation of synthesized retinol derivatives in vitro: Stability, toxicity, and activity

Retinol (vitamin A) is used as an antiwrinkle agent in the cosmetics industry. However, its photo-instability makes it unsuitable for use in general cosmetic formulations. To improve the photo-stability of retinol, three derivatives (3, 4, and 5) were synthesized and their biological activities were analyzed. 1H NMR and HPLC analysis indicated that derivatives 3 and 5 were much more stable than retinol under our sunlight exposure conditions. When human adult fibroblasts were treated, the IC(50) of derivative 3 was 96 microM, which is similar to that of retinol, as determined by the MTT assay. Derivatives 4 and 5 were 2.5 and 8 times more toxic than retinol, respectively. At 1 microM treatment, like retinol, derivatives 3 and 4 were specifically active for RARalpha out of six retinoid receptors (RAR/RXRalpha, beta, gamma). Dose-dependent analysis confirmed that derivative 4 was as active as retinol and the other two derivatives were less active for RARalpha. The effect of our derivatives on the expression of collagenase, an indicator of wrinkle formation, was measured using the transient co-expression of c-Jun and RT-PCR in HaCaT cells. Collagenase promoter activity, which is increased by c-Jun expression, was reduced 42% by retinol treatment. The other derivatives inhibited collagenase promoter activity similarly. These results were further confirmed by RT-PCR analysis of the collagenase gene. Taken together, our results suggest that retinol derivative 3 is a promising antiwrinkle agent based on its higher photo-stability, lower RARalpha activity (possibly indicating reduced side effects), and similar effect on collagenase expression.

Novel retinoic acid derivative ABPN has potent inhibitory activity on cell growth and apoptosis in cancer cells.

Retinoids are natural and synthetic derivatives of vitamin A that have great promise for cancer therapy and chemoprevention. Of the retinoids developed so far, 4‐(N‐hydroxyphenyl)retinamide (4‐HPR or fenretinide) appears to have the best therapeutic potential in vitro and in vivo and is currently being tested in clinical trials for cancer prevention and therapy. To develop other potentially potent antitumor agents, we synthesized 85 retinoid derivatives. In an initial screening of these synthetic retinoids using the HCT116 colon cancer cell line, we found that 4‐amino‐2‐(butyrylamino)phenyl(2E,4E,6E,8E)‐3,7‐dimethyl‐9‐(2,6,6‐trimethyl‐1‐cyclohexenyl)‐2,4,6,8‐nonatetraenoate (ABPN or CBG41) induced the greatest growth inhibition, with an IC50 value of 0.6 μM. Subsequent studies in other cancer cell lines indicated that ABPN was much more growth‐inhibitory than all‐trans retinoic acid or 4‐HPR. Compared to 4‐HPR, ABPN induced 5.5‐ to 70.0‐fold more growth inhibition in most cancer cells, with the exception of gynecologic cancer cells. In these cells, the antiproliferative effect was only 1.5‐ to 2.8‐fold more than 4‐HPR. We examined the molecular mechanism underlying the difference in growth inhibition between 4‐HPR and ABPN. DAPI staining, DNA fragmentation, FACS and Western blotting analyses suggest that ABPN induced apoptosis by activating caspase‐3 and ‐8, which may result in increased PARP cleavage. Unlike 4‐HPR, ABPN activated all 3 RAR isotypes to an extent similar to AtRA. In addition, ABPN significantly inhibited AP‐1 transcriptional activity and thus greatly suppressed the expression of the matrix metalloproteinase ‐1, ‐2 and ‐3 genes, which are involved in tumor invasion. These results suggest that ABPN may be a promising retinoid derivative offering not only enhanced cytotoxicity, but also increased inhibition of tumor invasiveness.

Potent effect of 5-HPBR, a butanoate derivative of 4-HPR, on cell growth and apoptosis in cancer cells

Fenretinide, 4‐(N‐hydroxyphenyl) retinamide (4‐HPR), has demonstrated anticancer activity associated with a favorable toxicity profile and is now being investigated in several clinical trials. However, its plasma levels in patients have been far lower than the effective concentration required to induce apoptosis (usually 10 μM). This result has led to the synthesis of derivatives with better efficacy. Sodium butyrates potential as an anticancer agent prompted us to synthesize a butanoate derivative of 4‐HPR, 5‐hydroxyphenyl butanoate retinamide (5‐HPBR) and compare it to the parent compound for antitumor potential in vitro. The cytotoxicity of 5‐HPBR was 2‐ to 6‐fold greater than that of 4‐HPR against cancer cell lines derived from various tissues. In premalignant bronchial cells (BEAS2B), 5‐HPBR exhibited about a 10‐fold stronger cytotoxicity than did 4‐HPR. Normal CHANG liver cells were unaffected by either 4‐HPR or 5‐HPBR. Subsequent assays using DNA fragmentation, DAPI staining, FACS and Western blotting suggested that the potent inhibitory effect of 5‐HPBR is mediated by apoptosis; the exact mechanism appears to differ among cancer cell types. In transcription assays with COS‐1 cells, 5‐HPBR selectively activated RARβ and RARγ but was a weaker ligand for all 3 subtypes of RAR than either all‐trans retinoic acid or 4‐HPR. Overall, these data suggest that 4‐BHPR may be a promising retinoid with enhanced antitumor activity and reduced toxicity.