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

Juyoung Lee; Jang-Hoon Lee; Youngchul Kim; Lee, Juyoung; Lee, Jang-Hoon; Kim, Youngchul

doi:10.13048/jkm.24046

JKM > Volume 45(3); 2024 > Article

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

Original Article

The Journal of Korean Medicine 2024; 45(3): 154-167.

Published online: September 1, 2024

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

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

Juyoung Lee¹^{, 2}

, Jang-Hoon Lee¹

, Youngchul Kim¹^{, *}

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 July 24, 2024 Revised August 16, 2024 Accepted August 19, 2024

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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