Codelivery of apigenin, FdUMP and CD276 antibody synergistic inhibit colorectal cancer by ferroptosis-apoptosis-pyroptosis and CD276 blockade

Abstract

Mitochondria provides adenosine triphosphate for multiple vital movements to ensure tumor cell proliferation. Compared to the broadly used method of inducing DNA replication arrest to kill cancer, inducing mitochondria damage to cause energy shortage is quite promising as it can inhibit tumor cell bioactivities, increase intracellular accumulation of toxic drugs, eventually sensitize chemotherapy and even reverse drug resistance. Breaking the balance of glutathione (GSH) and reactive oxygen species (ROS) contents have been proven efficient in destroying mitochondria respectively. Herein, apigenin, a GSH efflux reagent, and 2′-deoxy-5-fluorouridine 5′-monophosphate sodium salt (FdUMP) that could induce toxic ROS were co-delivered by constructed lipid nanoparticles, noted as Lip@AF. An immune-checkpoint inhibition reagent CD276 antibody was modified onto the surface of Lip@AF with high reaction specificity (noted as αCD276-Lip@AF) to enhance the recognition of immune cells to tumor. Results showed that the redox balance was destroyed, leading to severe injury to mitochondria and cell membrane. Furthermore, synergistic DNA/RNA replication inhibition caused by inhibiting the function of thymidylate synthase were observed. Eventually, significantly enhanced cytotoxicity was achieved by combining multiple mechanisms including ferroptosis, apoptosis and pyroptosis. In vivo, strengthen tumor growth inhibition was achieved by αCD276-Lip@AF with high biosafety, providing new sights in enhancing chemotherapy sensitiveness and achieving high-performance chemo-immunotherapy.

Keywords

Mitochondria damage / DNA replication arrest / Immune-checkpoint blockade / Chemo-immunotherapy / Colorectal cancer treatment

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (82173769), Tianjin Science Foundation for Distinguished Young Scholars (24JCJQJC00050), Applied Basic Research Multi-Investment Foundation of Tianjin (21JCYBJC01540), the National Natural Science Foundation of China (82300336), and the Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2019KJ178).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ajps.2025.101016. The figures and tables with "S" before the serial number are included in the Supplementary material.

References

[1] Ma P, Luo Z, Li Z, Lin Y, Li Z, Wu Z, et al. Mitochondrial artificial K⁺ channel construction using MPTPP@5F 8 nanoparticles for overcoming cancer drug resistance via disrupting cellular ion homeostasis. Adv Healthc Mater 2024; 13(2):e2302012.

[2] Wang M, Ma PA, Lin J. Nanoplatform-based cellular reactive oxygen species regulation for enhanced oncotherapy and tumor resistance alleviation. Chinese Chem Lett 2023; 34(9):108300.

[3] Ning P, Wang W, Chen M, Feng Y, Meng X. Recent advances in mitochondria- and lysosomes-targeted small-molecule two-photon fluorescent probes. Chinese Chem Lett 2017; 28(10):1943-51.

[4] Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 2021; 22(4):266-82.

[5] Jin X, Tang J, Qiu X, Nie X, Ou S, Wu G, et al. Ferroptosis: emerging mechanisms, biological function, and therapeutic potential in cancer and inflammation. Cell Death Discov 2024; 10(1):45.

[6] Catanzaro E, Demuynck R, Naessens F, Galluzzi L, Krysko DV. Immunogenicity of ferroptosis in cancer: a matter of context? Trends Cancer 2024; 10(5):407-16.

[7] Vodenkova S, Buchler T, Cervena K, Veskrnova V, Vodicka P, Vymetalkova V. 5-Fluorouracil and other fluoropyrimidines in colorectal cancer: past, present and future. Pharmacol Ther 2020; 206:107447.

[8] Bijnsdorp IV, Comijn EM, Padron JM, Gmeiner WH, Peters GJ. Mechanisms of action of FdUMP[10]: metabolite activation and thymidylate synthase inhibition. Oncol Rep 2007; 18(1):287-91.

[9] Xie Y, Kang R, Klionsky DJ, Tang D. GPX4 in cell death, autophagy, and disease. Autophagy 2023; 19(10):2621-38.

[10] Laberge RM, Karwatsky J, Lincoln MC, Leimanis ML, Georges E. Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress. Biochem Pharmacol 2007; 73(11):1727-37.

[11] Choi EJ, Kim GH. 5-Fluorouracil combined with apigenin enhances anticancer activity through induction of apoptosis in human breast cancer MDA-MB-453 cells. Oncol Rep 2009; 22(6):1533-7.

[12] Chan LP, Chou TH, Ding HY, Chen PR, Chiang FY, Kuo PL, et al. Apigenin induces apoptosis via tumor necrosis factor receptor- and Bcl-2-mediated pathway and enhances susceptibility of head and neck squamous cell carcinoma to 5-fluorouracil and cisplatin. Biochim Biophys Acta 2012; 1820(7):1081-91.

[13] Hu XY, Liang JY, Guo XJ, Liu L, Guo YB. 5-Fluorouracil combined with apigenin enhances anticancer activity through mitochondrial membrane potential ($\Delta \Psi \mathrm{m}$)-mediated apoptosis in hepatocellular carcinoma. Clin Exp Pharmacol P 2015; 42(2):146-53.

[14] Yang C, Song J, Hwang S, Choi J, Song G, Lim W. Apigenin enhances apoptosis induction by 5-fluorouracil through regulation of thymidylate synthase in colorectal cancer cells. Redox Biol 2021; 47:102144.

[15] Johnston PG, Lenz HJ, Leichman CG, Danenberg KD, Allegra CJ, Danenberg PV, et al. Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res 1995; 55(7):1407-12.

[16] Chu E, Koeller DM, Johnston PG, Zinn S, Allegra CJ. Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. Mol Pharmacol 1993; 43(4):527-33.

[17] Swain SM, Lippman ME, Egan EF, Drake JC, Steinberg SM, Allegra CJ. Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer. J Clin Oncol 1989; 7(7):890-9.

[18] Peters GJ, Backus HHJ, Freemantle S, Van Triest B, Codacci-Pisanelli G, Van Der Wilt CL, et al. Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism. Bba-Mol Basis Dis 2002; 1587(2-3):194-205.

[19] Zekri L, Lutz M, Prakash N, Manz T, Klimovich B, Mueller S, et al. An optimized IgG-based B7-H3xCD3 bispecific antibody for treatment of gastrointestinal cancers. Mol Ther 2023; 31(4):1033-45.

[20] Hu C, Wang S, Wang J, Ruan X, Wu L, Zhang Z, et al. B7-H3 enhances colorectal cancer progression by regulating HB-EGF via HIF-1 a. J Gastrointest Oncol 2024; 15(3):1035-49.

[21] Zhang T, Wang F, Wu JY, Qiu ZC, Wang Y, Liu F, et al. Clinical correlation of B7-H3 and B3GALT4 with the prognosis of colorectal cancer. World J Gastroenterol 2018; 24(31):3538-46.

[22] Varghese E, Samuel SM, Brockmueller A, Shakibaei M, Kubatka P, Büsselberg D. B7-H 3 at the crossroads between tumor plasticity and colorectal cancer progression: a potential target for therapeutic intervention. Cancer Metastasis Rev 2024; 43(1):115-33.

[23] Hao YY, Li H, Ge XY, Liu Y, Yin JL, Li X, et al. Site-specific nanoswitch circumventing immune resistance via activating TLR and inhibiting PD-L1/PD-1 axis. J Control Release 2023; 361:64-76.

[24] Hu X, Xu M, Hu Y, Li N, Zhou L. B7-H3, negatively regulated by miR-128, promotes colorectal cancer cell proliferation and migration. Cell Biochem Biophys 2021; 79(2):397-405.

[25] Jin H, Zhu M, Zhang D, Liu X, Guo Y, Xia L, et al. B7H3 increases ferroptosis resistance by inhibiting cholesterol metabolism in colorectal cancer. Cancer Sci 2023; 114(11):4225-36.

[26] Guo J, Yu Z, Sun D, Zou Y, Liu Y, Huang L. Two nanoformulations induce reactive oxygen species and immunogenetic cell death for synergistic chemo-immunotherapy eradicating colorectal cancer and hepatocellular carcinoma. Mol Cancer 2021; 20(1):10.

[27] Oliveira JP, Prado AR, Keijok WJ, Antunes PWP, Yapuchura ER, Guimarães MCC. Impact of conjugation strategies for targeting of antibodies in gold nanoparticles for ultrasensitive detection of 17$\beta$-estradiol. Sci Rep 2019; 9(1):13859.

[28] Yang H, Le QV, Shim G, Oh YK, Shin YK. Molecular engineering of antibodies for site-specific conjugation to lipid polydopamine hybrid nanoparticles. Acta Pharm Sin B 2020; 10(11):2212-26.

[29] Lwin TM, Hernot S, Hollandsworth H, Amirfakhri S, Filemoni F, Debie P, et al. Tumor-specific near-infrared nanobody probe rapidly labels tumors in an orthotopic mouse model of pancreatic cancer. Surgery 2020; 168(1):85-91.

[30] Su Z, Xiao D, Xie F, Liu L, Wang Y, Fan S, et al. Antibody-drug conjugates: recent advances in linker chemistry. Acta Pharmacol Sin B 2021; 11(12):3889-907.

[31] Cao W, Zhang X, Li R, Li Z, Lu A, Yu F, et al. Lipid core-shell nanoparticles co-deliver FOLFOX regimen and siPD-L1 for synergistic targeted cancer treatment. J Control Release 2024; 368:52-65.

[32] Cao W, Zhang X, Feng Y, Li R, Lu A, Li Z, et al. Lipid nanoparticular codelivery system for enhanced antitumor effects by ferroptosis-apoptosis synergistic with programmed cell death-ligand 1 downregulation. ACS nano 2024; 18(26):17267-81.