Jingchun Gao

Non‐steroidal anti‐inflammatory drugs inhibit cellular proliferation and upregulate cyclooxygenase‐2 protein expression in endometrial cancer cells

We determined the effects of several non‐steroidal anti‐inflammatory drugs (NSAIDs), aspirin (acetylsalicylic acid, ASA), indomethacin and a cyclooxygenase‐2 (COX‐2)‐selective inhibitor (NS398), on cellular proliferation and regulation of COX‐2 protein expression in endometrial cancer cells in vitro, and investigated their modes of action. All three NSAIDs markedly inhibited the proliferation of Ishikawa, HEC‐1A and AN3CA endometrial cancer cell lines in a time‐ and concentration‐dependent manner. ASA and indomethacin triggered apoptosis in cells of all three lines through release of cytosolic cytochrome c, activation of caspase‐9 and ‐3, and cleavage of poly(ADP‐ribose) polymerase (PARP), but NS398 induced minimal apoptosis only in Ishikawa cells. ASA altered the cell cycle distribution, with G2/M phase accumulation of cells, and induced overexpression of Ki‐67 protein. Both ASA and indomethacin reduced the protein levels of Bcl‐2 and Bcl‐xl, but upregulated those of Bax and Bcl‐xs. COX‐2 protein expression and PGE2 production were upregulated by ASA and indomethacin in all three cell lines. However, NS398 did not alter COX‐2 protein expression or PGE2 production in these cells. These results indicate that NSAIDs inhibit proliferation of endometrial cancer cells independently of the reduction of COX‐2 protein expression. A cytochrome c‐dependent apoptotic pathway and/or cell cycle arrest may contribute to the inhibitory effects of these NSAIDs.

Preventive Effects of Isoflavones, Genistein and Daidzein, on Estradiol-17β-related Endometrial Carcinogenesis in Mice

The effects of isoflavones (genistein and daidzein) on endometrial carcinogenesis in mice were investigated in two experiments. In the short‐term experiment (2 weeks), single subcutaneous (s.c.) administration of genistein [1 mg/30 g body weight (b.w.)] significantly decreased the levels of estradiol‐l7β (E2) (5 ppm in diet)‐induced expression of c‐jun, interleukin‐lα (IL‐lα) and tumor necrosis factor‐a (TNF‐a) mRNAs in the uteri of ovariectomized mice (P<0.005, P<0.05 and P<0.01, respectively). Daidzein significantly inhibited E2‐induced expression of c‐fos and IL‐lα (P<0.01, P<0.01 respectively). In the long‐term experiment (30 weeks), 140 female ICR mice were given N‐methyl‐N‐nitrosourea‐containing solution (1 mg/100 g b.w.) and normal saline (as controls) into their left and right uterine corpora, respectively. They were divided into six groups; group 1 was given E2 (in diet) alone. Group 2 was given E2 and genistein (1 mg/30 g b.w., s.c., every four weeks). Group 3 was exposed to E2 and daidzein (1 mg/30 g b.w., s.c., every four weeks). Groups 4 and 5 respectively received genistein and daidzein, and were kept on the basal diet. Group 6 was kept on the basal diet and served as a control. At the termination of the experiment, incidences of endometrial adenocarcinoma and atypical endometrial hyperplasia of the group given E2 and genistein or daidzein were significantly lower than of the group with E2 alone (P<0.01 and P<0.05, respectively). It is suggested that both genistein and daidzein have an inhibitory effect on estrogen‐related endometrial carcinogenesis in mice, possibly by suppressing expression of estrogen‐induced estrogen‐related genes c‐fos and c‐jun, and internal cytokines IL‐lα and TNF‐α through a cytokine and estrogen receptor‐mediated pathway.

Association of cellular apoptosis with anti-tumor effects of the Chinese herbal complex in endocrine-resistant cancer cell line

We previously reported the special herbal complex (Hoelen, Angelicae radix, Scutellariae radix and Glycyrrhizae radix) suppressed telomerase activity in chemo-endocrine-resistant cancer cell lines. The present study attempted to determine whether the above herbal complex induces apoptosis in endocrine-resistant AN3CA and adriamycin-resistant MCF7/ADR carcinoma cells. Exposure to the herbal complex decreased cell viability in a time- and dose-dependent manner in the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The agent induced cellular apoptosis was determined by DNA fragmentation and a nuclear staining assay. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed the decreased expression of apoptosis-related genes, bcl-2, c-myc and human telomerase catalytic subunit (hTERT). A decreased protein level of bcl-2 and c-myc was also determined by Western blot analysis. The data imply that the decreased expression of the genes via suppressing telomerase activity is involved in cellular apoptosis in endocrine-resistant AN3CA cells. Thus, it is suggested that the special herbal complex may be a promising candidate for the treatment of endocrine-resistant gynecologic carcinomas.

Shimotsu-to is the agent in Juzen-taiho-to responsible for the prevention of endometrial carcinogenesis in mice

We have found that Juzen-taiho-to has a preventive effect on endometrial carcinogenesis in mice (Carcinogenesis 22 (2001) 587). In the present study, the constituents of Juzen-taiho-to responsible for this effect were explored using a short-term experiment. Thirty female ICR mice were divided into five groups: Group 1 was given a diet containing 0.2% of Juzen-taiho-to and 5ppm estradiol-17beta (E(2)); Group 2 was given a diet containing Shimotsu-to (0.07%) and E(2) (5ppm); Group 3 received Shikunshi-to (0.08%) and E(2) (5ppm) in the diet; Group 4 was given 5ppm E(2) in the diet; and Group 5 served as a control. Exposure of Juzen-taiho-to or Shimotsu-to decreased E(2)-stimulated expression of estrogen-related gene c-fos mRNA (P<0.05), and the cytokines interleukin-1alpha mRNA and tumor necrosis factor alpha mRNA P<0.01). A similar trend was not found upon treatment with Shikunshi-to. These findings suggest that Shimotsu-to is responsible for the inhibitory effects of Juzen-taiho-to on the estrogen-related endometrial carcinogenesis in mice.

Inhibitory Effects of Toremifene on N-Methyl-N-nitrosourea and Estradiol-17β-induced Endometrial Carcinogenesis in Mice

Short‐ and long‐term experiments were designed to determine the effects of toremifene (TOR) on estrogen‐related endometrial carcinogenesis in mice. In the short‐term experiment, a single low dose of TOR (0.2 mg/30 g body weight) decreased expression of c‐fos, interleukin (IL)‐1α, estrogen receptor (ER)‐α mRNAs and corresponding proteins induced by estradiol‐l7β (E2), in the uteri of the ovariectomized mice. Expression of ER‐β mRNA was increased by the TOR treatment, compared with the control. In the long‐term experiment, 106 female ICR mice were given N‐methyl N‐nitrosourea (MNU) into their uterine corpora. The animals were divided into four groups as follows: group 1, E2 diet (5 ppm) plus TOR (0.2 mg/30 g body weight, subcutaneously, every four weeks); group 2, E2 diet alone; group 3, basal diet plus TOR. Group 4 served as the control. TOR treatment decreased the incidence of MNU and E2‐induced endometrial adenocarcinoma and atypical hyperplasia at the termination of the experiment (30 weeks after the start). These results suggest that TOR exerts preventive effects against estrogen‐related endometrial carcinogenesis in mice, through the suppression of c‐fos as well as IL‐1α expression induced by E2. Such suppressive effects of TOR may be related to the decreased ER‐α and increased ER‐β expressions.

Inhibitory Effects of Phytoestrogens and Related Herbal Extracts on Mouse Endometrial Carcinogenesis: A Review

The effects of isofiavones (genistein and daidzein) and related Kampo medicines (Juzen-taiho-to and Shimotsu-to) on endometrial carcinogenesis in mice were investigated separately in two experiments. In the short-term experiment (2 weeks), a single subcutaneous (s.c.) administration of genistein [(lmg/30g body weight (BW))] significantly decreased the levels of 17β-estradiol (E2; 5p.p.m. in diet)induced expression of c-jun, interleukin (IL)-1α and tumor necrosis factor (TNF)-α mRNAs in the uteri of ovariectomized mice (P < 0.005, P < 0.05, and P < 0.01, respectively). Daidzein significantly inhibited E2-induced expression of c-fos and IL-1α (P < 0.01 and P < 0.01, respectively). In the long-term experiment (30 weeks), the incidences of endometrial adenocarcinoma and atypical endometrial hyperplasia were significantly lower in the E2 group treated with either genistein or daidzein than in the corresponding control group (P < 0.01 and P < 0.05, respectively). In the shortterm experiment, Juzen-taiho-to or Shimotsu-to (Kampo formula) treatment (2 weeks) significantly decreased the levels of E2-stimulated expression of c-fos mRNA (P < 0.05). In the long-term experiment, Juzen-taiho-to and Shimotsu-to treatment significantly decreased the incidences of endometrial adenocarcinoma (P < 0.05) and other preneoplastic lesions under estrogenic stimulation. It is suggested that both genistein and daidzein, as well as Juzen-taiho-to, have an inhibitory effect on estrogen-related endometrial carcinogenesis in mice, indicated by the suppression of estrogen-related c-fos and c-jun, as well as the suppression of cytokine IL-1α and TNF-α-expression through a cytokine- and estrogen receptor-mediated pathway. Shimotsu-to is considered to be a key compounet of Juzen-taiho-to for the prevention of endometrial carcinoma.