PartTwo Echinacoside Induces Apoptotic Cancer Cell Death By Inhibiting The Nucleotide Pool Sanitizing Enzyme MTH1
Mar 03, 2022
Part Two How does echinacoside inhibit cell apoptosis and improve memory
For more information please contact ali.ma@wecistanche.com
Click here to part one
Discussion
Echinacoside is a natural compound isolated from the medicinal plants Cistanche and Echinacea. 51,52 It has been shown to possess versatile health promotional and disease preventive properties, including neural protection, hepatoprotection, and anti-inflammation, antifatigue, antisenescence, antidiabetes, and antitumor activities.53–58 The best known and accepted bioactivity of Echinacoside is its antioxidative and ROSscavenging action;44,45 however, it has also been shown to cause oxidative DNA damages in cancer cells, with the underlying mechanisms remaining unclear.58 In the present study, using a high-throughput in vitro screening assay, we found that Echinacoside effectively inhibited the MTH1-catalyzed enzymatic reaction. Increasing the amount of the MTH1 enzyme decreased the degree of inhibition while increasing the amount of the inorganic pyrophosphatase did not affect the inhibition, indicating that Echinacoside specifically inhibited the activity of MTH1 in the in vitro assay. Treatment of different human cancer cell lines with Echinacoside caused significant elevation of the cellular 8-oxoG level without changing the cellular ROS level. Given that Echinacoside itself is a potent antioxidant, these results suggested that the increased intracellular 8-oxoG level was likely resulted from the inhibition of cellular MTH1 by Echinacoside.
Click to Cistanche vitamin for memory
Treatment with Echinacoside caused extensive DNA damages and significant upregulation of the G1 /S-CDK blocker p21, which were followed by marked apoptotic cell death and suppression of cell proliferation specifically in cancer but not in the noncancer cells. Furthermore, a prominent loss of mitochondrial membrane potential after Echinacoside treatment indicated activation of the intrinsic apoptosis pathway. Echinacoside-induced DNA damages and upregulation of p21 were observed within 5 hours of treatment, while cancer cell apoptosis, disruption of mitochondrial membrane potential, and growth inhibition were observed 12 hours after initiation of treatment. These data support that cancer cell apoptosis and growth inhibition were the results of the extensive DNA damages caused by the inhibition of MTH1. Recent studies have shown that a reduction in the size of the cellular dNTP pool could also induce DNA replication stress and DNA damages.59,60 However, 200% increase in 8-oxoG level in the Echinacoside-treated cancer cells argues against the possibility of a reduced dNTP pool size, and Bcl2, a protein that reduces dNTP pool size,60 was significantly decreased in Echinacoside-treated SW480 cancer cells;58 moreover, our data clearly showed that Echinacoside directly inhibited MTH1, which could at least be partially responsible for the DNA damages and cellular effects caused by Echinacoside. The dNTP pool is a critical target of ROS, and oxidized dNTPs are important sources of oxidative DNA damages.3,10 Repair-associated DNA SSBs and DSBs can lead to cellular senescence and apoptosis, which are implicated in aging and aging-related diseases,61 and also serve as a barrier for tumorigenesis. Thus, reducing ROS-induced DNA damages by antioxidants is believed to be beneficial to overall health. However, several recent studies have shown that antioxidants could also promote the development of some types of cancer.62–64 Cancer cells generate much higher ROS and are critically dependent on the efficient elimination of oxidized nucleotides for survival and proliferation.3,30,32,33
Overexpression of MTH1 is the most important strategy cancer cells use to cope with the lethal burden of oxidized dNTPs.23,24,26–28 Inhibition of MTH1 efficiently killed cancer cells,3,29,30,32,33 while targeting MTH1 in normal cells had no impact on survival,32,33 and MTH1 knockout mice were largely normal.65 Consistently, we also showed that treatment with Echinacoside induced apoptosis and suppressed cell proliferation specifically in cancer but not in noncancer cells. Thus, unlike current chemotherapies and radiotherapies that target normal and tumor cells indiscriminately, the inhibition of MTH1 kills cancer cells highly selectively by using the abundant oxidized nucleotide precursors in tumors. On the other hand, therapies targeting genotype differences between normal and specific types of cancer in a personalized approach have shown impressive results, but they are also limited by the high degree of intratumor heterogeneity and high mutation rates in cancer cells. In contrast, inhibition of MTH1 targets a phenotype that distinguishes most cancer cells from normal cells and hence represents a novel anticancer strategy that is not limited by genetic adaptations. Interestingly, small molecule antagonists of the antiapoptosis protein Bcl266,67 and agonists of the proapoptosis Bax68 have been developed and were demonstrated to be promising novel anticancer agents. Given their complementary mechanisms of action, combining MTH1 inhibitors and apoptosis-promoting chemicals would create an exciting new generation of anticancer drugs. For the first time, we demonstrated a new function for Echinacoside as an anticancer natural compound. In the in vitro assay, Echinacoside inhibited MTH1 with an IC50 of 7.01 μM. This IC50 value is higher than that of the MTH1 inhibitors reported so far.32,33,69 Using (S)-crizotinib as a positive control, we showed that our assay is sevenfold less sensitive than that used by Huber et al.33 Thus, the actual IC50 of Echinacoside is likely to be lower. Nevertheless, to develop it as a therapeutic agent, the efficacy of the natural Echinacoside molecule will probably need to be improved. As a natural product that has been used as a herbal medicine for a long time, Echinacoside could serve as a good chemical scaffold for developing efficient and probably safe MTH1 inhibitors.37,70 Given that natural products have been a rich source of novel chemical scaffolds for rational structure-based drug design,70 approaches similar to what we used here, together with the vast interests in natural products for drug discovery, will be useful in finding target-based, effective, and safe new drugs.
Acknowledgment
This study was supported by a startup fund from Jilin University.
Disclosure
The authors report no conflicts of interest in this work.
References
1 Topal MD, Baker MS. DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells. Proc Natl Acad Sci U S A. 1982;79(7):2211–2215.
2. Ichikawa J, Tsuchimoto D, Oka S, et al. Oxidation of mitochondrial deoxynucleotide pools by exposure to sodium nitroprusside induces cell death. DNA Repair (Amst). 2008;7(3):418–430.
3. Rai P, Onder TT, Young JJ, et al. Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence. Proc Natl Acad Sci U S A. 2009;106(1):169–174.
4. Katafuchi A, Nohmi T. DNA polymerases involved in the incorporation of oxidized nucleotides into DNA: their efficiency and template base preference. Mutat Res. 2010;703(1):24–31.
5. Freudenthal BD, Beard WA, Perera L, et al. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature. 2015;517(7536):635–639.
6. Dizdaroglu M. Oxidatively induced DNA damage and its repair in cancer. Mutat Res Rev Mutat Res. 2015;763:212–245.
7. Nakabeppu Y. Cellular levels of 8-oxoguanine in either DNA or the nucleotide pool play pivotal roles in carcinogenesis and survival of cancer cells. Int J Mol Sci. 2014;15(7):12543–12557.
8. van Loon B, Markkanen E, Hubscher U. Oxygen as a friend and enemy: how to combat the mutational potential of 8-oxo-guanine. DNA Repair (Amst). 2010;9(6):604–616.
9. Pfeifer GP, Besaratinia A. Mutational spectra of human cancer. Hum Genet. 2009;125(5–6):493–506.
10. Russo MT, Blasi MF, Chiera F, et al. The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells. Mol Cell Biol. 2004;24(1): 465–474.
11. Kamiya H. Mutagenicity of oxidized DNA precursors in living cells: roles of nucleotide pool sanitization and DNA repair enzymes, and translesion synthesis DNA polymerases. Mutat Res. 2010;703(1): 32–36.
12. Nagy GN, Leveles I, Vértessy BG. Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool. FEB J. 2014;281(18):4207–4223.
13. McLennan AG. The Nudix hydrolase superfamily. Cell Mol Life Sci. 2006;63(2):123–143.
14. Fujikawa K, Kamiya H, Yakushiji H, Nakabeppu Y, Kasai H. Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP. Nucleic Acids Res. 2001;29(2):449–454. 15. Nakabeppu Y, Oka S, Sheng Z, Tsuchimoto D, Sakumi K. Programmed cell death triggered by nucleotide pool damage and its prevention by MutT homolog-1 (MTH1) with oxidized purine nucleoside triphosphatase. Mutat Res. 2010;703(1):51–58.
16. Dizdaroglu M. Oxidatively induced DNA damage: mechanisms, repair, and disease. Cancer Lett. 2012;327(1–2):26–47.
17. Bridge G, Rashid S, Martin SA. DNA mismatch repair and oxidative DNA damage: implications for cancer biology and treatment. Cancers (Basel). 2014;6(3):1597–1614. 18. Caldecott KW. Single-strand break repair and genetic disease. Nat Rev Genet. 2008;9(8):619–631.
19. Klement K, Goodarzi AA. DNA double-strand break responses and chromatin alterations within the aging cell. Exp Cell Res. 2014;329(1): 42–52.
20. Schulze A, Harris AL. How cancer metabolism is tuned for proliferation and vulnerable to disruption. Nature. 2012;491(7424):364–373. 21. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 2011;11(2):85–95.
22. Rai P. Human Mut T homolog 1 (MTH1): a roadblock for the tumor-suppressive effects of oncogenic Ras-induced ROS. Small GTPases. 2012;3(2):120–125.
23. Kennedy CH, Cueto R, Belinsky SA, Lechner JF, Pryor WA. Over-pression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells. FEBS Lett. 1998;429:17–20.
24. Lida T, Furuta A, Kawashima M, Nishida J, Nakabeppu Y, Iwaki T. Accumulation of 8-oxo-2′-deoxyguanosine and increased expression of hMTH1 protein in brain tumors. Neuro Oncol. 2001;3:73–81.
25. Coskun E, Jaruga P, Jemth AS, et al. Addiction to MTH1 protein results in intense expression in human breast cancer tissue as measured by liquid chromatography-isotope-dilution tandem mass spectrometry. DNA Repair (Amst). 2015;33:101–110. 26. Tudek B, Winczura A, Janik J, Simek A, Foksinski M, Olinski R. Involvement of oxidatively damaged DNA and repair in cancer development and aging. Am J Transl Res. 2010;2(3):254–284.
27. Spina E, Arczewska KD, Jackowski D, et al. Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non-smallcell lung cancer patients. J Natl Cancer Inst. 2005;97(5):384–395.
28. Kennedy CH, Pass HI, Mitchell JB. Expression of human MutT homolog (hMTH1) protein in primary non-small-cell lung carcinomas and histologically normal surrounding tissue. Free Radic Biol Med. 2003;34(11):1447–1457.
29. Rai P, Young JJ, Burton DG, Giribaldi MG, Onder TT, Weinberg RA. Enhanced elimination of oxidized guanine nucleotides inhibits oncogenic Ras-induced DNA damage and premature senescence. Oncogene. 2011;30(12):1489–1496.
30. Sakumi K, Tominaga Y, Furuichi M, et al. Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption. Cancer Res. 2003;63:902–905.
31. Patel A, Burton DG, Halvorsen K, et al. MutT homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways. Oncogene. 2015;34(20):2586–2596.
32. Gad H, Koolmeister T, Jemth AS, et al. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature. 2014; 508(7495):215–221.
33. Huber KV, Salah E, Radic B, et al. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature. 2014;508(7495): 222–227.
34. Giribaldi MG, Munoz A, Halvorsen K, Patel A, Rai P. MTH1 expression is required for effective transformation by oncogenic HRAS. Oncotarget. 2015;6(13):11519–11529.
35. Bauer A, Bronstrup M. Industrial natural product chemistry for drug discovery and development. Nat Prod Rep. 2014;31(1):35–60.
36. Orlova B, Legrand N, Panning J, Dicato M, Diederich M. Anti-inflammatory and anticancer drugs from nature. Cancer Treat Res. 2014; 159:123–143.
37. Li JW, Vederas JC. Drug discovery and natural products: end of an era or an endless frontier? Science. 2009;325(5937):161–165.
38. Liu EH, Qi LW, Wu Q, Peng YB, Li P. Anticancer agents derived from natural products. Mini Rev Med Chem. 2009;9(13):1547–1555.
39. Nobili S, Lippi D, Witort E, et al. Natural compounds for cancer treatment and prevention. Pharmacol Res. 2009;59(6):365–378.
40. Hsiao WL, Liu L. The role of traditional Chinese herbal medicines in cancer therapy – from TCM theory to mechanistic insights. Planta Med. 2010;76(11):1118–1131.
41. Baykov AA, Evtushenko OA, Avaeva SM. A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem. 1988;171(2):266–270.
42. Struthers L, Patel R, Clark J, Thomas S. Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine by avidin and its analogs. Anal Biochem. 1998;255(1):20–31.
43. Sheng Z, Oka S, Tsuchimoto D, et al. 8-Oxoguanine causes neurodegeneration during MUTYH-mediated DNA base excision repair. J Clin Invest. 2012;122(12):4344–4361.
44. Xiong Q, Kadota S, Tani T, Namba T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull. 1996;19(12): 1580–1585.
45. Hu C, Kitts DD. Studies on the antioxidant activity of Echinacea root extract. J Agric Food Chem. 2000;48(5):1466–1472.
46. Wallace SS, Murphy DL, Sweasy JB. Base excision repair and cancer. Cancer Lett. 2012;327(1–2):73–89. 47. Caldecott KW. DNA single-strand break repair. Exp Cell Res. 2014; 329(1):2–8.
48. Schultz LB, Chehab NH, Malikzay A, Halazonetis TD. p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol. 2000;151(7):1381–1390.
49. Li YY, Wang L, Lu CD. An E2F site in the 5′-promoter region contributes to serum-dependent up-regulation of the human proliferating cell nuclear antigen gene. FEBS Lett. 2003;544(1–3):112–118.
50. González Besteiro MA, Gottifredi V. The fork and the kinase: a DNA replication tale from a CHK1 perspective. Mutat Res Rev Mutat Res. 2015;763:168–180.
51. Wang Y, Hao H, Wang G, et al. An approach to identifying sequential metabolites of a typical phenylethanoid glycoside, echinacoside, based on liquid chromatography-ion trap-time of flight mass spectrometry analysis. Talanta. 2009;80(2):572–580.
52. Hudson JB. Applications of the phytomedicine Echinacea purpurea (purple coneflower) in infectious diseases. J Biomed Biotechnol. 2012; 2012:769896.
53. Mulani SK, Guh JH, Mong KK. A general synthetic strategy and the anti-proliferation properties on prostate cancer cell lines for natural phenylethanoid glycosides. Org Biomol Chem. 2014;12(18):2926–2937.
54. Kuo YY, Jim WT, Su LC, et al. Caffeic acid phenethyl ester is a potential therapeutic agent for oral cancer. Int J Mol Sci. 2015;16(5): 10748–10766.
55. Li X, Gou C, Yang H, Qiu J, Gu T, Wen T. Echinacoside ameliorates D-galactosamine plus lipopolysaccharide-induced acute liver injury in mice via inhibition of apoptosis and inflammation. Scand J Gastroen-terol. 2014;49(8):993–1000.
56. Gai XY, Tang F, Ma J, et al. Antiproliferative effect of echinacoside on rat pulmonary artery smooth muscle cells under hypoxia. J Pharmacol Sci. 2014;126(2):155–163.
57. Zhu M, Lu C, Li W. Transient exposure to echinacoside is sufficient to activate Trk signaling and protect neuronal cells from rotenone. J Neurochem. 2013;124(4):571–580.
58. Dong L, Yu D, Wu N, et al. Echinacoside induces apoptosis in human SW480 colorectal cancer cells by induction of oxidative DNA damages. Int J Mol Sci. 2015;16(7):14655–14668.
59. Bester AC, Roniger M, Oren YS, et al. Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell. 2011;145(3):435–446.
60. Xie M, Yen Y, Owonikoko TK, et al. Bcl2 induces DNA replication stress by inhibiting ribonucleotide reductase. Cancer Res. 2014;74(1): 212–223.
61. Ventura I, Russo MT, De Luca G, Bignami M. Oxidized purine nucleotides, genome instability, and neurodegeneration. Mutat Res. 2010; 703(1):59–65.
62. DeNicola GM, Karreth FA, Humpton TJ, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature. 2011;475(7354):106–109.
63. Santos MA, Faryabi RB, Ergen AV, et al. DNA-damage-induced differentiation of leukemic cells as an anti-cancer barrier. Nature. 2014;514(7520):107–111.
64. Sayin VI, Ibrahim MX, Larsson E, Nilsson JA, Lindahl P, Bergo MO. Antioxidants accelerate lung cancer progression in mice. Sci Transl Med. 2014;6(221):221ra215.
65. Egashira A, Yamauchi K, Yoshiyama K, et al. Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes. DNA Repair (Amst). 2002;1(11):881–893.
66. Vaillant F, Merino D, Lee L, et al. Targeting BCL-2 with the BH3 mimetic ABT-199 in estrogen receptor-positive breast cancer. Cancer Cell. 2013;24(1):120–129.
67. Han B, Park D, Li R, et al. Small-molecule Bcl2 BH4 antagonist for lung cancer therapy. Cancer Cell. 2015;27(6):852–863.
68. Xin M, Li R, Xie M, et al. Small-molecule Bax agonists for cancer therapy. Nat Commun. 2014;5:4935.
69. Streib M, Kraling K, Richter K, Xie X, Steuber H, Meggers E. An organometallic inhibitor for the human repair enzyme 7,8- dihydro-8-deoxyguanosine triphosphatase. Angew Chem Int Ed Engl. 2014;53(1):305–309.
70. Szychowski J, Truchon JF, Bennani YL. Natural products in medicine: transformational outcome of synthetic chemistry. J Med Chem. 2014;57(22):9292–9308.
