Semen podocarpi extract enhances radiosensitivity in nasopharyngeal carcinoma cells via Raf/MEK/ERK signaling pathway

Abstract

Objective: To investigate the chemical components of Semen podocarpi extract (SPE) and its effect on nasopharyngeal carcinoma cells and CNE-2R cells.

Methods: Chemical components in SPE were identified by UPLC-MS/MS. CCK-8 and cell cloning experiments were applied to evaluate the effects of SPE on the proliferation of CNE-2R cells, and a single-hit multitarget model was used to calculate the radiobiological parameters. Cell apoptosis and cell cycle were analyzed by flow cytometry, and the levels of genes and proteins of the Raf/MEK/ERK pathway were determined by RT-PCR and Western blotting.

Results: A total of 37 compounds from SPE were identified, and SPE with or without irradiation inhibited the proliferation of CNE-2R cells. SPE also promoted apoptosis, arrested cells in the G₂/M phase, and presented radiosensitizing effects. Compared with irradiation alone, the effects of SPE+irradiation on apoptosis and cell cycle distribution were not significantly different. In addition, SPE had no significant effect on MEK gene expression. SPE significantly increased the gene expression of C-Raf and significantly reduced the protein expression of C-Raf, as well as the gene and protein expression of ERK1 and ERK2. The protein levels of C-Raf, ERK1, and ERK2 were also significantly lower in cells treated with SPE+irradiation than in cells treated with irradiation alone.

Conclusions: The effects of SPE on inhibiting cell proliferation and promoting apoptosis are likely associated with cell cycle arrest and Raf/MEK/ERK pathway regulation, and the mechanism underlying radiosensitization by SPE may involve downregulating the protein expression of C-Raf, ERK1, and ERK2.

Keywords

Podocarpus macrophyllus / Nasopharyngeal carcinoma / Radiosensitization / Chemical composition / Inumakilactone / Podocarpus flavone

Conflict of interest statement

The authors declare that there is no conflict of interest.

Funding

This work was supported by the Project of Administration of Traditional Chinese Medicine of Guangxi Zhuang Autonomous

Region (grant number GZSY22-69), and by the Middle/Young aged Teachers’ Research Ability Improvement Project of Guangxi Higher Education (grant number 2024KY0120).

Data availability statement

The data supporting the findings of this study are available from the corresponding authors upon request.

Authors’s contributions

FYZ designed the study, carried out experiments, and prepared manuscript. ZQH participated in chemical composition data analysis, statistical analysis, and manuscript revision. YCS participated in experimental studies. XDZ revised the experimental protocol and reviewed the manuscript.

Publisher’s note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

[1] Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA-Cancer J Clin 2024; 74(3): 229-263.

[2] Gundog M, Basaran H, Bozkurt O, Eroglu C. A comparison of cisplatin cumulative dose and cisplatin schedule in patients treated with concurrent chemoradiotherapy in nasopharyngeal carcinoma. Braz J Otorhinolaryngol 2020; 86(6): 676-686.

[3] Chitapanarux I, Kittichest R, Tungkasamit T, Asakit T, Chomprasert K, Chakrabandhu S, et al. Two-year outcome of concurrent chemoradiation with carboplatin with or without adjuvant carboplatin/fluorouracil in nasopharyngeal cancer: A multicenter randomized trial. Curr Probi Cancer 2021; 45(1): 100620.

[4] Zhang Y, Chen L, Hu GQ, Zhang N, Zhu XD, Yang KY, et al. Gemcitabine and cisplatin induction chemotherapy in nasopharyngeal carcinoma. New Engl J Med 2019; 381(12): 1124-1135.

[5] He Y, Guo T, Wang J, Sun Y, Guan H, Wu S, et al. Which induction chemotherapy regimen followed by cisplatin-based concurrent chemoradiotherapy is the best choice among PF TP and TPF for locoregionally advanced nasopharyngeal carcinoma? Ann Transi Med 2019; 7(5): 104-118.

[6] Wu Y, Wei X, Yuan Z, Xu H, Li Y, Li Y, et al. Phase II study of induction chemotherapy followed by concurrent chemoradiotherapy with raltitrexed and cisplatin in locally advanced nasopharyngeal carcinoma. Chin J Cancer Res 2020; 32(5): 665-672.

[7] Zhu F, Wu Y, Wang H. Advance in integrating platinum-based chemotherapy with radiotherapy for locally advanced nasopharyngeal carcinoma. Front Oncol 2023; 13: 1259331.

[8] Wang L, Liu D, Wei D. Efficacy of cetuximab in nasopharyngeal carcinoma patients receiving concurrent cisplatin-radiotherapy a metaanalysis. Comput Math Method M 2022; 2022: 5145549.

[9] Chan AT, Lee VHF, Hong RL, Ahn MJ, Chong WQ, Kim SB, et al. Results of KEYNOTE-122: A phase III study of pembrolizumab (pembro) monotherapy vs chemotherapy (chemo) for platinum-pretreated, recurrent or metastatic (R/M) nasopharyngeal carcinoma (NPC). Ann Oncol 2021; 32(S5): S786.

[10] Kao HF, Ang MK, Ng QS, Tan DSW, Tan W, Rajasekaran T, et al. Combination ipilimumab and nivolumab in recurrent/metastatic nasopharyngeal carcinoma (R/M NPC): Updated efficacy and safety analysis of NCT03097939. Ann Oncol 2020; 31(S6): S1347.

[11] Mai HQ, Chen QY, Chen D, Hu C, Yang K, Wen J, et al. Final progression-free survival analysis of JUPITER-02, a randomized, double-blind, phase 3 study of toripalimab or placebo plus gemcitabine and cisplatin as first-line treatment for recurrent or metastatic nasopharyngeal carcinoma. Cancer Res 2022; 82(12S). doi: 10.1158/1538-7445.AM2022-CT226.

[12] Jia L, Ma S, Hou X, Wang X, Qased AB, Sun X, et al. The synergistic effects of traditional Chinese herbs and radiotherapy for cancer treatment. Oncol Lett 2013; 5(5): 1439-1447.

[13] Wang C, Yang Y, Sun L, Wang J, Jiang Z, Li Y, et al. Baicalin reverses radioresistance in nasopharyngeal carcinoma by downregulating autophagy. Cancer Cell Int 2020; 20(1): 1-8.

[14] Wang J, Yao Z, Lai X, Bao H, Li Y, Li S, et al. Tetrandrine sensitizes nasopharyngeal carcinoma cells toirradiation by inducing autophagy and inhibiting MEK/ERK pathway. Cancer Med 2020; 9(19): 7268-7278.

[15] Zhu D, Shao M, Yang J, Fang M, Liu S, Lou D, et al. Curcumin enhances radiosensitization of nasopharyngeal carcinoma via nediating regulation of tumor stem-like cells by a CircRNA network. J Cancer 2020; 11(8): 2360-2370.

[16] Itô S, Sunagawa M, Kodama M, Honma H, Takahashi T. Structures of inumakilactones B and C, lactones of diterpenoid origin. Chem Commun 1971; 2(2): 91-93.

[17] Kodama M, Kabuto C, Sunagawa M, Itô S. Structure of inumakilactone D. Tetrahedron Lett 1977; 18(33): 2909-2910.

[18] Park HS, Yoda N, Fukaya H, Aoyagi Y, Takeya K. Rakanmakilactones A-F, new cytotoxic sulfur-containing norditerpene dilactones from leaves of Podocarpus macrophyllus var maki. Tetrahedron 2004; 60(1): 171-177.

[19] Sato K, Sugawara K, Takeuchi H, Park HS, Akiyama T, Koyama T, et al. New cytotoxic nor-and bisnorditerpene dilactones makilactones A-D, from Podocarpus macrophyllus D. Don. Heterocycles 2009; 78(6): 1453-1462.

[20] Sato K, Inaba Y, Park HS, Akiyama T, Koyama T, Fukaya H, et al. Cytotoxic bisnor-and norditerpene dilactones having 7a,8a-epoxy-9,11-enolide substructure from Podocarpus macrophyllus D. Don. Chem Pharm Bull 2009; 57(7): 668-679.

[21] Feng ZL, Zhang LL, Zheng YD, Liu QY, Liu JX, Feng L, et al. Norditerpenoids and dinorditerpenoids from the seeds of Podocarpus nagi as cytotoxic agents and autophagy inducers. J Nat Prod 2017; 80(7): 2110-2117.

[22] Qi YY, Su J, Zhang ZJ, Li LW, Fan M, Zhu Y, et al. Two new anti-proliferative C₁₈-norditerpenes from the roots of Podocarpus macrophyUus. Chem Biodivers 2018; 15(4): e1800043.

[23] Zheng YD, Bai G, Tang C, Ke CQ, Yao S, Tong LJ, et al. 7α 8a-Epoxynagilactones and their glucosides from the twigs of Podocarpus nagi: Isolation, structures, and cytotoxic activities. Fitoterapia 2018; 125(7): 174-183.

[24] Benatrehina PA, Chen WL, Czarnecki AA, Kurina S, Chai HB, Lantvit DD, et al. Bioactivity-guided isolation of totarane-derived diterpenes from Podocarpus neriifolius and structure revision of 3-deoxy-2a-hydroxynagilactone E. Nat Prod Bioprospect 2019; 9(2): 157-163.

[25] Kim KH, Moon E, Kim SY, Choi SU, Son MW, Choi SZ, et al. Bioactive sesquiterpenes from the essential oil of Thuja orientalis. Planta Med 2013; 79(17): 1680-1684.

[26] Sturgeon CM, Craig K, Brown C, Rundle NT, Andersen RJ. Modulation of the G₂ cell cycle checkpoint by sesquiterpene lactones psilostachyins A and C isolated from the common ragweed Ambrosia artemisiifolia. Planta Med 2005; 71(10): 938-943.

[27] Lee KH, Ibuka T, Furukawa H, Kozuka M, Wu RY, Hall IH, et al. Antitumor agents XXXVIII: Isolation and structural elucidation of novel germacranolides and triterpenes from Elephantopus mollis. J Pharm Sci-US 1980; 68(9): 1050-1056.

[28] Matsumura E, Morita Y, Date T, Tsujibo H, Yasuda M, Okabe T, et al. Cytotoxicity of the hinokitiol-related compounds, γ-thujaplicin and β-dolabrin. Biol Pharm Bull 2001; 24(3): 299-302.

[29] Ji LL, Zhang M, Sheng YC, Wang ZT. Pyrrolizidine alkaloid divorine induces apoptosis in human normal liver L-02 cells and reduces the expression of p53 protein. Toxicol In Vitro 2005; 19(1): 41-46.

[30] Wang J, Chang L, Lai X, Li X, Wang Z, Huang Z, et al. Tetrandrine enhances radiosensitivity through the CDC25C/CDK1/cyclin B1 pathway in nasopharyngeal carcinoma cells. Cell Cycle 2018; 17: 671-680.

[31] Yang Y, Ren S, Huang W, Dong J, Guo J, Zhao J. Camptothecin delivery via tumor-derived exosome for radiosensitization by cell cycle regulation on patient-derived xenograft mice. Front Bioeng Biotechnol 2022; 10: 876641.

[32] Kang SH, Bak DH, Chung BY, Bai HW. Centipedegrass extract enhances radiosensitivity in melanoma cells by inducing G₂/M cell cycle phase arrest. Mol Biol Rep 2021; 48(2): 1081-1091.

[33] Paduano F, Gaudio E, Trapasso F. The tumour suppressor Fhit protein activates C-Raf ubiquitination and degradation in human melanoma cells by interacting with Hsp90. Biomedicines 2022; 10(10): 2551.

[34] Li HM, Ma H, Sun X, Li B, Cao C, Dai Y, et al. Anti-cancer properties of ginkgolic acids in human nasopharyngeal carcinoma cne-2z cells via inhibition of heat shock protein 90. Molecules 2021; 26(21): 6575.