Baicalein induces the apoptosis of HCT116 human colon cancer cells via the upregulation of DEPP/Gadd45a and activation of MAPKs.

Baicalein has efficient antitumor properties and has been reported to promote the apoptosis of several human cancer cell lines. Decidual protein induced by progesterone (DEPP), a transcriptional target of Forkhead Box O, was originally identified from the human endometrial stromal cell cDNA library. However, the expression and physiological functions of DEPP in human colon cancer cells remain to be fully elucidated. In the present study, it was reported that baicalein stimulated apoptosis and morphological changes of HCT116, A549 and Panc­1 cells in a dose-dependent manner. It also upregulated the mRNA and protein levels of DEPP and growth arrest and DNA damage-inducible 45α (Gadd45a). In addition, the overexpression of DEPP promoted mitogen-activated protein kinase (MAPK) phosphorylation. To further investigate the role of DEPP and Gadd45a in baicalein-induced apoptosis, HCT116 cells were transfected with small interfering RNA against either DEPP or Gadd45a as in vitro models. Through an Annexin V/PI double staining assay, it was observed that baicalein-induced apoptosis was impaired by the inactivation of either DEPP or Gadd45a, which in turn restricted the baicalein-induced activation of caspase­3 and caspase­9 and phosphorylation of MAPKs. In addition, the inhibition of c­Jun N­terminal kinase (JNK)/p38 activity with SP600125/SB203580 decreased the expression of Gadd45a, whereas the inactivation of extracellular signal-regulated kinase with SCH772984 had no effect on the expression of Gadd45a. Taken together, these results demonstrated that baicalein induced the upregulation of DEPP and Gadd45a, which promoted the activation of MAPKs with a positive feedback loop between Gadd45a and JNK/p38, resulting in a marked apoptotic response in human colon cancer cells. These results indicated that baicalein is a potential antitumor drug for the treatment of colon cancer.

Human herpesvirus-6-specific interleukin 10-producing CD4+ T cells suppress the CD4+ T-cell response in infected individuals.

Human herpesvirus-6 (HHV-6) infection normally persists for the lifetime of the host and may reactivate with immunosuppression. The mechanism behind HHV-6 latent infection is still not fully understood. In this study, we observed that decreased proliferation of CD4+ T cells and PBMCs but not CD8+ T cells from HHV-6-infected individuals was stimulated with HHV-6-infected cell lysates. Moreover, HHV-6-stimulated CD4+ T cells from HHV-6-infected individuals have suppressive activity on naïve CD4+ T and CD8+ T cells from HHV-6-uninfected individuals. However, no increased proportion of CD4+ CD25+ Treg cells from HHV-6-infected individuals contributed to the suppressive activity of the HHV-6-stimulated CD4+ T cells from HHV-6-infected individuals. Transwell experiments, ELISA and anti-IL-10 antibody blocking experiment demonstrated that IL-10 may be the suppressive cytokine required for suppressive activity of CD4+ T cells from HHV-6-infected individuals. Results of intracellular interleukin (IL)-10 and IL-4 further implicated the HHV-6-specific IL-10-producing CD4+ T cells in the suppressive activity of CD4+ T cells from HHV-6-infected individuals. Results of intracellular interferon (IFN)-gamma demonstrated a decreased frequency of HHV-6-specific IFN-gamma-producing CD4+ T, but not CD8+ T cells in HHV-6-infected individuals, indicating that it was the CD4+ Th1 responses in HHV-6-infected individuals that were selectively impaired. Our findings indicated that HHV-6-specific IL-10-producing CD4+ T cells from HHV-6-infected individuals possess T regulatory type 1 cell activity: immunosuppression, high levels of IL-10 production, with a few cells expressing IFN-gamma, but none expressing IL-4. These cells may play an important role in latent HHV-6 infection.

Detection and analysis of anti-latent membrane protein 2A antibodies in the sera of patients with Epstein-Barr virus associated malignancies.

BACKGROUND: Epstein-Barr virus (EBV) associated malignancies with a Type II latency gene expression pattern, such as Hodgkin's disease, and nasopharyngeal carcinoma (NPC), frequently express the EBV antigen latent membrane protein 2A (LMP2A). We expected to establish a highly expressing LMP2A yeast cell strain and get the high quality LMP2A protein, which was used for detection, analysis and characterization of its antibodies in various patients' sera of EBV associated malignancies. METHODS: The plasmid pPICZalphaA-LMP2A containing the full length of LMP2A cDNA was constructed and transformed to Pichia pastoris GS115 to express LMP2A protein. After fermentation and purification, the LMP2A protein was used as an antigen to detect anti-LMP2A antibodies (Abs) in the sera of patients with EBV-associated malignancies in enzyme linked immunosorbent assay (ELISA) or Western-blot. RESULTS: LMP2A was expressed successfully with an expected molecular weight of approximately 54 kD and Abs to LMP2A were strikingly specific to NPC. Two-thirds or more sera from NPC patients were positive for anti-LMP2A immunoglobulin G (IgG) Abs. The antibodies were absent from the sera of other EBV-associated diseases except a small fraction of the gastric carcinoma. Comparing anti-viral capsid Ags (VCA) IgA and LMP2A IgA titers in the sera from 76 NPC patients, only 55% were positive for anti-LMP2A IgA Abs while 70% were positive for anti-VCA IgA. However, we found that 3 sera negative for VCA IgA were positive for LMP2A IgA. CONCLUSION: The results suggested the potential significance of LMP2A specific Abs for the diagnosis of EBV-associated malignancies, especially NPC.

[Expression of HBcAg in eukaryotic cells by retroviral vector mediated gene transfer].

BACKGROUND: To construct recombinant retroviral vector expressing HBcAg in eukaryotic cells. METHODS: The HBV core gene fragment was amplified by using PCR from pADR which contains complement nucleotide sequence of HBV subtype adr and cloned into retroviral expression plasmid pLXSN, then transfected into packing cell (PT67) with lipofec AMINE. After 2-3 weeks selection with G418, large colonies were isolated and transferred to individual plates. Virus-containing medium was collected and used to infect NIH3T3, EL4 and mouse bone marrow derived dendritic cells(DC). DNA was extracted from infected cells and tested by PCR. Indirect immunofluorescence and FACS were used to detect the expression of HBcAg. Cell mediated immunity of immunized C57BL/6 mice with transduced DC was analyzed. RESULTS: The insertion of HBV core gene fragment in the recombinant retroviral plasmid was confirmed by PCR as well as enzyme digestion with EcoR1 and BamH2. The viral titer reached 3 x 10(5) CFU/ml. The result of PCR showed that the HBV core gene had been integrated into the genome of infected NIH3T3 cells. Indirect immunofluorescence and FACS analysis showed that HBcAg had been expressed in these cells. HBcAg specific CTL responses could be generated in mice immunized with retrovirus transduced DC. CONCLUSIONS: HBV core gene had been integrated into eukaryotic cells with retroviral vector and target gene had been expressed efficiently. These results may have some impact on gene therapy of chronic hepatitis B.

[Construction and identification of recombinant adenovirus vector containing thioredoxin reductase gene].

AIM: To construct recombinant adenovirus vector containing human thioredoxin reductase (TR) gene and to explore the correlation between antioxidant activity of TR and the degenerative neuropathy. METHODS: Full length TR cDNA was obtained from recombinant plasmid pGEM-TR via digestion with Apa I and Not I and was cloned into pShuttle vector and pShuttle-TR was recut with I-Ceu I and PI-Sce I. Fragment containing TR gene and CMV promoter was inserted into E1 and E3 deficient adeno-X virus DNA, and then the recombinant adenovirus vector was transfected into HEK 293 cells through lipofectamine and identified by PCR. The TR expression on and in cell lysate of CV1 cells infected with recombinant adenovirus was by immuno fluorescence assay and Western blot analysis. RESULTS: After replication of recombinant adenovirus Adeno-TR, the virus titer was about 4.4x10(11) pfu/L. The TR expression on CV1 cells was proved by fluorescent microscopy. Western blot analysis showed a band with relative molecular mass (M(r)) of 55,000. CONCLUSION: A recombinant adenovirus vector has been successfully constructed and TR is expressed on CV1 cells. This result lays the foundation for further study on function of TR and its correlation with degenerative neuropathy.

[Retroviral-mediated transfection of hepatitis B virus core gene into bone marrow-derived dendritic cells].

AIM: To evaluate the transfection efficiency of recombinant retrovirus vector bearing hepatitis B virus(HBV) core gene to bone marrow-derived dendritic cells(DCs) and the capability of these DCs to induce cytotoxic T lymphocyte(CTL) response. METHODS: C57BL/6 mice bone marrow cells were stimulated with recombinant mouse granulocyte-macrophage colony-stimulating factor(rmGM-CSF) and interleukin-4(rmIL-4) for 6 days. Expanding DC progenitors were transfected by retrovirus vector containing HBV core gene. Integration and transcription of HBV core gene were determined by PCR and RT-PCR, respectively. Expression of HBcAg was analyzed by fluorescence activated cell sorter (FACS) and Western blot. Cytokines were quantified by enzyme immunoassay. The expressions of CD80 and MHC class II molecules on DCs were measured by FACS. Generation of CTLs in mixed leukocyte reaction were determined by LDH release assays. RESULTS: Transfected bone marrow cells were capable of differentiating into DCs in-vitro at the presence of rmGM-CSF and rmIL-4. The result of PCR and RT-PCR showed that the HBV core gene was integrated into the genome of infected DCs. Western blot analysis showed that HBV core gene was expressed in DCs. Transfection rate was 28% determined by FACS. Retroviral transfection had no influence on expressions of CD80 and MHC class II molecules, as well as IL-12 production. HBcAg-specific CTLs could be generated by using transfected DCs as antigen presenting cell (APC). CONCLUSION: Retroviral transfected myeloid DC progenitors could efficiently express HBcAg, without significant change on development and function of DCs, which lays a solid foundation for immunotherapy of chronic hepatitis B.

[Modulation of lianbizi injection (andrographolide) on some immune functions].

OBJECTIVE: To study the effects of andrographolide on immune functions and the immune mechanism of its clinical application. METHOD: The amounts of IFN-alpha, IFN-gamma, TNF-alpha, IL-8 in the peripheral blood mononuclear cells(PBMCs) culture supernatants deal with by different concentrations of LianBiZhi (LBZ) injection made of andrographolide were detected by biological activity test or ELISA in vitro. The effects of LBZ injection on macrophage phagocytotic function and natural killer cells cytotoxicity were examined by means of macrophage to phagocytize cock erythrocyte and measurement of lactate dehydrogenase(LDH) activity released from the damaged cells, respectively. RESULT: The LBZ injection could promote IFN-alpha, IFN-gamma, TNF-alpha inductions of PBMCs, but had no effect on IL-8. At the same time, the LBZ injection could not only enhance the phagocytosis activity of peritoneal macrophage from guinea pig to phagocytosis cock erythrocyte, but also augment the cytotoxicity mediated by natural killer cells from PBMCs to damage the K562 cell lines. CONCLUSION: Andrographolide is an immunostimulant agent which can modulate both antigen specific and nonspecific immune function by means of its natural killer cells and macrophage and cytokines induction.