Yongli Guan

Low‐dose genistein induces cyclin‐dependent kinase inhibitors and G1 cell‐cycle arrest in human prostate cancer cells

Genistein, a naturally occurring isoflavone found chiefly in soy products, reportedly has antiprostate cancer effects, but the mechanisms underlying these effects are unknown. We studied the antiproliferative and apoptosis‐inducing effects of genistein in the androgen‐sensitive human prostate cancer cell line LNCaP. Viable cell number was assessed by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay; cell‐cycle progression and apoptosis were evaluated by flow cytometry; apoptosis was also assessed by a histone enzyme‐linked immunosorbent assay; and the expression of several cell‐cycle– and apoptosis‐related genes and their gene products was determined by northern blot analysis, western blot analysis, and/or assays based on polymerase chain reaction. Physiologic concentrations of genistein (≤ 20 μM) decreased LNCaP viable cell number in a dose‐dependent manner, induced a G1 cell‐cycle block, decreased prostate‐specific antigen mRNA expression, and increased p27KIP1 and p21WAF1 (mRNA and protein) but had no effect on apoptosis or the mRNA expression of the apoptosis‐ and cell‐cycle–related markers bcl‐2, bax, Rb, and proliferating cell nuclear antigen. Higher concentrations of genistein (> 20 μM) did induce apoptosis. We conclude that genistein (at physiologic concentrations) exerts potent antiproliferative effects on LNCaP cells by inducing a G1 cell‐cycle block. The antiproliferative effects of genistein may be mediated by increased levels of p27KIP1 and p21WAF1, which are negative cell‐cycle regulators that act as cyclin‐dependent kinase inhibitors and that have been recently linked with prostate carcinogenesis. These findings may provide insights into the mechanisms underlying the apparent antiprostate cancer effects of soy consumption observed in epidemiologic studies. Mol. Carcinog. 29:92–102, 2000.

EXPRESSION OF FIVE SELECTED HUMAN MISMATCH REPAIR GENES SIMULTANEOUSLY DETECTED IN NORMAL AND CANCER CELL LINES BY A NONRADIOACTIVE MULTIPLEX REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

Abnormalities in at least 1 of 5 mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2 and GTBP/hMSH6) are found in hereditary nonpolyposis colon cancer and sporadic colon cancers. We used a single-reaction multiplex reverse transcription (RT)-polymerase chain reaction (PCR), with the beta-actin gene as an internal control, to simultaneously evaluate expression of these 5 known human MMR genes in normal and tumor cell lines with known or uncharacterized mutations in MMR genes. The relative quantitation of the transcripts is demonstrated by controlling the number of PCR cycles and titrating cDNA with a dose-curve. The 13 normal cell lines tested were derived from normal lymphocytes, skin, thymus, breast, lung, colon, liver and kidney. The 26 cancer cell lines were derived from melanoma and cancers of the brain, breast, lung, colon, pancreas and prostate. All 5 MMR genes were ubiquitously expressed in all normal cell lines tested, suggesting their housekeeping roles. Aberrant MMR gene expression was only observed in the colon cancer cell lines. Two previously uncharacterized colon cancer cell lines did not express hMLH1. These data suggest that this nonradioactive multiplex RT-PCR assay for MMR gene expression may be useful for fast screening for genetic alterations that may affect gene expression and so may aid molecular analysis of MMR-related colon cancer.

N-(4-hydroxyphenyl)retinamide (4-HPR) modulates GADD45 expression in radiosensitive bladder cancer cell lines

We previously demonstrated that N-(4-hydroxyphenyl)retinamide (4-HPR) and gamma-irradiation, when used in combination, had a synergistic effect in inducing apoptosis in bladder cancer cells, suggesting that 4-HPR may increase radiosensitivity in bladder cancer cells. To unravel molecular correlates in this radiosensitizing effect of 4-HPR, we examined the baseline and 4-HPR-induced expression of GADD45 to elucidate possible mechanisms by which 4-HPR enhanced the effect of gamma-irradiation in three bladder cancer cell lines. To investigate the role of p53 in mediating the radiosensitizing effect of 4-HPR, we also examined mutations in exons 5-9 by using direct sequencing and the levels of p53 expression by using RT-PCR and Western blot, before and after treatment with 4-HPR in these bladder cancer cell lines. Two cell lines had low expression of GADD45, and a dose-dependent increase in GADD45 expression induced by 4-HPR was found in bladder cancer cell lines without p53 mutations in exons 5-9. A combination of gamma-irradiation and 4-HPR showed a significantly greater effect in enhancing GADD45 expression than either agent used alone. The results indicate that the combined treatment with 4-HPR and gamma-irradiation has a stronger effect on GADD45 expression than the treatment with either agent alone, which suggests that the two agents may have an additive/synergistic effect. However, a normal p53 function appears to be necessary for the dose-dependent induction of GADD45 by 4-HPR. Once our results are verified and replicated by other investigators, 4-HPR may have a potential clinical implication in effectively treating bladder cancer in combination with low-gamma-irradiation therapy.