The EDN1/EDNRA/β‑arrestin axis promotes colorectal cancer progression by regulating STAT3 phosphorylation

Lee,Yeo-Jin; Lee,Yeo-Jin; Jung,Eunsun; Jung,Eunsun; Choi,Jinhyeon; Choi,Jinhyeon; Hwang,Jin-Seong; Hwang,Jin-Seong; Jeong,Eun-Jeong; Jeong,Eun-Jeong; Roh,Yuna; Roh,Yuna; Ban,Hyun Seung; Ban,Hyun Seung; Kim,Sunhong; Kim,Sunhong; Kim,Seon-Kyu; Kim,Seon-Kyu; Kim,Seon-Young; Kim,Seon-Young; Min,Jeong-Ki; Min,Jeong-Ki; Han,Tae-Su; Han,Tae-Su; Kim,Jang-Seong; Kim,Jang-Seong

doi:10.3892/ijo.2022.5461

The EDN1/EDNRA/β‑arrestin axis promotes colorectal cancer progression by regulating STAT3 phosphorylation

Authors:
- Yeo-Jin Lee
- Eunsun Jung
- Jinhyeon Choi
- Jin-Seong Hwang
- Eun-Jeong Jeong
- Yuna Roh
- Hyun Seung Ban
- Sunhong Kim
- Seon-Kyu Kim
- Seon-Young Kim
- Jeong-Ki Min
- Tae-Su Han
- Jang-Seong Kim
View Affiliations

Affiliations: Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
Published online on: November 28, 2022 https://doi.org/10.3892/ijo.2022.5461
Article Number: 13
Copyright: © Lee et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Endothelin receptor A (EDNRA) has been reported to play various crucial physiological roles and has been shown to be associated with the pathology of several diseases, including colorectal cancer (CRC). However, the molecular mechanisms of EDNRA in the development of human CRC have not been fully elucidated to date. In this context, the present study was performed to investigate biological functions and novel downstream signaling pathways affected by EDNRA, during CRC progression. First, using public data repositories, it was observed that the EDRNA expression levels were markedly increased in CRC tissues, as compared to normal tissues. Patients with CRC with an increased EDNRA expression exhibited a significantly decreased survival rate in comparison with those with a lower EDNRA expression. Furthermore, a positive correlation between the levels of EDNRA and its ligand, EDN1, was found in CRC tissues. The ectopic expression of EDNRA or its ligand, EDN1, promoted, whereas the silencing of EDNRA or EDN1 decreased cell proliferation and migration in vitro. To elucidate the signaling pathways involved in the regulation of EDNRA expression in CRC cells, a phosphokinase array analysis was performed, and it was observed that the knockdown of EDNRA substantially suppressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in CRC cells. Of note, STAT3 silencing simultaneously decreased EDN1 and EDNRA expression, with the expression of EDN1 and/or EDNRA appearing to be directly regulated by binding STAT3 to their promoter region, according to chromatin immunoprecipitation and promoter assays, ultimately indicating a positive feedback loop in the expression of EDNRA and EDN1. It was also observed that treatment with an EDNRA antagonist (macitentan), alone or in combination with cisplatin, suppressed cell growth and migration ability, and induced cell apoptosis. Collectively, these data suggest a critical role of the EDN1/EDNRA signaling pathway in CRC progression. Thus, the pharmacological intervention of this signaling pathway may prove to be a potential therapeutic approach for patients with CRC.

View Figures

View References

1	Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 71:209–249. 2021. View Article : Google Scholar : PubMed/NCBI
2	Siegel RL, Miller KD, Sauer AG, Fedewa SA, Butterly LF, Anderson JC, Cercek A, Smith RA and Jemal A: Colorectal cancer statistics, 2020. CA Cancer J Clin. 70:145–164. 2020. View Article : Google Scholar : PubMed/NCBI
3	Murphy CC, Harlan LC, Lund JL, Lynch CF and Geiger AM: Patterns of colorectal cancer care in the United States: 1990-2010. J Natl Cancer Inst. 107:djv1982015. View Article : Google Scholar : PubMed/NCBI
4	Keum N and Giovannucci E: Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol. 16:713–732. 2019. View Article : Google Scholar : PubMed/NCBI
5	Sanchez-Gundin J, Fernandez-Carballido A M, Martinez-Valdivieso L, Barreda-Hernandez D and Torres-Suarez AI: New trends in the therapeutic approach to metastatic colorectal cancer. Int J Med Sci. 15:659–665. 2018. View Article : Google Scholar : PubMed/NCBI
6	Oh DY and Bang YJ: HER2-targeted therapies-a role beyond breast cancer. Nat Rev Clin Oncol. 17:33–48. 2020. View Article : Google Scholar
7	Ferguson FM and Gray NS: Kinase inhibitors: The road ahead. Nat Rev Drug Discov. 17:353–377. 2018. View Article : Google Scholar : PubMed/NCBI
8	Meric-Bernstam F and Mills GB: Overcoming implementation challenges of personalized cancer therapy. Nat Rev Clin Oncol. 9:542–548. 2012. View Article : Google Scholar : PubMed/NCBI
9	Sala E, Mologni L, Truffa S, Gaetano C, Bollag GE and Gambacorti-Passerini C: BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells. Mol Cancer Res. 6:751–759. 2008. View Article : Google Scholar : PubMed/NCBI
10	Chung CH, Mirakhur B, Chan E, Le QT, Berlin J, Morse M, Murphy BA, Satinover SM, Hosen J, Mauro D, et al: Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med. 358:1109–1117. 2008. View Article : Google Scholar : PubMed/NCBI
11	Cohen MH, Gootenberg J, Keegan P and Pazdur R: FDA drug approval summary: Bevacizumab plus FOLFOX4 as second-line treatment of colorectal cancer. Oncologist. 12:356–361. 2007. View Article : Google Scholar : PubMed/NCBI
12	Le XF, Pruefer F and Bast RC Jr: HER2-targeting antibodies modulate the cyclin-dependent kinase inhibitor p27Kip1 via multiple signaling pathways. Cell Cycle. 4:87–95. 2005. View Article : Google Scholar
13	Curigliano G and Criscitiello C: Successes and limitations of targeted cancer therapy in breast cancer. Prog Tumor Res. 41:15–35. 2014. View Article : Google Scholar : PubMed/NCBI
14	Holcmann M and Sibilia M: Mechanisms underlying skin disorders induced by EGFR inhibitors. Mol Cell Oncol. 2:e10049692015. View Article : Google Scholar
15	Welsh SJ and Corrie PG: Management of BRAF and MEK inhibitor toxicities in patients with metastatic melanoma. Ther Adv Med Oncol. 7:122–136. 2015. View Article : Google Scholar : PubMed/NCBI
16	Spaans JN and Goss GD: Drug resistance to molecular targeted therapy and its consequences for treatment decisions in non-small-cell lung cancer. Front Oncol. 4:1902014. View Article : Google Scholar : PubMed/NCBI
17	Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K and Masaki T: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 332:411–415. 1988. View Article : Google Scholar : PubMed/NCBI
18	Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K and Masaki T: The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA. 86:2863–2867. 1989. View Article : Google Scholar : PubMed/NCBI
19	Davenport AP, Hyndman KA, Dhaun N, Southan C, Kohan DE, Pollock JS, Pollock DM, Webb DJ and Maguire JJ: Endothelin. Pharmacol Rev. 68:357–418. 2016. View Article : Google Scholar : PubMed/NCBI
20	Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K and Masaki T: Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 348:732–735. 1990. View Article : Google Scholar : PubMed/NCBI
21	Rosanò L, Spinella F and Bagnato A: Endothelin 1 in cancer: Biological implications and therapeutic opportunities. Nat Rev Cancer. 13:637–651. 2013. View Article : Google Scholar : PubMed/NCBI
22	Bagnato A and Natali PG: Endothelin receptors as novel targets in tumor therapy. J Transl Med. 2:162004. View Article : Google Scholar : PubMed/NCBI
23	Rosanò L and Bagnato A: Endothelin therapeutics in cancer: Where are we? Am J Physiol Regul Integr Comp Physiol. 310:R469–R475. 2016. View Article : Google Scholar : PubMed/NCBI
24	Bondurand N, Dufour S and Pingault V: News from the endothelin-3/EDNRB signaling pathway: Role during enteric nervous system development and involvement in neural crest-associated disorders. Dev Biol. 444(Suppl 1): S156–S169. 2018. View Article : Google Scholar : PubMed/NCBI
25	Kohan DE, Rossi NF, Inscho EW and Pollock DM: Regulation of blood pressure and salt homeostasis by endothelin. Physiol Rev. 91:1–77. 2011. View Article : Google Scholar : PubMed/NCBI
26	Nelson JB, Udan MS, Guruli G and Pflug BR: Endothelin-1 inhibits apoptosis in prostate cancer. Neoplasia. 7:631–637. 2005. View Article : Google Scholar : PubMed/NCBI
27	Smith MP, Rowling EJ, Miskolczi Z, Ferguson J, Spoerri L, Haass NK, Sloss O, McEntegart S, Arozarena I, von Kriegsheim A, et al: Targeting endothelin receptor signalling overcomes heterogeneity driven therapy failure. EMBO Mol Med. 9:1011–1029. 2017. View Article : Google Scholar : PubMed/NCBI
28	Laurberg JR, Jensen JB, Schepeler T, Borre M, Orntoft TF and Dyrskjot L: High expression of GEM and EDNRA is associated with metastasis and poor outcome in patients with advanced bladder cancer. BMC Cancer. 14:6382014. View Article : Google Scholar : PubMed/NCBI
29	Minchenko DO, Tsymbal DO, Riabovol OO, Viletska YM, Lahanovska YO, Sliusar MY, Bezrodnyi BH and Minchenko OH: Hypoxic regulation of EDN1, EDNRA, EDNRB, and ECE1 gene expressions in ERN1 knockdown U87 glioma cells. Endocr Regul. 53:250–262. 2019. View Article : Google Scholar : PubMed/NCBI
30	Kawai K, Viars C, Arden K, Tarin D, Urquidi V and Goodison S: Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell line. Genes Chromosomes Cancer. 34:1–8. 2002. View Article : Google Scholar : PubMed/NCBI
31	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
32	Schust J, Sperl B, Hollis A, Mayer TU and Berg T: Stattic: A small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol. 13:1235–1242. 2006. View Article : Google Scholar : PubMed/NCBI
33	Gatfield J, Mueller Grandjean C, Sasse T, Clozel M and Nayler O: Slow receptor dissociation kinetics differentiate macitentan from other endothelin receptor antagonists in pulmonary arterial smooth muscle cells. PLoS One. 7:e476622012. View Article : Google Scholar : PubMed/NCBI
34	Sidharta PN, van Giersbergen PL, Halabi A and Dingemanse J: Macitentan: Entry-into-humans study with a new endothelin receptor antagonist. Eur J Clin Pharmacol. 67:977–984. 2011. View Article : Google Scholar : PubMed/NCBI
35	Hong IS, Coe HV and Catanzaro LM: Macitentan for the treatment of pulmonary arterial hypertension. Ann Pharmacother. 48:538–547. 2014. View Article : Google Scholar : PubMed/NCBI
36	Wang J, Gareri C and Rockman HA: G-protein-coupled receptors in heart disease. Circ Res. 123:716–735. 2018. View Article : Google Scholar : PubMed/NCBI
37	Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB and Gloriam DE: Trends in GPCR drug discovery: New agents, targets and indications. Nat Rev Drug Discov. 16:829–842. 2017. View Article : Google Scholar : PubMed/NCBI
38	Rosanò L, Cianfrocca R, Tocci P, Spinella F, Castro VD, Caprara V, Semprucci E, Ferrandina G, Natali PG and Bagnato A: Endothelin A receptor/β-arrestin signaling to the Wnt pathway renders ovarian cancer cells resistant to chemotherapy. Cancer Res. 74:7453–7464. 2014. View Article : Google Scholar
39	Kelland NF, Bagnall AJ, Morecroft I, Gulliver-Sloan FH, Dempsie Y, Nilsen M, Yanagisawa M, Maclean MR, Kotelevtsev YV and Webb DJ: Endothelial ET(B) limits vascular remodelling and development of pulmonary hypertension during hypoxia. J Vasc Res. 47:16–22. 2010. View Article : Google Scholar
40	Schneider MP, Boesen EI and Pollock DM: Contrasting actions of endothelin ET(A) and ET(B) receptors in cardiovascular disease. Annu Rev Pharmacol Toxicol. 47:731–759. 2007. View Article : Google Scholar
41	Wulfing C, Eltze E, Yamini J, Wülfing P, Bierer S, Böcker W, Hertle L, Semjonow A and Sievert KD: Expression of the endothelin axis in bladder cancer: Relationship to clinicopathologic parameters and long-term survival. Eur Urol. 47:593–600. 2005. View Article : Google Scholar : PubMed/NCBI
42	Fu W, Wu X, Yang Z and Mi H: The effect of miR-124-3p on cell proliferation and apoptosis in bladder cancer by targeting EDNRB. Arch Med Sci. 15:1154–1162. 2019. View Article : Google Scholar : PubMed/NCBI
43	Mahdi MR, Georges RB, Ali DM, Bedeer RF, Eltahry HM, Gabr AEHZ and Berger MR: Modulation of the endothelin system in colorectal cancer liver metastasis: Influence of epigenetic mechanisms? Front Pharmacol. 11:1802020. View Article : Google Scholar : PubMed/NCBI
44	Hsu LS, Lee HC, Chau GY, Yin PH, Chi CW and Lui WY: Aberrant methylation of EDNRB and p16 genes in hepatocellular carcinoma (HCC) in Taiwan. Oncol Rep. 15:507–511. 2006.PubMed/NCBI
45	Tao K, Wu C, Wu K, Li W, Han G, Shuai X and Wang G: Quantitative analysis of promoter methylation of the EDNRB gene in gastric cancer. Med Oncol. 29:107–112. 2012. View Article : Google Scholar
46	Yegnasubramanian S, Kowalski J, Gonzalgo ML, Zahurak M, Piantadosi S, Walsh PC, Bova GS, Marzo AM, Isaacs WB and Nelson WG: Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res. 64:1975–1986. 2004. View Article : Google Scholar : PubMed/NCBI
47	Soh BS, Ng SY, Wu H, Buac K, Park JHC, Lian X, Xu J, Foo KS, Felldin U, He X, et al: Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells. Nat Commun. 7:107742016. View Article : Google Scholar : PubMed/NCBI
48	Alvarez D, Briassouli P, Clancy RM, Zavadil J, Reed JH, Abellar RG, Halushka M, Fox-Talbot K, Barrat FJ and Buyon JP: A novel role of endothelin-1 in linking Toll-like receptor 7-mediated inflammation to fibrosis in congenital heart block. J Biol Chem. 286:30444–30454. 2011. View Article : Google Scholar : PubMed/NCBI
49	Cianfrocca R, Rosano L, Tocci P, Sestito R, Caprara V, Castro VD, Maria RD and Bagnato A: Blocking endothelin-1-receptor/beta-catenin circuit sensitizes to chemotherapy in colorectal cancer. Cell Death Differ. 24:1811–1820. 2017. View Article : Google Scholar : PubMed/NCBI
50	Wei W, Shi L, Chen W, Hu L, Chen D, Shi X, Xiang H, Guo C and Wu Z: miR-200c regulates the proliferation, apoptosis and invasion of gastric carcinoma cells through the downregulation of EDNRA expression. Int J Mol Med. 41:1619–1626. 2018.
51	Sestito R, Cianfrocca R, Tocci P, Rosanò L, Sacconi A, Blandino G and Bagnato A: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer. Commun Biol. 3:6772020. View Article : Google Scholar : PubMed/NCBI
52	Kamran MZ, Patil P and Gude RP: Role of STAT3 in cancer metastasis and translational advances. Biomed Res Int. 2013:4218212013. View Article : Google Scholar : PubMed/NCBI
53	Corvinus FM, Orth C, Moriggl R, Tsareva SA, Wagner S, Pfitzner EB, Baus D, Kaufmann R, Huber LA, Zatloukal K, et al: Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth. Neoplasia. 7:545–555. 2005. View Article : Google Scholar : PubMed/NCBI
54	Lin L, Liu A, Peng Z, Lin HJ, Li PK, Li C and Lin J: STAT3 is necessary for proliferation and survival in colon cancer-initiating cells. Cancer Res. 71:7226–7237. 2011. View Article : Google Scholar : PubMed/NCBI
55	Wendt MK, Balanis N, Carlin CR and Schiemann WP: STAT3 and epithelial-mesenchymal transitions in carcinomas. JAKSTAT. 3:e289752014.PubMed/NCBI
56	Thaete LG, Jilling T, Synowiec S, Khan S and Neerhof MG: Expression of endothelin 1 and its receptors in the hypoxic pregnant rat. Biol Reprod. 77:526–532. 2007. View Article : Google Scholar : PubMed/NCBI
57	Morikawa T, Baba Y, Yamauchi M, Kuchiba A, Nosho K, Shima K, Tanaka N, Huttenhower C, Frank DA, Fuchs CS and Ogino S: STAT3 expression, molecular features, inflammation patterns, and prognosis in a database of 724 colorectal cancers. Clin Cancer Res. 17:1452–1462. 2011. View Article : Google Scholar : PubMed/NCBI
58	Kankanamalage SG, Karra AS and Cobb MH: WNK pathways in cancer signaling networks. Cell Commun Signal. 16:722018. View Article : Google Scholar
59	Sato A, Shimizu M, Goto T, Masuno H, Kagechika H, Tanaka N and Shibuya H: WNK regulates Wnt signalling and β-Catenin levels by interfering with the interaction between β-catenin and GID. Commun Biol. 3:6662020. View Article : Google Scholar
60	Sulzmaier FJ, Jean C and Schlaepfer DD: FAK in cancer: Mechanistic findings and clinical applications. Nat Rev Cancer. 14:598–610. 2014. View Article : Google Scholar : PubMed/NCBI
61	Zhao J and Guan JL: Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Rev. 28:35–49. 2009. View Article : Google Scholar : PubMed/NCBI
62	Yang Y, Wang Y, Che X, Hou K, Wu J, Zheng C, Cheng Y, Liu Y, Hu X and Zhang J: Integrin alpha5 promotes migration and invasion through the FAK/STAT3/AKT signaling pathway in icotinib-resistant non-small cell lung cancer cells. Oncol Lett. 22:5562021. View Article : Google Scholar
63	Pei G, Lan Y, Chen D, Ji L and Hua ZC: FAK regulates E-cadherin expression via p-SrcY416/p-ERK1/2/p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway in B16F10 melanoma cells. Oncotarget. 8:13898–13908. 2017. View Article : Google Scholar : PubMed/NCBI
64	Pulido T, Adzerikho I, Channick RN, Delcroix M, Galiè N, Ghofrani HA, Jansa P, Jing ZC, Brun FOL, Mehta S, et al: Macitentan and morbidity and mortality in pulmonary arterial hypertension. N Engl J Med. 369:809–818. 2013. View Article : Google Scholar : PubMed/NCBI
65	Russignan A, Dal Collo G, Bagnato A, Tamassia N, Bugatti M, Belleri M, Lorenzi L, Borsi E, Bazzoni R, Gottardi M, et al: Targeting the endothelin-1 receptors curtails tumor growth and angiogenesis in multiple myeloma. Front Oncol. 10:6000252020. View Article : Google Scholar
66	Tocci P, Cianfrocca R, Di Castro V, Rosanò L, Sacconi A, Donzelli S, Bonfiglio S, Bucci G, Vizza E, Ferrandina G, et al: β-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancer. Nat Commun. 10:31962019. View Article : Google Scholar
67	Lee HJ, Hanibuchi M, Kim SJ, Yu H, Kim MS, He J, Langley RR, Lehembre F, Regenass U and Fidler IJ: Treatment of experimental human breast cancer and lung cancer brain metastases in mice by macitentan, a dual antagonist of endothelin receptors, combined with paclitaxel. Neuro Oncol. 18:486–496. 2016. View Article : Google Scholar : PubMed/NCBI
68	Kim SJ, Lee HJ, Kim MS, Choi HJ, He J, Wu Q, Aldape K, Weinberg JS, Yung WKA, Conrad CA, et al: Macitentan, a dual endothelin receptor antagonist, in combination with temozolomide leads to glioblastoma regression and long-term survival in mice. Clin Cancer Res. 21:4630–4641. 2015. View Article : Google Scholar : PubMed/NCBI
69	Damia G, Filiberti L, Vikhanskaya F, Carrassa L, Taya Y, D'incalci M and Broggini M: Cisplatinum and taxol induce different patterns of p53 phosphorylation. Neoplasia. 3:10–16. 2001. View Article : Google Scholar : PubMed/NCBI
70	Persons DL, Yazlovitskaya EM and Pelling JC: Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J Biol Chem. 275:35778–35785. 2000. View Article : Google Scholar : PubMed/NCBI
71	Dasari S and Tchounwou PB: Cisplatin in cancer therapy: Molecular mechanisms of action. Eur J Pharmacol. 740:364–378. 2014. View Article : Google Scholar : PubMed/NCBI
72	Tchounwou PB, Dasari S, Noubissi FK, Ray P and Kumar S: Advances in our understanding of the molecular mechanisms of action of cisplatin in cancer therapy. J Exp Pharmacol. 13:303–328. 2021. View Article : Google Scholar : PubMed/NCBI
73	Ohtani H, Arimoto Y, Nishio K, Kanamiya Y, Oba H, Adachi K, Shintani M, Nakamura R and Yui S: Efficacy and toxicity of fluorouracil, leucovorin plus oxaliplatin (FOLFOX4 and modified FOLFOX6) followed by fluorouracil, leucovorin plus irinotecan(FOLFIRI)for advanced or metastatic colorectal cancer-case studies. Gan To Kagaku Ryoho. 35:1769–1774. 2008.PubMed/NCBI
74	Gustavsson B, Carlsson G, Machover D, Petrelli N, Roth A, Schmoll HJ, Tveit KM and Gibson F: A review of the evolution of systemic chemotherapy in the management of colorectal cancer. Clin Colorectal Cancer. 14:1–10. 2015. View Article : Google Scholar : PubMed/NCBI