Telmisartan induces melanoma cell apoptosis and synergizes with vemurafenib in vitro by altering cell bioenergetics

Abstract

Objective: Despite recent advancements in targeted therapy and immunotherapies, prognosis for metastatic melanoma patients remains extremely poor. Development of resistance to previously effective treatments presents a serious challenge and new approaches for melanoma treatment are urgently needed. The objective of this study was to examine the effects of telmisartan, an AGTR1 inhibitor and a partial agonist of PPARγ, on melanoma cells as a potential agent for repurposing in melanoma treatment.

Methods: Expression of AGTR1 and PPARγ mRNA in melanoma patient tumor samples was examined in publicly available datasets and confirmed in melanoma cell lines by qRT-PCR. A panel of melanoma cell lines was tested in viability, apoptosis and metabolic assays in presence of telmisartan by flow cytometry and immunocytochemistry. A cytotoxic effect of combinations of telmisartan and targeted therapy vemurafenib was examined using the Chou-Talalay combination index method.

Results: Both AGTR1 and PPARγ mRNA were expressed in melanoma patient tumor samples and decreased compared to the expression in the healthy skin. In vitro, we found that telmisartan decreased melanoma cell viability by inducing cell apoptosis. Increased glucose uptake, but not utilization, in the presence of telmisartan caused the fission of mitochondria and release of reactive oxygen species. Telmisartan altered the cell bioenergetics, thereby synergizing with vemurafenib in vitro, and even sensitized vemurafenib-resistant cells to the treatment.

Conclusions: Given that the effective doses of telmisartan examined in our study can be administered to patients and that telmisartan is a widely used and safe antihypertensive drug, our findings provide the scientific rationale for testing its efficacy in treatment of melanoma progression.

Keywords: Melanoma; apoptosis; mitochondria; reactive oxygen species; targeted therapy; telmisartan.

Figures

1

Expression of PPARγ receptor in melanoma. Relative expression levels of PPARγ in melanoma tumors included in (A) GSE3189, (B) GSE46517, (C) GSE7553 and (D) GSE8401 datasets. Values represent mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA with Dunett’s multiple comparisons test. (E) High and low PPARγ expression in metastatic melanoma from dataset GSE19234 , ****P < 0.0001, unpaired t test. (F) Survival curves for 36 metastatic melanoma patients with tumors expressing highest (n = 5) and lowest (n = 31) PPARγ levels calculated using Kaplan-Meier analysis. (G) Relative mRNA levels of PPARγ in melanoma cell lines measured by qRT-PCR. Values represent mean ± SEM of three independent experiments.

2

Expression of AGTR1 receptor in melanoma. Relative expression levels of AGTR1 in melanoma tumors included in (A) GSE3189, (B) GSE46517, (C) GSE7553 and (D) GSE8401 datasets. Values represent mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA with Dunett’s multiple comparisons test. (E) Expression of AGTR1 in metastatic melanoma from dataset GSE19234 , red marks for values above standard deviation range. (F) Relative mRNA levels of AGTR1 in melanoma cell lines measured by qRT-PCR. Values represent mean ± SEM of three independent experiments.

3

Telmisartan reduces melanoma cell viability. Percent viability of melanoma cell lines after 72 h treatment with (A) telmisartan, (B) pioglitazone and (C) losartan determined by the MTT assay presented as log (inhibitor) vs. no. rmalized response and (D) best-fit IC50 values for each line, n = 4. (E) Representative graphs and (F) quantification of Annexin/PI assay for apoptosis in A375 cells after 24 h and 48 h of treatment with 50 μM telmisartan or 100 μM pioglitazone, values represent mean ± SD, ** P < 0.01, Two-way ANOVA, n = 3. (G) Flow cytometric analysis of cleaved caspase 3 in A375 cells after 48 h treatment with 50 μM telmisartan. Values represent mean ± SD, ***P < 0.001, unpaired t-test, n = 3.

4

Telmisartan induces generation of reactive oxygen species in melanoma cells. Representative graph (A) and quantification (B) of DCFDA fluorescence as a measure of ROS generation in A375 cells after 24 h treatment with 50 μM telmisartan, 100 μm pioglitazone or 100 μm losartan. Values represent mean ± SD, **P < 0.01, One way ANOVA, n = 3. (C) Flow cytometric analysis of phospho-p38 in A375 cells after 24 h treatment with 50 μM telmisartan. Values represent mean ± SD, **P < 0.01, unpaired t-test, n = 3. (D) Telmisartan-induced ROS generation in A375 cells in the presence of reduced 1 mM glutathione (GSH), 10 mM N-acetyl cysteine (NAC) or 100 μM apocynin (APO). Values represent mean ± SD, individual comparisons by unpaired t-test, n = 3. (E) Telmisartan-induced ROS generation in HTB140 cells in presence of 10 mM NAC. Values represent mean ± SD, individual comparisons by unpaired t-test, *P < 0.05, n = 3. (F) Representative graphs of measurement of mitochondrial potential of A375 cells by flow cytometry of Rhodamine123 in cells treated with 50 μM telmisartan or 100 μM pioglitazone, n = 2.

5

Telmisartan alters melanoma cell bioenergetics. (A) Representative graphs (i) and quantification (ii) of 2-NBDG uptake in A375 cells after 1 h or 24 h treatment with 50 μM telmisartan or pioglitazone, measured by flow cytometry. Values represent mean ± SD, One-way ANOVA, **P < 0.01, n = 3. Glucose consumption in (B) A375 cells and (E) HTB140 cells after 24 h treatment with 50 μM telmisartan or pioglitazone. Values represent mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001, individual comparisons by unpaired t-test, n = 3. Lactate excretion in (C) A375 cells and (F) HTB140 cells after treatment with 50 μM telmisartan or pioglitazone, measured in extracellular acidification assay, where fluorescence signal correlates with lactate production, n = 2, linear regression analysis. (D) MTT viability assay of A375 cells cultured in regular RPMI (11 mM glucose) or glucose enriched (25 mM) RPMI in the presence of 50 μM telmisartan, 5 mM 2DG or the combination. ** P < 0.0049, n = 3 in unpaired t-test.

6

Telmisartan induces mitochondrial fragmentation. (A) Immunocytochemistry of TOM20 (green) marker for mitochondria in telmisartan- or pioglitazone-treated A375 and HTB140 cells. Nuclei-DAPI-blue, scale bar 20 μm. Arrowheads point to the fragmented mitochondria. (B) MitoTracker Red CMXRos staining of the untreated and telmisartan treated A375 and HTB140 cells. White arrowheads point to the mitochondria that are presented in the zoomed-in inset (middle panel). Scale bar 20 μm.

7

Telmisartan has synergistic effects with vemurafenib in vitro. (A) 5 × 5 dose matrix for vemurafenib x telmisartan combination in A375 cells. Percent growth inhibition is visualized using a color scale. The data in matrix are average of three experiments. Isobologram analysis for the combination effect of (B) ED90 (C) ED75 and (D) ED50, single doses of vemurafenib and telmisartan were used to draw the line of additivity. Green triangles represent single doses needed for the combination effect ED. Representative graphs (E) and quantification of three independent experiments (F) for apoptosis in A375 cells treated with combination of vemurafenib and telmisartan. Two-way ANOVA, ****P < 0.0001. (G) MitoTracker Red CMXRos staining for mitochondria in telmisartan-, vemurafenib- or the combination-treated A375 cells. Arrowheads point to the fragmented mitochondria. Scale bar 20 μm.

8

Telmisartan is effective in vemurafenib-resistant melanoma cells. (A) Growth rates and (B) vemurafenib sensitivity of A375 parental and A375R vemurafenib resistant cell line. Non-linear fit, Two-way ANOVA, P = 0.005 for the 72 h time point in growth curves, ****P < 0.0001 for sensitivity across all values. (C) Cell cycle distribution of A375 parental and A375R cells after 72 h treatment with 0.5 μM vemurafenib, n = 3. (D) Percent viability of A375 and A375R cells after 72 h treatment with telmisartan, presented as log (inhibitor) vs. normalized response. (E) Relative ROS (DCFDA) in A375R cells after 24 h treatment with 50 μM telmisartan, values represent mean ± SD, *P < 0.05, t-test, n = 3. (F) Quantification and (G) representative graphs of apoptosis assay in A375R cells after 72 h treatment with telmisartan, vemurafenib or the combination. Values represent mean ± SD, Two-way ANOVA, ***P < 0.001, n = 3.

S1

Immunocytochemistry of TOM20 (green) marker for mitochondria in telmisartan-, vemurafenib- or the combination-treated A375 cells. Nuclei-DAPI-blue, arrowheads point to fragmented mitochondria.

S2

Schematic of the proposed mechanism of telmisartan action.