Hepatocyte Cell Adhesion Molecule Research Articles

Panobinostat reverses HepaCAM gene expression and suppresses proliferation by increasing histone acetylation in prostate cancer.

Increased expression of histone deacetylases (HDACs) affiliated to the epigenetic regulation is common aberration in prostate cancer (PCa). We have confirmed that hepatocyte cell adhesion molecule (hepaCAM), acting as a tumor suppressor gene, is rarely expressed in PCa previously, However, the mechanisms of which is still unknown. The level of histone acetylation reportedly may involve anti-oncogene transcription and expression. In this study, we investigated the effect of panobinostat, the broad-spectrum histone deacetylases inhibitor, on PCa LNCaP and DU145 cell growth, and observed re-expression of hepaCAM when treated with panobinostat. We demonstrated that intranuclear acetylation of lys9 of histone H3 (Ac-H3K9) were increased, while that of both mRNA and protein of HDAC1, HDAC3, and HDAC4 were decreased when the treating concentration of panobinostat increased. We confirmed the relationship between histone acetylation and the expression of hepaCAM and AR in prostate cancer tissues. We also confirmed that panobinostat could overcome the resistance for androgen deprivation therapy (ADT). Further, we combined panobinostat with Ad-hepaCAM, which resulted in significantly increased antitumor activity and significant attenuation of the proliferation-associated genes CCND1 and PCNA compared to each single treatment. In conclusion, panobinostat may enhance the acetylation of lys9 of histone 3 and reverse the hepaCAM expression through its inhibitory effect on HDACs activity in PCa LNCaP and DU145 cells; Ad-hepaCAM combined with panobinostat may synergistically inhibit the growth of LNCaP and DU145 cells, via a potential mechanism associated with the down-regulation of the expression of CCND1 and PCNA. These findings suggest that this therapeutic strategy should be further developed in clinical trials.

STAT3 phosphorylation is required for the HepaCAM-mediated inhibition of castration-resistant prostate cancer cell viability and metastasis.

Castration-resistant prostate cancer (CRPC) is an advanced disease that is difficult to treat, the mechanism of it is unclear. This study illustrated the function of hepatocyte cell adhesion molecule (HepaCAM) on CRPC cell viability and metastasis. The expression of HepaCAM and p-STAT3 in CRPC tissues were determined by immunohistochemistry and western blot analysis. Cell Counting Kit-8 and colony formation assays were deployed to analyze the growth ability of CRPC cells following the adenovirus-mediated re-expression of HepaCAM. CRPC cell migration and invasion capacity were investigated by wound healing and Matrigel-coated transwell assays, respectively. The messenger RNA or protein levels of p-STAT3, CyclinD1, cMyc, MMP2, MMP9, and VEGF were determined by reverse transcription (RT) followed by quantitative real-time polymerase chain reaction (RT-qPCR), and western blot analysis after either HepaCAM re-expression alone or in combination with IL-22 treatment. A CRPC orthotopic xenograft mouse model was applied to investigate the functional effect of HepaCAM on the metastasis of CRPC cells to the lungs. The expression levels of HepaCAM were decreased while those of p-STAT3 were elevated in CRPC cells compare with surrounding benign tissues (p < .001). The overexpression of HepaCAM in CRPC cells notably reduced proliferation, migration, and invasion by inhibiting the expression of p-STAT3, CyclinD1, cMyc, MMP2, MMP9, and VEGF (p < .05). In addition, the expression of HepaCAM significantly inhibited the IL-22/p-STAT3 axis and the metastasis of CRPC cells to the lungs. Our data suggested that HepaCAM suppressed the viability of CRPC cells via the IL-22/p-STAT3 axis and inhibited the metastasis of CRPC cells from the prostate to the lungs (p < .05).

HepaCAM inhibits cell proliferation and invasion in prostate cancer by suppressing nuclear translocation of the androgen receptor via its cytoplasmic domain

Hepatocyte cell adhesion molecule (HepaCAM) is a tumour suppressor. However, the mechanism of HepaCAM function in prostate cancer (PCa) remains unknown. In the present study, HepaCAM, androgen receptor (AR) and Ran were analysed in 46 PCa tissue samples using immunohistochemistry. Subsequently, the influence of HepaCAM and its cytoplasmic domain on cell proliferation, migration, and invasion, and associated proteins was examined using MTT, wound healing, Transwell and western blotting assays, respectively. Furthermore, nuclear translocation of AR and Ran was analysed using immunofluorescence and Western blot assays. The results demonstrated that HepaCAM expression was reduced in PCa, and there was an association between downregulation of HepaCAM and changes in the distribution of AR and Ran. Furthermore, HepaCAM, specifically the cytoplasmic domain, was involved in cell proliferation, migration and invasion. Nuclear translocation of AR was dependent on HepaCAM and its cytoplasmic domain. Additionally, HepaCAM suppression of the nuclear translocation of AR occurred via Ran. The results suggest that HepaCAM and its cytoplasmic domain suppress the nuclear translocation of AR via Ran in PCa. The cytoplasmic domain of HepaCAM may serve as a novel target for therapy in PCa.

HepaCAM Regulates Warburg Effect of Renal Cell Carcinoma via HIF-1α/NF-κB Signaling Pathway

ObjectiveTo investigate how hepatocyte cell adhesion molecule (hepaCAM) regulates cancer energy metabolism through hypoxia-inducible factor (HIF-1α) in renal cell carcinoma (RCC). Materials and MethodsThe expression of hepaCAM and HIF-1α in RCC tissue samples was examined by immunohistochemistry. Glucose consumption and lactate production assays were used to detect metabolic activity in RCC cell lines. P65 and IκB kinase (IKKβ) mRNA and protein expression were detected using quantitative real-time polymerase chain reaction and western blotting, respectively. Nuclear translocation of P65 was observed by immunofluorescence staining after re-expressing hepaCAM. The luciferase reporter assay was applied to validate the transcriptional activity of HIF-1α. ResultsHIF-1α expression was elevated and hepaCAM suppressed in RCC compared with adjacent normal tissues. Furthermore, hepaCAM re-expression significantly decreased glycolytic metabolism in RCC cell lines, and reduced HIF-1α, IKKβ, and P65 expression. The expression of HIF-1α, GLUT1, LDHA, and PKM2 were further reduced with combined hepaCAM overexpression and treatment with the NF-κB inhibitor BAY11-7082, compared to hepaCAM overexpression alone. Additionally, hepaCAM decreased the transcriptional activity of HIF-1α and blocked P65 nuclear translocation by the NF-κB pathway. ConclusionOur data suggest that hepaCAM suppresses the Warburg effect via the HIF-1α/NF-κB pathway in RCC, which is a facilitating factor in hepaCAM-reduced tumorigenesis.

HepaCAM inhibits the malignant behavior of castration-resistant prostate cancer cells by downregulating Notch signaling and PF-3084014 (a γ-secretase inhibitor) partly reverses the resistance of refractory prostate cancer to docetaxel and enzalutamide in vitro.

Castration-resistant prostate cancer (CRPC) continues to be a major challenge in the treatment of prostate cancer (PCa). The expression of hepatocyte cell adhesion molecule (HepaCAM), a novel tumor suppressor, is frequently downregulated or lost in PCa. Overactivated Notch signaling is involved in the development and progression of PCa, including CRPC. In this study, we found that the activities of Notch signaling were elevated, while HepaCAM expression was decreased in CRPC tissues compared with matched primary prostate cancer (PPC) tissues. In addition, HepaCAM negativity was found to be associated with a worse progression-free survival (PFS). Furthermore, the overexpression of HepaCAM induced by transfection with a HepaCAM overexpression vector (Ad-HepaCAM) exerted antitumor effects by decreasing the proliferation, and suppressing the invasion and migration of bicalutamide-resistant (Bica-R) cells and enzalutamide-resistant (Enza-R) cells. Importantly, we found that the antitumor effects of HepaCAM on the resistant cells were associated with the downregulation of Notch signaling. Moreover, we revealed that PF-3084014 (a γ-secretase inhibitor) re-sensitized Enza-R cells to enzalutamide, and sequential dual-resistant (E+D-R) cells to docetaxel. Additionally, the findings of this study demonstrated that the use of PF-3084014 alone exerted potent antitumor effect on the resistant cells in vitro. On the whole, this study indicates that HepaCAM potentially represents a therapeutic target and PF-3084014 may prove to a promising agent for use in the treatment of refractory PCa.

Overexpression of Hepatocyte Cell Adhesion Molecule (hepaCAM) Inhibits the Proliferation, Migration, and Invasion in Colorectal Cancer Cells.

Hepatocyte cell adhesion molecule (hepaCAM), a new type of CAM, belongs to the immunoglobulin superfamily. Recently, hepaCAM was reported to be implicated in cancer development, and many researchers investigated its biological function in the tumorigenesis of various cancers. However, what kind of role hepaCAM plays in colorectal cancer (CRC) remains unknown. In this study, we found that hepaCAM was downregulated in CRC tissues and cell lines. Overexpression of hepaCAM inhibited CRC cell proliferation, migration, and invasion in vitro. Furthermore, the tumorigenesis assay showed that increased expression of hepaCAM suppressed CRC tumor growth and metastasis in vivo. We also demonstrated that overexpression of hepaCAM reduced the protein expression levels of β-catenin, cyclin D1, and c-Myc, indicating its inhibitory effect on the Wnt/β-catenin signaling pathway. In conclusion, our study results suggest hepaCAM as a promising therapeutic target for CRC and provide a better understanding for the molecular basis of CRC progression.

Interleukin 6 induces cell proliferation of clear cell renal cell carcinoma by suppressing hepaCAM via the STAT3-dependent up-regulation of DNMT1 or DNMT3b

Interleukin 6 (IL-6), a tumor promoting cytokine, has been largely implicated in the development of renal cell carcinoma (RCC). Hepatocyte cell adhesion molecule (hepaCAM) is a novel tumor suppressor, which is lost or down-regulated in many cancer types including RCC. In the present study, we intensively investigated the connection between IL-6 and hepaCAM in RCC. Our analysis of RCC tissues, adjacent tissues and paired serum samples from RCC patients revealed that IL-6 was elevated in patient serum and RCC tissue, whereas hepaCAM was completely lost or significantly down-regulated. Furthermore, we observed an association between IL-6 increase and hepaCAM decrease in RCC tissue samples. In the section of cytological researches, we found in RCC cell lines that IL-6 was a direct upstream regulator of hepaCAM, and that hepaCAM down-regulation was involved in IL-6-driven cell proliferation. We also demonstrated that IL-6-mediated promoter hypermethylation largely accounted for the hepaCAM loss in RCC, and it was STAT3-dependent. Additionally, our data showed that DNMT1 up-regulation induced by IL-6/STAT3 signaling was indispensable for IL-6-mediated hepaCAM loss in RCC cell lines ACHN and 769-P, while DNMT3b up-regulation was crucial for hepaCAM loss in A498. Our findings provide a novel signal pathway regulating cell proliferation, potentially representing a therapeutic target for RCC.

Expression and relationship between hepaCAM protein and multidrug resistance protein in renal carcinoma

Objective To investigate the expression and relationship between hepatocyte cell adhesion molecule (hepaCAM) protein and some multidrug resistance proteins in renal carcinoma tissue. Methods Expressions of hepaCAM, multidrug resistance associated protein (MRP), P-glycoprotein (P-gp), lung resistance protein (LRP), and topoisomerase Ⅱ (TOPO Ⅱ) protein were detected by immunohistochemistry in different areas of human renal cell carcinoma tissues and their relationships were analyzed. Results In the peripheral zone of renal tumor, hepaCAM, MRP, P-gp and LRP protein were showed positive expression. In the central region of the renal tumor, the expressions of hepaCAM, P-gp and LRP were negative or weakly positive, while the expressions of MRP and TOPO Ⅱ protein were positive. The expressions of MRP and TOPO Ⅱ protein in the central region of tumor were stronger than those in the peripheral zone of tumor (31.23±5.67 vs. 23.89±4.56; 45.66±2.34 vs. 5.23±0.66), with statistically significant differences (t=-6.20, P=0.00; t=-100.16, P=0.00). While the expressions of other proteins (hepaCAM, P-gp and LRP) in the central region of tumor were weaker than those in the peripheral zone of tumor (3.21±1.12 vs. 27.25±2.23; 2.34±0.33 vs. 51.23±3.45; 4.22±1.78 vs. 44.23±1.45), with statistically significant differences (t=60.87, P=0.00; t=90.35, P=0.00; t=107.18, P=0.00). Correlation analysis showed that the expression of hepaCAM protein in the central region of renal carcinoma was related with the expression of MRP protein (r=0.94, P=0.01), but it was not related with the expressions of P-gp, LRP and TOPO Ⅱ protein (r=0.22, P=0.44; r=0.14, P=0.80; r=0.34, P=0.07). Conclusion The expression of hepaCAM protein in renal carcinoma may be related to tumor drug-resistance. Key words: Kidney neoplasms; Cell adhesion molecules; Multidrug resistance-associated proteins

The SMAD2/3 pathway is involved in hepaCAM-induced apoptosis by inhibiting the nuclear translocation of SMAD2/3 in bladder cancer cells.

The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-β1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-β1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-β1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.

5-azacytidine inhibits the proliferation of bladder cancer cells via reversal of the aberrant hypermethylation of the hepaCAM gene.

Hepatocyte cell adhesion molecule (hepaCAM), a tumor-suppressor gene, is rarely expressed in bladder carcinoma. However, little is known concerning the mechanisms of low hepaCAM expression in bladder cancer. Abnormal hypermethylation in the promoter plays a crucial role in cancer by silencing tumor-suppressor genes, which is catalyzed by DNA methyltransferases (DNMTs). In the present study, a total of 31 bladder cancer and 22 adjacent tissues were assessed by immunohistochemistry to detect DNMT3A/3B and hepaCAM expression. Methylation of hepaCAM was determined by methylation‑specific polymerase chain reaction (MSP). The mRNA and protein levels of DNMT3A/3B and hepaCAM were determined by RT-PCR and western blot analysis after treatment with 5-azacytidine (AZAC). Following AZAC treatment, the proliferation of bladder cancer cells was detected by CCK-8 and colony formation assays. Cell cycle distribution was examined by flow cytometry. To further evaluate the tumor‑suppressive roles of AZAC and the involved mechanisms, the anti-tumorigenicity of AZAC was tested in vivo. The expression of DNMT3A/3B protein was markedly increased in the bladder carcinoma tissues (P<0.05), and had a negative linear correlation with hepaCAM expression in the same patients according to Pearson's analysis (r=-0.7176/-0.7127, P<0.05). The MSP results indicated that the hepaCAM gene was hypermethylated in three bladder cancer cell lines. Furthermore, we found that downregulation of DNMT3A/3B expression, after treatment with AZAC, reversed the hypermethylation and expression of hepaCAM in bladder cancer cells. In addition, AZAC inhibited the proliferation of bladder cancer cells and arrested cells at the G0/G1 phase. The in vivo results showed that expression of DNMT3A/3B and hepaCAM as well as tumor growth of nude mice were markedly altered which corresponded with the in vitro results. Due to the ability to reactivate expression of hepaCAM and inhibit growth of bladder cancer cells, AZAC may represent an effective treatment for bladder cancer.

HEPACAM inhibited the growth and migration of cancer cells in the progression of non-small cell lung cancer.

Hepatocyte cell adhesion molecule (HEPACAM), a member of immunoglobulin superfamily, is an adhesion molecule. Although dysregulation of several adhesion molecules has been implicated in the progression of non-small cell lung cancer (NSCLC), the expression profile and functions of HEPACAM in NSCLC remains unknown. In this study, it was found that the expression of HEPACAM was downregulated in NSCLC tissues. Forced expression of HEPACAM in NSCLC cells inhibited the growth and migration of the cancer cells, while knocking down the expression of HEPACAM promoted cell growth, migration, and metastasis. In the molecular mechanism study, HEPACAM was found to be a negative regulator of beta-catenin/TCF signaling. Taken together, this study revealed the suppressive roles of HEPACAM in NSCLC and restoring the function of HEPACAM in NSCLC might be a promising strategy for the therapy.

Expression of hepaCAM inhibits bladder cancer cell proliferation via a Wnt/β-catenin-dependent pathway in vitro and in vivo

We previously established that hepatocyte cell adhesion molecule (hepaCAM), a typical structure of immunoglobulin (Ig)-like adhesion molecules, inhibited the proliferation and the progression of cultured human bladder cancer cells. As increasing evidence reveals that aberrant activation of canonical Wnt pathway is involved in the pathogenesis of bladder cancer, and β-catenin serves as a pivotal molecule of Wnt pathway. Then, we explored whether the anti-proliferation effect of hepaCAM was associated with Wnt/β-catenin pathway in human bladder cancer cells. The negative correlation between hepaCAM and β-catenin in transitional cell carcinoma of bladder (TCCB) was found. Follow by, studied the effect of hepaCAM on the key elements of Wnt pathway. Here, Our researches showed that hepaCAM played a central role in modulating the Wnt/β-catenin signaling pathway by interfering nuclear protein levels of β-catenin, leading to down-regulate transcriptional activity of LEF/TCF and its target genes c-Myc and cyclinD1. Mechanistically, we demonstrated that hepaCAM-activated GSK3β led to elevate the phosphorylation of β-catenin, contributing to the aberrant translocation of β-catenin. In addition, Anti-proliferation and associated molecular mechanisms of hepaCAM were demonstrated by using vivo experiment. In conclusion, our reports uncover that expression of hepaCAM suppresses the proliferation of bladder cancer cells through a Wnt/β-catenin-dependent signaling pathway in vitro and in vivo.

HepaCAM inhibits clear cell renal carcinoma 786-0 cell proliferation via blocking PKCε translocation from cytoplasm to plasma membrane

Hepatocyte cell adhesion molecule (HepaCAM) plays a crucial role in tumor progression and has been recognized as a novel tumor suppressor gene. The high protein expression level of protein kinase Cε (PKCε) has been discovered in many tumor types. In the present study, we determined HepaCAM and PKCε protein levels in human clear cell renal cell carcinoma (ccRCC) tissues and analyzed the correlation between them. We observed an inverse relationship in the expression of HepaCAM and PKCε in ccRCC and adjacent normal tissues. In ccRCC tissue, HepaCAM expression was undetectable while PKCε expression was high; the opposite was found in the adjacent normal tissue. Western blot analysis demonstrated that PKCε cytosolic protein levels increased while plasma membrane protein levels decreased without any change in total protein following infection of the ccRCC cell line 786-0 with adenovirus-GFP-HepaCAM (Ad-GFP-HepaCAM). Moreover, the application of Ad-GFP-HepaCAM combined with a PKCε-specific translocation inhibitor (εV1-2) effectively inhibited 786-0 cell growth. Ad-mediated expression of HepaCAM in 786-0 cells reduced the levels of phosphorylated AKT and cyclin D1 and inhibited cell proliferation. In summary, our studies point to interesting connections between HepaCAM and PKCε in tissues and in vitro. HepaCAM may prevent the translocation of PKCε from cytosolic to particulate fractions, resulting in the inhibition of 786-0 cell proliferation. Therapeutic manipulation of these novel protein targets may provide new ways of treating ccRCC.

The 786-0 renal cancer cell-derived exosomes promote angiogenesis by downregulating the expression of hepatocyte cell adhesion molecule

The aims of the current study were to determine whether 786‑0 renal cancer cell‑derived exosomes promote human umbilical vein endothelial cells (HUVECs) to form tubular structures and to uncover the underlying mechanisms associated with this process. Exosomes were extracted and purified using ultrafiltration and sucrose gradient centrifugation and characterized by transmission electron microscopy. Tubular structure formation was observed using the matrigel tubular assay. In addition, an adenovirus vector was used to transfect the hepatocyte cell adhesion molecule (hepaCAM) gene into renal cancer 786‑0 cells. The expression of hepaCAM and vascular endothelial growth factor (VEGF) mRNA and protein was determined by reverse transcription‑polymerase chain reaction and western blot analysis, respectively. Tumor cell‑derived exosomes were observed to significantly increase tubular formation in HUVECs. Following transfection with the hepaCAM gene, VEGF expression in 786‑0 cells was markedly decreased. In HUVECs, exosome treatment increased VEGF mRNA and protein expression, while hepaCAM expression was only decreased at the protein level. In the present study, renal cancer 786‑0 cell‑derived exosomes significantly promoted angiogenesis via upregulation of VEGF expression in HUVECs, which may be induced by the downregulation of hepaCAM.

Influence of hepatocyte cell adhesion molecule on gene expression profile of human bladder transitional cell carcinoma cell line

To investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule(hepaCAM) overexpression. Affymetrix Human Genome U133 Plus 2.0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein(GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells.Significant Analysis of Microarray(SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data. Compared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated.Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data.The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression. HepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.

Identification of hypermethylation in hepatocyte cell adhesion molecule gene promoter region in bladder carcinoma.

Background: Epigenetic regulation such as aberrant hypermethylation of CpG islands in promoter plays a key role in tumorigenesis. 5-Aza-2'-deoxycytidine (5-aza-CdR) which is a potent inhibitor of DNA methylation can reverse the abnormal hypermethylation of the silenced tumor suppressor genes (TSGs). It has been reported that hepatocyte cell adhesion molecule (hepaCAM) acts as a tumor suppressor gene and expression of its mRNA and protein were down-regulated in bladder cancer. Over-expression of hepaCAM can inhibit cancer growth and arrest renal cancer cells at G0/G1 phase. In this study, we investigated the methylation status of hepaCAM gene, as well as the influence of 5-aza-CdR on expression of hepaCAM gene in bladder cancer cells. Methods: CpG islands in hepaCAM promoter and methprimers were predicted and designed using bioinformatics program. Methylation status of hepaCAM promoter was evaluated in bladder cancer tissues and two cell lines (T24 and BIU-87) by Methylation-specific PCR; Western blot and Immunofluorescence were used to detect expression of hepaCAM protein after 5-aza-CdR treatment; Flow cytometry assay was performed to determine effectiveness of 5-aza-CdR on cell cycle profile. Results: CpG island in promoter of hepaCAM gene was hyper-methylated both in bladder carcinoma tissues and cell lines (T24 and BIU-87). Otherwise, aberrant methylation of its promoter was associated with its decreased expression. Hypermethylation of hepaCAM gene was reversed and expression of its mRNA and protein were re-activated in two cell lines by DNA methyltransferases inhibitor 5-aza-CdR. Flow cytometry assay demonstrated that 5-aza-CdR can inhibit growth of cancer cells by arresting cancer cells at G0/G1 phase. Conclusion: Abnormal hypermethylation in CpG island of hepaCAM promoter is involved in absence of hepaCAM gene expression when bladder cancer occurs. Re-activation of hepaCAM gene by 5-aza-CdR can inhibit growth of cancer cells and arrest cells at G0/G1 phase.

P.2.e.019 Remission of manic and depressive symptoms with asenapine in patients with mixed episodes

Interleukin 6 (IL-6), a tumor promoting cytokine, has been largely implicated in the development of renal cell carcinoma (RCC). Hepatocyte cell adhesion molecule (hepaCAM) is a novel tumor suppressor, which is lost or down-regulated in many cancer types including RCC. In the present study, we intensively investigated the connection between IL-6 and hepaCAM in RCC. Our analysis of RCC tissues, adjacent tissues and paired serum samples from RCC patients revealed that IL-6 was elevated in patient serum and RCC tissue, whereas hepaCAM was completely lost or significantly down-regulated. Furthermore, we observed an association between IL-6 increase and hepaCAM decrease in RCC tissue samples. In the section of cytological researches, we found in RCC cell lines that IL-6 was a direct upstream regulator of hepaCAM, and that hepaCAM down-regulation was involved in IL-6-driven cell proliferation. We also demonstrated that IL-6-mediated promoter hypermethylation largely accounted for the hepaCAM loss in RCC, and it was STAT3-dependent. Additionally, our data showed that DNMT1 up-regulation induced by IL-6/STAT3 signaling was indispensable for IL-6-mediated hepaCAM loss in RCC cell lines ACHN and 769-P, while DNMT3b up-regulation was crucial for hepaCAM loss in A498. Our findings provide a novel signal pathway regulating cell proliferation, potentially representing a therapeutic target for RCC.

HepaCAM induces G1 phase arrest and promotes c-Myc degradation in human renal cell carcinoma

Hepatocyte cell adhesion molecule (hepaCAM) encodes a generally inactive phosphorylated glycoprotein which mediates cancer cell proliferation, migration, and differentiation. We have reported that hepaCAM is down-regulated in renal cell carcinoma (RCC) and takes responsibility of cell growth inhibition. However, the precise mechanisms of hepaCAM inhibits cell growth is still unknown. In this study, we demonstrated that re-expression of hepaCAM can cause an accumulation in G0/G1 phase in 786-0 cells. This reaction was accompanied by a substantial reduction of c-Myc expression through using an ectopic hepaCAM expression system. Furthermore, we found a comparable decrease in proliferation and G0/G1 accumulation of 786-0 and RC-2 cells after treatment with a small molecule c-Myc inhibitor, 10058-F4. This indicated that the down regulation of c-Myc was an essential process in controlling growth inhibitory actions of hepaCAM. Nevertheless, re-expression of hepaCAM results in apparent reduction of c-Myc protein with no corresponding reduction of c-Myc mRNA. This suggests that this reaction might take place at a post-transcriptional level rather than transcriptional one. Consistent with these findings, hepaCAM decreased c-Myc stability by increasing the proportion of c-Myc phosphorylation on T58 which can be abrogated by a proteasomal inhibitor (MG132). Thus, our research implies that the decrease in c-Myc protein expression, resulting from ectopic expression of hepaCAM, may contribute to the inhibition of proliferation in these cells.

Expression and clinical significance of hepaCAM and VEGF in urothelial carcinoma

Investigate the expression of hepatocyte cell adhesion molecule (hepaCAM) and vascular endothelial growth factor (VEGF) mRNA in 55 cases of urothelial carcinoma to examine the potential relationship between hepaCAM and VEGF in urothelial carcinoma. Expression of hepaCAM and VEGF gene was determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) in 55 paired urothelial carcinoma specimens. T24 cells stably expressing hepaCAM gene were established by Lipofectamine 2000. RT-PCR and western blot analysis were used to detect gene and protein expression of hepaCAM and VEGF before and after transfection. MTT test was used to detect the effect of hepaCAM gene on the cell proliferation. RT-PCR showed that hepaCAM expression level was significantly lower, and VEGF was significantly higher in urothelial carcinoma tissues than in adjacent tissues (P < 0.05, P < 0.05). hepaCAM and VEGF were strongly correlated with tumor stage (P < 0.05, P < 0.05). Spearman correlation analysis showed lower hepaCAM level was associated with higher VEGF level (r = -0.277 P = 0.041). Experiments with T24 cells in vitro demonstrated the expression of VEGF mRNA and protein were significantly decreased after transfection of hepaCAM gene (P < 0.05, P < 0.05). Expression of hepaCAM resulted in a significant inhibition of T24 cells proliferation (P < 0.05). There is a close relationship between hepaCAM and VEGF in urothelial carcinoma. hepaCAM may be defined as a new target for diagnosis and anticancer therapy.

Expression of hepaCAM and Its Effect on Proliferation of Tumor Cells in Renal Cell Carcinoma

To evaluate hepaCAM (hepatocyte cell adhesion molecule) gene expression in patients with renal cell carcinoma (RCC) and to explore its effect on proliferation of 786-0 cells. hepaCAM is a tumor suppressor gene, which has been identified as a member of immunoglobulin superfamily cell adhesion molecule. Two-step reverse transcription-polymerase chain reaction was used to determine hepaCAM expression in 30 paired (RCC and the adjacent non-RCC) renal specimens. Transfection studies were carried out by expressing green fluorescent protein and green fluorescent protein-fused hepaCAM in 786-0 cells. Significant downregulation of hepaCAM was detected in 25 of 30 RCC patients tested. When transfected into 786-0 cells, the number of colony formation was reduced by 5-fold according to colony formation assay. MTT (3-diphenyltetrazolium bromide) showed the inhibition rates on the fourth, fifth, and sixth days of culturing were 26.5%, 38.1%, and 35.7%, respectively. Our data show that hepaCAM is frequently downregulated in RCC, and that exogenous hepaCAM exhibits antiproliferative effect on 786-0 cells, suggesting that silencing of hepaCAM may be associated with carcinogenesis of RCC.