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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

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.

Fig. 1
HPLC chromatograms of the representative standards (epicatechin gallate (ECG), quercetin and kaempferol)(A) and the O. japonicus EtOH extracts(B)
jkm-40-2-35f1.gif
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.
jkm-40-2-35f2.gif
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.
jkm-40-2-35f3.gif
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.)
jkm-40-2-35f4.gif
Fig. 5
Expression of apoptosis-related genes 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 mRNA levels were measured by real-time PCR. The crossing point of Bcl-2, Bax, Mcl-1, Survivin with β-actin was applied to the formula, 2-(target gene-β-actin), and relative amounts were quantified. The data are the mean±SD of triplicate samples. (*p<0.05 and **p<0.01 compared to the control.)
jkm-40-2-35f5.gif
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.)
jkm-40-2-35f6.gif
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.)
jkm-40-2-35f7.gif
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.)
jkm-40-2-35f8.gif
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.)
jkm-40-2-35f9.gif
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
5′ CCT TGG CAT GAG ATG CAG GA 3′

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

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

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

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

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

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