Skip to main content
PMC home page
Cellular & Molecular Biology Letters logoLink to Cellular & Molecular Biology Letters
. 2014 Sep 2;19(4):527–541. doi: 10.2478/s11658-014-0210-8

Downregulation of KDR expression induces apoptosis in breast cancer cells

Xiao Zhang 1,✉, Yin-Lin Ge 1,✉, Shu-Ping Zhang 2, Ping Yan 3, Run-Hua Tian 1
PMCID: PMC6276020  PMID: 25182240

Abstract

Angiogenesis plays a crucial role in the growth, invasion and metastasis of breast cancer. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the key regulators of tumor angiogenesis. VEGFR-2, known as the kinase insert domain receptor (KDR), is a key receptor involved in malignant angiogenesis. We previously showed that knocking down KDR with short interference RNA (KDR-siRNA) markedly decreased KDR expression and suppressed tumor growth in a xenograft model. However, the mechanisms underlying the anti-cancer effects of KDR-siRNA are not clearly understood. This study aimed to elucidate the molecular mechanisms that induce apoptosis in human breast cancer MCF-7 cells after transfection with KDR-siRNA. We studied the effects of KDR-siRNA on proliferation, apoptosis, antiapoptotic and pro-apoptotic proteins, mitochondrial membrane permeability, cytochrome c release and caspase-3 activity. The results indicated that KDR-siRNA treatment significantly inhibited the proliferation and induced the apoptosis of MCF-7 cells, reduced the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl, and increased the level of the pro-apoptotic protein Bax, resulting in a decreased Bcl-2/Bax ratio. KDR-siRNA also enhanced the mitochondrial membrane permeability, induced cytochrome c release from the mitochondria, upregulated apoptotic protease-activating factor-1 (Apaf-1), cleaved caspase-3, and increased caspase-3 activity in MCF-7 cells. Furthermore, KDR-siRNA-induced apoptosis in MCF-7 cells was blocked by the caspase inhibitor Z-VAD-FMK, suggesting a role of caspase activation in the induction of apoptosis. These results indicate that the Bcl-2 family proteins and caspase-related mitochondrial pathways are primarily involved in KDR-siRNAinduced apoptosis in MCF-7 cells and that KDR might be a potential therapeutic target for human breast cancer treatments.

Keywords: Apoptosis, Breast cancer, MCF-7 cells, Cytochrome c, caspase-3, Mitochondrial pathway, KDR, VEGF, Short interfering RNA, siRNA

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Abbreviations used

Apaf-1

apoptotic protease-activating factor-1

BSA

bovine serum albumin

DMEM

Dulbecco’s modified Eagle’s medium

KDR

kinase domain receptor

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-liumbromide

SDS

sodium dodecyl sulphate

siRNA

short interfering RNA

VEGF

vascular endothelial growth factor

VEGFR

vascular endothelial growth factor receptor

Contributor Information

Xiao Zhang, Email: zhx7605@126.com.

Yin-Lin Ge, Email: geyinlin@126.com.

References

  • 1.Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J. Clin. 2011;61:69–90. doi: 10.3322/caac.20107. [DOI] [PubMed] [Google Scholar]
  • 2.Linos E, Spanos D, Rosner BA, Linos K, Hesketh T, Qu JD, Gao YT, Zheng W, Colditz GA. Effects of reproductive and demographic changes on breast cancer incidence in China: a modeling analysis. J. Natl. Cancer Inst. 2008;100:1352–1360. doi: 10.1093/jnci/djn305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Dai M, Ren JS, Li N, Li Q, Yang L, Chen YH. Estimation and prediction on cancer related incidence and mortality in China, 2008. Zhonghua Liu. Xing. Bing. Xue. Za. Zhi. 2012;33:57–61. [PubMed] [Google Scholar]
  • 4.Hyder SM. Sex-steroid regulation of vascular endothelial growth factor in breast cancer. Endocr. Relat. Cancer. 2006;13:667–687. doi: 10.1677/erc.1.00931. [DOI] [PubMed] [Google Scholar]
  • 5.Takekoshi K, Isobe K, Yashiro T, Hara H, Ishii K, Kawakami Y, Nakai T, Okuda Y. Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas. Life Sci. 2004;74:863–871. doi: 10.1016/j.lfs.2003.07.036. [DOI] [PubMed] [Google Scholar]
  • 6.Roskoski R., Jr Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit. Rev. Oncol. Hematol. 2007;62:179–213. doi: 10.1016/j.critrevonc.2007.01.006. [DOI] [PubMed] [Google Scholar]
  • 7.Vieira JM, Santos SC, Espadinha C, Correia I, Vag T, Casalou C, Cavaco BM, Catarino AL, Dias S, Leite V. Expression of vascular endothelial growth factor (VEGF) and its receptors in thyroid carcinomas of follicular origin: a potential autocrine loop. Eur. J. Endocrinol. 2005;153:701–709. doi: 10.1530/eje.1.02009. [DOI] [PubMed] [Google Scholar]
  • 8.Guo S, Colbert LS, Fuller M, Zhang Y, Gonzalez-Perez RR. Vascular endothelial growth factor receptor-2 in breast cancer. Biochim. Biophys. Acta. 2010;1806:108–121. doi: 10.1016/j.bbcan.2010.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis and proliferation of human colon cancer. Cancer Res. 1995;55:3964–3968. [PubMed] [Google Scholar]
  • 10.Ryden L, Linderholm B, Nielsen NH, Emdin S, Jonsson PE, Landberg G. Tumor specific VEGF-A and VEGFR2/KDR protein are coexpressed in breast cancer. Breast Cancer Res. Treat. 2003;82:147–154. doi: 10.1023/B:BREA.0000004357.92232.cb. [DOI] [PubMed] [Google Scholar]
  • 11.Spratlin J. Ramucirumab (IMC-1121B): Monoclonal antibody inhibition of vascular endothelial growth factor receptor-2. Curr. Oncol. Rep. 2011;13:97–102. doi: 10.1007/s11912-010-0149-5. [DOI] [PubMed] [Google Scholar]
  • 12.Hammond SM, Bexnstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Dxosophila cells. Nature. 2000;404:293. doi: 10.1038/35005107. [DOI] [PubMed] [Google Scholar]
  • 13.Ge YL, Zhang JY, Zhang X, Hou L, Li Q, Xue ML. Chemically modified siRNA directed against the KDR gene inhibits the proliferation of breast cancer cells. Mol. Med. Rep. 2009;2:121–127. doi: 10.3892/mmr_00000072. [DOI] [PubMed] [Google Scholar]
  • 14.Kim EJ, Park SY, Lee JY, Park JH. Fucoidan present in brown algae induces apoptosis of human colon cancer cells. BMC Gastroenterol. 2010;10:96. doi: 10.1186/1471-230X-10-96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Minamino M, Sakaguchi I, Naka T, Iked N, Katoa Y, Tomiyasu I, Yano I, Kobayashi K. Bacterial ceramides and sphingophospholipids induce apoptosis of human leukaemic cells. Microbiology. 2003;149:2071–2081. doi: 10.1099/mic.0.25922-0. [DOI] [PubMed] [Google Scholar]
  • 16.Chen Q, Li L, Tu Y, Zheng LL, Liu W, Zuo XY, He YM, Zhang SY, Zhu W, Cao JP, Cui FM, Hou J. MiR-34a regulates apoptosis in liver cells by targeting the KLF4 gene. Cell. Mol. Biol. Lett. 2014;19:52–64. doi: 10.2478/s11658-013-0115-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Westphal S, Kalthoff H. Apoptosis: targets in pancreatic cancer. Mol. Cancer. 2003;2:6. doi: 10.1186/1476-4598-2-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kuo PC, Liu HF, Chao JI. Survivin and p53 modulate quercetininduced cell growth inhibition and apoptosis in human lung carcinoma cells. J. Biol. Chem. 2004;279:55875–55885. doi: 10.1074/jbc.M407985200. [DOI] [PubMed] [Google Scholar]
  • 19.Katiyar SK, Roy AM, Baliga MS. Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release and caspase activation. Mol. Cancer Ther. 2005;4:207–216. [PubMed] [Google Scholar]
  • 20.Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer Biol. Ther. 2005;4:139–163. doi: 10.4161/cbt.4.2.1508. [DOI] [PubMed] [Google Scholar]
  • 21.Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis pathways in cancer therapy. CA Cancer J. Clin. 2005;55:178–194. doi: 10.3322/canjclin.55.3.178. [DOI] [PubMed] [Google Scholar]
  • 22.Jagani H, Rao JV, Palanimuthu VR, Hariharapura RC, Gang S. A nanoformulation of siRNA and its role in cancer therapy: in vitro and in vivo evaluation. Cell. Mol. Biol. Lett. 2013;18:120–136. doi: 10.2478/s11658-012-0043-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Reed JC. Regulation of apoptosis by Bcl-2 family proteins and its role in cancer and chemoresistance. Curr. Opin. Oncol. 1995;7:541–546. doi: 10.1097/00001622-199511000-00012. [DOI] [PubMed] [Google Scholar]
  • 24.Adams JM, Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr. Opin. Immunol. 2007;19:488–496. doi: 10.1016/j.coi.2007.05.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kumari A, Kakkar P. Lupeol prevents acetaminophen-induced in vivo hepatotoxicity by altering the Bax/Bcl-2-and oxidative stress-mediated mitochondrial signaling cascade. Life Sci. 2012;90:561–570. doi: 10.1016/j.lfs.2012.01.012. [DOI] [PubMed] [Google Scholar]
  • 26.Chen YC, Shen SC, Lee WR, Hsu FL, Lin HY, Ko CH, Tseng SW. Emodin induces apoptosis in human promyelokukemic HL-60 cells accompanied by activation of caspase-3 cascade but independent of reactive oxygen species production. Biochem. Pharmacol. 2002;64:1713–1724. doi: 10.1016/S0006-2952(02)01386-2. [DOI] [PubMed] [Google Scholar]

Articles from Cellular & Molecular Biology Letters are provided here courtesy of BMC

RESOURCES