Psoralen inhibits malignant proliferation and induces apoptosis through triggering endoplasmic reticulum stress in human SMMC7721 hepatoma cells

Abstract

Background: Psoralen is a coumarin-like and coumarin-related benzofuran glycoside, which is a commonly used traditional Chinese medicine to treat patients with kidney and spleen-yang deficiency symptom. Psoralen has been reported to show estrogen-like activity, antioxidant activity, osteoblastic proliferation accelerating activity, antitumor effects and antibacterial activity. However, the antitumor mechanism of psoralen is not fully understood. This study aimed to investigate the therapeutic efficacy of psoralen in human hepatoma cell line SMMC7721 and the mechanism of antitumor effects.

Results: Psoralen inhibited proliferation of SMMC7721 in a dose- and time-dependent manner, and promoted apoptosis. Further, psoralen activated the ER stress signal pathway, including the expansion of endoplasmic reticulum, increasing the mRNA levels of GRP78, DDIT3, ATF4, XBP1, GADD34 and the protein levels of GDF15, GRP78, IRE1α, XBP-1s in a time-dependent manner. Psoralen induces cell cycle arrest at G1 phase by enhancing CyclinD1 and reducing CyclinE1 expression. Moreover, TUDC couldn't inhibit the psoralen-induced ER stress in SMMC7721 cells.

Conclusions: Psoralen can inhibit the proliferation of SMMC7721 cells and induce ER stress response to induce cell apoptosis, suggesting that psoralen may represent a novel therapeutic option for the prevention and treatment hepatocellular carcinoma.

Keywords: Apoptosis; Endoplasmic reticulum stress; Hepatocellular carcinoma; Psoralen; SMMC7721 cell.

Fig. 1

Chemical structure of psoralen. a The plant diagram of psoralea. b The chemical structure of psoralen

Fig. 2

Effects of psoralen on the proliferation of SMMC7721, L02 and HepG2. a Different doses of psoralen inhibit the proliferation of SMMC7721 for 24–72 h. The A570 value of SMMC7721 (b), L02 (c) and HepG2 (d) with the treatment of psoralen in 10–80 μM for 72 h. Values are mean ± SD (n = 4) and * is means compared to the Con group, *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Effects of psoralen on the morphology of SMMC7721. a SMMC7721 cells shrunk in the dose of 40 μM psoralen or 0.1 μM thapsigargin for 12–48 h. b Abnormalities of endoplasmic reticulum and cytoplasmic structure in SMMC7721 in the dose of 40 μM psoralen or 0.1 μM thapsigargin for 24 h. Yellow boxes represent the abnormal region

Fig. 4

Psoralen induces cell cycle arrest at G1 phase in SMMC7721. Flow cytometry (a) and Western blot (b) assays showing the effects of psoralen and thapsigargin on cell cycle progression and expression of G1/S transition-related proteins, respectively. Relative protein levels are showed on the right. ns no significant; ***P < 0.001

Fig. 5

Psoralen induces apoptosis in SMMC7721 hepatoma cells. AnnexinV-PI staining (a) and western blot (b) assays showing the effects of different doses of psoralen on cell apoptosis and the expression of apoptosis-related proteins

Fig. 6

Effects of psoralen on gene expression of ER-stress. a The mRNA levels of key gene in ER-stress by different doses of psoralen treatment for 48 h. b The mRNA levels of key gene in ER-stress by 40Μm psoralen for 6 h, 12 h and 24 h. c The mRNA levels of ER functional gene by 40Μm psoralen for 6 h, 12 h and 24 h. Values are mean ± SD (n = 3) and * is means compared to the Con group, *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

Effects of psoralen on ER-stress related protein expression in SMMC7721. a Western blot assays showing the effects of psoralen and thapsigargin on ER-stress related protein expression. b Western blot assays showing the effects of TUDC on the expression of ER-stress related protein induced by PSO. c The mRNA levels of GRP78 and DDIT3 with TUDC under PSO and TG treatment. Values are mean ± SD (n = 3) and * is means compared to the Con group, **P < 0.01