Hong-Sig Sin

Additional Sex Comb-like 1 (ASXL1), in Cooperation with SRC-1, Acts as a Ligand-dependent Coactivator for Retinoic Acid Receptor

Additional sex comb-like 1 (ASXL1, 170 kDa), a mammalian homolog of Drosophila ASX, was identified as a protein that interacts with retinoic acid receptor (RAR) in the presence of retinoic acid (RA). Systematic binding assays showed that the C-terminal nuclear receptor box (LVMQLL) of ASXL1 and the activation function-2 activation domain (AF-2 AD) core of the RAR are critical for ligand-dependent interaction. The interaction was confirmed using in vitro glutathione S-transferase pulldown and in vivo immunoprecipitation (IP) assays. Confocal microscopy revealed that ASXL1 localizes in the nucleus. In addition to the intrinsic transactivation function of ASXL1, its cotransfection together with an RA-responsive luciferase reporter increased the RAR activity. This ASXL1 activity appears to be mediated through the functional cooperation with SRC-1, as shown by GST pulldown, IP, chromatin IP, and transcription assays. In the presence of ASXL1, more acetylated histone H3 was accumulated on the RA-responsive promoter in response to RA. Finally, stable expression of ASXL1 increased the expression of endogenous RA-regulated genes and enhanced the antiproliferative potential of RA. Overall, these results suggest that ASXL1 is a novel coactivator of RAR that cooperates with SRC-1 and implicates it as a potential antitumor target of RA in RA-resistant cancer cells.

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome‐dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21WAF1. Here we show that p73 is also inactivated by HPV‐E6, but ubiquitin‐mediated proteolysis is not responsive. Yeast two‐hybrid and GST pull‐down assays indicate a physical interaction between p73 and either HPV‐16 or HPV‐11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co‐expression of E6 significantly inhibits the p73‐mdiated activation of p21WAF1 promoter in a p53‐defective C33A cell line. Using Gal4‐p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence‐specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino‐terminal activation domain. Co‐transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high‐ and low‐risk E6s and is not susceptible to E6‐dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV‐E6 may, at least in part, contribute to E6‐mediated transformation and hyperproliferation of cervical cells.

Antiproliferative mechanism of retinoid derivatives in ovarian cancer cells

Retinoid derivatives have been implicated for the growth regulation of ovarian cancer cells. However, the molecular mechanisms are not yet fully defined. To dissect detailed mechanisms of each derivative, four ovarian cancer cells (A2774, PA-1, OVCAR-3, SKOV-3) were treated with all-trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA), 13-cis RA, or 4-hydroxyphenyl retinamide (4-HPR). When treated with 1 microm, HPR inhibits most effectively the growth of all four cells. Depending on cell types treated, IC(50) values were 0.7-2.7 microm for 4-HPR, and 2.7-9.0 microm for other retinoid derivatives. DNA fragmentation assay indicated that the antiproliferative effect of HPR could be mediated by apoptosis. Transcription assays coupled with transient transfection in OVCAR-3 cells indicated that ATRA, 9-cis RA, and 13-cis RA were active for all RAR/RXR subtypes, whereas 4-HPR was only active for RARgamma. However, 4-HPR exerted the strongest suppression on AP-1 (c-Jun) activity. As expected from AP-1 data, in vitro invasion assays showed that HPR blocked effectively the migration of OVCAR-3 cells. Thus, 4-HPR showed not only more potent antiproliferative activity than any other retinoid derivatives used, but also effectively inhibited the invasion, probably through the suppression of AP-1 activity. Taken together coupled with its selective activity only for RARgamma, these results suggest that 4-HPR could be less toxic, and very effective anticancer drugs for late stage ovarian cancer.

Antitumor activity of spinasterol isolated from Pueraria roots

We purified phytoestrogens from Pueraria root (Pueraria mirifica from Thailand and Pueraria lobata from Korea), which is used as a rejuvenating folk medicine in Thailand and China. Dried, powdered plant material was extracted with 100% ethanol and further separated by concentration, filtration, and thin layer silica gel chromatography. Using the fractions obtained during separation, we first investigated their cytotoxicity in several cancer cell lines from various tissues. The ethanol-extracted components (PE1, PE4) had significant antiproliferative effects on breast cancer cell lines, including MCF-7, ZR-75-1, MDA-MB-231, SK-BR-3, and Hs578T. Second, we compared these results with the cytotoxic effects of known flavonoids, sterols, and coumarins from Pueraria root. The known compounds were not as effective, and occurred in a different polarity region on HPLC. Third, further separation resulted in the isolation of eight different components (Sub PE-A to -H). One of these, PE-D, affected the growth of some breast cancer cell lines (MCF-7, MDA-MB-231) in a dose- and time-dependent manner, as well as the growth of ovarian (2774) and cervical cancer cells (HeLa). Finally, a transfection assay showed that this component had an estrogenic effect similar to 17β-estradiol, which activates both estrogen receptor a (ER α) and ER β. The NMR analysis determined that spinasterol (stigmasta-7, 22-dien-3β-ol) is an active cytotoxic component of Pueraria root.

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.

The retinoic acid derivative, ABPN, inhibits pancreatic cancer through induction of Nrdp1

Combination chemotherapy for the treatment of pancreatic cancer commonly employs gemcitabine with an EGFR inhibitor such as erlotinib. Here, we show that the retinoic acid derivative, ABPN, exhibits more potent anticancer effects than erlotinib, while exhibiting less toxicity toward noncancerous human control cells. Low micromolar concentrations of ABPN induced apoptosis in BxPC3 and HPAC pancreatic cancer cell lines, concomitant with a reduction in phosphorylated EGFR as well as decreased ErbB3, Met and BRUCE protein levels. The degradation of ErbB3 is a result of proteasomal degradation, possibly due to the ABPN-dependent upregulation of Nrdp1. Administration of ABPN showed significant reductions in tumor size when tested using a mouse xenograft model, with higher potency than erlotinib at the same concentration. Analysis of the tumors demonstrated that ABPN treatment suppressed ErbB3 and Met and induced Nrdp1 in vivo. The data suggest that ABPN may be more suitable in combination chemotherapy with gemcitabine than the more widely used EGFR inhibitor, erlotinib.