Enhanced Apoptosis and Loss of Cell Viability in Melanoma Cells by Combined Inhibition of ERK and Mcl-1 Is Related to Loss of Mitochondrial Membrane Potential, Caspase Activation and Upregulation of Proapoptotic Bcl-2 Proteins

Targeting of MAP kinase pathways by BRAF inhibitors has evolved as a key therapy for BRAF-mutated melanoma. However, it cannot be applied for BRAF-WT melanoma, and also, in BRAF-mutated melanoma, tumor relapse often follows after an initial phase of tumor regression. Inhibition of MAP kinase pathways downstream at ERK1/2, or inhibitors of antiapoptotic Bcl-2 proteins, such as Mcl-1, may serve as alternative strategies. As shown here, the BRAF inhibitor vemurafenib and the ERK inhibitor SCH772984 showed only limited efficacy in melanoma cell lines, when applied alone. However, in combination with the Mcl-1 inhibitor S63845, the effects of vemurafenib were strongly enhanced in BRAF-mutated cell lines, and the effects of SCH772984 were enhanced in both BRAF-mutated and BRAF-WT cells. This resulted in up to 90% loss of cell viability and cell proliferation, as well as in induction of apoptosis in up to 60% of cells. The combination of SCH772984/S63845 resulted in caspase activation, processing of poly (ADP-ribose) polymerase (PARP), phosphorylation of histone H2AX, loss of mitochondrial membrane potential, and cytochrome c release. Proving the critical role of caspases, a pan-caspase inhibitor suppressed apoptosis induction, as well as loss of cell viability. As concerning Bcl-2 family proteins, SCH772984 enhanced expression of the proapoptotic Bim and Puma, as well as decreased phosphorylation of Bad. The combination finally resulted in downregulation of antiapoptotic Bcl-2 and enhanced expression of the proapoptotic Noxa. In conclusion, combined inhibition of ERK and Mcl-1 revealed an impressive efficacy both in BRAF-mutated and WT melanoma cells, and may thus represent a new strategy for overcoming drug resistance

Mcl-1 determines the Bax dependency of Nbk/Bik-induced apoptosis

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)–only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1–Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2

Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma

Although both Mcl-1 and Bcl-xL keep proapoptotic Bak in check, it is the loss of Mcl-1 that sensitizes cells to death receptor–mediated apoptosis

Mutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-xAK, a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-xAK may trigger apoptosis

Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway

Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-specific BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a single- vector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-deficient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wild-type HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x(L) and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not sufficient to confer sensitivity to Nbk

Bcl-x_AK-mediated apoptosis depends on Bax or Bak.

<p>(A, C) Expression of Bax and Bak is shown by Western blot analysis in subclones of HCT116 and DU145, respectively. Equal loading was confirmed by incubation with β-actin. Two independent series of protein extracts revealed largely comparable expression. (B) HCT116 parental cells (Bax⁺/Bak⁺) as well as subclones (Bax⁻/Bak⁺), (Bax⁺/Bak⁻) and (Bax⁻/Bak⁻) were transduced with AdV-AK (MOI=50) and cultured under OFF or ON conditions. Relative DNA fragmentation values (apoptosis ELISA) were normalized according to the values of parental cells under OFF conditions (set to 1). (D) DU145 parental cells (Bax⁻/EGFP-Bak⁻) as well as subclones (Bax⁻/EGFP-Bak⁺) and (EGFP-Bax⁺/EGFP-Bak⁻) were transduced with AdV-AK (MOI=50, 100) and cultured under OFF or ON conditions. The percentages of apoptotic cells (sub-G1 populations) are shown, as determined by flow cytometry at 48 h after transduction. (B, D) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Statistical significance as determined by Student's t-test is indicated by asterisks (*, p<0.05; **, p<0.005), when comparing parental cells and subclones under ON conditions.</p

Activation of caspases and mitochondria.

<p>(A) Processing of caspase-3, -8 and -9 is shown in Mel-2a cells at 24 h and at 48 h after transduction with AdV-AK (MOI=50). Expression of Bcl-x_AK was switched on in the absence of doxycycline (ON) or shut off with doxycycline (OFF). Equal protein loading (20 µg/lane) was confirmed by GAPDH. The whole experiment was performed twice. (B) Inhibition of apoptosis by preincubation with the pancaspase inhibitor zVAD-fmk (1 h, 10 µM) is shown. SK-Mel-13 cells had been transduced with AdV-AK (MOI=100, 48 h). Means and SDs of triplicate values of a representative experiment (one of two) are shown. (C) Decrease of the mitochondrial membrane potential (Δψ_m) is shown for Mel-2a cells at 48 h after transduction of AdV-AK, as determined by flow cytometry after JC-1 or TMRM⁺ staining. Cultures with doxycycline (OFF, grey) are compared to cultures grown in the absence of doxycycline (ON, open graphs). The experiment was performed three times, resulting in highly comparable results. (D) ROS levels were determined in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK under ON and OFF conditions (flow cytometry after H₂DCFDA staining). Below, parallel cultures were pre-treated for 1 h with 200 µM NAC before transduction. (E) Relative DNA-fragmentation rates (apoptosis) at 48 h with or without NAC were determined in parallel. Non-transduced cells (−/+NAC) are shown as additional controls (open bars). Values had been normalized with regard to non-treated controls, set to 1. Means and SDs of triplicate values of a representative experiment are shown (two independent experiments). (F) Expression levels of Bcl-2 proteins, of p53 and Survivin were determined by Western blot analysis in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK (ON and OFF conditions). Equal protein loading (20 µg/lane) was confirmed by GAPDH.</p

Efficient induction of cell death by Bcl-x_AK.

<p>(A) The structure of the adenoviral construct AdV-AK is shown. The adenoviral E1 region was replaced by the Bcl-x_AK cDNA driven by a tetracyclin-responsive promoter (P_TRE), and the E3 region was replaced by the tetracyclin-controlled transactivator (tTA) driven by a CMV promoter (P_CMV). The tTA mediates Tet-OFF regulation. Striped boxes indicate the poly(A)+ regions. (B) Bcl-x_AK expression as determined by Western blot analysis is shown in melanoma cell lines SK-Mel-13, A-375 and Mel-2a at 48 h after transduction with AdV-AK (MOI=50). Cells had received doxycycline (OFF condition) or were left without (ON condition). Equal protein loading was confirmed by β-actin. (C) Left, examples of cell cycle analysis after PI staining indicating sub-G1 apoptotic cell populations in Mel-2a at 48 h of transduction. Middle panel, detached and rounded cells indicating apoptosis are shown of Mel-2a at 48 h after transduction with AdV-AK under OFF and ON conditions. Right panel, chromatin condensation and nuclear fragmentation were visualized by bisbenzimide (DAPI) staining in Mel-2a at 48 h after AdV-AK transduction (MOI=50). D–F) Time course analyses of apoptosis (D, flow cytometry after PI staining), cytotoxicity (E, LDH release) and cell proliferation (F, WST-1 assay) are shown for SK-Mel-13, A-375 and Mel-2a cells at 24, 48 and 72 h after transduction with AdV-AK (50 MOI, +Dox=Off, −Dox=On). As positive controls for induced cytotoxicity, cell lines were completely lysed by triton X-100 (T=100%) or were treated with doxorubicin (D, 500 nM, 72 h). WST-1 values are expressed as percent of non-treated controls (=100%). (G) For comparison, apoptosis induction (sub-G1 cells) by AdV-Nbk is shown for Mel-2a cells at 24 h, 48 h and 72 h (MOI=50). AdV-Nbk shares the same backbone with AdV-AK. For induction, doxycycline was omitted (On). (H) A time course analysis of Bcl-x_AK expression (3–48 h) after AdV-AK transduction and promoter induction is shown for Mel-2a, as determined by Western blot analysis. (I) Cell survival was determined according to calcein staining in Mel-2a cells at 48 h of Bcl-x_AK induction. A shift to the left indicates calcein-negative (=non viable) cells. (J) Quantification of the calcein experiment. (D, E, F, G, J) Means and standard deviations of triplicate values of representative experiments are shown. A luciferase-encoding adenovirus (Ad5-CMV-Luc) applied at the same MOI served as mock control (M), for controlling adenovirus transduction. All experiments were performed at least twice, resulting in highly comparable results.</p

Bax and Bak activation after Bcl-x_AK overexpression.

<p>(A) For investigation of Bax and Bak clustering, DU145 cells stably transfected for expression of EGFP-Bax or EGFP-Bak were transduced with AdV-AK and cultured for 48 h under OFF or ON conditions. Doxorubicin-treated cells (2 µM, 24 h) were used as positive controls. Examples of fluorescence microscope images taken at 48 h after transduction and promoter induction are shown. (B) A quantitative evaluation of Bax and Bak clustering was performed (means and SDs of triplicate values of a representative experiment). A second experiment revealed comparable results. (C) Bax and Bak activation upon Bcl-x_AK expression was determined in Mel-2a at 48 after AdV-AK transduction (50 MOI), by flow cytometry after staining with conformation-specific antibodies against Bax and Bak N termini (Bax/Bak NT). The bars indicate the populations counted as positive for activated Bax and Bak, respectively. (D) A quantification of triplicate values (one experiment of two independent) is shown. Transduction with AdV-Nbk (50 MOI) is shown for comparison.</p