Fig. 1. Western analysis and confocal microscopy detection of AQP8 and

Mrp2: effect of E217G administration. Animals were treated with E217G

and liver samples examined 20 min later. (A) Twenty and 30 mg of

protein from plasma and intracellular membranes, respectively, were

loaded in the gels for AQP8 analysis, whereas 10 and 20 mg were loaded

for Mrp2 detection. Data on densitometric analysis (not shown) revealed

a significant increase (P!0.05, Student-t-test) in Mrp2 content in

intracellular membranes in response to E217G. In contrast, AQP8 levels

remained unchanged. (B) Confocal microscopy analysis of AQP8 and

occludin, a tight junction marker, indicated irregular distribution of the

water channel inside the canaliculus (see arrowheads) and that E217G

did not substantially change this pattern of staining. This study also

confirms preferential intracellular localization of AQP8 in rat

hepatocytes. In contrast, Mrp2 is mainly localized to the canalicular

space (see arrowheads), as delimited by ZO-1, and exhibited

internalization to the pericanalicular area in E217G group (see arrows).

Letters to the Editor / Journal of Hepatology 44 (2006) 232–235 233

Acknowledgements

The authors thank Drs Marcelo G. Roma and Fernando

A. Crocenzi for helpful discussions. This work was

supported by PHS grant GM55343 to Mary Vore and by

grants PICT 05-08418 (Aldo D. Mottino) and PICT 05-

10590 (Raul A. Marinelli) from Agencia Nacional de

Promocion Cientıfica y Tecnologica and by Consejo

Nacional de Investigaciones Cientıficas y Tecnicas,

Argentina.

Aldo D. Mottino1,2, Flavia I. Carreras1,

Sergio A. Gradilone1, Raul A. Marinelli1, Mary Vore2

1Institute of Experimental Physiology, National University

of Rosario, 2000-Rosario, Argentina,2Graduate Center for Toxicology, University of Kentucky,

306 Health Sciences Research Building,

Lexington, KY 40536-0305, USA

E-mail address: maryv@uky.edu

References

[1] Marinelli RA, Tietz P, LaRusso NF. Regulated vesicle trafficking of

membrane transporters in hepatic epithelia. J Hepatol 2005;42:

592–603.

[2] Huebert RC, Splinter PL, Garcıa F, Marinelli RA, LaRusso NF.

Expression and localization of aquaporin water channels in rat

hepatocytes. Evidence for a role in canalicular bile secretion. J Biol

Chem 2002;277:22710–22717.

[3] Mottino AD, Cao J, Veggi LM, Crocenzi F, Roma MG, Vore M.

Altered localization and activity of canalicular Mrp2 in estradiol-

17ß-D-glucuronide-induced cholestasis. Hepatology 2002;35:

1409–1419.

[4] Crocenzi FA, Mottino AD, Cao J, Veggi LM, Sanchez Pozzi EJ,

Vore M, et al. Estradiol 17-ß-D-glucuronide induces endocytic

internalization of Bsep in the rat. Am J Physiol Gastrointest Liver

Physiol 2003;17:G449–G459.

[5] Meyers M, Slikker W, Pascoe G, Vore M. Characterization of

cholestasis induced by estradiol-17b-D-glucuronide in the rat.

J Pharmacol Exp Ther 1980;214:87–93.

[6] Stieger B, Fattinger K, Madon J, Kullac-Ublick GA, Meier PJ. Drug-

and estrogen-induced cholestasis through inhibition of the hepatocel-

lular bile salt export pump (Bsep) of rat liver. Gastroenterology 2000;

118:422–430.

[7] Carreras FI, Gradilone SA, Mazzone A, Garcıa F, Ochoa JE, Tietz P,

et al. Rat hepatocyte aquaporin-8 water channels are down-regulated in

extrahepatic cholestasis. Hepatology 2003;37:1026–1033.

[8] Mottino AD, Crocenzi FA, Sanchez Pozzi EJ, Veggi LM, Roma MG,

Vore M. Role of microtubules in estradiol-17-ß-glucuronide-induced

alteration of canalicular Mrp2 localization and activity. Am J Physiol

Gastrointest Liver Physiol 2005;288:G327–G336.

doi:10.1016/j.jhep.2005.08.021

Induction of Angiopoietin-2 gene expression by COX-2: A novelrole for COX-2 inhibitors during hepatocarcinogenesis

To the Editor:

The possibility of preventing hepatitis B and C related

hepatocellular carcinoma (HCC) by anti-viral agents in

high-risk patients has become increasingly important given

the worldwide prevalence of this devastating disease [1]. In

this regard reported by Marquez-Rosado et al. [2], there is

also evidence that links cyclooxygenase-2 (COX-2) with

hepatopathogenesis. Indeed, a more recent report

Letters to the Editor / Journal of Hepatology 44 (2006) 232–235234

demonstrates that hepatitis C virus replicons enhance COX-

2 expression in human hepatocytes [3]. Although the precise

role of COX-2 in the pathogenesis of HCC remains to be

defined, there is accumulating evidence that ‘angiogenesis’

is one of the critical mechanisms important in the

progression of HCC. We have previously identified

Angiopoeitin-2 (Ang-2) as one of the angiogenic switch

genes in the pathogenesis of HCC [4–6]. Ang-2 was

extensively overexpressed in hypervascular HCCs, and

resulted in rapid tumor growth and hemorrhage in an animal

model system of HCC [4,6]. Furthermore, inhibition of

Ang-2 signaling induced vascular endothelial apoptosis,

leading to in vivo anti-angiogenic effects and suppression of

HCC tumor formation [5]. Although Ang-2 has a crucial

role in hepatocarcinogenesis [7,8], the effect of COX-2 on

Ang-2 expression in HCC cells has not been determined.

As shown in Fig. 1A, transient transfection of a COX-2

expressing cDNA enhanced Ang-2 expression in human

HCC cells; gene induction was dose-dependent. Next, the

effect of several inflammatory cytokines on Ang-2 gene

expression was analyzed as demonstrated by the RT-PCR

panel depicted in Fig. 1B. Prostaglandin E2 known to be

involved in COX-2 synthesis induced expression of the

Fig. 1. In vitro studies of human Hep3B HCC cells (A,B) and in vivo analysis of

Ang-2 was analyzed by RT-PCR in Hep3B cells transiently transfected with

COX-2 expression plasmid was kindly provided by Dr Timothy Hla (Univer

integrity of the mRNA in each sample. (B) RT-PCR analysis of Ang-2 express

interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, tumor growth fa

beta-actin demonstrated the integrity of the mRNA in each sample. (C) In vi

HCC. In brief, 2.5!104 MSH134 cells were inoculated subcutaneously in the

(0.5 mm3) developed 7-days post-inoculation and the mice were randomized to

treated mice were given NS-398 10 mg/kg (body weight) intraperitoneally tw

sacrificed on day 14 for examination of Ang-2 expression in tumor tissue. Equ

each sample. (D) Expression of CD31 protein in the endothelium of vessels su

tumor tissue were analyzed by immunohistochemistry using the anti-CD31 an

example of tumor treated with NS398 compared to a control. (E) Tumor grow

C3H-derived MH134 HCC cells. The volume of MH134 tumors was determin

observed on MH134 cell proliferation or apoptosis in vitro (data not shown), th

result of its anti-angiogenic properties.

Ang-2 gene. In contrast, Ang-2 expression was not induced

by stimulation with several inflammatory cytokines such as

interleukin-1b, interferon-g, tumor necrosis factor-a, or

tumor growth factor-b as controls. Finally, a specific

inhibitor of COX-2, namely NS-398, was used in the

murine model to study its effect of HCC tumor growth.

Administration of NS-398 attenuated gene expression of

Ang-2 (Fig. 1C), resulting in inhibition of tumor angiogen-

esis and growth in an animal model system of HCC (Fig. 1D

and E). Thus, COX-2 inhibitors might be considered as a

therapeutic agent to suppress Ang-2 expression and prevent

or reduce HCC progression by targeting this pathway.

The angiogenic switch mechanism involves sprouting of

new blood vessels from pre-existing ones and appears

essential for HCC development and progression [4,9]. The

evidence presented here suggest that COX-2 regulates Ang-2

expression in HCC cells in vitro and in vivo, and COX-2

inhibitors may serve as anti-tumor agents [3,10]. Finally, a

better understanding of the relationship between COX-2 and

Ang-2 gene expression will likely increase our knowledge

of the role of angiogenic activity during hepatic oncogenesis

as well as lead to new drug candidates for the treatment of

HCC.

murine HCC cells (MH134) in C3H mice (C–E). (A) Gene expression of

COX-2 expression plasmid (0.1, 1 mg) or mock plasmid (control). The

sity of Connecticut). Equal expression of beta-actin demonstrated the

ion in Hep3B cells following a 1hr-treatment of interleukin (IL)-1beta,

ctor (TGF)-beta, or prostaglandin (PG) E2 (5 mM). Equal expression of

vo expression of Ang-2 was analyzed by RT-PCR in a murine model of

flank of 6-week old syngeneic female C3H mice. Subcutaneous tumors

either control group (nZ5) or the NS-398 treatment group (nZ5). The

ice daily for 14 days. Tumor size was observed daily, and mice were

al expression of beta-actin demonstrated the integrity of the mRNA in

rrounding and penetrating a mouse HCC tumor. Sections of a MH134

tibody (x400). Note the lack of new vessel formation in a representative

th rate in the control and NS-398 treated group in a murine model using

ed every two days following treatment. While little effect of NS-398 was

ere was significant inhibition of HCC growth in vivo presumably as the

Letters to the Editor / Journal of Hepatology 44 (2006) 232–235 235

Shinji Tanaka1, Jack R. Wands2, Shigeki Arii1

1Department of Hepato-Biliary-Pancreatic Surgery,

Graduate School of Medicine,

Tokyo Medical and Dental University, 1-5-45 Yushima,

Bunkyo-ku, Tokyo 113-8519, Japan2Liver Research Center, Rhode Island Hospital and Brown

Medical School, Providence, RI 02903, USA

E-mail address: shinji.msrg@tmd.ac.jp

References

[1] Wands JR. Prevention of hepatocellular carcinoma. N Engl J Med

2004;351:1567–1570.

[2] Marquez-Rosado L, Trejo-Solis MC, Garcia-Cuellar CM, Villa-

Trevino S. Celecoxib, a cyclooxygenase-2 inhibitor, prevents

induction of liver preneoplastic lesions in rats. J Hepatol 2005;43:

653–660.

[3] Waris G, Siddiqui A. Hepatitis C virus stimulates the expression of

cyclooxygenase-2 via oxidative stress: role of prostaglandin E2 in

RNA replication. J Virol 2005;79:9725–9734.

[4] Tanaka S, Mori M, Sakamoto Y, Makuuchi M, Sugimachi K,

Wands JR. Biologic significance of angiopoietin-2 expression in

human hepatocellular carcinoma. J Clin Invest 1999;103:341–345.

[5] Tanaka S, Sugimachi K, Yamashita Yi Y, Ohga T, Shirabe K,

Shimada M, et al. Tie2 vascular endothelial receptor expression and

function in hepatocellular carcinoma. Hepatology 2002;35:861–867.

[6] Sugimachi K, Tanaka S, Taguchi K, Aishima S, Shimada M,

Tsuneyoshi M. Angiopoietin switching regulates angiogenesis and

progression of human hepatocellular carcinoma. J Clin Pathol 2003;

56:854–860.

[7] Mitsuhashi N, Shimizu H, Ohtsuka M, Wakabayashi Y, Ito H,

Kimura F, et al. Angiopoietins and Tie-2 expression in angiogenesis

and proliferation of human hepatocellular carcinoma. Hepatology

2003;37:1105–1113.

[8] Ogawa M, Yamamoto H, Nagano H, Miyake Y, Sugita Y, Hata T,

et al. Hepatic expression of ANG2 RNA in metastatic colorectal

cancer. Hepatology 2004;39:528–539.

[9] Tanaka S, Sugimachi K, Yamashita Yi Y, Shirabe K, Shimada M,

Wands JR, et al. Angiogenic switch as a molecular target of malignant

tumors. J Gastroenterol 2003;38:93–97.

[10] Koga H. Hepatocellular carcinoma: is there a potential for chemo-

prevention using cyclooxygenase-2 inhibitors? Cancer 2003;98:

661–667.

doi:10.1016/j.jhep.2005.09.012