The effect of andrographolide regulating STAT3/GPX4 pathway on proliferation and apoptosis of myeloma cells

doi:10.11958/20252757

Abstract

Abstract:

Objective To investigate the effect of andrographolide (Andro) on the proliferation and apoptosis of myeloma cells by regulating the signal transducer and activator of transcription 3 (STAT3)/glutathione peroxidase 4 (GPX4) pathway. Methods Human multiple myeloma U266 cells were divided into the myeloma group, the Andro low-concentration group, the Andro medium-concentration group, the Andro high-concentration group, the Stattic (STAT3 inhibitor) group and the Andro high-concentration + Colivelin (STAT3 activator) group. Cell proliferation was detected by CCK-8 assay and clone formation assay. Cell apoptosis was detected by flow cytometry. Mitochondrial morphology was observed by transmission electron microscopy. The levels of mitochondrial membrane potential, ferrous ion (Fe²⁺), 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and reduced glutathione (GSH) in cells were detected. The level of reactive oxygen species (ROS) in cells was detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Western blot assay was used to detect the proteins including proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax), solute carrier family 7 member 11 (SLC7A11), p-STAT3 and GPX4. Results Compared with the myeloma group, the mitochondria of U266 cells showed significant morphological changes in the Andro low-, medium- and high-concentration groups, the OD₄₅₀ value, clone formation rate, mitochondrial membrane potential, GSH level, and the protein levels of PCNA, Bcl-2/Bax, SLC7A11, p-STAT3/STAT3 and GPX4 were decreased, the cell apoptosis rate, the levels of Fe²⁺, 4-HNE, MDA and ROS were increased (P<0.05). Compared with the myeloma group, the changes in the corresponding indexes of U266 cells showed the same trend as above in the Stattic group (P<0.05). Colivelin reversed the promoting effect of high-concentration Andro on ferroptosis and apoptosis of U266 cells, as well as the inhibitory effect on cell proliferation. Conclusion Andro may induce ferroptosis by inhibiting the STAT3/GPX4 pathway, thereby inhibiting the proliferation and promoting the apoptosis of multiple myeloma cells.

Key words: andrographolide, multiple myeloma, ferroptosis, cell proliferation, apoptosis, STAT3 transcription factor, phospholipid hydroperoxide glutathione peroxidase

CLC Number:

R733.7

Figures/Tables 8

References 26

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组别	OD₄₅₀值	克隆形成率/%
骨髓瘤组	1.11±0.10	66.53±3.78
Andro低浓度组	0.91±0.09^a	55.46±2.93^a
Andro中浓度组	0.73±0.08^ab	41.77±2.56^ab
Andro高浓度组	0.41±0.05^abc	28.65±1.58^abc
Stattic组	0.36±0.04^a	25.44±1.39^a
Andro高浓度+Colivelin组	0.69±0.07^d	36.95±2.01^d
F	88.740^＊＊	239.300^＊＊
组别	细胞凋亡率/%	线粒体膜电位/%
骨髓瘤组	4.58±0.26	56.57±3.19
Andro低浓度组	8.99±0.43^a	47.78±2.75^a
Andro中浓度组	13.78±0.71^ab	37.76±2.44^ab
Andro高浓度组	23.44±1.38^abc	29.68±1.67^abc
Stattic组	24.06±1.41^a	27.71±1.83^a
Andro高浓度+Colivelin组	16.62±0.93^d	35.56±2.05^d
F	394.000^＊＊	129.800^＊＊

组别	OD₄₅₀值	克隆形成率/%
骨髓瘤组	1.11±0.10	66.53±3.78
Andro低浓度组	0.91±0.09^a	55.46±2.93^a
Andro中浓度组	0.73±0.08^ab	41.77±2.56^ab
Andro高浓度组	0.41±0.05^abc	28.65±1.58^abc
Stattic组	0.36±0.04^a	25.44±1.39^a
Andro高浓度+Colivelin组	0.69±0.07^d	36.95±2.01^d
F	88.740^＊＊	239.300^＊＊
组别	细胞凋亡率/%	线粒体膜电位/%
骨髓瘤组	4.58±0.26	56.57±3.19
Andro低浓度组	8.99±0.43^a	47.78±2.75^a
Andro中浓度组	13.78±0.71^ab	37.76±2.44^ab
Andro高浓度组	23.44±1.38^abc	29.68±1.67^abc
Stattic组	24.06±1.41^a	27.71±1.83^a
Andro高浓度+Colivelin组	16.62±0.93^d	35.56±2.05^d
F	394.000^＊＊	129.800^＊＊

组别		Fe²⁺/（nmol/10⁶ cell）		4-HNE/（mmol/g）
骨髓瘤组		0.23±0.03		0.45±0.05
Andro低浓度组		0.54±0.06^a		0.89±0.09^a
Andro中浓度组		0.95±0.10^ab		1.63±0.15^ab
Andro高浓度组		1.89±0.17^abc		2.44±0.19^abc
Stattic组		2.04±0.16^a		2.51±0.21^a
Andro高浓度+Colivelin组		1.08±0.12^d		1.53±0.14^d
F		224.300^＊＊		182.200^＊＊
组别	MDA/（μmol/g）		GSH/（μmol/g）		ROS/%
骨髓瘤组	20.33±1.58		9.86±0.51		4.38±0.25
Andro低浓度组	28.77±1.69^a		7.73±0.39^a		9.65±0.51^a
Andro中浓度组	37.73±1.98^ab		5.45±2.90^ab		16.69±0.97^ab
Andro高浓度组	46.65±2.82^abc		2.63±0.15^abc		35.76±1.89^abc
Stattic组	49.44±3.15^a		2.43±0.13^a		37.26±2.34^a
Andro高浓度+Colivelin组	35.51±1.87^d		4.88±0.26^d		18.88±1.06^d
F	139.600^＊＊		33.890^＊＊		572.000^＊＊

组别		Fe²⁺/（nmol/10⁶ cell）		4-HNE/（mmol/g）
骨髓瘤组		0.23±0.03		0.45±0.05
Andro低浓度组		0.54±0.06^a		0.89±0.09^a
Andro中浓度组		0.95±0.10^ab		1.63±0.15^ab
Andro高浓度组		1.89±0.17^abc		2.44±0.19^abc
Stattic组		2.04±0.16^a		2.51±0.21^a
Andro高浓度+Colivelin组		1.08±0.12^d		1.53±0.14^d
F		224.300^＊＊		182.200^＊＊
组别	MDA/（μmol/g）		GSH/（μmol/g）		ROS/%
骨髓瘤组	20.33±1.58		9.86±0.51		4.38±0.25
Andro低浓度组	28.77±1.69^a		7.73±0.39^a		9.65±0.51^a
Andro中浓度组	37.73±1.98^ab		5.45±2.90^ab		16.69±0.97^ab
Andro高浓度组	46.65±2.82^abc		2.63±0.15^abc		35.76±1.89^abc
Stattic组	49.44±3.15^a		2.43±0.13^a		37.26±2.34^a
Andro高浓度+Colivelin组	35.51±1.87^d		4.88±0.26^d		18.88±1.06^d
F	139.600^＊＊		33.890^＊＊		572.000^＊＊

组别	PCNA/GAPDH	Bcl-2/GAPDH	Bax/GAPDH	Bcl-2/Bax	SLC7A11/GAPDH	p-STAT3/STAT3	GPX4/GAPDH
骨髓瘤组	1.89±0.21	0.98±0.10	0.16±0.02	6.13±0.34	1.34±0.15	0.89±0.10	1.21±0.13
Andro低浓度组	1.61±0.18^a	0.75±0.08^a	0.34±0.04^a	2.21±0.12^a	1.02±0.11^a	0.71±0.08^a	0.95±0.10^a
Andro中浓度组	1.03±0.11^ab	0.51±0.06^ab	0.53±0.06^ab	0.96±0.10^ab	0.83±0.09^ab	0.54±0.06^ab	0.74±0.08^ab
Andro高浓度组	0.67±0.08^abc	0.23±0.03^abc	0.89±0.09^abc	0.26±0.03^abc	0.54±0.06^abc	0.26±0.03^abc	0.11±0.02^abc
Stattic组	0.69±0.09^a	0.26±0.03^a	0.91±0.10^a	0.29±0.03^a	0.50±0.06^a	0.22±0.03^a	0.13±0.02^a
Andro高浓度+Colivelin组	0.98±0.11^d	0.54±0.06^d	0.56±0.07^d	0.96±0.09^d	0.88±0.09^d	0.46±0.05^d	0.76±0.08^d
F	77.770^＊＊	117.100^＊＊	110.900^＊＊	1 200.000^＊＊	60.690^＊＊	99.060^＊＊	175.300^＊＊