<i>In vitro</i> anti-tumor effect and mechanism of clove extracts against radioresistant esophagus cancer cell_West China Medical Journal

Authors：

GONG Yingying ¹ , XIE Ling ² ,  ZHU Shengming ³ , WU Feng ³ , DUAN Qiwen ³ , DENG Xinzhou ³ , KE Qing ³ ,  LUO Zhiguo ³

1. Postgraduate Training Basement of Jinzhou Medicical University, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P. R. China;
2. Department of Dermatology, Taihe Hospital of Shiyan City, Affiliated Hospital of Hubei University of Medicine, Shiyan, Hubei 442000, P. R. China;
3. Department of Oncology, Taihe Hospital of Shiyan City, Affiliated Hospital of Hubei University of Medicine/Hubei Provincial Center for Precision Medicine of Cancer, Shiyan, Hubei 442000, P. R. China;

Corresponding author：

ZHU Shengming, Email: zsmxieling@sina.com; LUO Zhiguo, Email: camelwhite1973@163.com

Keywords：

Radioresistance; Clove; Eugenol; Esophagus cancer

DOI：

10.7507/1002-0179.202001193

Video：

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Objective To explore the in vitro anti-tumor effect of clove extracts (CEs) on radioresistant esophageal cancer cell KYSER and its mechanism.Methods Ethanol extracts of clove bud were prepared. Gas chromatography was used to identify the main active components of CEs. In vitro cell culture method was used to observe the effect of CEs at different concentrations on KYSER cell growth. Methyl thiazolyl tetrazolium (MTT) method was used to detect the effect of different concentration CEs on KYSER’s survival and its’ manner. Transmission electron microscope (TEM) was used to observe the changes of KYSER’s organelle microstructure after CEs treated. Transwell chamber method was used to detect the impact of CEs on KYSER’s migration ability. The apoptosis rate and cell cycle distribution of KYSER treated by CEs were quantitatively determined by flow cytometry. Clone formation experiment was used to detect the clone formation ability of KYSER treaded by CEs.Results The main components of CEs were eugenol, eugenol hydrocarbon, and eugenol acetate. In vitro cell culture showed that 0.4% CEs could inhibit KYSER growth. MTT assay showed that the concentration of CEs≥0.5% could inhibit the survival of KYSER in a dose-dependent manner. TEM assay showed that after treated by 0.5% CEs, KYSER’s microvilli became shorter and wider, ribosomes in the cytoplasm decreased, mitochondria atrophied, and a large number of autophagosomes were formed. Transwell migration assay showed that relative migration rates of KYSER after treated by 0.5% CEs and 0.6% CEs were (65±4)% and (41±3)%, respectively. Compared with the control group, the differences were statistically significant (P<0.001). Flow cytometry showed that the apoptosis rates of the control group, the 0.5% CEs treated group, and the 0.6% CEs treated group were (5.63±0.50)%, (11.77±0.42)%, and (19.44±0.19)%, respectively, and the differences between the control group and the two CEs treated groups were statistically significant (P<0.001). Flow cytometry showed that the G₁ phase ratios of cells in the control group, the 0.5% CEs treatment group, and the 0.6% CEs treatment group were (61.99±1.20)%, (75.38±1.50)%, and (78.81±1.00)%, respectively, and the differences between the control group and the two CEs treated groups were statistically significant (P<0.001). The clonal formation experiment showed that after 24 h of CEs treatment, the clonal formation rates of the control group, the 0.5% CEs treatment group, and the 0.6% CEs treatment group were (80.5±1.0)%, (18.1±0.8)%, and (5.0±0.5)%, respectively, and the differences between the control group and the two CEs treated groups were statistically significant (P<0.001).Conclusion CEs can exert anti-tumor effect on radioresistant esophageal cancer cells by inducing autophagy and apoptosis, promoting cell cycle arrest, inhibiting cell energy metabolism, and inhibiting migration.

Citation： GONG Yingying, XIE Ling, ZHU Shengming, WU Feng, DUAN Qiwen, DENG Xinzhou, KE Qing, LUO Zhiguo. In vitro anti-tumor effect and mechanism of clove extracts against radioresistant esophagus cancer cell. West China Medical Journal, 2021, 36(1): 66-75. doi: 10.7507/1002-0179.202001193 Copy

1.	黄清梅, 杜小波. 食管癌相关标志物研究进展. 华西医学, 2017, 32(10): 1620-1623.
2.	Sharifi-Rad J, Ozleyen A, Boyunegmez Tumer T, et al. Natural products and synthetic analogs as a source of antitumor drugs. Biomolecules, 2019, 9(11): 679.
3.	Hu Q, Zhou M, Wei S. Progress on the antimicrobial activity research of clove oil and eugenol in the food antisepsis field. J Food Sci, 2018, 83(6): 1476-1483.
4.	Chua LK, Lim CL, Ling APK, et al. Anticancer potential of syzygium species: a review. Plant Foods Hum Nutr, 2019, 74(1): 18-27.
5.	Vázquez-Fresno R, Rosana ARR, Sajed T, et al. Herbs and spices- biomarkers of intake based on human intervention studies: a systematic review. Genes Nutr, 2019, 14: 18.
6.	Zhu S, Wang J, Xie B, et al. Culture at a higher temperature mildly inhibits cancer cell growth but enhances chemotherapeutic effects by inhibiting cell-cell collaboration. PLoS One, 2015, 10(10): e0137042.
7.	Lin G, Yu B, Liang Z, et al. Silencing of c-jun decreases cell migration, invasion, and EMT in radioresistant human nasopharyngeal carcinoma cell line CNE-2R. Onco Targets Ther, 2018, 11: 3805-3815.
8.	Todorovic V, Prevc A, Zakelj MN, et al. Mechanisms of different response to ionizing irradiation in isogenic head and neck cancer cell lines. Radiat Oncol, 2019, 14(1): 214.
9.	Bezerra DP, Militão GCG, de Morais MC, et al. The dual antioxidant/prooxidant effect of eugenol and its action in cancer development and treatment. Nutrients, 2017, 9(12): 1367.
10.	Arunava D, Harshadha K, Dhinesh Kannan SK, et al. Evaluation of therapeutic potential of eugenol. A natural derivative of syzygium aromaticum on cervical cancer. Asian Pac J Cancer Prev, 2018, 19(7): 1977-1985.
11.	Vijayasteltar L, Nair GG, Maliakel B, et al. Safety assessment of a standardized polyphenolic extract of clove buds: subchronic toxicity and mutagenicity studies. Toxicol Rep, 2016, 3: 439-449.
12.	Dwivedi V, Shrivastava R, Hussain S, et al. Comparative anticancer potential of clove (Syzygium aromaticum)--an Indian spice--against cancer cell lines of various anatomical origin. Asian Pac J Cancer Prev, 2011, 12(8): 1989-1993.
13.	Elsyana V, Bintang M, Priosoeryanto BP. Cytotoxicity and antiproliferative activity assay of clove mistletoe (Dendrophthoe pentandra (L. ) Miq.) leaves extracts. Adv Pharmacol Sci, 2016, 2016: 3242698.
14.	Ryu B, Kim HM, Lee JS, et al. New flavonol glucuronides from the flower buds of Syzygium aromaticum (clove). J Agric Food Chem, 2016, 64(15): 3048-3053.
15.	Li C, Xu H, Chen X, et al. Aqueous extract of clove inhibits tumor growth by inducing autophagy through AMPK/ULK pathway. Phytother Res, 2019, 33(7): 1794-1804.
16.	Liu M, Zhao G, Zhang D, et al. Active fraction of clove induces apoptosis via PI3K/Akt/mTOR-mediated autophagy in human colorectal cancer HCT-116 cells. Int J Oncol, 2018, 53(3): 1363-1373.
17.	Liang WZ, Chou CT, Hsu SS, et al. The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells. Toxicol Lett, 2015, 232(1): 122-132.
18.	Júnior PL, Câmara DA, Costa AS, et al. Apoptotic effect of eugenol envolves G₂/M phase abrogation accompanied by mitochondrial damage and clastogenic effect on cancer cell in vitro. Phytomedicine, 2016, 23(7): 725-735.
19.	Ma M, Ma Y, Zhang GJ, et al. Eugenol alleviated breast precancerous lesions through HER2/PI3K-AKT pathway-induced cell apoptosis and S-phase arrest. Oncotarget, 2017, 8(34): 56296-56310.
20.	Kubatka P, Uramova S, Kello M, et al. Antineoplastic effects of clove buds (Syzygium aromaticum L. ) in the model of breast carcinoma. J Cell Mol Med, 2017, 21(11): 2837-2851.
21.	Liu HZ, Schmitz JC, Wei JT, et al. Clove extract inhibits tumor growth and promotes cell cycle arrest and apoptosis. Oncol Res, 2014, 21(5): 247-259.
22.	Pal D, Sur S, Roy R, et al. Epigallocatechin gallate in combination with eugenol or amarogentin shows synergistic chemotherapeutic potential in cervical cancer cell line. J Cell Physiol, 2019, 234(1): 825-836.
23.	Nam H, Kim MM. Eugenol with antioxidant activity inhibits MMP-9 related to metastasis in human fibrosarcoma cells. Food Chem Toxicol, 2013, 55: 106-112.

1. 黄清梅, 杜小波. 食管癌相关标志物研究进展. 华西医学, 2017, 32(10): 1620-1623.
2. Sharifi-Rad J, Ozleyen A, Boyunegmez Tumer T, et al. Natural products and synthetic analogs as a source of antitumor drugs. Biomolecules, 2019, 9(11): 679.
3. Hu Q, Zhou M, Wei S. Progress on the antimicrobial activity research of clove oil and eugenol in the food antisepsis field. J Food Sci, 2018, 83(6): 1476-1483.
4. Chua LK, Lim CL, Ling APK, et al. Anticancer potential of syzygium species: a review. Plant Foods Hum Nutr, 2019, 74(1): 18-27.
5. Vázquez-Fresno R, Rosana ARR, Sajed T, et al. Herbs and spices- biomarkers of intake based on human intervention studies: a systematic review. Genes Nutr, 2019, 14: 18.
6. Zhu S, Wang J, Xie B, et al. Culture at a higher temperature mildly inhibits cancer cell growth but enhances chemotherapeutic effects by inhibiting cell-cell collaboration. PLoS One, 2015, 10(10): e0137042.
7. Lin G, Yu B, Liang Z, et al. Silencing of c-jun decreases cell migration, invasion, and EMT in radioresistant human nasopharyngeal carcinoma cell line CNE-2R. Onco Targets Ther, 2018, 11: 3805-3815.
8. Todorovic V, Prevc A, Zakelj MN, et al. Mechanisms of different response to ionizing irradiation in isogenic head and neck cancer cell lines. Radiat Oncol, 2019, 14(1): 214.
9. Bezerra DP, Militão GCG, de Morais MC, et al. The dual antioxidant/prooxidant effect of eugenol and its action in cancer development and treatment. Nutrients, 2017, 9(12): 1367.
10. Arunava D, Harshadha K, Dhinesh Kannan SK, et al. Evaluation of therapeutic potential of eugenol. A natural derivative of syzygium aromaticum on cervical cancer. Asian Pac J Cancer Prev, 2018, 19(7): 1977-1985.
11. Vijayasteltar L, Nair GG, Maliakel B, et al. Safety assessment of a standardized polyphenolic extract of clove buds: subchronic toxicity and mutagenicity studies. Toxicol Rep, 2016, 3: 439-449.
12. Dwivedi V, Shrivastava R, Hussain S, et al. Comparative anticancer potential of clove (Syzygium aromaticum)--an Indian spice--against cancer cell lines of various anatomical origin. Asian Pac J Cancer Prev, 2011, 12(8): 1989-1993.
13. Elsyana V, Bintang M, Priosoeryanto BP. Cytotoxicity and antiproliferative activity assay of clove mistletoe (Dendrophthoe pentandra (L. ) Miq.) leaves extracts. Adv Pharmacol Sci, 2016, 2016: 3242698.
14. Ryu B, Kim HM, Lee JS, et al. New flavonol glucuronides from the flower buds of Syzygium aromaticum (clove). J Agric Food Chem, 2016, 64(15): 3048-3053.
15. Li C, Xu H, Chen X, et al. Aqueous extract of clove inhibits tumor growth by inducing autophagy through AMPK/ULK pathway. Phytother Res, 2019, 33(7): 1794-1804.
16. Liu M, Zhao G, Zhang D, et al. Active fraction of clove induces apoptosis via PI3K/Akt/mTOR-mediated autophagy in human colorectal cancer HCT-116 cells. Int J Oncol, 2018, 53(3): 1363-1373.
17. Liang WZ, Chou CT, Hsu SS, et al. The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells. Toxicol Lett, 2015, 232(1): 122-132.
18. Júnior PL, Câmara DA, Costa AS, et al. Apoptotic effect of eugenol envolves G₂/M phase abrogation accompanied by mitochondrial damage and clastogenic effect on cancer cell in vitro. Phytomedicine, 2016, 23(7): 725-735.
19. Ma M, Ma Y, Zhang GJ, et al. Eugenol alleviated breast precancerous lesions through HER2/PI3K-AKT pathway-induced cell apoptosis and S-phase arrest. Oncotarget, 2017, 8(34): 56296-56310.
20. Kubatka P, Uramova S, Kello M, et al. Antineoplastic effects of clove buds (Syzygium aromaticum L. ) in the model of breast carcinoma. J Cell Mol Med, 2017, 21(11): 2837-2851.
21. Liu HZ, Schmitz JC, Wei JT, et al. Clove extract inhibits tumor growth and promotes cell cycle arrest and apoptosis. Oncol Res, 2014, 21(5): 247-259.
22. Pal D, Sur S, Roy R, et al. Epigallocatechin gallate in combination with eugenol or amarogentin shows synergistic chemotherapeutic potential in cervical cancer cell line. J Cell Physiol, 2019, 234(1): 825-836.
23. Nam H, Kim MM. Eugenol with antioxidant activity inhibits MMP-9 related to metastasis in human fibrosarcoma cells. Food Chem Toxicol, 2013, 55: 106-112.

West China Medical Journal

In vitro anti-tumor effect and mechanism of clove extracts against radioresistant esophagus cancer cell

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