Article Figures & Data
Figures
- Fig. 1.
Effects of Dox and Paclitaxel on apoptotic protein expression/phosphorylation in H460 cells treated for 48 h. A, XIAP levels were analyzed by Western blot (top) and the amount of protein was quantitated by densitometry (bottom). XIAP levels were normalized to cellular actin levels and compared with untreated control levels; B, Western blot analysis of phosphorylation of Bcl-2 protein (p-Bcl-2) and wild type Bcl-2 (Bcl-2); C, Western blot analysis of Bcl-xL protein levels; bars, ±SD.
- Fig. 2.
Dox and Paclitaxel mediated caspase-3 and PARP activation in H460 cells treated for 48 h. A, effects of Dox and Taxol at different concentrations on the expression of pro-caspase-3 (Cas-3). B, the expression of PARP (full length, 116 kDa and processed 89 kDa). Protein expression was analyzed by Western blotting.
- Fig. 3.
Effects of serum starvation on the expression of XIAP protein and cell growth of H460 cells. A, reduction of XIAP protein expression and (B) reduction of growth rates of cells cultured in serum-free medium for 24 or 48 h compared with control cells (CNT) growing in 10% FBS medium. Enhanced reduction of XIAP protein (C) expression and PARP protein (D) expression (full length, Mr 116,000 and processed Mr 89,000) in H460 cells treated with Dox and Paclitaxel in serum-free medium for 24 h; bars, ±SD.
- Fig. 4.
Phosphorylation sites in phosphoproteins of protein kinases (listed in Table 1) in H460 cells; control (A), treated with 1 μm of Dox for 12 h (B), and cultured in serum-free medium for 24 h (C). Note: 1 = ERK1,2; 2=MEK1,2; 3=PKCα; 4=PKCα/β; 5 = SAPK/JNK (Mr 46,100); 6 = SAPK/JNK (Mr 38,600). The assay method was described in “Materials and Methods.”
- Fig. 5.
Effects of the MEK inhibitor U0126 and the protein kinase inhibitor STP on the induction of apoptosis in H460 cells. A, expression of XIAP (top) and Bcl-2 protein (bottom) analyzed by Western blotting; B, morphological changes of H460 cells stained with 4′,6-diamidino-2-phenylindole. Arrows indicate cells showing DNA condensation and nuclear fragmentation.
- Fig. 6.
MTT assay data plotted and analyzed by CalcuSyn software. A, C, E, and G, dose effect of Dox or Taxol alone or in combination with U0126 (U0) or STP. In plots B, D, F, and H data were analyzed for determination of CI. A CI index of <1 is synergistic,=1 is additive, and >1 is antagonistic. Strong synergism is indicated by CIs of 0.1–0.3, and values of 0.3–0.7 or 0.7–0.85 are considered to indicate synergism or moderate synergism, respectively. All of the U0126 or STP combinations tested with Dox or Taxol exhibited moderate to strong synergism (B, D, F, and H).
Tables
- Table 1
Thirty-one phosphorylation sites in phosphoproteins of protein kinases tested
Abbreviation Full name of protein Epitope(s)a Adducin Adducin α S662 CDK1 Cyclin-dependent kinase 1 (Cdc2) Y15 CREB cAMP responsive element binding protein S133 ERK1/2 Extracellular regulated kinase 1 T202/Y204 GSK3α Glycogen synthase kinase 3 α S21/Y279 GSK3β Glycogen synthase kinase 3 β S9/Y216 JUN Oncogene Jun S73 MEK1 MAP kinase kinase 1 S217/S221 MEK3 MAP kinase kinase 3 S189 MEK6 MAP kinase kinase 6 S207 MSK1 Mitogen and stress activated protein kinase 1 S376 NR1 N-methyl-d-aspartate glutamate receptor subunit 1 S896 p38α MAPK p38 alpha MAP kinase T180/Y182 p70 S6K S6 kinase p70 T389 PKBα (Akt1) Protein kinase B α T308/S473 PKCα Protein kinase C α S657 PKCα Protein kinase C α T638 PKCδ Protein kinase C δ T505 PKCε Protein kinase c epsilon S719 PKR dsRNA dependent protein kinase T451 RAF1 Oncogene Raf1 S259 RB1 Retinoblastoma S780 RB1 Retinoblastoma S807/S811 RSK1 Ribosomal S6 kinase 1 T360/S364 SAPK (JNK) Stress-activated protein kinase T183/Y185 SMAD1 SMA- and MAD-related protein 1 S463/S465 SRC Oncogene Src Y418 SRC Oncogene Src Y529 STAT1 Signal transducer and activator of transcription 1 Y701 STAT3 Signal transducer and activator of transcription 3 Y727 STAT5 Signal transducer and activator of transcription 5 Y694 -
↵a Y, tyrosine; S, serine; T, threonine.
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- Table 2
The reduced phosphorylation of protein kinases in H460 cells
No. Protein kinases Phos. epitope Molecular weight (Mr) Relative trace quantitya Control Dox (1 μm) Serum free 1 ERK1/2 T202/T204 42,000 1.0 0.64 0.20 2 MEK1/2 S221 41,000 1.0 0.73 0.15 3 PKCα S657 76,000 1.0 0.81 0.55 4 PKCα/β T638/641 76,000 1.0 0.71 0.31 5 SAPK/JNK T183/T185 46,000 1.0 0.38 0.30 6 SAPK/JNK T183/T185 39,000 1.0 0.44 0.56 -
↵a Trace quantity: The trace quantity of a band is measured by the area under its intensity profile curve. Units are intensity × mm. The relative trace quantities were calculated based on the cells cultured in 10% FBS medium in which the level of phosphorylation of protein kinase was set as 1. The data is average of two separate experiments. The difference of data from the two separate experiments is <15% of the average values.
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Volume 2, Issue 7
