Effect of <i>Orostachys japonicus</i> on Apoptosis and Autophagy in Human monocytic leukemia Cell line THP-1 via Inhibition of NF-<i>κ</i>B and Phosphorylation of p38 MAPK

Seonghee Joo; Eungyeong Jang; Youngchul Kim; Joo, Seonghee; Jang, Eungyeong; Kim, Youngchul

doi:10.13048/jkm.19015

JKM > Volume 40(2); 2019 > Article

Joo, Jang, and Kim: Effect of Orostachys japonicus on Apoptosis and Autophagy in Human monocytic leukemia Cell line THP-1 via Inhibition of NF-κB and Phosphorylation of p38 MAPK

Original Article

The Journal of Korean Medicine 2019; 40(2): 35-50.

Published online: June 30, 2019

DOI: https://doi.org/10.13048/jkm.19015

Effect of Orostachys japonicus on Apoptosis and Autophagy in Human monocytic leukemia Cell line THP-1 via Inhibition of NF-κB and Phosphorylation of p38 MAPK

Seonghee Joo

, Eungyeong Jang

, Youngchul Kim^*

Department of Internal Medicine, College of Korean Medicine, Kyung Hee University

Correspondence to: 김영철, 서울시 동대문구 경희대로 23 경희의료원 간장조혈내과, Tel: +82-2-958-9118, Fax: +82-2-958-9258, E-mail : yckim@khmc.or.kr

Received April 2, 2019 Revised May 23, 2019 Accepted May 23, 2019

Abstract

Objectives

Orostachys japonicas (O. japonicus) has been known for its anti-tumor effect. In the present study, it was investigated whether O. japonicus EtOH extracts could induce apoptosis and autophagy which are part of the main mechanism related to anti-tumor effect in THP-1 cells.

Methods

Cells were treated with various concentrations of O. japonicus EtOH extracts (0–300μg/ml) for 24, 48, and 72h. Cell viability was evaluated by MTS/PMS assay and apoptosis rate was examined by flow cytometry and ELISA assay. The mRNA expression of apoptosis-related genes (Bcl-2, Mcl-1, Survivin, Bax) and autophagy-related gene (mTOR) was evaluated using real-time PCR. The protein expression of Caspase-3, Akt, LC3II, Beclin-1, Atg5, NF-κB, p38, ERK was evaluated using western blot analysis.

Results

O. japonicus EtOH extracts inhibited cell proliferation and apoptosis rate was increased in both flow cytometry and ELISA assay. Bcl-2, Mcl-1, Survivin (anti-apoptosis factors) mRNA expressions were decreased and Bax (pro-apoptosis factor) mRNA level was increased. mTOR mRNA expressions was decreased and LC3II protein expressions was increased. Activation of NF-κB was decreased and phosphorylation of p38 was increased.

Conclusion

O. japonicus is regarded to inhibit cell proliferation, to induce apoptosis and to regulate autophagy-related genes in THP-1 cells via NF-κB and p38 MAPK signaling pathway. This suggests O. japonicus could be an effective herb in treating acute myeloid leukemia.

Keywords: Orostachys japonicus, THP-1, acute myeloid leukemia, apoptosis, autophagy

Fig. 1

HPLC chromatograms of the representative standards (epicatechin gallate (ECG), quercetin and kaempferol)(A) and the O. japonicus EtOH extracts(B)

Fig. 2

Effects of O. japonicus EtOH extracts on the cell viability in THP-1 cells

Cells were treated with various concentrations of O. japonicus EtOH extracts (0–500μg/ml) for 24, 48, and 72 h. Cell viability was evaluated using MTS/PMS assay. The control group was assigned a value of 100%. The data are the mean±SD of triplicate samples.

Fig. 3

The early and late apoptosis of O. japonicus EtOH extracts in THP-1 cells

Cells were treated with various concentration O. japonicus EtOH extracts (0, 100, 200, 300μg/ml) for 24, 48, and 72h. The apoptosis rate was analyzed using flow cytometry with Annexin V-FITC and PI staining.

Fig. 4

Apoptosis rate of O. japonicus EtOH extracts in THP-1 cells

Cells were treated with various concentration O. japonicus EtOH extracts (0, 100, 200, 300μg/ml) for 24, 48, and 72h. Apoptotic cells were measured using a cell death detection ELISA. The control group was assigned a value of 100%. The data are the mean±SD of triplicate samples. (*p<0.05 and **p<0.01 compared to the control.)

Fig. 5

Expression of apoptosis-related genes in O. japonicus extract treated THP-1 cells

Fig. 6

Expression of Caspase-3 and Akt in O. japonicus extract treated THP-1 cells

Cells were treated with various concentration O. japonicus EtOH extracts (0, 100, 200, 300μg/ml) for 24, 48, and 72h. The protein levels were measured by western blot analysis. Cells were lysed and 10μg of soluble protein was separated by electrophoresis on a 10 % SDS-PAGE gel. Densitometry analyses are presented as the relative ratios of caspase-3 and Akt to β-actin. The data are the mean±SD of triplicate samples. (*p<0.05 and **p<0.01 compared to the control.)

Fig. 7

Cell viability in the presence of caspase inhibitors in O. japonicus EtOH extracts treated THP-1 cells

Cells were treated with pan-caspase inhibitor (Z-VAD-FMK) (25–100μM) or caspase-3 inhibitor (Z-DEVD-FMK) (25–100μM) 2h before O. japonicus EtOH extracts treatment (300μg/ml). Cells were incubated for 72h and cell viability was measured by MTS/PMS assay. The data are the mean±SD of triplicate samples. (*p<0.05 and **p<0.01 compared to O. japonicus EtOH extracts-treated cells vs. caspase inhibitor-treated cells with O. japonicus EtOH extracts.)

Fig. 8

Expression of autophagy-related genes in O. japonicus EtOH extracts treated THP-1 cells

Cells were treated with various concentration O. japonicus EtOH extracts (0, 100, 200, 300μg/ml) for 24, 48, and 72h. The mRNA levels were measured by real-time PCR. The crossing point of mTOR with β-actin was applied to the formula, 2-(target gene-β-actin), and relative amounts were quantified. The protein levels were measured by western blot analysis. Cells were lysed and 10μg of soluble protein was separated by electrophoresis on a 10% SDS-PAGE gel. Densitometry analyses are presented as the relative ratios of LC3 II, Beclin-1 and Atg5 to β-actin. The data are the mean±SD of triplicate samples. (*p<0.05 and **p<0.01 compared to the control.)

Fig. 9

Expression of NF-κB and MAPK signaling pathway in O. japonicus EtOH extracts treated THP-1 cells

Cells were treated with various concentration O. japonicus EtOH extracts (0, 100, 200, 300μg/ml) for 24, 48, and 72h. The protein levels were measured by western blot analysis. Cells were lysed and 10μg of soluble protein was separated by electrophoresis on a 10% SDS-PAGE gel. Densitometry analyses are presented as the relative ratios of NF-B, p38 and ERK1/2 to β-actin. The data are shown as means±SD of three independent samples. (*p<0.05 and **p<0.01 compared to the control.)

Table 1

Primer Sequences for Real-Time PCR

Gene	Primer sequence	Size (bp)
Bcl-2	5′ GAT TGA TGG GAT CGT TGC CTT A 3′	200
Bcl-2	5′ CCT TGG CAT GAG ATG CAG GA 3′	200

Bax	5′ GGA TGC GTC CAC CAA GAA G 3′	216
Bax	5′ GCC TTG AGC ACC AGT TTG C 3′	216

Mcl-1	5′ CTC ATT TCT TTT GGT GCC TTT 3′	117
Mcl-1	5′ CCA GTC CCG TTT TGT CCT TAC 3′;	117

Survivin	5′ GGC CCA GTG TTT CTT CTG CTT 3′	91
Survivin	5′ GCA ACC GGA CGA ATG CTT T 3′	91

mTOR	5′ CCT GCC ACT GAG AGA TGA CA 3′	168
mTOR	5′ TCC GGC TGC TGT AGC TTA TT 3′	168

β-actin	5′ GCG AGA AGA TGA CCC AGA TC 3′	77
β-actin	5′ GGA TAG CAC AGC CTG GAT AG 3′	77

참고문헌

1. De Kouchkovsky, I, & Abdul-Hay, M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood cancer journal, (2016). 6(7), e441.

2. Park, EH, Lee, H, Won, YJ, Ju, HY, Oh, CM, & Ingabire, C, et al. Nationwide statistical analysis of myeloid malignancies in Korea: incidence and survival rate from 1999 to 2012. Blood research, (2015). 50(4), 204-217.

3. Lowenberg, B, Downing, JR, & Burnett, A. Acute myeloid leukemia. New England Journal of Medicine, (1999). 341(14), 1051-1062.

4. Shah, A, Andersson, TML, Rachet, B, Björkholm, M, & Lambert, PC. Survival and cure of acute myeloid leukaemia in England, 1971–2006: a population-based study. British journal of haematology, (2013). 162(4), 509-516.

5. Meyers, J, Yu, Y, Kaye, JA, & Davis, KL. Medicare fee-for-service enrollees with primary acute myeloid leukemia: an analysis of treatment patterns, survival, and healthcare resource utilization and costs. Applied health economics and health policy, (2013). 11(3), 275-286.

6. Ahn DG. Illustrated guide to clinical medicinal herbs. Seoul. Hyun-am-sa;(2012). p. 697-698.

7. Ryu, DS, Baek, GO, Kim, EY, Kim, KH, & Lee, DS. Effects of polysaccharides derived from Orostachys Japonicus on induction of cell cycle arrest and apoptotic cell death in human colon cancer cells. BMB reports, (2010). 43(11), 750-755.

8. Shin, DY, Lee, WS, Jung, JH, Hong, SH, Park, C, & Kim, HJ, et al. Flavonoids from Orostachys japonicus A. Berger inhibit the invasion of LnCaP prostate carcinoma cells by inactivating Akt and modulating tight junctions. International journal of molecular sciences, (2013). 14(9), 18407-18420.

9. Ryu, DS, Lee, HS, Lee, GS, & Lee, DS. Effects of the ethylacetate extract of Orostachys japonicus on induction of apoptosis through the p53-mediated signaling pathway in human gastric cancer cells. Biological and Pharmaceutical Bulletin, (2012). 35(5), 660-665.

10. Kim, YI, Park, SW, Choi, IH, Lee, JH, Woo, HJ, & Kim, Y. Effect of Orostachys japonicus on cell growth and apoptosis in human hepatic stellate cell line LX2. The American journal of Chinese medicine, (2011). 39(03), 601-613.

11. Koppula, S, Yum, MJ, Kim, JS, Shin, GM, Chae, YJ, & Yoon, T, et al. Anti-fibrotic effects of Orostachys japonicus A. Berger (Crassulaceae) on hepatic stellate cells and thioacetamide-induced fibrosis in rats. Nutrition research and practice, (2017). 11(6), 470-478.

12. Lee, HS, Ryu, DS, Lee, GS, & Lee, DS. Anti-inflammatory effects of dichloromethane fraction from Orostachys japonicus in RAW 264.7 cells: suppression of NF-κB activation and MAPK signaling. Journal of ethnopharmacology, (2012). 140(2), 271-276.

13. Lee, HS, Lee, GS, Kim, SH, Kim, HK, Suk, DH, & Lee, DS. Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway. BMB reports, (2014). 47(2), 98.

14. Gerl, R, & Vaux, DL. Apoptosis in the development and treatment of cancer. Carcinogenesis, (2005). 26(2), 263-270.

15. Mathew, R, Karantza-Wadsworth, V, & White, E. Role of autophagy in cancer. Nature Reviews Cancer, (2007). 7(12), 961.

16. Choi, ES, & Lee, JH. Orostachys japonicus DW and EtOH extracts Induce Apoptosis in Cholangiocarcinoma Cell Line SNU-1079. Journal of Korean Medicine, (2015). 36(4), 19-34.

17. Korean traditional herbal medicine standard book. Korea national legal information center;[cited 2018 December 10]. Available from: http://www.law/go.kr/admRulSc.do?tabMenuId=tab107

18. Sill, H, Olipitz, W, Zebisch, A, Schulz, E, & Wölfler, A. Therapy-related myeloid neoplasms: pathobiology and clinical characteristics. British journal of pharmacology, (2011). 162(4), 792-805.

19. Döhner, H, Estey, EH, Amadori, S, Appelbaum, FR, Büchner, T, & Burnett, AK, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood, (2010). 115(3), 453-474.

20. Vardiman, JW, Thiele, J, Arber, DA, Brunning, RD, Borowitz, MJ, & Porwit, A, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood, (2009). 114(5), 937-951.

21. Löwenberg, B, Zittoun, R, Kerkhofs, H, Jehn, U, Abels, J, & Debusscher, L, et al. On the value of intensive remission-induction chemotherapy in elderly patients of 65+ years with acute myeloid leukemia: a randomized phase III study of the European Organization for Research and Treatment of Cancer Leukemia Group. Journal of Clinical Oncology, (1989). 7(9), 1268-1274.

22. Cho, CS, & Cho, JG. A literature review of Orostachys japonicus. Collection of dissertations from Korean Medicine Laboratory, (1994). 2(2), 83-98.

23. Lee, GS, Lee, HS, Kim, SH, Suk, DH, Ryu, DS, & Lee, DS. Anti-cancer activity of the ethylacetate fraction from Orostachys japonicus for modulation of the signaling pathway in HepG2 human hepatoma cells. Food Science and Biotechnology, (2014). 23(1), 269-275.

24. Vivanco, I, & Sawyers, CL. The phosphatidylinositol 3-kinase-AKT pathway in human cancer. Nature Reviews Cancer, (2002). 2(7), 489.

25. Shin, DY, Lee, WS, Jung, JH, Hong, SH, Park, C, & Kim, HJ, et al. Flavonoids from Orostachys japonicus A. Berger inhibit the invasion of LnCaP prostate carcinoma cells by inactivating Akt and modulating tight junctions. International journal of molecular sciences, (2013). 14(9), 18407-18420.

26. Jung, CH, Ro, SH, Cao, J, Otto, NM, & Kim, DH. mTOR regulation of autophagy. FEBS letters, (2010). 584(7), 1287-1295.

27. Tanida I, Ueno T, Kominami E. LC3 and Autophagy. Autophagosome and Phagosome. Humana press;(2008). p. 77-88.

28. Kang, R, Zeh, H, Lotze, M, & Tang, D. The Beclin 1 network regulates autophagy and apoptosis. Cell death and differentiation, (2011). 18(4), 571.

29. Codogno, P, & Meijer, AJ. Atg5: more than an autophagy factor. Nature cell biology, (2006). 8(10), 1045.

30. Kim, A, Yim, NH, & Ma, JY. Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells. BMC complementary and alternative medicine, (2013). 13(1), 233.

31. Reed, JC. Apoptosis-targeted therapies for cancer. Cancer cell, (2003). 3(1), 17-22.

32. Murphy, LO, & Blenis, J. MAPK signal specificity: the right place at the right time. Trends in biochemical sciences, (2006). 31(5), 268-275.