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
<p>Loss of cell viability (48 h). Melanoma cell lines A-375, Mel-HO, MeWo, and SK-Mel-23 were seeded in 24-well plates, and were treated with S63845 (S63, 0.25, 0.5, 1.0 µM), SCH772984 (Sch, 0.1, 1.0, 10 µM), and vemurafenib (Vem, 10, 20, 30 µM), as indicated. (<b>A</b>) After 48 h, cell viability was determined by calcein-AM staining and flow cytometry. Values represent the percentage of cells with high calcein staining (=viable cells). Effects on cell viability were calculated as percentage of non-treated controls (100%). At least three independent experiments were performed, each one consisting of triplicate values. Mean values of all individual values (>8) are shown here. Statistical significance is indicated by asterisks (<span class="html-italic">p</span> < 0.05), and was calculated for S63845 treatment as compared to non-treated control cells. Statistical significance of the effects in combination-treated cells was calculated as compared to the respective single treatments (SCH772984 alone or vemurafenib alone, white bars). (<b>B</b>) Examples of flow cytometry measurements of cells treated for 48 h with (30 µM vemurafenib/1 µM S63845) or (10 µM SCH772984/1 µM S63845) are shown as overlays vs. controls (Ctr). Non-viable and viable cell populations are indicated.</p> "> Figure 2
<p>Decreased cell proliferation. Cell lines A-375 and MeWo were seeded in 96-well plates and were treated with increasing concentrations of S63845 (S63, 0.25, 0.5, 1.0 µM), SCH772984 (Sch, 0.1, 1.0, 10 µM) and vemurafenib (Vem, 10, 20, 30 µM), as indicated. Cell proliferation was quantified at 24 h and at 48 h of treatment by WST-1 assay. Effects on cell proliferation were calculated as percentage of non-treated controls (100%). Three independent experiments were performed, each one consisting of triplicate values. Mean values of all individual values (9) are shown here. Statistical significance is indicated by asterisks (<span class="html-italic">p</span> < 0.05), and was calculated for S63845 treatment as compared to non-treated control cells. Statistical significance of the effects of combination-treated cells was calculated as compared to the respective single treatments (SCH772984 alone or vemurafenib alone, white bars).</p> "> Figure 3
<p>Induction of apoptosis, as shown by cell cycle analysis. Melanoma cell lines A-375, Mel-HO, MeWo, and SK-Mel-23 were seeded in 24-well plates and were treated with S63845 (S63, 0.25, 0.5, 1.0 µM), SCH772984 (Sch, 0.1, 1.0, 10 µM), and vemurafenib (Vem, 10, 20, 30 µM), as indicated. (<b>A</b>) After 48 h, apoptotic cells were identified by flow cytometry after propidium iodide staining and flow cytometry as sub-G1 cells (cell cycle analyses). At least three independent experiments were performed, each one consisting of triplicate values. Mean values of all individual values (>8) are shown here. Statistical significance is indicated by asterisks (<span class="html-italic">p</span> < 0.05), and was calculated for S63845 treatment as compared to non-treated control cells. Statistical significance of the effects of combination-treated cells was calculated as compared to the respective single treatments (SCH772984 alone or vemurafenib alone, white bars). (<b>B</b>) Examples of flow cytometry measurements of cells treated for 48 h with (30 µM vemurafenib/1 µM S63845) or (10 µM SCH772984/1 µM S63845) are shown as compared to controls (Ctr). Cell cycle phases G1 (gap 1), S (synthesis), G2 (gap 2), as well as sub-G1 cells are indicated.</p> "> Figure 4
<p>Induction of apoptosis as shown by Annexin V/PI staining. A-375 and MeWo cells were treated with 1 µM SCH772984 (Sch), 1 µM S63845 (S63), or the combination. Cell death analyses by AnnV/PI staining and flow cytometry were performed after 24 h and 48 h. (<b>A</b>) Mean values and SDs of two cell death fractions, namely AnnV(+)/PI(−) and AnnV(+)/PI(+) cells, were calculated from four independent experiments, each one consisting of triplicate values, and are shown as %. Statistical significance of the effects of combination treatments, as compared to controls and as compared to single treatments, was calculated by <span class="html-italic">t</span>-test, and is indicated by asterisks for both cell death fractions (<span class="html-italic">p</span> < 0.01). (<b>B</b>) Representative flow cytometry histograms of treated and control cells, separated in four quadrants (according to PI and AnnV positivity), are shown below.</p> "> Figure 5
<p>Loss of mMP and ROS production. A-375 and MeWo cells were treated with S63845 (S63, 1 µM), SCH772984 (Sch, 1, 10 µM) and/or vemurafenib (Vem, 30 µM). (<b>A</b>) Mitochondrial membrane potential (mMP) was determined at 24 h by TMRM<sup>+</sup> staining and flow cytometry. Values represent the percentage of cells with loss of mMP. Examples of flow cytometry readings are shown on the right side for combination treatments of vemurafenib/S63845 and SCH772984/S63845 (overlays vs. controls). Cell populations with low and high (normal) mMP are indicated. (<b>B</b>) Cellular levels of ROS were determined by H<sub>2</sub>DCF-DA staining and flow cytometry. Values represent the percentage of cells with high ROS. Examples of flow cytometry readings are shown on the right side for combination treatments of S63845/vemurafenib and S63845/SCH772984 (overlays vs. controls). Cell populations with low (normal) and high ROS are indicated. (<b>A</b>,<b>B</b>) Mean values and SDs were calculated from three to four independent experiments, each one consisting of triplicate values, and are shown in %. Statistical significance, indicated by asterisks (<span class="html-italic">p</span> < 0.05), was calculated for S63845 as compared to non-treated control cells, whereas the effects of combination-treated cells were compared to the respective single treatments (SCH772984 or vemurafenib alone, white bars).</p> "> Figure 6
<p>Loss of mMP, as shown by JC-1 staining. A-375 and MeWo cells were treated with SCH772984 (Sch, 1 µM), S63845 (S63, 1 µM), or the combination. For microscopic visualization of loss of mMP, cells were stained with JC-1 and counterstained with Hoechst-33342 at 4, 8, 16, 24, and 48 h of treatment. Blue, nuclear staining; red, mitochondria with high (normal) mMP; faint green, JC-1-stained cytosol; bright green or turquoise, rounded and detached cells with loss of mMP. White scale bars are shown below, right (20 µm). One of two independent experiments, which revealed highly comparable results, is shown.</p> "> Figure 7
<p>Activation of proapoptotic pathways. A-375 and MeWo cells were treated with SCH772984 (Sch, 1 µM), S63845 (S63, 1 µM), or the combination. Protein extracts were analyzed by Western blotting for ERK phosphorylation (p-ERK) and total ERK expression (t-ERK) at 4 h (<b>A</b>), and at 24 h (<b>B</b>). Processing of caspase-3, caspase-8 and caspase-9 (Csp) was analyzed at 24 h. For caspase-3, two different antibodies against cleaved caspase-3 (<b>B</b>) and total caspase-3 (<b>C</b>) were applied. In addition, cleavage of PARP (89, 24 kDa), and phosphorylation of histone H2AX (γH2AX), were analyzed (<b>C</b>). For determination of mitochondrial cytochrome c release, cytosolic (cyto) and mitochondrial (mito) cell fractions were separately analyzed (<b>D</b>). Equal protein amounts (30 µg per lane) were separated by SDS-PAGE, and consistent blotting was proven by Ponceau staining, as well as by expression of GAPDH and β-actin, respectively. Molecular weights are indicated in kD. Each two independent series of protein extracts and Western blots revealed highly comparable results.</p> "> Figure 8
<p>Significant role of caspase activation. A-375 and MeWo cells were treated for 24 h with the combination of SCH772984 (Sch, 1 µM) and S63845 (S63, 1 µM). In addition, cells received the pan-caspase inhibitor QVD-Oph (QVD, 10 µM), when indicated; QVD-Oph was applied at 1 h before other treatments started. The inhibitory effects of QVD on apoptosis induction (<b>A</b>) and cell viability (<b>B</b>) were determined at 48 h, while the effects on mMP (<b>C</b>) and on ROS (<b>D</b>) were determined at 24 h. (<b>A</b>–<b>D</b>) Mean values (in %) and SDs were calculated from three independent experiments, each one consisting of triplicate values. Statistical significance of QVD treatments as compared to SCH772984/S63845 was calculated from all individual values (9), and is indicated by asterisks (<span class="html-italic">p</span> < 0.05). On the right side, example flow cytometry readings after SCH772984/S63845 combination treatment, +/− QVD, are shown as overlays with control cells.</p> "> Figure 9
<p>Regulation of Bcl-2 proteins. Cell lines MeWo and A-375 were treated with SCH772984 (Sch, 1 µM), S63845 (S63, 1 µM), or the combination. Protein extracts were analyzed at 24 h by Western blotting, for expression of Mcl-1 (41 kD), Bcl-2 (26 kD), Bax (22 kD), Bim<sub>EL</sub> (24 kD), Puma (23 kD), and Noxa (14 kD). Expression of phosphorylated and total Bad protein (pBad, t-Bad, 22 kD) was analyzed at 4 h of treatment. Equal protein amounts (30 µg per lane) were separated by SDS-PAGE, and consistent blotting was proven by Ponceau staining, as well as by evaluation of GAPDH expression. Molecular weights are indicated in kD. Two independent series of protein extracts and Western blots revealed highly comparable results.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Strongly Decreased Cell Viability by Combination of MAPK and Mcl-1 Inhibitors
2.2. Loss of Cell Proliferation in Parallel with Decreased Cell Viability
2.3. Decreased Cell Viability Correlates with Induction of Apoptosis
2.4. Loss of mMP and Production of ROS
2.5. Significant Role of Caspase Activation
2.6. Upregulation of Proapoptotic BH3-Only Proteins
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Treatment
4.2. Quantification of Cell Viability and Apoptosis
4.3. Cell Proliferation Assays
4.4. Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species (ROS)
4.5. Caspase Activation Assay and Cytochrome C Release
4.6. Western Blotting
4.7. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Peng, Z.; Gillissen, B.; Richter, A.; Sinnberg, T.; Schlaak, M.S.; Eberle, J. 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. Int. J. Mol. Sci. 2023, 24, 4961. https://doi.org/10.3390/ijms24054961
Peng Z, Gillissen B, Richter A, Sinnberg T, Schlaak MS, Eberle J. 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. International Journal of Molecular Sciences. 2023; 24(5):4961. https://doi.org/10.3390/ijms24054961
Chicago/Turabian StylePeng, Zhe, Bernhard Gillissen, Antje Richter, Tobias Sinnberg, Max S. Schlaak, and Jürgen Eberle. 2023. "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" International Journal of Molecular Sciences 24, no. 5: 4961. https://doi.org/10.3390/ijms24054961