Effect of sorafenib on des‑γ‑carboxyprothrombin secretion by a human hepatocellular carcinoma cell line

Ogasawara,Sachiko; Nakayama,Masamichi; Akiba,Jun; Kusano,Hironori; Yano,Hirohisa

doi:10.3892/ol.2017.6451

Effect of sorafenib on des‑γ‑carboxyprothrombin secretion by a human hepatocellular carcinoma cell line

Authors:
- Sachiko Ogasawara
- Masamichi Nakayama
- Jun Akiba
- Hironori Kusano
- Hirohisa Yano
View Affiliations

Affiliations: Department of Pathology, Kurume University School of Medicine, Kurume, Fukuoka 830‑0011, Japan, Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Fukuoka 830‑0011, Japan
Published online on: June 21, 2017 https://doi.org/10.3892/ol.2017.6451
Pages: 2170-2176
Copyright: © Ogasawara et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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

Patients with hepatocellular carcinoma (HCC) who respond to sorafenib have been reported to exhibit an increase in the level of des‑γ‑carboxyprothrombin (DCP) in the blood, subsequent to the initiation of sorafenib treatment. In the present study, the levels of secretion of DCP and DCP with more γ‑carboxyglutamic residues (NX‑DCP) and the effects of hypoxic conditions were examined in 13 liver cancer cell lines, and the presence of vitamin K and sorafenib, in the KYN‑2 cell line, which resulted in confirmed DCP and NX‑DCP secretion. DCP, NX‑DCP and prothrombin secretion were confirmed in 2/13 cell lines, KYN‑2 and KIM‑1. The level of secretions increased under hypoxic conditions. The addition of vitamin K suppressed cell proliferation, and DCP expression decreased to below detectable levels, however the level of prothrombin expression increased. Sorafenib treatment increased the level of apoptosis and suppressed cell proliferation, and decreased DCP and NX‑DCP. In contrast, levels of prothrombin and vascular endothelial growth factor (VEGF) expression exhibited a slight increase. When the same experiment was conducted under hypoxic conditions, DCP secretion significantly decreased in the presence of sorafenib. The level of DCP secretion increased by several fold in the sorafenib‑treated and non‑treated cells compared with the normoxic conditions. Prothrombin and VEGF values with normoxic conditions remained almost similar with hypoxic conditions. Under hypoxic conditions, NX‑DCP significantly decreased below the control values for the first 48 h subsequent to sorafenib treatment, but significantly increased at 72 h. In vivo experiments demonstrated that sorafenib inhibited angiogenesis and tumor proliferation, but the levels of DCP and NX‑DCP did not differ significantly from the controls. These findings indicate that the suppression of neovascularization by sorafenib promotes blood vessel ischemia, producing hypoxic conditions whereby vitamin K uptake and utilization efficiency is reduced.

View Figures

View References

1	Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, Wilhelm S, Lynch M and Carter C: Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 66:11851–11858. 2006. View Article : Google Scholar : PubMed/NCBI
2	Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, et al: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 359:378–390. 2008. View Article : Google Scholar : PubMed/NCBI
3	Escudier B, Eisen T, Stanler WM, Szczylik C, Oudard S, Staehler M, Negrier S, Chevreau C, Desai AA, Rolland F, et al: Sorafenib for treatment of renal cell carcinoma: Final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. J Clin Oncol. 27:3312–3318. 2009. View Article : Google Scholar : PubMed/NCBI
4	Carlomagno F, Anaganti S, Guida T, Salvatore G, Troncone G, Wilhelm SM and Santoro M: BAY 43-9006 inhibition of oncogenic RET mutants. J Natl Cancer Inst. 98:326–334. 2006. View Article : Google Scholar : PubMed/NCBI
5	Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, et al: BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 64:7099–7109. 2004. View Article : Google Scholar : PubMed/NCBI
6	Liebman HA, Furie BC, Tong MJ, Blanchard RA, Lo KJ, Lee SD, Coleman MS and Furie B: Des-gamma-carboxy (abnormal) prothrombin as a serum marker of primary hepatocellular carcinoma. N Engl J Med. 310:1427–1431. 1984. View Article : Google Scholar : PubMed/NCBI
7	Sakon M, Monden M, Gotoh M, Kanai T, Umeshita K, Nakano Y, Mori T, Sakurai M and Wakasa K: Relationship between pathologic prognostic factors and abnormal levels of des-gamma-carboxy prothrombin and alpha-fetoprotein in hepatocellular carcinoma. Am J Surg. 163:251–256. 1992. View Article : Google Scholar : PubMed/NCBI
8	Suehiro T, Sugimachi K, Matsumata T, Itasaka H, Taketomi A and Maeda T: Protein induced by vitamin K absence or antagonist II as a prognostic marker in hepatocellular carcinoma. Comparison with alpha-fetoprotein. Cancer. 73:2464–2471. 1994. View Article : Google Scholar : PubMed/NCBI
9	Sakamoto N: NX-PVKA assay, a conventional but refined prognostic biomarker for hepatocellular carcinoma. J Gastroenterol Hepatol. 28:755–756. 2013. View Article : Google Scholar : PubMed/NCBI
10	Sumi A, Akiba J, Ogasawara S, Nakayama M, Nomura Y, Yasumoto M, Sanada S, Nakashima O, Abe T and Yano H: Des-γ-carboxyprothrombin (DCP) and NX-DCP expressions and their relationship with clinicopathological features in hepatocellular carcinoma. PLoS One. 10:e01184522015. View Article : Google Scholar : PubMed/NCBI
11	Kim BK, Ahn SH, Seong JS, Park JY, Kim DY, Kim JK, Lee DY, Lee KH and Han KH: Early α-fetoprotein response as a predictor for clinical outcome after localized concurrent chemoradiotherapy for advanced hepatocellular carcinoma. Liver Int. 31:369–376. 2011. View Article : Google Scholar : PubMed/NCBI
12	Johnson PJ: The role of serum alpha-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma. Clin Liver Dis. 5:145–159. 2001. View Article : Google Scholar : PubMed/NCBI
13	Zhou L, Liu J and Luo F: Serum tumor markers for detection of hepatocellular carcinoma. World J Gastroenterol. 12:1175–1181. 2006. View Article : Google Scholar : PubMed/NCBI
14	Malaguarnera G, Giordano M, Paladina I, Berretta M, Cappellani A and Malaguarnera M: Serum markers of hepatocellular carcinoma. Dig Dis Sci. 55:2744–2755. 2010. View Article : Google Scholar : PubMed/NCBI
15	Kuzuya T, Asahina Y, Tsuchiya K, Tanaka K, Suzuki Y, Hoshioka T, Tamaki S, Kato T, Yasui Y, Hosokawa T, et al: Early decrease in α-fetoprotein, but not des-γ-carboxy prothrombin, predicts sorafenib efficacy in patients with advanced hepatocellular carcinoma. Oncology. 81:251–258. 2011. View Article : Google Scholar : PubMed/NCBI
16	Nakazawa T, Hidaka H, Shibuya A and Koizumi W: Rapid regression of advanced hepatocellular carcinoma associated with elevation of des-gamma-carboxy prothrombin after short-term treatment with sorafenib-a report of two cases. Case Rep Oncol. 3:298–303. 2010. View Article : Google Scholar : PubMed/NCBI
17	Ueshima K, Kudo M, Takita M, Nagai T, Tatsumi C, Ueda T, Kita S, Ishikawa E, Yada N, Inoue T, et al: Des-γ-carboxyprothrombin may be a promising biomarker to determine the therapeutic efficacy of sorafenib for hepatocellular carcinoma. Dig Dis. 29:321–325. 2011. View Article : Google Scholar : PubMed/NCBI
18	Murakami T: Establishment and characterization of human hepatoma cell line (KIM-1). Acta Hepatol Jpn. 25:532–539. 1984. View Article : Google Scholar
19	Yano H, Kojiro M and Nakashima T: A new human hepatocellular carcinoma cell line (KYN-1) with a transformation to adenocarcinoma. In Vitro Cell Dev Biol. 22:637–646. 1986. View Article : Google Scholar : PubMed/NCBI
20	Yano H, Maruiwa M, Murakami T, Fukuda K, Ito Y, Sugihara S and Kojiro M: A new human pleomorphic hepatocellular carcinoma cell line, KYN-2. Acta Pathol Jpn. 38:953–966. 1988.PubMed/NCBI
21	Murakami T, Maruiwa M, Fukuda K, Kojiro M, Tanaka M and Tanikawa K: Establishment and characterization of a new human hepatoma cell line (KYN-3) derived from the ascites of the hepatoma paitient. Proceedings of the Japanese Cancer Association. Jpn J Cancer Res. 292:1988;
22	Yano H, Iemura A, Fukuda K, Mizoguchi A, Haramaki M and Kojiro M: Establishment of two distinct human hepatocellular carcinoma cell lines from a single nodule showing clonal dedifferentiation of cancer cells. Hepatology. 18:320–327. 1993. View Article : Google Scholar : PubMed/NCBI
23	Haramaki M, Yano H, Iemura A, Momosaki S, Ogasawara S, Inoue M, Yamaguchi R, Kusaba A, Utsunomiya I and Kojiro M: A new human hepatocellular carcinoma cell line (HAK-2) forms various structures in collagen gel matrices. Hum Cell. 10:183–192. 1997.PubMed/NCBI
24	Utsunomiya I, Iemura A, Yano H, Akiba J and Kojiro M: Establishment and characterization of a new human hepatocellular carcinoma cell line, HAK-3, and its response to growth factors. Int J Oncol. 15:669–675. 1999.PubMed/NCBI
25	Murakami T, Yano H, Maruiwa M, Sugihara S and Kojiro M: Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice. Hepatology. 7:551–556. 1987. View Article : Google Scholar : PubMed/NCBI
26	Yano H, Iemura A, Haramaki M, Momosaki S, Ogasawara S, Higaki K and Kojiro M: A human combined hepatocellular and cholangiocarcinoma cell line (KMCH-2) that shows the features of hepatocellular carcinoma or cholangiocarcinoma under different growth conditions. J Hepatol. 24:413–422. 1996. View Article : Google Scholar : PubMed/NCBI
27	Shah DV, Engelke JA and Suttie JW: Abnormal prothrombin in the plasma of rats carrying hepatic tumors. Blood. 69:850–854. 1987.PubMed/NCBI
28	Shah DV, Zhang P, Engelke JA, Bach AU and Suttie JW: Vitamin K-dependent carboxylase activity, prothrombin mRNA, and prothrombin production in two cultured rat hepatoma cell lines. Thromb Res. 70:365–373. 1993. View Article : Google Scholar : PubMed/NCBI
29	Ono M, Ohta H, Ohhira M, Sekiya C and Namiki M: Measurement of immunoreactive prothrombin, des-gamma-carboxy prothrombin, and vitamin K in human liver tissues: Overproduction of immunoreactive prothrombin in hepatocellular carcinoma. Am J Gastroenterol. 85:1149–1154. 1990.PubMed/NCBI
30	Yamagata H, Nakanishi T, Furukawa M, Okuda H and Obata H: Levels of vitamin K, immunoreactive prothrombin, des-gamma-carboxy prothrombin and gamma-glutamyl carboxylase activity in hepatocellular carcinoma tissue. J Gastroenterol Hepatol. 10:8–13. 1995. View Article : Google Scholar : PubMed/NCBI
31	Wang Z, Wang M, Finn F and Carr BI: The growth inhibitory effects of vitamins K and their actions on gene expression. Hepatology. 22:876–882. 1995. View Article : Google Scholar : PubMed/NCBI
32	Okuda H, Obata H, Nakanishi T, Furukawa R and Hashimoto E: Production of abnormal prothrombin (des-gamma-carboxy prothrombin) by hepatocellular carcinoma. A clinical and experimental study. J Hepatol. 4:357–363. 1987. View Article : Google Scholar : PubMed/NCBI
33	Sakon M, Monden M, Gotoh M, Kobayashi K, Kanai T, Umeshita K, Endoh W and Mori T: The effects of vitamin K on the generation of des-gamma-carboxy prothrombin (PIVKA-II) in patients with hepatocellular carcinoma. Am J Gastroenterol. 86:339–345. 1991.PubMed/NCBI
34	Kuzuya T, Tsuchiya K, Tanaka K, Suzuki Y, Hoshioka T, Tamaki S, Kato T, Yasui Y, Tanaka T, Hosokawa T, et al: Significance of PIVKA-II in sorafenib therapy for advanced hepatocellular carcinoma. Kanzo. 51:403–404. 2010. View Article : Google Scholar
35	Ueshima K and Kudo M: PIVKA-II is a predictive marker in the treatment response of sorafenib to hepatocellular carcinoma. Kanzo. 51:681–683. 2010. View Article : Google Scholar
36	Llobet D, Eritja N, Yeramian A, Pallares J, Sorolla A, Domingo M, Santacana M, Gonzalez-Tallada FJ, Matias-Guiu X and Dolcet X: The multikinase inhibitor Sorafenib induces apoptosis and sensitises endometrial cancer cells to TRAIL by different mechanisms. Eur J Cancer. 46:836–850. 2010. View Article : Google Scholar : PubMed/NCBI
37	Fernando J, Sancho P, Fernández-Rodriguez CM, Lledó JL, Caja L, Campbell JS, Fausto N and Fabregat I: Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli. J Cell Physio. 227:1319–1325. 2012. View Article : Google Scholar
38	Suzuki M, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Nakanishi Y, Koike K, Takaki A and Shiratori Y: Des-gamma-carboxy prothrombin is a potential autologous growth factor for hepatocellular carcinoma. J Biol Chem. 280:6409–6415. 2005. View Article : Google Scholar : PubMed/NCBI
39	Gao CF and Woude GF Vande: HGF/SF-Met signaling in tumor progression. Cell Res. 15:49–51. 2005. View Article : Google Scholar : PubMed/NCBI
40	Murata K, Suzuki H, Okano H, Oyamada T, Yasuda Y and Sakamoto A: Hypoxia-induced des-gamma-carboxy prothrombin production in hepatocellular carcinoma. Int J Oncol. 36:161–170. 2010.PubMed/NCBI