Article Figures & Data
Figures
- Figure 1.
Effects of AZD6244 on cell viability; proliferation; phosphorylation of MEK, ERK1/2, and p90RSK; and apoptosis in primary 2-1318 cells. Primary cells from 2-1318 tumors were isolated as described in Materials and Methods. They were grown and treated with 0.1% DMSO or escalating doses of AZD6244 in serum-free MEM. Cell proliferation and cell viability were determined by bromodeoxyuridine incorporation and MTT assay, respectively, as described in Materials and Methods. Cell proliferation (A) and cell viability (B) at 24 and 48 h. Experiments were done in quadruplicate. Columns, mean; bars, SE. C, Western blot analysis was done as described in Materials and Methods with cells treated as described above. Blots were incubated with the indicated antibodies and representative samples were shown. D, apoptotic cells were determined by TUNEL assay; the rate of apoptosis was expressed as a percentage of the total cells counted. Similar results were obtained when 26-1004 and 4-1318 cells were used. Columns with different letters are significantly different from one another (P < 0.01) as determined by ANOVA. Columns, mean; bars, SE.
- Figure 2.
Time-dependent cleavage of caspase-3 and caspase-7 and PARP in primary HCC cells, and caspase-dependent AZD6244-induced apoptosis. Primary HCC cells were isolated and cultured as described in Materials and Methods. A, cells were treated with serum-free MEM containing 1 μmol/L AZD6244 for various times. B, cells were treated with 1 μmol/L AZD6244 in the presence or absence of 50 μmol/L Z-VAD-FMK for 12 h. Western blot analysis was done as described in Materials and Methods. Blots were incubated with the indicated antibodies. Experiments were repeated thrice with similar results.
- Figure 3.
Growth behavior of seven HCC xenografts and their basal levels of MEK1, Cdk-5, ERK1/2, phosphorylated ERK1/2, and phosphorylated MEK1 at Ser218/222 and Thr286. The indicated lines of HCC xenografts were established as described in Materials and Methods. A, the growth rate of each line of xenografts shown was previously presented (30). B, tumors from each line of xenografts were collected and Western blotting was done as described in Materials and Methods. Blots were incubated with the indicated antibodies. C, basal MEK kinase activity for each line of xenografts was done as described in Materials and Methods; the [32P]MBP levels were presented. Note that the numbers in parentheses for the curves in A correspond to the columns in B.
- Figure 4.
Effects of AZD6244 on growth rate and tumor weight of HCC xenografts. 4-1318 and 5-1318 xenografts were s.c. implanted on the right side of male SCID mice as described in Materials and Methods. Mice bearing xenografts were p.o. given twice a day either with vehicle or AZD6244 for 21 d. A and B, 50 mg (AZD50) and 100 mg (AZD100) AZD6244 per kilogram of body weight were used for 4-1318 xenografts. C and D, only the 50-mg dose (AZD50) was used for 5-1318. Treatment started on day 7 after tumor cell injection. Tumor growth was measured and calculated as described in Materials and Methods. Tumor volume at a given time for control and AZD6244-treated 4-1318 (A) and 5-1318 (C) xenografts is plotted. Each data point represents six to eight tumor samples. Tumor weight at sacrifice for control and AZD6244-treated 4-1318 (B) and 5-1318 (D) xenografts are shown. Columns with different letters were statistically significant (P < 0.01) as analyzed by ANOVA. Experiments were repeated at least twice with similar results.
- Figure 5.
Effects of AZD6244 on tumor weight of 2-1318, 26-1004, and 29-1104 xenografts. Indicated lines of HCC xenografts were implanted as described above. Mice bearing xenografts were p.o. given twice a day either with vehicle or 50 mg/kg of AZD6244 for 21 d. Treatment started on day 7 after tumor cell injection. Tumor growth was measured and calculated as described in Materials and Methods. Tumor weight at sacrifice for control and AZD6244-treated xenografts, 2-1318 (A), 26-1004 (B), and 29-1104 (C). Columns with different letters were statistically significant (P < 0.01) as analyzed by ANOVA. Experiments were repeated at least twice with similar results.
- Figure 6.
Effects of AZD6244 on the levels of B-Raf; phosphorylation of MEK1, ERK1/2, and p90RSK; and apoptosis in HCC xenografts. Indicated HCC xenografts were implanted as described in Materials and Methods. Mice bearing indicated xenografts were p.o. given, twice daily, with either vehicle or 50 mg AZD6244 per kilogram for 21 d. Lysates from 4-1318, 2-1318, and 5-1318 (A), and 26-1004 (C) tumors were subjected to Western blot analysis as described in Materials and Methods. Blots were incubated with indicated antibodies. B, basal MEK1 kinase activity for vehicle- and AZD6244-treated 4-1318 xenografts was done as described in Materials and Methods, and the [32P]MBP levels are shown. Representative samples. Experiments were repeated at least twice with similar results.
Volume 6, Issue 1
