Impact of shikonin on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway

doi:10.11958/20221929

Abstract

Abstract:

Objective To investigate the impact of shikonin (SHI) on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway. Methods Western blot assay was used to detect the expression of Notch, Hairy mitosis-related enhancer-1 (Hes1), hairy-related transcription factor-1 (HEY1) protein in liver cancer tissue, paracancerous tissue, hepatoma cells (HepG2 cells, Hep3B cells, HCCLM3 cells, Huh-7 cells and SMMC-7721 cells) and normal liver cells (HL-7702 cells). Huh-7 cells were divided into the control group, the L-SHI group (1 μmol/L SHI), the M-SHI group (2 μmol/L SHI), the H-SHI group (4 μmol/L SHI), the DAPT group (50 μmol/L Notch signal inhibitor DAPT) and the H-SHI+VPA group [4 μmol/L SHI and 8 mmol/L Notch pathway activator Valproic acid (VPA)]. The proliferation of Huh-7 cells was detected by CCK-8 method and plate cloning test. The apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry. Cell scratch test and Transwell invasion test were used to detect migration and invasion of Huh-7 cells. Western blot assay was used to detect the expression of epithelial-mesenchymal transformation (EMT) and apoptosis related proteins. Results The expression levels of Notch, HES1 and HEY1 were obviously increased in liver cancer tissue and cells, and Huh-7 cells showed the most obvious difference, therefore, Huh-7 cells were taken as the research object. Compared with the control group, the protein levels of Notch, HES1, HEY1 and Bcl-2 decreased, and the proportions of S phase and G2 phase cells, OD₄₅₀ value, number of clones, migration rate, number of invasive cells and levels of N-cadherin and Vimentin decreased significantly in the L-SHI group, the M-SHI group, the H-SHI group and the DAPT group (P<0.05). The proportion of G1/G0 phase cells, apoptosis rate and levels of Bax, cleaved Casase-3, and E-cadherin increased obviously (P<0.05). The effect of SHI was dose-dependent. Compared with the H-SHI group, the above indexes showed the opposite trend in the H-SHI+VPA group. VPA attenuated the effect of SHI on reducing the malignant biological activity of liver cancer cells. Conclusion SHI may inhibit the proliferation, migration and invasion of Huh-7 cells and promote apoptosis of Huh-7 cells by inhibiting Notch signal pathway.

Key words: Shikonin, carcinoma, hepatocellular, receptors, Notch, cell proliferation, cell movement, apoptosis

CLC Number:

R735.7

Figures/Tables 18

References 19

[1]	CAO W, CHEN H D, YU Y W, et al. Changing profiles of cancer burden worldwide and in China:a secondary analysis of the global cancer statistics 2020[J]. Chin Med J (Engl), 2021, 134(7):783-791. doi:10.1097/CM9.0000000000001474.
[2]	CHIDAMBARANATHAN-REGHUPATY S, FISHER P B, SARKAR D. Hepatocellular carcinoma(HCC):epidemiology,etiology and molecular classification[J]. Adv Cancer Res, 2021, 149:1-61. doi:10.1016/bs.acr.2020.10.001.
[3]	WALLACE M C, PREEN D B, SHORT M W, et al. Hepatocellular carcinoma in Australia 1982-2014:increasing incidence and improving survival[J]. Liver Int, 2019, 39(3):522-530. doi:10.1111/liv.13966.
[4]	ZHANG S, GAO Q, LI W, et al. Shikonin inhibits cancer cell cycling by targeting Cdc25s[J]. BMC Cancer, 2019, 19(1):20. doi:10.1186/s12885-018-5220-x.
[5]	BAO C, LIU T, QIAN L, et al. Shikonin inhibits migration and invasion of triple-negative breast cancer cells by suppressing epithelial-mesenchymal transition via miR-17-5p/PTEN/Akt pathway[J]. J Cancer, 2021, 12(1):76-88. doi:10.7150/jca.47553.
[6]	XU Z, HUANG L, ZHANG T, et al. Shikonin inhibits the proliferation of cervical cancer cells via FAK/AKT/GSK3β signalling[J]. Oncol Lett, 2022, 24(3):304. doi:10.3892/ol.2022.13424.
[7]	LIU T, LI S, WU L, et al. Experimental study of hepatocellular carcinoma treatment by Shikonin through regulating PKM2[J]. J Hepatocell Carcinoma, 2020, 7:19-31. doi:10.2147/JHC.S237614.
[8]	LUIKEN S, FRAAS A, BIEG M, et al. NOTCH target gene HES5 mediates oncogenic and tumor suppressive functions in hepatocarcinogenesis[J]. Oncogene, 2020, 39(15):3128-3144. doi:10.1038/s41388-020-1198-3.
[9]	SHI J, HAN G, WANG J, et al. Matrine promotes hepatic oval cells differentiation into hepatocytes and alleviates liver injury by suppression of Notch signalling pathway[J]. Life Sci, 2020, 261:118354. doi:10.1016/j.lfs.2020.118354.
[10]	LEE J H, HAN S H, KIM Y M, et al. Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis[J]. Biosci Rep, 2021, 41(1):BSR20203834. doi:10.1042/BSR20203834.
[11]	XIANG Z, MIAO Q, ZHANG J, et al. AB4 inhibits Notch signaling and promotes cancer cell apoptosis in liver cancer[J]. Oncol Rep, 2021, 45(6):112. doi:10.3892/or.2021.8063.
[12]	YANG X, LIU J, LIANG Q, et al. Valproic acid reverses sorafenib resistance through inhibiting activated Notch/Akt signaling pathway in hepatocellular carcinoma[J]. Fundam Clin Pharmacol, 2021, 35(4):690-699. doi:10.1111/fcp.12608.
[13]	ARMENGOL C, SARRIAS M R, SALA M. Hepatocellular carcinoma:present and future[J]. Med Clin (Barc), 2018, 150(10):390-397. doi:10.1016/j.medcli.2017.08.010.
[14]	MARKOWITSCH S D, JUETTER K M, SCHUPP P, et al. Shikonin reduces growth of docetaxel-resistant prostate cancer cells mainly through necroptosis[J]. Cancers (Basel), 2021, 13(4):882. doi:10.3390/cancers13040882.
[15]	TSAI M F, CHEN S M, ONG A Z, et al. Shikonin induced program cell death through generation of reactive oxygen species in renal cancer cells[J]. Antioxidants (Basel), 2021, 10(11):1831-1844. doi:10.3390/antiox10111831.
[16]	LI M Y, MI C, WANG K S, et al. Shikonin suppresses proliferation and induces cell cycle arrest through the inhibition of hypoxia-inducible factor-1α signaling[J]. Chem Biol Interact, 2017, 274:58-67. doi:10.1016/j.cbi.2017.06.029.
[17]	LI Y, ZHANG T, QIN S, et al. Effects of UPF1 expression on EMT process by targeting E-cadherin,N-cadherin,Vimentin and Twist in a hepatocellular carcinoma cell line[J]. Mol Med Rep, 2019, 19(3):2137-2143. doi:10.3892/mmr.2019.9838.
[18]	LI X, LIU W, GENG C, et al. Ginsenoside Rg3 suppresses epithelial-mesenchymal transition via downregulating Notch-Hes1 signaling in colon cancer cells[J]. Am J Chin Med, 2021, 49(1):217-235. doi:10.1142/S0192415X21500129.
[19]	ZHANG H S, ZHANG Z G, DU G Y, et al. Nrf2 promotes breast cancer cell migration via up-regulation of G6PD/HIF-1α/Notch1 axis[J]. J Cell Mol Med, 2019, 23(5):3451-3463. doi:10.1111/jcmm.14241.

组别	Notch	HES1	HEY1
癌旁组织	0.78±0.09	0.63±0.07	0.39±0.05
癌组织	1.41±0.13	1.24±0.11	1.37±0.14
t	17.819^＊＊	10.633^＊＊	29.481^＊＊

组别	Notch	HES1	HEY1
癌旁组织	0.78±0.09	0.63±0.07	0.39±0.05
癌组织	1.41±0.13	1.24±0.11	1.37±0.14
t	17.819^＊＊	10.633^＊＊	29.481^＊＊

组别	Notch	HES1	HEY1
HL-7702细胞	0.60±0.06	0.51±0.05	0.35±0.04
HepG2细胞	1.41±0.13^a	1.05±0.09^a	1.16±0.12^a
Hep3B细胞	1.33±0.12^a	1.13±0.12^a	1.22±0.13^a
HCCLM3细胞	1.35±0.14^a	1.07±0.11^a	1.26±0.14^a
SMMC-7721细胞	1.37±0.15^a	1.09±0.13^a	1.24±0.16^a
Huh-7细胞	1.60±0.17^a	1.23±0.14^a	1.42±0.17^a
F	67.663^＊＊	52.880^＊＊	81.609^＊＊

组别	Notch	HES1	HEY1
HL-7702细胞	0.60±0.06	0.51±0.05	0.35±0.04
HepG2细胞	1.41±0.13^a	1.05±0.09^a	1.16±0.12^a
Hep3B细胞	1.33±0.12^a	1.13±0.12^a	1.22±0.13^a
HCCLM3细胞	1.35±0.14^a	1.07±0.11^a	1.26±0.14^a
SMMC-7721细胞	1.37±0.15^a	1.09±0.13^a	1.24±0.16^a
Huh-7细胞	1.60±0.17^a	1.23±0.14^a	1.42±0.17^a
F	67.663^＊＊	52.880^＊＊	81.609^＊＊

组别	Notch	HES1	HEY1
对照组	1.63±0.19	1.26±0.14	1.48±0.17
L-SHI组	1.31±0.15^a	1.03±0.12^a	1.15±0.13^a
M-SHI组	0.98±0.10^ab	0.80±0.09^ab	0.79±0.09^ab
H-SHI组	0.64±0.06^abc	0.63±0.08^abc	0.38±0.04^abc
DAPT组	0.72±0.07^a	0.39±0.03^a	0.89±0.09^a
H-SHI+VPA组	1.39±0.14^d	1.11±0.14^d	1.27±0.15^d
F	96.630^＊＊	91.860^＊＊	106.778^＊＊