Epigenetic inactivation of LHX6 mediated microcystin-LR induced hepatocarcinogenesis via the Wnt/ß-catenin and P53 signaling pathways.

Microcystins (MCs) have been shown to be carcinogenic by animal and cellular experiments and found to be associated with the development of human hepatocellular carcinoma (HCC) through epidemiological studies. However, the molecular mechanism of microcystin-LR (MC-LR) induced HCC is still unclear. This study is determined to clarify the role and mechanism of LHX6 in MC-LR-induced hepatocarcinogenesis. Using the previously established MC-LR-induced malignant transformation model in L02 cells, we screened out LHX6, homeobox gene that was significantly changed. We found that LHX6 was significantly down-regulated in MC-LR treated L02 cells and the liver tissue of rats treated for 35 weeks with 10 µg/kg body weight of MC-LR. Expression of LHX6 in human tumor tissue was significantly down-regulated in high MC-LR-exposure group. LHX6 was hypermethylated in MC-LR treated L02 cells and up-regulated after treatment with 10 µM of 5-aza-2'-deoxycytidine. Furthermore, overexpression of LHX6 inhibited proliferation, invasion and migration of malignantly transformed L02 cells in vitro and in vivo, while knockdown of LHX6 resulted in an opposite phenotype. In addition, we found that up-regulation of P53 and Bax resulted in apoptosis, and that down-regulation of CTNNB1 and MMP7 led to migration of MC-LR treated L02 cells. Blockade of P53 and CTNNB1 by its inhibitor significantly diminished the effect of LHX6. These genes were working together during the process of MC-LR-induced hepatocarcinogenesis. Our study demonstrated for the first time that LHX6 gene expression is regulated by DNA methylation and can inhibit the proliferation, invasion and migration through Wnt/ß-catenin and P53 signaling pathways during the MC-LR-induced hepatocarcinogenesis. This result may suggest that LHX6 gene can be used as a potential target gene and a biomarker for liver cancer treatment.

Epigenetic silencing of ALX4 regulates microcystin-LR induced hepatocellular carcinoma through the P53 pathway.

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.

Experimental comparison of the reproductive outcomes and early development of the offspring of rats given five common types of drinking water.

Tap water (unfiltered), filtered tap water and processed bottled water (purified water, artificial mineralized water, or natural water) are now the five most widely consumed types of drinking water in China. However, the constituents (organic chemicals and inorganic ingredients) of the five waters differ, which may cause them to have different long-term health effects on those who drink them, especially sensitive children. In order to determine which type of water among the five waters is the most beneficial regarding reproductive outcomes and the developmental behaviors of offspring, two generations of Sprague-Dawley rats were given these five waters separately, and their reproductive outcomes and the developmental behaviors of their offspring were observed and compared. The results showed that the unfiltered tap water group had the lowest values for the maternal gestation index (MGI) and offspring's learning and memory abilities (OLMA); the lowest offspring survival rate was found in the purified water group; and the highest OLMA were found in the filtered tap water group. Thus, the best reproductive and offspring early developmental outcomes were found in the group that drank filtered tap water, which had the lowest levels of pollutants and the richest minerals. Therefore, thoroughly removing toxic contaminants and retaining the beneficial minerals in drinking water may be important for both pregnant women and children, and the best way to treat water may be with granular activated carbon and ion exchange by copper zinc alloy.

[Research on phytoestrogenic effects and its mechanism of psoralen].

OBJECTIVE: To observe and evaluate the phytoestrogenic effects and its mechanism of psoralen in estrogen receptor (ER) alpha and beta positive T47D and ishikawa cells. METHOD: The proliferation rate of T47D influenced by 1 x 10(-5) mol x L(-1) to 1 x 10(-9) mol x L(-1) psoralen and that of Ishikawa influenced by 1 x 10(-6) mol x L(-1) and 1 x 10(-7) mol x L(-1) psoralen were analyzed by MTT assay. PR mRNA expression in T47D was quantified by RT-PCR assay. Estrogen receptor antagonist ICI 182, 780 was employed as a tool. ER-alpha and ER-beta expression of T47D was measured by flow cytometry. RESULT: The proliferation rates of T47D cells treated with 1 x 10(-5) mol x L(-1) to 1 x 10(-7) mol x L(-1) psoralen and ishikawa cells treated with 1 x 10(-6) mol x L(-1) to 1 x 10(-7) mol x L(-1) psoralen were increased significantly. The RT-PCR result showed that 1 x 10(-7) mol x L(-1) and 1 x 10(-6) mol x L(-1) psoralen could increase PR expression in T47D cells. The above effects could be blocked by ICI 182,780. Psoralen could also induce the augment of ER-alpha and ER-beta expression in T47D cells significantly. CONCLUSION: Psoralen has phytoestrogenic effects. The effects are attained through ER pathway.

[Evaluation on phytoestrogen effects of ten kinds of Chinese medicine including flos carthami].

OBJECTIVE: To explore the phytoestrogenic effects of ten kinds of Chinese medicine including flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus ligustri lucidi, fructus lycii, radix clycyrrhizae, herba cistanches, herba epimedii, fructus psoraleae and semen cuscutae. METHOD: 240 female Kunming mice weighting 9 - 12 g were randomly divided into two main groups A and B. A group was divided into 12 small groups: 1 solvent control group, 1 diethylstilbestrol control group and 10 Chinese medicine groups. B group was also divided into 12 small groups: 1 solvent control group, 1 diethylstilbestrol control group and 10 Chinese medicine antagonistic groups. Mice in ten antagonistic groups were administered both Chinese medicine and diethylstilbestrol everyday. After administered(op) for 4 days, blood was collected and serum was separated. The effect of the pharmacological serum on proliferation rate of MCF-7 (ER+) was analyzed by MTT-assay. RESULT: In A group, proliferation rates of MCF-7 cells treated with serum from eight Chinese medicine groups including flos carthami, radix cyathulae, radix salviae miltiorrhizae, fructus lycii, herba cistanches, herba epimedii, fructus psoraleae and semen cuscutae were coued markedly increase respectively. While serum from fructus ligustri lucidi group could markedly decrease the proliferation rate of MCF-7 cells. In B group, the increased proliferation rate of MCF-7 cells caused by diethylstilbestrol was significantly reduced in seven Chinese medicine antagonistic groups including flos carthami, radix cyathulae, radix salviae miltiorrhizae, radix clycyrrhizae, herba epimedii, fructus psoraleae and semen cuscutae. While the increased proliferation rate could be markedly enhanced in herba cistanches group. CONCLUSION: Six kinds of Chinese medicine such as flos carthami, radix cyathulae, radix salviae miltiorrhizae, herba epimedii, fructus psoraleae and semen cuscutae show both estrogenic effects (when administered indepently) and antiestrogenic effects (when administered together with diethylstilbestrol). Such bidirectional effects depends on the internal estrogen level.

[Experimental study of estrogenic activities of five kinds of Chinese herbal medicines].

OBJECTIVE: To evaluate the estrogenic activity of several kinds of Chinese herbal medicines. METHOD: Use zoopery and reporter gene technique to study the estrogenic activity of five Chinese herbal medicines. Zoopery: weanling female Kunming mice weight 9-12 g were administrated botanical extracts of Selaginella tamariscina, Pinus Massoniana, Corallodiscus flabellate, Dryopteris sublaeta and Leonurus heterophyllus, the positive control group with Nilestriol tablets and control group with water, respectiely. On the eighth day, the animals were sacrificed and the uteri were separated solely and weighed. Reporter gene technique: Induce the expression of reporter gene controlled by ERE and measure the activity of luciferase on cell's clear supernatant. RESULT: The botanical extracts of S. tamariscina can increase weights of mice (P < 0.01); In the expression of reporter gene controlled by ERE, Either ERalpha or ERbeta's has estrogenic activity (P < 0.01). Follow in the zoopery we find the water part and the n-butanol part of S. tamariscina are the two active parts. CONCLUSION: S. tamariscina and it's water part and n-butanol part have estrogenic activities, effect on ERbeta is greater than ERalpha.

[Rat alveolar type II injured by bleomycin].

OBJECTIVE: To explore dysfunction mechanism of rat alveolar type II (AT-II) injured by bleomycin (BLM). METHODS: SD rats were injected with a single intratracheal dose of bleomycin or control saline. On day 7, 14, and 28 following intratracheal bleomycin or saline instillation, animals were killed under overdose of 1.5% sodium pentobarbital (0.25 ml/100 g, i.p.) and bronchoalveolar lavage fluid (BALF) from the lung was tested for the activity of pulmonary surfactant (PS) by the Whihelmy Film Balance. Several concentrations of bleomycin stimulated the culture of rat AT-II cells, and surfactant protein (SP) A, B, and aquaporin-1 (AQP) mRNA were analyzed by fluorescent quantitative polymerase chain reaction (FQ-PCR). RESULTS: The activity of PS and hypoxemia significantly decreased on day 7 and improved on day 14 and completely recovered to normal status on day 28. SP-A, B, and AQP-1 mRNA expression in BLM-stimulated group were significantly lower than those in the control group (P<0.001). CONCLUSION: BLM-injured AT-II cells decrease the levels of SP-A, B, and AQP-1 mRNA and cause PS dysfunction, resulting in hypoxemia and pneumonedema.

Effects of Radix Puerariae flavones on liver lipid metabolism in ovariectomized rats.

AIM: To study the effects of Radix Puerariae flavones (RPF) on liver lipid metabolism in ovariectomized (OVX) rats. METHODS: Forty adult female Wistar rats were randomly divided into four groups: OVX group; sham-OVX group; OVX+estrogen group and OVX+RPF group. One week after operation rats of the first two groups were treated with physiological saline, rats of OVX+estrogen group with estrogen (1 mg/kg.b.w.) and rats of OVX+RPF group with RPF (100 mg/kg.b.w.), respectively for 5 weeks. After the rats were killed, their body weight, the weight of the abdominal fat and uterus were measured, and the levels of total cholesterol (TC) and triglyceride (TG) in liver homogenate were determined. RESULTS: Compared with the sham-OVX group, the body mass of the rats in OVX group was found increased significantly; more abdominal fat in store; TC and TG in liver increased and uterine became further atrophy. As a result, the RPF was found to have an inhibitive action on those changes of various degrees. CONCLUSION: RPF has estrogen-like effect on lipid metabolism in liver and adipose tissue.

[Influence of compound biejia ruangan prescription on extracelluar matrix in bleomycin induced pulmonary fibrosis rats].

OBJECTIVE: To study the influence of compound Biejia Ruangan Prescription (CBRP) on extracelluar matrix in bleomycin induced pulmonary fibrosis rats. METHOD: 54 male Sprague-Dawley rats were randomly divided into 6 groups (9 rats in each group). Rats in the model control group, positive medicine group, and high, moderate and low CBRP groups were injected with bleomycin A5 by trachea, and rats in sham-model control group with same volume normal saline. 29 days after the injection, CBRP solution of different dosages (1.4 g x kg(-1), 0.7 g x kg(-1), 0.35 g x kg(-1)) was respectively given to rats in the high, moderate and low CBRP group by gavage, while equal volume of normal saline was given to those in the sham-model control group and model control group, and an equal volume of prednisone (0.56 mg x kg(-1)) was given to those in positive medicine control group. On the 80th day, the levels of III-collagen, IV-collagen, laminin and hyaluronic acid in the serum were determined, the determination of hydroxyproline in lung homogenates was analyzed, and the right lung was incised to make pathological sections which were stained with Hematoxylin-Eosin (HE) and Masson staining for pathological diagnosis. RESULT: CBRP could decrease the levels of III-collagen, IV-collagen, laminin and hyaluronic acid in the serum. CONCLUSION: CBRP may play its therapeutic role by leveling down the content of extracellular matrix in rats with pulmonary fibrosis induced by Bleomycin A5.

[Establishment of drug screening model based on transcriptional regulation of estrogen responsive element].

OBJECTIVE: AIM To establish a drug screening model based on transcriptional regulation of estrogen responsive element (ERE) and use it to screen compounds for discovering new ligands of estrogen receptor (ER) subtypes. METHOD: A recombinant reporter vector pERE-TAL-SEAP was constructed by inserting a synthetic sequence composed of five tandem copies of EREs upstream of promoter of the reporter vector pTAL-SEAP. The pERE-TAL-SEAP and the internal control plasmid pCMV were transiently co-transfected into Hela cells expressing ER subtype or ER subtype, and the effects of pure ER agonists 17estradiol, phytoestrogen genistein and pure ER antagonist ICI182, 780 on reporter gene SEAP expression were observed. RESULT: In the Hela cells expressing ER alpha or ER beta subtype, the expression of SEAP gene were induced in a dose dependent manner by 17-estrodiol with a maximal effect at approximately 10 nmol.L-1 and with EC50 of (80.58 +/- 8.51) pmol.L-1 and (103.90 +/- 5.29) pmol.L-1, respectively, so done by phytoestrogen genistein with a maximal effect at 1 mumol.L-1 and with EC50 of (10.86 +/- 0.75) nmol.L-1 and (39.38 +/- 2.26) nmol.L-1, respectively. The maximal level induced by estrodiol and genistein were about 7-14 fold higher than that of vehicle. The pure antiestrogen ICI182, 780 at concentration of 1 mumol.L-1 completely blocked the inductions of 17-estrodiol and genistein. CONCLUSION: The cellular drug screening model can be established by transfecting reporter vector pERE-TAL-SEAP in Hela cell lines expressing ER alpha or ER beta. The cell lines can be used to screen compounds with estrogenicity by testing SEAP activity in the culture media of cells growing in microtitier wells. The system should provide an efficient model for screening and analyzing the activity of large numbers of ligands of ER.