Skip to main content

Advertisement

SpringerLink
Log in

Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling

  • Original Article
  • Published:
Cancer Chemotherapy and Pharmacology Aims and scope Submit manuscript

Abstract

Purpose

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Intriguingly, KRAS-mutant cancers are particularly vulnerable to ferroptosis. Osthole is a natural coumarin extracted from Cnidium spp. and other Apiaceous plants. In the present study, we explored the antitumor potential of osthole in KRAS-mutant colorectal cancer (CRC) cells.

Methods

Cell viability assay, EdU incorporation assay, flow cytometry, tumor xenograft model, western blot, immunochemistry staining, immunofluorescence, transcriptome RNA sequencing and quantitative reverse transcription-PCR were performed to evaluate the influence of osthole treatment on KRAS-mutant CRC cells.

Results

We found that osthole treatment suppressed proliferation and tumor growth of KRAS-mutant CRC cell lines HCT116 and SW480. Moreover, osthole treatment increased ROS production and induced ferroptosis. Osthole treatment also promoted autophagy, but inhibition of autophagy by ATG7 knockdown or 3-MA showed no influence on osthole-induced ferroptosis. In comparison, osthole increased lysosomal activation, and co-treatment with lysosome inhibitor Baf-A1 attenuated osthole-induced ferroptosis. Besides, osthole treatment reduced the phosphorylation of AMPK, Akt and mTOR in HCT116 and SW480 cells, while restored AMPK signaling by AMPK agonist AICAR partially abrogated ferroptosis induced by osthole treatment. Finally, co-treatment with osthole increased the cytotoxicity of cetuximab in KRAS-mutant CRC cells in vitro and in vivo.

Conclusion

Our results suggested that the natural product osthole exerted its anticancer effects in KRAS-mutant CRC cells via inducing ferroptosis, and this was partially through inhibiting AMPK/Akt/mTOR signaling. Our results may expand our current knowledge for the use of osthole as an anticancer agent.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA003938) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J Clin. 71(3):209–249. https://doi.org/10.3322/caac.21660

    Article  PubMed  Google Scholar 

  2. Miller KD, Nogueira L, Mariotto AB, Rowland JH, Yabroff KR, Alfano CM, Jemal A, Kramer JL, Siegel RL (2019) Cancer treatment and survivorship statistics, 2019. CA: Cancer J Clin. 69(5):363–385. https://doi.org/10.3322/caac.21565

    Article  PubMed  Google Scholar 

  3. Brenner H, Kloor M, Pox CP (2014) Colorectal cancer. Lancet 383(9927):1490–1502. https://doi.org/10.1016/S0140-6736(13)61649-9

    Article  PubMed  Google Scholar 

  4. Serebriiskii IG, Connelly C, Frampton G, Newberg J, Cooke M, Miller V, Ali S, Ross JS, Handorf E, Arora S, Lieu C, Golemis EA, Meyer JE (2019) Comprehensive characterization of RAS mutations in colon and rectal cancers in old and young patients. Nat Commun 10(1):3722. https://doi.org/10.1038/s41467-019-11530-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Arrington AK, Heinrich EL, Lee W, Duldulao M, Patel S, Sanchez J, Garcia-Aguilar J, Kim J (2012) Prognostic and predictive roles of KRAS mutation in colorectal cancer. Int J Mol Sci 13(10):12153–12168. https://doi.org/10.3390/ijms131012153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shen Q, Liang M, Yang F, Deng YZ, Naqvi NI (2020) Ferroptosis contributes to developmental cell death in rice blast. New Phytol 227(6):1831–1846. https://doi.org/10.1111/nph.16636

    Article  CAS  PubMed  Google Scholar 

  7. Distefano AM, Martin MV, Cordoba JP, Bellido AM, D’Ippolito S, Colman SL, Soto D, Roldan JA, Bartoli CG, Zabaleta EJ, Fiol DF, Stockwell BR, Dixon SJ, Pagnussat GC (2017) Heat stress induces ferroptosis-like cell death in plants. J Cell Biol 216(2):463–476. https://doi.org/10.1083/jcb.201605110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Stockwell BR (2022) Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. Cell 185(14):2401–2421. https://doi.org/10.1016/j.cell.2022.06.003

    Article  CAS  PubMed  Google Scholar 

  9. Mukhopadhyay S, Vander Heiden MG, McCormick F (2021) The metabolic landscape of RAS-driven cancers from biology to therapy. Nat Cancer 2(3):271–283. https://doi.org/10.1038/s43018-021-00184-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bartolacci C, Andreani C, El-Gammal Y, Scaglioni PP (2021) Lipid metabolism regulates oxidative stress and ferroptosis in ras-driven cancers: a perspective on cancer progression and therapy. Front Mol Biosci 8:7066. https://doi.org/10.3389/fmolb.2021.706650

    Article  CAS  Google Scholar 

  11. Dolma S, Lessnick SL, Hahn WC, Stockwell BR (2003) Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3(3):285–296. https://doi.org/10.1016/s1535-6108(03)00050-3

    Article  CAS  PubMed  Google Scholar 

  12. Yang WS, Stockwell BR (2008) Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol 15(3):234–245. https://doi.org/10.1016/j.chembiol.2008.02.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149(5):1060–1072. https://doi.org/10.1016/j.cell.2012.03.042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sun M, Sun M, Zhang J (2021) Osthole: an overview of its sources, biological activities, and modification development. Med Chem Res 30(10):1767–1794. https://doi.org/10.1007/s00044-021-02775-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shokoohinia Y, Jafari F, Mohammadi Z, Bazvandi L, Hosseinzadeh L, Chow N, Bhattacharyya P, Farzaei MH, Farooqi AA, Nabavi SM, Yerer MB, Bishayee A (2018) Potential anticancer properties of osthol: a comprehensive mechanistic review. Nutrients. https://doi.org/10.3390/nu10010036

    Article  PubMed  PubMed Central  Google Scholar 

  16. Huangfu M, Wei R, Wang J, Qin J, Yu D, Guan X, Li X, Fu M, Liu H, Chen X (2021) Osthole induces necroptosis via ROS overproduction in glioma cells. FEBS Open Bio 11(2):456–467. https://doi.org/10.1002/2211-5463.13069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Liang L, Yang B, Wu Y, Sun L (2021) Osthole suppresses the proliferation and induces apoptosis via inhibiting the PI3K/AKT signaling pathway of endometrial cancer JEC cells. Exp Ther Med 22(4):1171. https://doi.org/10.3892/etm.2021.10605

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhou XH, Kang J, Zhong ZD, Cheng Y (2021) Osthole induces apoptosis of the HT-29 cells via endoplasmic reticulum stress and autophagy. Oncol Lett 22(4):726. https://doi.org/10.3892/ol.2021.12987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huang SM, Tsai CF, Chen DR, Wang MY, Yeh WL (2014) p53 is a key regulator for osthole-triggered cancer pathogenesis. Biomed Res Int 2014:175247. https://doi.org/10.1155/2014/175247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, Fulda S, Gascon S, Hatzios SK, Kagan VE, Noel K, Jiang X, Linkermann A, Murphy ME, Overholtzer M, Oyagi A, Pagnussat GC, Park J, Ran Q, Rosenfeld CS, Salnikow K, Tang D, Torti FM, Torti SV, Toyokuni S, Woerpel KA, Zhang DD (2017) Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171(2):273–285. https://doi.org/10.1016/j.cell.2017.09.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Petrat F, de Groot H, Rauen U (2000) Determination of the chelatable iron pool of single intact cells by laser scanning microscopy. Arch Biochem Biophys 376(1):74–81. https://doi.org/10.1006/abbi.2000.1711

    Article  CAS  PubMed  Google Scholar 

  22. Hou W, Xie Y, Song X, Sun X, Lotze MT, Zeh HJ 3rd, Kang R, Tang D (2016) Autophagy promotes ferroptosis by degradation of ferritin. Autophagy 12(8):1425–1428. https://doi.org/10.1080/15548627.2016.1187366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gao M, Monian P, Pan Q, Zhang W, Xiang J, Jiang X (2016) Ferroptosis is an autophagic cell death process. Cell Res 26(9):1021–1032. https://doi.org/10.1038/cr.2016.95

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Collier JJ, Guissart C, Olahova M, Sasorith S, Piron-Prunier F, Suomi F, Zhang D, Martinez-Lopez N, Leboucq N, Bahr A, Azzarello-Burri S, Reich S, Schols L, Polvikoski TM, Meyer P, Larrieu L, Schaefer AM, Alsaif HS, Alyamani S, Zuchner S, Barbosa IA, Deshpande C, Pyle A, Rauch A, Synofzik M, Alkuraya FS, Rivier F, Ryten M, McFarland R, Delahodde A, McWilliams TG, Koenig M, Taylor RW (2021) Developmental consequences of defective ATG7-mediated autophagy in humans. N Engl J Med 384(25):2406–2417. https://doi.org/10.1056/NEJMoa1915722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Senol E, Ersoy A, Erdinc S, Sarandol E, Yurtkuran M (2008) Oxidative stress and ferritin levels in haemodialysis patients. Nephrol Dial Transplant 23(2):665–672. https://doi.org/10.1093/ndt/gfm588

    Article  CAS  PubMed  Google Scholar 

  26. Settembre C, Zoncu R, Medina DL, Vetrini F, Erdin S, Erdin S, Huynh T, Ferron M, Karsenty G, Vellard MC, Facchinetti V, Sabatini DM, Ballabio A (2012) A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. EMBO J 31(5):1095–1108. https://doi.org/10.1038/emboj.2012.32

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, Tyagi S, Ma L, Westbrook TF, Steinberg GR, Nakada D, Stockwell BR, Gan B (2020) Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol 22(2):225–234. https://doi.org/10.1038/s41556-020-0461-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Li C, Dong X, Du W, Shi X, Chen K, Zhang W, Gao M (2020) LKB1-AMPK axis negatively regulates ferroptosis by inhibiting fatty acid synthesis. Signal Transduct Target Ther 5(1):187. https://doi.org/10.1038/s41392-020-00297-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yi J, Zhu J, Wu J, Thompson CB, Jiang X (2020) Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Sci USA 117(49):31189–31197. https://doi.org/10.1073/pnas.2017152117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu D, Tebbutt NC, Simes RJ, Chalchal H, Shapiro JD, Robitaille S, Price TJ, Shepherd L, Au HJ, Langer C, Moore MJ, Zalcberg JR (2008) K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 359(17):1757–1765. https://doi.org/10.1056/NEJMoa0804385

    Article  CAS  PubMed  Google Scholar 

  31. Kordulewska NK, Topa J, Tanska M, Cieslinska A, Fiedorowicz E, Savelkoul HFJ, Jarmolowska B (2020) Modulatory effects of osthole on lipopolysaccharides-induced inflammation in caco-2 cell monolayer and co-cultures with THP-1 and THP-1-derived macrophages. Nutrients. https://doi.org/10.3390/nu13010123

    Article  PubMed  PubMed Central  Google Scholar 

  32. Duan J, Yang Y, Liu H, Dou PC, Tan SY (2016) Osthole ameliorates acute myocardial infarction in rats by decreasing the expression of inflammatory-related cytokines, diminishing MMP-2 expression and activating p-ERK. Int J Mol Med 37(1):207–216. https://doi.org/10.3892/ijmm.2015.2402

    Article  CAS  PubMed  Google Scholar 

  33. Peng KY, Chou TC (2022) Osthole exerts inhibitory effects on hypoxic colon cancer cells via EIF2[Formula: see text] phosphorylation-mediated apoptosis and regulation of HIF-1[Formula: see text]. Am J Chin Med 50(2):621–637. https://doi.org/10.1142/S0192415X22500240

    Article  CAS  PubMed  Google Scholar 

  34. Farooq S, Banday JA, Hussain A, Nazir M, Qurishi MA, Hamid A, Koul S (2019) Synthesis and biological evaluation of novel osthol derivatives as potent cytotoxic agents. Med Chem 15(2):138–149. https://doi.org/10.2174/1573406414666180911161047

    Article  CAS  PubMed  Google Scholar 

  35. Zhang ZR, Leung WN, Cheung HY, Chan CW (2015) Osthole: a review on its bioactivities, pharmacological properties, and potential as alternative medicine. Evid Based Complement Alternat Med: eCAM 2015:919616. https://doi.org/10.1155/2015/919616

    Article  PubMed  PubMed Central  Google Scholar 

  36. Chen P, Li X, Zhang R, Liu S, Xiang Y, Zhang M, Chen X, Pan T, Yan L, Feng J, Duan T, Wang D, Chen B, Jin T, Wang W, Chen L, Huang X, Zhang W, Sun Y, Li G, Kong L, Chen X, Li Y, Yang Z, Zhang Q, Zhuo L, Sui X, Xie T (2020) Combinative treatment of beta-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation. Theranostics 10(11):5107–5119. https://doi.org/10.7150/thno.44705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yan WY, Cai J, Wang JN, Gong YS, Ding XB (2022) Co-treatment of betulin and gefitinib is effective against EGFR wild-type/KRAS-mutant non-small cell lung cancer by inducing ferroptosis. Neoplasma 69(3):648–656. https://doi.org/10.4149/neo_2022_211103N1568

    Article  CAS  PubMed  Google Scholar 

  38. Wang J, Huangfu M, Li X, Han M, Liu G, Yu D, Zhou L, Dou T, Liu Y, Guan X, Wei R, Chen X (2022) osthole induces apoptosis and caspase-3/GSDME-dependent pyroptosis via NQO1-mediated ROS generation in hela cells. Oxid Med Cell Longev 2022:8585598. https://doi.org/10.1155/2022/8585598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sumorek-Wiadro J, Zajac A, Langner E, Skalicka-Wozniak K, Maciejczyk A, Rzeski W, Jakubowicz-Gil J (2020) Antiglioma potential of coumarins combined with sorafenib. Molecules. https://doi.org/10.3390/molecules25215192

    Article  PubMed  PubMed Central  Google Scholar 

  40. Zheng X, Yu Y, Shao B, Gan N, Chen L, Yang D (2019) Osthole improves therapy for osteoporosis through increasing autophagy of mesenchymal stem cells. Exp Anim 68(4):453–463. https://doi.org/10.1538/expanim.18-0178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pierzynowska K, Rintz E, Gaffke L, Wegrzyn G (2021) Ferroptosis and Its modulation by autophagy in light of the pathogenesis of lysosomal storage diseases. Cells. https://doi.org/10.3390/cells10020365

    Article  PubMed  PubMed Central  Google Scholar 

  42. Wang ZX, Ma J, Li XY, Wu Y, Shi H, Chen Y, Lu G, Shen HM, Lu GD, Zhou J (2021) Quercetin induces p53-independent cancer cell death through lysosome activation by the transcription factor EB and reactive oxygen species-dependent ferroptosis. Br J Pharmacol 178(5):1133–1148. https://doi.org/10.1111/bph.15350

    Article  CAS  PubMed  Google Scholar 

  43. Mai TT, Hamai A, Hienzsch A, Caneque T, Muller S, Wicinski J, Cabaud O, Leroy C, David A, Acevedo V, Ryo A, Ginestier C, Birnbaum D, Charafe-Jauffret E, Codogno P, Mehrpour M, Rodriguez R (2017) Salinomycin kills cancer stem cells by sequestering iron in lysosomes. Nat Chem 9(10):1025–1033. https://doi.org/10.1038/nchem.2778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

None.

Funding

None.

Author information

Author notes

  1. Xinghua Zhou, Jian Kang have contributed equally to this article.

Authors and Affiliations

  1. Department of Anorectal Diseases, Hospital of Chengdu University of Traditional Chinese Medicine, No. 39 Shiqiao Road, Jinniu District, Chengdu, 610075, China

    Xinghua Zhou, Jian Kang, Liangliang Zhang & Yue Cheng

Authors
  1. Xinghua Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liangliang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors guaranteed the integrity of the entire study. The experiments were conducted by XZ and JK. Data was analyzed by LZ and YC. Manuscript was prepared and reviewed by YC. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Yue Cheng.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal studies and all protocols for animal studies were reviewed and approved by the ethical standards of the ethics committee of the Hospital of Chengdu University of Traditional Chinese Medicine (Approval no. 2020KL-012).

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

280_2023_4549_MOESM1_ESM.jpg

Supplementary Figure 1. Validation of KRAS mutations in colorectal cancer cell lines by sanger sequencing. A, KRAS G13D mutation in HCT116 cells. B, KRAS G12V mutation in SW480 cells. Supplementary file1 (JPG 611 KB)

280_2023_4549_MOESM2_ESM.jpg

Supplementary Figure 2. Osthole and/or cetuximab treatment in HCT116 tumor xenografts. A, osthole and/or cetuximab treatment showed no influence on body weight of Balb/c nude mice. B, C, phosphorylated EGFR and SLC7A11 were evaluated by IHC staining in HCT116 tumor xenografts. Data were analyzed by unpaired Student’s t-test or one-way ANOVA (Tukey's post-hoc test). *P<0.05 between compared groups. Supplementary file2 (JPG 921 KB)

280_2023_4549_MOESM3_ESM.jpg

Supplementary Figure 3. Osthole increases the antitumor effects of cetuximab in MC38 orthotopic tumors. A-D, MC38 cells (1×106) were orthotopically injected into the cecum of 6-week-old female C57BL/6J mice, then treated with 20 mg/kg osthole, 10 mg/kg cetuximab or equal volume of corn oil intraperitoneally as indicated once every other day for 3 weeks. Tumor growth (A), representative image (B), tumor weight (C) and mice body weight (D) were shown. E-F, TUNEL positive cells, phosphorylated EGFR, and phosphorylated Akt, transferrin and SLC7A11 were evaluated by IHC staining in MC38 tumors. Representative images (E) and relative positive cells (F) were shown. Data were analyzed by unpaired Student’s t-test or one-way ANOVA (Tukey's post-hoc test). *P<0.05 between compared groups. Supplementary file3 (JPG 2395 KB)

Supplementary file4 (XLSX 53 KB)

Supplementary file5 (DOCX 14 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, X., Kang, J., Zhang, L. et al. Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling. Cancer Chemother Pharmacol 92, 119–134 (2023). https://doi.org/10.1007/s00280-023-04549-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00280-023-04549-0

Keywords

Search

Navigation