Anticancer effects of punicalagin and 5-fluorouracil on laryngeal squamous cell carcinoma: an in vitro study

Published: 22 February 2024
Abstract Views: 420
PDF: 167
HTML: 42
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.


The purpose of this study was to assess the apoptotic effects of punicalagin alone and in combination with 5-fluorouracil (5-FU) on laryngeal squamous cell carcinoma (Hep-2) cell line. Hep-2 cells were cultured and divided into four groups: Group 1 received no therapy and served as control, Group 2 received 5-FU only, Group 3 received punicalagin only, and Group 4 received a combination of 5-FU and punicalagin. After 48 hours of incubation, cellular changes were examined under an inverted microscope. The methyl thiazolyl tetrazolium assay, caspase-3 gene level, and vascular endothelial growth factor (VEGF) level were assessed. The control group showed the highest mean value of cancer cell proliferation rate (1.595±0.58), followed by the punicalagin group (1.263±0.447), then the 5-FU group (0.827±0.256), while the combination group showed the lowest proliferation rate (0.253±0.111). The combination group showed the highest mean value of caspase-3 concentration (3.177±0.736), followed by the 5-FU group (1.830±0.646), and punicalagin group (0.741±0.302), while the control group showed the lowest mean value (0.359±0.117). Regarding VEGF levels, the control group had a statistically significant higher mean value, followed by the punicalagin and 5-FU groups, and finally, the combination group which showed the lowest value. Punicalagin exerts an anticancer effect through anti-proliferative action and induction of apoptosis on Hep-2 cell line. Combining punicalagin with 5-FU potentiates its anti-proliferative, apoptotic, and anti-angiogenic actions. It, further, helps in mitigating the putative side effects of 5-FU by reducing the dose required for its therapeutic effects.



PlumX Metrics


Download data is not yet available.


Johnson DE, Burtness B, Leemans CR, et al. Head and neck squamous cell carcinoma. Nat Rev Dis Primer 2020;6. DOI:
Gao P, Gong L, Wang X. Induction chemotherapy in patients with resectable laryngeal cancer: A meta-analysis. Mol Clin Oncol 2018;9:155-62. DOI:
Li Z, Li T, Zhang P, Wang X. A practical online prediction platform to predict the survival status of laryngeal squamous cell carcinoma after 5 years. Am J Otolaryngol 2024;45:104209. DOI:
Gamez ME, Blakaj A, Zoller W, et al. Emerging Concepts and Novel Strategies in Radiation Therapy for Laryngeal Cancer Management. Cancers 2020;12:1651. DOI:
Megwalu UC, Sikora AG. Survival outcomes in advanced laryngeal cancer. JAMA Otolaryngol Head Neck Surg 2014;140:855-60. DOI:
Haigentz MJr, Silver CE, Hartl DM, et al. Chemotherapy regimens and treatment protocols for laryngeal cancer. Expert Opin Pharmacother 2010;11:1305-16. DOI:
Iizuka Y, Yoshimnura M, Inokuchi H, et al. Recurrence patterns after postoperative radiotherapy for squamous cell carcinoma of the pharynx and larynx. Acta Otolaryngol (Stockh.) 2015;135:96-102. DOI:
Wang D, Yu D, Liu X, et al. Targeting laryngeal cancer cells with 5-fluorouracil and curcumin using mesoporous silica nanoparticles. Technol Cancer Res Treat 2020;19:1533033820962114 DOI:
Echanique KA, Evans LK, Han AY, et al. Cancer of the Larynx and Hypopharynx. Head Neck Cancer 2021;35:933-47. DOI:
Demain AL, Vaishnav P. Natural products for cancer chemotherapy. Microb Biotechnol 2011;4:687-99. DOI:
Iqbal J, et al. Plant-derived anticancer agents: A green anticancer approach. Asian Pac J Trop Biomed 2017;7:1129-50. DOI:
Sharma P, McClees SF, Afaq F. Pomegranate for Prevention and Treatment of Cancer: An Update. Molecules 2017;22:177. DOI:
Syed DN, Chamcheu JC, Adhami VM, Mukhtar H. Pomegranate extracts and cancer prevention: Molecular and cellular activities. Anticancer Agents Med Chem 2013;13:1149-61. DOI:
Lin LT, Chen TY, Lin SC, et al. Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry. BMC Microbiol 2013;13:187. DOI:
Tang J, Li B, Hong S, et al. Punicalagin suppresses the proliferation and invasion of cervical cancer cells through inhibition of the β-catenin pathway. Mol Med Rep 2017;161439-44.
Lee CJ, Chen LG, Liang WL, Wang CC. Anti-inflammatory effects of Punica granatum Linne in vitro and in vivo. Food Chem 2010;118:315-22. DOI:
Bialonska D, Ramnani P, Kasimsetty SG, et al. The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota. Int J of Food Microbiol 2010;140:175-82. DOI:
Adams LS, Seeram NP, Aggarwal BB, Y, et al. Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signaling in colon cancer cells. J Agric Food Chem 2006;54:980-5. DOI:
Overbergh L, Giulietti A, Valckx D, et al. The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech 2003;1:33-43.
Kulkarni AP, Mahal HS, Kapoor S, Aradhya SM. In Vitro Studies on the Binding, Antioxidant, and Cytotoxic Actions of Punicalagin. J Agric Food Chem 2007;55:1491-500. DOI:
Seeram NP, Adams LS, Henning SM, et al. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. J Nutr Biochem 2005;16:360-67. DOI:
Liu W, Ou Y, Yang Y, et al. Inhibitory Effect of Punicalagin on Inflammatory and Angiogenic Activation of Human Umbilical Vein Endothelial Cells. Front Pharmacol 2021;12. DOI:
Cao D, Zhu G-Y, Lu Y, et al. Luteolin suppresses epithelial-mesenchymal transition and migration of triple-negative breast cancer cells by inhibiting YAP/TAZ activity. Biomed Pharmacother 2020;129:110462. DOI:
Mirzaei S, Zarrabi A, Hashemi F, et al. Nrf2 Signaling Pathway in Chemoprotection and Doxorubicin Resistance: Potential Application in Drug Discovery. Antioxidants 2021;10. DOI:
Odell E, et al. European Laryngological Society position paper on laryngeal dysplasia Part I: aetiology and pathological classification. Eur Arch Otorhinolaryngol 2021;278:1717-22. DOI:
Berdowska I, Matusiewicz M, Fecka I. Punicalagin in Cancer Prevention—Via Signaling Pathways Targeting. Nutrients 2021;13. DOI:
Souchon EA, Copeland EM, Watson P, Dudrick SJ. Intravenous hyperalimentation as an adjunct to cancer chemotherapy with 5-fluorouracil. J Surg Res 1975;18:451-4. DOI:
Zhang L, Chinnathambi A, Alharbi SA, et al. Punicalagin promotes the apoptosis in human cervical cancer (ME-180) cells through mitochondrial pathway and by inhibiting the NF-kB signaling pathway. Saudi J Biol Sci 2020;27:1100-6. DOI:
Anitha A, Sreeranganathan M, Chennazhi KP, et al. In vitro combinatorial anticancer effects of 5-fluorouracil and curcumin loaded N,O-carboxymethyl chitosan nanoparticles toward colon cancer and in vivo pharmacokinetic studies. Eur J Pharm Biopharm 2014;88:238-51. DOI:
Ghiringhelli F, Apetoh L. Enhancing the anticancer effects of 5-fluorouracil: current challenges and future perspectives. Biomed J 2015;38:111-6. DOI:
Huang T, Zhang X, Wang H. Punicalagin inhibited proliferation, invasion and angiogenesis of osteosarcoma through suppression of NF‑κB signaling. Mol Med Rep 2020;22:2386-94. DOI:
Suruli D, Mirza FBS, Kottaiswamy GJCRA, et al. Naringin and 5-fluorouracil suppress inflammatory Cytokines in human skin cancer cell line. Int J Res Pharm Sci 2021;12:729-33. DOI:
Yan C, et al. Punicalagin attenuates palmitate-induced lipotoxicity in HepG2 cells by activating the Keap1-Nrf2 antioxidant defense system. Mol Nutr Food Res 2016;60:1139-49. DOI:

How to Cite

Taha, A. M., Abd-Elshafy, E., Khalifa, A. M., Dar-Odeh, N., Elsayed, S., Alharkan, D. S., Alhodhodi, A., Sapri, A. M., Ragheb, Y. S., & Balbola, G. A. (2024). Anticancer effects of punicalagin and 5-fluorouracil on laryngeal squamous cell carcinoma: an <i>in vitro</i> study. Italian Journal of Medicine, 18(1).