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. 2023 May 24;15(11):2447.
doi: 10.3390/nu15112447.

Furanocoumarin Notopterol: Inhibition of Hepatocellular Carcinogenesis through Suppression of Cancer Stemness Signaling and Induction of Oxidative Stress-Associated Cell Death

Ting-Yun Huang  1   2   3 , Ching-Kuo Yang  4 , Ming-Yao Chen  5   6 , Vijesh Kumar Yadav  5   6 , Iat-Hang Fong  5   6 , Chi-Tai Yeh  5   6   7 , Yih-Giun Cherng  8   9
Affiliations

Furanocoumarin Notopterol: Inhibition of Hepatocellular Carcinogenesis through Suppression of Cancer Stemness Signaling and Induction of Oxidative Stress-Associated Cell Death

Ting-Yun Huang et al. Nutrients. .

Abstract

Background: Hepatocellular carcinoma (HCC) remains an aggressive malignancy with a poor prognosis and a leading cause of cancer-related mortality globally. Cumulative evidence suggests critical roles for endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in chronic liver diseases. However, the role of ER stress in HCC pathogenesis, aggressiveness and therapy response remains unclear and understudied.

Objectives: Against this background, the present study evaluated the therapeutic efficacy and feasibility of notopterol (NOT), a furanocoumarin and principal component of Notopterygium incisum, in the modulation of ER stress and cancer stemness, and the subsequent effect on liver oncogenicity.

Methods: An array of biomolecular methods including Western blot, drug cytotoxicity, cell motility, immunofluorescence, colony and tumorsphere formation, flow-cytometric mitochondrial function, GSH/GSSG ratio, and tumor xenograft ex vivo assays were used in the study.

Results: Herein, we demonstrated that NOT significantly suppresses the viability, migration, and invasion capacity of the human HCC HepJ5 and Mahlavu cell lines by disrupting ATF4 expression, inhibiting JAK2 activation, and downregulating the GPX1 and SOD1 expression in vitro. NOT also markedly suppressed the expression of vimentin (VIM), snail, b-catenin, and N-cadherin in the HCC cells, dose-dependently. Treatment with NOT significantly attenuated cancer stem cells (CSCs)-like phenotypes, namely colony and tumorsphere formation, with the concomitant downregulation of stemness markers OCT4, SOX2, CD133, and upregulated PARP-1 cleavage, dose-dependently. We also demonstrated that NOT anticancer activity was strongly associated with increased cellular reactive oxidative stress (ROS) but, conversely, reduced mitochondrial membrane potential and function in the HepJ5 and Mahlavu cells in vitro. Our tumor xenograft studies showed that compared with sorafenib, NOT elicited greater tumor growth suppression without adverse changes in mice body weights. Compared with the untreated control and sorafenib-treated mice, NOT-treated mice exhibited markedly greater apoptosis ex vivo, and this was associated with the co-suppression of stemness and drug-resistance markers OCT4, SOX2, ALDH1, and the upregulation of endoplasmic reticulum stress and oxidative stress factors PERK and CHOP.

Conclusions: In summary, we demonstrated for the first time that NOT exhibits strong anticancer activity via the suppression of cancer stemness, enhanced endoplasmic reticulum stress and increased oxidative stress thus projecting NOT as a potentially effective therapeutic agent against HCC.

Keywords: HCC; anticancer treatment; cancer stemness; endoplasmic reticulum stress; hepatocellular carcinoma; notopterol; oxidative stress.

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Conflict of interest statement

The authors declare that they have no potential financial competing interests that may in any way, gain or lose financially from the publication of this manuscript at present or in the future. Additionally, no non-financial competing interests are involved in the manuscript.

Figures

Figure 1
Figure 1
Notopterol suppresses hepatocellular carcinoma cell viability by inhibiting JAK2 activation and enhancing oxidative stress. (A) Chemical structure of notopterol with a molecular weight of 354.4 g/mol. (B) Line graphs showing the effect of 0–100 μM notopterol on the viability of HepJ5 and Mahlavu cell lines at 24, 48, and 72 h. (C) Photo images showing the effect of 48 h treatment with 30 μM notopterol on the viability and morphology of HepJ5 and Mahlavu cell lines at 4× and 10× magnification. Scale bar = 50 μm. (D) Representative Western blot images showing the effect of 30 μM notopterol on the expression levels of ATF4, p-JAK2, JAK2, GPX1, CAT and SOD1 in HepJ5 or Mahlavu cell lines at 0, 24, and 48 h. GAPDH was loading control. * p < 0.05, *** p < 0.001.
Figure 2
Figure 2
Notopterol significantly attenuates HCC cell migration and invasive capacity. Photo-images and histograms of the effect of 0–30 μM notopterol on the (A) migration and (B) invasion capabilities of HepJ5 and Mahlavu cell lines at Day 0 and Day 2 after wound-scratch. (C) Representative Western blot images showing the effect of 0–30 μM notopterol on the expression levels of VIM, Snail, β-catenin, and N-cadherin in HepJ5 or Mahlavu cell lines at 0, 24, and 48 h. GAPDH was loading control. VIM, vimentin; β-cat, β-catenin; N-cad; N-cadherin. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
Notopterol effectively inhibits the cancer stem cells (CSCs)-like phenotypes of HCC cells. Photo-images and histograms of the effect of 0–30 μM notopterol on the (A) colony formation and (B) tumorsphere formation capabilities of HepJ5 and Mahlavu cell lines. (C) Representative Western blot images showing the effect of 0–30 μM notopterol on the expression levels of OCT4, CD133, and SOX2 in HepJ5 or Mahlavu cell lines. (D) Representative Western blot images showing the effect of 30 μM notopterol on the expression levels of BCL-2, PARP, and c-PARP in HepJ5 or Mahlavu cell lines after 24 or 48 h treatment, compared with untreated control. GAPDH was loading control. c-PARP, cleaved PARP. ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
Notopterol anticancer activity is mediated by increased intracellular reactive oxygen species (ROS) activity, mitochondrial dysfunction and oxidative stress induction in HCC cells. Photo-images and histograms showing the (A) fluorescence signal of DCFH-DA (red) and (B) MitoTracker staining (green) in HepJ5 or Mahlavu cell lines treated with 0–30 μM NOT. DAPI (blue) served as a nuclear marker. (C) Histograms showing the effect of 0–6 h treatment with 15 or 30 μM NOT on GSH concentration in HepJ5 and Mahlavu cell lines. NOT, notopterol; GSH, reduced glutathione; ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
Notopterol inhibits HCC tumor development and growth by disrupting stemness signals with endoplasmic reticulum and oxidative stress induction, ex vivo. (A) Schema of the mice HCC tumor xenograft experimental design using BALB/c nude mice inoculated with Mahlavu cells pre-treated with 30 μM NOT or 30 μM Sora. Line graphs comparing the effects of 30 μM NOT and 30 μM Sora on the (B) tumor sizes, and (C) changes in body weights of xenografted mice over 22 days. (D) Flow cytometry analysis showing the effect of pre-treatment with 30 μM NOT or 30 mM Sora on Hoechst 33342 staining in cells dissociated from tumors harvested from the 30 μM NOT or 30 μM Sora xenografted mice group, in the absence or presence of Vera. The SP cells were gated and shown as a percentage as indicated. (E) Histograms showing the effect of pre-treatment with 30 μM NOT or 30 μM Sora on OCT4, SOX2, ALDH1, SOD1, GPX1, CHOP, or PERK mRNA expression levels. (F) Representative Western blot images showing the effect of 30 μM NOT and 30 μM Sora on the expression levels of OCT4, SOX2, ALDH1, SOD1, GPX1, CHOP and PERK in an ex vivo mice model. GAPDH was loading control. Sora, sorafenib; NOT, notopterol; Vera, verapamil; SP, side-population; * p < 0.05, *** p < 0.001.
Figure 6
Figure 6
In the schematic figure, we demonstrated for the first time that NOT exhibits strong anticancer activity in vitro and ex vivo via suppression of cancer stemness, enhanced endoplasmic reticulum stress and increased oxidative stress.
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