Pristimerin induces Noxa-dependent apoptosis by activating the FoxO3a pathway in esophageal squamous cell carcinoma

Abstract

Pristimerin, which is one of the compounds present in Celastraceae and Hippocrateaceae, has antitumor effects. However, its mechanism of action in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aims to investigate the efficacy and mechanism of pristimerin on ESCC in vitro and in vivo. The inhibitory effect of pristimerin on cell growth was assessed using trypan blue exclusion and colony formation assays. Cell apoptosis was evaluated by flow cytometry. Gene and protein expressions were analyzed through quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry. RNA sequencing (RNA-Seq) was employed to identify significantly differentially expressed genes (DEGs). Cell transfection and RNA interference assays were utilized to examine the role of key proteins in pristimerin’s effect. Xenograft models were established to evaluate the antitumor efficiency of pristimerin in vivo. Pristimerin inhibited cell growth and induced apoptosis in ESCC cells. Upregulation of Noxa was crucial for pristimerin-induced apoptosis. Pristimerin activated the Forkhead box O3a (FoxO3a) signaling pathway and triggered FoxO3a recruitment to the Noxa promoter, leading to Noxa transcription. Blocking FoxO3a reversed pristimerin-induced Noxa upregulation and cell apoptosis. Pristimerin treatment suppressed xenograft tumors in nude mice, but these effects were largely negated in Noxa-KO tumors. Furthermore, the chemosensitization effects of pristimerin in vitro and in vivo were mediated by Noxa. This study demonstrates that pristimerin exerts an antitumor effect on ESCC by inducing AKT/FoxO3a-mediated Noxa upregulation. These findings suggest that pristimerin may serve as a potent anticancer agent for ESCC treatment.

Keywords

ESCC / Pristimerin / FoxO3a / Noxa / Apoptosis

References

[1] Sung H, Ferlay J, Siegel RL, et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3):209-249. https://doi.org/10.3322/caac.21660.

[2] Zeng HM, Zheng RS, Guo YM, et al.Cancer survival in China, 2003-2005: a population-based study. Int J Cancer. 2015; 136(8):1921-1930. https://doi.org/10.1002/ijc.29227.

[3] Testa U, Castelli G, Pelosi E. Esophageal cancer: genomic and molecular characterization, stem cell compartment and clonal evolution. Medicines (Basel). 2017; 4(3):67.

[4] Watanabe M, Toh Y, Ishihara R, et al.Comprehensive registry of esophageal cancer in Japan, 2015. Esophagus. 2023; 20(1):1-28. https://doi.org/10.1007/s10388-022-00950-5.

[5] He YT, Liang D, Du LB, et al. Clinical characteristics and survival of 5283 esophageal cancer patients: a multicenter study from eighteen hospitals across six regions in China. Cancer Commun (Lond). 2020; 40(10):531-544. https://doi.org/10.1002/cac2.12087.

[6] Cory S, Adams JM. The Bcl 2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer. 2002; 2(9):647-656. https://doi.org/10.1038/nrc883.

[7] Dubecz A, Gall I, Solymosi N, et al. Temporal trends in long-term survival and cure rates in esophageal cancer. J Thorac Oncol. 2012; 7(2):443-447. https://doi.org/10.1097/JTO.0b013e3182397751.

[8] Ajani JA, D'Amico TA, Bentrem DJ, et al. Esophageal and esophagogastric junction cancers, version 2.2019, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2019; 17(7):855-883. https://doi.org/10.6004/jnccn.2019.0033.

[9] Okumura H, Uchikado Y, Matsumoto M, et al. Prognostic factors in esophageal squamous cell carcinoma patients treated with neoadjuvant chemoradiation therapy. Int J Clin Oncol. 2013; 18(2):329-334. https://doi.org/10.1007/s10147-012-0383-y.

[10] Sewastjanow MD, Rogers JE, Hofstetter WL, et al. Esophageal cancer: is the CROSS strategy ready for history books? J Thorac Cardiovasc Surg. 2023; 165(3):901-905. https://doi.org/10.1016/j.jtcvs.2021.12.054.

[11] Petronelli A, Pannitteri G, Testa U. Triterpenoids as new promising anticancer drugs. Anticancer Drugs. 2009; 20(10):880-892. https://doi.org/10.1097/CAD.0b013e328330fd90.

[12] Figueiredo JN, Räz B, Séquin U. Novel quinone methides from with in vitro antimalarial activity. J Nat Prod. 1998; 61(6):718-723. https://doi.org/10.1021/np9704157.

[13] Nagpal K, Garg M, Arora D, et al. An extensive review on phytochemistry and pharmacological activities of Indian medicinal plant Willd. Phytother Res. 2022; 36(5):1930-1951. https://doi.org/10.1002/ptr.7424.

[14] Tian MX, Peng S, Wang SS, et al.Pristimerin reduces dextran sulfate sodium-induced colitis in mice by inhibiting microRNA-155. Int Immunopharmacol. 2021;94:107491. https://doi.org/10.1016/j.intimp.2021.107491.

[15] Lei XP, Zhong YH, Huang LJ, et al. Identification of a novel tumor angiogenesis inhibitor targeting Shh/Gli1 signaling pathway in non-small cell lung cancer. Cell Death Dis. 2020; 11(4):232. https://doi.org/10.1038/s41419-020-2425-0.

[16] Yousef BA, Hassan HM, Zhang LY, et al. Pristimerin exhibits and anticancer activities through inhibition of nuclear factor-κB signaling pathway in colorectal cancer cells. Phytomedicine. 2018; 40:140-147. https://doi.org/10.1016/j.phymed.2018.01.008.

[17] Tang YB, Chen J, Li JQ, et al. Pristimerin synergistically sensitizes conditionally reprogrammed patient derived-primary hepatocellular carcinoma cells to sorafenib through endoplasmic reticulum stress and ROS generation by modulating Akt/FoxO1/p27 signaling pathway. Phytomedicine. 2021;86:153563. https://doi.org/10.1016/j.phymed.2021.153563.

[18] Chen RZ, Yang F, Zhang M, et al. Cellular and molecular mechanisms of pristimerin in cancer therapy: recent advances. Front Oncol. 2021;11:671548. https://doi.org/10.3389/fonc.2021.671548.

[19] Li JJ, Yan YY, Sun HM, et al. Anti-cancer effects of pristimerin and the mechanisms: a critical review. Front Pharmacol. 2019;10:746. https://doi.org/10.3389/fphar.2019.00746.

[20] Tu YQ, Tan FX, Zhou JF, et al. Pristimerin targeting NF-κB pathway inhibits proliferation, migration, and invasion in esophageal squamous cell carcinoma cells. Cell Biochem Funct. 2018; 36(4):228-240. https://doi.org/10.1002/cbf.3335.

[21] Ehrhardt H, Höfig I, Wachter F, et al. NOXA as critical mediator for drug combinations in polychemotherapy. Cell Death Dis. 2012; 3(6):e327. https://doi.org/10.1038/cddis.2012.53.

[22] Wang J, Thomas HR, Li Z, et al. Puma noxa, p53, and p63 differentially mediate stress pathway induced apoptosis. Cell Death Dis. 2021; 12(7):659. https://doi.org/10.1038/s41419-021-03902-6.

[23] Feehan RP, Nelson AM, Shantz LM. Inhibition of mTORC2 enhances UVB-induced apoptosis in keratinocytes heck fo through a mechanism dependent on the FOXO3a transcriptional target NOXA but independent of TRAIL. Cell Signal. 2018; 52:35-47. https://doi.org/10.1016/j.cellsig.2018.08.018.

[24] Wang Y, Poon RYC. MARCH5 regulates mitotic apoptosis through MCL1-dependent and independent mechanisms. Cell Death Differ. 2023; 30(3):753-765. https://doi.org/10.1038/s41418-022-01080-2.

[25] Huang AS, Chin HS, Reljic B, et al. Mitochondrial E 3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins. Cell Death Differ. 2023; 30(3):632-646. https://doi.org/10.1038/s41418-022-01067-z.

[26] Park SY, Jeong MS, Jang SB. In vitro binding properties of tumor suppressor p53 with PUMA and NOXA. Biochem Biophys Res Commun. 2012; 420(2):350-356. https://doi.org/10.1016/j.bbrc.2012.03.001.

[27] Huang M, Jiao JZ, Cai H, et al. C-C motif chemokine ligand 5 confines liver regeneration by down-regulating reparative macrophage-derived hepatocyte growth factor in a forkhead box O 3a-dependent manner. Hepatology. 2022; 76(6):1706-1722. https://doi.org/10.1002/hep.32458.

[28] Jiang ZZ, Zhao Y, Liu YQ. Pristimerin synergizes with gemcitabine through abrogating Chk1/53BP1-mediated DNA repair in pancreatic cancer cells. Food Chem Toxicol. 2021;147:111919.

[29] Eum DY, Byun JY, Yoon CH, et al. Triterpenoid pristimerin synergizes with taxol to induce cervical cancer cell death through reactive oxygen species-mediated mitochondrial dysfunction. Anti-Cancer Drug. 2011; 22(8):763-773. https://doi.org/10.1097/CAD.0b013e328347181a.

[30] Eyck BM, Hulshof MCCM, et al. Ten-year outcome of neoadjuvant chemoradiotherapy plus surgery for esophageal cancer: the randomized controlled CROSS trial. J Clin Oncol. 2021; 39(18):1995-2004. https://doi.org/10.1200/JCO.20.03614.

[31] Liu SL, Wen J, Yang H, et al.Recurrence patterns after neoadjuvant chemoradiotherapy compared with surgery alone in oesophageal squamous cell carcinoma: results from the multicenter phase III trial NEOCRTEC5010. Eur J Cancer. 2020; 138:113-121. https://doi.org/10.1016/j.ejca.2020.08.002.

[32] Wang GY, Zhang L, Geng YD, et al. β-Elemene induces apoptosis and autophagy in colorectal cancer cells through regulating the ROS/AMPK/mTOR pathway. Chin J Nat Med. 2022; 20(1):9-21.

[33] DU BX, Lin P, Lin J. EGCG and ECG induce apoptosis and decrease autophagy the AMPK/mTOR and PI3K/AKT/mTOR pathway in human melanoma cells. Chin J Nat Med. 2022; 20(4):290-300.

[34] Yu DP, Wang Q, Zhang Q, et al. Molecular mechanisms of ferroptosis and its antitumor applications in natural products. Acta Biochim Biophys Sin (Shanghai). 2023; 55(9):1337-1347. https://doi.org/10.3724/abbs.2023120.

[35] Yan F, Liao R, Silva M, et al. Pristimerin-induced uveal melanoma cell death via inhibiting PI3K/Akt/FoxO3a signalling pathway. J Cell Mol Med. 2020; 24(11):6208-6219. https://doi.org/10.1111/jcmm.15249.

[36] Xie XS, Xie SS, Xie CY, et al. Pristimerin attenuates cell proliferation of uveal melanoma cells by inhibiting insulin-like growth factor-1 receptor and its downstream pathways. J Cell Mol Med. 2019; 23(11):7545-7553. https://doi.org/10.1111/jcmm.14623.

[37] Jin S, Cojocari D, Purkal JJ, et al. 5-azacitidine induces NOXA to prime AML cells for venetoclax-mediated apoptosis. Clin Cancer Res. 2020; 26(13):3371-3383. https://doi.org/10.1158/1078-0432.CCR-19-1900.

[38] Calissi G, Lam EW, Link W, et al. Therapeutic strategies targeting FOXO transcription factors. Nat Rev Drug Discov. 2021; 20(1):21-38. https://doi.org/10.1038/s41573-020-0088-2.

[39] Liu Y, Wang YG, Li X, et al. FOXO3a in cancer drug resistance. Cancer Lett. 2022;540:215724. https://doi.org/10.1016/j.canlet.2022.215724.

[40] Sun TY, Zhang JG, Deng B, et al. FOXO1 and FOXO3a sensitize non-small-cell lung cancer cells to cisplatin-induced apoptosis independent of Bim. Acta Biochim Biophys Sin (Shanghai). 2020; 52(12):1348-1359.

[41] Zhou X, Dong S, Zhou Y, et al. EMX2 inhibits clear cell renal cell carcinoma progress via modulating Akt/FOXO3a pathway. Mol Carcinog. 2024; 63(5):951-961. https://doi.org/10.1002/mc.23700.

[42] Yin LY, Cao RX, Liu ZQ, et al. FUNDC2, a mitochondrial outer membrane protein, mediates triple-negative breast cancer progression via the AKT/GSK3β/GLI1 pathway. Acta Biochim Biophys Sin (Shanghai). 2023; 55(11):1770-1783.