Abstract
Hepatocellular carcinoma (HCC) is a tough opponent. HCC contributes to 14.8% of all cancer mortality in Egypt. There are many choices for management of HCC; however tumor relapse has been reported in animal and clinical studies. This study was conducted to investigate the impact of low dose γ-irradiation (LDR) and combretastatin A-4 disodium phosphate (CA-4DP) on HCC recurrence. HCC was induced in male Wistar albino rats by oral administration of N-nitrosodiethylamine (NDEA) for 17 weeks. We evaluated the expression of the endothelial cell marker (CD31) by immunostaining. Expression of Rho Associated Coiled-Coil Containing Protein Kinase 1(ROCK1) and Vascular endothelial growth factor (VEGF) expression was assessed by real-time PCR after (6, 24 and 48 h). Our results showed that expression of CD31 and gene expression of ROCK1 and VEGF was significantly repressed at all-time intervals by combination therapy ofLDR and CA-4DP as compared with untreated NDEA/HCC group and NDEA/HCC groups treated with either LDR or CA-4DP alone, (P < 0.05). Our study demonstrated the additive effect of LDR in combination with CA-4DP in suppression of HCC.
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Balogh J, Victor D, Asham EH, Burroughs SG, Boktour M, Saharia A, Li X, Ghobrial RM, Monsour HP (2016) Hepatocellular carcinoma: a review. J Hepatocell Carcinoma 3:41–53
Johnston MP, Khakoo SI (2019) Immunotherapy for hepatocellular carcinoma: current and future. World J Gastroenterol 24:2977–2989
Yapali S, Tozun N (2018) Epidemiology and viral risk factors for hepatocellular carcinoma in the Eastern Mediterranean countries. Hepatoma Res 4:24
Abdel-Wahab M, El-Ghawalby N, Mostafa M, Sultan A, El-Sadany M, Fathy O, Salah T, Ezzat F (2007) Epidemiology of hepatocellular carcinoma in lower Egypt.&nbsp;Mansoura Gastroenterology Center. Hepato-gastroenterology 73:157–162
El-Serag HB (2001) Epidemiology of hepatocellular carcinoma. Clin Liver Dis 1:87–107
Hecht SS (1997) Approaches to cancer prevention based on an understanding of N-nitrosamine carcinogenesis. Proc Soc Exp Biol Med 2:181–191
Saenger W (1984) Physical properties of nucleotides: charge densities, pK values, spectra and tautomerism. Springer, New York, p 106
Verna L, Whysner J, Williams GM (1996) N-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation. Pharmacol Ther 1–2:57–81
Park YN, Kim YB, Yang KM, Park C (2000) Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch Pathol Lab Med 124:1061–1065
Holmes DI, Zachary I (2005) The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biol 2:209
Pradeep CR, Sunila ES, Kuttan G (2005) Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in tumor angiogenesis and malignancies. Integr Cancer Ther 4:315–321
Righi L, Deaglio S, Pecchioni C, Gregorini A, Horenstein AL, Bussolati G, Sapino A, Malavasi F (2003) Role of CD31/platelet endothelial cell adhesion molecule-1 expression in in vitro and in vivo growth and differentiation of human breast cancer cells. Am J Pathol 4:1163–1174
Stacchini A, Chiarle R, Antinoro V, Demurtas A, Novero D, Palestro G (2003) Expression of the CD31 antigen in normal B-cells and non-Hodgkin’s lymphomas. J Biol Regul Homeost Agents 4:308–315
Zhang YY, Kong LQ, Zhu XD, Cai H, Wang CH, Shi WK, Cao MQ, Li XL, Li KS, Zhang SZ, Chai ZT, Ao JY, Ye BG, Sun HC (2018) CD31 regulates metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma via the ITGB1-FAK-Akt signaling pathway. Cancer Lett 429:29–40
Müller AM, Hermanns MI, Skrzynski C, Nesslinger M, Müller KM, Kirkpatrick CJ (2002) Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro. Exp Mol Pathol 3:221–229
Morgan-Fisher M, Wewer UM, Yoneda A (2013) Regulation of ROCK activity in cancer. J Histochem Cytochem 3:185–198
Chin VT, Nagrial AM, Chou A, Biankin AV, Gill AJ, Timpson P, Pajic M (2015) Rho-associated kinase signalling and the cancer microenvironment: novel biological implications and therapeutic opportunities. Expert Rev Mol Med 17:e17
Lane J, Martin TA, Watkins G, Mansel RE, Jiang WG (2008) The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer. Int J Oncol 3:585–593
Xue F, Takahara T, Yata Y, Xia Q, Nonome K, Shinno E, Kanayama M, Takahara S, Sugiyama T (2008) Blockade of Rho/Rho-associated coiled coil-forming kinase signaling can prevent progression of hepatocellular carcinoma in matrix metalloproteinase-dependent manner. Hepatol Res 8:810–817
Li J, Bharadwaj SS, Guzman G, Vishnubhotla R, Glover SC (2015) ROCK I has more accurate prognostic value than MET in predicting patient survival in colorectal cancer. Anticancer Res 6:3267–3273
Zhang P, Lu Y, Liu XY, Zhou YH (2015) Knockdown of Rho-associated protein kinase 1 suppresses proliferation and invasion of glioma cells. Tumour Biol 1:421–428
Pettit GR, Singh SB, Hamel E, Lin CM, Alberts DS, Garcia-Kendal D (1989) Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4. Experientia 2:209–211
Young SL, Chaplin DJ (2004) Combretastatin A4 phosphate: background and current clinical status. Expert Opin Investig Drugs 9:1171–1182
Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4:253–265
Chaplin D, Hill S (2002) The development of combretastatin A4 phosphate as a vascular targeting agent. Int J Radiat Oncol Biol Phys 5:1491–1496
Galbraith SM, Maxwell RJ, Lodge MA, Tozer GM, Wilson J, Taylor NJ, Stirling JJ, Sena L, Padhani AR, Rustin GJ (2003) Combretastatin A4 phosphate has tumor antivascular activity in rat and man as demonstrated by dynamic magnetic resonance imaging. J Clin Oncol 15:2831–2842
Nelkin BD, Ball DW (2001) Combretastatin A-4 and doxorubicin combination treatment is effective in a preclinical model of human medullary thyroid carcinoma. Oncol Rep 8:157–160
Murata R, Siemann DW, Overgaard J, Horsman MR (2001) Interaction between combretastatin A4 disodium phosphate and radiation in murine tumours. Radiother Oncol 60:155–161
Zhou L, Zhang X, Li H, Niu C, Yu D, Yang G, Liang X, Wen X, Li M, Cui J (2018) Validating the pivotal role of the immune system in low-dose radiation-induced tumor inhibition in Lewis lung cancer-bearing mice. Cancer Med 4:1338–1348
Ren H, Shen J, Tomiyama-Miyaji C, Watanabe M, Kainuma E, Inoue M, Kuwano Y, Abo T (2006) Augmentation of innate immunity by low-dose irradiation. Cell Immunol 1:50–56
Kim CS, Kim JK, Nam SY, Yang KH, Jeong M, Kim HS, Kim CS, Jin YW, Kim J (2007) Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt. Mol Cells 3:424–430
Lin CC, Wang TE, Liu CY, Lin CP, Liu TP, Chen MJ, Chang WH, Lin JC, Chang KM, Chu CH, Shih SC, Chao KS, Chen YJ (2008) Potentiation of the immunotherapeutic effect of autologous dendritic cells by pretreating hepatocellular carcinoma with low-dose radiation. Clin Invest Med 3:E150–E159
Wang GJ, Cai L (2000) Induction of cell-proliferation hormesis and cell-survival adaptive response in mouse hematopoietic cells by whole-body low-dose radiation. Toxicol Sci 2:369–376
Sakai K, Hoshi Y, Nomura T, Oda T, Iwasaki T, Fujita K, Yamada T, Tanooka H (2003) Suppression of carcinogenic processes in mice by chronic low dose rate gamma-irradiation. Int J Low Radiat 1:142–146
Ina Y, Sakai K (2004) Prolongation of life span associated with immunological modification by chronic low-dose-rate irradiation in MRL-lpr/lpr mice. Radiat Res 2:168–173
Hosoi Y, Sakamoto K (1993) Suppressive effect of low dose total body irradiation on lung metastasis: dose dependency and effective period. Radiother Oncol 2:177–179
Ju GZ, Liu SZ, Li XY, Liu WH, Fu HQ (1995) Effect of high versus low dose radiation on the immune system. In: Hagen U, Harder D, Jung H, Streffer C (eds) Radiation research 1895–1995. Proc of the Tenth Int Congress of Radiation Research, Würzburg
Cai L (1999) Research of the adaptive response induced by low-dose radiation: where have we been and where should we go? Hum ExpToxicol 7:419–425
Hashimoto S, Shirato H, Hosokawa M, Nishioka T, Kuramitsu Y, Matushita K, Kobayashi M, Miyasaka K (1999) The suppression of metastases and the change in host immune response after low-dose total-body irradiation in tumor-bearing rats. Radiat Res 6:717–724
Nowosielska EM, Cheda A, Wrembel-Wargocka J, Janiak MK (2008) Modulation of the growth of pulmonary tumour colonies in mice after single or fractionated low-level irradiations with X-rays. Nukleonika 1:S9–S15
Alghzzawy ZM, Elmaghraby TK, Hagag SA, Awwad MH (2019) Effect of vascular disrupting agent (CA-4DP) and ionizing radiation on tumor growth in rats. J Basic Env Sci 6:97–103
Darwish HA, El-Boghdady NA (2011) Possible involvement of oxidative stress in diethylnitrosamine induced hepatocarcinogenesis: chemopreventive effect of curcumin. J Food Biochem 37:353–361
Cheda A, Wrembel-Wargocka J, Lisiak E, Nowosielska EM, Marciniak M, Janiak MK (2004) Single low doses of X rays inhibit the development of experimental tumor metastases and trigger the activities of NK cells in mice. Radiat Res 3:335–340
Wang H, Sun X, Chen F, De Keyzer F, Yu J, Landuyt W, Vandecaveye V, Peeters R, Bosmans H, Hermans R, Marchal G, Ni Y (2009) Treatment of rodent liver tumor with combretastatin a4 phosphate: noninvasive therapeutic evaluation using multiparametric magnetic resonance imaging in correlation with microangiography and histology. Invest Radiol 1:44–53
Banchroft JD, Stevens A, Turner DR (1996) Theory and practice of histological techniques, 4th edn. Churchill Livingstone, New York
Wang D, Stockard CR, Harkins L, Lott P, Salih C, Yuan K, Buchsbaum D, Hashim A, Zayzafoon M, Hardy RW, Hameed O, Grizzle W, Siegal GP (2008) Immunohistochemistry in the evaluation of neovascularization in tumor xenografts. Biotech Histochem 3–4:179–189
Chomczynski PA (1993) Reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 3:532–534
Chen J, Li Q, Dong R, Gao H, Peng H, Wu Y (2014) The effect of the Ras homolog gene family (Rho), member A/Rho associated coiled-coil forming protein kinase pathway in atrial fibrosis of type 2 diabetes in rats. Exp Ther Med 3:836–840
Gómez R, Simón C, Remohí J, Pellicer A (2002) Vascular endothelial growth factor receptor-2 activation induces vascular permeability in hyperstimulated rats, and this effect is prevented by receptor blockade. Endocrinology 11:4339–4348
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative pcr and the 2–∆∆ct method. Methods 25:402–408
Abe H, Kamai T, Tsujii T, Nakamura F, Mashidori T, Mizuno T, Tanaka M, Tatsumiya K, Furuya N, Masuda A, Yamanishi T, Yoshida K (2008) Possible role of the RhoC/ ROCK pathway in progression of clear cell renal cell carcinoma. Biomed Res 3:155–161
Gilkes DM, Xiang L, Lee SJ, Chaturvedi P, Hubbi ME, Wirtz D, Semenza GL (2014) Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells. Proc Natl Acad Sci USA 3:E384–E393
Benitah SA, Valerón PF, van Aelst L, Marshall CJ, Lacal JC (2004) Rho GTPases in human cancer: an unresolved link to upstream and downstream transcriptional regulation. Biochim Biophys Acta 1705:121–132
Ellenbroek SI, Collard JG (2007) Rho GTPases: functions and association with cancer. Clin Exp Metastasis 8:657–672
Ma W, Wong CC, Tung EK, Wong CM, Ng IO (2013) RhoE is frequently down-regulated in hepatocellular carcinoma (HCC) and suppresses HCC invasion through antagonizing the Rho/Rho-kinase/myosin phosphatase target pathway. Hepatology 1:152–161
Liu H, Li W, Chen C, Pei Y, Long X (2015) MiR-335 acts as a potential tumor suppressor miRNA via downregulating ROCK1 expression in hepatocellular carcinoma. Tumour Biol 8:6313–6319
Ding W, Tan H, Zhao C, Li X, Li Z, Jiang C, Zhang Y, Wang L (2016) MiR-145 suppresses cell proliferation and motility by inhibiting ROCK1 in hepatocellular carcinoma. Tumour Biol 5:6255–6260
Pajonk F, McBride WH (2001) Ionizing radiation affects 26 s proteasome function and associated molecular responses, even at low doses. Radiother Oncol 2:203–212
Chen W, Xu X, Bai L, Padilla MT, Gott KM, Leng S, Tellez CS, Wilder JA, Belinsky SA, Scott BR, Lin Y (2012) Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen. Carcinogenesis 7:1368–1374
Slinger E, Maussang D, Schreiber A, Siderius M, Rahbar A, Fraile-Ramos A, Lira SA, Söderberg-Nauclér C, Smit MJ (2010) HCMV-encoded chemokine receptor US28 mediates proliferative signaling through the IL-6-STAT3 axis. Sci Signal 133:ra58
Sanz-Moreno V, Gaggioli C, Yeo M, Albrengues J, Wallberg F, Viros A, Hooper S, Mitter R, Féral CC, Cook M, Larkin J, Marais R, Meneguzzi G, Sahai E, Marshall CJ (2011) ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma. Cancer Cell 2:229–245
Wang Y, van Boxel-Dezaire AH, Cheon H, Yang J, Stark GR (2013) STAT3 activation in response to IL-6 is prolonged by the binding of IL-6 receptor to EGF receptor. Proc Natl Acad Sci USA 42:16975–16980
Wilson GS, Tian A, Hebbard L, Duan W, George J, Li X, Qiao L (2013) Tumoricidal effects of the JAK inhibitor Ruxolitinib (INC424) on hepatocellular carcinoma in vitro. Cancer Lett 2:224–230
Mohan CD, Bharathkumar H, Bulusu KC, Pandey V, Rangappa S, Fuchs JE, Shanmugam MK, Dai X, Li F, Deivasigamani A, Hui KM, Kumar AP, Lobie PE, Bender A, Basappa, Sethi G, Rangappa KS (2014) Development of a novel azaspirane that targets the Janus kinase-signal transducer and activator of transcription (STAT) pathway in hepatocellular carcinoma in vitro and in vivo. J Biol Chem 49:34296–34307
Poon RT, Lau CP, Cheung ST, Yu WC, Fan ST (2003) Quantitative correlation of serum levels and tumor expression of vascular endothelial growth factor in patients with hepatocellular carcinoma. Cancer Res 12:3121–3126
Choi SB, Han HJ, Kim WB, Song TJ, Choi SY (2017) VEGF overexpression predicts poor survival in hepatocellular carcinoma. Open Med 12:430–439
Torimura T, Ueno T, Kin M, Harada R, Taniguchi E, Nakamura T, Sakata R, Hashimoto O, Sakamoto M, Kumashiro R, Sata M, Nakashima O, Yano H, Kojiro M (2004) Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma. J Hepatol 5:799–807
Yasuda S, Arii S, Mori A, Isobe N, Yang W, Oe H, Fujimoto A, Yonenaga Y, Sakashita H, Imamura M (2004) Hexokinase II and VEGF expression in liver tumors: correlation with hypoxia-inducible factor 1 alpha and its significance. J Hepatol 1:117–123
Lavarone M, Lampertico P, Iannuzzi F, Manenti E, Donato MF, Arosio E, Bertolini F, Primignani M, Sangiovanni A, Colombo M (2007) Increased expression of vascular endothelial growth factor in small hepatocellular carcinoma. J Viral Hepat 14:133–139
Su M, Huang J, Liu S, Xiao Y, Qin X, Liu J, Pi C, Luo T, Li J, Chen X, Luo Z (2016) The anti-angiogenic effect and novel mechanisms of action of Combretastatin A-4. Sci Rep 6:28139
Zhang J, Lu A, Beech D, Jiang B, Lu Y (2007) Suppression of breast cancer metastasis through the inhibition of VEGF-mediated tumor angiogenesis. Cancer Ther 5:273–286
Wang S, Liu H, Ren L, Pan Y, Zhang Y (2008) Inhibiting colorectal carcinoma growth and metastasis by blocking the expression of VEGF using RNA interference. Neoplasia 4:399–409
Zou Y, Guo CG, Zhang MM (2015) Inhibition of human hepatocellular carcinoma tumor angiogenesis by siRNA silencing of VEGF via hepatic artery perfusion. Eur Rev Med Pharmacol Sci 24:4751–4761
Bancroft CC, Chen Z, Dong G, Sunwoo JB, Yeh N, Park C, Van Waes C (2001) Coexpression of proangiogenic factors IL-8 and VEGF by human head and neck squamous cell carcinoma involves coactivation by MEK-MAPK and IKK-NF- κB signal pathways. Clin Cancer Res 7:435–442
Huang S, Robinson JB, Deguzman A, Bucana CD, Fidler IJ (2000) Blockade of nuclear factor-kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin-8. Cancer Res 1:5334–5339
Huang S, Pettaway CA, Uehara H, Bucana CD, Fidler IJ (2001) Blockade of NF-kappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene 31:4188–4197
Zhao L, Xu G, Zhou J, Xing H, Wang S, Wu M, Lu YP, Ma D (2006) The effect of RhoA on human umbilical vein endothelial cell migration and angiogenesis in vitro. Oncol Rep 5:1147–1152
Strilić B, Kucera T, Eglinger J, Hughes MR, McNagny KM, Tsukita S, Dejana E, Ferrara N, Lammert E (2009) The molecular basis of vascular lumen formation in the developing mouse aorta. Dev Cell 4:505–515
van der Meel R, Symons MH, Kudernatsch R, Kok RJ, Schiffelers RM, Storm G, Gallagher WM, Byrne AT (2011) The VEGF/Rho GTPasesignalling pathway: a promising target for anti-angiogenic/anti-invasion therapy. Drug Discov Today 5–6:219–228
Dangerfield J, Larbi KY, Huang MT, Dewar A, Nourshargh S (2002) PECAM-1 (CD31) homophilic interaction up-regulates alpha6beta1 on transmigrated neutrophils in vivo and plays a functional role in the ability of alpha6 integrins to mediate leukocyte migration through the perivascular basement membrane. J Exp Med 9:1201–1211
Charpin C, Dales JP, Garcia S, Carpentier S, Djemli A, Andrac L, Lavaut MN, Allasia C, Bonnier P (2004) Tumor neoangiogenesis by CD31 and CD105 expression evaluation in breast carcinoma tissue microarrays. Clin Cancer Res 10(17):5815–5819
Fitzgibbons PL, Dillon DA, Alsabeh R, Berman MA, Hayes DF, Hicks DG, Hughes KS, Nofech-Mozes S (2014) Template for reporting results of biomarker testing of specimens from patients with carcinoma of the breast. Arch Pathol Lab Med 5:595–601
Obuchowski NA (2004) How many observers are needed in clinical studies of medical imaging? AJR Am J Roentgenol 4:867–869
Wang JY, Xu XY, Jia JH, Wu CH, Ge RW (2010) Expressions of SE-1, CD31 and CD105 in the vascular endothelial cells and serum of rat with hepatocellular carcinoma. Chin Med J 6:730–733
Srivastava S, Wong KF, Ong CW, Huak CY, Yeoh KG, Teh M, Luk JM, Salto-Tellez M (2012) A morpho-molecular prognostic model for hepatocellular carcinoma. Br J Cancer 2:334–339
Sapino A, Bongiovanni M, Cassoni P, Righi L, Arisio R, Deaglio S, Malavasi F (2001) Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast. J Pathol 2:254–261
Croft DR, Sahai E, Mavria G, Li S, Tsai J, Lee WM, Marshall CJ, Olson MF (2004) Conditional ROCK activation in vivo induces tumor cell dissemination and angiogenesis. Cancer Res 64:8994–9001
Acknowledgements
The authors gratefully acknowledge Dr Adel M. Bakeer Professor of Pathology, Faculty of Veterinary Medicine, Cairo University, and Dr Osama M. Mostafa Lecturer of Pathology, Faculty of Medicine, Al-Azhar University, Egypt for their input in this work.
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Alghzzawy, Z.M., Elmaghraby, T.K., El-Hamid Hagag, S.A. et al. Combretastatin A-4 disodium phosphate and low dose gamma irradiation suppress hepatocellular carcinoma by downregulating ROCK1 and VEGF gene expression. Mol Biol Rep 47, 1883–1893 (2020). https://doi.org/10.1007/s11033-020-05282-0
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DOI: https://doi.org/10.1007/s11033-020-05282-0