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The innate immune system plays an important role as the first line of defense against many types of microbes. Accumulating reports suggest that human β-defensins (hBDs) are expressed by and have certain roles in some cancer cells. In this study, we investigated the roles of hBD-3 in colon cancer cells. The expression of hBD-3 was examined by reverse transcriptase-polymerase chain reaction analysis of colon cancer cell lines and immunohistochemical staining of colon cancer tissues. The effect of hBD-3 on proliferation of colon cancer was assessed using the MTT assay and a real-time cell analyzer, and the effect of hBD-3 on the migration of colon cancer cells was also examined. The results showed that hBD-3 is not expressed in colon cancer cells but is produced by tumor-infiltrating monocytes. Migration of colon cancer cells was significantly inhibited by hBD-3 in a dose-dependent manner, although proliferation of colon cancer cells was not affected by administration of hBD-3. Moreover, reduced expression of metastasis-associated 1 family, member 2 (MTA2) mRNA in colon cancer cells was associated with exposure to hBD-3. In conclusion, progression of colon cancer was inhibited by hBD-3 in a paracrine fashion. Therefore, hBD-3 may be a potent new agent for treating colon cancer.

Although several therapeutic options are available for hepatocellular carcinoma (HCC), the outcome is still very poor. One reason is the complexity of signal transduction in the pathogenesis of HCC. The aim of this study was to iden- tify new HCC-related genes and to investigate the functions of these genes in the pathogenesis and progression of HCC. Whole genomes of 15 surgically resected HCC specimens were examined for copy number alterations with compara- tive genomic hybridization. Gene expression was compared between HCC and normal liver tissues. The roles of the new genes in the progression of HCC were studied using cultured cell lines. Copy number gain in chromosome 8q was detected in 53% of HCC tissues examined. The gene that coded for collagen triple helix repeat containing 1 (CTHRC1), located at chromosome 8q22.3, was overexpressed in HCC compared with normal or liver cirrhosis tissues and identified as a new HCC-related gene. CTHRC1 deletion with short hairpin RNA significantly reduced proliferation, migration and invasion of HepG2 and Huh7 cells. In addition, mRNA of integrins β-2 and β-3 was downregulated, with deletion of CTHRC1 in these cells. Immunohistochemical staining on resected HCC tissues showing positive staining areas for CTHRC1 was significantly greater in poorly-differentiated HCC compared with well-differentiated HCC. Moreover, some cases showed strong staining for CTHRC1 in invasive areas of HCC. CTHRC1 has the potential to be a new biomarker for the aggressive HCC, and to be a new therapeutic target in treating HCC.

Altered epigenetic control of gene expression plays a substantial role in tumor development and progression. Accumulating studies suggest that somatic mutations of CREB binding proteins (CBP)/p300 occur in some cancer cells. CBP/p300 possess histone acetyltransferase (HAT) activity, and are involved in many cellular processes. In this study, we investigated the expression and functional role of CBP/p300 in hepatocellular carcinoma (HCC) using the specific inhibitor C646 of CBP/p300 HAT activity. We examined its effect on several apoptosis-related proteins and invasion-related genes. The results showed that CBP/p300 were highly expressed in HCC tissues and that expression of p300, but not of CBP, was strongly correlated with the malignant character of HCC. C646 inhibited proliferation of HCC cell lines in a dose dependent manner. C646 significantly augmented TRAIL-induced apoptotic sensitivity, which was accompanied by reduced levels of survivin, in HepG2, HLE and SK-HEP1 cells. C646 significantly inhibited invasion of Huh7, HLE and SK-HEP1 cells. The level of matrix metallopeptidase 15 (MMP15) mRNA expression was significantly reduced, whereas the level of laminin alpha 3 (LAMA3) and secreted phosphoprotein 1 (SPP1) mRNA expression was significantly increased in Huh7 cells following exposure to C646. In conclusion, our results suggest that CBP/p300 HAT activity has an important role in malignant transformation, proliferation, apoptotic sensitivity and invasion in HCC. CBP/p300 could be a promising therapeutic target in HCC.

A multi-kinase inhibitor, sorafenib, was recently approved and is currently recommended for the treatment of advanced hepatocellular carcinoma (HCC). However, HCC treatment outcomes are still poor and necessitate improvement. Therefore, we investigated the influence of sorafenib in combination with each of cytotoxic chemotherapy agents, hypoxia or tumor necrosis factor (TNF)-related apoptosis‑inducing ligand (TRAIL), on cytotoxicity to determine which is the better adjuvant. Additive cytotoxicity of sorafenib to chemotherapy agents, hypoxia and TRAIL, to HCC cells was assessed using cell viability assay. Intracellular levels of anti-apoptotic proteins were determined using western blot analysis. Activation of Wnt/β-catenin signaling was assessed using a luciferase reporter gene assay. Sorafenib significantly and synergistically enhanced the cytotoxicity of TRAIL to HCC cells and 4',6-diamidino-2-phenylindole (DAPI) staining showed increased apoptosis among cells treated with sorafenib and TRAIL. This augmentation in cytotoxicity was derived from sorafenib-mediated downregulation of anti-apoptotic proteins. However, sorafenib did not enhance the cytotoxicity of chemotherapy agents (cisplatin, 5-FU or doxorubicin) or hypoxic treatment to HCC. Moreover, hypoxic treatment induced Wnt/β-catenin signaling activation. Our data showed that in combination TRAIL and sorafenib had a synergistic cytokilling effect on HCC cells and that this effect derived from sorafenib-mediated downregulation of anti-apoptotic proteins.

Toll-like receptor 9 (TLR9) is a pattern-recognition receptor that is involved in immune signaling and plays a crucial role in cell survival through recognition of various bacterial and viral components including unmethylated CpG-DNA. TLR9 expression and function in cancer cells are not well understood. We investigated the expression of TLR9, and the function of TLR9 signaling, in hepatocellular carcinoma (HCC) cells following stimulation with CpG-oligodeoxynucleotides (ODNs). Positive immunohistochemical staining for TLR9 was observed in 85.7% of HCC tissues. Western blot analysis revealed that TLR9 was expressed both on the cell membrane and in the cytoplasm of HCC cell lines. Full-length TLR9 was predominantly expressed on the membrane rather than in the cytoplasm, whereas multiple cleaved forms of TLR9 were predominantly expressed in the cytoplasm rather than on the membrane. Cell surface stimulation of TLR9 promoted cell proliferation, and, furthermore, the TLR9 agonists, CpG-ODNs, reduced the cytotoxicity of the anti-cancer drug adriamycin (ADM) via up-regulation of apoptosis inhibitors such as survivin, Bcl-xL, XIAP and cFLIP, in HCC cell lines. Although cell surface stimulation of TLR9 did not activate either the NF-kappaB signaling pathway or the type-I IFN secretion pathway, gene chip microarray analysis indicated that TLR9 agonists closely regulated multiple oncology-related genes and transcription factors involved in tumorigenesis and cancer progression. In conclusion, our results indicate that functional cell surface expression of TLR9 in human HCC may play an important role in tumorigenesis and cancer progression.

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). TLR3 expression and function in cancer cells are not well understood. We investigated the expression of TLR3 in hepatocellular carcinoma (HCC) cells and the function of TLR3 signaling by stimulation and transfection with polyinosinic-polycytidylic acid (Poly I:C), a synthetic form of dsRNA. TLR3 mRNA was expressed in HCC tissues as well as in non-tumor tissues. Positive immunohistochemical staining for TLR3 was observed in 52.7% of HCC tissues, and in HCC cells we found both membranous and cytoplasmic expression of TLR3. While cell surface stimulation of TLR3 with Poly I:C did not affect cell viability, it did activate NF-kappaB levels. In contrast, cytoplasmic stimulation with transfected Poly I:C significantly induced apoptosis accompanied by the down-regulation of anti-apoptotic protein. Transfected Poly I:C also synergistically augmented TRAIL-induced apoptosis, but only with low levels of transfected Poly I:C was IFN-beta production not observed. In conclusion, our results indicate that TLR3 expression in HCC plays an important role with regard to cell survival and proapoptotic activity. Endogenously expressed TLR3 may provide new clinical prospects for TLR3 agonists as cytotoxic agents in HCC.

In this article, Fig. 2 is incorrect. The corrected Fig. 2 is shown using data from the tissue array samples. The new figure demonstrates the same findings as the original figure. Accordingly, in the paragraph of Materials and methods, the sentence '...surgically resected colon cancer tissues' and 'This study was...research committee' and in the paragraph of Results, the sentence 'Similar results were...tissue array samples' should be deleted. The above changes do not alter the original conclusions of this study. [the original article was published in the International Journal of Oncology 45: 1059-1064, 2014 DOI: 10.3892/ijo.2014.2507].

TNF-related apoptosis-inducing ligand (TRAIL), as well as Fas ligand, plays a pivotal role in lymphocyte cytotoxicity and the maintenance of immunological homeostasis in various tissues, but its physiological role in immune evasion of cancer cells remains unknown. We have previously shown strong resistance to TRAIL-induced cytotoxicity in human hepatocellular carcinomas (HCCs). The current study investigates the expression of TRAIL in HCCs. We found that three HCC cells, HepG2, Hep3B and Huh7 cells, constitutively express TRAIL mRNA and protein, as detected by reverse transcriptase PCR and Western blotting. Four of 10 human HCC tissues demonstrated positive staining for TRAIL, whereas non-tumor tissues showed little detectable staining. TRAIL expression on tumor cells was detected by flow cytometry and was dramatically induced after the addition of doxorubicin, a chemotherapeutic agent, or cytokine stimulation with TNF-alpha, IL-1beta or IL-18. This expression was induced principally via the NF-kappaB activation pathway, since IkappaB transfection significantly reduced TRAIL expression. In addition, the expressed TRAIL was functional. The TRAIL on HCC cells induced apoptosis in Jurkat cells that are sensitive to TRAIL-mediated apoptosis, and this process was specifically inhibited by recombinant TRAIL-receptors:Fc which binds to TRAIL. In conclusion, TRAIL expressed on the surface of HCC cells by cytokines or cytostatic drugs might contribute to an alternative mechanism that enables tumors to evade immune surveillance by inducing apoptosis of activated human lymphocytes.

The TNF-like weak inducer of apoptosis (TWEAK) can induce diverse cellular responses, including cell death, inflammation, migration, and proliferation in various transformed cell lines. We investigated TWEAK sensitivity, TWEAK effects on nuclear factor-kappaB activation, and expression of TWEAK in the HT-29, LS180, SK-CO-1 and SW480 human colonic adenocarcinoma cell lines, all of which express the TWEAK receptor (Fn14). TWEAK alone induced cell death in SW480 cells and induced cell death of HT-29 cells after addition of IFN-gamma, actinomycin D or cycloheximide. TWEAK did not affect cell viability of LS-180 or SK-CO-1 cells. Activation of NF-kappaB was not obviously influenced by TWEAK in any of the cell lines. All four human colonic adenocarcinoma cell lines constitutively expressed TWEAK mRNA, protein and membrane-bound TWEAK antigen, as detected by RT-PCR, Western blotting and flow cytometry. Stimulation by an anticancer drug (camptothecin) augmented cell surface expression of TWEAK and all human colonic adenocarcinoma tissue samples studied (n=59) demonstrated positive staining for TWEAK antigen. Soluble TWEAK was detected in culture medium of these cell lines by ELISA and conditioned medium from SW480 cells incubated with anti-TWEAK antibody significantly inhibited endothelial cell tube formation in Matrigels. Thus, functional expression of TWEAK from human colonic adenocarcinoma cells may contribute to neovascularization.

A staging system for hepatocellular carcinoma was reported from Italy (CLIP). In this study, we evaluate the CLIP scoring system and establish a new scoring system for predicting the prognosis of patients with hepatocellular carcinoma. Patients (n=141) who were diagnosed and who underwent initial treatment at our single institution were recruited retrospectively into this study. We evaluated markers for prognosis, using a stratified Cox proportional hazard regression model and Kaplan-Meier survival analysis. CLIP score differentiated patients with different survival experiences by Kaplan-Meier estimated survival analysis. However, with respect the CLIP score, more than two thirds of patients were included in the early stage (CLIP 0-1), and the group with better prognosis than the survival rate of all patients was the only one with CLIP 0. Multivariate analysis revealed that des-gamma-carboxy prothrombin (DCP) >/=100 mAU/ml (relative risk, 2.06; P=0.0218) was statistically significant as a predictor of poor survival. A new prognostic scoring system included DCP classified patients to 6 well-balanced groups (score 0-5). The new prognostic scoring system 0 group (14.9% of the cohort) and the CLIP score 0 group (34.0% of the cohort) had a median survival of 66.9 and 61.6 months. The new prognostic scoring system performs better for prediction of survival than either the CLIP score or the Child-Pugh stage. In conclusion, the described scoring system provides more accurate prognostic information than the CLIP scoring system. It may help physicians decide more appropriate clinical and therapeutic management.

A staging system for hepatocellular carcinoma was reported from Italy (CLIP). In this study, we evaluate the CLIP scoring system and establish a new scoring system for predicting the prognosis of patients with hepatocellular carcinoma. Patients (n=141) who were diagnosed and who underwent initial treatment at our single institution were recruited retrospectively into this study. We evaluated markers for prognosis, using a stratified Cox proportional hazard regression model and Kaplan-Meier survival analysis. CLIP score differentiated patients with different survival experiences by Kaplan-Meier estimated survival analysis. However, with respect the CLIP score, more than two thirds of patients were included in the early stage (CLIP 0-1), and the group with better prognosis than the survival rate of all patients was the only one with CLIP 0. Multivariate analysis revealed that des-gamma-carboxy prothrombin (DCP)/=100 mAU/ml (relative risk, 2.06; P=0.0218) was statistically significant as a predictor of poor survival. A new prognostic scoring system included DCP classified patients to 6 well-balanced groups (score 0-5). The new prognostic scoring system 0 group (14.9% of the cohort) and the CLIP score 0 group (34.0% of the cohort) had a median survival of 66.9 and 61.6 months. The new prognostic scoring system performs better for prediction of survival than either the CLIP score or the Child-Pugh stage. In conclusion, the described scoring system provides more accurate prognostic information than the CLIP scoring system. It may help physicians decide more appropriate clinical and therapeutic management.

Ethanol injection into HCC tumors is an effective therapy and percutaneous ethanol injection therapy (PEIT) is performed on many HCC patients. However, there are cases in which PEIT becomes difficult because the HCC could not be detected by ultrasonography or the tumor is located in an area where it is impossible to perform PEIT. Nine patients with HCC underwent laparoscopic ethanol injection therapy (LEIT) in our institution. Their tumors were located on the liver surface and could be visualized by laparoscopic examination. Ethanol injection was performed under laparoscopic direct visualization. The total injected ethanol volume required ranged from 4 to 15 ml and in most cases both tumor size and alpha-fetoprotein (AFP) levels decreased after LEIT. Three cases showed a transient complication of abdominal pain or/and portal vein damage. Other severe complications were not observed. All cases required additional therapies, including transcatheter arterial chemoembolization (TAE) or PEIT to complete the tumor necrosis. In conclusion, LEIT is a safe and effective therapy for HCC located on the liver surface, but should be combined with other therapies to facilitate its effect against HCC.

We performed interventional treatments on 50 patients with hepatocellular carcinoma (HCC) and analyzed the relationship between these treatments and the exacerbation of liver function after treatment. The different treatments included transcatheter arterial embolization (TAE), percutaneous ethanol injection therapy (PEIT), selective segmental sclerotherapy (SSS), combined TAE and PEIT, or transcatheter arterial chemo-injection (TAI). Thirteen patients showed an exacerbation of liver function after treatment. The laboratory data on admission, showed the lower levels of serum albumin and cholinesterase in this group. In comparison to patients who did not show any exacerbation of liver function, these 13 patients had undergone combined TAE and PEIT. An analysis of cases after TAE and PEIT treatment revealed that the time from TAE to PEIT was shorter in the exacerbation group than in the non-exacerbation group, however, there was no significant difference in the amount of injected ethanol between the two groups. It is assumed that the values of albumin and cholinesterase before treatment, or the period from TAE to PEIT are related to liver failure after treatment. Combining TAE and PEIT treatment may be effective for HCC, however, we should pay special attention to liver failure after treatment.

There are few reports describing cavernous hepatic hemangiomas with alteration of ultrasonographical imaging during examinations. We performed ultrasonographic examination of 64 cavernous hepatic hemangiomas and recognized 26 cases (41%) with an alteration of echogenicity during the examinations. We refer to this alteration of echogenicity of cavernous hepatic hemangioma as a "variable echo sign". We performed angiography of the cavernous hepatic hemangiomas with variable echo sign. Most of these imaging patterns showed mild or moderate pooling, suggesting that the alteration of echogenicity might be based on a slow blood flow exchange. We suggest that a variable echo sign is specific to ultrasonographic imaging with cavernous hepatic hemangioma and may be useful to differentiate cavernous hepatic hemangioma from other tumors.

The effect of segmental transcatheter arterial chemoembolization (TAE) on serum amino acid levels and liver function were studied in 23 patients with HCC associated with hepatitis virus C (22 patients) or alcoholism (1 patient), with compensated liver cirrhosis (Child A 18 patients, Child B 5 patients). Serum levels of branched-chain amino acids (BCAA), tyrosine, branched-chain amino acids to tyrosine ratio (BTR), ammonia, total bilirubin and albumin, and prothrombin times were measured before and after TAE (24 h, 7 and 14 days). The BTR was increased significantly 24 h after TAE (p

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). TLR3 expression and function in cancer cells are not well understood. We investigated the expression of TLR3 in hepatocellular carcinoma (HCC) cells and the function of TLR3 signaling by stimulation and transfection with polyinosinic-polycytidylic acid (Poly I:C), a synthetic form of dsRNA. TLR3 mRNA was expressed in HCC tissues as well as in non-tumor tissues. Positive immunohistochemical staining for TLR3 was observed in 52.7% of HCC tissues, and in HCC cells we found both membranous and cytoplasmic expression of TLR3. While cell surface stimulation of TLR3 with Poly I:C did not affect cell viability, it did activate NF-kappaB levels. In contrast, cytoplasmic stimulation with transfected Poly I:C significantly induced apoptosis accompanied by the down-regulation of anti-apoptotic protein. Transfected Poly I:C also synergistically augmented TRAIL-induced apoptosis, but only with low levels of transfected Poly I:C was IFN-beta production not observed. In conclusion, our results indicate that TLR3 expression in HCC plays an important role with regard to cell survival and proapoptotic activity. Endogenously expressed TLR3 may provide new clinical prospects for TLR3 agonists as cytotoxic agents in HCC.