The cucurbitacins D, E, and I from Ecballium elaterium (L.) upregulate the LC3 gene and induce cell-cycle arrest in human gastric cancer cell line AGS

Document Type : Original Article


1 Department of Cell & Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran

2 Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran


Objective(s): Cucurbitacins exhibit a range of anti-cancer functions. We investigated the effects of cucurbitacins D, E, and I purified from Ecballium elaterium (L.) A. Rich fruits on some apoptotic and autophagy genes in human gastric cancer cell line AGS.
Materials and Methods: Using quantitative reverse transcription PCR (qRT-PCR), the expression of LC3, VEGF, BAX, caspase-3, and c-MYC genes were quantified in AGS cells 24 hr after treatment with cucurbitacins D, E, and I at concentrations 0.3, 0.1 and 0.5 μg/ml, respectively. Cell cycle and death were analyzed by flowcytometry.
Results: Purified cucurbitacins induced sub-G1 cell-cycle arrest and cell death in AGS cells and upregulated LC3mRNA effectively, but showed a very low effect on BAX, caspase-3, and c-MYC mRNA levels. Also after treatment with cucurbitacin I at concentration 0.5 μg/ml, VEGF mRNA levels were increased about 4.4 times. Pairwise comparison of the effect of cucurbitacins D, E, and I on LC3 mRNA expression showed that the cucurbitacin I effect is 1.3 and 1.1 times that of cucurbitacins E and D, respectively; cucurbitacin D effect is 1.2 times that of cucurbitacin E (P-value <0.05). In silico analysis showed that among autophagy genes, LC3 has an important gastric cancer rank relation.
Conclusion: Cucurbitacins D, E, and I purified from E. elaterium fruits upregulate LC3 and induce sub-G1 cell-cycle arrest and cell death in human gastric cancer cell line AGS. Cucurbitacin I effect on LC3 mRNA expression is significantly more than that of cucurbitacins E and D.


Main Subjects

1. Sporn MB, Suh N. Chemoprevention of cancer. Carcinogenesis 2000; 21: 525-530.
2. Chen JC, Chiu MH, Nie RL, Cordell GA, Qiu SX. Cucurbitacins and cucurbitane glycosides: structures and biological activities. Nat Prod Rep 2005; 22: 386-399.
3. Ríos JL, Escandell JM, Recio MC. New insights into the bioactivity of cucurbitacins. In: Atta-ur-Rahman, editor. Studies in Natural Products Chemistry Volume 32: Bioactive Natural Products Part L. Netherlands: Elsevier Science; 2005. p. 429-469.
4. Lee DH, Iwanski GB, Thoennissen NH. Cucurbitacin: ancient compound shedding new light on cancer treatment. Sci World J 2010; 10: 413-418.
5. Hamasaki M, Furuta N, Matsuda A, Nezu A, Yamamoto A, Fujita N, et al. Autophagosomes form at ER mitochondria contact sites. Nature 2013; 495: 389-393.
6. Bernard A, Klionsky DJ. Autophagosome formation: tracing the source. Dev Cell 2013; 25: 116-117.
7. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011; 27: 107-132.
8. Ryter SW, Choi AM. Regulation of autophagy in oxygen-dependent cellular stress. Curr Pharm Des 2013; 19: 2747-2756.
9. Ryter SW, Cloonan SM, Choi AM. Autophagy: A critical regulator of cellular metabolism and homeostasis. Mol Cells 2013; 36: 7-16.
10. Brech A, Ahlquist T, Lothe RA, Stenmark H. Autophagy in tumour suppression and promotion. Mol Oncol 2009; 3: 366-375.
11. Schmukler E, Grinboim E, Schokoroy S, Amir A, Wolfson E, Kloog Y, et al. Ras inhibition enhances autophagy, which partially protects cells from death. Oncotarget 2013; 4: 142-152.
12. Cufí S, Vazquez-Martin A, Oliveras-Ferraros C, Corominas-Faja B, Urruticoechea A, Martin-Castillo B, et al.  Autophagy-related gene 12 (ATG12) is a novel determinant of primary resistance to HER2-targeted therapies: utility of transcriptome analysis of the autophagy interactome to guide breast cancer treatment. Oncotarget 2012; 3: 1600-1614.
13. Gewirtz, D.A. Cytoprotective and nonprotective autophagy in cancer therapy. Autophagy 2013; 9: 1263-1265.
14. Chu SC, Hsieh YS, Yu CC, Lai YY, Chen PN. Thymoquinone induces cell death in human squamous carcinoma cells via caspase activation-dependent apoptosis and LC3-II activation-dependent autophagy. PLoS One 2014; 9: e101579.
15. Hoffman B, Liebermann DA. Apoptotic signaling by c-MYC. Oncogene 2008; 27: 6462-6472.
16. Tsuneoka M, Umata T, Kimura H, Koda Y, Nakajima M, Kosai K, et al. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell Struct Funct 2003; 28:195-204.
17. Luna-Vargas MP, Chipuk JE. Physiological and pharmacological control of BAK, BAX, and beyond. Trends Cell Biol 2016; 26: 906-917.
18. Ishdorj G, Johnston JB, Gibson SB. Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B leukemic cells. BMC cancer 2011; 11: 268-279.
19. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015; 136: E359-386.
20. Jafargholizadeh N, Zargar SJ, Yassa N, Tavakoli S.  Purifcation of Cucurbitacins D, E, and I from Ecballium Elaterium (L.) A. Rich Fruits and Study of Their Cytotoxic Effects on the AGS Cell Line. Asian Pac J Cancer Prev 2016; 17: 4631-4635.
21. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin 2015; 65: 87-108.
22. Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res 2009; 37(suppl_2): W305-W311.
23. Aerts S, Lambrechts D, Maity S, Van Loo P, Coessens B, De Smet F, et al. Gene prioritization   through genomic data fusion. Nat Biotechnol 2006; 24: 537-544.
24. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012; 75: 311-335.
25. Attard E, Scicluna-Spiteri A. The cultivation and cucurbitacin content of Ecballium elaterium (L.) A. Rich. Rep Cucurbit Genet Coop 2003; 26: 66-69.
26. Ríos JL, Andújar I, Escandell JM, Giner RM, Recio MC. Cucurbitacins as inducers of cell death and a rich source of potential anticancer compounds. Curr Pharm Des 2012; 18: 1663-1676.
27. Wlodkowic D, Skommer J, Darzynkiewicz Z. Cytometry in cell necrobiology revisited. Recent advances and new vistas. Cytometry A 2010; 77: 591-606.
28. Gurusamy N, Das DK. Detection of cell death by autophagy. Methods Mol Biol 2009; 559:  95-103.
29. Zha QB, Zhang XY, Lin QR, Xu LH, Zhao GX, Pan H, et al. Cucurbitacin E induces autophagy via downregulating mTORC1 signaling and upregulating AMPK activity. PloS one. 2015; 10: e0124355.
30. Zhang T, Li Y, Park KA, Byun HS, Won M, Jeon J, et al. Cucurbitacin induces autophagy through mitochondrial ROS production which counteracts to limit caspase-dependent apoptosis. Autophagy. 2012; 8: 559-576.
31. Yuan G, Yan SF, Xue H, Zhang P, Sun JT, Li G. Cucurbitacin I induces protective autophagy in glioblastoma in vitro and in vivo. J Biol Chem 2014; 289: 10607-10619.
32. Deng C, Zhang B, Zhang S, Duan C, Cao Y, Kang W, et al. Low nanomolar concentrations of Cucurbitacin-I induces G2/M phase arrest and apoptosis by perturbing redox homeostasis in gastric cancer cells in vitro and in vivo. Cell Death Dis 2016; 7:e2106.
33. Sun T, Li D, Wang L, Xia L, Ma J, Guan Z, et al. c-Jun NH2-terminal kinase activation is essential for up-regulation of LC3 during ceramide-induced autophagy in human nasopharyngeal carcinoma cells. J Transl Med 2011; 9: 161-170.
34. Young MM, Takahashi Y, Khan O, Park S, Hori T, Yun J, et al. Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis. J Biol Chem 2012; 287:12455-12468.