Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism

doi:10.3969/j.issn.1004-1524.2022.05.12

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (5): 974-983.DOI: 10.3969/j.issn.1004-1524.2022.05.12

• Horticultural Science • Previous Articles Next Articles

Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism

ZHENG Yuanyua¹^,²(), YU Zheping², ZHANG Shuwen², LI Yougui³, SUN Li², ZHENG Xiliang², QI Xingjiang¹^,²^,^*()

1. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Institute of Sericultural and Tea, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2021-11-03 Online:2022-05-25 Published:2022-06-06
Contact: QI Xingjiang

Abstract

Abstract:

Chinese bayberry (Myrica rubra) has high medicinal values. This study was performed to evaluate the effect of alcohol extracts of Myrica rubra branch extract (MRBE) on human malignant melanoma cells A375. MTT assay, flow cytometry based on PI, Annexin V/PI double staining and quantitative reverse-transcription PCR (qRT-PCR) were used to dissect the potential mechanism of the arrest and apoptosis of A375 cells induced by MRBE. The results revealed that MRBE inhibited the proliferation of human A375 cells in a dose-dependent way, and the inhibition rate was up to 49.46%. Flow cytometry analyses suggested that 48 h treatment of MRBE induced both S-phase cell cycle arrest and apoptotic death in A375 cells. The results of qRT-PCR further showed that MRBE induced S phase arrest by promoting the upregulation of p21 and decreasing the expression of CyclinA, CyclinD, CDK2, CDK4, CDK6 and E2F1, and induced apoptosis by decreasing the expression of Bcl-w and Raf. This study revealed the action mechanism of MRBE on the induction of S-phase arrest and apoptosis of A375 cells, which could provide scientific evidence for the development of potential anti-melanom drugs by using MRBE, and help to improve the medicinal added value of Chinese bayberry branches and broaden the diversified development of its industry chain.

Key words: Chinese bayberry, alcohol extracts of branch extract, human melanoma A375 cells, cell cycle, cell apoptosis

CLC Number:

ZHENG Yuanyua, YU Zheping, ZHANG Shuwen, LI Yougui, SUN Li, ZHENG Xiliang, QI Xingjiang. Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism[J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 974-983.

Figures/Tables 5

Fig.1 Effect of MRBE on proliferation of A375 cells A, Effects of different concentrations of MRBE on the inhibition rate of A375 cells after 48 h treatment. B, Effects of different concentrations of MRBE on cell morphology of A375 cells after 48 h treatment. * represented 0.01<P<0.05, ** represented P<0.01. The same as below.

Fig.2 Effect of MRBE on A375 cell cycle A, Effects of different concentrations of MRBE on A375 cell cycle analysed by PI flow cytometry; B, Percentage of cells in different cell cycle phases.

Fig.3 Effect of MRBE on apoptosis of A375 cells A, Effects of different concentrations of MRBE on A375 cell apoptosis analysed by Annexin V-PI flow cytometry, Lower left quadrant showed normal cells, lower right quadrant showed early apoptotic cells, upper right quadrant showed late apoptotic cells, upper left quadrant showed necrotic cells. B, Percentages of cells in different death types.

Fig.4 Effect of MRBE on expression of cycle related genes in A375 cells

Fig.5 Effect of MRBE on expression of apoptosis related genes in A375 cells

References 53

[1]	刘路贤. 栽培杨梅谱系地理、驯化起源及品种(系)间关系的研究[D]. 杭州: 浙江大学, 2016.
	LIU L X. Diverse geographic origins, domestication origins, and inter varietal relationships of cultivated myricetum species[D]. Hangzhou: Zhejiang University, 2016. (in Chinese with English abstract)
[2]	孔美兰, 刘谋泉, 张福平, 等. 超声波提取杨梅色素及其清除超氧阴离子作用[J]. 食品工业, 2015, 36(1): 58-62.
	KONG M L, LIU M Q, ZHANG F P, et al. Ultrasound pigment extraction from waxberry and ability to scavenge superoxide radicals[J]. The Food Industry, 2015, 36(1): 58-62. (in Chinese with English abstract)
[3]	尹洁, 蒋健, 刘文静, 等. GC-O和GC-MS结合鉴定杨梅叶精油中的关键香气成分[J]. 中国南方果树, 2017, 46(3): 64-71.
	YIN J, JIANG J, LIU W J, et al. Combined GC-O and GC-MS for identification of key aroma components in essential oil of Chinese bayberry leaves[J]. South China Fruits, 2017, 46(3): 64-71. (in Chinese)
[4]	何新华, 陈力耕, 胡西琴. 杨梅属植物共生结瘤固氮研究进展[J]. 果树学报, 2002, 19(5): 351-355.
	HE X H, CHEN L G, HU X Q. Progress in research of root nodule and nitrogen-fixing symbiosis with actinomycetal endophytes of Myrica[J]. Journal of Fruit Science, 2002, 19(5): 351-355. (in Chinese with English abstract)
[5]	梁森苗, 郑锡良, 戚行江, 等. 杨梅精准修剪方案的确定及其对生长结果和果实品质的影响[J]. 浙江农业科学, 2017, 58(4): 615-618.
	LIANG S M, ZHENG X L, QI X J, et al. Impact of a precision pruning protocol for Myrica rubra on growth outcomes and fruit quality[J]. Journal of Zhejiang Agricultural Sciences, 2017, 58(4): 615-618. (in Chinese)
[6]	颜丽菊, 戚行江, 蒋芯, 等. 不同修剪方式对杨梅树冠、产量及品质的影响研究[J]. 农学学报, 2018, 8(12): 84-87.
	YAN L J, QI X J, JIANG X, et al. Effect of pruning methods on crown, yield and quality of Myrica rubra[J]. Journal of Agriculture, 2018, 8(12): 84-87. (in Chinese with English abstract)
[7]	黄士文. 杨梅栽培研究综述[J]. 中国园艺文摘, 2015, 31(6): 41-45.
	HUANG S W. A review of studies on the cultivation of Myrica rubra[J]. Chinese Horticulture Abstracts, 2015, 31(6): 41-45. (in Chinese)
[8]	方波, 武峥, 杨丽, 等. 杨梅果实生物活性物质研究进展[J]. 南方农业, 2018, 12(28): 29-34, 66.
	FANG B, WU Z, YANG L, et al. Research progress on bioactive substances in waxberry[J]. South China Agriculture, 2018, 12(28): 29-34, 66. (in Chinese)
[9]	傅燕玲. 杨梅非果部位有效成分的抗氧化活性和减肥功效研究[D]. 杭州: 浙江大学, 2010.
	FU Y L. Studies on antioxidant capacity and body-weight control effect of extracts from the leaves, branches and barks of Myrica rubra[D]. Hangzhou: Zhejiang University, 2010. (in Chinese with English abstract)
[10]	李罗明, 黄亚芳, 李宗军, 等. 响应面法优化杨梅核仁油浸提工艺及其脂肪酸组成分析[J]. 中国粮油学报, 2016, 31(4): 113-117.
	LI L M, HUANG Y F, LI Z J, et al. Optimization of Myrica rubra seed oil extraction conditions with response surface methodology and fatty acid analysis[J]. Journal of the Chinese Cereals and Oils Association, 2016, 31(4): 113-117. (in Chinese with English abstract)
[11]	孙莎, 吴伟杰, 郜海燕, 等. 杨梅果实抗氧化物质的提取及其稳定性研究[J]. 中国食品学报, 2018, 18(8): 185-193.
	SUN S, WU W J, GAO H Y, et al. Studies on the extraction efficiency and stability of antioxidant substances of Chinese bayberry (Myrica rubra) fruit[J]. Journal of Chinese Institute of Food Science and Technology, 2018, 18(8): 185-193. (in Chinese with English abstract)
[12]	卢赛赛, 许凤, 王鸿飞, 等. 杨梅叶中总黄酮提取及其抗氧化能力研究[J]. 果树学报, 2015, 32(3): 460-468.
	LU S S, XU F, WANG H F, et al. Extraction and antioxidant activities of total flavonoids from Myrica rubra leaves[J]. Journal of Fruit Science, 2015, 32(3): 460-468. (in Chinese with English abstract)
[13]	佟岩. 杨梅素的抗炎免疫调节作用及机制研究[D]. 沈阳: 沈阳药科大学, 2009.
	TONG Y. Studies on anti-inflammatory and immunoregulatory effects of myricetin and its mechanism[D]. Shenyang: Shenyang Pharmaceutical University, 2009. (in Chinese with English abstract)
[14]	王潞, 周云英. 杨梅素抗感染、抗炎及抗氧化活性研究进展[J]. 中草药, 2019, 50(3): 778-784.
	WANG L, ZHOU Y Y. Research progress on anti-infective, anti-inflammatory, and anti-oxidant activities of myricetin[J]. Chinese Traditional and Herbal Drugs, 2019, 50(3): 778-784. (in Chinese with English abstract)
[15]	朱丽云, 张春苗, 高永生, 等. 杨梅叶精油成分分析及其对肺癌A549细胞增殖的抑制作用[J]. 果树学报, 2018, 35(7): 836-844.
	ZHU L Y, ZHANG C M, GAO Y S, et al. Identification of essential oils in Myrica rubra leaves and their inhibitory effect on the proliferation of lung cancer A549 cell[J]. Journal of Fruit Science, 2018, 35(7): 836-844. (in Chinese with English abstract)
[16]	ZHANG Y, CHEN S G, WEI C Y, et al. Flavonoids from Chinese bayberry leaves induced apoptosis and G₁ cell cycle arrest via Erk pathway in ovarian cancer cells[J]. European Journal of Medicinal Chemistry, 2018, 147: 218-226. DOI URL
[17]	张加干, 唐伟敏, 刘哲, 等. 杨梅果渣醇提物降糖活性组分筛选及其功能评价[J]. 中国食品学报, 2021, 21(2): 128-135.
	ZHANG J G, TANG W M, LIU Z, et al. Screening of hypoglycemic bioactive components and evaluation from ethanol extract of Chinese bayberry pomace[J]. Journal of Chinese Institute of Food Science and Technology, 2021, 21(2): 128-135. (in Chinese with English abstract)
[18]	张小东, 高永生, 朱丽云, 等. 杨梅核仁油的成分、抗氧化活性及其降血脂功能分析[J]. 中国南方果树, 2018, 47(2): 64-68, 70.
	ZHANG X D, GAO Y S, ZHU L Y, et al. Composition, antioxidant activity and antihyperlipidemic activity of bayberry (Myrica rubra) kernel oil in mice[J]. South China Fruits, 2018, 47(2): 64-68, 70. (in Chinese)
[19]	MATSUDA H, MORIKAWA T, TAO J, et al. Bioactive constituents of Chinese natural medicines. VII. Inhibitors of degranulation in RBL-2H3 cells and absolute stereostructures of three new diarylheptanoid glycosides from the bark of Myrica rubra[J]. Chemical & Pharmaceutical Bulletin, 2002, 50(2): 208-215.
[20]	徐容容. 杨梅树皮有效成分抗肿瘤活性的体外研究[D]. 杭州: 浙江大学, 2012.
	XU R R. Studies on the anti-tumor effect of active components from bayberry bark in vitro[D]. Hangzhou: Zhejiang University, 2012. (in Chinese with English abstract)
[21]	戴关海, 童晔玲, 任泽明, 等. 杨梅树皮提取物体外抗人肺腺癌A549作用的实验研究[J]. 中华中医药学刊, 2013, 31(1): 81-82.
	DAI G H, TONG Y L, REN Z M, et al. Effect of extracts from bark of Myrica rubra on resisting human lung adenocarcinoma A549 cells in vitro[J]. Chinese Archives of Traditional Chinese Medicine, 2013, 31(1): 81-82. (in Chinese with English abstract)
[22]	周洁, 汪仕韬, 孙石磊, 等. 杨梅果实提取物抗小鼠腹泻作用的研究[J]. 中国微生态学杂志, 2009, 21(4): 323-326.
	ZHOU J, WANG S T, SUN S L, et al. The prevention and antidiarrheal activity of Chinese bayberry fruit extract on mice diarrhea[J]. Chinese Journal of Microecology, 2009, 21(4): 323-326. (in Chinese with English abstract)
[23]	林建原, 陶志华, 朱钐汕. 杨梅叶中总黄酮提取工艺及其抗氧化活性研究[J]. 食品科学, 2011, 32(20): 26-29.
	LIN J Y, TAO Z H, ZHU S S. Extraction and antioxidant activity evaluation of total flavonoids from bayberry (Myrica rubra Sieb.et Zucc.) leaves[J]. Food Science, 2011, 32(20): 26-29. (in Chinese with English abstract) DOI URL
[24]	徐敏, 余陈欢, 蒋剑平, 等. 杨梅核醇提取物体外抗氧化活性研究[J]. 中华中医药杂志, 2012, 27(10): 2716-2719.
	XU M, YU C H, JIANG J P, et al. Antioxidant activity of Myrica rubra kernel extract in vitro[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2012, 27(10): 2716-2719. (in Chinese with English abstract)
[25]	沈胜楠. 杨梅树皮、杨梅叶和杨梅果体外抗氧化物质基础研究[D]. 北京: 北京协和医学院, 2015.
	SHEN S N. Study on antioxidative substance of Chinese bayberry bark, leaves and fruits[D]. Beijing: Peking Union Medical College, 2015. (in Chinese with English abstract)
[26]	KOZOVSKA Z, GABRISOVA V, KUCEROVA L. Malignant melanoma: diagnosis, treatment and cancer stem cells[J]. Neoplasma, 2016, 63(4): 510-517. DOI URL
[27]	KALYAN S, ADAM B, SUKUMAR A J, et al. Epidemiology of melanoma[J]. Medical Sciences (Basel, Switzerland), 2021, 9(4): 63. DOI URL
[28]	HOMET B, RIBAS A. New drug targets in metastatic melanoma[J]. The Journal of Pathology, 2014, 232(2): 134-141. DOI URL
[29]	LUKE J J, FLAHERTY K T, RIBAS A, et al. Targeted agents and immunotherapies: optimizing outcomes in melanoma[J]. Nature Reviews Clinical Oncology, 2017, 14(8): 463-482. DOI URL
[30]	LUTHER C, SWAMI U, ZHANG J, et al. Advanced stage melanoma therapies: detailing the present and exploring the future[J]. Critical Reviews in Oncology/Hematology, 2019, 133: 99-111. DOI URL
[31]	FONTANA F, RAIMONDI M, DI DOMIZIO A, et al. Unraveling the molecular mechanisms and the potential chemopreventive/therapeutic properties of natural compounds in melanoma[J]. Seminars in Cancer Biology, 2019, 59: 266-282. DOI URL
[32]	YU T H, ZHONG S, SUN Y Q, et al. Aqueous extracts of Sanghuangporus vaninii induce S-phase arrest and apoptosis in human melanoma A375 cells[J]. Oncology Letters, 2021, 22(2): 628. DOI URL
[33]	ZHONG S, JIN Q S, YU T H, et al. Phellinus gilvus derived protocatechualdehyde induces G₀/G₁ phase arrest and apoptosis in murine B16 F10 cells[J]. Molecular Medicine Reports, 2020, 21(3): 1107-1114.
[34]	刘霞, 戴关海, 童晔玲, 等. 东魁杨梅树皮不同提取物体内外抗肿瘤作用的实验研究[J]. 中国中医药科技, 2015, 22(5): 512-514.
	LIU X, DAI G H, TONG Y L, et al. Comparative study on anti-tumor effects of different extracts from bark of Dongkui Yangmei (Dongkui Indigenious Myrica rubra)[J]. Chinese Journal of Traditional Medical Science and Technology, 2015, 22(5): 512-514. (in Chinese with English abstract)
[35]	RODRIGUEZ I, ODY C, ARAKI K, et al. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis[J]. The EMBO Journal, 1997, 16(9): 2262-2270. DOI URL
[36]	DONG H, STROME S E, SALOMAO D R, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion[J]. Nature Medicine, 2002, 8(8): 79-800.
[37]	MALUMBRES M, BARBACID M. Cell cycle, CDKs and cancer: a changing paradigm[J]. Nature Reviews Cancer, 2009, 9(3): 153-166. DOI URL
[38]	SHERR C J, ROBERTS J M. CDK inhibitors: positive and negative regulators of G₁-phase progression[J]. Genes & Development, 1999, 13(12): 1501-1512. DOI URL
[39]	XU X Y, GU J M, DING X G, et al. LINC00978 promotes the progression of hepatocellular carcinoma by regulating EZH2-mediated silencing of p21 and E-cadherin expression[J]. Cell Death & Disease, 2019, 10(10): 752.
[40]	YU C P, RAO D A, ZHU H, et al. TDO2 was downregulated in hepatocellular carcinoma and inhibited cell proliferation by upregulating the expression of p21 and p27[J]. BioMed Research International, 2021, 2021: 4708439.
[41]	WILLIAMS A B, SCHUMACHER B. p53 in the DNA-damage-repair process[J]. Cold Spring Harbor Perspectives in Medicine, 2016, 6(5): a026070. DOI URL
[42]	PERVEEN S, ASHFAQ H, AMBREEN S, et al. Methanolic extract of Citrullus colocynthis suppresses growth and proliferation of breast cancer cells through regulation of cell cycle[J]. Saudi Journal of Biological Sciences, 2021, 28(1): 879-886. DOI URL
[43]	XUE Q, LIU Z H, FENG Z Z, et al. Penfluridol: an antipsychotic agent suppresses lung cancer cell growth and metastasis by inducing G₀/G₁ arrest and apoptosis[J]. Biomedicine & Pharmacotherapy, 2020, 121: 109598. DOI URL
[44]	YANG E, ZHA J P, JOCKEL J, et al. Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces bax and promotes cell death[J]. Cell, 1995, 80(2): 285-291. DOI URL
[45]	JAN R, CHAUDHRY G E. Understanding apoptosis and apoptotic pathways targeted cancer therapeutics[J]. Advanced Pharmaceutical Bulletin, 2019, 9(2): 205-218. DOI URL
[46]	PISTRITTO G, TRISCIUOGLIO D, CECI C, et al. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies[J]. Aging, 2016, 8(4): 603-619. DOI URL
[47]	KALE J, OSTERLUND E J, ANDREWS D W. BCL-2 family proteins: changing partners in the dance towards death[J]. Cell Death and Differentiation, 2018, 25(1): 65-80. DOI URL
[48]	ADAMS C M, MITRA R, VOGEL A N, et al. Targeting BCL-W and BCL-XL as a therapeutic strategy for Hodgkin lymphoma[J]. Leukemia, 2020, 34(3): 947-952. DOI URL
[49]	MARSHALL M. Interactions between Ras and Raf: key regulatory proteins in cellular transformation[J]. Molecular Reproduction and Development, 1995, 42(4): 493-499. DOI URL
[50]	KARASARIDES M, CHILOECHES A, HAYWARD R, et al. B-RAF is a therapeutic target in melanoma[J]. Oncogene, 2004, 23(37): 6292-6298. DOI URL
[51]	SCHMIDT C A, OETTLE H, LUDWIG W D, et al. Overexpression of the raf-1 proto-oncogene in human myeloid leukemia[J]. Leukemia Research, 1994, 18(6): 409-413. DOI URL
[52]	HWANG Y H, CHOI J Y, KIM S, et al. Over-expression of c-raf-1 proto-oncogene in liver cirrhosis and hepatocellular carcinoma[J]. Hepatology Research, 2004, 29(2): 113-121. DOI URL
[53]	MCPHILLIPS F, MULLEN P, MACLEOD K G, et al. Raf-1 is the predominant Raf isoform that mediates growth factor-stimulated growth in ovarian cancer cells[J]. Carcinogenesis, 2005, 27(4): 729-739. DOI URL

Effect of alcohol extracts from Chinese bayberry branch on proliferation and apoptosis of A375 cells and its molecular mechanism

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 5

References 53

Related Articles 8

Recommended Articles

Metrics

Comments

[1]	MA Xiaohua, YU Zheping, ZHENG Xiliang, HU Xiaojin, ZHANG Shuwen, QI Xingjiang, MA Jingyan. Introduction experiment and phenotype cluster analysis of Chinese bayberry in Jingzhou [J]. , 2020, 32(11): 1987-1993.
[2]	ZHANG Shuwen, LIANG Senmiao, ZHU Tingting, REN Haiying, ZHENG Xiliang, QI Xingjiang. Cold tolerance of different Chinese bayberry varieties [J]. , 2020, 32(10): 1772-1779.
[3]	LIANG Senmiao, GUO Xiuzhu, ZHENG Xiliang, ZHANG Shuwen, WEN Luhua, HUANG Pinhu, QI Xingjiang. Mineral nutritional characteristics of different organs in fruit-bearing tree of Myrica rubra Lour. [J]. , 2017, 29(10): 1669-1677.
[4]	YANG Min, HUANG Qiao, CHEN Liang-biao. Construction of type Ⅲ antifreeze protein eukaryotic expression plasmid and expression in zebrafish cell line [J]. , 2016, 28(11): 1862-1866.
[5]	XU Yun-huan, LIANG Sen-miao, ZHENG Xi-liang, REN Hai-ying, QI Xing-jiang. Effects of foliar nutrition on fruit yield and quality of Chinese bayberry (Myrica rubra Sieb. et Zucc.) [J]. , 2016, 28(10): 1711-1717.
[6]	CHEN Wei1， LIANG Sen\\|miao2， GUO Xiu\\|zhu1， HUANG Pin\\|hu1， QIU Ying\\|ying2， QI Xing\\|jiang2，*. Effects of foliar nutrition on fruit quality and postharvest storage of Chinese bayberry [J]. , 2014, 26(6): 1491-.
[7]	CAO Xue-dan;QI Xing-jiang;FANG Xiu-gui;ZHAO Kai. Drying properties and quality of Chinese bayberry fruit [J]. , 2010, 22(3): 0-369.
[8]	GUO Xiu-zhu;Qiu Ying-ying;HUANG Pin—hu;WANG Kang-qiang;ZENG Ai-ping;QI Xing-Jiang;*. Effect of diferent fertilization methods on quality of Chinese bayberry fruit(Myrica Rubra Sieb．＆ Zucc．) [J]. , 2009, 21(4): 0-361.