Anti-proliferative and anti-invasive effects of garcinol from Garcinia indica on gallbladder carcinoma cells.

Garcinol, a natural histone acetyltransferase inhibitor, has been reported to exhibit significant anti-proliferative activity in various cancer cell types. However, no information is available about the anti-cancer effects of garcinol on gallbladder carcinoma cells (GBC). In this study, GBC cells (GBC-SD and NOZ) were treated by garcinol and subjected to Cell Counting Kit-8 (CCK-8), and GBC-SD cells were selected for further transwell chamber assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Our results indicated that garcinol could significantly inhibit the growth of GBC cells in a dose- and time-dependent manner. It also inhibited the invasion of GBC-SD cells in a dose-dependent manner. Garcinol treatment decreased the activity of matrix metalloproteinase 2 (MMP2) and MMP9 by the downregulation of mRNA levels, and these two enzymes are critical to tumor invasion. Treatment with garcinol also decreased Stat3 and Akt activation in GBC-SD cells. Taken together, the effects of garcinol on GBC-SD cells may be associated with the suppression of Stat3 and Akt signaling pathways, which may contribute to inhibiting their downstream targets such as mRNA levels of MMP2 and MMP9.

In vitro activity of cepharanthine hydrochloride against clinical wild-type and lamivudine-resistant hepatitis B virus isolates.

Hepatitis B virus (HBV) infection causes major public health problems worldwide. The clinical limitation of current antiviral drugs for HBV, such as lamivudine, is the emergence of drug-resistant viral strains during prolonged antiviral therapy. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has been reported to possess potent activity against various viruses. The present study was performed to evaluate the in vitro activity of CH against clinical wild-type and lamivudine-resistant HBV isolates in transiently transfected cells. HBV DNA was extracted from serum samples collected both before lamivudine therapy and at the time of viral breakthrough and was amplified by polymerase chain reaction (PCR). The amplicons were cloned into a novel expression vector, pHY106, which can initiate the intracellular HBV replication cycle after cell transfection. Following transfection of the cloned amplicon into HepG2 cells, a drug susceptibility assay was performed. The level of viral antigen, HBeAg, was determined by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (Q-PCR) was used for determining the amount of intracellular HBV DNA. Heat stress cognate 70 (Hsc70), a host protein required for HBV replication, was also analyzed by reverse transcription PCR (RT-PCR) to explore the possible antiviral mechanism of CH. The results showed that CH inhibited replication and HBeAg production by either wild-type or lamivudine-resistant HBV clinical isolates in a dose-dependent manner. The Hsc70 mRNA was also downregulated significantly. In conclusion, CH is active against both wild-type and lamivudine-resistant HBV clinical isolates, and its activity may be associated with its inhibition of host Hsc70.

[Screening and analysis of genes encoding hepatocellular carcinoma associated tumor antigens].

OBJECTIVES: To screen and clone the genes encoding hepatocellular carcinoma associated tumor antigens. METHODS: A hepatocellular carcinoma cDNA express library was constructed with ZAP vector and analyzed by serological analysis of recombinant cDNA expression library (SEREX) with sera from autologous and allogenous patients. Monoclonalized positive phage clones were converted into pBK-CMV phagemid forms by in vivo excision. The cDNA inserts were determined by restriction endonuclease digestion with EcoR I and Xho I. The cDNA inserts were sequenced and analyzed with bioinformatics. LIMS1 insert was cut from the clone HCL5-70 and constructed into pQE 31 express vector. The recombinant LIMS1 was expressed in M15 and analyzed with SDS-PAGE and Western blot. RESULTS: Fourteen genes were cloned from autologous screening and eleven genes were obtained with allogeneous analysis. One gene, kinectin, was identified in both autologous and allogeneous screening. Eight of the total twenty-four genes were unknown for their functions; the other sixteen genes can be classified into eight groups according to their established or putative function. Recombinant LIMS1 was expressed in M15. CONCLUSION: The identification of hepatocellular carcinoma associated tumor antigens provides potential targets for immunotherapy of hepatocellular carcinoma patients and will help in the understanding of the carcinogenesis of hepatocellular carcinoma.

Immunohistochemical analysis of the expression of FATE/BJ-HCC-2 antigen in normal and malignant tissues.

FATE/BJ-HCC-2 is a newly identified cancer/testis (CT) antigen, which was detected in tumor tissues and testis. As previous studies of FATE/BJ-HCC-2 expression pattern were mainly based on messenger RNA (mRNA) analysis, it is necessary to investigate its actual protein expression pattern in tumor tissues for the evaluation of its application value. In this study, we produced specific polyclonal antibody (pAb) to the recombinant FATE/BJ-HCC-2 protein and analyzed the FATE/BJ-HCC-2 antigen expression in normal and malignant tissues by the immunohistochemical approach. The results showed that there was no detectable FATE/BJ-HCC-2 antigen expressed in normal tissues except testis. In hepatocellular carcinoma (HCC) tissues, the FATE/BJ-HCC-2 antigen was detected in 20% (7/35) specimens. All samples that expressed the FATE/BJ-HCC-2 antigen were of poorly or moderately differentiated HCC. The stained antigen was located in the cytoplasm and the staining pattern showed heterogeneity from focal to more than 40% of the tumor cells. The FATE/BJ-HCC-2 antigen was also expressed in other tumor tissues. The results of [3H]thymidine incorporation showed that FATE/BJ-HCC-2 protein enhanced tumor cell proliferation after transfection of FATE/BJ-HCC-2 gene in HCC cell line (P<0.01). This effect could be specifically blocked by anti-FATE/BJ-HCC-2 pAb. Serological screening showed that the antibody specific to the FATE/BJ-HCC-2 antigen was detected in 7.7% (4/52) patients. Notably, the four positive patients bore poorly or moderately differentiated HCC. FATE/BJ-HCC-2 mRNA transcript was detected in the peripheral blood mononuclear cells (PBMCs) of 46.67% patients whose resected HCC tissue samples were positive for FATE/BJ-HCC-2 mRNA, which implicated tumor cell dissemination in blood circulation and may relate to the metastasis of HCC. Thus, FATE/BJ-HCC-2 may be a valuable candidate CT antigen for polyvalent vaccines in tumor immunotherapy and an assisting diagnostic marker for prognosis of the disease.

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.

Detection of antibodies against SARS-CoV in serum from SARS-infected donors with ELISA and Western blot.

Recombinant fragments of S proteins from the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARA-CoV) were generated and used in a Western blot (WB) assay that was compared to a commercial SARS ELISA method. In 85% of confirmed SARS cases (n = 20), the S2 recombinant fragment based WB was positive and this was comparable to the commercial ELISA using heat killed SARS-CoV. WB using the other four recombinant fragments in confirmed SARS cases generated lower rates of detection (S1--75%, S1-N--25%, S1-C--55%). Evaluation of sera from healthy controls (n = 60) resulted in two weakly positive ELISA results with the remainder being negative while the S2 protein WB demonstrated three positive results from the 20 controls with a history of SARS contact and no positive results in 40 noncontact controls. A discrepancy between the ELISA and S2 WB arose when evaluating per-2003 sera from individuals (n = 10) with SARS-like symptoms (ELISA--100% positive, S2 WB--30% positive). These data suggest that the S2 WB assay may be particularly useful in ELISA-negative SARS cases and in some ELISA-positive non-SARS cases.

[The cloning, expression, purification and identification of SARS virus S2 gene and study on its immunological characteristics].

AIM: To express S2 protein of SARS virus fused with Trx and then detect its reactivity to the sera from convalescent SARS patients. METHODS: The Trx-S2 fusion protein was expressed in E.coli. After purification, the Trx-S2 fusion protein was detected by Western blot with 6 serum samples of convalescent SARS patients and 6 serum samples of healthy donors. RESULTS: According to the SDS-PAGE analysis, the relative molecular mass (M(r)) of the Trx-S2 fusion protein is about 76 x 10(3). The fusion protein could react with all the sera from convalescent SARS patients but not with the sera from healthy donors. CONCLUSION: The Trx-S2 fusion protein provides a basis for the research on its role in the course of SARS virus infection of host cells and preparation of recombinant vaccine against SARS virus.

[Expression and purification of recombinant N-terminal protein of SARS S1 subunit expressed in E. Coli].

OBJECTIVE: To express and purify the recombinant N-terminal protein of SARS virus S1 subunit and to study its role in SARS immune response. METHODS: The gene encoding N-terminal 334 amino acid residuals of SARS virus S1 subunit was cloned and expressed in E. Coli. After purification, the recombinant protein was identified by anti-SARS positive sera from recovered SARS patients. The sera from health donors, which were collected before the out-break of SARS, were used as negative control in the study. RESULTS: Sequencing analysis confirmed that the desired DNA sequence in recombinant plasmid was correct and had the same sequence of natural N-terminal of SARS virus S1 subunit. The molecular weight of recombinant fusion protein is about 64 000. The recombinant S1 protein could react with three antibody positive samples from recovered SARS patients, which showed specific bands at 64 000, but not with the control samples according to results of western blot. CONCLUSION: The recombinant N-terminal protein of SARS virus S1 subunit displays specific reaction with SARS antibody and may provide a good tool for further research of immune response to SARS virus.

Purification and refolding of a novel cancer/testis antigen BJ-HCC-2 expressed in the inclusion bodies of Escherichia coli.

BJ-HCC-2 is one of the cancer/testis antigens that may be the most promising targets for tumor immunotherapy. To investigate the expression of BJ-HCC-2 protein in tumor cells and its capacity to elicit CTL response, the recombinant protein of BJ-HCC-2 was expressed in the inclusion bodies in Escherichia coli. The inclusion bodies were solubilized effectively with 0.3% N-lauroyl sarcosine in alkaline buffer. Under this denatured form, the BJ-HCC-2 protein carrying 6x histidine tag was purified with Ni-NTA affinity chromatography in a single step with a purity of over 97%. The yield of the purified protein was about 78%. The purified recombinant protein was refolded in a simple way. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The recovery rate of refolded protein was 92.1%. The renatured protein displayed its immunoreactivity with the antibodies to BJ-HCC-2 protein by Western blotting. This method of protein purification and refolding is easy to manipulate and may be applicable to the hydrophobic proteins that are unable to be purified by other methods.