Multi-Attribute Method for Quality Control of Therapeutic Proteins.

Recent advances in high resolution mass spectrometry (MS) instrumentation and semi-automated software have led to a push toward the use of MS-based methods for quality control (QC) testing of therapeutic proteins in a cGMP environment. The approach that is most commonly being proposed for this purpose is known as the multi-attribute method (MAM). MAM is a promising approach that provides some distinct benefits compared to conventional methods currently used for QC testing of protein therapeutics, such as CEX, HILIC, and CE-SDS. Because MS-based methods have not been regularly used in this context in the past, new scientific and regulatory questions should be addressed prior to the final stages of implementation. We have categorized these questions into four major aspects for MAM implementation in a cGMP environment for both new and existing products: risk assessment, method validation, capabilities and specificities of the New Peak Detection (NPD) feature, and comparisons to conventional methods. This perspective outlines considerations for each of these main points and suggests approaches to help address potential issues.

Fc receptor-like 5 promotes B cell proliferation and drives the development of cells displaying switched isotypes.

The biological roles of B cell membrane proteins in the FCRL family are enigmatic. FCRL proteins, including FCRL5, were shown to modulate early BCR signaling, although the subsequent, functional consequences of receptor engagement are poorly understood. We found that FCRL5 surface protein itself was induced temporarily upon BCR stimulation of human, naive B cells, indicating precise control over timing of FCRL5 engagement. Cross-linking of FCRL5 on cells induced to express FCRL5 enhanced B cell proliferation significantly. This enhancement required costimulation of the BCR and TLR9, two signals required for optimal proliferation of naive B cells, whereas T cell help in the form of anti-CD40 and IL-2 was dispensable. In addition, we found that FCRL5 stimulation generated a high proportion of cells displaying surface IgG and IgA. Optimal development of cells expressing switched isotypes required T cell help, in addition to stimuli found necessary for enhanced proliferation. Surprisingly, cells that developed upon FCRL5 stimulation simultaneously displayed surface IgM, IgG, and IgA. Cells expressing multiple Ig isotypes were described in hairy cell leukemia, a disease in which FCRL5 is overexpressed. Enhanced proliferation and downstream isotype expression upon FCRL5 stimulation could reflect a physiological role for FCRL5 in the expansion and development of antigen-primed B cells. In addition, FCRL5 may promote growth of malignant cells in hairy cell leukemia and other FCRL5-expressing tumors.

Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4⁺ T cells to T-regulatory cells.

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However, it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells, designated tumor-evoked Bregs (tBreg), phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-ß-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs, 4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications, as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis.

Single round of antigen receptor signaling programs naive B cells to receive T cell help.

To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and the transcription factor NF-kappaB activation indistinguishably from conventional continuous IgM crosslinking but did not induce G1 progression. However, single round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling.

B cell receptor-mediated sustained c-Rel activation facilitates late transitional B cell survival through control of B cell activating factor receptor and NF-kappaB2.

Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21(int)-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-kappaB2). Sustained c-Rel activation required de novo c-Rel gene transcription and translation via Btk-dependent mechanisms. Like T1 cells, mature B cells from Btk- and c-Rel-deficient mice also failed to activate these genes. These findings suggest that the gain of survival potential within transitional B cells is dependent on the ability to produce a long-term c-Rel response, which plays a critical role in T2 B cell survival and differentiation in vivo by inducing anti-apoptotic genes, BAFF-R and NF-kappaB2, an essential component for BAFF-R survival signaling. Thus, acquisition of resistance to apoptosis during transitional B cell maturation is achieved by integration of BCR and BAFF-R signals.

Aberrant expression of connexin 26 is associated with lung metastasis of colorectal cancer.

PURPOSE: Connexin 26 (Cx26) is one of the gap junction-forming family members classically considered to be tumor suppressors. However, recent studies show association of elevated expression of Cx26 with poor prognosis in several human malignancies. Furthermore, Cx26 has been observed to be indispensable to spontaneous metastasis of melanoma cells. Here, we assessed Cx26 expression in primary colorectal cancer (CRC) and the metastatic lesions to elucidate its role in metastasis. EXPERIMENTAL DESIGN: Cx26 expression was assessed in 25 adenomas, 167 CRCs, and normal mucosa, together with the metastatic lesions. RESULTS: Normal mucosa and adenomatous tissue expressed Cx26 mainly in the plasma membrane, whereas cancer cells mostly contained Cx26 in the cytoplasm. The incidence of aberrant Cx26 expression varied widely in CRC (mean, 49.5 +/- 35.5%), and the expression levels were confirmed by Western blot and quantitative reverse transcription-PCR. Clinicopathologic survey revealed association of high expression with less differentiated histology and venous invasion (P = 0.0053 and P = 0.0084, respectively). Notably, high Cx26 expression was associated with shorter disease-free survival and shorter lung metastasis-free survival in 154 curatively resected CRC sets (P = 0.041 and P = 0.028, respectively). Survey of metastatic lesions revealed that lung metastasis, but not liver and lymph nodes metastases, expressed higher Cx26 than the CRC series or corresponding primary CRCs (P < 0.0001 and P = 0.0001, respectively). CONCLUSIONS: These findings suggest that aberrant expression of Cx26 plays an essential role in lung metastasis. Thus, Cx26 is a promising therapeutic target, particularly for CRC patients who develop lung metastasis.

Combined intra-arterial 5-fluorouracil and subcutaneous interferon-alpha therapy for highly advanced hepatocellular carcinoma.

Because of the difficulties of low sensitivity for anticancer agents and giving sufficient dose because of poor liver function, chemotherapy may not play a central role for treatment of hepatocellular carcinoma (HCC) patients, especially those with liver cirrhosis. However, chemotherapy must be one of the important possibilities of multimodal treatment for advanced HCC, for which hepatic resection, percutaneous ablation, transcatheter arterial embolization and other general therapies would not be effective or even possible. Also, intra-arterial perfusion chemotherapy is a common therapy for HCC and it is not difficult to maintain; but the effective rate is not sufficient. Recently, the combination therapy of s.c. interferon (IFN)-alpha and intra-arterial 5-fluorouracil (5-FU) showed an outstandingly effective rate for intractable HCC (with portal vein thrombosis). In addition,recent preclinical and clinical studies have revealed that the mechanism of combination therapy may concern direct antitumor effects (through cell-cycle arrest and induction of apoptosis) and indirect actions (through immunocompetent cells and anti-angiogenic effect). For the further advance of HCC treatment and prognosis, this therapy might be a promising treatment modality and is expected to develop. In this review, we summarize recent clinical and preclinical data regarding IFN-alpha and 5-FU combination therapy and discuss the further prospects of this therapy.

Combination therapy of interferon-alpha and 5-fluorouracil inhibits tumor angiogenesis in human hepatocellular carcinoma cells by regulating vascular endothelial growth factor and angiopoietins.

We recently reported that interferon-alpha (IFN-alpha) and 5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC) achieved excellent clinical results. However, the mechanism underlying this combination therapy remains to be elucidated. In this study, we examined the anti-tumor effects of IFN-alpha and 5-FU combination therapy in vivo and aimed to reveal its anti-angiogenic effects by investigating the expression of vascular endothelial growth factor (VEGF) and angiopoietins (Ang-1 and Ang-2). Human HCC cells, HuH7, were subcutaneously injected in nude mice. Ten days later, groups of mice received treatment with IFN-alpha alone, 5-FU alone, or with a combination of IFN-alpha and 5-FU for four weeks. Immunohistochemical examinations of proliferating cell nuclear antigen (PCNA), cell differentiation antigen 34 (CD34), Ang-1, -2 and VEGF, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and quantification of VEGF, Ang-1 and-2 mRNA using real-time RT-PCR were performed. Results showed that IFN-alpha and 5-FU combination therapy significantly inhibited the growth of human HCC cells compared with the control group or single agent treatment. The combination therapy decreased PCNA-positive cells as well as microvessel density (MVD) and induced apoptosis of (TUNEL-positive cells) more than other treatment groups. Immunohistochemical analysis revealed that the combination therapy significantly decreased the expression of VEGF and Ang-2 and increased that of Ang-1. The ANG2/ANG1 mRNA expression ratio was significantly lower in the combination therapy group. In conclusion, our results suggested that IFN-alpha and 5-FU combination therapy has anti-proliferative and anti-angiogenic effects and can induce apoptosis in vivo. The synergistic and anti-angiogenic effects may in part be attributable to the regulation of Ang-1, -2 and VEGF.

Complete remission of hepatocellular carcinoma with portal vein tumor thrombus and lymph node metastases by arterial infusion of 5-fluorouracil and interferon-alpha combination therapy following hepatic resection.

We report two cases of hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT) and lymph node (LN) metastases successfully treated by hepatic arterial infusion of 5-fluorouracil (5-FU) combined with systemic injection of interferon (IFN)-alpha following hepatic resection for the liver tumor. Complete remission was obtained. Case 1 was a 51-year-old man who had HCC in the right lobe of the liver with PVTT and multiple intrahepatic metastases. He also had abdominal and mediastinal LN metastases. Case 2 was a 53-year-old man who had diffuse-type HCC in the right lobe of the liver with PVTT and intrahepatic metastases. A chest computed tomography scan revealed lymph nodes enlarged to 1.0 cm from the mediastinum to the left supraclavicular space. Both patients underwent the hepatectomy to reduce the tumor volumes and remove the PVTT to relieve portal vein obstruction. Following the surgery, the patients underwent IFN-alpha/5-FU combination therapy. Three months after this combined therapy, tumor markers (both alpha-fetoprotein and protein induced by vitamin K absence or antagonist II) returned to the normal range and residual tumors in the liver disappeared. The patients are alive without any recurrence more than 1 year after initial treatment. IFN-alpha/5-FU combined therapy following hepatic resection is a promising modality for the treatment of advanced HCC with LN metastasis.

Successful treatment of multiple hepatocellular carcinoma with tumor thrombi in the major portal branches by intraarterial 5-fluorouracil perfusion chemotherapy combined with subcutaneous interferon-alpha and hepatectomy.

We experienced a patient who received successful treatment for multiple hepatocellular carcinoma (HCC) nodules, with tumor thrombi in the major portal branches, with intraarterial 5-fluorouracil perfusion chemotherapy combined with subcutaneous interferon-alpha administration. The patient was a 50-year-old man with hepatitis C virus and HCC. The tumors consisted of a 5-cm main nodule in the right lobe (segment 8) and multiple intrahepatic metastases. The tumor also involved portal vein thrombosis throughout the right portal branch. After two cycles of interferon-alpha/5-fluorouracil combination chemotherapy, tumor markers demonstrated a decreasing tendency. Nine months after the initiation of this therapy, the tumors were limited to the right lobe and were surgically removed by S8 subsegmentectomy, S5 partial hepatectomy, and portal thrombectomy. The serum levels of both alpha-fetoprotein and protein induced by vitamin K absence II fell to normal levels after hepatic resection. Fifty-eight months after the first treatment, he is alive with several recurrent nodules in the liver. In conclusion, the interferon-alpha/5-fluorouracil combination therapy is a useful treatment for HCC in patients who have multiple intrahepatic metastases and portal vein thrombosis. In addition to this therapy, combined modality therapy including, for example, surgical resection, can sometimes have a dramatic therapeutic effect, shown by tumor markers reverting to normal levels.

Overexpression of MT3-MMP in hepatocellular carcinoma correlates with capsular invasion.

BACKGROUND/AIMS: Extracellular matrix-degrading matrix metalloproteinases (MMPs) are invariably up-regulated in epithelial cancers and are key agonists of angiogenesis, invasion and metastasis. Recent studies have shown high levels of various MMPs, including MT1-MMP, MMP-1, MMP-2 and MMP-9, and their involvement in tumor progression in human hepatocellular carcinoma (HCC). However, the expression and role of MT3-MMP in HCC remains unclear. METHODOLOGY: We examined the immunohistochemical expression of MT3-MMP in surgically resected HCCs (n=58), hepatitis C virus (HCV) and hepatitis B virus (HBV)-related chronic hepatitis (n=34) and cirrhosis (n=24). RESULTS: MT3-MMP expression was observed in all non-cancerous liver tissues. In HCCs, 52% (30/58) of patients showed high MT3-MMP expression while the remaining 48% (28/58) of patients showed low expression. A clinicopathological survey demonstrated a significant correlation between high MT3-MMP expression and capsular invasion of carcinoma (p = 0.034) although there was no correlation between high MT3-MMP expression in HCC and overall survival or disease-free survival. CONCLUSIONS: MT3-MMP was expressed not only in chronic hepatitis and liver cirrhosis, but also in HCC, and high MT3-MMP expression correlated significantly with capsular invasion of carcinoma.

Interferon alpha receptors are important for antiproliferative effect of interferon-alpha against human hepatocellular carcinoma cells.

AIM: Interferon (IFN)-alpha is a promising drug for the prevention and treatment of hepatocellular carcinoma (HCC). We reported that responders to IFN-alpha/5-fluorouracil combination therapy expressed higher IFN alpha receptor (IFNAR)2 in tumor. Herein we studied involvement of IFNARs in response to IFN-alpha in HCC cells. METHODS: IFN-alpha sensitivity and expression of IFNARs were studied in six HCC cell lines (HuH7, PLC/PRF/5, HLE, HLF, HepG2, Hep3B) using growth-inhibitory and RT-PCR, Western blot assays. Short interfering RNAs (SiRNAs) against IFNAR1 and 2 were used to analyze the role of the IFNARs in IFN-alpha's effect and signal transduction. RESULTS: The expressions of IFNAR1 and 2c mRNAs were higher in PLC/PRF/5 cells than those in other cell lines, and PLC/PRF/5 cells expressed abundant IFNAR2c on their cell membrane. When we examined the sensitivity of the HCC cell lines to the growth-inhibitory effect of IFN-alpha, PLC/PRF/5 exhibited a significant response, while the other cells were much more resistant. Knockdown of either IFNAR1 or 2 using siRNAs suppressed the IFN-alpha's signal transduction (2.5-fold), and decreased the growth-inhibitory effect (down by 69.9% and 67.3%). CONCLUSION: The results suggest that the expression of IFNAR1 and IFNAR2c independently are important for the antiproliferative effect of IFN-alpha in HCC cells.

Patterns and clinicopathologic features of extrahepatic recurrence of hepatocellular carcinoma after curative resection.

BACKGROUND: Little is known about the metastatic pattern in patients with extrahepatic metastasis after the removal of primary hepatocellular carcinoma (HCC). The aim of the present study was to determine the clinicopathologic characteristics and prognosis of patients with extrahepatic metastasis from HCC according to the recurrence pattern. METHODS: Among the patients who underwent hepatic resection for HCC between 1981 and 2001, 80 patients had no recurrence; 221 patients had intrahepatic recurrence, and 47 patients experienced extrahepatic metastasis within a mean follow-up period of 4.8 +/- 3.7 years (+/-SD; range, 2-15 years). The pattern of extrahepatic metastasis after hepatic resection was divided into pattern I (first recurrence in the liver and then spread outside the liver after repetitive intrahepatic recurrences and repetitive locoregional treatments), pattern II (simultaneous recognition of intrahepatic and extrahepatic recurrences), and pattern III (extrahepatic, but no intrahepatic, lesions at first recurrence). RESULTS: There were significant differences in proportions of patients with invasion of the portal vein, hepatic vein, or inferior vena cava, intrahepatic metastases, and tumor stage between patients with intra- and extrahepatic metastases. The disease-free survival and extrahepatic metastasis-free survival in pattern I were better than pattern II. Survival after extrahepatic metastasis did not correlate with the 3 patterns. CONCLUSION: Although long-term overall survival was better in patients with pattern I of extrahepatic recurrences, prognosis was poor in all patterns once extrahepatic metastasis developed.

Stronger growth-inhibitory effect of interferon (IFN)-beta compared to IFN-alpha is mediated by IFN signaling pathway in hepatocellular carcinoma cells.

Interferon (IFN) is a promising drug for prevention and treatment of hepatocellular carcinoma (HCC) in combination with chemotherapeutic agents. We previously reported that the spectra of antiproliferative activity and synergistic effect of IFN-beta when combined with anticancer drugs are more potent than those of IFN-alpha in HCC cells. However, the mechanism of the diverse antitumor effects of the IFNs is not understood yet. We studied the expression of IFN alpha receptor 2 (IFNAR2), STATs, and IFN-alpha, IFN-beta's growth-inhibitory effect, signal transduction and binding to IFNAR2 on three HCC cell lines and a tumor xenografted mouse model (12 animals/group). From the results, IFN-beta showed a significantly stronger growth-inhibitory effect than IFN-alpha on the HuH7 cell line (expressing low IFNAR2), however it was similarly high on PLC/PRF/5 and weak on HLE. In the nude mouse tumor xenograft model, IFN-beta injection significantly suppressed tumor volume relative to vehicle injection, while IFN-alpha showed weaker growth-inhibition. IFN signal transduction (phosphorylated-STAT1, 3) induced by IFN-beta was higher than that by IFN-alpha in HuH7 and tumor xenografts. Pretreatment of hepatoma cells with anti-IFNAR2 antibody blocked the IFN signaling, more for IFN-alpha. IFN-alpha's antiproliferative effect was reduced by the antibody in lower concentrations compared to that of IFN-beta. Taken together, the HCC cells that express low IFNAR2 and are resistant to IFN-alpha were sensitive to the growth-inhibitory effect of IFN-beta, which might be mediated by stronger IFN signal transduction and distinct binding to IFNAR compared to IFN-alpha.

Role of the Fas/FasL pathway in combination therapy with interferon-alpha and fluorouracil against hepatocellular carcinoma in vitro.

BACKGROUND/AIMS: Several studies have reported the efficacy of combination therapy of interferon (IFN) alpha and 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). However, the mechanism underlying the clinical anti-tumor effects of this treatment is not well understood. The aim of this study was to determine the role of Fas/FasL signaling in the anti-tumor effect of this combination therapy. METHODS AND RESULTS: We used six human hepatoma cell lines, three of which are known Fas-expressing cells. Growth of Fas-positive hepatoma cell lines was inhibited by an agonistic anti-Fas antibody in a dose-dependent manner, and these effects were enhanced by IFNalpha or 5-FU alone, but even more so by combination therapy using both agents. Annexin-V assay implicated apoptosis as the main mechanism underlying these growth inhibitory effects, although changes in Fas expression regulated by IFNalpha and/or 5-FU did not correlate with increased apoptosis. Caspase-3 activation was exclusively increased by IFNalpha/5-FU combination treatment, which was compatible with enhancement of the synergistic apoptotic effect, and other caspases and apoptotic factors (FLIP, BCL-xl, and Bax) were also regulated by IFNalpha/5-FU. 51Cr-release assay revealed that pretreatment with IFN activated cytotoxicity of peripheral blood mononuclear cells (PBMCs) against HCC cells. The largest interaction was observed when both PBMC and HCC cells were pretreated with the combination of IFNalpha/5-FU. These cytotoxicities were markedly inhibited by a neutralizing anti-Fas antibody. CONCLUSIONS: Our results indicated that IFNalpha/5-FU combination treatment enhances the induction of apoptosis and the cytotoxic effect of PBMCs via the Fas/FasL pathway. The Fas/FasL pathway seems, at least in part, to contribute to the anti-tumor effects of IFNalpha/5-FU against HCCs.

A case of hepatocellular carcinoma with multiple lung, spleen, and remnant liver metastasis successfully treated by combination chemotherapy with the novel oral DPD-inhibiting chemotherapeutic drug S-1 and interferon-alpha.

A 54-year-old man was admitted Osaka University Hospital for hepatocellular carcinoma (HCC) with portal vein thrombus and multiple intrahepatic metastases that extended to the bilateral lobes of the liver. He underwent multimodal therapy, including extended left lobectomy followed by intra-arterial 5-fluorourcil (5-FU) infusion chemotherapy combined with subcutaneous interferon-alpha (IFN-alpha) to treat the lesions in the residual liver. Seven months after the initial resection, recurrent tumors in the spleen, lung, and residual liver were detected by follow-up examination. We started a new regimen of per oral administration of S-1 and subcutaneous IFN-alpha injection, because the combined therapy with intra-arterial 5-FU infusion was not considered effective for distant metastases. After two cycles of S-1 and IFN-alpha, the metastatic tumor in the spleen and the recurrence in the residual liver had disappeared, and the diagnosis was complete remission with no adverse effect; the pulmonary metastasis showed a partial response, and was finally resected. This patient is still alive with no recurrence 32 months after initial hepatic resection. This outcome suggests that combination therapy with S-1 and IFN-alpha may be a promising treatment modality against advanced HCC with distant metastasis.

Antisense to cyclin D1 inhibits vascular endothelial growth factor-stimulated growth of vascular endothelial cells: implication of tumor vascularization.

PURPOSE: Our aim was to determine the effects of cyclin D1 inhibition on tumor-associated neovascularization and endothelial cell growth. EXPERIMENTAL DESIGN: We have generated adenovirus system for antisense to cyclin D1 (AS CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1 was also used to analyze cyclin D1 inhibition-associated vascular endothelial growth factor (VEGF) regulation. RESULTS: The xenografts treated with adenoviral AS CyD1 showed less vessel density and displayed smaller tumor size in colon cancer cell lines HCT116 and DLD1. In vitro studies indicated that AS CyD1 decreased VEGF protein expression in DLD1 but not in HCT116. Cyclin D1 small interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1. A modest decrease was noted in the VEGF promoter activity, with inactivation of the STAT3 transcription factor through dephosphorylation. On the hand, the cyclin D1 inhibition plus STAT3 inhibitor markedly decreased VEGF expression in HCT116, although VEGF did not change by the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC), VEGF augmented cyclin D1 expression and cell growth. AS CyD1 significantly inhibited HUVEC growth even in the presence of VEGF. AS CyD1 also significantly suppressed in vitro tube formation in VEGF-treated HUVEC and in vivo macroaneurysm formation in VEGF-treated Matrigel plug. CONCLUSIONS: Our results suggest that cyclin D1 may play a role in the maintenance of VEGF expression and that AS CyD1 could be potentially useful for targeting both cancer cells and their microenvironment of tumor vessels.

Expression of type I interferon receptor as a predictor of clinical response to interferon-alpha therapy of gastrointestinal cancers.

Interferon (IFN) is used in the treatment of many malignancies and viral disorders. We recently reported a significant correlation between the efficacy of IFN-alpha combined with chemotherapy in the treatment of advanced hepatocellular carcinoma (HCC) and IFN-alpha/type I IFN receptor (IFNAR2) expression. It is possible that the expression of IFNAR2 in gastrointestinal cancerous tissue, apart from HCC, may predict the efficacy of IFN-alpha combination therapy. We investigated the expression of IFNAR2 in 100 gastrointestinal cancerous tissues. IFNAR2 expression was examined using immunohistochemistry, in surgically resected tissue samples (20 esophageal, 20 gastric, 20 colorectal, 20 cholangiocarcinoma, and 20 pancreatic samples). The expression rate of IFNAR2 was 35.0% (7/20), 25.0% (5/20), 20.0% (4/20), 45.0% (9/20), and 25.0% (5/20) in esophageal, gastric cancer, colorectal, cholangiocarcinoma and pancreatic cancer samples, respectively. In our previous report, the expression rate of IFNAR2 in HCC samples was 64.8% (59/91). Thus, the expression rates of IFNAR2 in the five types of gastrointestinal cancers tested here were low, compared with HCC. The clinical efficacy of IFN-alpha mono- or combination therapies in patients with gastrointestinal neoplasms is expected to be lower than in patients with HCC based on the expression level of IFNAR2.

Vitamin K2 has growth inhibition effect against hepatocellular carcinoma cell lines but does not enhance anti-tumor effect of combination treatment of interferon-alpha and fluorouracil in vitro.

Several studies have recently reported the efficacy of combination therapy of interferon (IFN) alpha and 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). However, the clinical effect of this treatment was not complete. The new therapeutic modality should be necessary to rise up this clinical response rate. Recently, the anti-tumor effect of Vitamin K2 has been reported in terms of decreased recurrence rate of HCC patients. The aim of this study was to explore the additive or synergistic effect of Vitamin K2 to combined therapy of interferon (IFN) alpha and 5-fluorouracil (5-FU) against hepatocellular carcinoma (HCCs). The study was conducted using three hepatoma cell lines (PLC/PRF/5, Hep3B and HepG2). The 3-(4-5-dimethylthiazol-2-yl)-2, 5-dyphenyl tetrazolium bromide (MTT) assay (48h) revealed anti-tumor effect of IFNalpha and 5-FU. Cell growth assay (3-7 days) showed growth inhibitory effect of Vitamin K2 on three cell lines after day 5. But additional effect of combination treatment of Vitamin K2 and IFNalpha/5-FU was not observed in any time course from 48h to 7 days. Cell cycles were assessed with flowcytometry. Although either Vitamin K2 or IFNalpha/5-FU alone has the influence to the cell cycles, no significant change was shown in the combination of Vitamin K2 and IFNalpha/5-FU. In conclusion, Vitamin K2 itself has potentially growth inhibitory effect for HCC cell lines, but does not enhance the anti-tumor effect of IFNalpha and 5-FU.

Expression pattern of angiogenic factors and prognosis after hepatic resection in hepatocellular carcinoma: importance of angiopoietin-2 and hypoxia-induced factor-1 alpha.

BACKGROUND: Hepatocellular carcinoma (HCC) is a hypervascular tumor and angiogenesis plays an important role in its progression. Angiogenesis is regulated by a balance between pro and antiangiogenic molecules. The aim of this study was to investigate the expressions of angiogenic factors and elucidate their roles in angiogenesis in HCC. METHODS: We investigated immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), hypoxia-induced factor-1alpha (HIF-1alpha) and thrombospondin-1 (TSP-1) in 60 specimens of surgically resected HCC. We investigated the relationship between their expressions and clinicopathological factors or prognosis. RESULTS: Ang-2 staining had a significant correlation with the grade of differentiation of HCC cells (P=0.0082). VEGF and Ang-2 expression correlated positively with microvessel density (MVD) (P=0.0061 and 0.0374, respectively). MVD of well-differentiated HCC were significantly lower than those of moderately and poorly differentiated HCC. The disease-free survival time of patients with high Ang-2 and/or HIF-1alpha expression was significantly shorter than that of the low expression group (P=0.0278 and 0.0374, respectively). CONCLUSION: Our study showed that the expression of VEGF and Ang-2 correlated with MVD. Strong Ang-2 expression and/or high nuclear expression of HIF-1alpha is a significant predictive factor for recurrence after curative resection in HCC patients.