The Effect of Rhus verniciflua Stokes Extract on Apoptosis and Autophagy in human leukemic cell line (MOLT-4)

Article information

J Korean Med. 2024;45(3):154-167

Publication date (electronic) : 2024 September 1

doi : https://doi.org/10.13048/jkm.24046

Juyoung Lee ¹^,²

, Jang-Hoon Lee ¹

, Youngchul Kim ¹^,

¹Dept. of Internal Medicine, College of Korean Medicine, Kyung Hee University

²Dept. of Clinical Korean Medicine Graduate School, Kyung Hee University

Correspondence to: 김영철, 서울시 동대문구 경희대로 23 경희대학교 한방병원 간장ㆍ조혈내과, Tel: 02-958-9118, FAX: 02-958-9258, E-mail: yckim@khmc.or.kr

Received 2024 July 24; Revised 2024 August 16; Accepted 2024 August 19.

Abstract

Objectives

This study was performed to investigate the effects of Rhus verniciflua Stokes (RVS) on apoptosis and autophagy in the human leukemic cell line, MOLT-4.

Methods

Cell viability was measured by MTS/PMS assay, and cell cycle distribution was analyzed by flow cytometry. The expression levels of mRNA implicated in apoptosis and ER-stress were investigated with RT-qPCR. Lastly, apoptosis- and autophagy-related protein expressions were measured by Western blot analysis.

Results

RVS inhibited proliferation of MOLT-4 in a dose-dependent manner over 24, 48 and 72 hours. RVS treatment also induced an increase in subG1 phase. Exposure to RVS increased the expression of the mRNAs encoding Bax and caspase-3, while decreasing the expression of Bcl-2 mRNA, suggesting that RVS induced apoptosis in MOLT-4 cells. Additionally, RVS extract up-regulated ER-stress related mRNAs such as IRE1α, CHOP, PERK and ATF6. Changes in RVS extract-induced apoptosis and autophagy proteins on MOLT-4 cells were also investigated. The level of Bcl-2 was decreased, whereas the levels of Bax, caspase-3, AMPK, Beclin-1, Atg5, p62, and LC3II were increased.

Conclusion

These results suggest that RVS would be beneficial in the treatment of Acute Lymphoblastic Leukemia.

Keywords: Rhus verniciflua Stokes; Rhois Vernicifluae Cortex; MOLT-4; Acute Lymphoblastic Leukemia; Apoptosis; Autophagy

Fig. 1

HPLC chromatograms of the representative standard (Fisetin) (A) and the 30% EtOH extract of roasted RVS (B)

Fig. 2

Effects of RVS extract on the cell viability in MOLT-4 cells.

Cells were treated with various concentrations of RVS extract (0–500μg/mL) for 24, 48, and 72 h. Cell viability was measured by MTS/PMS assay. The data are the mean±SD of three independent samples.

Fig. 3

RVS induces subG1 Phase Cell cycle Arrest in MOLT-4 Cells

MOLT-4 cells were treated with RVS extract (0, 100, 200, and 300μg/mL) for 72 h and subjected to cell cycle analysis.

Fig. 4

Effects of RVS extract on the expression of pro- and anti-apoptotic, Caspase-3 mRNAs in MOLT-4 cells.

Cells were cultured either with different concentrations of RVS extract (0, 100, 200, and 300μg/mL) for 72 h. The mRNA levels were measured by real-time PCR. The crossing point of Bax, Bcl-2 and Caspase-3 with β-actin was applied to the formula, 2-(target gene-β-actin), and relative amounts were quantified. The data represent the mean±SD of three independent samples. *p<0.05 and **p<0.01 compared to the control.

Fig. 5

Effects of RVS extract on the expression of ER stress-related gene mRNAs in MOLT-4 cells.

Cells were cultured either with different concentrations of RVS extract (0, 100, 200, and 300μg) for 72 h. The mRNA levels were measured by real-time PCR. The crossing point of ER Stress related-genes with β-actin was applied to the formula, 2-(target gene-β-actin), and relative amounts were quantified. The data represent the mean±SD of three independent samples. *p<0.05 and **p<0.01 compared to the control.

Fig. 6

Effects of RVS extract on the expression of pro- and anti-apoptotic, Caspase-3 protein in MOLT-4 cells.

Expression of protein was examined by western blot. Densitometric analyses are presented as the relative rations of Bax, Bcl-2 and Caspase-3 to β-actin. The data represent the mean±SD of three independent samples. *p<0.05 and **p<0.01 compared to the control.

Fig. 7

Effects of RVS extract on the expression of Autophagy related protein in MOLT-4 cells.

Expression of protein was examined by western blot. Densitometric analyses are presented as the relative rations of AMPK, Beclin-1, Atg5, Atg3, p62 and LC3II to β-actin. The data represent the mean±SD of three independent samples. *p<0.05 and **p<0.01 compared to the control.

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