Pseudo‑Ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway

Wang,Yuchen; Xu,Huali; Lu,Zeyuan; Yu,Xiaofeng; Lv,Chen; Tian,Yuan; Sui,Dayun

doi:10.3892/etm.2018.6067

Pseudo‑Ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway

Authors:
- Yuchen Wang
- Huali Xu
- Zeyuan Lu
- Xiaofeng Yu
- Chen Lv
- Yuan Tian
- Dayun Sui
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Affiliations: Department of Pharmacology, School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin 130021, P.R. China
Published online on: April 13, 2018 https://doi.org/10.3892/etm.2018.6067
Pages: 4916-4924
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Ginsenoside Rh2, a major effective constituent of ginseng, has been suggested to have a pro‑apoptotic effect in a variety of cancer cells. Pseudo‑Ginsenside‑Rh2 (pseudo‑G‑Rh2) is a novel derivative of ginsenoside Rh2. The aim of the present study was to evaluate the effect of pseudo‑G‑Rh2 on the apoptosis of lung adenocarcinoma A549 cells. The cytotoxicity of pseudo‑G‑Rh2 on A549 cells was evaluated using an MTT assay. Apoptosis was detected using DAPI staining and flow cytometry. The expression of apoptosis associated proteins was identified by western blot analysis. The results demonstrated that pseudo‑G‑Rh2 inhibits the proliferation of A549 cells in a dose‑dependent manner. DAPI staining revealed topical morphological changes in apoptotic bodies following pseudo‑G‑Rh2 treatment. Flow cytometric analysis revealed that the percentage of Annexin V‑fluorescein isothiocyanate‑positive cells, which are apoptotic, increased with pseudo‑G‑Rh2 treatment in a dose‑dependent manner. Furthermore, treatment with pseudo‑G‑Rh2 increased the level of reactive oxygen species in A549 cells as well as the activation of caspase‑9, caspase‑3 and poly ADP‑ribose polymerase. Pseudo‑G‑Rh2 treatment was observed to induce mitochondrial membrane potential loss. Furthermore, the results of western blotting revealed that B‑cell lymphoma 2 (Bcl‑2) expression was significantly decreased while Bcl‑2‑associated X protein expression was significantly upregulated in A549 cells with pseudo‑G‑Rh2 treatment. Pseudo‑G‑Rh2‑induced apoptosis was accompanied by sustained phosphorylation of Ras, Raf, extracellular signal‑regulated kinase (ERK) and p53. In conclusion, the results of the present study suggest that pseudo‑G‑Rh2 induces mitochondrial apoptosis in A549 cells and is responsible for excessive activation of the Ras/Raf/ERK/p53 pathway.

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1	Chen W, Zheng R, Zeng H and Zhang S: Epidemiology of lung cancer in China. Thorac Cancer. 6:209–215. 2015. View Article : Google Scholar : PubMed/NCBI
2	Burstein HJ and Schwartz RS: Molecular origins of cancer. N Engl J Med. 358:5272008. View Article : Google Scholar : PubMed/NCBI
3	Uramoto H and Tanaka F: Recurrence after surgery in patients with NSCLC. Transl Lung Cancer Res. 3:242–249. 2014.PubMed/NCBI
4	Rittmeyer A: Quality of life in patients with NSCLC receiving maintenance therapy. Cancers (Basel). 7:950–962. 2015. View Article : Google Scholar : PubMed/NCBI
5	Leidinger P, Brefort T, Backes C, Krapp M, Galata V, Beier M, Kohlhaas J, Huwer H, Meese E and Keller A: High-throughput qRT-PCR validation of blood microRNAs in non-small cell lung cancer. Oncotarget. 7:4611–4623. 2016. View Article : Google Scholar : PubMed/NCBI
6	Balmanno K and Cook SJ: Tumour cell survival signalling by the ERK1/2 pathway. Cell Death Differ. 16:368–377. 2009. View Article : Google Scholar : PubMed/NCBI
7	Cox AD and Der CJ: The dark side of Ras: Regulation of apoptosis. Oncogene. 22:8999–9006. 2003. View Article : Google Scholar : PubMed/NCBI
8	Cagnol S and Chambard JC: ERK and cell death: Mechanisms of ERK-induced cell death-apoptosis, autophagy and senescence. FEBS J. 277:2–21. 2010. View Article : Google Scholar : PubMed/NCBI
9	Randhawa H, Kibble K, Zeng H, Moyer MP and Reindl KM: Activation of ERK signaling and induction of colon cancer cell death by piperlongumine. Toxicol In Vitro. 27:1626–1633. 2013. View Article : Google Scholar : PubMed/NCBI
10	Bacus SS, Gudkov AV, Lowe M, Lyass L, Yung Y, Komarov AP, Keyomarsi K, Yarden Y and Seger R: Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene. 20:147–155. 2001. View Article : Google Scholar : PubMed/NCBI
11	Lv C, Hong Y, Miao L, Li C, Xu G, Wei S, Wang B, Huang C and Jiao B: Wentilactone A as a novel potential antitumor agent induces apoptosis and G2/M arrest of human lung carcinoma cells, and is mediated by HRas-GTP accumulation to excessively activate the Ras/Raf/ERK/p53-p21 pathway. Cell Death Dis. 4:e9522013. View Article : Google Scholar : PubMed/NCBI
12	Liu T, Zhao L, Zhang Y, Chen W, Liu D, Hou H, Ding L and Li X: Ginsenoside 20(S)-Rg3 targets HIF-1α to block hypoxia-induced epithelial-mesenchymal transition in ovarian cancer cells. PLoS One. 9:e1038872014. View Article : Google Scholar : PubMed/NCBI
13	Kiefer D and Pantuso T: Panax ginseng. Am Fam Physician. 68:1539–1542. 2003.PubMed/NCBI
14	Zhang C, Yu H and Hou J: Effects of 20 (S)-ginsenoside Rh2 and 20 (R)-ginsenoside Rh2 on proliferation and apoptosis of human lung adenocarcinoma A549 cells. Zhongguo Zhong Yao Za Zhi. 36:1670–1674. 2011.(In Chinese). PubMed/NCBI
15	Oh M, Choi YH, Choi S, Chung H, Kim K, Kim SI, Kim DK and Kim ND: Anti-proliferating effects of ginsenoside Rh2 on MCF-7 human breast cancer cells. Int J Oncol. 14:869–875. 1999.PubMed/NCBI
16	Liu YF, Yuan HN, Bi XL, Piao HR, Cao JQ, Li W, Wang P and Zhao YQ: 25-Methoxylprotopanaxadiol derivatives and their anti-proliferative activities. Steroids. 78:1305–1311. 2013. View Article : Google Scholar : PubMed/NCBI
17	Qian G, Wang Z, Zhao J, Li D, Gao W, Wang B, Sui D, Qu X and Chen Y: Synthesis and anti-cancer cell activity of pseudo-ginsenoside Rh2. Steroids. 92:1–6. 2014. View Article : Google Scholar : PubMed/NCBI
18	Qu X, Qu S, Yu X, Xu H, Chen Y, Ma X and Sui D: Pseudo-G-Rh2 induces mitochondrial-mediated apoptosis in SGC-7901 human gastric cancer cells. Oncol Rep. 26:1441–1446. 2011.PubMed/NCBI
19	Xu HL, Yu XF, Qu SC, Zhang R, Qu XR, Chen YP, Ma XY and Sui DY: Anti-proliferative effect of Juglone from Juglans mandshurica Maxim on human leukemia cell HL-60 by inducing apoptosis through the mitochondria-dependent pathway. Eur J Pharmacol. 645:14–22. 2010. View Article : Google Scholar : PubMed/NCBI
20	Matin MM, Nakhaeizadeh H, Bahrami AR, Iranshahi M, Arghiani N and Rassouli FB: Ferutinin, an apoptosis inducing terpenoid from Ferula ovina. Asian Pac J Cancer Prev. 15:2123–2128. 2014. View Article : Google Scholar : PubMed/NCBI
21	Chen HY, Zhang X, Chen SF, Zhang YX, Liu YH, Ma LL and Wang LX: The protective effect of 17β-estradiol against hydrogen peroxide-induced apoptosis on mesenchymal stem cell. Biomed Pharmacother. 66:57–63. 2012. View Article : Google Scholar : PubMed/NCBI
22	Deeb D, Gao X, Jiang H, Janic B, Arbab AS, Rojanasakul Y, Dulchavsky SA and Gautam SC: Oleanane triterpenoid CDDO-Me inhibits growth and induces apoptosis in prostate cancer cells through a ROS-dependent mechanism. Biochem Pharmacol. 79:350–360. 2010. View Article : Google Scholar : PubMed/NCBI
23	Wang H and Joseph JA: Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 27:612–616. 1999. View Article : Google Scholar : PubMed/NCBI
24	Feng X, Yu W, Zhou F, Chen J and Shen P: A novel small molecule compound diaporine inhibits breast cancer cell proliferation via promoting ROS generation. Biomed Pharmacother. 83:1038–1047. 2016. View Article : Google Scholar : PubMed/NCBI
25	Trachootham D, Alexandre J and Huang P: Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach? Nat Rev Drug Discov. 8:579–591. 2009. View Article : Google Scholar : PubMed/NCBI
26	Lee YJ, Cho HN, Soh JW, Jhon GJ, Cho CK, Chung HY, Bae S, Lee SJ and Lee YS: Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation. Exp Cell Res. 291:251–266. 2003. View Article : Google Scholar : PubMed/NCBI
27	Zhang P, Wang YZ, Kagan E and Bonner JC: Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK. J Biol Chem. 275:22479–22486. 2000. View Article : Google Scholar : PubMed/NCBI
28	Ringer L, Sirajuddin P, Tricoli L, Waye S, Choudhry MU, Parasido E, Sivakumar A, Heckler M, Naeem A, Abdelgawad I, et al: The induction of the p53 tumor suppressor protein bridges the apoptotic and autophagic signaling pathways to regulate cell death in prostate cancer cells. Oncotarget. 5:10678–10691. 2014. View Article : Google Scholar : PubMed/NCBI
29	Tsuruo T, Naito M, Tomida A, Fujita N, Mashima T, Sakamoto H and Haga N: Molecular targeting therapy of cancer: Drug resistance, apoptosis and survival signal. Cancer Sci. 94:15–21. 2003. View Article : Google Scholar : PubMed/NCBI
30	Abraham MC and Shaham S: Death without caspases, caspases without death. Trends Cell Biol. 14:184–193. 2004. View Article : Google Scholar : PubMed/NCBI
31	Shi C, Zheng DD, Fang L, Wu F, Kwong WH and Xu J: Ginsenoside Rg1 promotes nonamyloidgenic cleavage of APP via estrogen receptor signaling to MAPK/ERK and PI3K/Akt. Biochim Biophys Acta. 1820:453–460. 2012. View Article : Google Scholar : PubMed/NCBI
32	Choi YJ, Yoon JH, Cha SW and Lee SG: Ginsenoside Rh1 inhibits the invasion and migration of THP-1 acute monocytic leukemia cells via inactivation of the MAPK signaling pathway. Fitoterapia. 82:911–919. 2011. View Article : Google Scholar : PubMed/NCBI
33	Zhang C, Zhu H, Yang X, Lou J, Zhu D, Lu W, He Q and Yang B: P53 and p38 MAPK pathways are involved in MONCPT-induced cell cycle G2/M arrest in human non-small cell lung cancer A549. J Cancer Res Clin Oncol. 136:437–445. 2010. View Article : Google Scholar : PubMed/NCBI
34	Shih A, Davis FB, Lin HY and Davis PJ: Resveratrol induces apoptosis in thyroid cancer cell lines via a MAPK- and p53-dependent mechanism. J Clin Endocrinol Metab. 87:1223–1232. 2002. View Article : Google Scholar : PubMed/NCBI
35	Wang X, Martindale JL and Holbrook NJ: Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem. 275:39435–39443. 2000. View Article : Google Scholar : PubMed/NCBI
36	Waris G and Ahsan H: Reactive oxygen species: Role in the development of cancer and various chronic conditions. J Carcinog. 5:142006. View Article : Google Scholar : PubMed/NCBI
37	Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M, Kalyanaraman B, Mutlu GM, Budinger GR and Chandel NS: Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci USA. 107:8788–8793. 2010. View Article : Google Scholar : PubMed/NCBI
38	Wu CL, Huang AC, Yang JS, Liao CL, Lu HF, Chou ST, Ma CY, Hsia TC, Ko YC and Chung JG: Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC)-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of caspase-3, mitochondria dysfunction and nitric oxide (NO) in human osteogenic sarcoma U-2 OS cells. J Orthop Res. 29:1199–1209. 2011. View Article : Google Scholar : PubMed/NCBI
39	Ge G, Yan Y and Cai H: Ginsenoside Rh2 inhibited proliferation by inducing ROS mediated ER stress dependent apoptosis in lung cancer cells. Biol Pharm Bull. 40:2117–2124. 2017. View Article : Google Scholar : PubMed/NCBI
40	Singh DV, Agarwal S, Singh P, Godbole MM and Misra K: Curcumin conjugates induce apoptosis via a mitochondrion dependent pathway in MCF-7 and MDA-MB-231 cell lines. Asian Pac J Cancer Prev. 14:5797–5804. 2013. View Article : Google Scholar : PubMed/NCBI
41	Matsunaga Y, Kawai Y, Kohda Y and Gemba M: Involvement of activation of NADPH oxidase and extracellular signal-regulated kinase (ERK) in renal cell injury induced by zinc. J Toxicol Sci. 30:135–144. 2005. View Article : Google Scholar : PubMed/NCBI
42	Ramachandiran S, Huang Q, Dong J, Lau SS and Monks TJ: Mitogen-activated protein kinases contribute to reactive oxygen species-induced cell death in renal proximal tubule epithelial cells. Chem Res Toxicol. 15:1635–1642. 2002. View Article : Google Scholar : PubMed/NCBI
43	Woessmann W, Chen X and Borkhardt A: Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines. Cancer Chemother Pharmacol. 50:397–404. 2002. View Article : Google Scholar : PubMed/NCBI
44	DeHaan RD, Yazlovitskaya EM and Persons DL: Regulation of p53 target gene expression by cisplatin-induced extracellular signal-regulated kinase. Cancer Chemother Pharmacol. 48:383–388. 2001. View Article : Google Scholar : PubMed/NCBI
45	Kim GS, Hong JS, Kim SW, Koh JM, An CS, Choi JY and Cheng SL: Leptin induces apoptosis via ERK/cPLA2/cytochrome c pathway in human bone marrow stromal cells. J Biol Chem. 278:21920–21929. 2003. View Article : Google Scholar : PubMed/NCBI
46	Li DW, Liu JP, Mao YW, Xiang H, Wang J, Ma WY, Dong Z, Pike HM, Brown RE and Reed JC: Calcium-activated RAF/MEK/ERK signaling pathway mediates p53-dependent apoptosis and is abrogated by alpha B-crystallin through inhibition of RAS activation. Mol Biol Cell. 16:4437–4453. 2005. View Article : Google Scholar : PubMed/NCBI
47	Liu J, Mao W, Ding B and Liang CS: ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes. Am J Physiol Heart Circ Physiol. 295:H1956–H1965. 2008. View Article : Google Scholar : PubMed/NCBI
48	Wu Z, Wu LJ, Tashiro S, Onodera S and Ikejima T: Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis. Chin Med J (Engl). 118:671–677. 2005.PubMed/NCBI