The power of kindness: curative treatment with metronomic combination in advanced hepatocellular carcinoma.

The administration of approved systemic treatments for advanced hepatocellular carcinoma (HCC) is limited to patients with preserved liver function (Child-Pugh A/B7) and performance status. Conversely, metronomic chemotherapy can be safely administered to patients with poor clinical conditions and severe liver impairment. The metronomic schedule demonstrated to exert different anticancer mechanisms compared to that of the same agent administered at its standard schedule, including immune stimulation and the inhibition of angiogenesis and vasculogenesis. Nevertheless, metronomic chemotherapy is a nearly neglected option for the treatment of advanced HCC patients, even among those who cannot afford standard treatments. Herein, we report the case of a 40-year-old patient affected by HBV-HDV-related cirrhosis who was diagnosed with advanced HCC. The severe liver impairment (Child-Pugh B9) did not allow to administer first-line treatment with tyrosine kinase inhibitors so that the patient received metronomic capecitabine as upfront therapy. Due to the suspect of progressive disease at the first radiologic assessment, metronomic cyclophosphamide was added to capecitabine aiming to enhance its efficacy. After 4 months of treatment, complete tumor response, alpha-fetoprotein (AFP) normalization and the recovery of a Child-Pugh A were achieved. The patient was then able to undergo liver transplantation, and, after 18 months from the diagnosis, he is still free of disease recurrence. This experience emphasizes the reliability of metronomic capecitabine as a well-tolerated and effective treatment when patient's conditions prevent the administration of standard first-line treatments. In fact, metronomic capecitabine demonstrated its effectiveness in advanced HCC in retrospective and prospective analyses, leading to median progression-free survival and overall survival of, respectively, 6.03 and 14.47 months in phase II single-arm trial. Moreover, in consideration of the raising interest in immune-checkpoint inhibition in HCC, we believe that the immunomodulating effects of metronomic chemotherapy, either capecitabine or cyclophosphamide, warrant future trials exploring its combination with immunotherapy.

MicroRNA-26a systemic administration attenuates tumor formation in hepatocellular carcinoma mouse model.

MicroRNA (miRNA)-26a is one of the tumor suppressor genes that has been down regulated during the development of hepatocellular carcinoma (HCC). This work was conducted to evaluate the possible preventive effect of exogenous miRNA-26a administration on diethylnitrosamine (DEN)-mediated HCC. Balb/C mice were intraperitoneally injected with saline (Normal group), DEN (HCC group) or miRNA-26a (HCC+miRNA-26a group). On week 8, 12, 16 and 20, the concentrations of alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), the levels of helper T cells-associated cytokines, and the vascular endothelial growth factor (VEGF), were measured. Flow cytometry determined the frequencies of regulatory T (Treg) cells. The concentrations of AFP, DCP and VEGF, as well as the frequency of Treg cells showed significantly lower values following miRNA-26a administration than in HCC group. miRNA-26a administration has reduced the levels of IL (interleukin)-2 and TNF (tumor necrosis factor)-α, in contrast, IL-10 level was markedly elevated in comparison to HCC model at all experimental time points. The restore of miRNA-26a function significantly (P<0.001) down regulated the expression levels of survivin & caspase-3 compared to HCC group. The obtained data introduce an evidence for the suppressive impact of miRNA-26a on liver tumor formation and its possible manipulation as a therapeutic design for HCC.

Treatment of inoperable hepatocellular carcinoma with immunotherapy.

In the USA, mortality associated with hepatocellular carcinoma (HCC) continues to rise. Globally, HCC is the third most common cause of cancer-related death. In early stages of HCC, hepatic resection or liver transplantation are the preferred treatment options with a high probability of recurrence-free postoperative course. However, ineffective screening of chronic liver diseases in high-risk populations, poor linkage to care and suboptimal HCC surveillance has led to increasing rates of late-stage HCC at clinical presentation or diagnosis amenable only to palliative and experimental treatment options. Our case is a 66-year-old man with chronic hepatitis C virus infection complicated by cirrhosis and inoperable HCC which was non-responsive to selective intrahepatic trans-arterial chemoembolisation by interventional radiology. Therefore, he was treated with nivolumab immunotherapy and demonstrated normalisation of previously elevated alpha-fetoprotein levels suggestive of at least a partial response to immunotherapy. No adverse events related to nivolumab immunotherapy were encountered.

A Phase 2 Study of Galunisertib (TGF-ß1 Receptor Type I Inhibitor) and Sorafenib in Patients With Advanced Hepatocellular Carcinoma.

INTRODUCTION: Inhibition of tumor growth factor-ß (TGF-ß) receptor type I potentiated the activity of sorafenib in preclinical models of hepatocellular carcinoma (HCC). Galunisertib is a small-molecule selective inhibitor of TGF-ß1 receptor type I, which demonstrated activity in a phase 2 trial as second-line HCC treatment. METHODS: The combination of galunisertib and sorafenib (400 mg BID) was tested in patients with advanced HCC and Child-Pugh A liver function without prior systemic therapy. Galunisertib dose was administered 80 or 150 mg b.i.d. orally for 14 days every 28 days in safety lead-in cohorts; in the expansion cohort, all patients received galunisertib 150 mg b.i.d. Objectives included time-to-tumor progression, changes in circulating alpha fetoprotein and TGF-ß1, safety, overall survival (OS), response rate, and pharmacokinetics (PK). RESULTS: Patients (n = 47) were enrolled from 5 non-Asian countries; 3 and 44 patients received the 80 mg and 150 mg b.i.d. doses of galunisertib, respectively. The pharmacokinetics and safety profiles were consistent with monotherapy of each drug. For the 150 mg b.i.d. galunisertib cohort, the median time-to-tumor progression was 4.1 months; the median OS was 18.8 months. A partial response was seen in 2 patients, stable disease in 21, and progressive disease in 13. TGF-ß1 responders (decrease of >20% from baseline) vs nonresponders had longer OS (22.8 vs 12.0 months, P = 0.038). DISCUSSION: The combination of galunisertib and sorafenib showed acceptable safety and a prolonged OS outcome.

Zygomatic bone metastasis from hepatocellular carcinoma and the therapeutic efficacy of apatinib: A case report and literature review.

RATIONALE: Hepatocellular carcinoma (HCC) metastases to the zygomatic bone are extremely uncommon, and the treatment of target drugs against such case is unknown. PATIENT CONCERNS: A 48-year-old male patient was admitted to our hospital under suspicion of an advanced liver tumor due to an increase in levels of alpha-fetoprotein (AFP) after radiofrequency ablation for independent nodule in his liver 1 month before. He had a hepatitis B virus (HBV) history for 20 years without treatment. DIAGNOSIS AND INTERVENTIONS: A diagnosis of primary HCC was made based on pathological examination following right hepatectomy. Seven months after the surgery, a mass in S8 was identified and treated by ARF. Twenty days later, a right zygomatic mass was observed and the incisional biopsy revealed metastasis from HCC. Due to side effects of chemotherapy, the metastatic zygomatic mass was treated with radioactive seed implantation. Despite these interventions, there was steady increase in AFP values as well as increase in size of the zygomatic mass. Hence, the patient was started on apatinib with a dose of 500 mg/day from 1 to 28 days per cycle for a duration of 10 months. OUTCOMES: The AFP values were significantly decreased but the size of the zygomatic mass continued to increase indicating progression of disease. But the progression-free survival was more than 10 months. The patient exhibited adverse reactions which were controllable by symptomatic treatments. As of last follow-up, the patient is unwell with pain in the face, blurred vision in the right eye, dyscrasia, and exhibited difficulty in opening his mouth. LESSONS: HCC metastases to the zygomatic bone are very aggressive with a very low incidence and immunohistochemistry is useful diagnostic indicators. Still now, there is no optimal treatment strategy for these patients. Apatinib may be a promising drug in the treatment of HCC metastases to the zygomatic bone.

Oncostatin M in William's E medium is suitable for initiation of hepatocyte differentiation in human induced pluripotent stem cells.

William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α­fetoprotein (AFP) and albumin were examined by reverse transcription­quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all­trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all­trans retinoic acid and insulin­transferrin­selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post­stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.

Significance of the Signal Intensity of Gadoxetic Acid-enhanced MR Imaging for Predicting the Efficacy of Hepatic Arterial Infusion Chemotherapy in Hepatocellular Carcinoma.

PURPOSE: We attempted to clarify the relationship between the signal intensity (SI) in the hepatobiliary phase of gadoxetic acid-enhanced magnetic resonance (MR) imaging and the efficacy of hepatic arterial infusion chemotherapy (HAIC) in hepatocellular carcinomas (HCCs). METHODS: We enrolled 14 patients with HCCs who underwent gadoxetic acid-enhanced MR imaging prior to HAIC using cisplatin and 5-fluorouracil. In the hepatobiliary phase, we calculated the SI of the HCCs and the background liver. In cases with multiple HCCs, we calculated the SI of the largest lesion. Patients were classified into high (n = 7) and low intensity (n = 7) groups based on the median value of the SI ratio (SI of the tumor/SI of the background liver). We analyzed progression-free survival using the Kaplan-Meier method and the log-rank test. In the 5 patients with a history of HCC surgery, we compared the expression of immunohistochemical organic anion-transporting polypeptide (OATP) 8 between the high and low intensity groups by chi-square test. RESULTS: The SI ratios were 0.568 ± 0.093 (mean ± standard deviation) in the high intensity group and 0.251 ± 0.086 in the low intensity group. Compared to the group with low signal intensity, the group with high signal intensity demonstrated significantly lower serum levels of alpha fetoprotein (AFP) (P = 0.0350), significantly higher progression-free survival (P = 0.0108), better differentiation of tumor grade at histologic examination (P = 0.0253), and significantly higher OATP8 expression (P = 0.0253). CONCLUSION: Patients with HCCs of high SI ratio in the hepatobiliary phase of gadoxetic acid-enhanced MR imaging can respond better to HAIC.

Up-regulation of thy-1 promotes invasion and metastasis of hepatocarcinomas.

Increasing evidence has revealed that thy-1 was a potential stem cell marker of liver cancer, but no data have been shown on how thy-1 regulates the pathophysiology of liver cancer, such as proliferation, apoptosis, invasion and migration. We previously demonstrated that thy-1 was expressed in about 1% of hepg2 cells, thy-1+ hepg2 cells, but not thy-1-, demonstrating high tumorigenesis on inoculation 0.5x105 cells per BACA/LA mouse after 2 months. In the present study, our results showed that higher expression of thy-1 occurs in 72% (36/50 cases) of neoplastic hepatic tissues as compared to 40% (20/50 cases) of control tissues, and the expression of thy-1 is higher in poorly differentiated liver tumors than in the well-differentiated ones. In addition, thy-1 expression was detected in 85% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis or hepatitis. There was a significant negative correlation between thy-1 expression and E-cadherin expression (a marker of invasion and migraton), but not between thy-1 expression and AFP expression in all the liver cancer and blood samples. We further investigated the relationship between thy-1 and E- cadherin in liver cancer hepg2 cell line which was transfected with pReceiver-M29/thy-1 eukaryotic expression vector followed by aspirin treatment. Lower expression of E- cadherin but higher expressions of thy-1 were detected in hepg2 cells transfected with pReceiver-M29/thy-1. Taken together, our study suggested that thy-1 probably regulates liver cancer invasion and migration.

Proteomic profiling of k-11706 responsive proteins.

Erythropoietin promotes the production of red blood cells. Recombinant human erythropoietin is illicitly used to improve performance in endurance sports. Expression of the ERYTHROPOIETIN gene is negatively controlled by the transcription factor GATA-binding protein (GATA). Specific GATA inhibitors have recently been developed as novel drugs for the management of anemia. These drugs could, therefore, be illicitly used like recombinant human erythropoietin to improve performance in sports. To examine alterations in levels of plasma protein after administration of GATA inhibitors, proteomic analyses were conducted on mouse plasma samples treated with the potent GATA inhibitor K-11706. The analysis based on gel electrophoresis identified 41 protein spots differentially expressed when compared with normal plasma. Each spot was identified with liquid chromatography coupled to tandem mass spectrometry and 2 of them, fetuin-B and prothrombin, were verified by Western blotting. The results showed that the expression of fetuin-B in mice plasma was increased by K-11706, but not by recombinant human erythropoietin or hypoxia. These results suggest the potential of proteomic-based approaches as tools to identify biomarkers for the illegal use of novel drugs (e.g., GATA inhibitors). Also, fetuin-B could be a sensitive marker for the detection of abuse of GATA inhibitors.

Comparison of different tumor response criteria in patients with hepatocellular carcinoma after systemic therapy with the multikinase inhibitor sorafenib.

RATIONALE AND OBJECTIVES: The aim of this study was to compare tumor changes in patients with hepatocellular carcinoma receiving sorafenib using evaluation criteria of the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) as opposed to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. MATERIALS AND METHODS: Twenty-five patients with inoperable hepatocellular carcinoma receiving oral sorafenib underwent magnetic resonance imaging at baseline and follow-up every 8 weeks (range, 2-19 weeks; mean, 7.6 weeks). Data were evaluated retrospectively. Survey time until progression ranged from 5 to 102 weeks (mean, 25.6 weeks), with a total of 54 target lesions being monitored. Additionally, evaluation of serum α-fetoprotein was performed at follow-up. RESULTS: The best response at follow-up using RECIST resulted in rates of 4% objective response (complete remission or partial remission), 24% (progressive disease), and 72% (stable disease). In contrast, AASLD and EASL criteria identified objective responses in 28% and 48%. Twenty percent of all patients classified as having progressive disease by RECIST were identified as having "pseudo"-progression due to extensive necrosis. Eleven percent of patients classified as having stable disease by RECIST were disclosed as essentially progressive. AASLD area and AASLD diameter disclosed 36% and 40% of patients as having partial remission, respectively, whereas EASL criteria discovered only 24%. There was no significant correlation between serum α-fetoprotein progression and AASLD, EASL, or RECIST evaluation criteria. CONCLUSIONS: Response monitoring via functional criteria such as AASLD or EASL criteria is likely to more accurately reflect vital tumor burden in hepatocellular carcinoma compared to RECIST.

Molecular link(s) between hepatoblastoma pathogenesis and exposure to di-(2-ethylhexyl)phthalate: a hypothesis.

Hepatoblastoma (HB) is the most important primary liver cancer in children, accounting for up to 1% of all paediatric malignancies. It affects mostly infants and young children between the age of 6 months and 3 years. Predisposing genetic factors for HB have been identified and scientific evidence clearly points out HB as a multi-factorial condition based on both genetic and environmental factors. Nevertheless, a clear understanding of the pathogenesis is yet lacking. The present review will focus on the impact of exposure to environmental chemicals, such as di-(2-ethylhexyl)phthalate (DEHP), and recognized risk factors, such as intrauterine growth retardation (IUGR), on HB establishment via altered signalling pathways (Wnt and IGF). The hypothesis linking the impairment of IGF2 foetal/adult switching and exposure to DEHP is discussed as a way forward to support HB prevention and early diagnosis.

A decrease in AFP level related to administration of interferon in patients with chronic hepatitis C and a high level of AFP.

It is known that there is a very high incidence of hepatocellular carcinoma (HCC) among patients with type C chronic hepatitis and cirrhosis, and alpha -fetoprotein (AFP) has been widely used as a diagnostic marker for HCC. However, there are some patients showing continuous high AFP values but no evidence of HCC, and some studies have defined such patients as a high-risk group for HCC. In vitro study has shown that interferon (IFN) inhibits cell proliferation and enhances apoptosis as well as specific cytotoxic T lymphocytes against HCC, resulting in direct anticancer actions. In this study, we investigated the effect of IFN on AFP changes in chronic hepatitis C patients. Of 40 patients with chronic hepatitis C in whom diagnostic imaging confirmed the absence of HCC, 24 patients showed high pretreatment AFP values (high AFP group: AFP level > 10 ng/dl; mean +/- SD, 46.3 +/- 41.5 ng/dl) and 16 showed low pretreatment AFP values (low AFP group: pretreatment AFP level < or = 10 ng/dl; mean +/- SD, 5.3 +/- 2.2 ng/dl). Pretreatment clinical parameters were statistically evaluated in relation to the AFP value. In the high AFP group, the platelet count, albumin level, and prothrombin (%) were significantly lower (P = 0.047, P = 0.0002, and P = 0.044, respectively), suggesting that AFP value increases with advancing liver disease. Subsequently 27 patients were administered IFN (IFN group), and the remaining 13 patients were administered Stronger Neo-minophagen C (SNMC), a glycyrrhizin preparation (SNMC group), as a control group receiving liver-protective therapy. Alanine aminotransferase was reduced in both the IFN and the SNMC group (mean, 132.56 to 60.07 mg/ml [P < 0001] and 147.85 to 56.23 mg/ml [P = 0.0240], respectively). AFP was significantly reduced in the IFN group (mean, 30.03 to 12.65 ng/ml; P = 0.0034), but there was no significant change in AFP in the SNMC group (mean, 29.70 to 39.17 ng/ml). AFP is useful for diagnosing HCC; however, some patients show a persistently high AFP level in the absence of HCC, and these patients have been described as a high-risk group for HCC. In this study, we found that IFN therapy but not SNMC universally reduced the AFP baseline. Since AFP is a significant predictor for HCC, therapeutic strategies for hepatitis C, e.g., long-term low-dose IFN treatment, may reduce hepatocarcinogenesis.

Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens.

Two clearly opposing views exist on the function of alpha-fetoprotein (AFP), a fetal plasma protein that binds estrogens with high affinity, in the sexual differentiation of the rodent brain. AFP has been proposed to either prevent the entry of estrogens or to actively transport estrogens into the developing female brain. The availability of Afp mutant mice (Afp(-/-)) now finally allows us to resolve this longstanding controversy concerning the role of AFP in brain sexual differentiation, and thus to determine whether prenatal estrogens contribute to the development of the female brain. Here we show that the brain and behavior of female Afp(-/-) mice were masculinized and defeminized. However, when estrogen production was blocked by embryonic treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione, the feminine phenotype of these mice was rescued. These results clearly demonstrate that prenatal estrogens masculinize and defeminize the brain and that AFP protects the female brain from these effects of estrogens.

Redifferentiation of human hepatoma cells induced by synthesized coumarin.

The effects of synthesized 7-OH-4-CH(3)-coumarin on proliferation and differentiation of human hepatoma carcinoma cell line, SMMC-7721, were examined. Results showed that 7-OH-4-CH(3)-coumarin suppressed the proliferation of the SMMC-7721 cells in a dose-dependent manner, with an IC(50) value of 356 +/- 1.8 .M, while concentrations< or =200 mM could trigger differentiation. After treatment with this compound at 100 mM, the growth curve of human hepatoma cells decreased markedly. When treated with 50 and 100 mM, the cells' electrophoresis rate decreased from 2.2 mm/s/V/cm in the control group to 1.5 and 1.8 mm/s/V/cm, respectively, and the alpha-fetoprotein content decreased from 123 ng/mg in the control group to 68 and 45 ng/mg, respectively. The microvilli on the surface of treated cells were also reduced. All the above indexes related to cell malignancy were alleviated significantly. Results showed that 7-OH-4-CH(3)-coumarin could reverse human hepatoma cells' malignant phenotypic characteristics and induce redifferentiation.

Growing Teratoma syndrome treated by interferon alpha-2beta: case report and literature review.

A 14-year old girl was diagnosed as suffering from an immature teratoma. As removal of the teratoma was technically unfeasible, the patient was started on interferon alpha2beta, 3 MU/day, 5 days per week. On this therapy, the growth of the teratoma was stopped and the patient has been well for the past 50 months. She continues to be treated with interferon alpha2beta, with almost no side effects and no restrictions of her everyday activities, with a good quality of life. She is able to attend school regularly, travels abroad frequently, and participates fully in everyday activities normal for her age and social conditions. Her Karnofsky performance status is 100. This case demonstrates the efficacy of interferon alpha2beta therapy in certain instances of growing teratoma syndrome, even when the tumorous mass is initially quite large.

Impact of interferon-alpha therapy on the serum level of alpha-fetoprotein in patients with chronic viral hepatitis.

PURPOSE: Assessment of the long-term effect of interferon-alpha (IFN-alpha) treatment on the serum level of hepatocarcinogenesis marker--alpha-fetoprotein (AFP), in patients (pts) with chronic viral hepatitis (c.v.h.) type B and C. MATERIAL AND METHODS: Thirty seven pts (21 with HCV and 16 with HBV infection; 20 women, 17 men, aged 24-62) were included in the study. The pts were administered IFN-alpha in the dose of 9-15 MU per week, thrice weekly, for 16 weeks (HBV group) and 24-52 weeks (HCV group). The effectiveness of IFN-alpha treatment was evaluated on the basis of the HBV DNA and HCV RNA level in the blood. The serum AFP values were determined before and 4-7 years after IFN-alpha treatment. RESULTS: The baseline serum AFP level was increased in 26 out of 37 pts (70%) (14/21 from HCV group; 12/16 from HBV group). After the 4-7 years' follow-up it remained elevated only in 2 out of 37 pts (5%). AFP values significantly decreased after IFN-alpha treatment (17.58 +/- 19.09 IU/ml vs 7.95 +/- 21.78 IU/ml; p < 0.05; normal range 0-5 IU/ml) in both HBV and HCV, responder and non-responder groups. CONCLUSIONS: IFN-alpha therapy significantly decreases the serum AFP level in patients with chronic viral hepatitis B and C. Its beneficial clinical effects have been observed both in responders and in non-responders. It could diminish the risk of liver carcinogenesis, however further studies are required to elucidate this issue.

The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes.

We explored the effect of extracellular matrix (ECM) produced by fetal and adult hepatocytes on tissue-specific gene expression and proliferation of fetal and adult hepatocytes. Adult hepatocytes ECM strongly induced expression of both albumin and HNF-4 in adult hepatocytes. In contrast, fibroblast ECM reduced the expression of mRNAs for albumin and alpha-fetoprotein in fetal hepatocytes. Adult hepatocytes ECM also increased the activity of liver-specific enzymes of adult hepatocytes (DPP IV and glucose-6-phosphatase) in both fetal and adult hepatocytes, while fetal hepatocyte-derived ECM increased activity of the fetal hepatocyte enzyme GGT in fetal hepatocytes. Fibroblast ECM was inhibitory for the activity of all enzymes assayed. Removal of heparin chains from the various matrices by pretreatment of the ECM with heparinase resulted in reduction of glucose-6-phosphatase and DPP IV in adult hepatocytes. Removal of chondroitin sulfate chains from fetal hepatocyte-derived ECM resulted in loss of induction of GGT in the fetal cells. Fetal hepatocytes proliferated best on adult hepatocyte-derived ECM. Adult hepatocytes showed only modest proliferation on both fetal and adult hepatocytes ECM and their growth was inhibited by fibroblast ECM. In conclusion, adult hepatocyte ECM better supports the expression of adult genes, whereas fetal hepatocyte ECM induced expression of fetal genes. Fibroblast derived-ECM was inhibitory for both proliferation and tissue-specific gene expression in fetal and adult hepatocytes. The data support a role for heparan sulfate being the active element in adult ECM, and chondroitin sulfate being the active element in fetal ECM.

Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein.

In earlier studies we have found that incubation of low density lipoprotein (LDL) with autologous blood plasma-derived serum leads to a loss of sialic acid from lipoprotein particles. In this study we demonstrated that sialic acid removed from LDL was transferred to glycoconjugates of lipoproteins, glycoproteins and sphingolipids of human serum. This showed that human serum contained the trans-sialidase activity. Gel-filtration chromatography of human blood serum demonstrated the presence of trans-sialidase activity in lipoprotein subfractions as well as in lipoprotein-deficient serum. Trans-sialidase (about 65 kDa) was isolated from lipoprotein-deficient serum using affinity chromatography carried out with Neu5Acalpha2-8Neu5Ac-sepharose FF-6. Optimal pH values for the trans-sialidase were 3.0, 5.0 and 7.0. Calcium and magnesium ions stimulated the enzyme activity at millimolar concentrations. Isolated enzyme can remove sialic acid from LDL, IDL, VLDL, and HDL particles (in decreasing rate order). Serum trans-sialidase transferred sialic acid from glycoconjugates of plasma proteins (fetuin, transferrin) and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Sialylated glycoconjugates of human blood erythrocytes also served as substrate for serum trans-sialidase. We have found that sialic acid can also be removed from N- and O-linked glycans, sialylated Le(x) and Le(a), oligosialic acids, and sphingolipid carbohydrate chains. The rate of sialic acid release decreased in the following order: alpha2,6>alpha2,3>>alpha2,8. Transferred molecule of sialic acid can form alpha2,6, alpha2,3 and to a lesser degree alpha2,8 linkage with galactose, N-acetyl-galactosamine or sialic acid of acceptors. The glycoconjugates of erythrocytes, lipoprotein particles, plasma proteins, neutral sphingolipids and gangliosides may serve as acceptors of transferred sialic acid. Trans-sialidase-treated native LDL becomes desialylated and then can induce cholesteryl ester accumulation in human aortic intimal smooth muscle cells. Thus, trans-sialidase may be involved in the early stages of atherogenesis characterized by foam cell formation.

Establishment of a testicular carcinoma cell line producing alpha-fetoprotein.

OBJECTIVE: To characterize a newly established human testicular carcinoma cell line that continuously produces alpha-fetoprotein (AFP), and to investigate the effects of retinoic acid on AFP production. MATERIALS AND METHODS: A 24-year-old man underwent a radical orchidectomy for a right testicular tumour and was found to have two separate metastatic lesions in the lungs, both of which were removed surgically. The cancer cells were isolated from one of the tumours, which was composed of undifferentiated germ cells and produced AFP; the cells were cultured in a monolayer. This cell line was designated as KU-MT. RESULTS: The cell line was successfully maintained both in athymic nude mice and in culture. Histological examination showed that the xenografted tumours were composed of cells in the reticular, solid and glandular patterns of a yolk sac tumour, and of embryonal carcinoma cells. These cells immunostained positively for AFP. On electron microscopy, the extracellular deposition of a basement lamina-like substance, a typical feature of yolk sac tumour, was detected. The AFP production in mice correlated well with the tumour weight of the xenograft. The cultured KU-MT cells were oval to polygonal in morphology and grew exponentially, with a population doubling time of approximately 2 days. Chromosomal analysis showed a modal number of 57 with consistent structural abnormalities of +add(1)(p13), del(1)(q32), del(2)(q31), add(6) (q21), +add(9)(p22), add(11)(p15), and add(14)(p11). Reverse-transcription polymerase chain reaction analysis showed that the retinoic acid receptors (RAR)-alpha, RAR-gamma, and retinoid X receptor-alpha were present in the cells. The expression of AFP mRNA was up-regulated in response to all-trans-retinoic acid; treatment with this agent caused morphological changes and induced apoptosis in the cells. CONCLUSIONS: This newly established cell line provides a reproducible model system that should offer a good insight into the differentiation of testicular carcinoma.