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Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line

Coricovac, Dorina, Dehelean, Cristina Adriana, Pinzaru, Iulia, Mioc, Alexandra, Aburel, Oana-Maria, Macasoi, Ioana, Draghici, George Andrei, Petean, Crina, Soica, Codruta, Boruga, Madalina, Vlaicu, Brigitha, Muntean, Mirela Danina·2021·International Journal of Molecular Sciences
DOI 10.3390/ijms22094870PMC PMC8125295
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Abstract

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 μM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

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Figure 1 In vitro viability evaluation of BA (1, 5, 10, 20, 25 and 50 μM) in HaCaT cells at 24 h post-stimulation by MTT assay. The results are expressed as cell viability percentage (%) normalized to control (DMSO-stimulated) cells. The data represent the mean values ± SD of three independent experiments performed in triplicate. One-way ANOVA analysis was applied to determine the statistical differences in rapport with DMSO followed by Dunnett’s multiple comparisons post-test (* p < 0.05, ** p < 0.01 and **** p < 0.0001).
Figure 1 In vitro viability evaluation of BA (1, 5, 10, 20, 25 and 50 μM) in HaCaT cells at 24 h post-stimulation by MTT assay. The results are expressed as cell viability percentage (%) normalized to control (DMSO-stimulated) cells. The data represent the mean values ± SD of three independent experiments performed in triplicate. One-way ANOVA analysis was applied to determine the statistical differences in rapport with DMSO followed by Dunnett’s multiple comparisons post-test (* p < 0.05, ** p < 0.01 and **** p < 0.0001).CC BY · PubMed Central
Figure 2 Cell nuclei staining using Hoechst 33342 in HaCaT cells after treatment with BA (10, 20 and 50 µM) and DMSO for 24 h. The pictures were taken at 24 h post-treatment. Staurosporine solution (5 µM) represents the positive control for apoptotic changes at nuclear level and Triton X-100 solution (0.5%) for necrosis. The yellow arrows represent signs of apoptosis as nuclear shrinkage or nuclear fragmentation, and the red arrow indicates signs of necrosis as cellular membrane disruption. The scale bar was 10 µm.
Figure 2 Cell nuclei staining using Hoechst 33342 in HaCaT cells after treatment with BA (10, 20 and 50 µM) and DMSO for 24 h. The pictures were taken at 24 h post-treatment. Staurosporine solution (5 µM) represents the positive control for apoptotic changes at nuclear level and Triton X-100 solution (0.5%) for necrosis. The yellow arrows represent signs of apoptosis as nuclear shrinkage or nuclear fragmentation, and the red arrow indicates signs of necrosis as cellular membrane disruption. The scale bar was 10 µm.CC BY · PubMed Central
Figure 3 In vitro viability evaluation of BA (1, 5, 10, 20, 25 and 50 μM) in A375 cells at 24 h post-stimulation by MTT assay. The results are expressed as cell viability percentage (%) normalized to control (DMSO-stimulated) cells. The data represent the mean values ± SD of three independent experiments performed in triplicate. One-way ANOVA analysis was applied to determine the statistical differences in rapport with DMSO followed by Dunnett’s multiple comparisons post-test (** p < 0.01 and **** p < 0.0001).
Figure 3 In vitro viability evaluation of BA (1, 5, 10, 20, 25 and 50 μM) in A375 cells at 24 h post-stimulation by MTT assay. The results are expressed as cell viability percentage (%) normalized to control (DMSO-stimulated) cells. The data represent the mean values ± SD of three independent experiments performed in triplicate. One-way ANOVA analysis was applied to determine the statistical differences in rapport with DMSO followed by Dunnett’s multiple comparisons post-test (** p < 0.01 and **** p < 0.0001).CC BY · PubMed Central
Figure 4 Representative images of the morphological aspect of A375 cells after treatment for 24 h with BA and DMSO (5, 10 and 50 μM). The scale bar was 50 μm.
Figure 4 Representative images of the morphological aspect of A375 cells after treatment for 24 h with BA and DMSO (5, 10 and 50 μM). The scale bar was 50 μm.CC BY · PubMed Central
Figure 5 Nuclear staining using Hoechst 33342 in A375 cells after treatment with BA (10, 20 and 50 µM) and DMSO for 24 h. The pictures were taken at 24 h post-treatment. Staurosporine solution (5 µM) represents the positive control for apoptotic changes at nuclear level and Triton X-100 solution (0.5%) for necrosis. The yellow arrows represent signs of apoptosis as nuclear shrinkage or nuclear fragmentation, and the red arrow indicates signs of necrosis as cellular membrane disruption. The scale bar was 10 µm.
Figure 5 Nuclear staining using Hoechst 33342 in A375 cells after treatment with BA (10, 20 and 50 µM) and DMSO for 24 h. The pictures were taken at 24 h post-treatment. Staurosporine solution (5 µM) represents the positive control for apoptotic changes at nuclear level and Triton X-100 solution (0.5%) for necrosis. The yellow arrows represent signs of apoptosis as nuclear shrinkage or nuclear fragmentation, and the red arrow indicates signs of necrosis as cellular membrane disruption. The scale bar was 10 µm.CC BY · PubMed Central
Figure 6 Mitochondrial membrane potential evaluation using JC-1 mitochondrial staining in A375 cells after treatment with BA (10 and 50 µM) and DMSO for 24 h. The pictures were taken at 24 h post-treatment. CCCP (50 µM) represents the positive control for mitochondrial depolarization. The graph represents the quantification of fluorescence intensity expressed as JC-1 aggregates (red)/JC-1 monomers (green) ratio. The data represent the mean values ± SD of four independent measurements. One-way ANOVA analysis was applied to determine the statistical differences in rapport with control group followed by Dunnett’s multiple comparisons post-test (*** p < 0.001 and **** p < 0.0001).
Figure 6 Mitochondrial membrane potential evaluation using JC-1 mitochondrial staining in A375 cells after treatment with BA (10 and 50 µM) and DMSO for 24 h. The pictures were taken at 24 h post-treatment. CCCP (50 µM) represents the positive control for mitochondrial depolarization. The graph represents the quantification of fluorescence intensity expressed as JC-1 aggregates (red)/JC-1 monomers (green) ratio. The data represent the mean values ± SD of four independent measurements. One-way ANOVA analysis was applied to determine the statistical differences in rapport with control group followed by Dunnett’s multiple comparisons post-test (*** p < 0.001 and **** p < 0.0001).CC BY · PubMed Central
Figure 7 Relative fold expression of mRNA expression of pro- and anti-apoptotic mitochondrial markers in A375 cells after stimulation with BA (10 µM) for 24 h. The expressions were normalized to GAPDH and DMSO was used as control. Data represent the mean values ± SD of three independent experiments. One-way ANOVA with Dunnett’s post-test was applied to determine the statistical differences in rapport with DMSO stimulated cells (* p < 0.05, ** p < 0.01, and **** p < 0.0001).
Figure 7 Relative fold expression of mRNA expression of pro- and anti-apoptotic mitochondrial markers in A375 cells after stimulation with BA (10 µM) for 24 h. The expressions were normalized to GAPDH and DMSO was used as control. Data represent the mean values ± SD of three independent experiments. One-way ANOVA with Dunnett’s post-test was applied to determine the statistical differences in rapport with DMSO stimulated cells (* p < 0.05, ** p < 0.01, and **** p < 0.0001).CC BY · PubMed Central
Figure 8 The impact of BA (10 µM) 24 h stimulation in A375 cells on: (a) nuclei—DAPI staining (blue), (b) F-actin fibers—Phalloidin (red) and (c) COX IV mitochondrial marker (green). The pictures were taken using 40× objective at a scale bar of 10 µm.
Figure 8 The impact of BA (10 µM) 24 h stimulation in A375 cells on: (a) nuclei—DAPI staining (blue), (b) F-actin fibers—Phalloidin (red) and (c) COX IV mitochondrial marker (green). The pictures were taken using 40× objective at a scale bar of 10 µm.CC BY · PubMed Central
Figure 9 Summary traces of the bioenergetic profile of A375 cells: Control—untreated cells; BA 5—cells treated with 5 µM betulinic acid; BA 10—cells treated with 10 µM betulinic acid; BA 50—cells treated with 50 µM betulinic acid. OCR was expressed in units of nmoles/min/no of cells, ECAR in mpH/min/no of cells, and PPR in pmoles/min/no of cells.
Figure 9 Summary traces of the bioenergetic profile of A375 cells: Control—untreated cells; BA 5—cells treated with 5 µM betulinic acid; BA 10—cells treated with 10 µM betulinic acid; BA 50—cells treated with 50 µM betulinic acid. OCR was expressed in units of nmoles/min/no of cells, ECAR in mpH/min/no of cells, and PPR in pmoles/min/no of cells.CC BY · PubMed Central
Figure 10 The effects of a 24 h treatment with BA (5, 10 and 50 µM) on OCR, ECAR, and PPR (n = 14–20/group. * p < 0.05 vs. untreated controls, ** p < 0.01, *** p < 0.001).
Figure 10 The effects of a 24 h treatment with BA (5, 10 and 50 µM) on OCR, ECAR, and PPR (n = 14–20/group. * p < 0.05 vs. untreated controls, ** p < 0.01, *** p < 0.001).CC BY · PubMed Central
Figure 11 Respiration of permeabilized human melanoma cells—A375 following 24 h stimulation with BA (10 μM) and DMSO. Data represent the mean values ± SD of three independent experiments. Unpaired t test with Welch’s correction was applied to determine the statistical differences vs. DMSO-incubated cells (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). The results were normalized to cells stimulated with DMSO (the vehicle used for BA solubilization). The following respiratory parameters were assessed: Routine—respiration of cells suspended in a substrate-free media; State 2CI—the respiration in basal conditions, OXPHOSCI—active respiration through CI; OXPHOSCI+II—maximal active respiration driven by CI and CII; State 4CI+II—basal respiration dependent on both CI and CII; ETSCI+II—maximal respiratory capacity of the electron transport system after FCCP titrations; ETSCII—electron transport system dependent only on CII.
Figure 11 Respiration of permeabilized human melanoma cells—A375 following 24 h stimulation with BA (10 μM) and DMSO. Data represent the mean values ± SD of three independent experiments. Unpaired t test with Welch’s correction was applied to determine the statistical differences vs. DMSO-incubated cells (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). The results were normalized to cells stimulated with DMSO (the vehicle used for BA solubilization). The following respiratory parameters were assessed: Routine—respiration of cells suspended in a substrate-free media; State 2CI—the respiration in basal conditions, OXPHOSCI—active respiration through CI; OXPHOSCI+II—maximal active respiration driven by CI and CII; State 4CI+II—basal respiration dependent on both CI and CII; ETSCI+II—maximal respiratory capacity of the electron transport system after FCCP titrations; ETSCII—electron transport system dependent only on CII.CC BY · PubMed Central

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Metadata

Authors
Coricovac, D., Dehelean, C. A., Pinzaru, I., Mioc, A., Aburel, O.-M., Macasoi, I., Draghici, G. A., Petean, C., Soica, C., Boruga, M., Vlaicu, B., & Muntean, M. D.
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2021-05-04
Volume
22
Issue
9
Pages
4870
ISSN
1422-0067
DOI
10.3390/ijms22094870
PMC
PMC8125295
Cited by
56
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI (PN-III-P1-1.1-PD-2016-1982)
  • Romanian National Authority for Scientific Research and Innovation, CNCS –UEFISCDI (PN-III-P1-1.1-TE-2019-2134)

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Coricovac, D., Dehelean, C. A., Pinzaru, I., Mioc, A., Aburel, O.-M., Macasoi, I., Draghici, G. A., Petean, C., Soica, C., Boruga, M., Vlaicu, B., & Muntean, M. D. (2021). Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms22094870

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