Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 1 Rottlerin acts synergistically with imatinib to induce apoptosis of K562 cells. (a) K562 cells were cultured for 24 h with or without 1 mM imatinib in the presence or absence of 2 mM rottlerin, as indicated. Cells were stained with Annexin V-FITC as well as PI and analysed by flow cytometry. (b) K562 cells were cultured for 24 h with indicated concentrations of imatinib and rottlerin. Apoptotic cells were analysed by flow cytometry, and percentages of cells staining positive for Annexin V are plotted. (c) K562 cells were cultured for 24 h with 1 mM imatinib, 5 mM etoposide (VP16), or 10 ng/ml bortezomib in the presence or absence of 2 mM rottlerin, as indicated. Cells were stained with Trypan blue, and percentages of non-viable cells stained with the dye were plotted. Each data point represents the mean of triplicate determination, with error bars indicating s.e.

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Cell Culture, Staining, Cytometry

Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 2 Synergistic enhancement of imatinib-induced apoptosis by rottlerin is not through inhibition of PKCd. (a) COS7 cells were transfected with 1 mg of an empty vector or expression vector for PKCd, as indicated. Two days after transfection, cells were treated with or without 25 mM rottlerin for 1 h, as indicated, and harvested. Cell lysates were subjected to Western blot analysis with anti-PKCd (PKCd) and antiphospho-Thr505-PKCd (p-PKCd) as well as to the in vitro kinase assay for PKCd (IVK), as indicated. (b) COS7 cells were transfected with 1 mg of an empty vector or expression vector for PKCd, as indicated. Two days after transfection, cells were harvested for the in vitro PKCd kinase assay (IVK). The immunoprecipitates were reacted in vitro in the absence (Control) or presence of 25 mM rottlerin or 10 mM Go6850, as indicated. (c) Ton.B210 cells were cultured for 8 h in the presence or absence of 100 ng/ml TPA, as indicated, and a portion of cells was harvested. Cells were further cultured for 24 h in the presence or absence of 1 mM imatinib or 1 mM rottlerin, as indicated, and harvested. Cell lysates were subjected to Western blot analysis with anti-PKCd. (d, e, f, g) Ton.B210 cells were cultured in the absence of 100 ng/ml TPA in (d–e) or in its presence in (f–g) for 8 h. Cells were further cultured for 24 h in the presence or absence of 1 mM imatinib and 1 mM rottlerin, as indicated. Cells were stained with Annexin V-FITC as well as PI and analysed by flow cytometry (d, f). The percentages of cells staining positive for Annexin V (e, g).

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Western Blot, In Vitro, Kinase Assay, Control, Cell Culture, Staining, Cytometry

Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 3 Rottlerin does not inhibit the BCR/ABL kinase activity. K562 (a) or Ton.B210 (b) cells were cultured for indicated times in the presence or absence of 1 mM imatinib and 1 mM rottlerin, as indicated, and harvested. Cell lysates were subjected to Western blot analysis with antiphosphotyrosine (PY) followed by sequential reprobing with antibodies against Abl, phospho-STAT5 (p- STAT5), and STAT5, as indicated.

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Activity Assay, Cell Culture, Western Blot

Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 4 Mitochondrial uncouplers, FCCP and DNP, also synergistically enhance imatinib-induced apoptosis of K562 cells. (a, b, c, d) K562 cells were cultured for 24 h with or without 1 mM imatinib and 10 mM FCCP or 0.5 mM DNP, as indicated. Cells were stained with Annexin V-FITC as well as PI and analysed by flow cytometry. Percentages of cells staining positive for Annexin V are plotted (b, d). (e, f) K562 cells were cultured for indicated times with 2 mM rottlerin, 10 mM FCCP, or 0.5 mM DNP, as indicated, and subjected to the flow cytometric analysis for Dcm as described in ‘Materials and methods’. (g) K562 cells were cultured for indicated times with 2 mM rottlerin or 0.5 mM DNP in the presence or absence of 1 mM imatinib, as indicated, and subjected to the flow cytometric analysis for Dcm. Each data points represent relative Dcm compared with the pretreatment control level. (h) K562 cells were cultured for 24 h with 2 mM rottlerin or 0.5 mM DNP in the presence or absence of 1 mM imatinib, as indicated, or left untreated as control and subjected to the flow cytometric analysis for Dcm.

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Cell Culture, Staining, Cytometry, Control

Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 5 Synergistic induction of apoptosis in K562 cells by imatinib and rottlerin is induced through MMP leading to the cytochrome c release and activation of caspases-3 and -9. (a) K562 cells were treated for indicated times with 1 mM imatinib, 2 mM rottlerin, or 10 mM FCCP, as indicated. Cells were subjected to the flow cytometric analysis for ROS by using the fluorescent probe HE, as described in ‘Materials and methods’. The mean fluorescent intensity, normalized by that of cells treated without any reagent for 2 h, is plotted. (b, c) K562 cells were preincubated with calcein-AM and CoCl2 for 15 min and subsequently cultured for indicated times (b) or for 16 h (c) with 2 mM rottlerin, 0.5 mM DNP, or 1 mM imatinib, as indicated. Cells were then subjected to the flow cytometric analysis for calcein fluorescence in mitochondria, as described in ‘Materials and methods’, and percentages of cells undergoing MMP and exhibiting the decreased fluorescence are plotted in b. (d, e) K562 cells were treated with 1 mM imatinib or 2 mM rottlerin, as indicated, for 24 cells. Cytoplasmic extracts (d) or total cell lysates (e) were harvested and subjected to Western blot analysis with antibodies against indicated proteins. Positions of full-length or cleaved proteins are indicated with arrows or asterisks, respectively. (f) K562 cells were left untreated for control or treated with 1 mM imatinib and 2 mM rottlerin in the presence of indicated concentrations of z-VAD- fmk for 24 h. Cells were then stained with Annexin V-FITC as well as PI and analysed by flow cytometry. (g) K562 cells were treated with 1 mM imatinib, 2 mM rottlerin, and 25 or 50 mM z-VAD-fmk, as indicated, for 24 h, and percentages of cells staining positive for Annexin V are plotted.

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Activation Assay, Cell Culture, Western Blot, Control, Staining, Cytometry

Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 6 Effects of imatinib and rottlerin on viability of primary leukemic cells expressing BCR/ABL. Primary leukemic cells from a patient with CML in blastic crisis (a), Ph-positive ALL (b), or Ph-positive biphenotypic acute leukemia (c) were cultured in the presence or absence of 1 mM of rottlerin and 1 mM (a) or 0.5 mM (b, c) of imatinib, as indicated, for 48 h (a) or 24 h (b, c). Viable cell numbers were counted after staining with Trypan blue, and the decreases in viable cell number from control cells, culture in the absence of imatinib and rottlerin, were calculated and plotted. Each data point represents the mean of triplicate determination, with error bars indicating s.e.

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Expressing, Cell Culture, Staining, Control

Journal: Oncogene

Article Title: Rottlerin synergistically enhances imatinib-induced apoptosis of BCR/ABL-expressing cells through its mitochondrial uncoupling effect independent of protein kinase C-delta.

doi: 10.1038/sj.onc.1210117

Figure Lengend Snippet: Figure 7 Rottlerin and imatinib synergistically induce apoptosis of cells expressing the imatinib-resistant E255K mutant of BCR/ ABL. (a) Ton.B210 (WT) or Ton.B210/E255K (E255K) cells were treated with indicated concentrations of imatinib for 2 h and harvested. Cell lysates were subjected to Western blot analysis with antiphosphotyrosine (PY) followed by reprobing with antibodies against Abl, phospho-STAT5 (p-STAT5), or STAT5, as indicated. (b) Ton.B210/E255K cells were cultured for indicated times in the presence or absence of 1 mM imatinib and 1 mM rottlerin, as indicated, and harvested. Cell lysates were subjected to Western blot analysis with antiphosphotyrosine (PY) followed by reprobing with antibodies against Abl, phospho-STAT5 (p-STAT5), and STAT5. (c, d) Ton.B210/E255K cells were cultured for 24 h with indicated concentrations of imatinib in the presence or absence of 1 mM rottlerin, as indicated. Cells were stained with Annexin V-FITC as well as PI and analysed by flow cytometry in c, and percentages of cells staining positive for Annexin V are plotted in d. (e) Ton.B210 (WT), Ton.B210/E255K (E255K), or Ton.B210/T315I (T315I) cells were treated with 1 mM imatinib for 2 h and harvested for immunoprecipitation with anti-STAT5. Total cell lysates (TCL) or anti- STAT5 immunoprecipitates (STAT5 IP) were subjected to Western blot analysis with antiphosphotyrosine (PY) followed by reprobing with antibodies against Abl or STAT5, as indicated.

Article Snippet: The ABL kinase inhibitor imatinib was kindly provided by Novartis (Basel, Switzerland).

Techniques: Expressing, Mutagenesis, Western Blot, Cell Culture, Staining, Cytometry, Immunoprecipitation