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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cholangiocarcinoma (CCA) is a biliary cancer arising from damaged bile ducts. Epithelial-mesenchymal transition (EMT) occurs as epithelial cells begin to resemble mesenchymal cells leading to increased invasion potential as the extracellular matrix (ECM) degrades. Histamine exerts its effects by way of four receptors (H1-H4 HRs). Clobenpropit, a potent H4HR agonist, inhibits mammary adenocarcinoma growth. We have shown that (1) cholangiocytes and CCA cells express H1-H4 HRs and (2) the H3HR decreases CCA proliferation. We evaluated the effects of clobenpropit on CCA proliferation, invasion, EMT phenotypes, and ECM degradation. In vitro, we used CCA cell lines to study proliferation, signaling pathways, and the morphological invasive potential. Gene and protein expression of the hepatobiliary epithelial markers CK-7, CK-8, and CK-19, the focal contact protein paxillin, and the mesenchymal markers fibronectin, s100A4, and vimentin were evaluated. Cell invasion across an ECM layer was quantitated and matrix metalloproteinase-1, -2, -3, -9, and -11 gene and protein expression was examined. Evaluation of the specific role of H4HR was performed by genetic knockdown of the H3HR and overexpression of H4HR. Proliferation was evaluated by proliferating cellular nuclear antigen immunoblotting. In vivo, xenograft tumors were treated with either vehicle or clobenpropit for 39 days. Tumor volume was recorded every other day. Clobenpropit significantly decreased CCA proliferation by way of a Ca2+-dependent pathway and altered morphological development and invasion. Loss of H3HR expression or overexpression of H4HR significantly decreased CCA proliferation. In vivo, clobenpropit inhibited xenograft tumor growth compared with controls. Conclusion: Modulation of H4HR by clobenpropit disrupts EMT processes, ECM breakdown, and invasion potential and decreases tumor growth. Interruption of tumorigenesis and invasion by histamine may add to therapeutic advances for CCAs. (HEPATOLOGY 2011;)

Cholangiocarcinoma (CCA) is a devastating cancer arising from both intra- and extrahepatic bile ducts.1 Because of the location of this tumor, diagnosis is difficult, treatment options are limited, and patient prognosis is dismal.1, 2 Biliary tumors are able to metastasize into other organs of the body including lungs, bones, adrenal glands, and brain, which significantly reduce survival.3 CCA is associated with altered expression of inflammation-associated cytokines and mediators.1, 2, 4 Elucidating the mechanisms regulating biliary tumor growth is critical to finding new treatment strategies.

Histamine by way of the histamine receptors (H1-H4 HRs) regulates the growth of numerous cancers including breast and colon.5 We have recently shown that (1) CCA cells express H1-H4HRs and (2) stimulation with the H3HR agonist, (R)-(α)-(-)-methylhistamine dihydrobromide (RAMH), decreases cholangiocarcinoma growth by activation of the Ca2+-dependent protein kinase C alpha (PKCα).6 Besides playing a role in inflammation, H4HR has been implicated in the mediation of cellular chemotaxis by way of inhibition of cyclic adenosine monophosphate (cAMP) synthesis and Ca2+ mobilization.7-10 The specific H3 agonist/H4 antagonist, clobenpropit, inhibits the spread of mammary adenocarcinoma by decreasing invasion potential.11

Epithelial to mesenchymal transition (EMT) is a feature of proliferating cells including CCA. Cellular EMT is characterized by alterations in morphology, adhesion, and migratory capacity.12, 13 During EMT, breakdown of the extracellular matrix (ECM) occurs by up-regulation of matrix metalloproteinases (MMPs).14 With degradation of the ECM, tumors begin to spread and invade other tissues and are often coupled with the overexpression of mesenchymal markers like paxillin,15 vimentin,16 fibronectin,16 and s100A4.17, 18 After establishing themselves in another tissue, cancer cells begin the transition back to an epithelial-type cell to allow for proliferation and tumor spreading.19

Studies have noted that the H4 receptor decreases the rate of metastasis and growth in other tumors4, 11 and due to CCA being a fatal malignancy with limited treatment options that is able to metastasize, causing further destruction, we aimed to determine if activation of H4HR with clobenpropit decreases CCA growth and metastatic potential.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

All reagents were obtained from Sigma Chemical (St. Louis, MO) unless stated otherwise. Antibodies for immunoblots, immunofluorescence, and immunohistochemistry were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture reagents and media were obtained from Invitrogen (Carlsbad, CA). Human sequenced primers and real-time polymerase chain reaction (PCR) assay materials were obtained from SABiosciences (Frederick, MD).

Methods

Details of cell culture maintenance and all routinely used methodologies are provided in Supporting Appendix A.

In Vitro Studies

Cultured Cell Lines.

We used multiple intra- and extrahepatic CCA cell lines along with nonmalignant cells. All cell lines were loaned by Dr. Gianfranco Alpini (Texas A&M HSC COM, Temple, TX) and were originally obtained from the sources found in Supporting Appendix A.

Immunofluorescence.

For immunofluorescence, cells were seeded on coverslips in a 6-well plate (500,000 cells/well) and allowed to adhere overnight. Immunofluorescence was performed as described using specific antibodies described in Supporting Appendix A. Images were obtained using an inverted Olympus IX-71 confocal microscope (Tokyo, Japan).6, 20

Real-Time PCR.

Gene expression was evaluated in messenger RNA (mRNA) (1 μg) from all CCA and nonmalignant cell lines as well as in mRNA extracted from tumors. RNA was extracted using the Qiagen RNeasy mini kit (Valencia, CA). Human primers and the RT2 Real-time assay kit were obtained from SABiosciences. See Supporting Table 1 for all sequence information.

Immunoblotting.

Cells were trypsinized and lysed for immunoblots.6, 20 Lysed cells were prepared for immunoblotting using 10 μg of protein. Band intensity was determined by scanning video densitometry using the phospho-imager, Storm 860 (GE Healthcare, Piscataway, NJ) and the ImageQuant TLV 2003.02 (Little Chalfont, Buckinghamshire, UK) software.

Immunohistochemistry in Human Tumor Biopsies.

Immunoreactivity was performed in AccuMax tissue arrays (obtained from XpressBio) by immunohistochemistry using a specific antibody for H4HR (1:50).6 Details are provided in Supporting Appendix A.

MTS Proliferation Assays.

Cells were plated into 96-well plates and stimulated with clobenpropit (1-50 μm)4, 11 for up to 48 hours to determine optimal dose and stimulation time. Cell proliferation was evaluated using the CellTiter 96 Aqueous One Solution cell Proliferation Assay and the absorbance was measured at 490 nm on a microplate spectrophotometer (Versamax, Molecular Devices).6, 21 Data are expressed as fold change of treated cells as compared with basal-treated controls.

Small Interfering RNA (siRNA) Knockdown of H3HR and Overexpression of H4HR.

CCA cells were transiently transfected with a human siRNA targeting H3HR (Santa Cruz Biotechnology) to knockdown H3HR expression. Cells were plated and transfected as described.6 After establishing knockdown efficiency (≈50%), CCA cells ± H3HR siRNA (40 nM) were stimulated with basal or clobenpropit (10 μM, 6 hours) and proliferation was assessed by proliferating cellular nuclear antigen (PCNA) immunoblotting as described above. The specific antiproliferative role of H4HR in CCA growth was also evaluated by overexpression of H4HR in CCA cells. Using a human complementary DNA (cDNA) vector for H4HR (Origene, Rockville, MD), we performed amplification and transfected this DNA according to instructions provided by the vendor into CCA cells. Proliferation was assessed by PCNA immunoblotting.

Secondary Signaling Mechanisms.

Mz-ChA-1 cells were stimulated with RAMH (10 μM) or clobenpropit (10 μM) and intracellular cAMP and IP3 levels were evaluated by RIA.22 MTS proliferation assays were performed in Mz-ChA-1 cells stimulated with clobenpropit (10 μM) in the absence or presence of the intracellular calcium chelator, BAPTA/AM, the PKA inhibitor, RpcAMPs, or the PKC inhibitor Gö6976. Details of stimulations can be found in Supporting Appendix A.

Annexin V Labeling.

Cells were seeded into 6-well plates (500,000 cells/well) containing sterile coverslips on the bottom of each well. After overnight adherence, cells were stimulated and images were obtained using an inverted Olympus IX-71 confocal microscope. The number of Annexin V-positive cells were counted and expressed as a percentage of total cells in five random fields for each treatment group.

Matrigel Outgrowth Assay.

Mz-ChA-1 cells were added to the upper chamber of the Boyden chamber coated with Matrigel inserts (BDPharmingen, San Diego, CA) and the lower chamber contained 10% serum-containing medium. Cells were incubated for up to 48 hours. Invasion of cells through to the underside of the Matrigel-coated membrane was detected by visualizing the cells under a light microscope. Cells were counted in four random fields (magnification ×100) and results were expressed in morphologic images as well as the form of a bar graph.23

ECM Cell Invasion Assay.

ECM cell invasion was assessed across a solid gel of basement membrane proteins prepared from the Engelbreth Holm-Swarm mouse tumor using the QCM 96-well cell invasion assay kit as recommended by the instructions provided by the vendor (Chemicon International, Temecula, CA).

Cellular Viability.

To evaluate cell viability, Mz-ChA-1 cells were plated in Matrigel and using an Alamar blue kit obtained from Invitrogen and transferase-mediated dUTP nick end labeling (TUNEL) staining using TACS.XL DAB In Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD), viable cells were evaluated in the absence/presence of clobenpropit (10 μM, 6 hours). Specific details and stimulations are found in Supporting Appendix A.

In Vivo Studies

Model and Treatment Schedule.

We used a xenograft mouse model to evaluate the effects of clobenpropit on tumor growth.6 Animals were injected with Mz-ChA-1 cells and, after tumor establishment, were treated with and without the H4 agonist, clobenpropit. Tumor volume was recorded and tumor mRNA was collected. Full details can be found in Supporting Appendix A. All experimental procedures were conducted under the guidelines of the Scott & White and Texas A&M Health Science Center Institutional Animal Care and Use Committee.

Morphological Analysis.

Tumor samples were excised and fixed in 10% buffered formalin for 2-4 hours, embedded in low-temperature fusion paraffin, and sectioned (4-5 μm) for immunohistochemistry analysis.6, 21

EMT and ECM Marker Analysis.

We measured the expression of EMT (paxillin, fibronectin, s100A4 and vimentin) and hepatobiliary epithelial markers (CK-7, CK-8 and CK-19) and ECM degradation (MMPs) markers by real-time PCR in whole tumor mRNA (1 μg). See Supporting Appendix A for all details regarding in vivo studies.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

H4HR Expression.

By immunofluorescence (Fig. 1A), all cell lines express H4HR. Specific immunoreactivity is seen in red and the nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI), shown in blue (Fig. 1A). H4HR mRNA gene (Fig. 1B, top panel) and protein expression (Fig. 1B, bottom panel) is increased in numerous CCA cells compared with nonmalignant cells. Immunohistochemistry in human cholangiocarcinoma tissue samples showed that there is increased H4HR immunoreactivity in CCA tissues compared with nonmalignant biopsies (Fig. 1C).

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Figure 1. H4 histamine receptor expression was evaluated by (A) immunofluorescence, (B) real-time PCR and immunoblotting, and (C) immunohistochemistry in human biopsy samples. (A) By immunofluorescence we found that all CCA and nonmalignant cell lines are positive for the H4HR. Positive H4HR staining is found in red and nuclei are stained with DAPI, seen in blue. Original magnification ×20. (B) By real-time PCR (top left panel) and immunoblotting (bottom left panel) we found that several CCA cell lines have a higher gene and protein expression compared with nonmalignant cell lines. *P < 0.05 versus nonmalignant mRNA and data are SEM ± 3 experiments. *P < 0.05 versus nonmalignant protein and data are SEM ± 6 experiments. (C) Using Accumax tissue arrays we found that the H4HR immunoreactivity is highly increased in human CCA biopsies compared with nonmalignant biopsy samples. *P < 0.05 versus nonmalignant biopsy samples.

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Clobenpropit Effects on CCA Proliferation and Apoptosis.

Clobenpropit inhibited CCA proliferation in a dose-dependent manner (Fig. 2). Significant inhibition of the growth of numerous cell lines was found at doses of 5-50 μM after 48 hours of stimulation (Fig. 2). Using 10 μM, cells were stimulated from 6-48 hours to determine time effects. Supporting Fig. 1 shows that clobenpropit decreases CCA proliferation as early as 6 hours. In nonmalignant cells, clobenpropit began to decrease cellular proliferation at dosages as low as 10 μM at 48 hours (Supporting Fig. 2, top panels). When clobenpropit was used at 10 μM for 6 hours, there was no significant change in nonmalignant cell proliferation (Supporting Fig. 2, lower panels); therefore, we chose this dose (10 μM) and time period (6 hours) to perform the remainder of our experiments. Using Pearson's Correlation Coefficient analysis in several of our CCA cell lines, we failed to find that the expression of H3HR contributes to cell viability (or cell proliferation) seen after clobenpropit treatment and no significant correlation was discovered (Supporting Fig. 3). Further, using the same analysis we assessed the correlation between H4HR expression and cell proliferation. We found there was no significant correlation between H4HR gene expression and the effects of clobenpropit treatment (Supporting Fig. 3). Clobenpropit had no effects on CCA and nonmalignant cellular apoptosis (not shown).

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Figure 2. Effects of clobenpropit on CCA proliferation was evaluated by MTS proliferation assays. (A) Dose effects of clobenpropit was evaluated in CCA cell lines stimulated with clobenpropit 1-50 μM at 48 hours. Clobenpropit significantly decreased proliferation in most CCA cell lines at 5, 10, 25, and 50 μM compared with basal treatment. *P < 0.05 versus basal and data are SEM ± 12 experiments.

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Effects of Clobenpropit on Proliferation After H3HR Knockdown or H4HR Overexpression.

In H3HR siRNA transiently transfected Mz-ChA-1 cells, clobenpropit significantly decreased CCA growth to a greater extent when compared with cells that were mock-transfected, emphasizing the specific role of H4HR in our study (Supporting Fig. 4A,B). The antiproliferative effect of clobenpropit in H3HR knockdown cells was also greater than the effects of RAMH (the H3HR agonist, Supporting Fig. 4B). When Mz-ChA-1 cells were overexpressed with H4HR (2.5 μg, Supporting Fig. 4C), there was a reduction in CCA proliferation compared with LacZ control cells as shown by PCNA protein expression (Supporting Fig. 4D).

Clobenpropit Effects on Downstream Signaling Mediators: Intracellular cAMP and IP3 Levels.

Clobenpropit significantly increases intracellular IP3 levels (Supporting Fig. 5A), but has no effect on cAMP synthesis (Supporting Fig. 5B) suggesting that, in CCA cells, H4HR works primarily through a Gαo/Ca2+-mediated pathway. Further supporting evidence was obtaining using MTS proliferation assays. Supporting Fig. 5C shows that clobenpropit inhibits Mz-ChA-1 proliferation that is blocked by BAPTA/AM and Gö6976 but not RpcAMPs, confirming that H4HR activates a Ca2+-dependent pathway during CCA inhibition. Inhibitors alone had no effect on CCA proliferation (not shown).

EMT Phenotypes and Epithelial Preservation Induced by Clobenpropit.

By immunofluorescence, the expression of the focal contact protein, paxillin, and the mesenchymal markers, fibronectin, s100A4, and vimentin was decreased in clobenpropit-treated cholangiocarcinoma cells compared with basal (Fig. 3A, top panel). Real-time PCR revealed that gene expression for paxillin, fibronectin, s100A4, and vimentin was decreased after clobenpropit treatment relative to controls (Fig. 3A, bottom panel). Protein expression of the hepatobiliary epithelial markers, CK-7, CK-8, and CK-19, was maintained after clobenpropit treatment relative to controls (Fig. 3B, top panel). Furthermore, gene expression of the hepatobiliary epithelial markers CK-7, CK-8, and CK-19 was increased in CCA cells after clobenpropit treatment compared with basal (Fig. 3B, bottom panel). These results suggest that H4HR disrupts EMT-related proteins enabling CCA cells to retain epithelial-like qualities.

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Figure 3. Clobenpropit effects of on EMT markers were evaluated by immunofluorescence and real-time PCR in Mz-ChA-1 cells. (A) By immunofluorescent staining (top panel) clobenpropit treatment (10 μM, 6 hours) decreased the protein expression of paxillin, fibronectin, s100A4, and vimentin in CCA cells. Positive protein staining is found in red and nuclei are stained with DAPI, seen in blue. Original magnification ×20. By real-time PCR, clobenpropit (10 μM) significantly decreased the gene expression of paxillin, fibronectin, s100A4, and vimentin compared with basal treatment (bottom panel). (B) CK-7, CK-8, and CK-19 protein expression was highly up-regulated in clobenpropit-treated cells compared with basal treatment as shown by immunofluorescent staining. Positive protein staining is found in red and nuclei are stained with DAPI, seen in blue (top panel). Original magnification ×20. By real-time PCR, clobenpropit (10 μM) significantly increased CK-7, CK-8, and CK-19 expression compared with basal treatment (bottom panel). *P < 0.05 versus basal mRNA and data are SEM ± 3 experiments.

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Clobenpropit Effects on ECM Degradation Enzymes.

Figure 4 shows that clobenpropit treatment induces a downregulation of MMP-1, -2, -3, -9, and -11 protein expression compared with basal-treated cells. Consistent with the immunofluorescence studies, real-time PCR revealed that the gene expression for MMP-1, -2, -3, -9, and -11 is significantly reduced after clobenpropit treatment compared with control (Supporting Fig. 6). These data demonstrate that H4HR retains the stability of the ECM by silencing ECM degrading components during cholangiocarcinogenesis.

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Figure 4. Clobenpropit effects on ECM degradation enzymes were evaluated by immunofluorescence in Mz-ChA-1 cells. By immunofluorescence we found, in Mz-ChA-1 cells, that clobenpropit treatment decreased the expression of MMPs-1, -2, -3, -9, and -11. Positive protein staining is found in red and nuclei are stained with DAPI, seen in blue. Original magnification ×50.

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Morphological, Invasion, and Metastatic Potential Effects Induced by Clobenpropit.

We observed Mz-ChA-1 cells by light microscopy to examine morphological changes of CCA cells. Movements of CCA cells in the Matrigel were associated with the formation of peripheral ruffles. Compared with control cells, clobenpropit-treated Mz-ChA-1 cells were observed with fewer membrane ruffles and cells were significantly less invasive (Fig. 5A). Using the QCM 96-well cell Invasion assay kit in vitro we evaluated the invasion potential of CCA cells treated with clobenpropit (Fig. 5B). Graphic representation of clobenpropit-treated Mz-ChA-1 cells is found in Fig. 5B and were observed to be significantly less invasive through the ECM gel (Fig. 5B) than respective basal-treated cells. Representative images for Calcein AM-labeled invading cells are found in Fig. 5B. In Supporting Fig. 7, alamar blue (top) and TUNEL staining (bottom) were used to detect cell viability and apoptosis in Mz-CHA-1 cells cultured in Matrigel. There was no difference in cell viability or in the number of apoptotic cells in the Matrigel between the basal- and clobenpropit-treated group with either alamar blue assay (top) or TUNEL staining (Supporting Fig. 7, bottom panel, viable cells are labeled by red, apoptotic cells are labeled by blue). These results suggest that the H4HR agonist reduces invasive potential in cholangiocarcinoma cells without subjecting the cells to apoptosis.

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Figure 5. (A) Light microscopy was used to document morphologic differences between the clobenpropit and control treated Mz-ChA-1 cells. Mz-ChA-1 cells displayed an aggressive phenotype, which became elongated and formed the leading edge of pseudopodia. Clobenpropit treatment significantly reduced the percentage of cells with invasive phenotypes. (B) Cell invasion was assessed in clobenpropit treated Mz-ChA-1 cells with relative controls. Cell invasion was assessed in 96-well plates precoated with ECM. The mean and standard error from four separate experiments are graphically illustrated in the top panel, whereas the representative images are displayed in the bottom panel. *P < 0.05 when compared with relative controls.

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In Vivo Effects of Chronic Clobenpropit Treatment.

Clobenpropit significantly decreased tumor volume beginning after day 12 of treatment and continuing throughout the treatment schedule compared with vehicle-treated tumor volume (Fig. 6A, top panel). Morphologically, tumors from clobenpropit-treated mice maintained a relatively high expression of CK-7-positive cells compared with tumors from vehicle-treated mice (Fig. 6A, middle panel). In mRNA from tumors extracted from clobenpropit-treated mice gene expression for CK-7, CK-8, and CK-19 gene expression was increased compared with vehicle-treated mice tumors (Fig. 6A, lower panel). Further, we discovered that clobenpropit decreased the expression of EMT markers, paxillin, fibronectin, s100A4, and vimentin compared with vehicle treatment (Fig. 6B). Consistent with our in vitro findings, we demonstrated that the expression of MMPs (-1, -2, -3, -9, and -11) also decreased in tumors from the clobenpropit-treated groups compared with vehicle (Fig. 6C). These in vivo findings further support our conclusion that clobenpropit maintains the epithelial characteristics of cholangiocytes and inhibits EMT proteins and ECM breakdown during cholangiocarcinogenesis.

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Figure 6. In vivo effects of clobenpropit were evaluated in a xenograft tumor model. (A) Clobenpropit treatment significantly decreased tumor growth beginning at day 12 and continuing throughout the treatment schedule. *P < 0.05 versus vehicle-treated xenograft tumor volume (top panel). Analysis was performed by 2-way analysis of variance (ANOVA). Representative images are seen for immunohistochemistry for CK-7 in tumors extracted from both vehicle (left image) and clobenpropit (right image) treated mice (middle panel). Original magnification ×60. Real-time PCR data revealed that in mRNA from tumors from clobenpropit-treated mice CK-7, CK-8, and CK-19 gene expression increased compared with vehicle treatment (lower panel). (B) Real-time PCR data revealed that, in mRNA from tumors from clobenpropit-treated mice, there was a downregulation of paxillin, fibronectin, s100A4 and vimentin gene expression compared with vehicle treatment. (C) Gene expression of MMP-1, -2, -3, -9, and -11 was downregulated in clobenpropit-treated mice tumor mRNA compared with vehicle-treated tumors. *P < 0.05 versus vehicle mRNA and data are SEM ± 3 experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In our study we found that the H4HR agonist inhibits CCA progression by disrupting tumor metastatic- and growth-promoting behaviors including EMT and breakdown of the ECM. In vitro, the H4HR agonist clobenpropit decreased CCA proliferation that was coupled with a decrease in numerous proteins associated with both EMT processes and ECM breakdown. Clobenpropit increased CK-7, CK-8, and CK-19 expression, suggesting that this compound preserves epithelial characteristics in CCA cells. Clobenpropit prevented the development of protrusions and acinar bridges and decreased migration/invasion potential in CCA cells. Also, we performed in vivo experiments and found that clobenpropit decreased malignant tumor volume. Xenograft tumor studies in vivo revealed that clobenpropit prevented tumor growth and induced a decrease in the aforementioned proteins involved in EMT and ECM degradation.

Similar to other malignancies,24 we found that H4HR is present. Human mammary tissue and adenocarcinoma express both the H3 and H4 HRs that induce a differential effect on this cancer.11 Levels of H4 expression were found to be increasingly high in malignant cells and human tumor biopsy samples versus nonmalignant. This finding is consistent with other observations showing an increased level of histamine receptor expression in a malignant state.25 Previously, we found that the H3 histamine receptors are up-regulated during biliary carcinogenesis compared with nonmalignant cells6 and the H2 and H3 HRs are up-regulated in pancreatic carcinoma.26, 27

Similar to our previous study using the H3HR agonist,6 we demonstrated that clobenpropit decreased CCA proliferation by way of an IP3/PKC-dependent pathway. These findings, however, could not be linked to cellular apoptosis, as treatment with clobenpropit did not induce apoptosis. Further, the H4HR agonist did not have any effect on normal cholangiocyte proliferation or apoptosis, making clobenpropit a potentially attractive therapeutic compound. In mammary cancer, clobenpropit was also found to decrease the growth of this tumor at a similar dosage.11 H4 (but not H3) HR inhibits the growth of pancreatic carcinoma, although the exact mechanism is unknown.27 Although we acknowledge that our studies have uncovered somewhat conflicting results with regard to our studies involving H3HR, we attribute our findings not to the antagonistic ability of clobenpropit on H3HR, but its activation of H4HR. It has already been shown that clobenpropit stimulates H4HRs at a much higher affinity compared with its antagonistic ability on H3HR.28 Furthermore, it has reported that clobenpropit increases calcium levels by interacting with a novel GPCR, GPRv53 (later to become known as H4HR) and not by inhibition of H3HR, which is consistent with our current study.29 To further confirm that our effects are solely mediated by a clobenpropit[LEFT RIGHT ARROW]H4HR interaction, we genetically knocked out the H3HR and evaluated the effects of clobenpropit in these cells. After H3HR knockdown we found that H4HR significantly decreases CCA proliferation to a greater extent than in cells with H3HR expression, thus demonstrating that our stimulation and effect is mediated primarily by activation of H4HR and not by inhibition of H3HR. Further confirmation and similar results were found by overexpression of H4HR. Our study has demonstrated the presence of H4HR and inhibitory effect of clobenpropit on CCA.

EMT is observed during aggravated cellular proliferation in cancers such as pancreatic,30 gastric,31 and CCA.33 H4HR has been shown to decrease migratory and invasion potential in mammary cancer, making this a potential therapeutic target to block EMT and metastasis.11 In our study we demonstrated that the H4HR agonist clobenpropit decreases the expression of numerous EMT markers, suggesting that clobenpropit disrupts these processes. Histamine and histamine receptors participate in the regulation of cellular adhesion and migratory events. Histamine increases fibronectin-induced migration capacity of lung fibroblasts, an action that is inhibited by pharmacologically blocking H4HR.34 In isolated coronary venular endothelial cells, histamine exerts an increase in the phosphorylation of paxillin demonstrating the ability of histamine to have an influence on focal adhesion proteins.35 s100A4 is known to be an inducer of EMT and it has been shown that targeting this mediator may lead to a decrease in head and neck cancer growth.36 In colon cancer, the expression of vimentin is up-regulated and a potential contributing factor in increasing EMT.37 Our study demonstrates that clobenpropit targets components such as s100A4, vimentin, fibronectin, and paxillin, decreasing their expression, thus disrupting EMT.

Breakdown of the ECM must occur for tumors to become mobile and relocate themselves. In keratinocytes, histamine increases the expression of MMP-9 by way of the H1HR38 and contributes to the active migratory potential through the basement membrane.39 A correlation between MMPs and pancreatic cancer has been demonstrated and in this study histamine, by way of H1 and H2 HRs, increased the tumoral progression of pancreatic cancer.26 In our study we found that H4HR (which has an inhibitory role) decreases the expression of numerous MMPs, protecting the ECM from degradation and other organs from tumor invasion.

Although our findings in the present study suggest that clobenpropit decreases migratory movement in CCA, clobenpropit has also been shown to increase chemotaxis in other cell types including dendritic cells.40 Further, H4HR has been found to inhibit the chemoattractant, interleukin 12 in monocyte-derived dendritic cells, and block the chemotactic property of histamine in human eosinophils.10 During carcinogenesis there is an increase in chemoattractants like histamine and interleukin that can potentiate tumor spreading and growth. Agents like clobenpropit have the potential to decrease chemotactic activities including that are found in CCA.

Histamine has both trophic and antitrophic effects on cancer growth both in vitro and in vivo.4 In a number of cancers, the H3 and H4 HRs have an inhibitory role whereas, the H1 and H2 HRs mediate a stimulatory mitotic action.4 A recent study involving thyroid cancer demonstrated that histamine exerted a self-directed effect on the growth of this cancer potentially mediated by the H1 and H2 HRs.41 The differential effects of histamine (by acting through the four receptors) have been found in many cancers including colon, pancreatic, and breast.4 Our current studies offer further evidence of the ability of histamine (solely by way of H4HR) to play a critical role in the inhibition of cancer progression both in vitro and in vivo.

In summary, these experiments define the role of the H4HR in CCA growth, EMT, and invasion potential. By inhibiting critical components and proteins involved in EMT, adhesion, and ECM breakdown we have shown that activation of H4HR may be a potential therapeutic target for human CCA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. Gianfranco Alpini for advice regarding the development of the article, Julie Venter for assistance with animal studies and cell culture, Kimberly Baker and Mellanie White for technical assistance, and the Texas A&M Microscopy Center for assistance with confocal microscopy.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24573_sm_suppfig1.tif488KSupporting Figure 1 Using the lowest dose of clobenpropit found to be inhibitory in all CCA cell lines (10 μM), we found that clobenpropit decreased CCA proliferation beginning at 6 hours and up to 48 hours compared to basal treatment. *p < 0.05 versus basal and data are SEM ± 12 experiments.
HEP_24573_sm_suppfig2.tif448KSupporting Figure 2 MTS assays in nonmalignant cell lines revealed that clobenpropit had no effect at doses of 10 μM stimulated for 6 hours, but began to decrease normal cell proliferation at higher doses (25 and 50 μM) and longer times (48 hrs). *p < 0.05 versus basal and data are SEM ± 12 experiments.
HEP_24573_sm_suppfig3.tif314KSupporting Figure 3 Using the Pearson's Correlation Coefficient analysis in several of our CCA cell lines, we found that the expression of H3HR does not contribute to the cell viability (or cell proliferation) seen after clobenpropit treatment and no significant correlation was detected. Further, using the same analysis we have assessed the correlation between H4HR expression and cell proliferation. Here we found no significant correlation between histamine receptor gene expression and the anti-proliferative effects of clobenpropit.
HEP_24573_sm_suppfig4.tif424KSupporting Figure 4 (a) siRNA transfection resulted in the genetic knockdown of H3HR in Mz-ChA-1 cells at a 50% efficiency rate as shown by real-time PCR for H3HR gene expression. (b) PCNA proliferation by immunoblots in Mz-ChA-1 cells stimulated with clobenpropit in both mock- and H3HR-siRNA transfected cells was performed. The H3HR agonist, RAMH was added as a positive control. In mock-transfected cells, RAMH and clobenpropit significantly decreased CCA growth. In Mz-ChA-1 cells transiently silenced for the H3HR by siRNA (40 nM), clobenpropit significantly inhibited CCA growth cells to a greater extent than in Mz-ChA-1 cells with normal H3HR expression. (c) Overexpression of H4HR was performed in Mz-ChA-1 cells treated with and without clobenpropit (10 μM). Gene and protein expression for H4HR was significantly increased after H4HR transfection compared to LacZ controls. (d) By PCNA protein expression, overexpression of H4HR (2.5 μg) significantly decreased Mz-ChA-1 proliferation compared to LacZ controls. *p < 0.05 versus basal treatment (or scrambled siRNA or LacZ controls) and data are SEM ± 6 experiments for immunoblots and SEM ± 6 for PCR.
HEP_24573_sm_suppfig5.tif248KSupporting Figure 5 Intracellular cAMP and IP3 signaling mechanisms were evaluated in Mz-ChA-1 cells stimulated with clobenpropit (10 μM). By RIA we found that clobenpropit (10 μM) significantly increased intracellular IP3 levels (a), but had no significant impact on cAMP levels (b). *p < 0.05 versus basal treatment and data are SEM ± 12 experiments. (c) MTS assays in Mz-ChA-1 cells stimulated with clobenpropit (10 μM) in the absence or presence of BAPTA/AM, Gö6976 or RpcAMPs demonstrated that BAPTA/AM and Gö6976, but not RpcAMPs block clobenpropit-induced CCA inhibition. *p < 0.005 versus basal treatment and #p < 0.05 versus clobenpropit treatment. Data are SEM ± 12 experiments.
HEP_24573_sm_suppfig6.tif234KSupporting Figure 6 By real-time PCR, clobenpropit significantly decreased the gene expression of MMP -1, -2, -3, -9 and -11 in CCA compared to basal treatment. *p < 0.05 versus basal mRNA and data are SEM ± 3 experiments.
HEP_24573_sm_suppfig7.tif949KSupporting Figure 7 Alamar blue (top) and TUNEL staining (bottom) were used to detect cell viability and apoptosis in Mz-CHA-1 cells cultured in matrigel. There was no difference in cell viability or in the number of apoptotic cells in the matrigel between the basal- and clobenpropit-treated group with either alamar blue assay (top) or TUNEL staining (bottom, viable cells are labeled by red, apoptotic cells are labeled by blue). Alamar blue fluorescence was recorded using a fluorescence microplate reader using 560/590 nm ex/em filter settings, not significant p > 0.05 relative to basal-treatment. TUNEL was evaluated by BrdU incorporation by terminal deoxynucleotidyl transferase at the site of DNA fragmentation and detected by a highly specific and sensitive biotinylated anti-BrdU antibody and visualized by a streptavidin-horseradish peroxidase conjugate. The cells were counterstained with methyl green.
HEP_24573_sm_suppinfo.doc161KSupporting Information.
HEP_24573_sm_supptable1.doc44KSupporting Table 1.

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