In Vitro Modulation of Telomerase Activity, Telomere Length and Cell Cycle in MKN45 Cells by Verbascoside

• Abstract
• Abbreviations
• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Chemical and reagents
• Cell culture and drugs treatment
• Assay of telomerase activity
• Telomere length analysis
• Cell cycle analysis
• Results and Discussion
• Acknowlegements
• References

Abstract

Screening of natural products with anti-tumor activity as telomerase inhibitor is a new subject in the field of tumor therapy. Using telomerase PCR ELISA, telomere DNA hybridization and flow cytometry analysis, the effects of verbascoside, a phenylpropanoid glucoside extracted from Pedicularis striata Pall, on telomerase activity, telomere length and cell cycle of human gastric carcinoma cells MKN45 was examined in vitro. After being treated with a 50 % inhibition concentration of verbascoside (17.8 μg/ml), telomerase activity in the cells was significantly inhibited but not in the cellular supernatant, the average telomere length became remarkably short, and the sub-G0 /G1 peak and G2/M arrest were also displayed when compared to the control cells. These results suggest that verbascoside mediated-cell differentiation and apoptosis may be affected by telomere-telomerase-cell cycle dependent modulation. Thus, the antitumor mechanism of verbascoside is demonstrated once more by its inhibiting effect on telomerase activity in tumor cells, and the telomerase assay may provide a valuable screening method for antitumor activity of natural products.

Abbreviations

PCR:polymerase chain reaction

ELISA:enzyme-linked immunosorbent assay

DDW:disinfected distillation water

TRAP:telomere repeat amplification protocol

PPG:phenylpropanoid glycoside

RT:room temperature

OD:optical density

^telIC₅₀:50 % inhibition of the telomerase activity

TRF:telomeric restriction fragments

FCM:flow cytometry

NC:nitrocellulose

Abstract

Screening of natural products with anti-tumor activity as telomerase inhibitor is a new subject in the field of tumor therapy. Using telomerase PCR ELISA, telomere DNA hybridization and flow cytometry analysis, the effects of verbascoside, a phenylpropanoid glucoside extracted from Pedicularis striata Pall, on telomerase activity, telomere length and cell cycle of human gastric carcinoma cells MKN45 was examined in vitro. After being treated with a 50 % inhibition concentration of verbascoside (17.8 μg/ml), telomerase activity in the cells was significantly inhibited but not in the cellular supernatant, the average telomere length became remarkably short, and the sub-G0 /G1 peak and G2/M arrest were also displayed when compared to the control cells. These results suggest that verbascoside mediated-cell differentiation and apoptosis may be affected by telomere-telomerase-cell cycle dependent modulation. Thus, the antitumor mechanism of verbascoside is demonstrated once more by its inhibiting effect on telomerase activity in tumor cells, and the telomerase assay may provide a valuable screening method for antitumor activity of natural products.

Abbreviations

PCR:polymerase chain reaction

ELISA:enzyme-linked immunosorbent assay

DDW:disinfected distillation water

TRAP:telomere repeat amplification protocol

PPG:phenylpropanoid glycoside

RT:room temperature

OD:optical density

^telIC₅₀:50 % inhibition of the telomerase activity

TRF:telomeric restriction fragments

FCM:flow cytometry

NC:nitrocellulose

Introduction

Normal human somatic cells progressively lose telomeric repeat sequences with each successive cell division and with aging due to the ”end replication” problem [1]. However, telomere length can be maintained in immortal and cancer cells by expressing the enzyme telomerase, an RNA-dependent DNA polymerase that elongates the 3’ ends of telomere [2]. Telomere shortening and telomerase activity have been detected in almost all human tumors but not in normal somatic tissues [2] [3] [4]. Thus, telomerase may not only play a critical role in regulating the life span of normal cells and in tumorigenesis but also serve as an important target for anti-cancer drug.

Verbascoside (Fig. [1]) was extracted from Pedicularis striata Pall [5], a Chinese herbal medicine which exhibits extensive biological anti-tumor effects [6], [7]. The relation between verbascoside and telomerase had not been examined. We found that verbascoside is able to inhibit telomerase, to shorten telomere length and to induce apoptosis in human gastric carcinoma cells MKN45.

Materials and Methods

Chemical and reagents

Verbascoside (purity >98 %) was isolated from Pedicularis striata Pall in our laboratory [5]. Hydroxycamptothecine (HCPT) was purchased from Huangshi Pharmaceutical Factory (Hubei, China). HinfI, RsaI,T4 polynucleotide kinase, Klenow, λ DNA/Hind III and DNA isolated kit were purchased from Promega Co., [γ-³²P] ATP, [α-P³²] dCTP were obtained from Ya-Hui Biotech Co (Beijin, China), (TTAGGG)₄ were synthesized by Shanghai Sangon Biotech Co (Shanghai, China), Telomerase PCR ELISA kit was purchased from Boehringer Mannheim (Germany).

Cell culture and drugs treatment

Human gastric carcinoma cell line MKN45 was obtained from Fourth Military Medical University, Xi’an, China and cultured in RPMI1640 medium (Sigma Chemical Co, St. Louis, MO) supplemented with 10 % fetal calf serum at 37 °C in 5 % CO₂. The cells of logarithmic growth were harvested as suspension of single cells, incubated in 6-well tissues culture plates at a cell density of 4 × 10⁴ cells/ml for 6 h. The culture medium was aspirated and these cells were directly exposed to serial dilutions of verbascoside dissolved in DDW and the control cells were only cultured in equivalent volumes of DDW without verbascoside for 48 h. The cellular viability was measured by the trypan blue stain method. The cellular viability had no remarkably change after treatment, and the pelleted cells were stored at -80 °C.

Assay of telomerase activity

Preparation of extracts from the cells: The pelleted cells were thawed and resuspended in 200 μl lysis reagent and incubated on ice for 30 min, the lyse cells were centrifuged at 1600 × g for 20 min at 4 °C. Human embryonic kidney cell line 293 was used as a telomerase-positive control, human skin fibroblast was used as a telomerase-negative control and 293 cell extracts treated with RNase (+RNase) was used as an internal negative control.

TRAP reaction: 25 μl of reaction mixture were transferred into a tube suitable for PCR amplification. 2 μl of cell extract were added and sterile water was added to a final volume of 50 μl. The tubes were transferred to a thermal cycler (PTC 100^TM, MJ Research INC, USA) and a TRAP reaction was performed by the following protocol: primer elongation for 30 min at 25 °C, telomerase inactivation for 5 min at 94 °C, products amplification by repeat of 32 cycles (denaturation for 30 sec at 94 °C, annealing for 30 sec at 50 °C and polymerization for 90 sec at 72 °C), then 72 °C/10 min for one cycle and 4 °C hold.

Hybridization and ELISA procedure: 5 μl of the amplification product were incubated in 20 μl of denaturation reagent at RT for 10 min. 225 μl of hybridization buffer were added and mixed. 100 μl of the mixture were transferred per well of the precoated MTP modules supplied with the kit and the wells covered with the self-adhesive cover foil. The MTP modules were incubated at 37 °C on a shaker for 2 h. The hybridization solutions were removed and washed 3 times with 250 μl of washing buffer. 100 μl anti-DIG-POD working solution were added per well, the MTP modules were covered with a cover foil and incubated at RT for 30 min while shaking at 300 rpm. The solutions were removed and rinsed 5 times with 250 μl of washing buffers per well for 30 sec each, and the washing buffers were removed. 100 μl of TMB substrate solution were added and prewarmed to RT per well, the wells were covered with cover foil and incubated for color development at RT for 15 min while shaking at 300 rpm. 100 μl of stop reagent were added per well. The absorbance of the samples (OD) was measured at 450 nm (with a reference wavelength of 690 nm) within 30 min after addition of the stop reagent by using a microtiter plate reader (Nanjin, China). The ^telIC₅₀ was obtained by linear regression of logarithmic value of the drug concentrations and telomerase inhibited rate [(1 - OD treated /OD control) × 100] in the treated cells and the control cells.

Telomere length analysis

Cells treated with verbascoside and DNA isolation: 4 × 10⁴/ml cells were maintained in complete medium containing the ^telIC₅₀ of verbascoside or DDW for 8 weeks, based on the telomerase assay in the treated cells. The cell pellets were resuspended in lysis buffer, and their genomic DNA was isolated. The DNA was resuspended in 100 μl TE buffer and determined from absorbance at 260 nm to 280 nm by an UV spetrophotometer (HP8435, USA).

Cleavage of DNA and labeling of (TTAGGG)4 probes [4], [8]: The genomic DNA was digested by using 4 U of Ras I/Hinf I enzyme mixture per μg of DNA in enzyme reaction buffer for 12 h at 37 °C. Individual volumes of the following were added to a microcentrifuge tube: 1 μl (TTAGGG)₄ (μg/μl), 3 μl [γ-³²P] ATP (10 μCi/μl),1 μl 10 × kinase buffer, 1 μl T₄ polynucleotide kinase (10 U/L) and H₂O for a final volume of 10 μl. The mixture was centrifuged and incubated at 37 °C for 30 min. 240 μl of 5M sodium acetate, 40 μl of H₂O and 750 μl of ice-cold dehydrated ethanol were added at 4 °C bath for 30 min and centrifuged, 0.5 ml of 70 % ice-cold ethanol were added and mixed, centrifuged, dried and stored at -20 °C in TE. Individual volumes of the following were added to a microcentrifuge tube: 2 μl λ DNA/Hind III (0.5 μg/μl), 2 μl 10 × buffer, 1 μl 2mmol dATP, dTTP, dUTP, 1 μl [α-³²P]dCTP (10 μCi/μl), 1 μl Klenow, H₂O for a final volume of 19 μl at RT for 15 min, 1 μl 2 mM dCTP were added at RT for 15 min and at 70 °C for 5 min, ethanol precipitated DNA fragments, that were dried and stored at -20 °C in TE.

Hybridization of telomeric DNA and TRF length analysis [8], [9]: Equal aliquots (5 μg of digested DNA or labled λ DNA/Hind III) were loaded onto 0.6 % agarose gels and electrophoresed for 12 h at 1 V/cm, EB stained the gels and observed in a UV transilluminator. The gel was soaked in 0.25 M HCl for 15 min and rinsed with dH₂O and repeated. The gel was soaked in 0.4 N NaOH for 15 min and repeated. The separated DNA was transferred onto a NC membrane in 0.5 mol NaOH and 1.5 mol NaCl at RT for overnight. The membrane was rinsed in 2 × SSC and dried, the filter was soaked in 2 × SSC, and when completely wet, transferred into pre-hybridization buffer and shaken at 42 °C for 6 h. The labeled telomere probe was added in the hybridization buffer to the membranes and shaken at 42 °C, overnight. The filter was then washed twice in 2 × SSC/0.1 % SDS at RT for 5 min. The filter was then washed twice in prewarmed 0. 2 × SSC/0.1 % SDS at 42 °C for 15 min. After being wrapped between two pieces of transparent plastic sheet, the filter was autoradiographed on preflashed Kodak X-ray films for 48 h at -20 °C. Each lane on the photographed picture was scanned with a densitometer (CX-900, Japan) and the data were used to determine the amount of telomeric DNA and the TRF length. The mean TRF length for each sample was calculated by integrating the signal intensity above background over the entire TRF distribution as a function of TRF length using the formula, L = Σ (ODiLi)/Σ (ODi), where ODi and Li were the signal intensity and TRF length respectively at position i on the gel image.

Cell cycle analysis

After being treated with verbascoside, HCPT (0.012 mg/ml) as a positive control or DDW as a negative control for 72 h, 1 × 10⁵ cells/ml were digested, washed twice with cold PBS, and fixed in 70 % ethanol at -20 °C overnight. The cells were centrifuged at 500 × g for 6 min, 1 ml of DNase-free RNase (200 μg/ml) and propidium iodide (50 μg/ml) mixture (1 : 1) were added for 30 min at RT, then a minimum of 10⁴ cells per sample were analysed on a FCM (FACScan, LYSYS II software, Becon Diekinson). The relative percentages of cells in the G0/G1, S and G2/M phases of cell cycle, and cells with a lower DNA content than those of G0/G1 referred to as apoptotic cells (sub-G0/G1), were determined [10].

Results and Discussion

We found that the ^telIC₅₀ value of verbascoside on telomerase in MKN45 cells was 17.8 ± 7.2 (μg/ml). The result of the untreated cellular supernatant treated with the verbascoside was the same as that of the DDW treated cells. Therefore, verbascoside could not directly inhibit telomerase activity in extraction of the untreated cells (Fig. [2]). It is indicated that the loss of telomerase activity is due to a cellular function, but not due to chemical or direct biochemical effects.

To determine whether the telomerase inhibition may result in telomere shortening, we measured the TRF length in the MKN45 cell line before and after eight weeks of the ^telIC₅₀ verbascoside treatment. The results showed that the average TRF length in MKN45 cells was shortened significantly when compared with that of control. Shortening of telomere length in verbascoside treated cells is shown in Table [1] and Fig. [3]. The results were the average values obtained in three independent experiments. The telomere length shortening seems to be consistent with telomerase inhibition of the verbascoside treated cells, perhaps indicating that telomerase inhibition by verbascoside may involve more or less in telomere-length regulation.

The biological data showed that stabilization of TRF length by expressing telomerase may result in apoptosis inhibition, cellular immortalization and carcinogenesis [1], [2], [11]. Many factors, such as high temperature, radiation, cytokine, anti-tumor chemotherapy agents, antibody, gene, etc. [12], [13], have a certain effect on modulating tumor cell cycle. Although verbascoside can arrest tumor cell growth, repair DNA oxidative damage and induce cell apoptosis and differentiation [6], [7], [14], [15], whether telomerase inhibition by verbascoside may lead to tumor cell apoptosis, and whether all the cell cycle changes exist as a common mechanism is not known. Human gastric cancer cells induced to differentiation with verbascoside at the ^telIC₅₀ of telomerase inhibition were analyzed for DNA content by flow cytometry. A subdiploid peak of DNA can be observed in apoptotic cells by FCM. Fig. [4] shows the changes of the sub-G0/G1 DNA content among the untreated, treated and positive control cells. The apoptotic rates of the ^telIC₅₀ of verbascoside treated cells and the IC₅₀ of HCPT treated cells were about 18.3 % (Fig. [4] B) and 19.7 % (Fig. [4] C), respectively, when compared with the negative control cells. The G2/M cells reduced from 15.4 % to 7 % in the verbascoside treated MKN45 cells (Fig. [4] B), implying that verbascoside may induce tumor cell apoptosis by telomerase inhibition and telomere shortening.

Our results showed that verbascoside exhibited telomerase inhibiting, telomere shortening and apoptosis inducing effects in MKN45 cells. The cell apoptosis might be associated with the telomerase inhibiting and telomere shortening. Thus, the antitumor mechanism of verbascoside is demonstrated once more to be its inhibiting effect on telomerase activity in tumor cells, and the telomerase assay may provide a valuable screening method for antitumor activity of natural products.

Acknowlegements

This project was partly supported by the National Natural Science Foundation of China (No 29 972 017) and partly by China Postdoctoral Science Foundation.

References

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  PubMedSearch in Google Scholar

Prof. Zhong-Jian Jia

Department of Chemistry

University Lanzhou 730 000

People’s Republic of China

Email: zhangfx@lz.gs.cninfo.net

Fax: +86-0931-891258

References

• 1 Mera S L. The role of telomere in ageing and cancer.  Br J Biomed Sci. 1998;  55 221-5
  PubMedSearch in Google Scholar
• 2 Rhyu M S. Telomere, telomerase, and immortality.  J National Cancer Institute. 1995;  87 884-94
  PubMedSearch in Google Scholar
• 3 Autexier C, Greider C W. Telomerase and cancer: revisiting the telomere hypothesis.  TIBS. 1996;  21 387-91
  PubMedSearch in Google Scholar
• 4 Zhang F X, Zhang X Y, Fan D M, Den Z Y, Yan Y. Expression of telomere and telomerase in human primary gastric carcinoma.  Chinese J Pathol. 1998;  27 429-32
  PubMedSearch in Google Scholar
• 5 Liu Z M, Jia Z J. Phenylpropanoid and iridoid glycoside from Pedicularis striata .  Phytochemistry. 1991;  30 1341-4
  CrossrefPubMedSearch in Google Scholar
• 6 Li J, Zheng Y, Zheng R L, Liu Z M, Jia Z J. Antitumor effects of phenylpropanoid glycosides.  Chinese Pharmaceut J. 1995;  30 269-71
  PubMedSearch in Google Scholar
• 7 Li J, Zheng Y, Zhou H, Su B N, Zheng R L. Differentiation of human gastric adenocarcinoma cell line MGC803 induced by verbascoside.  Planta Med. 1997;  63 499-502
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Davis L G, Kuehl W M, Battey J F, editors. Basic Methods in Molecular Biology. Appleton & Lange Norwalk, Connecticut; 1997: 27-288
  Search in Google Scholar
• 9 Allsopp R C, Vaziri H, Patterson C, Goldstein S, Younglai E V, Futcher A E,. et al . Telomere length predicts replicative capacity of human fibroblasts.  Proc Natl Acad Sci. 1992;  89 10 114-8
  PubMedSearch in Google Scholar
• 10 Tounekti O, Belehradek J, Mir L M. Relationships between DNA fragmentation, chromatin condensation, and changes in flow cytometry profiles detected during apoptosis.  Exp Cell Res.. 1995;  217 506-16
  CrossrefPubMedSearch in Google Scholar
• 11 Fu W M, Begley J G, Killen M W, Mattson M P. Anti-apoptotic role of telomerase in pheochromocytoma cells.  J Biol Chem. 1999;  274 7264-71
  CrossrefPubMedSearch in Google Scholar
• 12 Zhang F X, Zhang X Y, Fan D M, Deng Z Y, Yan Y, Wu H P,. et al . Antisense telomerase RNA induced human gastric cancer cell apoptosis.  World J Gastroenterol. 2000;  6 430-2
  PubMedSearch in Google Scholar
• 13 Lichtsteiner S P, Lebkowski J S, Vasserot A P. Telomerase. A target for anticancer therapy.  Ann N Y Acad Sci. 1999;  886 1-11
  PubMedSearch in Google Scholar
• 14 Jia Z J, Gao J J. Phenylpropanoid glycosides from Pedicularis striata Pall ssp. arachnoidea .  Phytochemistry. 1994;  34 1188-90
  PubMedSearch in Google Scholar
• 15 Li J, Ge R C, Zheng R L, Liu Z M, Jia Z J. Antioxidative and chelating activities of phenylpropanoid glycosides from Pedicularis strata .  Acta Pharmacol Sin. 1997;  18 77-80
  PubMedSearch in Google Scholar

Prof. Zhong-Jian Jia

Department of Chemistry

University Lanzhou 730 000

People’s Republic of China

Email: zhangfx@lz.gs.cninfo.net

Fax: +86-0931-891258