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. 2021 Jun;11(6):1513-1525.
doi: 10.1016/j.apsb.2021.05.006. Epub 2021 May 13.

Identification of ferroptosis as a novel mechanism for antitumor activity of natural product derivative a2 in gastric cancer

Ying Liu  1 , Zan Song  1 , Yajie Liu  1 , Xubin Ma  1 , Wang Wang  1 , Yu Ke  1 , Yichao Xu  1 , Dequan Yu  2 , Hongmin Liu  1
Affiliations

Identification of ferroptosis as a novel mechanism for antitumor activity of natural product derivative a2 in gastric cancer

Ying Liu et al. Acta Pharm Sin B. 2021 Jun.

Abstract

Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, thus stimulating ferroptosis may be a potential strategy for treating gastric cancer, therapeutic agents against which are urgently required. Jiyuan oridonin A (JDA) is a natural compound isolated from Jiyuan Rabdosia rubescens with anti-tumor activity, unclear anti-tumor mechanisms and limited water solubility hamper its clinical application. Here, we showed a2, a new JDA derivative, inhibited the growth of gastric cancer cells. Subsequently, we discovered for the first time that a2 induced ferroptosis. Importantly, compound a2 decreased GPX4 expression and overexpressing GPX4 antagonized the anti-proliferative activity of a2. Furthermore, we demonstrated that a2 caused ferrous iron accumulation through the autophagy pathway, prevention of which rescued a2 induced ferrous iron elevation and cell growth inhibition VSports手机版. Moreover, a2 exhibited more potent anti-cancer activity than 5-fluorouracil in gastric cancer cell line-derived xenograft mice models. Patient-derived tumor xenograft models from different patients displayed varied sensitivity to a2, and GPX4 downregulation indicated the sensitivity of tumors to a2. Finally, a2 exhibited well pharmacokinetic characteristics. Overall, our data suggest that inducing ferroptosis is the major mechanism mediating anti-tumor activity of a2, and a2 will hopefully serve as a promising compound for gastric cancer treatment. .

Keywords: 5-FU, 5-fluorouracil; Autophagy; CDX, cell line-derived xenograft; DCFH-DA, dichlorodihydro-fluorescein diacetate; DCM, dichloromethane; Ferroptosis; Ferrous iron; GPX4; Gastric cancer; IKE, imidazole ketone erastin; JDA derivative; JDA, Jiyuan oridonin A; Jiyuan Rabdosia rubescens; KEGG, Kyoto Encyclopedia of Genes and Genomes; NAC, N-acetylcysteine; PARP, poly ADP-ribose polymerase; PDX, patient-derived tumor xenograft; PK, pharmacokinetic; Papp, apparent permeability coefficient; ROS; ROS, reactive oxygen species; RTV, relative tumor volume; Verp, verapamil; qRT-PCR, quantitative real time PCR. V体育安卓版.

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Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Compound a2 selectively inhibited the growth of gastric cancer cells. (A) Structural formulas of JDA and a2. (B) GI50 values of a2 in selected cells for 72 h. (C) MGC-803 cells were treated with a2 at indicated doses and then were imaged by the bright-field microscopy at specific point in time. (D) MGC-803 cells were treated with a2 at indicated concentrations for different time duration and then cell viability was detected by SRB staining. (E) MGC-803 cells were treated with a2 for 48 h, cells were then stained with PI and analyzed by flow cytometry and quantified. Data are presented as the mean ± SD (n = 3) from three independent experiments with biological duplicates in (B, D, E). Statistics differences were analyzed by two-way ANOVA analysis (E): ∗P < 0.05, ∗∗∗P < 0.005 vs. the control.
Figure 2
Figure 2
Compound a2 induced mitochondria-dependent apoptosis in gastric cancer cells. MGC-803 cells were treated with a2 at indicated concentrations for 24 h (A and B) and 48 h (C and D), cells were then stained by PI and Annexin V-FITC and analyzed by flow cytometry. (E and F) MGC-803 cells were treated with a2 for 24 h, cells were then stained by JC-1 and analyzed by flow cytometry. (G) MGC-803 cells were treated with a2 for 24 h with illustrated concentrations, indicated proteins were tested by Western blot. (H) MGC-803 cells were treated with a2 alone or in combination with the indicated agents for 72 h, cell viability was then tested by SRB assay. Data are presented as the mean ± SD (n = 3) from three independent experiments with biological duplicates in (B, D, F, H). Statistics differences were analyzed by two-way ANOVA analysis (B, D, H) or one-way ANOVA analysis (F): ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.001 vs. the control (B, D, F). ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001 vs. the a2 treated samples (H).
Figure 3
Figure 3
Identification of ferroptosis induced by compound a2 in gastric cancer cells. (A) Heatmap of differentially expressed mRNAs after incubation of MGC-803 cells with a2 (10 μmol/L) for 24 h (RNA-seq, n = 3), P < 0.05, basemean of read > 100. (B) KEGG pathways of differentially expressed mRNAs in a2 treated vs. control MGC-803 cells. Bar color represents statistical significance of the enrichment, length of bar indicates gene number. (C) Heatmap of differentially expressed mRNAs in ferroptosis pathway in a2 treated versus control MGC-803 cells. (D) MGC-803 cells were treated with a2 for 6 h, cells were then stained by C11-BODIPY581/591 and analyzed by flow cytometry. (E) MGC-803 cells were treated with a2 in the absence or presence of NAC for 24 h, cells were then stained with DCFH-DA and analyzed by flow cytometry. (F) MGC-803 cells were treated with a2 in the absent or present of NAC for 72 h, relative inhibitory rates of compounds were determined with SRB assay. (G) MGC-803 cells were treated with a2 for 6 h, cells were then observed by transmission electron microscopy. Data are presented as the mean ± SD (n = 3) from three independent experiments with biological duplicates in (D and F). Statistics differences were analyzed by one-way ANOVA analysis (D) or two-way ANOVA analysis (E and F): ∗∗P < 0.01 vs. the control (D). ∗∗∗P < 0.005, ∗∗∗∗P < 0.001 vs. the a2 treated samples (E and F).
Figure 4
Figure 4
Compound a2 induced ferroptosis via decreasing GPX4. MGC-803 (A) and MKN-45 (B) cells were treated with a2 at indicated doses for 24 h, indicated mRNAs were then determined by qRT-PCR. (C) MGC-803 and MKN-45 cells were treated with a2 for 24 h, indicated proteins were measured. GPX4 were overexpressed in MGC-803 (D) and MKN-45 (E) cells, relative inhibitory rates of a2 in indicated cells for 24 h were tested. MGC-803 (F) and MKN-45 (G) cells were treated with a2 alone or combined with IKE for 72 h, cells were then examined by SRB assay. Data are presented as the mean ± SD (n = 3) from three independent experiments with biological duplicates in (A–B, D–G). Statistics differences were analyzed by one-way ANOVA analysis (A and B) or two-way ANOVA analysis (D–G): ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.001 vs. the control (A–B, D–E). ∗P < 0.05, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001 vs. the a2 treated samples (F and G).
Figure 5
Figure 5
Compound a2 induced ferroptosis through accumulation of ferrous iron. (A) Ferrous iron contents were measured in MGC-803 and MKN-45 cells treated with a2 for 24 h. Relative inhibitory effects of a2 without or with deferoxamine on MGC-803 (B) and MKN-45 (C) cells (72 h). MGC-803 (D) and MKN-45 (E) cells were treated with a2 alone or combined with 3-MA for 24 h, ferrous iron contents were then examined. MGC-803 (F) and MKN-45 (G) cells were treated with indicated compounds for 72 h, relative inhibitory rates were tested. Data are presented as the mean ± SD (n = 3) from three independent experiments with biological duplicates in (A–G). Statistics differences were analyzed by two-way ANOVA analysis: ∗∗P < 0.05, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001 vs. the control (A). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.005, ∗∗∗∗P < 0.001 vs. the a2 treated samples (B–G).
Figure 6
Figure 6
Compound a2 inhibited tumor growth in both CDX and PDX models of gastric cancer. (A) Male mice bearing MGC-803 xenograft were treated with indicated agents once a day for 21 days, tumor volume was measured periodically. Data are presented as the mean ± SD (n = 6). (B) Average tumor weights with indicated agents at the end of treatment. Data are presented as the mean ± SD (n = 6). Statistics differences were analyzed by one-way ANOVA analysis: ∗P < 0.05, ∗∗P < 0.01 vs. the saline group. (C) Tumor growth inhibition T/C ratio of indicated compounds were measured at the end of treatment in mice bearing specific gastric cancer patient derived xenografts. (D) Expression of GPX4 in tumor tissues from PDX modes was determined by IHC.
Figure 7
Figure 7
Pharmacokinetic characteristics of compound a2. (A) Microsomal metabolic stability of a2 (1 μmol/L) in liver microsomes from indicated species. Verapamil (Verp) was selected as a positive control. Data are presented as the mean ± SD (n = 2). (B) Mean plasma concentration–time profile of a2 after an intravenous administration of a2 (20 mg/kg) to male SD rats. Data are presented as the mean ± SD (n = 3).
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"V体育官网入口" References

    1. Bray F., Ferlay J., Soerjomataram I., Siegel R.L., Torre L.A., Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. - PubMed
    1. Smyth E.C., Nilsson M., Grabsch H.I., van Grieken N.C., Lordick F. Gastric cancer. Lancet. 2020;396:635–648. - PubMed
    1. Liu H.M., Zhu C.G., Wang Q.D., Ke Y., Liu Z.Z., Yan X.B. 2011 Dec 27. inventors. Novel ent-kaurene diterpene compound and its derivatives, their preparation and their use. US patent US8084430B2.
    1. Zhang Y., Wang S., Dai M., Nai J., Zhu L., Sheng H. Solubility and bioavailability enhancement of oridonin: a review. Molecules. 2020;25:332–355. - PMC - PubMed
    1. Sarwar M.S., Xia Y.X., Liang Z.M., Tsang S.W., Zhang H.J. Mechanistic pathways and molecular targets of plant-derived anticancer ent-kaurane diterpenes. Biomolecules. 2020;10:144–161. - PMC - PubMed

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