Diosgenin Inhibits hTERT Gene Expression in the A549 Lung Cancer Cell Line

Human telomerase is a ribonucleoprotein that adds TTAGGG repeats to telomere ends of linear chromosomes in eukaryotes (Sato et al., 2012). Telomerase activity is not evident in normal cells, with elimination of germ cells and regeneration tissues (Buseman et al., 2012). Though, it is reactive currently in almost 80-90% of human cancer. Telomerase includes three main constituents including hTR (human telomerase RNA component), tp1 (telomerase associate protein) and hTERT (human telomerase reverse transcriptase) (Noel and Wellinger, 2012). Between these three constituents, hTERT plays a vital role in telomerase activation. Telomerase inhibitors can be divided into two groups (A sprouse et al., 2012). One group binds to telomerase and blocks its activity, although others are suppressors of gene expression of hTERT (Hsin et al., 2010). Complementary and alternative treatment use is common among cancer patients. In many studies, herbal medicines are among the most normally used group of treatments. Herbal medicines are believed by the general public to be safe, cause less side effects and less likely to cause dependency (Olaku and White, 2011). Diosgenin is a main bioactive constituent of fenugreek (Trigonellafoenum-graecum Linn) that is structurally similar to cholesterol and other steroids (Nagore et al.,


Introduction
Human telomerase is a ribonucleoprotein that adds TTAGGG repeats to telomere ends of linear chromosomes in eukaryotes (Sato et al., 2012). Telomerase activity is not evident in normal cells, with elimination of germ cells and regeneration tissues (Buseman et al., 2012). Though, it is reactive currently in almost 80-90% of human cancer. Telomerase includes three main constituents including hTR (human telomerase RNA component), tp1 (telomerase associate protein) and hTERT (human telomerase reverse transcriptase) (Noel and Wellinger, 2012). Between these three constituents, hTERT plays a vital role in telomerase activation. Telomerase inhibitors can be divided into two groups (A sprouse et al., 2012). One group binds to telomerase and blocks its activity, although others are suppressors of gene expression of hTERT (Hsin et al., 2010). Complementary and alternative treatment use is common among cancer patients. In many studies, herbal medicines are among the most normally used group of treatments. Herbal medicines are believed by the general public to be safe, cause less side effects and less likely to cause dependency (Olaku and White, 2011). Diosgenin is a main bioactive constituent of fenugreek (Trigonellafoenum-graecum Linn) that is structurally similar to cholesterol and other steroids (Nagore et al., 2012). Basically, diosgenin [(25R)-spirost-5-en-3b-ol] is a spirostanolsaponin containing of a hydrophilic sugar moiety connected to a hydrophobic steroid aglycone ( Figure 1) (Miyoshi et al., 2011). Study effect of diosgenin on human hepatocellular carcinoma cells was showed that diosgenin can induce the expression of Src homology 2 phosphatase 2 (SH-PTP2) that correlated with down-regulation of constitutive STAT3 activation. Diosgenin also down-regulatedthe expression of various STAT3-regulated gene products, inhibited proliferation and potentiated the apoptotic effects of paclitaxel and doxorubicin (Li et al., 2010). Diosgenin induced apoptosis in HT-29 cells at least in part by inhibition of Bcl-2 and by induction of caspase-3 protein expression (Raju et al., 2004). diosgenin, a steroidal saponin, inhibits migration and invasion of human prostate cancer PC-3 cells by reducing matrix metalloproteinases expression (Chen et al., 2011). Lung cancer is the most widespread type of cancer and is also one of the major causes of death in the world (Athey et al., 2012). Lung cancer between many types of cancer such as breast cancer, lung cancer, ovarian cancer, colon cancer and stomach cancer, is the leading cause of cancer deaths in the world. The two major forms of lung cancer are non-small-cell lung cancer (about 85% of all lung cancers) and small-cell lung cancer (about 15%). Despite advances in early detection and standard treatment, non-small-cell lung cancer (NSCLC) is often diagnosed at an advanced stage and has a poor prognosis. This type of lung cancer can be divided into three major histologic subtypes: squamous-cell carcinoma, adenocarcinoma, and large-cell lung cancer (Herbst et al., 2008). There are many molecular targets for prevention or treatment of none-small cell lung cancer (Hamilton et al., 2005). From these molecular targets, telomerase is the best because it is active in almost 85% of cancers including non-small cell lung cancer. Therefore, it is an appropriate molecular target. So the goal of current study is to define the effect of diosgenin on gene expression regulation of hTERT gene in A549 lung cancer cell line.

Materials and Methods
Pure diosgenin obtained from Sigma co, A549 lung cancer cell lines prepared from Cell Bank of Pasteur Institute of Iran (Code: C137) were cultured in RPMI-1640 medium (Invitrogen, UK) include 10% of Heat-inactivated Fetal Bovine Serum (FBS) (Gibco, Invitrogen, Uk) with Penicillin G (Serva Co, Germany) and Streptomycin (Merck Co, Germany).

MTT-assay
Cytotoxicity of diosgenin on A549 lung cancer cell line evaluates by MTT assay which is based on the reduction of MTT (catalogue number M2128, sigma Co, Germany) to MTT-formazan by mitochondorial enzymes. Cells were harvested and resuspended in growth media and counted by Trypan blue 0.4% solution, to make a stock cell suspension containing 5,000 cells/ml. 200μl of this stock cell suspension was added to the each wells of a 96-well plate without blank wells. After 24h incubation, cells were treated with diosgenin in ethanol (0, 10, 20, 30, 40, 50, 60μM, three wells per concentration) for 24, 48 and 72h, after exchanging medium, 50μl of MTT solution (2 mg/mL) was added to each well. After 4 h incubation in dark and 37˚C, medium with MTT empty and 200μl DMSO and 25μl of Sorensen buffer were added to each wells, and the absorbance was measured in an ELISA reader (Micro plate reader STAT FAX 2100) at 570nm wave length, with 630nm wave lengths as a reference value. The cell viability ratio was calculated by the subsequent formula: Inhibitory ratio (%)=(OD control−OD treated)/OD control×100%. Cytotoxicity was expressed as the concentration of diosgenin inhibiting cell growth by 50% (IC 50 value).

Cell treatment
After determination of IC 50 , 1×10 6 cells were treated with 5μM of Diosgenin solution. The ethanol without diosgenin solution was added to flask of control cells. Then, culture flasks were incubated in 37°C containing 5% CO 2 with humidified atmosphere incubator for 24, 48 and 72h.

RNA extraction and cDNA synthesis
Total RNA was isolated using the Total RNA isolation TRIZOL reagent protocol (Cinnagene, Iran). Since sample purity and integrity are not associated, both should be evaluated to determine that the RNA sample meets minimal acceptance standards for the downstream workflow. Purity of spectrophotometrically by measuring the OD260/280 ratio in nanodrop ND1000. An OD260/280 of 1.7-2.1 was acceptable (Table 1).The sample with respect to protein contaminant and it's concentration can be measured. However, no RNA integrity information can be obtained from a spectrophotometric reading. RNA integrity can be assessed using gel electrophoresis on a formaldehyde agarose gel in the presence of a fluorescent dye such as ethidium bromide. Observation of two sharp bands for the large (28S rRNA) and the small (18S rRNA) subunits of ribosomal RNAs (rRNA) with the intensity of the larger band being about twice that of the smaller band is indicative of intact RNA (Figure 2).
Complementary DNA (cDNA) was synthesised using reverse transcriptase (RT) using the First Strand cDNA Synthesis Kit (Fermentase, K1622). For cDNA synthesis

Quantitative Real-Time PCR (qRT-PCR)
For real-time PCR, according to previous study (Kazemi et al., 2011), hTERT primers (Genbank accession: NM_ 198255) and beta actin primers (Genbank accession: NM_001101) were used (Table 2). According to manufacture, the total volume of PCR reaction was 20µl that contained: 5 pmole of the forward and reverse PCR primers of hTERT or for beta actin, 2× PCR Master Mix Syber Green I, and 2μl of the cDNA was used. The hTERT and beta-actin gene were amplified by real-time PCR as triplicate. Beta actin and hTERT for each sample at the same run was done, 20μl of PCR reaction mixture contained 10µl of SYBER Ex Taq II (2×) PCR master mix, 2μl template cDNA, 6.4μl dH 2 O water per reaction. Negative controls were prepared each time with 2μl ddH 2 O instead of the cDNA template. Real time PCR amplification was performed using a Corbett (Rortor Gene 6000) system with the following setting as manufacture protocol. The reaction mixture was incubated under the following conditions: 95˚C, 2 minutes, 1 cycle (Holding step); 65˚C, 20 seconds, 45 cycles (Annealing); 72˚C, 20 seconds, 40 cycles (Extension);72˚C, 5min, 1cycle and 75-95˚C, 1 cycle (Melting).

Data analysis
Changes in the hTERT gene expression levels between the control and treated A549 cells were calculated by the 2 -ΔΔCt method. The formulas for the calculation were as follows: ΔCT=CT (target) -CT (reference) ; ΔΔCT=ΔCT (test sample)

-ΔCT (control sample)
The fold for target, relative to a calibrator sample, is calculated by: 2 -ΔΔCt .

Results
The MTT results for different concentrations of diosgenin at different times for the treatment of A549 cell line showed that IC 50 for diosgenin were 47, 44 and 43 μM after 24, 48, and 72 hours of treatment, respectively ( Figure 3). RNA extracts quantity and qualify by nanodrop and agarose gel electrophoresis. The MTT results showed that diosgenin effect on A549 cells has time-dependent and dose-dependent manner, and it's best effect was in 72h after treatment.
Analysis of real-time PCR for evaluation of hTERT gene expression inhibition with 2 -ΔΔCt values showed that diosgenin inhibited hTERT gene expression and decrease hTERT mRNA level in comparison with control group (Figure 4A, B). Fold of decreasing were 0.352, 0.200, 0.092 for 24, 48 and 72h, respectively ( Figure 5).

Discussion
Since most cancer cells hold telomerase activity, one possible gain of telomerase targeted therapy would be its specificity on telomerase positive tumor cells, because most human somatic tissues are telomerase negative. On the base of these comments various types of telomerase inhibitors have been discovered and advanced. Modulators of mRNA expression of telomerase components (hTERT) have been regarded as one of type of anti telomeral agents. These include all transretonoic acid (Xiao et al., 2005), 5, 6-trans-16 -ene vitamin D3 (Holysz et al., 2013) ceramide (Goldkom et al., 2013) and curcumin (Kazemi et al., 2011). These complexes act as inhibitors of hTERT mRNA expression which results in decreased telomerase activity. According to the prior studies there is a strong association among the expression of hTERT mRNA and telomerase activity in extract after culture cells and tissues (Wojtyla et al., 2011). Modulators of hTERTgene expression are regarded as anti telomeral agents. In recent years, various dietary components that can potentially be used for the prevention and treatment of cancer have been identified. One of the most compounds that founded in fenugreek is Diosgenin with anticancer activity.The study effect of diosgenin on osteosarcoma cells were showed that diosgenin treatment caused an inhibition of 1547 cell growth with a cycle arrest in G1 phase and apoptosis induction and moreover exist a correlation between p53, p21 mRNA expression and nuclear factor-kappaB activation (Moalic et al., 2001). Diosgenin induces cell cycle arrest and apoptosis in HEL cells with increase in intracellular calcium level, activation of cPLA2 and COX-2 overexpression (Leger et al.,2004).
Another study about diosgenin effect on colon cancer cells showed that diosgenin induces death receptor-5 through activation of p38 pathway and promotes TRAIL-induced apoptosis (Lepage et al., 2011). In vitro and in vivo effect of steroid saponins of Paris polyphylla var. yunnanensis against lung adenocarcinoma cell line were showed that this compounds have anticancer activity and their effect was dependent on structure of compounds in a certain grade (Yan et al., 2009). Also, another study by Shishodia and Aggarwal (2006) showed that diosgenin could inhibits cmyc gene expression that cmyc acts as a upregulator for hTERT gene expression. Therefore, based on these studies were showed this fact that diosgenin have anticancer activity and exert this property by several mechanisms probably with downregulation of cmyc gene expression.
Since the relationship between cancer and high expression of hTERT gene, our results are especially interesting in demonstrating that Diosgenin downregulates hTERT gene expression in A549 cell line. In this study, for the first time, we studied the effect of diosgenin on hTERT gene expression. Result of Real-time PCR indicated that diosgenin cause decreased hTERT gene expression and inhibits growth of A549 cell line. Therefore, dioggenin could be a good antocancer candidate for cancer therapy in the future.
In conclusion, according to the results, diosgenin by antioxidant activity affect the growth of A549 lung cancer cell line and these cytotoxic effects were time-dependent manner, and diosgenin could inhibit hTERT gene expression as time dependent manner. The results showed that diosgenin could be a candidate subject for lung cancer therapy because natural products like genistein, curcumin, and retinoic acid are proved as chemopreventive agents.