Study On The Anti-cancer Effect Of Guidemycin

Geldanamycin(GA) is a naturally occurring low molecular weight compound produced by microorganisms and is the fast ansamycin-derivative of benzoquinone isolated from medium of streptomycete.GA was found to possess many kinds of biological acitivity.Currently,it has been confirmed that the target of GA is heat shock protein 90(Hsp90).GA can inhibit the function of Hsp90 that required for the folding and the activity of client proteins.Many molecules involved in survival and proliferation of tumor cells such as transmembrane tyrosine kinase,signal transduction protein,oncogenic and anti-oncogenic protein,chimeric protein,steroid hormone receptor and cyclin are associated with Hsp90.Treatment of cells with GA generally induces not only the inactivation of client proteins but also the reduction in the concentrations of these proteins in the cell.In addition,the level of Hsp90 in the tumor cell is higher than that in the normal cell.Therefore,many researchers have been actively engaged in the development of specific Hsp90 inhibitor.Cinnamic Acid(CA) is a widely existed natural product.CA has been listed in GRAS due to non-toxic in limited doses.CA can reduce the size of many solid tumors in experimental animals by inhibiting proliferation and derivation of tumor cell.In the present study,a new derivative of GA named(GUM) was obtained by introducing the CA group into the 17-site of GA.Furthermore,the anti-cancer effects of GUM in vitro and in vivo were evaluated. 1.Effects of GUM on tumor cells cultured in vitroInhibition of cell proliferation was measured by the MTT assays.A remarkable difference in chemosensitivity to GUM was found in six cell lines.The IC₅₀ value of GUM for MCF-7,HepG2,H460,HCT116, SW1990 and U266 cells was 13.6±1.6μg/ml,63.56±5.7μg/ml,67.46±7.1μg/ml,283.8±11.8μg/ml,16.8±1.8μg/ml and 17.8±2.7μg/ml, respectively.Flow cytometry combined with FITC-Annexin V/PI staining showed that GUM could induce earlier apoptosis in four cell lines(MCF-7,HepG2, SW1990,U266).The rate of apoptosis incubated with 10μg/ml,1 g/ml and 0.1μg/ml GUM for 24 h was 5.21%±0.41%,2.7%±0.21%,2.86 %±0.25%and 2.15%±0.16%(Control) in MCF-7 cells,7.63%±0.56%,5.53%±0.44%,3.85%±0.31%and 2.48%±0.28% (Control) in SW1990 cells,7.68%±0.59%,6.92%±0.61%,3.72%±0.33%and 3.08%±0.32%(Control) in HepG2 cells,9.33%±0.81%, 4.92%±0.52%,3.93%±0.37%and 3.44%±0.31%(Control) in H460 cells,11.22%±0.89%,5.75%±0.47%,4.99%±0.41%and 2.63%±0.31%(Control) in U266 cells,respectively.The rate of apoptosis incubated with 10μg/ml,1μg/ml and 0.1μg/ml for 48 h was 23.16%±0.97%,7.35%±0.51%,4.96%±0.37%and 3.17%±0.24 %(Control) in MCF-7 cells,20.47%±0.91%,8.1%±0.73%,4.11%±0.38%and 2.71%±0.31%(Control) in SW1990 cells,27.55%±1.02%,8.38%±0.69%,3.17%±0.29%and 2.8%±0.31%(Control) in HepG2 cells,22.21%±1.27%,12.06%±0.74%,3.9%±0.41% and 2.98%±0.29%(Control) in H460 cells,25.22%±1.13%,8.0%±0.63%,2.48%±0.29%and 2.98%±0.24%(Control) in U266 cells, respectively.After exposure to GUM for 48 h,most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus by Hoechst33258 staining.Some condensed nuclei were observed when cells exposed to higher concentrations of GUM. The effect of GUM and CA on the migration of MCF-7,HepG2,H460 and SW1990 cells was examined using a transwell cell migration chamber assay.A dose-dependent reduction in cell migration in four cell lines was found after exposure to GUM.After treatment with 10μg/ml,1μg/ml and 0.1μg/ml GUM for 8 h,the inhibit rate of tumor cell migration was 79.78 %±4.82%,37.42%±2.14%and 8.17%±1.12%in MCF-7 cells, 66.62%±3.62%,31.08%±1.96%and 7.69%±1.21%in HepG2 cells,33.51%±2.28%,15.13%±1.41%and6.48%±1.38%inH460 cells,72.89%±6.49%,36.25%±3.02%and 8.18%±1.07%in SW1990 cells.The inhibit rate of tumor cell migration showed significant differences between control and GUM groups.The levels of RAF-1,EGFR,AKT,CDK4 and HER-2 were determined by Western blot analysis.MCF-7,HepG2 and H460 cells treated with GUM showed a dose-dependent decrease in the levels of RAF-1,EGFR,AKT, CDK4 and HER-2.The levels of RAF-1,EGFR,AKT,CDK4 and HER-2 were markedly reduced by 10μg/ml GUM.2.In vivo antitumor activity of GUMToxic assay of GUM and GA.The mice were received by i.p.injection either GUM or GA once a day for 10 days.The doses of GUM were 10mg/kg,20mg/kg and 40mg/kg.The doses of GA were 1.25mg/kg, 2.5mg/kg and 5mg/kg.After 10 days injection,there were no mice survival treated with GA at the dose of 5mg/kg.The survival numbers of treatment with GA at the dose of 2.5mg/kg and 1.25mg/kg was 1 and 3,respectively. There were no mice dead treated with GUM at the dose of 10mg/kg, 20mg/kg and 10mg/kg.Transplantable murine hepatoma22 model was used to evaluate the antitumor activity of GUM in vivo.Animals bearing s.c.tumors received by i.p.injection(200μl) either water(vehicle control) or GUM once a days for 10 days.The doses of GUM were 40 mg/kg,20 mg/kg and 10 mg/kg, respectively.The mice were weighed and tumor sizes were measured with a vernier caliper and recorded every day.Tumor volume was calculated using the formula:length×width²Ã—0.5.Growth of the established s.c. tumors in mice was decreased significantly when treated with GUM over a period of 10 days compared with vehicle control treated animals. Treatment with GUM at the dose of 40 mg/kg,20 mg/kg and 10 mg/kg inhibited the growth of H22 by 61.8%,38.9%and 25.7%,respectively. The antitumor activity of GUM is remarkable.The animals' weights showed no significant difference between control and treated groups.Athymic female BALB/c(nu/nu) mice were used for MCF-7 tumor xenografts.Mice bearing s.c.tumors received by i.p.injection(200μl) either water(vehicle control) or GUM once per 2 days for 5 weeks.The doses of GUM were 40 mg/kg,20 mg/kg and 10 mg/kg,respectively.The mice were weighed and tumor sizes were measured with a vernier caliper and recorded per 5 days.Tumor volume was calculated using the formula: length×width²Ã—0.5.Growth of the established s.c.tumors in nude mice was decreased significantly when treated with GUM over a period of 5 weeks compared with vehicle control treated animals.Treatment with GUM at the dose of 40 mg/kg,20 mg/kg and 10 mg/kg inhibited the growth of MCF-7 xenografts by 65.8%,45.9%and 15.4%,respectively. The antitumor activity of GUM is remarkable.The animals' weights showed no significant difference between control and treated groups.Above all,the results show that GUM can inhibit proliferation,induce apoptosis and limit migration of the tumor cells;it also can decrease the levels of Hsp90 client proteins such as RAF-1,EGFR,AKT,CDK4 and HER-2. Meanwhile,GUM inhibits the growth of tumor in H22 allografts and MCF-7 xenografts models.