Efficacy of Bee Products (Anzer Honey, Pollen and Propolis) in Detection and Healing of Damage Induced by Antidiabetic Drug Vildagliptin/Metformin Hydrochloride in Healthy Human Pancreatic Cells: Cytotoxic, Genotoxic and Biochemical Studies.

BACKGROUND AND OBJECTIVE: Although drugs are powerful therapeutic agents, they have a range of side effects. These side effects are sometimes cellular and not clinically noticeable. Vildagliptin/metformin hydrochloride is one of the most widely used oral antidiabetic drugs with two active ingredients. In this study, we investigated its harmful effects on the metabolic activation system in healthy human pancreatic cells "hTERT-HPNE", and we aimed to improve these harmful effects by natural products. To benefit from the healing effect, we used the unique natural products produced by the bees of the Anzer Plateau in the Eastern Black Sea Region of Turkey. METHODS: Cytotoxic and genotoxic effects of the drug were investigated by different tests, such as MTT, flow cytometry-apoptosis and comet assays. Anzer honey, pollen and propolis were analyzed by gas chromatography/mass spectrometry (G/C-MS). A total of 19 compounds were detected, constituting 99.9% of the samples. RESULTS: The decrease in cell viability at all drug concentrations was statistically significant compared to the negative control (P<0.05). A statistically significant decrease was detected in the apoptosis caused by vildagliptin/metformin hydrochloride with the supplementation of Anzer honey, pollen and propolis in hTERT-HPNE cells (P<0.05). CONCLUSION: This study can contribute to other studies testing the healing properties of natural products against the side effects of oral antidiabetics in human cells. In particular, Anzer honey, pollen and propolis can be used as additional foods to maintain cell viability and improve heal damage and can be evaluated against side effects in other drug studies.

Associations of meditation with telomere dynamics: a case-control study in healthy adults.

Introduction: Telomeres are protective end caps of chromosomes which naturally shorten with each cell division and thus with age. Short telomeres have been associated with many age-related diseases. Meditation has come to the fore as a mind-body practice which could influence the telomere dynamics underlying these phenomena. We previously reported meditation to be associated with higher telomerase levels, mindfulness and quality of life. Here, reporting on the same study population, we describe associations between long-term meditation and telomere length (TL), expression of hTERT and hTR genes and methylation of the promoter region of hTERT gene. Methods: Thirty healthy meditators and matched non-meditators were recruited. TL was measured using quantitative PCR, gene expression was assessed using reverse transcriptase PCR, and methylation level was quantified by bisulfite-specific PCR followed by Sanger sequencing. Comparisons between meditators and controls were carried out using t-tests, while Pearson correlation was used to identify correlations, and regression was used to identify predictors. Results: Males comprised 63.4% of each group with an average age of 43 years. On average, they had meditated daily for 5.82 h (±3.45) for 6.8 years (±3.27). Meditators had longer relative TLs (p = 0.020), and TL decreased with age (p < 0.001) but was not associated with other socio-demographic variables. Regression analysis showed that age (p < 0.001) and duration of meditation (p = 0.003) significantly predicted TL. The meditators showed higher relative expression of hTERT (p = 0.020) and hTR (p = 0.029) genes while the methylation level of the promoter region of hTERT gene was significantly lower when compared to non-meditators (p < 0.001). Negative correlations were identified between the methylation level of the promoter region of hTERT gene and the expression of the hTERT gene (p = 0.001) and duration of meditation (p = 0.001). Conclusion: The findings suggest that meditation as a lifestyle practice has multi-level beneficial effects on telomere dynamics with potential to promote healthy aging.

Combinatorial Effect of Arsenic and Herbal Compounds in Telomerase-Mediated Apoptosis Induction in Liver Cancer.

Tumour illness and its resistance against existing anticancer therapies pose a serious health concern globally despite the progressive advancement of therapeutic options. The prevailing treatment of HCC using numerous antitumor agents has inflated long-lived complete remissions, but a percentage of individuals still die due to disease recurrence, indicating a need for further exploration of possible anti-tumour regimes. We aim to boost the effectiveness of the HCC treatment by conducting current investigations evaluating the effect of arsenic trioxide (ATO) with different herbal compounds like quercetin and aloe-emodin against liver tumour via inhibition of telomerase, a pro-cancer enzyme. The anticancer activity of ATO with herbal compounds was investigated in human control liver cell line (Wrl-68) and cancer liver cell line (HepG2) at different time intervals. Viability and cytotoxicity in response to combinatorial drugs were assessed in vitro by trypan blue dye exclusion assay and MTT and WST assay. Apoptosis was analysed by annexin V/PI assay, and the expression of telomerase and apoptosis-regulating proteins was evaluated by immunoblotting and qRT-PCR. Arsenic trioxide in combination with quercetin and aloe-emodin reduced cell viability in cancerous cells compared to normal cells by inducing apoptosis, downregulating telomerase and Bcl-2 (anti-apoptotic protein) and upregulating the expression of Bax (pro-apoptotic protein). ATO exhibited significant anticancer effects due to the synergistic effects of quercetin and aloe-emodin in liver tumour cells. The current study data collectively suggest that a successful inhibition of cancer growth by the combination of ATO and tested herbal medicines against liver tumour growth is via the inhibition of telomerase activity.

Preconception nutraceutical food supplementation can prevent oxidative and epigenetic DNA alterations induced by ovarian stimulation for IVF and increases pregnancy rates.

BACKGROUND: It is hypothesized that oxidative and epigenetic alterations to DNA induced by ovarian stimulation for in vitro fertilization (IVF) may be associated with an increased risk of diseases and cancer in the offspring and could possibly be attenuated by preconception food supplementation. METHODS: In a prospective randomised open-label trial, 62 patients were randomly assigned to either 30 days of preconception treatment with the nutraceutical Fertility woman ® duo (Nutriphyt, Beernem, Belgium) (group 1), this nutraceutical complemented with selenomethionine (group 2), or folic acid only (group 3). Biochemical and epigenetic effects and pregnancy rates were assessed. RESULTS: In all 3 groups the level of DNA oxidative damage, estimated by the concentration of 8-hydroxy- 2-deoxyguanosine over creatinine in early morning urine, and the concentration of homocysteine in the blood decreased after treatment. In group 2, the degree of methylation of the cancer-associated CpG2 dinucleotide of the human Telomerase Reverse Transcriptase (hTERT) promoter region, assessed by pyrosequence in follicular cells obtained at oocyte pick-up, was 18% lower than that of group 3. The pregnancy rate, including the transfer of fresh and frozen embryos, was significantly higher in group 2 (50%) than in group 3 (6%) with the result in group 1 being intermediate (30%). CONCLUSION: The results suggest that preconception food supplementation using a specific nutraceutical significantly reduces oxidative and epigenetic DNA changes to follicular cells of women treated by IVF, and may optimize gene expression in the oocytes, thus increasing the pregnancy rate per cycle of ovarian stimulation.

The Role of Alternative RNA Splicing in the Regulation of hTERT, Telomerase, and Telomeres: Implications for Cancer Therapeutics.

Alternative RNA splicing impacts the majority (>90%) of eukaryotic multi-exon genes, expanding the coding capacity and regulating the abundance of gene isoforms. Telomerase (hTERT) is a key example of a gene that is alternatively spliced during human fetal development and becomes dysregulated in nearly all cancers. Approximately 90% of human tumors use telomerase to synthesize de novo telomere repeats and obtain telomere-dependent cellular immortality. Paradigm shifting data indicates that hTERT alternative splicing, in addition to transcription, plays an important role in the regulation of active telomerase in cells. Our group and others are pursuing the basic science studies to progress this emerging area of telomerase biology. Recent evidence demonstrates that switching splicing of hTERT from the telomerase activity producing full-length hTERT isoform to alternatively spliced, non-coding isoforms may be a novel telomerase inhibition strategy to prevent cancer growth and survival. Thus, the goals of this review are to detail the general roles of telomerase in cancer development, explore the emerging regulatory mechanisms of alternative RNA splicing of the hTERT gene in various somatic and cancer cell types, define the known and potential roles of hTERT splice isoforms in cancer cell biology, and provide insight into new treatment strategies targeting hTERT in telomerase-positive cancers.

Complex context relationships between DNA methylation and accessibility, histone marks, and hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all-trans retinoic acid in cancer therapy.

Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of hTERT gene promoter is important, we still lack a clear understanding of the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epigenetic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investigate the integrated epigenetic events that promote hTERT promoter transition from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core promoter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activating hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between hTERT promoter methylation, chromatin accessibility, and histone modifications that force the revisiting of previously proposed concepts regarding hTERT epigenetic regulation. They represent, therefore, a major advance in predicting sensitivity to retinoid-induced hTERT repression and, more generally, in the potential development of therapies targeting hTERT expression in cancers.

Calebin-A induced death of malignant peripheral nerve sheath tumor cells by activation of histone acetyltransferase.

BACKGROUND: Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. The malignant peripheral nerve sheath tumor (MPNST), transformed from NF1 related plexiform neurofibroma, is a rapidly growing and highly invasive tumor. No effective chemotherapeutic agent is currently available. Calebin-A is a derivative from turmeric Curcuma longa. Given the anti-inflammatory and anticancer potentials of curcumin, whether Calebin-A also had the tumoricidal effect upon MPNST cells is still elusive. PURPOSE: To determine whether Calebin-A has the potential for anti-MPNST effect. METHODS: The MTT and FACS analysis of normal Schwann (HSC) and MPNST cells have been employed to determine the tumoricidal effect of Calebin-A. The expression of the signal pathway molecules was assessed by Western blotting. The CHIP with quantitative PCR assay was performed to quantify the promoter DNA binding to acetylated histone 3 (acetyl H3). The enzyme activities of histone acetyltransferase (HAT) and deacetylase (HDAC) have been evaluated by commercial kits. The measurements of tumor size of the xenograft mouse model were also performed. RESULTS: Calebin-A inhibited the proliferation of MPNST and primary neurofibroma cells in a dose-dependent manner. The flow cytometry analysis of the MPNST cells after treatment of 25 µm of Calebin-A demonstrated an increase of population in the G0/G1 phase but decrease in G2/M phase. Before treatment, the expression of Axl, Tyro3, and acetyl H3 was significantly higher in MPNST cells when compared to HSC. The expression of phosphorylated-AKT, -ERK1/2, survivin, hTERT, and acetyl H3 proteins were reduced after treatment. The CHIP assay shows the promoter DNA copies of survivin (BRIC5) and hTERT genes are significantly reduced post-treatment. The enzyme activity of HAT was significantly reduced, but not that of HDAC. Two HAT inhibitors, epigallocatechin-3-gallate (EGCG) and anacardic acid (AA) have also demonstrated a significant inhibitory effect on MPNST cells. Finally, the measurements of tumor size showed a significant reduction of the xenograft tumors after treatment of Calebin-A. CONCLUSION: Both in vitro and in vivo studies showed Calebin-A could inhibit the proliferation of MPNST with suppression of survivin and hTERT. The reduced expression of these two factors might be through the epigenetic histone modification resulting from the decreased activity of HAT.

Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review.

Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

The herbal medicine Melissa officinalis extract effects on gene expression of p53, Bcl-2, Her2, VEGF-A and hTERT in human lung, breast and prostate cancer cell lines.

INTRODUCTION: Earlier, we verified that Melissa officinalis extract (MOE) elicits potent antiproliferative effects on different human cancer cells. To gain insights into the molecular mechanisms accounting for the cytotoxic effects of MOE, we assessed the expression patterns of several prominent molecules with therapeutic potential in cancer by Quantitative PCR (Q-PCR). METHODS: A549, MCF-7 and PC3 cancer cells were grown in complete RPMI 1640 and seeded in 24 well micro plates. After incubation for 72h, 100µg/ml of MOE was added and the cells were further incubated for 72h. Afterwards, the cells were subjected to RNA extraction for the means of Q-PCR. RESULTS: Our results indicated that in PC3 cancer cells, MOE resulted in a significant downregulation of VEGF-A (0.0004 fold), Bcl-2 (0.001 fold), Her2 (0.02 fold), and hTERT (0.023 fold) compared to the untreated control. In addition, VEGF-A and hTERT mRNA were significantly downregulated in MCF-7 and A549 cancer cells, as well. Notably, high anti-angiogenic activity was closely associated with a high anti-telomerase activity of MOE in studying cancer cells. The decrease in VEGF-A expression was significantly superior than that of hTERT downregulation, as PC3 cancer cells with the highest hTERT down regulation (0.023) presented the highest anti VEGF activity (0.0004 fold), whereas MCF-7 cells with the lowest hTERT inhibition (0.213) showed the lowest VEGF inhibition(0.0435) among the three studied cancer cells. We noticed that the modulation of VEGF-A and hTERT gene expression can be considered as a common target, accounting for the therapeutic potential of MOE on human breast, lung and prostate cancer cells. CONCLUSION: Altogether, it is suggested that the potent antiproliferative activity of the hydroalcoholic extract of Melissa officinalis is somehow explainable by its high potency to inhibit expression of the prominent oncogenes Bcl2, Her2, VEGF-A and hTERT in prostate cancer. In tumors with functional p53, including MCF-7 and A549 cancer cells, the role of p53, Bcl2 and Her2 is less significant. It appears that MOE exerts its antiproliferative effects in these cancer cells partly via concurrent downregulation of VEGF-A and hTERT. Additional studies are needed to clarify the role of other active molecules in cancer cells harboring functional p53.

Chinese herbal extracts inhibit the proliferation and induce the apoptosis of human lung cancer A549 cells / 医学研究生学报

Objective Cancer, a disease induced by abnormally regulated cell growth and apoptosis, is imposing a global threat to human health.This study was to explore the effects of Chinese herbal extracts ( CHE) in inducing the apoptosis and inhibiting the proliferation of human lung cancer cells. Methods Human lung cancer A549 cells were divided into a negative control, a high-dose CHE (680 ng/mL), a medium-dose CHE (340 ng/mL), and a low-dose CHE (170 ng/mL) group.The inhibitory effect of CHE on the proliferation of the lung cancer cells was detected by CCK8 and LDH assays, the apoptosis of the cells was assessed by fluorescence microscopy and flow cytometry, and the expressions of hTERT mRNA, cleaved caspase-3 and cleaved PARP were deter-mined by RT-PCR and Western blot. Results CHE inhibited the proliferation of the A549 cells with an IC50 value of 510 ng/mL. Treatment with high-dose CHE for 48 hours significantly suppressed the proliferation of the cells, induced the release of LDH, and promo-ted the apoptosis of the cells by 72.3%.RT-PCR and Western blot showed that 24-hour treatment with medium-dose CHE reduced the expression of hTERT mRNA by 4 times that of the negative control and up-regulated the expressions of cleaved caspase-3 and cleaved PARP. Conclusion Chinese herbal extracts can induce cell apoptosis by decreasing the expression of hTERT mRNA and increasing those of the cleaved caspase 3 and cleaved PARP proteins.

Decreased expression of PinX1 protein predicts poor prognosis of colorectal cancer patients receiving 5-FU adjuvant chemotherapy.

Previous studies suggest that Pin2/TRF1 interacting protein X1 (PinX1) is an intrinsic telomerase inhibitor and a putative tumor suppressor gene in human cancers. The aims of this study were to investigate PinX1 expression status in colorectal cancer (CRC) specimens and to clarify its clinical significance. A total of 83 CRC patients treated with radical resection and 5-fluorouracil (5-FU) based adjuvant chemotherapy were enrolled in this study. Immunohistochemistry was used to detect PinX1 and human telomerase reverse transcriptase (hTERT) protein expression in paired tumor and adjacent normal tissues. Results showed that PinX1 expression was significantly reduced in tumor tissues as compared to normal tissues, the rate of PinX1 protein low/negative expression in CRC and normal tissues was 43.4% (36/83) and 9.6% (8/83), respectively (P<0.001), while hTERT protein expression was upregulated in CRC and negative correlated with PinX1 expression. Although no correlations with clinicopathological features, PinX1 downregulation was significantly associated with adverse 5-year overall survival (OS) and disease-free survival (DFS). Cox proportional hazards model further revealed that PinX1 expression was an independent factor in predicting OS and DFS for CRC patients, apart from lymph metastasis. In conclusion, PinX1 protein expression is decreased in CRC, which may be a new promising tumor marker for CRC prognosis and 5-FU chemosensitivity.

Role of diallyl disulfide-mediated cleavage of c-Myc and Sp-1 in the regulation of telomerase activity in human lymphoma cell line U937.

OBJECTIVE: Garlic (Allium sativum) has been considered a wonder herb for years with a reputation of disease prevention. Telomerase, a ribonucleoprotein enzyme responsible for telomere integrity, is strongly up-regulated in different types of cancers. The aim of this study was to reveal the role of diallyl disulfide (DADS), an organosulfur component of garlic, on telomerase activity in human lymphoma with an emphasis on key transcription factors c-Myc and Sp-1. METHODS: Human lymphoma cell line U937 was used as model cell line. Telomerase activity was measured by telomerase repeat amplification protocol assay, levels of related proteins and mRNAs were measured by Western blot and reverse transcriptase polymerase chain reaction, respectively. Moreover, in vitro binding assay was performed using radiolabeled double-stranded DNA having specific sequences to detect involvement of transcription factors in DADS-dependent modulation of telomerase activity. RESULTS: The present study demonstrated DADS-mediated decrease in telomerase activity in U937 cells with concomitant transcriptional down-regulation of human telomerase reverse transcriptase (hTERT) that is caused by reduced binding of c-Myc and Sp-1 to their respective binding sites on hTERT promoter. Lowering of DNA-binding activity of c-Myc and Sp-1 due to DADS treatment is caused by the deactivation of these transcription factors due to cleavage. Additionally, Mad1-the repressor protein of hTERT expression-is also overexpressed in DADS-treated U937 cells. CONCLUSIONS: These findings strongly suggest that DADS down-regulate telomerase activity through c-Myc-, Sp-1-, and Mad1-dependent transcriptional down-regulation of hTERT.

The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC.

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti-cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4-methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose-dependent switch to anti-survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC-mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC-induced DNA damage but impacts signalling processes upstream of apoptosis execution level.

Effects of Zhiling capsule on apoptosis and expressions of hTERT,CD44 in small cell lung cancer cell line NCI-H446 / 中国药理学通报

Aim To explore the mechanisms of the apoptosis induction and the effects of adhesion suppression of Zhiling capsule (ZLJN) in small cell lung cancer cell line NCI-H446.Methods According to the different components of ZLJN,NCI-H446 cells were treated with traditional Chinese medicine,western medicine and ZLJN composite groups.Apoptotic cells were tested by light microscopy,Hochest33258 staining method.The mRNA and protein expressions of bcl-2,bax and hTERT were analyzed by RT-PCR and Western blot respectively.The expressions of CD44 were detected by flow cytometry.Results After NCI-H446 cells were treated with different drug groups,The morphological changes of apoptotic cells were found by light microscopy and Hochest33258 staining method.The mRNA and protein expressions of bcl-2 were down-regulated while the expressions of bax were up-regulated compared to the control groups(P

Protein kinase C modulates telomerase activity in human cervical cancer cells

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Go6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Go6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Go6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of beta-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.