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Division of Cellular and Molecular Pathogenesis, Department of Pathology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298-0297

² To whom requests for reprints should be addressed, at Department of Pathology, Medical College of Virginia Campus, Virginia Commonwealth, University, P. O. Box 980297, Richmond, VA 23298-0297. Phone: (804) 828-9549; Fax: (804) 828-9749; E-mail: asirica{at}hsc.vcu.edu

	Abstract

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Emodin, a tyrosine kinase inhibitor, effectively blocked tyrosine phosphorylation of p185^neu overexpressed in cultured rat C611B cholangiocarcinoma (ChC) cells and in neu-transformed WB-F344 rat-liver epithelial stem-like cells (WBneu cells). Celecoxib, a cyclooxygenase-2 inhibitor, markedly decreased prostaglandin (PG) levels overproduced by these respective neoplastically transformed liver cell types but was without effect in inhibiting PG production by untransformed WB-F344 cells that do not express detectable cyclooxygenase-2 protein. Notably, in combination, emodin (30 µM) and celecoxib (35 µM) acted synergistically to significantly suppress anchorage-dependent and -independent growth of C611B ChC cells and of WBneu cells over treatments with either agent alone. This prominent suppression of cell growth correlated with significant increases in the activation of caspases-9 and -3 and induction of apoptosis in the combination-treated cells, which was associated with an enhanced suppression of Akt activation. Here it is important to note that the concentration of celecoxib needed to suppress growth and induce apoptosis in the C611B and WBneu cells was markedly higher than that needed to effectively inhibit PG production by these malignant cell types. Thus, our data indicate that celecoxib is acting independently of its ability to inhibit cyclooxygenase-2 activity in suppressing growth of C611B and WBneu cells in vitro. Furthermore, our findings strongly suggest that increased inhibition of the antiapoptotic kinase Akt activation produced by the emodin/celecoxib combination treatment plays a key role in the mechanism by which this drug combination acts to enhance cell growth suppression and apoptosis in cultured C611B ChC cells and WBneu cells.

	Introduction

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ChC,³ which arises from the epithelial lining cells of intra- and extrahepatic bile ducts, is a devastating cancer with a generally poor prognosis and high mortality rates (1–3). These cancers are difficult to manage clinically because they usually present in an advanced stage, for which there is no currently effective treatment (4, 5). Recently, we demonstrated a strong positive correlation between ERBB-2 protein (p185^neu) overexpression and COX-2 up-regulation in human ChCs and related risk conditions (6). In addition, we have previously shown COX-2 mRNA and protein to be markedly up-regulated in the neoplastic epithelium of furan-induced rat ChCs constitutively overexpressing activated p185^neu (7–10). C611B, a novel tumorigenic ChC cell line established by us from a furan-derived transplantable rat ChC, also exhibited concomitant overexpression of tyrosine-phosphorylated p185^neu and COX-2 compared with untransformed hyperplastic bile duct/ductular epithelial cells obtained from the liver of bile duct-ligated rats (9–11). Furthermore, we have recently demonstrated that neoplastic transformation of WB-F344 rat liver epithelial stem-like cells in culture with oncogenic neu, like C611B ChC cells, yields tumorigenic transformants (WBneu cells) that also overexpress tyrosine-phosphorylated p185^neu and express up-regulated COX-2 compared with untransformed WB-F344 cells (10, 11).

In this study, we tested the p185^neu tyrosine kinase inhibitor emodin (12–14) in combination with the COX-2 inhibitor celecoxib (15, 16) for their ability to act synergistically to suppress the growth of both rat C611B ChC cells and WBneu cells in culture. We also investigated potential molecular mechanisms for this effect, with an emphasis on determining whether the combination treatment enhanced inhibition of the antiapoptotic kinase Akt, leading to increased activation of caspase-mediated apoptosis. In addition, our findings indicate that celecoxib acts through a COX-2-independent mechanism.

	Materials and Methods

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Drugs and Reagents.
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) and DMSO were purchased from Sigma-Aldrich Co. (St. Louis, MO). Celecoxib was generously provided by Pharmacia Corp. (St. Louis, MO). The PI3K inhibitor LY294002 was purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Cell Lines.
The rat C611B ChC and WBneu cell lines used in this study were established in our laboratory, and their tumorigenic properties and aberrant p185^neu and COX-2 expression profiles have been described previously (9–11). Untransformed WB-F344 rat liver epithelial stem-like cells were a kind gift from Dr. Joe Grisham’s laboratory (Department of Pathology and Laboratory Medicine, University of North Carolina- Chapel Hill, Chapel Hill, NC). Dr. James E. Trosko (Department of Pediatrics and Human Development, Michigan State University, East Lansing, MI) kindly provided the retroviral construct containing the transforming rat neu oncogene used by us to generate the WBneu cell line from infected WB-F344 cells. Our standard conditions for culturing these respective cell lines have been published previously (9). Experimental details of specific in vitro treatments of the cell lines are given in the figure legends.

Western Blot Analysis and Primary Antibodies.
Western blot analysis was performed essentially as described previously (8, 9) using each of the following primary antibodies: (a) Neu (C-18), sc-284, an affinity-purified rabbit polyclonal antibody raised against an epitope mapping at the COOH terminus of human p185^neu and cross-reactive with rat furan-induced rat ChC p185^neu (7, 8); (b) caspase-3 (H-277), sc-7148, a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1–277 representing the full-length precursor form of human caspase-3; (c) COX-2 (M-19), sc-1747, an affinity-purified goat polyclonal antibody raised against a peptide mapping at the COOH terminus of rat COX-2 and reactive with rat C611B ChC COX-2 (Refs. 10 and 11; antibodies in a-c were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); (d) Akt antibody, 9272, an affinity-purified rabbit polyclonal antibody produced against a synthetic peptide corresponding to amino acids 466–479 of mouse Akt; (e) phospho-Akt (Ser473) antibody, 9271, an affinity-purified rabbit polyclonal antibody raised against a synthetic phospho-473 peptide corresponding to residues around Ser⁴⁷³ of mouse Akt; and (f) cleaved caspase-9 (Asp353) rat specific antibody, 9507, an affinity-purified rabbit polyclonal antibody produced against a synthetic peptide corresponding to amino acids mapping NH₂-terminal to Asp³⁵³ (antibodies in d-f were obtained from Cell Signaling Technology, Inc., Beverly, MA).

Assays.
PG concentration in medium was assayed using the PG Screening ACE Enzyme Immunoassay Kit (catalogue number 514012) purchased from Cayman Chemical Company, Ann Arbor, MI. Anchorage-dependent cell growth on plastic substratum was measured using the CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation Assay from Promega (Madison, WI). The assay for anchorage-independent growth involved an initial seeding of 3000 C611B ChC cells or 2000 WBneu cells in culture medium containing 0.3% agar (Sigma-Aldrich) over a 0.6% soft agar layer in tissue culture wells, as demonstrated previously (10). The cultures were then exposed every 2 days for a period of 2 or 3 weeks to either 30 µM emodin alone, 35 µM celecoxib alone, or 30 µM emodin in combination with 35 µM celecoxib. DMSO at a final concentration of 0.1% was used as the solvent. Cell colony counts per individual culture wells were made under phase contrast at the end of the treatment period. Tyrosine phosphorylation of p185^neu was detected using the enhanced chemiluminescence phosphorylation detection system (RPN 2220) purchased from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). Akt kinase activity was assayed using the Akt Kinase Assay Kit (catalogue number 9840) from Cell Signaling Technology. Activated caspase-3 activity was measured using the ApoAlert Caspase-3 Colorometric Assay Kit from BD Biosciences Clontech, (Palo Alto, CA), and activated caspase-9 activity was determined using the Caspase-9 Colorimetric Assay Kit (catalogue number 218824) purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Apoptosis was assessed by standard DNA laddering. For the cell growth experiments with emodin and celecoxib, the culture medium contained 10% fetal bovine serum, which was omitted from the medium in experiments assessing caspase activation, DNA laddering, and Akt phosphorylation.

Statistical Analysis.
Student’s two-tailed t test was used to determine Ps. For evaluation of the combined effect of emodin and celecoxib, three methods were used to determine synergy as opposed to additivity. In Method 1, the fractional inhibition method (17) was used. With this method, synergism occurs when i_1,2 > i₁ + i₂ as opposed to being additive when i_1,2 = i₁ + i₂. In Method 2, we used the procedure described by Zhang et al. (13), in which observed values are compared with predicted values (c) calculated from the equation c = a x b/100, where a and b represent survival values for a single agent. Observed values <70% of predicted ones were considered synergistic (13). Method 3 was according to the classic isobologram equation where the CI = D₁/Dx₁ + D₂/Dx₂ (18). With this method, a CI value <1 is considered synergistic, whereas a CI value = 1 is additive. Additional details are given in the figure legends and in "RESULTS AND DISCUSSION."

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
In agreement with the initial findings of Zhang et al. (12) for p185^neu-overexpressing human breast cancer cell lines, we also found using immunoprecipitation and Western blotting that in vitro exposure of cultured C611B ChC and WBneu cells to emodin did not affect expressed p185^neu band levels but did induce a dramatic inhibition in the level of tyrosine phosphorylation exhibited by this constitutively activated tyrosine kinase in both malignant neoplastic cell types (Fig. 1, A and B). Also, as expected and shown in Fig. 1C, the selective COX-2 inhibitor celecoxib elicited a significant inhibition of PG production from arachidonic acid in treated C611B ChC and WBneu cells overexpressing COX-2 mRNA and protein (10, 11) but did not affect the basal concentration of PG produced by untransformed WB-F344 cells not expressing detectable COX-2 mRNA or protein (10, 11). Fig. 1, D and E, depicts representative dose-response curves for emodin and celecoxib alone on suppressing anchorage-dependent growth of C611B ChC and WBneu cells cultured on plastic substratum. Note that emodin and celecoxib each yielded very similar dose-response curves relevant to anchorage-dependent growth suppression when each was independently evaluated in the C611B ChC versus WBneu cell cultures.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, Western blot demonstrating emodin (80 µM for 12 h) to have no effect on the levels of detectable p185neu expressed by cultured C611B ChC and WBneu cell lines compared with respective solvent (0.1% DMSO) controls cultured in the absence of emodin. B, Western blot demonstrating prominent inhibition of tyrosine phosphorylation of p185neu by emodin under the same treatment conditions as described in A in cultured C611B ChC and WBneu cells compared with respective solvent control cultures. C, significant inhibition of PG production from arachidonic acid by celecoxib in cultured C611B ChC and WBneu cell lines overexpressing COX-2, but not in cultured untransformed WB-F344 cells, which do not express detectable COX-2 (10, 11). , cells cultured under our standard conditions in the presence of 35 µM celecoxib in 0.1% DMSO for a period of 4 days before being assayed for "total" PG in medium per total cell protein. , solvent control cultures containing 0.1% DMSO without celecoxib. Each value represents the mean ± SD, with n = 5 (*, P 0.001). D, dose-response curves demonstrating concentration-dependent suppression of anchorage-dependent growth of C611B ChC and WBneu cells on plastic substratum by emodin. Cultured cells were exposed to emodin for 4 days at the indicated concentrations before assaying for the number of living cells per culture with the Promega Cell Titer 96 AQueous Cell Proliferation Assay. Each data point represents the mean ± SD, with n = 10. E, dose-dependent suppression of anchorage-dependent growth of C611B ChC and WBneu cultured cells by celecoxib at the indicated concentrations. Culture and in vitro treatment conditions were essentially identical to those described in D. Each data point represents the mean ± SD, with n = 8. F, effect of 4 days of treatment with emodin (30 µM ) or celecoxib (35 µM ), alone or in combination , on suppressing anchorage-dependent growth of C611B ChC and WBneu cell lines. The combination treatment was determined to be synergistic by Methods 1, 2, and 3 described in "Materials and Methods." Each value represents the mean ± SD, with n = 8 for C611B ChC and n = 5 for WBneu. *, P 0.001.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Effect of emodin (30 µM) and celecoxib (35 µM) alone and in combination on suppressing anchorage-independent growth in soft agar of C611B ChC and WBneu cell lines overexpressing tyrosine-phosphorylated p185neu together with COX-2 (9–11). Experimental conditions are described in "Materials and Methods." Each value represents the mean ± SD determined from three separate experiments in which the number of soft agar cultures analyzed per individual experiment ranged between three and six. The combination treatment was determined to be synergistic by Methods 1 and 2 described in "Materials and Methods." *, P 0.001.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. A, Western blot demonstrating a marked increase in cleaved caspase-9 (p17) in cultured C611B ChC and WBneu cells exposed for 10 h to 35 µM celecoxib and 30 µM emodin in combination compared with cells exposed to corresponding concentrations of either agent alone or to 0.1% DMSO solvent without drug. B, Western blot data showing cleaved caspase-3 to be prominently increased in cultured C611B ChC and WBneu cells exposed for 14 h to 35 µM celecoxib and 30 µM emodin in combination compared with those exposed to corresponding concentrations of either agent alone or to 0.1% DMSO. In A and B, LyB refers to lysis buffer (noncellular) control. C and D, caspase-9 and -3 activities, respectively, are significantly increased in cultured C611B ChC and WBneu cells subjected to combination treatment with 35 µM celecoxib and 30 µM emodin , compared with those treated with either 35 µM celecoxib alone or to 30 µM emodin alone . Each value represents the mean ± SD, with n = 4. *, P 0.001. E and F, representative DNA ladders for cultured C611B ChC and WBneu cells, respectively, reflecting significant apoptosis produced by the in vitro combination treatment with 35 µM celecoxib plus 30 µM emodin, compared with those cultures exposed to corresponding concentrations of either agent alone or to 0.1% DMSO solvent only.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. A, Western blot demonstrating that 12-h in vitro exposure of C611B ChC cells to 30 µM emodin in combination with 35 µM celecoxib markedly suppressed phosphorylation of Akt in these cultured cells over that produced by comparable treatments with either agent alone or the DMSO control (DMSO CT). Phospho-Akt was detected using the phospho-Akt (Ser473) antibody. Note that expressed Akt protein levels did not appear to be affected by the various drug treatments but that the PI3K inhibitor LY294002 elicited a comparable degree of inhibition of Akt phosphorylation as that produced by the combination treatment. B, Western blot showing that combination treatment of cultured C611B ChC cells with 30 µM emodin in combination with 35 µM celecoxib was equally effective in inhibiting Akt kinase activity in these cells as was LY294002 and clearly more effective in blocking this kinase activity than either emodin or celecoxib alone, compared with DMSO-CT cells. phospho-GSK-3/ß, phosphorylated glycogen synthase kinase and ß. C, Western blot demonstrating the lack of effect of 30 µM emodin and 35 µM celecoxib, alone or in combination, on COX-2 protein expression in cultured C611B ChC cells. ß-Actin was used to normalize the blot. RIPA refers to negative buffer control without cells.

The dose-response curves for emodin on the growth inhibition of cultured C611B and WBneu cells, respectively, as shown in Fig. 1D, are quite comparable with those obtained by Zhang et al. (12) for emodin on the growth suppression of a number of different p185^neu-overexpressing human breast cancer cell lines. These investigators further reported that emodin at high concentrations can also repress epidermal growth factor receptor tyrosine kinase activity (13). However, this is unlikely to be a factor in our results because we have observed by Western blotting that p185^neu is the dominant ERBB family receptor tyrosine kinase expressed in C611B ChC and WBneu cells and that expression of epidermal growth factor receptor in these respective transformed cell types is greatly reduced compared with that of untransformed control cells. In addition, we could not detect evidence of constitutively activated epidermal growth factor receptor in either theC611B ChC or WBneu cell lines (data not shown).

In conclusion, our findings indicate that the p185^neu tyrosine kinase inhibitor emodin in combination with the COX-2 inhibitor celecoxib acts synergistically to suppress anchorage-dependent and -independent growth of both rat ChC cells and neu-transformed rat liver epithelial stem-like cells expressing activated p185^neu together with COX-2. They further indicate that this effect is the result of a synergistic action to enhance apoptosis through a mechanism involving inhibition of Akt activation and kinase activity. Consistent with this interpretation, we recently obtained preliminary data to suggest that apoptosis induced by celecoxib alone at a 50 µM concentration in cultured C611B ChC cells was independent of Bcl₂ and Bcl-X_L but also correlated with the effect of this agent on suppressing Akt phosphorylation and kinase activity. This, in turn, correlated with Bax translocation to mitochondria, cytochrome c release, and activation of caspase-9 and -3.⁴ These latter results are currently being confirmed and expanded.

In addition to providing an underlying mechanism for the synergistic effects exhibited by emodin and celecoxib in combination, our data support a potentially novel therapeutic strategy for biliary cancer treatment as well as that of certain other malignant adenocarcinoma types. This would involve specific drug targeting of activated p185^neu overexpressed in ChC cells with more clinically relevant p185^neu tyrosine kinase inhibitors than emodin given in combination with a select PI3K inhibitor also preferentially acting upstream to suppress Akt phosphorylation. Here the very recent findings of Klejman et al. (23) are highly relevant. These investigators demonstrated that PI3K inhibitors (i.e., wortmannin) significantly enhance the antileukemia effect of ST1571. Wortmannin given in combination with gemcitabine was also recently found to induce a 5-fold increase in apoptosis in orthotopic human pancreatic cancer xenografts in immunodeficient mice compared with either agent alone (24).

We are currently testing this strategy preclinically in vitro against C611B ChC and WBneu cells and in vivo against tumorigenic C611B cells orthotopically transplanted into rat liver. Also, we are currently investigating the potential antitumor and chemopreventative effects of select COX-2 inhibitors in combination with other inhibitors of p185^neu tyrosine kinase activity than emodin in a rat model of ChC to optimize an in vivo therapeutic effect in a tumor model having relevance to the human disease.

Because the celecoxib concentrations used in the current in vitro study are about 10-fold higher than those reasonably achieved in vivo in the plasma of chronically treated mice and humans, we anticipate that there will be a lack of correlation between our in vitro and in vivo models of ChC with respect to the mechanism underlying celecoxib-induced tumor growth suppression in vivo in a manner similar to that reported previously by Williams et al. (21). However, this does not preclude that COX-2 inhibitors such as celecoxib or rofecoxib might not still have a significant therapeutic impact on ChC development and growth in vivo when given in combination with select inhibitors of p185^neu tyrosine kinase activity. In this context, the findings of Mann et al. (25) are encouraging. These investigators recently demonstrated that combination treatment with celecoxib and the humanized anti-p185^neu antibody Herceptin of athymic mice bearing s.c. HCA-7 human colorectal carcinoma xenografts resulted in additive effects that yielded almost complete inhibition of tumor growth. In an even more recent preliminary report, Discafani et al. (26) have also shown that treatment of C57BL/6J-Min/+ mice with the selective epidermal growth factor receptor antagonist EKB-569 in combination with the COX-2 inhibitor rofecoxib reduced the development of colorectal polyps and significantly increased the survival of these animals over those treated with either agent alone. Testing such a strategy in vivo against ChC concomitantly overexpressing activated p185^neu and COX-2 is thus clearly warranted, even though the potential mechanism of tumor growth suppression may be different from that currently demonstrated in vitro; hopefully, this will result in data relevant to the development of an effective adjuvant therapy for such a devastating human cancer as ChC.

	Acknowledgments

 
We thank Dr. Timothy J. Mazaisz (Pharmacia Corp., St. Louis, MO) for critical review of this work.

	Footnotes

 
¹ Supported by Grants RO1 CA 39225 and RO1 CA 83650 (to A. E. S.) from the National Cancer Institute, NIH.

³ The abbreviations used are: ChC, cholangiocarcinoma; COX, cyclooxygenase; PG, prostaglandin; PI3K, phosphatidylinositol 3'-kinase; CI, combination index.

⁴ Z. Zhang and A. E. Sirica, unpublished data.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/17/02; revised 10/ 2/02; accepted 1/14/03.

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