Knockdown of mutated H-Ras V12 expression induces chemosensitivity of hepatocellular carcinoma cells to cisplatin treatment in vitro and in nude mouse xenografts

Meng,Fanjie; Cao,Bin; Feng,Zengli; Ma,Shunmao; Wang,Haigang; Li,Yanshu; Li,Hui

doi:10.3892/or.2014.3466

Knockdown of mutated H-Ras V12 expression induces chemosensitivity of hepatocellular carcinoma cells to cisplatin treatment in vitro and in nude mouse xenografts

Authors:
- Fanjie Meng
- Bin Cao
- Zengli Feng
- Shunmao Ma
- Haigang Wang
- Yanshu Li
- Hui Li
View Affiliations

Affiliations: Department of General Surgery, North-China Oil Field General Hospital, Hebei Medical University, Renqiu 062552, P.R. China, Department of General Surgery, North-China Oil Field General Hospital, Hebei Medical University, Renqiu 062552, P.R. China
Published online on: September 5, 2014 https://doi.org/10.3892/or.2014.3466
Pages: 2023-2030

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Ras mutations contribute to human cancer development. The present study assessed the Ras V12 mutation in hepatocellular carcinoma (HCC) cells and the role of its silencing in vitro and in nude mouse xenografts. HCC BEL7402 cells expressed mutations of V12 (Val/Gly) and wild-type H-Ras, whereas SMMC7721 cells only expressed wild-type H-Ras. The shRNA constructs specifically silenced mutated H-Ras expression in BEL7402 cells, but did not affect wild-type Ras expression in SMMC7721 cells. Silencing of mutated H-Ras expression reduced BEL7402 cell viability, induced apoptosis and arrested cells at the G0/G1 phase of the cell cycle. Expression of phospho-Akt was also significantly decreased, while several apoptotic proteins were also activated. Furthermore, sensitivity of stably H1-shRNA and H2-shRNA transfected BEL7402 cells to cisplatin treatment was increased by 7.19- and 5.39-fold, respectively, compared to that in the negative control cells, and apoptosis-related gene expression was also altered. Intraperitoneal administration of cisplatin led to a substantial reduction in HCC xenograft growth by 81 and 77% in the H1-shRNA and H2-shRNA transfected tumor xenografts, respectively, compared to a 37% reduction in mice bearing negative control tumor cells. These data demonstrate that knockdown of mutated H-Ras V12 expression induced chemosensitivity of HCC cells to cisplatin treatment in vitro and in vivo.

View Figures

View References

1	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global Cancer Statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar
2	Saxena N, Lahiri SS, Hambarde S and Tripathi RP: RAS: target for cancer therapy. Cancer Invest. 26:948–955. 2008. View Article : Google Scholar : PubMed/NCBI
3	Boguski MS and McCormick F: Proteins regulating Ras and its relatives. Nature. 366:643–654. 1993. View Article : Google Scholar : PubMed/NCBI
4	Bos JL: ras oncogenes in human cancer: a review. Cancer Res. 49:4682–4689. 1989.PubMed/NCBI
5	Bollag G and McCormick F: Regulators and effectors of ras proteins. Annu Rev Cell Biol. 7:601–632. 1991. View Article : Google Scholar
6	Sui G, Ma X, Liu S, Niu H and Dong Q: Study of the correlation between H-ras mutation and primary hepatocellular carcinoma. Oncol Lett. 4:779–782. 2012.PubMed/NCBI
7	Lim KH and Counter CM: Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance. Cancer Cell. 8:381–392. 2005. View Article : Google Scholar : PubMed/NCBI
8	Rotblat B, Ehrlich M, Haklai R and Kloog Y: The Ras inhibitor farnesylthiosalicylic acid (Salirasib) disrupts the spatiotemporal localization of active Ras: a potential treatment for cancer. Methods Enzymol. 439:467–489. 2008. View Article : Google Scholar : PubMed/NCBI
9	Reuter C, Morgan M and Bergmann L: Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies? Blood. 96:1655–1669. 2000.PubMed/NCBI
10	Méndez-Sánchez N, Vásquez-Fernández F, Zamora-Valdés D and Uribe M: Sorafenib, a systemic therapy for hepatocellular carcinoma. Ann Hepatol. 7:46–51. 2008.PubMed/NCBI
11	Njoroge FG, Doll RJ, Vibulbhan B, et al: Discovery of novel nonpeptide tricyclic inhibitors of Ras farnesyl protein transferase. Bioorg Med Chem. 5:101–113. 1997. View Article : Google Scholar : PubMed/NCBI
12	Yang G, Thompson JA, Fang B and Liu J: Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumor growth in a model of human ovarian cancer. Oncogene. 22:5694–5701. 2003.
13	Hansen MB, Nielsen SE and Berg K: Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 119:203–210. 1989. View Article : Google Scholar : PubMed/NCBI
14	Paoluzzi L, Gonen M, Bhagat G, et al: The BH3-only mimetic ABT-737 synergizes the antineoplastic activity of proteasome inhibitors in lymphoid malignancies. Blood. 112:2906–2916. 2008. View Article : Google Scholar : PubMed/NCBI
15	Wang H, Rayburn ER, Wang W, Kandimalla ER, Agrawal S and Zhang R: Chemotherapy and chemosensitization of non-small cell lung cancer with a novel immunomodulatory oligonucleotide targeting Toll-like receptor 9. Mol Cancer Ther. 5:1585–1592. 2006. View Article : Google Scholar : PubMed/NCBI
16	Sepp-Lorenzino L, Rands E, Mao X, et al: A novel orally bioavailable inhibitor of kinase insert domain-containing receptor induces antiangiogenic effects and prevents tumor growth in vivo. Cancer Res. 64:751–756. 2004. View Article : Google Scholar
17	Crowder C, Kopantzev E, Williams K, Lengel C, Miki T and Rudikoff S: An unusual H-Ras mutant isolated from a human multiple myeloma line leads to transformation and factor-independent cell growth. Oncogene. 22:649–659. 2003. View Article : Google Scholar : PubMed/NCBI
18	Johnston SR: Farnesyl transferase inhibitors: a novel targeted tnerapy for cancer. Lancet Oncol. 2:18–26. 2001. View Article : Google Scholar : PubMed/NCBI
19	Tang Z, Qin L, Wang X, et al: Alterations of oncogenes, tumor suppressor genes and growth factors in hepatocellular carcinoma: with relation to tumor size and invasiveness. Chin Med J. 111:313–318. 1998.PubMed/NCBI
20	Iida M, Iwata H, Inoue H, Enomoto M, Horie N and Takeishi K: Correlation between Bcl-2 overexpression and H-ras mutation in naturally occurring hepatocellular proliferative lesions of the B6C3F1 mouse. Toxicol Sci. 56:297–302. 2000.
21	Fleming JB, Shen GL, Holloway SE, Davis M and Brekken RA: Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy. Mol Cancer Res. 3:413–423. 2005.PubMed/NCBI
22	Adjei AA: Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst. 93:1062–1074. 2001. View Article : Google Scholar : PubMed/NCBI
23	Roberts PJ and Der CJ: Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 26:3291–3310. 2007. View Article : Google Scholar : PubMed/NCBI
24	Friday BB and Adjei AA: Advances in targeting the Ras/Raf/MEK/Erk mitogen-activated protein kinase cascade with MEK inhibitors for cancer therapy. Clin Cancer Res. 14:342–346. 2008. View Article : Google Scholar : PubMed/NCBI
25	Diehl JA, Cheng M, Roussel MF and Sherr CJ: Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 12:3499–3511. 1998.
26	Gesbert F, Sellers WR, Signoretti S, Loda M and Griffin JD: BCR/ABL regulates expression of the cyclin-dependent kinase inhibitor p27^Kip1 through the phosphatidylinositol 3-kinase/AKT pathway. J Biol Chem. 275:39223–39230. 2000. View Article : Google Scholar : PubMed/NCBI
27	Pugazhenthi S, Nesterova A, Sable C, et al: Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem. 275:10761–10766. 2000. View Article : Google Scholar : PubMed/NCBI
28	Bernhard EJ: Pancreatic cancer inhibition by specific knockdown of K-ras mutant allele expression. Cancer Biol Ther. 6:293–294. 2007. View Article : Google Scholar : PubMed/NCBI
29	Ming L, Wang P, Bank A, Yu J and Zhang L: PUMA dissociates Bax and Bcl-X_L to induce apoptosis in colon cancer cells. J Biol Chem. 281:16034–16042. 2006.PubMed/NCBI
30	Jiang M, Wei Q, Wang J, Du Q, Yu J, Zhang L and Dong Z: Regulation of PUMA-α by p53 in cisplatin-induced renal cell apoptosis. Oncogene. 25:4056–4066. 2006.
31	Hemann MT, Zilfou JT, Zhao Z, Burgess DJ, Hannon GJ and Lowe SW: Suppression of tumorigenesis by the p53 target PUMA. Proc Natl Acad Sci USA. 101:9333–9338. 2004. View Article : Google Scholar : PubMed/NCBI
32	Villunger A, Michalak EM, Coultas L, et al: p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science. 302:1036–1038. 2003. View Article : Google Scholar : PubMed/NCBI
33	Tsuruya K, Yotsueda H, Ikeda H, et al: Involvement of p53-transactivated Puma in cisplatin-induced renal tubular cell death. Life Sci. 83:550–556. 2008. View Article : Google Scholar : PubMed/NCBI