PEG-PLA diblock copolymer micelle-like nanoparticles as all-trans-retinoic acid carrier: in vitro and in vivo characterizations.

The purpose of this study was to characterize the properties in vitro, i.e. release, degradation, hemolytic potential and anticancer activity, and in vivo disposition of all-trans-retinoic acid (ATRA) in rats after administration of ATRA-loaded micelle-like nanoparticles. The amphiphilic block copolymers consisted of a micellar shell-forming mPEG block and a core-forming PLA block. The mPEG-PLA nanoparticles prepared by an acetone volatilization dialysis procedure were identified as having core-shell structure by (1)H NMR spectroscopy. Critical association concentration, drug contents, loading efficiency, particle size and xi potential were evaluated. The release of ATRA from the nanoparticles and the degradation of PLA were found to be mostly associated with the compositions of the nanoparticles. ATRA release was faster at smaller molecular weight of copolymer and lower drug contents. In vitro, the incorporation of ATRA in mPEG-PLA nanoparticles reduced the hemolytic potential of ATRA. Furthermore, anticancer activity of ATRA against HepG2 cell was increased by encapsulation, which showed an enhancement of tumor treatment of ATRA. In vivo, after intravenous injection to rats, the levels of ATRA in the blood stream and the bioavailability were higher for ATRA-loaded mPEG-PLA nanoparticles than those for ATRA solution. In conclusion, the structure of the mPEG-PLA diblock copolymer could be modulated to fit the demand of in vitro and in vivo characterizations of nanoparticles. The mPEG-PLA nanoparticles' loading ATRA have a promising future for injection administration.

Possibility of hepatic resection in patients on maintenance hemodialysis.

A review of seven hepatic resections in six patients undergoing maintenance hemodialysis is presented. These cases consisted of hepatocellular carcinoma in four, and cholangiocellular carcinoma, myelolipoma and focal nodular hyperplasia in one each. The last preoperative hemodialysis was undertaken within 24 h prior to the operation with heparin. Intraoperatively, infused solutions containing no potassium, along with strict attention to preventing overhydration, allowed us to manage the patients without hemodialysis on the day of the operation. No specific intra-operative complications related to hemodialysis were noted. Postoperative hemodialysis was performed on the first or second day after operation, using nafamstat mesilate, a synthetic protease-inhibiting agent. The morbidity rate in the hemodialyzed patients was 85.7% (6/7), which was significantly higher than that in the non-hemodialyzed patients who underwent hepatic resections in our hospital. Fluid collection in the pleural and/or peritoneal cavities was frequent and difficult to control, but transient. Our experience suggests that hepatic resection is an acceptable procedure for hemodialyzed patients, when used in conjunction with careful perioperative management.

Adhesive properties of hepatoma cells to collagen IV coated surfaces.

OBJECTIVES: To quantitatively study the adhesive properties of hepatoma cells to collagen IV coated artificial basement membrane and to investigate the relevance of cell adhesive forces to the concentration of collagen IV. METHODS: Synchronous G1 and S phase cells were achieved using thymine-2-desoxyriboside and cochicine sequential blockage method and double thymine-2-desoxyriboside blockage method respectively. The adhesive forces of hepatoma cells were investigated by micropipette aspiration technique. RESULTS: The adhesive forces of hepatoma cells to artificial basement membrane were (107.78+/-65.44)x10(-10)N, (182.60+/-107.88)x10(-10)N, (298.91+/-144.13)x10(-10)N when the concentration of the membrane coated by 1, 2, 5 microg/ml collagen IV respectively (P<0.001). The adhesive forces of G1 and S phases hepatoma cells to artificial basement membrane were (275.86+/-232.80)x10(-10)N and (161.16+/-120.40)x10(-10)N respectively when the concentration of the membrane coated by 5 microg/ml collagen IV (P<0.001). CONCLUSIONS: The adhesive forces of hepatoma cells to artificial basement membrane in direct proportion to the concentration of collagen IV suggests that the increase of basement membrane might be conducive to the chemotactic motion and adhesiveness of tumor cells. G1 phase cells are more capable of adhering to basement membrane than S phase cells. Hepatoma cells, especially G1 phase cells, may survive in blood circulation, and sequest and adhere in microcirculation, and get through basement membrane for remote metastasis.

["Partial wrapping therapy" for recurrent hepatocellular carcinoma by transthoracic route].

Two patients with recurrent hepatocellular carcinoma close to the diaphragma, underwent TAE following decollateralization using silicone rubber sheeting with thoracotomy. The patients suffered from severe liver cirrhosis. Laparotomy was not carried out for the preservation of hepatic function. After wrapping therapy, serum AFP level was reduced to almost the normal range in one case, but in another case to half because of bilateral lung metastasis. The maximum serum level of postoperative serum total bilirubin in these 2 cases was 2.0 mg/dl and 1.6 mg/dl, respectively. We think wrapping therapy with thoracotomy is readily applicable for patients with severe cirrhosis.

["Wrapping therapy" for advanced hepatic cancer with arterial and intraportal infusion chemotherapy].

A new treatment method to intercept collaterals using silicone rubber sheeting was used for 5 patients with advanced hepatic cancer. The therapy was carried out to prevent new collaterals. This procedure was followed by arterial chemoembolization, arterial infusion chemotherapy and intraportal infusion chemotherapy. The results were complete response in 2 patients, partial response in 2 patients, and no change in 1 patient. The overall survival time was 7-54 months after wrapping. Although a randomized control study is necessary to assess the true value of this method, the new therapy is considered worth using as an adjuvant treatment for advanced hepatic malignancies uncontrolled by arterial chemotherapy or chemoembolization.

[Arterial infusion of SMANCS-Lipiodol for advanced hepatocellular carcinoma].

Twenty-four patients were treated with arterial infusion of SMANCS dissolved in Lipiodol. Twenty of these patients had HCC with the main trunks of portal vein occluded by tumor, and four patients had severe cirrhosis and multiple HCC. The actual dose of SMANCS administered each patient ranged from 4 to 6 mg. Side effects occurred in 50%. Severe side effects such as shock and shivering-chilliness were observed in 18%. The differences between the values of hepatic functional serum indexes obtained before and after treatment with SMANCS were small and transient. With regard to the therapeutic response of the arterial infusion of SMANCS, the mean survival time was approximately 2.8 months. It was suggested that the more effective administration of SMANCS was combination of the arterial infusion of SMANCS-Lipiodol with TAE at the level of the right hepatic artery of left hepatic artery for multiple HCC.

The effect of 2-methoxyestradiol liposome on growth inhibition, angiogenesis and expression of VEGF and Ki67 in mice bearing H22 hepatocellular carcinoma.

AIMS AND BACKGROUND: 2-methoxyestradiol (2-ME), an endogenous metabolite of estrogen, has very low water solubility. It is currently in phase II clinical trials as both a chemopreventive and chemotherapeutic agent and has been orally administered to cancer patients. However, the poor oral absorption of the compound is one of the major obstacles for 2-ME development. Based on the molecular features of 2-ME, liposome can be considered an attractive formulation approach. Our purpose in this study is to research the antitumor efficacy of 2-methoxyestradiol liposome (2-ME-L) in mice bearing H 22 tumors. METHODS: Murine H22 hepatocarcinoma served as an ectopic solid tumor model. The effects of antitumor therapy were evaluated by testing tumor growth, measuring the tumor inhibition rates in terms of weight and volume, and staining the tissues by hematoxylin and eosin. The synergistic mechanism of 2-ME-L therapy was elucidated by detecting changes in the expression of pathognostic factors in the tumor microenvironment. RESULTS: 2-ME-L significantly suppressed tumor growth. The morphological changes in the tumors indicated that the tumors in the treatment groups were effectively confined with little surrounding angiogenesis. Tumor cells of the treatment groups had abundant areas of necrosis with few nuclei in the mitotic phase. It was found that there was less immunohistochemical expression of vascular endothelial growth factor (VEGF), Ki67 and CD31 in the treatment groups and the efficacy of 2-ME-L was better than that of 2-ME solution (2-ME-S). This research demonstrated that 2-ME-L inhibited the growth of H 22 tumors in a concentration-dependent manner and was more effective than 2-ME-S. CONCLUSIONS: 2-ME-L can suppress the growth of H22 solid tumors and has antiproliferative, proapoptotic and antiangiogenic activity. 2-ME-L could be of potential use in the treatment of hepatocellular carcinoma.

Does chemotherapy prevent HBV-related hepatocellular carcinoma? Pros.

Studies have shown that hepatitis B virus (HBV) replication is the key driver of disease progression, including development of cirrhosis and hepatocellular carcinoma (HCC), in patients with chronic HBV infection. Among the currently available anti-HBV drugs, the most extensive and longest experience has been gained with conventional interferon alpha (IFN) and lamivudine. Both controlled studies and meta-analyses have shown that a finite course of IFN therapy has long-term benefit in achieving cumulative response and corresponding reduction of cirrhosis and/or HCC. Maintained virological response to lamivudine therapy has similar long-term benefits in reducing disease progression. Although emergence of lamivudine drug resistance may negate therapeutic effect, rescue drugs are now available to overcome the adverse effect of drug resistance. Pegylated IFN and newer nucleos(t)ide analogs may have even better long-term outcomes because of better therapeutic efficacy and/or much lower risk of drug resistances. However, the treatment outcomes are still far from satisfactory. The development of safe and affordable anti-HBV agents/strategies is needed to further improve outcomes.

Does chemotherapy prevent HBV-related hepatocellular carcinoma? Cons.

The retrospective scrutiny of studies that were originally designed to assess the antiviral activity of interferon (IFN) and nucleos(t)ide analogues (NUC) suggested reduced incidence of hepatocellular carcinoma (HCC) in responders. The interpretation of these studies, however, is questioned by the heterogeneity of patient referral, adoption of surrogate end-points, lack of control arms and, overall, by the lack of power to capture enough hard end-points of the natural history of hepatitis B, including HCC. Another point of criticism is that above all, IFN studies could have been affected by study enrolment skewed towards patients with less advanced liver disease, who had a better predicted compliance to therapy but a lower risk of developing HCC in the short-term. In my opinion, these constraints coupled with the lack of patient stratification by HCC predictors, make the evaluation of the prophylactic activity of IFN and NUC even more difficult. Overall, while single studies provide some evidence for a reduced HCC incidence in virological responders, particularly in those with moderate liver fibrosis, we still lack confirmation that anti-HBV therapy prevents HCC in patients with an established cirrhosis, too. Finally, tertiary prevention with anti-HBV treatments is controversial, due to the existence of a few, methodologically flawed studies.

Survivin downregulation by siRNA/cationic liposome complex radiosensitises human hepatoma cells in vitro and in vivo.

PURPOSE: To investigate the effect of survivin-short hairpin RNA (shRNA) on the proliferation, apoptosis and radiosensitivity of human hepatoma SMMC-7721 cells. MATERIALS AND METHODS: Survivin-targeted small interfering RNA (siRNA) expression vector was constructed and transfected into SMMC-7721 cells mediated by cationic liposome. Survivin mRNA and protein expression were analysed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell cycle and apoptosis were measured by flow cytometry (FCM) assay. Radiosensitivity of SMMC-7721 cells was examined using a colony-forming assay. Mice subcutaneously implanted with SMMC-7721 cells were monitored for tumour growth and survival after treatment, and tumours were analysed for proliferation, apoptosis, and angiogenesis biomarkers by immunohistochemistry staining. RESULTS: After transfection, the mRNA and protein expression of survivin gene in SMMC-7721 cells downregulated, which led to significant cell growth inhibition, cell arrest in G2/M phase, increased apoptotic rate and radiosensitivity. Survivin-shRNA in combination with radiotherapy was more effective than radiotherapy or survivin-shRNA therapy alone in suppressing tumour growth and extending survival duration. Combined therapy inhibited cell proliferation and tumour angiogenesis and increased apoptosis in tumour xenografts. CONCLUSION: Survivin downregulation by siRNA/cationic liposome inhibited proliferation, induced apoptosis and enhanced radiosensitivity in human hepatoma cells in vitro and in vivo.

The biocompatibility of quantum dot probes used for the targeted imaging of hepatocellular carcinoma metastasis.

Semiconductor quantum dots (QDs) have several photo-physical advantages over organic dyes making them good markers in biomedical application. We used CdSe/ZnS QDs with maximum emission wavelength of 590nm (QD590) linked to alpha-fetoprotein (AFP) monoclonal antibody (Ab) to detect AFP in cytoplasm of human hepatocellular carcinoma (HCC) cell line HCCLM6. For the in vivo studies, we used QD-AFP-Ab probes for targeted imaging of human HCC xenograft growing in nude mice by injecting them into the tail vein. In addition, the cytotoxicity in vitro, the acute toxicity in vivo, the hemodynamics and tissue distribution of these probes were also investigated. The results in vitro and in vivo indicate that our QD-based probes have good stability, specificity and biocompatibility for ultrasensitive fluorescence imaging of molecular targets in our liver cancer model system.

Stepwise formation of patterned cell co-cultures in silicone tubing.

Here, we describe a method for producing patterned cell adhesion inside silicone tubing. A platinum (Pt) needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with layer-by-layer deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The utility of this approach was demonstrated by patterning co-cultures of hepatocytes or endothelial cells with fibroblasts. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

Dacron-covered stent therapy for portal vein tumor thrombus in hepatocellular carcinoma.

A tumor thrombus of the portal vein is refractory to therapy and constitutes a serious prognostic factor in hepatocellular carcinoma. For the purpose of treating portal vein tumor thrombus by restoring the blood flow and preventing recurrent ingrowth of tumor, we devised a metallic stent partially covered with a Dacron mesh sheet, and a coaxial percutaneous delivery system. One half of the wall of a Gianturco Z-stent was covered with a sheet of 0.25-mm-thick Dacron mesh, fixed to the stent wall with nylon threads. The covered stent was implanted in a patient with severe main portal vein stenosis due to tumor thrombus protruding from the left portal vein branch. Immediately after stent placement the tumor stenosis was effectively dilated, the portal blood flow restored, and the portal hypertension relieved. CT and angiography after 8 months still showed complete portal vein patency. Intrahepatic tumor dissemination or other complications were not observed. Intraportal placement of a covered metallic stent appears to be an efficacious therapy of major portal tumor thrombi.

Transforming growth factor-beta1 triggers hepatocellular carcinoma invasiveness via alpha3beta1 integrin.

Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express alpha3beta1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-beta1 stimulates alpha3-integrin expression at a transcriptional level in noninvasive HCC cells, causing transformation into a motile and invasive phenotype. Such activities are inhibited by neutralizing anti-alpha3- but not anti-alpha6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-beta1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-beta1. In this way, the cells express alpha3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-beta1-neutralizing antibody reduces alpha3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-beta1 serum concentrations and alpha3-integrin expression are strongly correlated. The integrin, absent in normal and peritumoral liver parenchyma, is abundantly expressed in HCC primary and metastatic tissue. In particular, patients with metastasis show higher levels of TGF-beta1 serum concentrations and stronger expression of TGF-beta1 and alpha3-integrin in HCC tissues. In conclusion, TGF-beta1 may play an important role in HCC invasiveness by stimulating alpha3-integrin expression, and could therefore be an important target for new therapies.