Sains Malaysiana 46(8)(2017): 1289–1297

http://dx.doi.org/10.17576/jsm-2017-4608-15

 

A Gain of Function p53 Gene Mutant Promotes Growth Suppression in Human Liver Cancer Cells

(Pemulihan Fungsi Mutan Gen p53 Menggalakkan Penindasan Pertumbuhan Sel Kanser Hati Manusia)

 

NOR ADZIMAH JOHDI*, SITI NURMI NASIR & RAHMAN JAMAL

 

UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

Received: 18 August 2016/Accepted: 11 January 2017

 

ABSTRACT

Primary liver cancer is one of the most common cancer in the world with highest cancer mortality rate. The most common type of primary liver cancer is hepatocellular carcinoma (HCC). There are many risk factors for liver cancer and currently available treatments for HCC are largely inadequate. Gene mutation and dysfunction of p53 are common and is recognized as an important molecular event in hepatocarcinogenesis. Therefore, replacement of the aberrant p53 gene is an attractive approach in the treatment of HCC providing an alternative treatment for primary HCC. In this study, we assessed whether the transfection with wild-type p53 gene is able to restore the pro-apoptotic effects and evaluate the feasibility of gene therapy in fixing a faulty p53 molecule. We established a non-viral cationic lipid-based p53 gene delivery into two human HCC cell lines namely HLF and PLC/PRF/5 cells. Both cell lines have mutations in the p53 gene. We compared the results with the normal liver cell line, WRL68, that constitutively expresses the wild-type p53 gene. In this study, the introduction of wild-type p53 gene into HLF and PLC/PRF/5 cells resulted in an increased of p53 gene expression, protein expression and cells growth inhibition shown in MTS reduction cell viability assay, FITC-Annexin V and PI apoptosis assay, western blot and caspase activity assay. In summary, the study provides a promising therapeutic approach for p53 gene delivery into HCC patients. The p53 gene delivery can be instituted together with chemotherapy as a combination treatment to induce apoptosis.

 

Keywords: Apoptosis; cell lines; hepatocellular carcinoma; p53 gene; transfection

 

ABSTRAK

Kanser hati utama adalah salah satu kanser umum di dunia dengan kadar kematian kanser tertinggi. Jenis yang paling biasa untuk kanser hati utama adalah karsinoma hepatoselular (HCC). Terdapat banyak faktor risiko untuk kanser hati utama dan rawatan untuk HCC sebahagian besarnya tidak mencukupi. Mutasi gen dan kehilangan fungsi gen p53 adalah faktor yang paling biasa dan gen p53 diiktiraf sebagai antara rantaian molekul yang penting dalam proses pembentukan kanser hati. Oleh itu, pemulihan mutan gen p53 yang defektif adalah satu pendekatan yang menarik dalam rawatan HCC sebagai rawatan alternatif untuk HCC utama. Dalam kajian ini, penilaian dilakukan sama ada transfeksi gen p53 normal ke atas titisan sel HCC mampu mengembalikan kesan pro-apoptotik dan potensi terapi gen dalam pemulihan molekul p53 yang defektif. Ini melibatkan transfeksi gen p53 normal menggunakan teknik lipid kationik kepada dua jenis titisan sel HCC manusia iaitu HLF dan PLC/PRF/5. Kedua-dua jenis titisan sel ini mempunyai mutasi dalam gen p53. Keputusan kajian dibandingkan dengan titisan sel hati normal, WRL68, yang mengekspres gen p53 normal. Transfeksi gen p53 normal ke dalam titisan sel HLF dan PLC/PRF/5 menyebabkan peningkatan ekspresi gen p53, pertambahan ekspresi protein p53 dan perencatan pertumbuhan titisan sel-sel yang ditunjukkan dalam assai MTS sel pengurangan daya maju, assai apoptosis FITC-Annexin V & PI, pemendapan barat dan aktiviti caspase. Secara ringkasnya, kajian ini menyediakan pendekatan terapeutik yang boleh digunakan untuk menyalurkan gen p53 ke dalam sel pesakit HCC. Penyampaian gen p53 boleh dimulakan bersama dengan kemoterapi sebagai rawatan kombinasi untuk mendorong aktiviti apoptosis.

 

Kata kunci: Apoptosis; gen p53; hepatoselular karsinoma; titisan sel; transfeksi

REFERENCES

Algaba, F., Trias, I., Santinelli, A. & Montironi, R. 2003. TP53 in urologic tumors. Anal. Quant. Cytol. Histol.25(3): 123-130.

Cheng, A.L., Kang, Y.K., Chen, Z., Tsao, C.J., Qin, S., Kim, J.S., Luo, R., Feng, J., Ye, S., Yang, T.S., Xu, J., Sun, Y., Liang, H., Liu, J., Wang, J., Tak, W.Y., Pan, H., Burock, K., Zou, J., Voliotis, D. & Guan, Z. 2009. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: A phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 10(1): 25-34. doi: 10.1016/S1470-2045(08)70285-7.

Chuang, S.C., La Vecchia, C. & Boffetta, P. 2009. Liver cancer: Descriptive epidemiology and risk factors other than HBV and HCV infection. Cancer Lett. 286(1): 9-14. doi: 10.1016/j. canlet.2008.10.040.

de Moura Gallo, C.V., Azevedo E Silva Mendonça, G., de Moraes, E., Olivier, M. & Hainaut, P. 2005. TP53 mutations as biomarkers for cancer epidemiology in Latin America: Current knowledge and perspectives. Mutat Res. 589(3): 192-207. doi: 10.1016/j.mrrev.2005.01.002.

Ferraz da Costa, D.C., Casanova, F.A., Quarti, J., Malheiros, M.S., Sanches, D., Dos Santos, P.S., Fialho, E. & Silva, J.L. 2012. Transient transfection of a wild-type p53 gene triggers resveratrol-induced apoptosis in cancer cells. PLoS One 7(11): e48746. doi: 10.1371/journal.pone.0048746.

Habib, N., Salama, H., Abd El Latif Abu Median, A., Isac Anis, I., Abd Al Aziz, R.A., Sarraf, C., Mitry, R., Havlik, R., Seth, P., Hartwigsen, J., Bhushan, R., Nicholls, J. & Jensen, S. 2002. Clinical trial of E1B-deleted adenovirus (dl1520) gene therapy for hepatocellular carcinoma. Cancer Gene Ther. 9(3): 254-259. doi: 10.1038/sj.cgt.7700431.

He, G., Siddik, Z.H., Huang, Z., Wang, R., Koomen, J., Kobayashi, R., Khokhar, A.R. & Kuang, J. 2005. Induction of p21 by p53 following DNA damage inhibits both Cdk4 and  Cdk2 activities. Oncogene 24(18): 2929-2943. doi: 10.1038/ sj.onc.1208474.

Hsu, I.C., Tokiwa, T., Bennett, W., Metcalf, R.A., Welsh, J.A., Sun, T. & Harris, C.C. 1993. p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines. Carcinogenesis 14(5): 987-992.

Li, L.Y., Dai, H.Y., Yeh, F.L., Kan, S.F., Lang, J., Hsu, J.L., Jeng, L.B., Chen, Y.H., Sher, Y.P., Lin, W.C. & Hung, M.C. 2011. Targeted hepatocellular carcinoma proapoptotic BikDD gene therapy. Oncogene 30(15): 1773-1783. doi: 10.1038/ onc.2010.558.

Llovet, J.M., Ricci, S., Mazzaferro, V., Hilgard, P., Gane, E., Blanc, J.F., de Oliveira, A.C., Santoro, A., Raoul, J.L., Forner, A., Schwartz, M., Porta, C., Zeuzem, S., Bolondi, L., Greten, T.F., Galle, P.R., Seitz, J.F., Borbath, I., Häussinger, D., Giannaris, T., Shan, M., Moscovici, M., Voliotis, D., Bruix, J. & SHARP Investigators Study Group. 2008. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359(4): 378-390. doi: 10.1056/NEJMoa0708857.

Lu, Q., Teng, G.J., Zhang, Y., Niu, H.Z., Zhu, G.Y., An, Y.L., Yu, H., Li, G.Z., Qiu, D.H. & Wu, C.G. 2008. Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery. Cancer Biol. Ther. 7(2): 218-224.

Ministry of Health, Malaysia. 2011. National Cancer Registry Report, Malaysia Cancer Statistics-Data and Figure.

Morgan, T.R., Mandayam, S. & Jamal, M.M. 2004. Alcohol and hepatocellular carcinoma. Gastroenterology 127(5 Suppl 1): S87-S96.

Naccarati, A., Polakova, V., Pardini, B., Vodickova, L., Hemminki, K., Kumar, R. & Vodicka, P. 2012. Mutations and polymorphisms in TP53 gene--an overview on the role in colorectal cancer. Mutagenesis 27(2): 211-218. doi: 10.1093/ mutage/ger067.

Ozdemir, F.T., Tiftikci, A., Sancak, S., Eren, F., Tahan, V., Akın, H., Gündüz, F., Kedrah, A.E., Ustündağ, Y., Avşar, E., Tözün, N. & Ozdoğan, O. 2010. The prevalence of the mutation in codon 249 of the P53 gene in patients with hepatocellular carcinoma (HCC) in Turkey. J. Gastrointest. Cancer 41(3): 185-189. doi: 10.1007/s12029-010-9140-5.

Park, J.W., Finn, R.S., Kim, J.S., Karwal, M., Li, R.K., Ismail, F., Thomas, M., Harris, R., Baudelet, C., Walters, I. & Raoul, J.L. 2011. Phase II, open-label study of brivanib as first-line therapy in patients with advanced hepatocellular carcinoma. Clin. Cancer Res. 17(7): 1973-1983. doi: 10.1158/1078-0432. CCR-10-2011.

Parkin, D.M. 2006. The global health burden of infection-associated cancers in the year 2002. Int. J. Cancer 118(12): 3030-3044. doi: 10.1002/ijc.21731.

Ravizza, R., Gariboldi, M.B., Passarelli, L. & Monti, E. 2004. Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin. BMC Cancer 4: 92. doi: 10.1186/1471-2407-4-92.

Rejeeth, C. & Kannan, S. 2014. p53 gene therapy of human breast carcinoma: Using a transferrin-modified silica nanoparticles. Breast Cancer. doi: 10.1007/s12282-014-0537-z.

Rivlin, N., Brosh, R., Oren, M. & Rotter, V. 2011. Mutations in the p53 tumor suppressor gene: Important milestones at the various steps of tumorigenesis. Genes Cancer 2(4): 466-474. doi: 10.1177/1947601911408889.

Sakakima, Y., Hayashi, S., Yagi, Y., Hayakawa, A., Tachibana, K. & Nakao, A. 2005. Gene therapy for hepatocellular carcinoma using sonoporation enhanced by contrast agents. Cancer Gene Ther. 12(11): 884-889. doi: 10.1038/sj.cgt.7700850.

Senzer, N., Nemunaitis, J., Nemunaitis, D., Bedell, C., Edelman, G., Barve, M., Nunan, R., Pirollo, K.F., Rait, A. & Chang, E.H. 2013. Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Mol. Ther. 21(5): 1096-1103. doi: 10.1038/mt.2013.32.

Shariff, M.I., Cox, I.J., Gomaa, A.I., Khan, S.A., Gedroyc, W. & Taylor-Robinson, S.D. 2009. Hepatocellular carcinoma: Current trends in worldwide epidemiology, risk factors, diagnosis and therapeutics. Expert Rev. Gastroenterol. Hepatol.3(4): 353-367. doi: 10.1586/egh.09.35.

Shu, K.X., Li, B., Liang, Y.L., Xie, Y.F., Zhang, J.C. & Wei, J.M. 2006. Effects of exogenous p53 transfection on the gene expression in the human brain glioma cell line U251. Colloids Surf. B Biointerfaces 47(2): 126-131. doi: 10.1016/j. colsurfb.2005.12.005.

Stewart, C.P. & Wild, B.W. 2014. World Cancer Report.

Wang, W., Yao, J., Zhou, J.P., Lu, Y., Wang, Y., Tao, L. & Li, Y.P. 2008. Urocanic acid-modified chitosan-mediated p53 gene delivery inducing apoptosis of human hepatocellular carcinoma cell line HepG2 is involved in its antitumor effect in vitro and in vivo. Biochem. Biophys. Res. Commun. 377(2): 567-572. doi: 10.1016/j.bbrc.2008.10.023.

Yang, Z.X., Wang, D., Wang, G., Zhang, Q.H., Liu, J.M., Peng, P. & Liu, X.H. 2010. Clinical study of recombinant adenovirus-p53 combined with fractionated stereotactic radiotherapy for hepatocellular carcinoma. J. Cancer Res. Clin. Oncol.136(4): 625-630. doi: 10.1007/s00432-009- 0701-6.

 

 

*Corresponding author; email: adzimah@ppukm.ukm.edu.my