Prolonged exposure of colon cancer cells to the epidermal growth factor receptor inhibitor gefitinib(Iressa^(TM))and to the antiangiogenic agent ZD6474:Cytotoxic and biomolecular effects

AIM： To analyze the biological effects of prolonged in vitro exposure of HT-29 and LoVo colon cancer cell lines to gefitinib （Iressa^TM）, an inhibitor of epidermal growth factor receptor （EGFR） activity, and ZD6474, an inhibitor of both KDR and EGFR activities. METHODS： Cells were treated with each drug for up to 2 wk using either a continuous or an intermittent （4 d of drug exposure followed by 3 d of washout each week） schedule. RESULTS： In both cell types, prolonged exposure （up to 14 d） to gefitinib or ZD6474 produced a similar inhibition of cell growth that was persistent and independent of the treatment schedule. The effects on cell growth were associated with a pronounced inhibition of p-EGFR and/ or p-KDR expression. Treatment with gefitinib or ZD6474 also inhibited the expression of EGFR downstream signal molecules, p-Erkl/2 and p-Akt, although the magnitude of these effects varied between treatments and cell lines. Furthermore, expression of the drug resistance-related protein ABCG2 was shown to significantly increase after 14 d of continuous exposure to the two drugs. CONCLUSION： We conclude that long-term exposure of colon cancer cells to gefitinib and ZD6474 does not modify their cytotoxic effects but it might have an effect on sensitivity to classical cytotoxic drugs.

Protein kinases are potential targets to treat inflammatory bowel disease

Protein kinases play a crucial role in the pathogenesis of inflammatory bowel disease(IBD), the two main forms of which are ulcerative colitis and Crohn's dis-ease. In this article, we will review the mechanisms of involvement of protein kinases in the pathogenesis of and intervention against IBD, in terms of their effects on genetics, microbiota, mucous layer and tight junc-tion, and the potential of protein kinases as therapeutic targets against IBD.

Role of p38 MAPK in lipopolysaccharide-induced iNOS expression by endothelial cells

Objective:To examine the role of p38 mitogen-activated protein kinase (MAPK) in NO production and Inos expression in human endothelial cells stimulated by lipopolysaccharide (LPS). Methods: The NO level in the supernatant of the cell culture media was measured with Griess method, expressions of Inos protein and Mrna in vitro cultured endothelial cell line ECV304 were detected with immunofluorescence analysis and reverse transcriptase-PCR respectively. Immunokinase assay was employed to measure P38mapk activity. Results: Compared with the basal level of Inos expression and NO production, the NO level and the expressions of Inos Mrna and protein in the cells were increased after LPS stimulation. P38mapk activity in ECV304 cells exhibited a marked increase at 15 min after LPS stimulation, lasting for about 45 min before gradually decline. The Inos protein and Mrna expressions induced by LPS stimulation was significantly inhibited by SB203580 [4-(4-fluorophenyl)-2-(4- methylsulfinylphenyl)-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK. Conclusion: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and the inhibition of the signal transduction pathway can be effective to reduce the production of iNOS and other cytokines, and therefore constitutes a useful strategy for treating septic shock or inflammation.

Aqueous extract of Taxus Chinensis (Pilger) Rehd inhibits lung carcinoma A549 cells through the epidermal growth factor receptor/mitogen-activated protein kinase pathway in vitro and in vivo

OBJECTIVE： To explore the anticancer mechanism of aqueous extract of Taxus Chinensis （Pilger） Rehd （AETC）. METHODS： The serum pharmacological method was used to avoid interference from administration of the crude medicinal herbs. Eight purebred NewZealand rabbits were used for preparation of serum containing various concentrations of AETC. For- ty-eight Balb/c-nu mice were used for in vivo experi- ments. The effects of serum containing AETC on the proliferation of A549 cells and expression levels of the epidermal growth factor receptor/mito- gen-activated protein kinase （EGFR/MAPK） path- way-related proteins in vitro were investigated. Ad- ditionally, the effects on the growth of A549 xeno- grafts in nude mice, and expression levels of the EG- FR/MAPK pathway-related proteins in the xeno- grafts, were investigated. RESULTS： 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl- tetrazolium bromide （MTT） assay revealed that the serum containing AETC significantly decreased the viability of A549 cells in a dose-dependent manner. Western blot showed that the serum containing various concentrations of AETC strongly reduced the levels of phospho-Jun N-terminal kinase （p-JNK） and phospho-extracellular signal-regulated kinasel/2 （ERK1/2） while it increased the level of p-p38. However, no significant effects on the ex- pression levels of JNK, ERK1/2, and p38 MAPK were found. In addition, an anticancer effect from AETC was observed in vivo in the Balb/c-nu mice bearing A549 xenografts.CONCLUSION： AETC has significant effects on the growth of A549 xenografts and on the activity of the EGFR/MAPK pathway. Therefore, AETC may be beneficial in lung carcinoma treatment.

Cytoskeleton-associated protein tyrosine phosphorylation involved in induction of differentiation in mouse melanoma cells

The malignancy of a cancer is due partly to its poor differentiation. Genistein, a protein tyrosine kinase inhibitor, is found to induce the highly malignant B16-BL6 mouse melanoma cells to differentiate to mature phenotypes. When Triton X-100 insoluble fraction of the differentiated cells is prepared and analyzed, tyrosine phosphorylation levels of three cytoskeleton-associated proteins (65, 60 and 53 ku respectively) are found to decrease dramatically. But no any change is found when phosphotyrosine contents of the cytosol fraction or the total cellular protein preparations are evaluated. It is concluded that cytoskeleton-associated protein tyrosine phosphorylation may be involved in the control of differentiation of cancer cells. The decrease of phosphotyrosine contents of cytoskeleton-associated proteins may be one of the important mechanisms underlying the differentiation induction of cancer cells by anticancer agents.