Figure 8
Figure 8. Bcl-2 BH3 peptides and silencing or functional inhibition of Rac1 inhibit intramitochondrial O2∸ levels and enhance sensitivity in Bcl-2-overexpressing cells to chemotherapeutic agents. (A) CEM/Neo and CEM/Bcl-2 cells were incubated with various doses of Bcl-2 BH3 peptides and control peptides for 2 hours. MitoSOX dye was used to assess mitochondrial O2∸ levels as described in “Determination of O2∸.” (B) CEM/Neo and CEM/Bcl-2 cells were preincubated for 1 hour with 10μM Bcl-2 BH3 peptide followed by a 24-hour incubation with various doses of etoposide (Eto) or vincristine (Vin). Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay as described in “Determination of cell viability.” Data are mean ± SD of at least 3 independent experiments performed in triplicate. P < .05 and P < .005 compared with the drug treatment alone. (C) HeLa/Bcl-2 and CEM/Bcl-2 cells were preincubated with Rac1 inhibitor (RI) for 1 hour followed by 24-hour incubation with various doses of etoposide (Eto), vincristine (Vin), or daunorubicin (Dau). Cell viability was assessed as described in “Determination of cell viability.” Data are mean ± SD of at least 3 independent experiments performed in triplicate. P < .05 and P < .005 compared with the drug treatment alone. (D) Western blot analysis of Rac1 and β-actin expression in HeLa/Bcl-2 cells transfected with nonspecific scrambled siRNA (Negsi) or siRNA against Rac1 (Rac1si). (E) HeLa/Bcl-2 cells silenced with nonspecific scrambled siRNA (Negsi) or siRNA against Rac1 (Rac1si) were treated with various doses of etoposide (Eto) or vincristine (Vin) for 24 hours. Cell viability was assessed by the crystal violet assay as described in “Determination of cell viability.” Data are mean ± SD of at least 3 independent experiments performed in triplicate. P < .005 and P < .05 compared with the drug treatment alone. (F) Western blot analysis for poly(ADP-ribose) polymerase (PARP; both full length and cleaved) on exposure to 50nM vincristine (Vin) or 5μM etoposide (Eto) was performed to show the proapoptotic phenotypes of CEM/Bcl-2 cells. (G) Intramitochondrial O2∸ levels were determined following cell loading with MitoSOX as described in “Determination of O2∸.” The histograms are representative of 3 independent experiments. (H) CEM cells overexpressing Bcl-2 (60 × 106) were treated with 50nM vincristine (Vin) or 5μM etoposide (Eto) for 16 hours, lysed, and fractionated into purified heavy membrane (HM), LM, and cytosolic (S) fractions. The fraction lysates were then immunoprecipitated with anti-Bcl-2 and probed with anti-Rac1. (I) The fraction lysates were probed for Rac1, Bcl-2, VDAC1, Flotillin, and Cu/Zn SOD.

Bcl-2 BH3 peptides and silencing or functional inhibition of Rac1 inhibit intramitochondrial O2∸ levels and enhance sensitivity in Bcl-2-overexpressing cells to chemotherapeutic agents. (A) CEM/Neo and CEM/Bcl-2 cells were incubated with various doses of Bcl-2 BH3 peptides and control peptides for 2 hours. MitoSOX dye was used to assess mitochondrial O2∸ levels as described in “Determination of O2∸.” (B) CEM/Neo and CEM/Bcl-2 cells were preincubated for 1 hour with 10μM Bcl-2 BH3 peptide followed by a 24-hour incubation with various doses of etoposide (Eto) or vincristine (Vin). Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide assay as described in “Determination of cell viability.” Data are mean ± SD of at least 3 independent experiments performed in triplicate. P < .05 and P < .005 compared with the drug treatment alone. (C) HeLa/Bcl-2 and CEM/Bcl-2 cells were preincubated with Rac1 inhibitor (RI) for 1 hour followed by 24-hour incubation with various doses of etoposide (Eto), vincristine (Vin), or daunorubicin (Dau). Cell viability was assessed as described in “Determination of cell viability.” Data are mean ± SD of at least 3 independent experiments performed in triplicate. P < .05 and P < .005 compared with the drug treatment alone. (D) Western blot analysis of Rac1 and β-actin expression in HeLa/Bcl-2 cells transfected with nonspecific scrambled siRNA (Negsi) or siRNA against Rac1 (Rac1si). (E) HeLa/Bcl-2 cells silenced with nonspecific scrambled siRNA (Negsi) or siRNA against Rac1 (Rac1si) were treated with various doses of etoposide (Eto) or vincristine (Vin) for 24 hours. Cell viability was assessed by the crystal violet assay as described in “Determination of cell viability.” Data are mean ± SD of at least 3 independent experiments performed in triplicate. P < .005 and P < .05 compared with the drug treatment alone. (F) Western blot analysis for poly(ADP-ribose) polymerase (PARP; both full length and cleaved) on exposure to 50nM vincristine (Vin) or 5μM etoposide (Eto) was performed to show the proapoptotic phenotypes of CEM/Bcl-2 cells. (G) Intramitochondrial O2∸ levels were determined following cell loading with MitoSOX as described in “Determination of O2∸.” The histograms are representative of 3 independent experiments. (H) CEM cells overexpressing Bcl-2 (60 × 106) were treated with 50nM vincristine (Vin) or 5μM etoposide (Eto) for 16 hours, lysed, and fractionated into purified heavy membrane (HM), LM, and cytosolic (S) fractions. The fraction lysates were then immunoprecipitated with anti-Bcl-2 and probed with anti-Rac1. (I) The fraction lysates were probed for Rac1, Bcl-2, VDAC1, Flotillin, and Cu/Zn SOD.

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