Periplocymarin regulates STAT3 to inhibit proliferation and invasion of human pancreatic cancer cell lines

doi:10.11958/20260112

Abstract

Abstract:

Objective To reveal the effect of periplocymarin (PPM) on the proliferation and invasion of human pancreatic cancer cell lines, and to analyze whether it exerts anti-cancer effects by regulating signal transducer and activator of transcription 3 (STAT3). Methods (1) In vitro experiments: SW1990 cells were divided into the control group, the 0.05 PPM group, the 0.1 PPM group and the 0.2 PPM group (cultured with 0.05, 0.1 and 0.2 μmol/L PPM for 48 h, respectively), the control lentivirus (NC-oe)+0.2 PPM group (NC-oe+0.2 PPM) and the STAT3 overexpression lentivirus (STAT3-oe)+0.2 PPM group (STAT3-oe+0.2 PPM). The control group was cultured with DMEM medium for 48 h. The NC-oe+0.2 PPM group and the STAT3-oe+0.2 PPM group were first transfected with NC-oe and STAT3-oe into SW1990 cells using Lipofectamine 3000, and then the cells were cultured with 0.2 μmol/L PPM for 48 h. Cell viability was detected by MTT, cell proliferation was detected by EdU staining, cell apoptosis was detected by TUNEL, and cell invasion was detected by Transwell assay. (2) In vivo experiments: Nude mice were randomly divided into the model group, the low (L-PPM) group, the high-dose PPM group (H-PPM), the NC-oe+H-PPM group and the STAT3-oe+H-PPM group, with 6 mice in each group. The model group, the L-PPM group and the H-PPM group were subcutaneously injected with normal SW1990 cells. The NC-oe+H-PPM group and the STAT3-oe+H-PPM group were subcutaneously injected with SW1990 cells transfected with NC-oe and STAT3-oe lentivirus, respectively. After 10 days of inoculation, the model group was intraperitoneally injected with normal saline at a dose of 0.1 mL/10 g, the L-PPM and H-PPM groups were intraperitoneally injected with 1.0 and 3.0 mg/(kg·d) PPM solution, respectively. And the NC-oe+H-PPM group and the STAT3-oe+H-PPM group were intraperitoneally injected with 3.0 mg/(kg·d) PPM solution. The administration cycle was 28 days. The morphology of tumor tissue was observed by HE staining. The expressions of p-STAT3, Ki-67 and Bcl-2 in tumor tissue were detected by immunohistochemistry, and the levels of STAT3 mRNA in cells and tumor tissue were detected by qRT-PCR. The protein expression of p-STAT3 (Tyr705), STAT3, Bcl-2, Bax, matrix metalloproteinase (MMP)-2 and MMP-9 in SW1990 cells and tumor tissue were detected by Western blot assay. Results (1) In vitro experiments: compared with the control group, the levels of STAT3 mRNA and p-STAT3 (Tyr705) protein were all decreased in SW1990 cells in the 0.05, 0.1 and 0.2 PPM groups (P<0.05). Relative cell viability, EdU positive rate, the number of invasion cells and the protein levels of Bcl-2, MMP-2 and MMP-9 decreased (P<0.05), while the positive rate of TUNEL and the protein level of Bax both increased (P<0.05). Compared with the NC-oe+0.2 PPM group, the levels of STAT3 mRNA and p-STAT3 (Tyr705) protein in SW1990 cells were increased in the STAT3-oe+0.2 PPM group (P<0.05). Relative cell viability, EdU positive rate, the number of invasion cells and the protein levels of Bcl-2, MMP-2 and MMP-9 increased (P<0.05), while the positive rate of TUNEL and the protein level of Bax both decreased (P<0.05). (2) In vivo experiments: compared with the model group, the tumor volume, STAT3 mRNA level, p-STAT3 (Tyr705) protein level and the positive rates of p-STAT3, Ki-67 and Bcl-2 in nude mice of the L-PPM group and the H-PPM group were all decreased (P<0.05). Compared with the NC-oe+H-PPM group, the tumor volume, STAT3 mRNA level, p-STAT3 (Tyr705) protein level and the positive rates of p-STAT3, Ki-67 and Bcl-2 in nude mice of the STAT3-oe+H-PPM group were all increased (P<0.05). Conclusion PPM has anti-pancreatic cancer activity, which inhibits the proliferation and invasion of pancreatic cancer cells by regulating STAT3.

Key words: pancreatic neoplasms, cell line, tumor, mice, inbred BALB C, Periplocin, STAT3 transcription factor, Periplocymarin

CLC Number:

R735.9

Figures/Tables 15

References 21

[1]	闫玲新, 李森, 郭改莉, 等. 异牡荆素通过miR-339-5p/HSPA8轴调节胰腺癌细胞的生物学行为[J]. 天津医药, 2025, 53(3):230-235.
	Yan L X, Li S, Guo G L, et al. Isovitexin regulates proliferation,migration and invasion of pancreatic cancer cells via the miR-339-5p/HSPA8 axis[J]. Tianjin Med J, 2025, 53(3):230-235. doi:10.11958/20242056.
[2]	Hamel Z, Sanchez S, Standing D, et al. Role of STAT3 in pancreatic cancer[J]. Explor Target Antitumor Ther, 2024, 5(5):20-34. doi:10.37349/etat.2024.00202.
[3]	Li X, Jiang W, Dong S, et al. STAT3 inhibitors:a novel insight for anticancer therapy of pancreatic cancer[J]. Biomolecules, 2022, 12(10):1450. doi:10.3390/biom12101450.
[4]	Petrocca F, Lieberman J. Promise and challenge of RNA interference-based therapy for cancer[J]. J Clin Oncol, 2011, 29(6):747-754. doi:10.1200/JCO.2009.27.6287.
[5]	Cheng Y, Wang G, Zhao L, et al. Periplocymarin induced colorectal cancer cells apoptosis via impairing PI3K/AKT pathway[J]. Front Oncol, 2021, 11:753598. doi:10.3389/fonc.2021.753598.
[6]	Hao Y, Song T, Wang M, et al. Dual targets of lethal apoptosis and protective autophagy in liver cancer with periplocymarin elicit a limited therapeutic effect[J]. Int J Oncol, 2023, 62(3):44. doi:10.3892/ijo.2023.5492.
[7]	Ke A, Yang W, Zhang W, et al. The cardiac glycoside periplocymarin sensitizes gastric cancer to ferroptosis via the ATP1A1-Src-YAP/TAZ-TFRC axis[J]. Phytomedicine, 2025, 142:156804. doi:10.1016/j.phymed.2025.156804.
[8]	Fan C, Liang S, Ye M, et al. Periplocymarin alleviates pathological cardiac hypertrophy via inhibiting the JAK2/STAT3 signalling pathway[J]. J Cell Mol Med, 2022, 26(9):2607-2619. doi:10.1111/jcmm.17267.
[9]	邢雪, 郭晓, 任晓亮, 等. 杠柳毒苷、杠柳次苷及杠柳苷元生物药剂学分类研究[J]. 中草药, 2019, 50(5):1082-1087.
	Xing X, Guo X, Ren X L, et al. Biopharmaceuticals classification system (BCS) of periplocin,periplocymarin and periplogenin[J]. Chinese Traditional and Herbal Drugs, 2019, 50(5):1082-1087. doi:10.7501/j.issn.0253-2670.2019.05.009.
[10]	Martey O N K, He X, Xing H, et al. Periplocymarin is a potential natural compound for drug development:highly permeable with absence of P-glycoprotein efflux and cytochrome P450 inhibitions[J]. Biopharm Drug Dispos, 2014, 35(4):195-206. doi:10.1002/bdd.1884.
[11]	Chen H, Bian A, Yang L, et al. Targeting STAT3 by a small molecule suppresses pancreatic cancer progression[J]. Oncogene, 2021, 40(8):1440-1457. doi:10.1038/s41388-020-01626-z.
[12]	董赟, 冯一中, 李峰. 应用组织芯片研究Pim-3、p-STAT3在胰腺癌中的表达及临床意义[J]. 临床与实验病理学杂志, 2008, 24(3):291-295.
	Dong Y, Feng Y Z, Li F. Expression and clinical significance of pim-3 and p-STAT3 in pancreatic cancer with tissue microarray[J]. Chinese Journal of Clinical and Experimental Pathology, 2008, 24(3):291-295. doi:10.3969/j.issn.1001-7399.2008.03.010.
[13]	Bai L, Zhou H, Xu R, et al. A potent and selective small-molecule degrader of STAT3 achieves complete tumor regression in vivo[J]. Cancer Cell, 2019, 36(5):498-511.e17. doi:10.1016/j.ccell.2019.10.002.
[14]	Qin A, Yu Q, Gao Y, et al. Inhibition of STAT3/cyclinD1 pathway promotes chemotherapeutic sensitivity of colorectal caner[J]. Biochem Biophys Res Commun, 2015, 457(4):681-687. doi:10.1016/j.bbrc.2015.01.048.
[15]	Zhang S, Li J, Xie P, et al. STAT3/c-Myc Axis-mediated metabolism alternations of inflammation-related glycolysis involve with colorectal carcinogenesis[J]. Rejuvenation Res, 2019, 22(2):138-145. doi:10.1089/rej.2018.2089.
[16]	Minus M B, Wang H, Munoz J O, et al. Targeting STAT3 anti-apoptosis pathways with organic and hybrid organic-inorganic inhibitors[J]. Org Biomol Chem, 2020, 18(17):3288-3296. doi:10.1039/c9ob02682g.
[17]	Xu Y, Feng S, Niu B. Silencing Stat3 inhibits viability and induces apoptosis in BGC-823 human gastric cancer cell line[J]. Biotech Histochem, 2021, 96(1):76-81. doi:10.1080/10520295.2020.1770332.
[18]	Bhattacharya S, Ray R M, Johnson L R. STAT3-mediated transcription of Bcl-2,Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells[J]. Biochem J, 2005, 392(Pt 2):335-344. doi:10.1042/BJ20050465.
[19]	Guha P, Gardell J, Darpolor J, et al. STAT3 inhibition induces Bax-dependent apoptosis in liver tumor myeloid-derived suppressor cells[J]. Oncogene, 2019, 38(4):533-548. doi:10.1038/s41388-018-0449-z.
[20]	Xiang T, Xia X, Yan W. Expression of matrix metalloproteinases-2/-9 is associated with microvessel density in pancreatic cancer[J]. Am J Ther, 2017, 24(4):e431-e434. doi:10.1097/MJT.0000000000000424.
[21]	Zhang F, Wang Z, Fan Y, et al. Elevated STAT3 signaling-mediated upregulation of MMP-2/9 confers enhanced invasion ability in multidrug-resistant breast cancer cells[J]. Int J Mol Sci, 2015, 16(10):24772-24790. doi:10.3390/ijms161024772.

组别	STAT3 mRNA	p-STAT3（Tyr705）/STAT3
对照组	1.00±0.06	1.00±0.05
0.05 PPM组	0.70±0.06^a	0.65±0.08^a
0.1 PPM组	0.45±0.04^ab	0.43±0.02^ab
0.2 PPM组	0.24±0.02^abc	0.20±0.02^abc
NC-oe+0.2 PPM组	0.23±0.02	0.19±0.02
STAT3-oe+0.2 PPM组	2.25±0.13^d	1.66±0.16^d
F	762.529^＊＊	342.966^＊＊

组别	STAT3 mRNA	p-STAT3（Tyr705）/STAT3
对照组	1.00±0.06	1.00±0.05
0.05 PPM组	0.70±0.06^a	0.65±0.08^a
0.1 PPM组	0.45±0.04^ab	0.43±0.02^ab
0.2 PPM组	0.24±0.02^abc	0.20±0.02^abc
NC-oe+0.2 PPM组	0.23±0.02	0.19±0.02
STAT3-oe+0.2 PPM组	2.25±0.13^d	1.66±0.16^d
F	762.529^＊＊	342.966^＊＊

组别		相对细胞活力/%		EdU阳性率/%
对照组		100.00±3.23		100.00±5.11
0.05 PPM组		77.81±6.51^a		82.70±6.47^a
0.1 PPM组		60.35±3.62^ab		64.46±4.11^ab
0.2 PPM组		38.46±3.49^abc		43.12±3.57^abc
NC-oe+0.2 PPM组		39.22±2.96		39.93±2.77
STAT3-oe+0.2 PPM组		69.91±4.79^d		68.63±11.78^d
F		183.597^＊＊		78.132^＊＊
组别	TUNEL阳性率/%		Bcl-2/GAPDH		Bax/GAPDH
对照组	4.82±0.32		1.00±0.06		1.00±0.07
0.05 PPM组	15.35±1.26^a		0.81±0.06^a		2.79±0.36^a
0.1 PPM组	26.91±2.56^ab		0.65±0.04^ab		4.25±0.39^ab
0.2 PPM组	34.58±4.22^abc		0.41±0.03^abc		5.11±0.66^abc
NC-oe+0.2 PPM组	35.41±3.76		0.40±0.02		5.28±0.70
STAT3-oe+0.2 PPM组	18.92±2.33^d		0.62±0.05^d		3.53±0.24^d
F	111.874^＊＊		152.983^＊＊		73.371^＊＊

组别		相对细胞活力/%		EdU阳性率/%
对照组		100.00±3.23		100.00±5.11
0.05 PPM组		77.81±6.51^a		82.70±6.47^a
0.1 PPM组		60.35±3.62^ab		64.46±4.11^ab
0.2 PPM组		38.46±3.49^abc		43.12±3.57^abc
NC-oe+0.2 PPM组		39.22±2.96		39.93±2.77
STAT3-oe+0.2 PPM组		69.91±4.79^d		68.63±11.78^d
F		183.597^＊＊		78.132^＊＊
组别	TUNEL阳性率/%		Bcl-2/GAPDH		Bax/GAPDH
对照组	4.82±0.32		1.00±0.06		1.00±0.07
0.05 PPM组	15.35±1.26^a		0.81±0.06^a		2.79±0.36^a
0.1 PPM组	26.91±2.56^ab		0.65±0.04^ab		4.25±0.39^ab
0.2 PPM组	34.58±4.22^abc		0.41±0.03^abc		5.11±0.66^abc
NC-oe+0.2 PPM组	35.41±3.76		0.40±0.02		5.28±0.70
STAT3-oe+0.2 PPM组	18.92±2.33^d		0.62±0.05^d		3.53±0.24^d
F	111.874^＊＊		152.983^＊＊		73.371^＊＊

组别	侵袭细胞数/个	MMP-2/GAPDH	MMP-9/GAPDH
对照组	128.17±6.91	1.00±0.02	1.00±0.06
0.05 PPM组	107.17±12.02^a	0.79±0.06^a	0.69±0.05^a
0.1 PPM组	79.33±10.61^ab	0.52±0.03^ab	0.55±0.03^ab
0.2 PPM组	49.67±8.04^abc	0.25±0.02^abc	0.31±0.02^abc
NC-oe+0.2 PPM组	53.50±3.02	0.24±0.02	0.29±0.02
STAT3-oe+0.2 PPM组	88.67±3.14^d	0.62±0.03^d	0.59±0.04^d
F	85.943^＊＊	487.081^＊＊	243.020^＊＊