[Effect of Angelica sinensis polysaccharide on expression of telomere, telomerase and P53 in mice aging hematopoietic stem cells].

OBJECTIVE: To observe the effect of Angelica sinensis polysaccharides (ASP) on the length of telomere, the activity of telomerase and the expression of P53 protein in mice hematopoietic stem cells (HSCs), and explore ASP's potential mechanism for regulating HSC aging. METHOD: C57BL/6J mice were randomly divided into the normal group, the aging group and the intervention group. The aging group was radiated with X ray to establish the mice aging HSC model. The intervention group was orally administered with ASP during X-ray irradiation, while the normal group was orally administered with NS. Their HSCs were isolated by immunomagnetic beads. Cell cycles analysis and senescence-associated beta-galactosidase (SA-beta-Gal) staining were used to detect changes in aging HSCs. The expression of P53 was determined by western blot analysis. The length of telomere and the vitality of telomerase were analyzed by southern blot and TRAP-PCR, respectively. RESULT: Compared with the normal group, X-ray irradiation could significantly increase the cell ratio of in HSC G1 stage, rate of SA-beta-Gal positive cells and expression of P53 protein, and reduce the length of telomere and the vitality of telomerase. Compared with the aging group, ASP could significantly inhibit the cell ratio of in HSC G1 stage and the increase in the number of SA-beta-Gal positive cells, down-regulate the expression of P53 protein, and increase the length of telomere and the vitality of telomerase in HSCs. CONCLUSION: ASP could antagonize X-ray-induced aging of HSCs, which may be related to the increase in the length of telomere and the activity of telomerase, as well as the down-regulation of the expression of P53 protein.

Retinoids induce differentiation and downregulate telomerase activity and N-Myc to increase sensitivity to flavonoids for apoptosis in human malignant neuroblastoma SH-SY5Y cells.

Human malignant neuroblastoma is characterized by poor differentiation and uncontrolled proliferation of immature neuroblasts. Retinoids such as all-trans-retinoic acid (ATRA), 13-cis-retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses are capable of inducing differentiation, while flavonoids such as (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST) at relatively high dose can induce apoptosis. We used combination of retinoid and flavonoid for controlling growth of malignant neuroblastoma SH-SY5Y cells. Cells were treated with a retinoid (1 microM ATRA, 1 microM 13-CRA, or 0.5 microM 4-HPR) for 7 days and then with a flavonoid (25 microM EGCG or 25 microM GST) for 24 h. Treatment of cells with a low dose of a retinoid for 7 days induced neuronal differentiation with downregulation of telomerase activity and N-Myc but overexpression of neurofilament protein (NFP) and subsequent treatment with a relatively high dose of a flavonoid for 24 h increased apoptosis in the differentiated cells. Besides, retinoids reduced the levels of inflammatory and angiogenic factors. Apoptosis was associated with increases in intracellular free [Ca2+], Bax expression, cytochrome c release from mitochondria and activities of calpain and caspases. Decreases in expression of calpastatin (endogenous calpain inhibitor) and baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins (endogenous caspase inhibitors) favored apoptosis. Treatment of SH-SY5Y cells with EGCG activated caspase-8, indicating induction of the receptor-mediated pathway of apoptosis. Based on our observation, we conclude that combination of a retinoid and a flavonoid worked synergistically for controlling the malignant growth of human neuroblastoma cells.

Effects of Shugansanjie Tang on matrix metalloproteinases 1, 3 and 9 and telomerase reverse transcriptase expression in human breast cells in vitro.

BACKGROUND: The traditional Chinese medication Shugansanjie Tang (SGT), whose active component is Akebia Trifoliate Koidz, possesses potential anti-tumor and immunostimulatory effects especially for breast cancer. The invasive processes of such cancers have been attributed to matrix metalloproteinases (MMPs) which may be the key factor mediating this process. Telomerase reverse transcriptase (TERT) catalyze the lengthening of telomeres, which prolongs cell life and interrupts natural cell death. The aim of this study is to determine the effects of Shugansanjie Tang on MMP levels and TERT activity using breast cancer cell lines. MATERIALS AND METHODS: We used the breast cancer cell lines MDA-MB-231 and BT-483. Cell inhibition rate was measured by WST-1 reagent, cell apoptosis by Apoptotic DNA Ladder Kit, mRNA expression of MMP-1, MMP-3 and MMP-9 by PCR and TERT by immunohistochemistry stain. RESULTS: Comparing to the control group, the test group showed lower cell growth rate, decreased mRNA expression of MMP-1, MMP-3 and MMP-9 production and less intense staining of MMPs with diaminobenzidine tetrahydrochloride. CONCLUSION: This study demonstrated that Shugansanjie Tang inhibits the growth of breast cancer cells by apoptosis and lowers the level of certain matrix metalloproteinases and activity of telomerase reverse transcriptase in breast cancer in vitro.

[Effects of selenium on expression of TERT, c-Myc and p53 induced by cadmium in rat liver].

OBJECTIVE: To study the effects of sodium selenite on expression of telomerase reverse transcriptase mRNA, c-Myc and p53 induced by cadmium chloride in rat liver. METHODS: Male SD rats were divided randomly into 6 groups, each group had 5 animals. The groups comprised the control group, Se group (5 micromol/kg sodium selenite), 5 micromol/kg cadmium chloride group, 10 micromol/kg cadmium chloride group, Se (5 micromol/kg sodium selenite) + 5 micromol/kg cadmium chloride group, Se (5 micromol/kg sodium selenite) + 10 micromol/kg cadmium chloride group. After 48 hours of the first injection, the expression of TERT mRNA was measured with RT-PCR and c-Myc, and p53 proteins were measured by immunohistochemistry method. RESULTS: Compared with control group, the expression of TERT was increased in 5 micromol/kg Cd group and 10 micromol/kg Cd group, c-Myc protein was increased in 10 micromol/kg Cd group, and the expression of p53 protein was increased in 5 micromol/kg group and 10 micromol/kg Cd group. TERT expression in Se + 10 micromol/kg Cd group was lower than that of 10 micromol/kg Cd group significantly. c-Myc protein was decreased in Se + 10 micromol/kg Cd group compared with 10 micromol/kg Cd group. p53 protein of Se + 5 micromol/kg Cd group and Se + 10 micromol/kg Cd group were decreased significantly compared with 5 micromol/kg Cd group and 10 micromol/kg Cd group respectively. CONCLUSION: The cadmium at the doses of between 5 and 10 micromol/kg can activate TERT and up-regulate c-Myc and p53 proteins. The selenium at the dose of 5 micromol/kg has the antagonistic effect on expression of TERT, c-Myc and p53 induced by cadmium in rat liver.

Inhibitory effect of the tea polyphenol, (-)-epigallocatechin gallate, on growth of cervical adenocarcinoma cell lines.

To investigate the effect of (-)-epigallocatechin gallate (EGCG) on cervical adenocarcinoma, we performed a cell proliferation assay. TRAP assay is used for telomerase activity, flow cytometry analysis and pKi-67 immunofluoroscein staining in cervical adenocarcinoma cell lines (OMC-4, TMCC-1). Our results showed that EGCG inhibited the proliferation assay, TRAP assay for telomerase activity of both cell lines. Although cell apoptosis was induced, we observed that the expression of pKi-67 was suppressed. Our data suggest that EGCG may be effective for the treatment of cervical adenocarcinoma. The mechanisms of the anti-tumor effects were revealed to be the inhibition of telomerase activity, the induction of apoptosis and cell cycle dysregulation.

The tea polyphenols EGCG and EGC repress mRNA expression of human telomerase reverse transcriptase (hTERT) in carcinoma cells.

Tea polyphenols have inhibitive effects for carcinogenesis. A reporter system controlled by hTERT promoter was constructed to evaluate the effects of tea polyphenols, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocatechin (EGC) on the repression of hTERT transcription. The hTERT promoter activity was selectively repressed by 20-40 microM EGCG and EGC in a dose- and time-dependent manner. Real-time RT-PCR confirmed that the endogenous hTERT mRNA level was decreased in H1299, OECM-1 and SAS cells treated with EGCG or EGC. Our results identified the repression activities of EGCG and EGC toward telomerase expression that might be linked to inhibition of carcinoma cell growth. This cell-based reporter system is useful for screening drugs targeting hTERT repression.

[Change of Bcl-2 expression and telomerase during apoptosis induced by oridonin on human hepatocelluar carcinoma cells].

OBJECTIVE: To investigate the change of Bcl-2 expression and telomerase during the apoptosis induced by oridonin on human hepatocelluar carcinoma BEL7402 cells. METHOD: BEL-7402 cells in culture medium were given 8,16,24,32 micromol x L(-1) different concentrations of oridonin. The cell apoptotic rate was detected by flow cytometry (FCM), morphology of cell apoptosis was observed by Hoechst 3325 staining. Bcl-2 and Bax expressions were detected by Western blotting. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR enzyme-linked immunosorbent assay (ELISA) were used to detect hTERT mRNA expression and telomerase activity. RESULT: Oridonin induced BEL-7402 cells apoptosis significantly, and the apoptosis rate was both in time-and dose-dependent manner. Marked morphological changes of cell apoptosis were observed very clearly by Hoechst 33258 staining after the cells exposed to oridonin for 60 hours; Western blotting showed that Bcl-2 expression was down-regulated and Bax expression up-regulated concurrently along with the apoptotic process, and the expression of hTERT mRNA as well as activity of telomerase were decreased concurrently. CONCLUSION: Oridonin could decrease the expression of hTERT mRNA and telomerase activity as well as down-regulation of Bcl-2 and up-regulation of Bax expression during the apoptosis of BEL-7402 cells.

Induction of apoptosis and inhibition of telomerase activity by aqueous extract from Platycodon grandiflorum in human lung carcinoma cells.

The objective of the present study was to investigate the effect of aqueous extract from the root of Platycodon grandiflorum (AEPG) on the cell growth and apoptosis in human lung carcinoma cell line A549. Exposure of A549 cells to AEPG resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy and flow cytometry analysis. This increase in apoptosis was associated with a decrease in Bcl-2 expression, an increase of Bax and an activation of caspase-3. AEPG treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by AEPG treatment. These findings suggest that the apoptotic events by AEPG were associated with the diminished telomerase activity and down-regulation of Bcl-2 expression.

[Effect of telomerase on ginsenoside Rh2-induced differentiation of hepatocarcinoma cell line SMMC-7721].

BACKGROUND & OBJECTIVE: Recently we found that 20 microg/ml of ginsenoside Rh2 (G-Rh(2)) can inhibit growth, and induce differentiation of hepatocarcinoma cell line SMMC-7721. Re-activation of telomerase may play an important role in carcinogenesis, and cellular immortalization. This study was to explore the mechanism of G-Rh(2)-induced differentiation of SMMC-7721 cells by detecting telomerase activity. METHODS: After treated with 20 mug/ml of G-Rh(2), telomerase activity in SMMC-7721 cells was detected by polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) coupled with enzyme-linked immune sorbent assay(ELISA); mRNA levels of human telomerase reverse transcriptase (hTERT), and p21 were measured by reverse transcriptase-polymerase chain reaction (RT-PCR); changes of cell cycle, and expression of Cyclin D1, Cyclin E,P16 protein were detected by flow cytometry (FCM). RESULTS: G-Rh(2) inhibited telomerase activity of SMMC-7721 in a time- dependent manner: the activity decreased from 1.105 to 0.765 (P< 0.01) after 1-day treatment, while from 1.152 to 0.326 (P< 0.01) after treated for 5-day treatment. The mRNA expression of hTERT was significantly reduced by 20 microg/ml of G-Rh(2). Cell cycle assay showed that 20 microg/ml of G-Rh(2) increased the proportion of SMMC-7721 cells in G1 phase, and decreased those in S, and G2/M phases, the increase of G1 phase after 1-day treatment was the most apparent (from 60.85% to 78.53%,P< 0.05). Furthermore,G-Rh(2) weakened the expressions of positive-regulating factors(Cyclin D1,Cyclin E),and increased the expressions of negative-regulating factors (P16 protein, p21 gene) in SMMC- 7721 cells. CONCLUSIONS: G-Rh(2) may effectively reduce telomerase activity through affecting transcription level of hTERT,and arresting cell cycle progression. The down- regulation of telomerase activity in SMMC-7721 cells may be closely related to G-Rh(2)-induced differentiation.

[Experimental study on effect of epimedium flavonoids in protecting telomere length of senescence cells HU].

OBJECTIVE: To investigate the mechanism of senescence delay of human diploid fibroblast (2BS) and protecting telomere length by epimedium flavonoids (EF). METHODS: The drug sera of EF were used to treat the 2BS. The population doublings of 2BS cells were observed, the mRNA expression of p16 gene were determined by fluorescence real-time quantitative RT-PCR, the telomerase activation of 2BS cells were determined by TRAP-Hyb, the total retinoblastoma (Rb) and phosphorated Rb protein content were detected by ELISA, the telomere length of 2BS cells were determined by telomere restriction fragment (TRF) Southern blot assay. RESULTS: EF could significantly extend the population doublings of 2BS cells, the expression of p16 mRNA was decreased and the content of phosphorated Rb protein were increased by EF. The telomere lengthening of 2BS cells were improved by EF, but the telomerase was not activated. CONCLUSION: In senescence human fibroblasts 2BS cells, p16 gene mRNA expression increased, content of phosphorated Rb protein decreased and the telomere length of 2BS shortened, EF might delay the aging of cells through inhibiting the p16 gene expression, promoting the production of phosphorated Rb protein and to protect the length of telomere, but not activating the telomerase.

[Inhibitory effect of yiqijiedu powder to telomerase and telomerase RNA in nasopharyngeal tumorigenesis of rats].

OBJECTIVE: Telomerase activation is a common event in malignant transformation, and is associated with tumorigenesis. This has been clarified in nasopharyngeal carcinoma. YIQIJIEDU powder (Chinese traditional medicine) is used to treated the patients with nasopharyngeal carcinoma (NPC) after radio-therapy and patients with precancerous lesion of NPC. We investigated whether YIQIJIEDU powder inhibits the telomerase activity in nasopharyngeal carcinogenesis. METHODS: Nasopharyngeal carcinomas in rats were induced by N, N' Dinitrosopiperazine (DNP), and some of them were treated with YIQIJIEDU powder. Telomerase and telomerase RNA in the rats' nasopharyngeal tissue were assayed by using PCR-ELISSA and Nested-PCR, and were pathologically diagnosed. RESULTS: YIQIJIEDU powder could inhibit telomerase activity and nasopharyngeal carcinogenesis in the nasopharyngeal tissue of the rats treated, but no telomerase RNA. CONCLUSION: The findings suggest that the anticancer effect of YIQIJIEDU powder links with their inhibitory effects on telomerase.

Longevity of lobsters is linked to ubiquitous telomerase expression.

Mammals have high growth rates in embryonic and juvenile phases and no growth in adult and senescent phases. We analyzed telomerase activity in a fundamentally different animal which grows indeterminately. Lobsters (Homarus americanus) grow throughout their life and the occurrence of senescence is slow. A modified TRAP assay was developed and the lobster telomeric repeat sequence TTAGG was determined. We detected telomerase activities which were dependent on RNA and protein components, required dGTP, dATP and dTTP, but not dCTP. Telomerase products with a five nucleotide periodicity were generated. High telomerase activities were detected in all lobster organs. We conclude that telomerase activation is a conserved mechanism for maintaining long-term cell proliferation capacity and preventing senescence, not only in cellular models or embryonic life stages but also in adult multicellular organisms.