Effects of royal jelly protein <i>in vitro</i> digestion products on proliferation and apoptosis of gastric cancer cell SGC-7901 and its possible mechanism

doi:10.3785/j.issn.1008-9209.2019.01.221

Journal of Zhejiang University (Agriculture and Life Sciences)

2019, Vol. 45

Issue (5): 533-541 DOI: 10.3785/j.issn.1008-9209.2019.01.221

Biological sciences & biotechnology

Effects of royal jelly protein in vitro digestion products on proliferation and apoptosis of gastric cancer cell SGC-7901 and its possible mechanism

Tianshi WANG¹(

),Xinmeng WANG²,Li FU¹(

)

1. College of Food Science and Engineering, Jinzhou Medical University, Jinzhou 121000, Liaoning, China
2. Department of Biochemistry and Molecular Biology, Jinzhou Medical University, Jinzhou 121000, Liaoning, China

Download:

HTML

HTML ( )

PDF(2525KB)
Export: BibTeX | EndNote (RIS)

Abstract

To study the effects of in vitro digestion products of royal jelly protein on the proliferation and apoptosis of gastric cancer cell SGC-7901 and to explore its possible mechanism so as to provide an experimental basis for the royal jelly protein in the treatment of gastric cancer, the royal jelly protein was extracted by alkali extraction and acid precipitation. The in vitro digestion products of royal jelly protein were obtained by in vitro simulated digestion experiments. The gastric cancer cells SGC-7901 were cultured in vitro with different concentrations of royal jelly protein in vitro digestion products, and the influence on gastric cancer cells were observed by an inverted microscope. The colony formation ability of gastric cancer cells after induction was tested. The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect the inhibition against proliferation of the gastric cancer cells; the cell cycle and apoptosis were detected by flow cytometry; and the expression levels of p53 and PARP-1 proteins in the gastric cancer cells were detected by Western blotting. The results showed that the in vitro digestion products of royal jelly protein leaded to the shrinkage of gastric cancer cells, decreased cell density and colony formation ability (P＜0.05), and inhibited the proliferation of gastric cancer cell SGC-7901. When the in vitro digestion product concentration was 0.2 mg/mL, the inhibition rates at 24 h and 48 h were (57.58±3.48)% and (62.84±1.98)%, respectively (P＜0.05). Compared with the blank control group, the in vitro digestion products of royal jelly protein inhibited the proliferation of SGC-7901 cells in a dose-dependent manner, and the S phase cells decreased, which leading to the apoptosis. After induction by in vitro digestion products of royal jelly protein, the expression of p53 increased in the gastric cancer cells (P＜0.05), and the expression level of PARP-1 protein decreased (P＜0.05). The above results show that the in vitro digestion products of royal jelly protein can inhibit the proliferation of gastric cancer cell SGC-7901 and induce its apoptosis. The mechanism may be related to the up-regulation of p53 expression and the down-regulation of PARP-1 expression.

Key words： royal jelly protein in vitro digestion products gastric cancer cell proliferation cell cycle apoptosis

Received: 22 January 2019 Published: 05 December 2019

CLC:

TS 201.1

Corresponding Authors: Li FU E-mail: a297248362@163.com;fuli1979119@126.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Tianshi WANG
	Xinmeng WANG
	Li FU

Cite this article:

Tianshi WANG,Xinmeng WANG,Li FU. Effects of royal jelly protein in vitro digestion products on proliferation and apoptosis of gastric cancer cell SGC-7901 and its possible mechanism. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 533-541.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2019.01.221 OR http://www.zjujournals.com/agr/Y2019/V45/I5/533

蜂王浆蛋白体外消化产物对胃癌细胞SGC-7901增殖和凋亡的影响及其可能机制

通过研究蜂王浆蛋白体外消化产物对胃癌细胞SGC-7901增殖、凋亡的影响，并探讨其可能的作用机制，为蜂王浆蛋白辅助治疗胃癌奠定实验基础。采用碱提酸沉法提取蜂王浆蛋白，并通过体外模拟消化实验得到蜂王浆蛋白体外消化产物，然后用不同质量浓度的蜂王浆蛋白体外消化物诱导体外培养的胃癌细胞SGC-7901，通过倒置显微镜观察其对胃癌细胞形态的影响，并通过集落形成实验检测诱导后胃癌细胞的集落形成能力；采用3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐（MTT）法检测其对胃癌细胞增殖的抑制作用，通过流式细胞术检测细胞周期及凋亡，并通过蛋白质印迹法检测其诱导后胃癌细胞中p53和PARP-1蛋白的表达情况。结果表明：蜂王浆蛋白体外消化产物使胃癌细胞形态发生皱缩，细胞密度降低，集落形成能力降低（P＜0.05），对胃癌细胞SGC-7901的增殖具有抑制作用。0.2 mg/mL体外消化产物经24 h和48 h培养后对胃癌细胞抑制率分别达到（57.58±3.48）%和（62.84±1.98）%（P＜0.05）。与空白对照组相比，蜂王浆蛋白体外消化产物对SGC-7901细胞增殖的抑制作用呈剂量依赖性，S期细胞减少，从而引起细胞凋亡；且经蜂王浆蛋白体外消化产物诱导后，胃癌细胞中p53蛋白表达量增加（P＜0.05），PARP-1蛋白表达量减少（P＜0.05）。说明蜂王浆蛋白体外消化产物能抑制胃癌细胞SGC-7901的增殖并诱导其发生凋亡，其机制可能与p53表达上调和PARP-1表达下调有关。

关键词： 蜂王浆蛋白体外消化产物, 胃癌, 细胞增殖, 细胞周期, 细胞凋亡

Fig. 1 Effect of different concentrations of royal jelly protein in vitro digestion products on cell morphologyA₀. Control group of 293T cells treated by 0.2 mg/mL protein in vitro digestion products; A. Control group of SGC-7901 cells treated by DMSO; B. Experimental group of SGC-7901 cells treated by 0.05 mg/mL protein in vitro digestion products; C. Experimental group of SGC-7901 cells treated by 0.1 mg/mL protein in vitro digestion products; D. Experimental group of SGC-7901 cells treated by 0.2 mg/mL protein in vitro digestion products.

Fig. 2 Effect of different concentrations of royal jelly protein in vitro digestion products on cell colony forming abilityPlease see the footnote of Fig. 1 for the details of A₀-D treatments.

Fig. 3 Effect of different concentrations of royal jelly protein in vitro digestion products on cell numberPlease see the footnote of Fig. 1 for the details of A₀-D treatments. Single asterisk (*) or double asterisks (**) above bars indicate significant or highly significant differences from the DMSO treatment (control group) at the 0.05 or 0.01 probability level, respectively; n=3.

Table 1 Effect of different concentrations of royal jelly protein in vitro digestion products on proliferation inhibition rate of gastric cancer cells

Fig. 4 Cycles of gastric cancer cells induced by different concentrations of royal jelly protein in vitro digestion products for 48 h by flow cytometryA. Control group of SGC-7901 cells treated by DMSO; B. Experimental group of SGC-7901 cells treated by 0.05 mg/mL protein in vitro digestion products; C. Experimental group of SGC-7901 cells treated by 0.1 mg/mL protein in vitro digestion products; D. Experimental group of SGC-7901 cells treated by 0.2 mg/mL protein in vitro digestion products. PI-A: Fluorescence intensity value by propidium iodide staining.

Table 2 Effects of different concentrations of royal jelly protein in vitro digestion products on cycle of gastric cancer cells induced for 48 h

Fig. 5 Apoptosis of gastric cancer cells induced by different concentrations of royal jelly protein in vitro digestion products for 48 h by flow cytometryPlease see the footnote of Fig. 4 for the details of A-D treatments. Q₁: Necrotic cells; Q₂: Late apoptotic cells; Q₃: Living Cells; Q₄: Early apoptotic cells. 7-AAD-A: Fluorescence intensity value by 7-amino-actinomycin D staining; PE-A: Fluorescence intensity value by annexin V-PE staining. Single asterisk (*) above bars indicates significant differences from the DMSO treatment (control group) at the 0.05 probability level; n=3.

Fig. 6 Effect of different concentrations of royal jelly protein in vitro digestion products on expression levels of p53 and PARP-1 in gastric cancer cellsPlease see the footnote of Fig. 4 for the details of A-D treatments. Single asterisk (*) above bars indicates significant differences from the DMSO treatment (control group) at the 0.05 probability level; n=3.


[1]	YUNG J B, ERIC V C, ANDREA F, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA):a phase 3, open-label, randomised controlled trial. Lancet, 2010,376:687-697.

[2]	尚海霞,杨弘,靳祎祎,等.八宝丹抑制胃癌细胞增殖与诱导细胞凋亡的实验研究.福建中医药,2018,49(1):29-31. SHANG H X, YANG H, JIN Y Y, et al. Experimental study on inhibitory effect of Babaodan on proliferation and apoptosis of gastric cancer cells. Journal of Fujian University of Traditional Chinese Medicine, 2018,49(1):29-31. (in Chinese)

[3]	彭涛,刘丽波,马建军.抗肿瘤食品的研究.食品研究与开发,2004,25(1):61-65. PENG T, LIU L B, MA J J. Study on antitumor food. Food Research and Development, 2004,25(1):61-65. (in Chinese with English abstract)

[4]	于张颖.蜂王浆主蛋白(MRJPs)替代牛血清(FBS)培养人体细胞应用技术研究.杭州:浙江大学,2014:1-2. YU Z Y. Studies on the applied technologies of major royal jelly proteins (MARJPs) in culturing of human cells to replace fetal bovine serum (FBS). Hangzhou: Zhejiang University, 2014:1-2. (in Chinese with English abstract)

[5]	陈波,徐德平.蜂王浆醇溶性成分分析及其抗氧化性研究.食品与发酵工业,2012,38(7):124-127. CHEN B, XU D P. Structure and antioxidation studies of alcohol soluble components in royal jelly. Food and Fermentation Industries, 2012,38(7):124-127. (in Chinese with English abstract)

[6]	KAMAKURA M, FUKADA T, FUKUSHIMA M, et al. Storage-dependent degradation of 57-kDa protein in royal jelly: a possible marker for freshness. Journal of the Agricultural Chemical Society of Japan, 2001,65(2):277-284.

[7]	HADDADIN M S Y, HADDADIN J, BENGUIAR R. The effect of royal jelly on growth and short-chain fatty acid production of probiotic bacteria and activity of bacterial procarcinogenic enzymes in rat faeces. Polish Journal of Food and Nutrition Sciences, 2012,62(4):251-258.

[8]	INOUE Y, HARA H, MITSUGI Y, et al. Hydroperoxy-2-decenoic acid ethyl ester protects against 6-hydroxydopa-mine-induced cell death via activation of Nrf2-ARE and eIFza-ATF4 pathways. Neurochemisity International, 2018,112:288-296.

[9]	吴雨祺,陈伊凡,游蒙蒙,等.2017年国内外蜂王浆研究概况.中国蜂业,2018,69(7):66-70. WU Y Q, CHEN Y F, YOU M M, et al. Research status of royal jelly in 2017. Apiculture of China, 2018,69(7):66-70. (in Chinese with English abstract)

[10]	GUENDOUZ M, HADDI A, GRAR H, et al. Preventive effects of royal jelly against anaphylactic response in a murine model of cow’s milk allergy. Pharmaceutical Biology, 2017,55(1):2145-2152.

[11]	刘合生.蜂王浆活性蛋白的分离、纯化、生物活性及结构初步表征.武汉:华中农业大学,2009:1-2. LIU H S. Study on isolation, purification, bio-activity and preliminary structure identification of royal jelly active protein. Wuhan: Huazhong Agricultural University, 2009:1-2. (in Chinese with English abstract)

[12]	ZAMANZAD B, SHIRZAD H, SHAHINFARD N, et al. Effect of royal jelly on fibrosarcoma tumor in BALB/c mice. Journal of Shahrekord University of Medical Sciences, 2009,10(4):64-70.

[13]	KIM J, KIM Y, YUN H J, et al. Royal jelly enhances migration of human dermal fibroblasts and alters the levels of cholesterol and sphinganine in an in vitro wound healing mode. Nutrition Research and Practice, 2010,4(5):362-368.

[14]	孟超,肖凯文,郑双艳,等.蜂王浆主蛋白对小鼠的抗疲劳作用.中国食品学报,2017,17(10):23-29. MENG C, XIAO K W, ZHENG S Y, et al. Anti-fatigue activity of MRJPs from fresh royal jelly in mice. Journal of Chinese Institute of Food Science and Technology, 2017,17(10):23-29. (in Chinese with English abstract)

[15]	JIANG C M, LIU X, LI C X, et al. Anti-senescence effect and molecular mechanism of the major royal jelly proteins on human embryonic lung fibroblast (HFL-Ⅰ) cell line. Journal of Zhejiang University: Science B (Biomedicine & Biotechnology), 2018,19(12):960-972.

[16]	彭瑜,陈梓嘉,何腾飞.蜂王浆冻干粉对小鼠脾细胞增殖的影响.中国蜂业,2014,65(增刊3):40-42. PENG Y, CHEN Z J, HE T F. Royal jelly producing effects on mice spleen cell proliferation. Apiculture of China, 2014,65(Suppl. 3):40-42. (in Chinese with English abstract)

[17]	王楠,王杰,姜龙达,等.甲鱼碱溶蛋白肽的分离及抗肿瘤活性研究.中国食品学报,2017,17(4):98-102. WANG N, WANG J, JIANG L D, et al. Separation and antitumor activity evaluation of turtle alkali-soluble protein peptide. Journal of Chinese Institute of Food Science and Technology, 2017,17(4):98-102. (in Chinese with English abstract)

[18]	蓝瑞阳,朱威,季文静,等.蜂王浆蛋白质提取工艺研究.蜜蜂杂志,2008,28(3):18-20. LAN R Y, ZHU W, JI W J, et al. Study on the protein extracting technology of royal jelly. Journal of Bee, 2008,28(3):18-20. (in Chinese with English abstract)

[19]	佟立涛,陈倩倩,钱海峰,等.大米蛋白体外消化产物抗氧化活性的研究.现代食品科技,2015,31(9):92-97. TONG L T, CHEN Q Q, QIAN H F, et al. Antioxidant activity of in vitro rice protein digest. Modern Food Science and Technology, 2015,31(9):92-97. (in Chinese with English abstract)

[20]	贺雄辉,邹科见,叶木林,等.胃癌细胞HGC-27与SGC-7901中基质金属蛋白酶基因表达水平和细胞迁移能力比较.山东医药,2018,58(2):28-30. HE X H, ZOU K J, YE M L, et al. Comparison of matrix metalloproteinase gene expression and cell migration between HGC-27 and SGC-7901 in gastric cancer cells. Shandong Medicine Journal, 2018,58(2):28-30. (in Chinese)

[21]	曲凯,董士苍,常庆勇,等.慢病毒介导的TLR4基因沉默对U251细胞增殖与迁移的影响.解剖科学进展,2018,24(4):344-346. QU K, DONG S C, CHANG Q Y, et al. The effect of lentivirus mediated TLR4 gene silencing on the proliferation and migration of U251 cells. Advances in Anatomical Science, 2018,24(4):344-346. (in Chinese with English abstract)

[22]	朱昌毫,孙城谊,喻超,等.青藤碱对胰腺癌细胞系SW1990增殖及凋亡的影响.贵州医科大学学报,2018,43(8):879-883. ZHU C H, SUN C Y, YU C, et al. Effect of sinomenine on pancreatic cancer cell SW1990 proliferation and apoptosis. Journal of Guizhou Medical University, 2018,43(8):879-883. (in Chinese with English abstract)

[23]	曾杰,陈佳,邵邻相,等.葡萄糖饥饿对HeLa细胞形态与结构的影响.浙江师范大学学报(自然科学版),2018,41(1):92-96. ZENG J, CHEN J, SHAO L X, et al. The effects of glucose deprivation on morphology and structure of HeLa cells. Journal of Zhejiang Normal University (Natural Sciences), 2018,41(1):92-96. (in Chinese with English abstract)

[24]	尤亚林,李慧,潘思轶,等.细香葱提取物对人胃癌细胞SGC7901抑制作用.食品科学,2017,38(3):176-181. YOU Y L, LI H, PAN S Y, et al. Inhibitory effect of chive extract on the proliferation of human gastric cancer cell line SGC7901. Food Science, 2017,38(3):176-181. (in Chinese with English abstract)

[25]	汪国玉,张蕾,耿亚迪,等.β-榄香烯诱导人结肠癌细胞DLD-1的增殖抑制和凋亡研究.安徽医科大学学报,2018,53(4):491-497. WANG G Y, ZHANG L, GENG Y D, et al. Study of β-elemene on tumor inhibition and apoptosis of human colon cancer DLD-1 cells. Acta Universitatis Medicinalis Anhui, 2018,53(4):491-497. (in Chinese with English abstract)

[26]	FERLAY J, SOERJOMATARAM I, DIKSHIT R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer, 2015,36(5):359-386.

[27]	王勇姿,凌昌全,黄雪强,等.生物蜂王浆抑制腹水型肝癌细胞H22生长的实验研究.第二军医大学学报,2001,22(4):357-359. WANG Y Z, LING C Q, HUANG X Q, et al. Experimental study of the inhibitory effects of biotic royal jelly on the growth of ascitic hepatoma cell H22 in mice. Journal of the Second Military Medical University, 2001,22(4):357-359. (in Chinese with English abstract)

[28]	陈立军,彭瑜,朱荃.蜂王浆冻干粉对B16-BL6黑色素瘤生长的影响.中国医药指南,2011,9(5):55-57. CHEN L J, PENG Y, ZHU Q. Effect of royal jelly freeze-drying powder on the growth of B16-BL6 melanoma. Guide to Chinese Medicine, 2011,9(5):55-57. (in Chinese with English abstract)

[29]	任汐鹰,刘勤献,王海英.p53与细胞死亡.中国生物化学与分子生物学报,2018,34(6):588-594. REN X Y, LIU Q X, WANG H Y. p53 and cell death. Chinese Journal of Biochemistry and Molecular Biology, 2018,34(6):588-594. (in Chinese with English abstract)

[1]	Peng JIAO, Ying XIAO, Zhen LIU, Ruiting CHEN, Yan LU, Huhu XIN, Yungen MIAO . BmDaxx regulates apoptosis in cells and larval tissues of the silkworm ( Bombyx mori )[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(4): 472-483.

[2]	YU Mengfei, WANG Wenlu, YI Jianming. Effects of 5- azacytidine-2’- deoxycytidine on the proliferation, cell cycle, and apoptosis of fetal bovine fibroblast cells[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(6): 727-734.

[3]	QIAN Haocheng, JIANG Chenmin, CHEN Huacai, SHEN Lirong. Effect and mechanism of major royal jelly proteins as an alternative of fetal bovine serum to culture Chinese hamster ovary cell CHO-K1[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(1): 1-9.

[4]	CHEN Siyuan, HU Ji’an. Progress and application of salinomycin-induced cell autophagy in anti-cancer treatment[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(6): 694-702.

[5]	Yu Zhangying, Chen Di, Wang Yiran, Shen Lirong. Effect of major royal jelly proteins (MRJPs) on proliferation activity of Chang's liver cell line and their mechanism of action[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(1): 7-14.

[6]	ZHOU Fangzhen, ZHANG Xiaoyuan, SUN Fenyong, GUO Yong. MDA-MB-231 cell apoptosis in vitro induced by dihydromyricetin.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2012, 38(3): 293-298.

[7]	NING Ying,YU Hai-ning,ZHOU Xin-mei,WANG Wei,JIANG Feng,PAN Xiao-yan,SHEN Sheng-r. Optimal process for semibionic extraction of bioactive compounds against gastric cancer from water chestnutshell by uniform designation[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2011, 37(3): 332-337.

Viewed

Full text

Abstract

Cited

Shared

Discussed