Secondary Logo

Journal Logo

Two isomers of HDTIC isolated fromAstragali Radixdecrease the expression of p16 in 2BS cells

WANG, Pei-chang; ZHANG, Zong-yu; ZHANG, Jian; TONG, Tan-jun

Section Editor(s): SHE, NXi-bin

Original article
Free
SDC

Background Astragali Radix, the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), is a crude drug considered as one of the effective traditional Chinese anti-ageing material. The two isomers of 4-hydroxy-5-hydroxymethyl-[1,3]dioxolan-2,6'-spirane-5′,6′,7′,8′-tetrahydro-indolizine-3′-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were first extracted from the herb in 2002. We demonstrated previously that 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2 strongly delay replicative senescence of human fetal lung diploid fibroblasts (2BS). In this study, we chose them to investigate their effects on the expression of senescence-associated genes to explore the mechanism of how HDTIC delays replicative senescence.

Methods The effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed in vitro by RT-PCR and Western blot. The anti-oxidative activities of the compounds were also observed by phenotype alteration after treatment with antioxidants.

Results There was an obvious expression of p16 in the control senescent cells. However, in the 2BS cells, after 56 population doublings (PDs) grown from PD28 in 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2, there was a weak mRNA expression of p16 and no protein expression of p16 was observed. The expression level of p21 increased with cell ageing. Moreover, there was no difference between the expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds. The results also showed that 2BS cells exposed to 100 μmol/L H2O2 for 5 minutes return to their non-senescent phenotype and continue to be confluent after incubating the damaged cells with HDTIC-1 (1.0 μmol/L ) or HDTIC-2 (10 μmol/L ) for 1 hour.

Conclusions Expression of p16 by 2BS cells was strongly inhibited by HDTIC compounds, which could contribute to their delayed replicative senescence by the way of p16INK4a/Rb/MAPK. The anti-oxidative activities of HDTIC-1 and HDTIC-2, described in this study for the first time, might be indirectly related to their inhibition of p16 expression.

Edited by SHENXi-bin

Department of Medical Laboratory, Xuanwu Hospital of Capital Medical University, Beijing 100053, China (Wang PC and Zhang J) Ageing Researching Center, Medical and Health Center of Peking University, Beijing 100083, China (Zhang ZY and Tong TJ)

Correspondence to: Dr. WANG Pei-chang, Department of Medical Laboratory, Xuanwu Hospital, Capital Medical University, Beijing 100053, China (Tel: 86-10-83198688. Fax: 86-10-83154745. Email: peichangwang@yahoo.com)

This work was supported by the grants from the National Nature Science Foundation of China (No. 30672469) and Beijing Nature Science Foundation (No. 7062030).

(ReceivedAugust 22, 2007)

Cyclin dependent kinase inhibitors (CDKIs), one kind of negative regulator of cyclin dependent kinase (CDK), are associated with cell ageing.1-3 CDKIs include the INK4 family and cip/kip family. p16INK4a, p15INK4b, p18INK4c and p19INK4d are members of INK4 family and the cip/kip family includes p21waf1/cip1/sdi1 etc. Previous studies indicated that p16INK4a/Rb/MAPK and p19ARF/p53/p21cip1 could be two important steps of replicative senescence.4-6 Moreover, the high levels of the expression of p21 and p16 initiate or promote cell ageing.7,8

Astragali Radix is the root of Astragalus membranceus (Fish) Bunge Var. mongholicus (Bge), a Chinese herb considered to be an effective traditional anti-ageing material.9 The two isomers of 4-hydroxy-5-hydroxymethyl-[1,3]dioxolan-2,6′-spirane-5′,6′,7′,8′-tetr ahydro-indolizine-3′-carbaldehyde (HDTIC), HDTIC-1 and HDTIC-2, were extracted from Astragali Radix and were demonstrated in 2003 to delay senescence of human fetal lung diploid fibroblasts (2BS) when characterized by morphology, population doublings (PDs), senescence-associated β-galactosidase (SA-β-gal) positive rate, potentials of growth and proliferation, cell cycle profile and advanced glycation end products (AGEs).10 However, little is known about the biological characteristics of the new compounds. To investigate the mechanism by which HDTIC compounds delay the replicative senescence of 2BS cells, this study examined the effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 in 2BS cells. The anti-oxidative activities of the compounds were also observed to try to reveal the role of the compounds in the relation between the decrease in p21 and p16.

Back to Top | Article Outline

METHODS

Cells

The 2BS cells were previously isolated from female fetal lung fibroblast tissue and have been fully characterized.11,12 The 2BS cell line was originally established at the National Institute of Biological Products (Beijing, China). The 2BS cells are considered to be young at PD30 or below and to be fully senescent at PD55 or above.

Back to Top | Article Outline

HDTIC-1 and HDTIC-2

HDTIC-1 and HDTIC-2 (Figure 1) were isolated from Astragali Radix that was kindly provided as a gift by Dr. TU Peng-fei (Modern Research Center for Traditional Chinese Medicine, Peking University, China). The structure of the molecules was identified by Mass Spectrum (MS), 1H-NMR (Nuclear Magnetic Resonance) and 13C-NMR. HDTIC-1 and HDTIC-2 were kept as 1 mmol/L stock solutions in DMSO. They were then diluted to the required concentrations using DMEM.

Figure 1.

Figure 1.

Back to Top | Article Outline

Cell culture

Cells were grown in DMEM (Life Technologies Inc., Grand Island, NY, USA) supplemented with 10% fetal calf serum and with 60 μg/ml penicillin and 100 μg/ml streptomycin in an incubator at 37°C with 5% CO2. The cumulative population doublings (CPDs) were calculated as log2 (D/D0), where D and D0 are defined as the density of cells at the time of harvesting and seeding, respectively.

Back to Top | Article Outline

Semi-quantitative RT-PCR analysis

Total RNA was extracted using the Totally RNA Kit (QIAGEN), and cDNA was prepared using RT Kit (Promega). The PCR system (20 μl) consisted of 1 μg cDNA, 2 μl 10×Taq DNA polymerase buffer, 1.6 μl 25 mmol/L MgCl2, 0.4 μl 10 mmol/L dNTP mixture, 0.7 μl 10 μmol/L upper primer, 0.7 μl 10 μmol/L lower primer and 1 U Taq DNA polymerase. The number of amplification cycles was 30. Each cycle included incubation at 95°C for 20 seconds, 56-60°C 1 minute and 72°C 30 seconds. The annealing temperatures of the amplification for p16, p21 and GADPH were 55°C, 56°C and 60°C, respectively. Amplicons of the expected size (401 bp for p16, 401 bp for p21 and 320 bp for GAPDH transcripts) were obtained and quantified by scanning densitometry using the Molecular Analyst Software (BioRad). Results were normalized relative to the intensity of the GAPDH control band. Primer sequences: p16 upper primer: 5’-GAATAGTTACGGTCGGAG-3’, p16 lower primer: 5’-CGGTGACTGATGATCTAAG-3’; p21 upper primer: 5’-GAGGAAGACCATGTGGAC-3’, p21 lower primer: 5’-CAGCACTCTTAGGAACCTC-3’; GAPDH upper primer: 5’-CGAGTCAACGGATT-TGGTGGTAT-3’, GAPDH lower primer: 5′-AGCCTTC-TCCATGGTGAAGAC-3′.

Back to Top | Article Outline

Western blot

Protein expression was detected by western blotting as previously described but with minor modifications.13 Briefly, 2BS cells were grown from PD28 in DMEM supplemented with or without HDTIC. The cells were lysed, at PD45 and PD55, in lysis buffer (50 mmol/L Tris-HCl, 250 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 0.15% Igepal CA-630 and 1.5 mmol/L PMSF). Equal amounts of proteins (120 μg) were size fractionated on 9%-15% SDS-PAGE. The antibodies used were anti-p16INK4a (DCS-50.1/A7, NEOMARKERS, Fremont, CA, USA, 1 μg/ml), anti-p21waf1/cip1 (F-5, Santa Cruz, CA, USA, 1 μg/ml) and anti-actin (I-19, Santa Cruz, CA, USA, 2 μg/ml).

Back to Top | Article Outline

Protection against oxidative stress

PD28 2BS cells were damaged by 100 μmol/L H2O2 for 5 minutes in the dark. Then, the cells were washed twice with cold PBS and continually cultured with DMEM containing HDTIC-1 (1.0 μmol/L) or HDTIC-2 (10.0 μmol/L) for 1 hour. The phenotype of the cells was determined microscopically.

Back to Top | Article Outline

Statistical analysis

The significance of the differences between the control groups and experimental groups were analyzed by t test using SAS software. A P value less than 0.05 was considered statistically significant. Data were obtained from three independent experiments.

Back to Top | Article Outline

RESULTS

Effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 mRNA levels

The mRNA levels of p16 and p21 were observed using RT-PCR in the 2BS cells at PD45 and PD56. The results listed in Figures 2 and 3 showed that there was a slight expression of p16 in the middle-aged control cells, but an obviously increased expression of p16 in the senescent control cells. However, the p16 bands of PD45 cells, grown from PD28 in 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2, could hardly be seen. The p16 mRNA level of PD56 cells cultured with HDTIC compounds was almost half that of PD55 control cells. The mRNA expression of p21 have been detected both in PD45 cells and in PD56 cells, whereas, the mRNA expression level of p21 in PD56 cells was significantly higher than in PD45 cells. There is no significant difference between the mRNA expression level of p21 in the control cells and that in the same PD cells cultured with HDTIC compounds (P>0.05).

Figure 2.

Figure 2.

Figure 3.

Figure 3.

Back to Top | Article Outline

Effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 in protein level

The protein levels of p16 and p21 in 2BS cells were determined using Western blot. The results listed in Figures 4 and 5 showed that there was a weak expression of p16 in PD46 control cells and a strong expression of p16 in PD55 control cells. No expression of p16 protein was observed even in PD56 cells that were grown from PD28 in 0.1 μmol/L HDTIC-1 or in 1.0 μmol/L HDTIC-2. The expression of p21 protein was detected in the control cells and in the HDTIC-cultured cells at PD45 and PD55. However, the expression level of p21 protein in the control cells was similar to that in the same passage of the HDTIC-cultured cells.

Figure 4.

Figure 4.

Figure 5.

Figure 5.

Back to Top | Article Outline

HDTIC-1 and HDTIC-2 reverse senescent phenotype induced by H2O2

PD28 2BS cells were damaged by exposure to 100 μmol/L H2O2 for 5 minutes in the dark. The cells were then continually cultured with DMEM supplemented with or without HDTIC for 1 hour. The results showed that the damaged cells returned to a normal phenotype after they were treated with 1.0 μmol/L HDTIC-1 or 10.0 μmol/L HDTIC-2 for 1 hour, while the control cells remained seriously damaged even after they were cultured in normal DMEM for 1 hour (Figure 6). These results indicated that HDTIC compounds might be anti-oxidative and had the potential for removing free radicals and reactive oxygen species.

Figure 6.

Figure 6.

Back to Top | Article Outline

DISCUSSION

Astragali Radix is a crude drug used as one of the effective traditional Chinese anti-ageing materials.9 Recent studies show that Astragali Radix has a significant mitogenic activity as well as the function of improving the metabolism and survival time of cells.9,14,15 The extract of Astragali Radix was found by Chinese scientists in the 1970s to have the potential to increase the cumulative population doublings of human fetal lung diploid fibroblasts and human fetal kidney cells in vitro by at least 20-30 PDs, furthermore, the delaying effect of the crude drug on ageing was initially identified in rats.16

Astragali Radix mainly consists of flavonoids, isoflavonoids, saponins, polysaccharides and amino acids.17 Their biological properties are well-known, but they are not responsible for the effect of the crude drug in laboratory. HDTIC is a novel trace component separated from Astragali Radix in 2002 and has two isomers: HDTIC-1 and HDTIC-2. We previously reported that both HDTIC-1 and HDTIC-2 maintain a non-senescent phenotype of 2BS cells even at late population doubling and increase CPDs by at least 15-20 PDs. HDTIC also improves cell growth and proliferation and promotes the entry of 2BS cells from G0 or G1 phase to S-phase. In addition, the delaying effects of HDTIC compounds on replicative senescence were characterized by AGEs and SA-β-Gal, two senescence-associated biomarkers.10 Up to now, HDTIC is the only component reported to be responsible for the delaying effect of Astragali Radix on replicative senescence. Unfortunately, its biological properties, including the mechanism of its delaying senescence, are not well understood.

What’s the exact mechanism of replicative senescence? There is, at present, no definitive answer to the question. However, in the last decade considerable progress has been made in the understanding of the regulation of CDKIs as well as of senescence-associated genes and of the biological consequences of cellular senescence. p16INK4a and p21cip1/WAF1, two critically important inhibitors of cyclin-dependent kinase, induce cell cycle arrest in G1 phase and have been proposed to be involved in the dependent pathways of p16INK4a/Rb/MAKP and p19ARF/p53/p21cip14-8,18 A progressive increase in the expression levels of p16INK4a and p21cip have been shown to correlate with cellular senescence while the inactivation of p16INK4a or p21cip1 by antisense strategy allows primary human keratinocytes and 2BS cells to escape replicative senescence.19-21 We assume that the effects of HDTIC compounds on replicative senescence are correlated with the senescence-associated genes.

In this study, the effects of HDTIC-1 and HDTIC-2 on the expression of p16 and p21 were observed using RT-PCR and Western blot. The results showed that the expression level of p21 in the control cells was similar to that in the same PD cells cultured with HDTIC compound. There was a slight increase of mRNA expression of p16 in the middle-aged control cells, but an obvious increase in the expression of p16 mRNA and protein in the senescent control cells could be seen. However, the protein expression of p16INK4a in the senescent cells grown from PD28 in 0.1 μmol/L HDTIC-1 or 1.0 μmol/L HDTIC-2 was not observed. The results mentioned above demonstrated that HDTIC strongly inhibits the expression of p16INK4a but does not influence the expression of p21 in 2BS cells.

Previous studies demonstrate that the expression level of p21 increases initially with cellular senescence; the high level of p16 expression will follow. When cellular senescence is initiated the cells maintain a high level of p16INK4a, while the p21 expression decreases gradually.2,22,23 Therefore, it is considered that replicative senescence is initiated by p21 and continued by p16. Studies by Macip show that the accumulation of free radicals (FR) and reactive oxygen species (ROS) are down-stream events following p21 expression. Removal of FR and ROS by anti-oxidative agents inhibits the initiation of cellular senescence induced by p21 over-expression.24 These results indicate that the accumulation of FR and ROS might be the medium between p21 expression and p16 expression. In this study, the senescent phenotype of 2BS cells induced by H2O2 was reversed by HDTIC compounds. This indicates that the compounds may have a strong potential for anti-oxidative activity and, that the removal of the accumulation of FR and ROS by HDTIC could contribute to its inhibition of p16 expression.

The regulation mechanism of p16INK4a is very complex.25-27 We have little knowledge of the detailed mechanisms of HDTIC inhibiting p16 expression, whereas, we can draw the conclusion that HDTIC blocks the pathway of p16/Rb/MAPK by inhibiting p16 expression.

In summary, HDTIC-1 and HDTIC-2 inhibit the expression of p16 but do not affect p21 expression. The results of our study show that HDTIC compounds can reverse the senescent phenotype of 2BS cells induced by H2O2, which indicates that HDTIC-1 and HDTIC-2 might have potential as anti-oxidative stress factors. It is likely that the anti-oxidative activities of HDTIC compounds block the initiation of p16 expression through the removal of FR and ROS. The results of this study support the view that HDTIC blocks the pathway of p16/Rb/MAPK thereby delaying replicative senescence of 2BS cells. The difference in the optimal concentrations of HDTIC-1 (0.1 μmol/L) and HDTIC-2 (1.0 μmol/L) for inhibiting p16 expression indicate that the structure of the HDTICs is a factor in their biological activities.

Back to Top | Article Outline

REFERENCES

1. Stein GH, Drullinger LF, Soulard A, Dulic V. Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 1999; 19: 2109-2117.
2. Alcorta DA, Xiong Y, Phelps D, Hannon G, Beach D, Barrett JC. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci U S A 1996; 93: 13742-13747.
3. Pajalunga D, Mazzola A, Salzano AM, Biferi MG, De Luca G, Crescenzi M. Critical requirement for cell cycle inhibitors in sustaining nonproliferative states. J Cell Biol 2007; 176: 807-818.
4. Cox LS. Multiple pathways control cell growth and transformation; overlapping and independent activities of p53 and p21cip1/waf1/sdi1. J Pathol 1997; 183: 134-140.
5. Takahashi A, Ohtani N, Yamakoshi K, Iida S-I, Tahara H, Nakayama K, et al. Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol 2006; 8: 1291-1297.
6. Beausejour CM. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J 2003; 22: 4212-4222.
7. Dai CY, Enders GH. p16INK4a can initiate an autonomous senescence program. Oncogene 2000; 19: 1613-1622.
8. Tahara H, Sato E, Noda A, Ide T. Increase in expression level of p21sdi1/cip1/waf1 with increasing division age in both normal and SV40-transformed human fibroblasts. Oncogene 1995; 10: 835-840.
9. Chen K, Li C. Recent advances in studies on traditional Chinese anti-ageing medica. J Tradit Chin Med 1993; 13: 223-226.
10. Wang PC, Zhang ZY, Ma XF, Huang Y, Liu XW, Tu PF, et al. HDTIC-1 and HDTIC-2, two compounds extracted from Astragali Radix, delay replicative senescence of human diploid fibroblasts. Mech Ageing Dev 2003; 124: 1025-1034.
11. Wang PC, Zhang ZY, Tong TJ. Aminoguanidine delays the replicative senescense of human diploid fibroblasts. Chin Med J 2007; 120: 2028-2035.
12. Li JH, Zhang ZY, Tong TJ. The proliferative response and anti-oncogene expression in old 2BS cells after growth factor stimulation. Mech Ageing Dev 1995; 80: 25-34.
13. Zhu WG, Lakshmanan RR, Beal MD, Otterson GA. DNA Methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 2001; 61: 1327-1333.
14. Yamada H, Kiyohara H, Takemoto N, Zhao JF, Kawamura H, Komatsu Y, et al. Mitogenic and complement activating activities of the herbal components of juzen-taiho-to. Planta Med 1992; 58: 166-170.
15. Cai Q, Li XM, Wang HY. Astragali and Angelica protect the kidney against ischemia and reperfusion injury and accelerate recovery. Chin Med J 2001; 114: 119-123.
16. Wang XJ, Haruyo I, Tetsuya K. Antioxidant potential of qizhu tang, a Chinese herbal medicine, and the effect on cerebral oxidative damage after ischemia reperfusion in rats. Biol Pharm Bull 2001; 24: 558-563.
17. Ma XQ, Shi Q, Duan JA, Dong TT, Tsim KW. Chemical analysis of Astragali (Huangqi) in china: a comparison with its adulterants and seasonal variations. J Agric Food Chem 2002; 50: 4861-4866.
18. Jarrard DF, Sarkar S, Shi Y, Yeager TR, Magrane G. p16/pRb pathway alterations are required for bypassing senescence in human prostate epithelial cells. Cancer Res 1999; 59: 2957-2964.
19. Duan JM, Zhang ZY, Tong TJ. Senescence delay of human diploid fibroblast induced by anti-sense p16INK4a expression. J Biol Chem 2001; 276: 48325-48331.
20. Maurelli R, Zambruno G, Guerra L, Abbruzzese C, Dimri G, Gellini M, et al. Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment. FASEB J 2006; 20: 1516-1518.
21. Huang Y, Corbley MJ, Tang Z, Peng Y, Zhang ZY, Tong TJ. Down-regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: involvement of retinoblastoma protein phosphorylation and delay of cellular aging. J Cell Physiol 2004; 201: 483-491.
22. Hara E, Smith R, Parry D, Tahara H, Stone S, Peters G. Regulation of p16 expression and its implications for cell immortalization and senescence. Mol Cell Biol 1996; 16: 859-867.
23. Xue L, Wu J, Zheng W, Wang P, Li J, Zhang ZY, et al. Sp1 is involved in the transcriptional activation of p16(INK4) by p21(Waf1) in Hela cells. FEBS Lett 2004; 564: 199-204.
24. Macip S, Igarashi M, Fang L, Chen A, Pan ZQ, Lee SW, et al. Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence. EMBO J 2002; 21: 2180-2188.
25. Zheng WJ, Wang HY, Xue LX, Zhang ZY, Tong TJ. Regulation of cellular senescence and p16INK4a expression by Id1 and E47 proteins in human diploid fibroblast. J Biol Chem 2004; 279: 31524-31532.
26. Wang W, Wu JF, Zhang ZY, Tong TJ. Characterization of regulatory elements on the promoter region of p16(INK4a) that contribute to overexpression of p16 in senescent fibroblasts. J Biol Chem 2001; 276: 48655-48661.
27. Kim WY, Sharpless NE. The regulation of INK4/ARF in cancer and aging. Cell 2006; 127: 265-275.
Keywords:

HDTIC; Astragali Radix; replicative senescence; fibroblast; gene expression

© 2008 Chinese Medical Association