br caption on next page br Fig Rac induced S
(caption on next page)
Fig. 3. Rac1-induced S70 phosphorylation of Bcl-2 is ROS-dependent.
(a–b) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 24-h treatment of increasing doses of Tiron in CEM/Bcl-2 and Jurkat cells. n = 4. Bar charts showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. (c) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 12-h treatment of increasing doses of DPI in Jurkat cells. n = 4. Bar chart showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. (d) Measurement of O2.-production in M14 Melanoma LY 379268 stably expressing the Myc-tagged constitutively active Rac1 mutant, V12, or empty vector, pIRES. n = 3. (e) Measurement of O2.-production following 24-h treatment of 10 mM Tiron in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12. n = 3. (f) Western Blot analysis showing S70pBcl-2, Bcl-2, Rac1 and β-actin following 24-h treatment of 10 mM Tiron in M14 Melanoma cells stably expressing the Myc-tagged constitutively active Rac1 mutant, V12. pIRES served as control. n = 4. Bar chart showing fold change in densitometry of S70pBcl-2 normalized to Bcl-2. All bar charts displaying mean and SD. Paired T-test was used for experiments with 2 samples. One way-ANOVA and Tukey's multiple comparisons tests were used for experiments with more than 2 samples. * and ** indicate P-value < 0.05 and < 0.02 respectively.
interaction between Rac1 and Bcl-2.
3.5. Pharmacological disruptions of the Active Rac1-S70pBcl-2 feedforward loop sensitizes cancer cell to apoptosis
To understand the functional relevance of our findings in the con-text of cancer cell fate, we next investigated the effect of disrupting this feedforward loop on cancer cell response to apoptotic stimuli. Indeed, in silico RAC1 knockdown, a computational simulation generated by Cellworks™, or in vitro Rac1 inhibition (NSC23766, 24 h) resulted in a significant reduction in viability of CEM/Bcl-2 and Jurkat cells (Fig. 6a–b, S9a-b, S10). Alternatively, Rac1V12 expression endowed M14 cells the ability to resist staurosporine-induced apoptosis (Fig. 6c). As Rac1V12 upregulates S70pBcl-2 via an increase in intracellular O2.-, we then evaluated the effect of scavenging O2.- on drug sensitivity. Indeed, a priori treatment of Rac1V12 cells with tiron for 1 h significantly increased the sensitivity of cells to staurosporine (24 h) (Fig. 6c). As we showed that ABT199 could similarly reduce S70pBcl-2, a similar pre-treatment with non-cytotoxic concentration of ABT199 (1 μM) for 1 h synergized with non-cytotoxic concentration of staurosporine (50 nM, 24 h) in decreasing the viability of CEM/Bcl-2 cells (Fig. 6d). Re-ciprocally, transient overexpression of S70E and to a lesser extent WT Bcl-2, but not S70A, protected M14 cells from staurosporine-induced cell death (Fig. 6e). These data were further corroborated by assaying the ability of 2000 cells to survive, proliferate and form 3D-spheroids in 72 h following similar treatments. 3D-spheroid formed by Rac1V12-ex-pressing M14 cells was minimally affected by staurosporine. However, pre-treatment with tiron resulted in a significant reduction in spheroid-forming ability, thereby providing testimony to the involvement of in-tracellular O2.- in apoptosis resistance induced by active Rac1 (Fig. 6f–g). More importantly, the effect of staurosporine on 3D-spheroid formation was significantly blocked by transient expression of S70E, whereas S70A mutant had no significant effect in M14 cells (Fig. 6h–i). We also verified the status of S70pBcl-2 under all treatment conditions. Notably, scavenging O2.- (tiron) or preventing Rac1/Bcl-2 interaction (ABT199) resulted in a significant reduction in the elevated
levels of S70pBcl-2 seen in M14 Rac1V12 or CEM/Bcl-2 cells, respec-tively, while staurosporine treatment had only modest to no effect (Fig. 6j–k). As expected, S70E mutant expression showed much higher S70pBcl-2 expression (Fig. S11). Taken together, targeting cellular redox state and/or physical interaction between active Rac1 and S70pBcl-2 in the feedforward loop could have therapeutic implications, particularly in cancer cells exhibiting reliance on active Rac1 and/or Bcl-2.