[Effects of beta-sitosterol on microtubular systems in cervical cancer cells].

OBJECTIVE: To investigate beta-sitosterol's inhibitory effects on SiHa cells' growth, and the effects on microtubular system in SiHa cell. METHODS: Proliferation inhibition of SiHa cell line was evaluated by MTT assay. Cell cycle of SiHa cells treated with beta-sitosterol was analyzed by flow cytometry. The expression and distribution of microtubule and microtubule associated protein 2 in SiHa cells were investigated by confocal microscopy. Immunoblotting analysis was used to determine tubulin alpha, microtubule associated protein 2, and the proportion of polymerization of tubulin. RESULTS: beta-sitosterol could obviously inhibit the proliferation of SiHa cells, and induce the accumulation of cells in S phase (rather than the G2/M phase) and mitotic arrest in the cell cycle. Confocal microscopy showed an abnormal microtubular network in SiHa cell treated with beta-sitosterol for 5 days, and the expression of microtubule associated protein 2 was marked down-regulated. Further analysis by immunoblotting confirmed the down-regulation of beta-sitosterol on the expression for both microtubule associated protein 2 and tubulin alpha. Moreover, beta-sitosterol reduced the proportion of polymerization of microtubule in a time-dependent manner. CONCLUSION: beta-sitosterol could down-regulate the expression of tubulin alpha and microtubule associated protein 2 in SiHa cells, and inhibit the microtubular polymerization. Our results suggested an anti-microtubule characteristic of beta-sitosterol which might contribute to the proliferation inhibition of SiHa cells.

Role of interleukin 18 in acute lung inflammation induced by gut ischemia reperfusion.

AIM: To study the changes of endogenous interleukin 18 (IL-18) levels and evaluate the role of IL-18 on lung injury following gut ischemia/reperfusion. METHODS: A superior mesenteric artery occlusion model was selected for this research. The mice were randomly divided into four groups: Sham operation (sham), ischemia (0.5 h) followed by different times of reperfusion (I/R), and I/R pretreated with exogenous IL-18 (I/R+IL-18) or IL-18 neutralizing antibody (I/R+IL-18Ab) 15 min before ischemia. Serum IL-18 levels were detected by Western blot and ELISA, and the levels of IL-18 in lung tissue were evaluated by immunohistochemical staining. For the study of pulmonary inflammation, the lung myeloperoxidase (MPO) contents and morphological changes were evaluated. RESULTS: Gut ischemia/reperfusion induced rapid increase of serum IL-18 levels, peaked at 1 h after reperfusion and then declined. The levels of IL-18 in lung tissue were gradually enhanced as the progress of reperfusion. Compared with I/R group, exogenous administration of IL-18 (I/R+IL-18) further remarkably enhanced the pulmonary MPO activity and inflammatory cell infiltration, and in I/R+IL-18Ab group, the content of MPO were significantly reduced and lung inflammation was also decreased. CONCLUSION: Gut ischemia/reperfusion induces the increase of IL-18 expression, which may make IL-18 act as an important proinflammatory cytokine and contribute to gut ischemia/reperfusion-induced lung inflammation.

The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv.

A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors' laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A-1 and SPC-A-1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.

HPV E6 down-regulation and apoptosis induction of human cervical cancer cells by a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott in vitro.

AIM OF THE STUDY: To evaluate the cytotoxicity and apoptosis induction effects of a novel lipid-soluble extract (PE) from Pinellia pedatisecta Schott on CaSki, HeLa and HBL-100 cells. Particularly, the effect of PE on HPV E6 gene expression was tested, and the mechanism of its apoptosis induction effect was also studied. MATERIALS AND METHODS: Cell viability was measured by the MTT assay. DAPI staining and flow cytometric analysis (FCM) were used to identify apoptotic cells in PE-treated CaSki, HeLa, and HBL-100 cells. Expression of the HPV E6 gene in CaSki and HeLa cells was detected by real-time RT-PCR and western blot analysis. Apoptosis-associated genes were examined by RT-PCR and western blot analysis in CaSki cells. RESULTS: PE inhibited the growth of CaSki and HeLa cells in a time- and dose-dependent manner, but it had no obvious inhibiting effect on HBL-100 cells except at a relatively high dose (500 µg/mL). PE could induce apoptosis in CaSki and HeLa cells in a time-dependent manner but not in HBL-100 cells. HPV E6 mRNA and protein were decreased significantly by PE. Caspase-8, caspase-3, Bax, P53 and P21 mRNAs as well as proteins were increased while Bcl-2 mRNA and protein were decreased significantly by 24 h of PE treatment. CONCLUSIONS: PE can function as a tumor suppressor by inducing apoptosis in human cervical cancer cells but it has little side effect on normal cells. It probably acts via mitochondria-dependent and death receptor-dependent apoptotic pathways. HPV E6 may be the key target of its action.

Role of interleukin-18 in the development of acute pulmonary injury induced by intestinal ischemia/reperfusion and its possible mechanism.

BACKGROUND AND AIMS: Lung injury is an important target for the systemic inflammatory response associated with intestinal ischemia/reperfusion (I/R). In the present study, the role of interleukin (IL)-18 in the development of acute pulmonary injury induced by intestinal I/R and its possible mechanism in relation to the increased activity of inducible nitric oxide synthase and tumor necrosis factor (TNF)-alpha were investigated. METHODS: Mice were randomly divided into three groups: normal control group without operation; sham group with sham operation; and I/R group in which mice underwent superior mesenteric artery occlusion for 30 min followed by reperfusion for 3 h. Each group received pretreatment with exogenous IL-18, anti-IL-18 neutralizing antibody or L-NIL, the selective inhibitor of inducible nitric oxide synthase, 30 min before ischemia. The expression of TNF-alpha was detected by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Lung injury was evaluated by means of Evans blue dye (EBD) concentration, myeloperoxidase (MPO) activity and morphological analysis. RESULTS: The experimental results showed that both in the sham-operated and I/R groups of animals, pretreatment with exogenous IL-18 clearly enhanced pulmonary MPO activity, microvascular leakage and the expression of TNF-alpha mRNA and protein. In contrast, IL-18 did not increase the TNF-alpha level and degree of lung injury, although it clearly enhanced the pulmonary MPO activity in normal animals. Meanwhile, IL-18 antibody given prior to ischemia led to a reduction in the sequestration of neutrophils, extravasation of EBD and downregulation of the serum level of TNF-alpha in the I/R group of animals. In addition, selective inhibition of inducible nitric oxide synthase (iNOS) that inhibited plasma extravasation and pulmonary injury without affecting the MPO activity could be demonstrated in all treated animals. CONCLUSIONS: These data suggested a role of IL-18 in the activation and sequestration of neutrophils in lungs. Our results were consistent with the hypothesis that increased sequestration of neutrophils and microvascular leakage might, respectively, relate to the increased IL-18 level and the elevation of TNF-alpha/iNOS activity, and these two aspects might synergically contribute to intestinal I/R-induced pulmonary dysfunction.

[The modification of culture and cryopreservation methods for human junctional epithelium cells].

PURPOSE: To study the methods for isolating, culturing and cryopreserving junctional epithelium(JE) cells. METHODS: JE tissue samples were obtained from human periodontally healthy premolars which were extracted for orthodontic reasons. The sulcular epithelium was removed at a distance of 1 mm from the gingival margin. JE tissue was detached from the extracted teeth by using 11 sterile blade and minced into small fragments. The cultured JE cells were incubated with a freezing medium consisting of 90% calf serum and 10% DMSO. The freezing protocol was applied using a computer-controlled freezer to freeze the medium to -80 degrees centigrade before cells were plunged into liquid nitrogen and stored. The above procedures were optimized to further study the morphology, proliferation, determination of JE cells and measure the viability after thawing. RESULTS: 0.1% trypsin containing 0.02% ethylenediamine tetraacetate (EDTA) digestion was used to isolate JE cells successfully without dispase. The viability of thawing JE cells was(93.87+/-3.11)%, and the morphology was similar to that of the second passage JE cells. CK19 and vimentin were both positive in JE cells immunocytochemically. CONCLUSION: The methods could be applied into practice. The phenotype of JE cells in vitro is not always consistent with that in vivo. It might be associated with the substrate which cells grow in contact with.

Cloning and characterization of a new isoform of mouse interleukin-18.

Interleukin-18 (IL-18) is a novel proinflammatory cytokine with potent interferon (IFN)-lambda inducing activity that plays an important biological role in the enhancement of the activity of natural killer cells and cytotoxic T lymphocytes. In this study, we have identified a novel short form of IL-18 in mouse, named IL-18s. IL-18s might be an alternative splicing variant of IL-18 and its cDNA contains a 57 bp in-frame deletion. Like IL-18, IL-18s is also widely expressed in mouse tissues. It was suggested that IL-18s might have a caspase-1-dependent mechanism for maturation and secretion similar to that of IL-18: when transfected in COS-7 cells, pro-IL-18s (22 kDa) could be detected, and the mature IL-18s (16 kDa) could also be detected when combined with caspase-1. We observed that recombinant mouse IL-18s did not show any IL-18-like function, and IL-18s could enhance the ability of IL-18 to increase IFN-lambda production by approximately 40% in mouse splenocytes. This effect was observed primarily at relative low concentrations of IL-18, suggesting that IL-18s might regulate the activity of IL-18 in the physiological conditions.

[Culture and comparison of the biological characteristics of human junctional epithelium and gingival epithelium].

PURPOSE: To study the different biological characteristics of human junctional epithelium (JE) and gingival epithelium (GE). METHODS: Human JE cells were cultured and identified by the cell culture and immunohistochemistry, and the biological characteristics of JE cells and GE cells were compared. RESULTS: The morphology of cultured JE cells was various and unequal, the arrangement of the cells was loose and mitosis was common, while GE colony was consisted of equal and closely packed epithelial-like cells in a paving stone arrangement. CK-Pan staining was positive in all JE and GE cells. CK19 was strongly stained in all JE cells but only moderately stained in some GE cells, and most GE cells were negative which was obviously different from JE cells. JE cells had longer latent period (7 days) than GE cells (4 days) during the cell growth period, and then the cells proliferated rapidly (4 days) to attain the maximum and descended rapidly in the declining period. While GE cells ascended evenly (7 days) to attain the maximum and descended slowly in the declining period. Proliferation study demonstrated the doubling time of JE cells was 48 to 60 hours and that of GE cells was 72 to 96 hours. It was possible to subculture JE cells up to 5 times serially, and that of GE cells was up to 7 times. CONCLUSIONS: The human JE cells are a kind of unique non-differentiated epithelial cells different from GE cells. In this experimental culture condition the subculture times of JE cells were less than GE cells, which affects JE cells, so the culture methods and conditions should be improved.