Photodynamic priming modulates cellular ATP levels to overcome P-glycoprotein-mediated drug efflux in chemoresistant triple-negative breast cancer

Abstract

P-glycoprotein (P-gp, ABCB1) is a well-researched ATP-binding cassette (ABC) drug efflux transporter linked to the development of cancer multidrug resistance (MDR). Despite extensive studies, approved therapies to safely inhibit P-gp in clinical settings are lacking, necessitating innovative strategies beyond conventional inhibitors or antibodies to reverse MDR. Photodynamic therapy is a globally approved cancer treatment that uses targeted, harmless red light to activate non-toxic photosensitizers, confining its cytotoxic photochemical effects to disease sites while sparing healthy tissues. This study demonstrates that photodynamic priming (PDP), a sub-cytotoxic photodynamic therapy process, can inhibit P-gp function by modulating cellular respiration and ATP levels in light accessible regions. Using chemoresistant (VBL-MDA-MB-231) and chemosensitive (MDA-MB-231) triple-negative breast cancer cell lines, we showed that PDP decreases mitochondrial membrane potential by 54.4% ± 30.4 and reduces mitochondrial ATP production rates by 94.9% ± 3.46. Flow cytometry studies showed PDP can effectively improve the retention of P-gp substrates (calcein) by up to 228.4% ± 156.3 in chemoresistant VBL-MDA-MB-231 cells, but not in chemosensitive MDA-MB-231 cells. Further analysis revealed that PDP did not alter the cell surface expression level of P-gp in VBL-MDA-MB-231 cells. These findings indicate that PDP can reduce cellular ATP below the levels that is required for the function of P-gp and improve intracellular substrate retention. We propose that PDP in combination with chemotherapy drugs, might improve the efficacy of chemotherapy and overcome cancer MDR.

Keywords: ABC transporter; P‐glycoprotein; cellular ATP levels; drug delivery; multidrug resistance; photodynamic priming.

FIGURE 1

Chemoresistant cancer cells show increased cellular respiration and ATP production. Flow cytometry was used to analyze MDA‐MB‐231 (chemosensitive) and VBL‐MDA‐MB‐231 (chemoresistant) cells for cell surface P‐gp expression by P‐gp‐specific MRK16 antibody (fluorescent signal from FITC‐labeled secondary antibody) compared to the IgG2a negative isotype control (A–C) and P‐gp function using rhodamine 123 (D, E). (F) Oxygen consumption rate (OCR) and (G) extracellular acidification rate (ECAR) in response to sequential injection of oligomycin (O) and rotenone with antimycin A (R/AA). (H, I) Mitochondrial and glycolytic ATP production rates in MDA‐MB‐231 and VBL‐MDA‐MB‐231 cells were determined by Seahorse XF96 extracellular flux analyzer. VBL‐MDA‐MB‐231 cells exhibit increased ATP production rates compared to those of MDA‐MB‐231 cells. (J) Analysis of total intracellular ATP content by ATPLite luminescence assay reveals higher intracellular ATP content in VBL‐MDA‐MB‐231 cancer cells. Data presented as mean ± SD values of three independent experiments (*p ≤ 0.05, **p ≤ 0.01, two‐tailed t‐test).

FIGURE 2

Photodynamic priming induces mitochondrial membrane potential depolarization. Cells were treated with BPD for 90 min prior to the PDP treatment. Cells were irradiated from the bottom up at 1 J/cm² and then allowed to incubate for 2 h. Cytotoxicity, intracellular BPD concentration, and TMRE accumulations were measured 2‐h post‐PDP. Cytotoxicity was measured for (A) MDA‐MB‐231 and (B) VBL‐MDA‐MB‐231 cells using an MTT assay. BPD uptake was measured for both cell lines (C, D), with and without the P‐gp inhibitor, tariquidar, showing that BPD is a substrate for P‐gp. Mitochondrial membrane potential depolarization was measured in both cell lines (E, F) using a TMRE assay 2‐h post‐illumination (690 nm, 10 mW/cm², 1 J/cm²). Data presented as mean ± SD values (n ≥ 3), (*p ≤ 0.05, **p ≤ 0.01, two‐tailed t‐test).

FIGURE 3

Photodynamic priming reduces mitochondrial and glycolytic ATP production rates. Cells were treated with BPD for 90 min prior to the PDP treatment. Cells were irradiated from the bottom up at 1 J/cm² and then allowed to incubate for 2 h. Mitochondrial and glycolytic ATP production rates were measured using the Agilent Seahorse Real Time ATP Assay 2 h post‐PDP. Mitochondrial (A, B) and glycolytic (C, D) ATP production rates in MDA‐MB‐231 and VBL‐MDA‐MB‐231 cells were determined by Seahorse XF96 extracellular flux analyzer. Data presented as mean ± SD values (n ≥ 3, *p ≤ 0.05, **p ≤ 0.01, two‐tailed t‐test).

FIGURE 4

Photodynamic priming does not cause significant changes in the cell surface expression level of P‐gp. Flow cytometry was used to measure P‐gp expression using human P‐gp‐specific MRK16 antibody and IgG2a as a negative isotype control 2‐h post‐illumination (690 nm, 10 mW/cm², 1 J/cm²) (A, B). MDA‐MB‐231 and VBL‐MDA‐MB‐231 cells exhibit no significant changes in fluorescence of MRK16 binding due to PDP (C, D). MRK16 binding was measured using a BD FACS Canto II flow cytometer and FITC fluorescence was analyzed using FlowJo software. Data presented as histogram, 2 μM BPD, 0 J/cm² in red and 1 J/cm² in orange for MDA‐MB‐231, 0 J/cm² in light blue and 1 J/cm² in dark blue for VBL‐MDA‐MB‐231, and values are given as mean ± SD (n > 3), ordinary one‐way ANOVA.

FIGURE 5

Photodynamic priming increases P‐gp substrate accumulation in chemoresistant cells. Flow cytometry was used to measure intracellular accumulation of rhodamine 123 and calcein generated from calcein‐AM. Accumulation of rhodamine 123 (A, B) and calcein (F, G) both increased in VBL‐MDA‐MB‐231 cells 2‐h post‐illumination. DAPI staining shows membrane permeability due to PDP treatment at 2 μM BPD and 1 J/cm² (D (rhodamine 123), I calcein) compared to 2 μM BPD and 0 J/cm² (C (rhodamine 123), H (calcein)). Overlapping histograms show substrate retention in cell populations with nuclear staining with DAPI (E (rhodamine 123), J (calcein)). Substrate accumulation was measured using a Sony ID7000 spectral analyzer and fluorescence was analyzed using FlowJo software. Data presented as histograms, with 2 μM BPD, 0 J/cm² in black and 1 J/cm² in blue and mean ± SD (n > 3), *p ≤ 0.05, **p ≤ 0.01, ordinary one‐way ANOVA.

FIGURE 6

Photodynamic priming increases membrane permeability and decreases P‐gp substrate accumulation in chemosensitive cells. Flow cytometry was used to measure intracellular accumulation of rhodamine 123 and calcein. Accumulation of rhodamine 123 (A, B) and calcein (F, G) both decreased in MDA‐MB‐231 cells 2‐h post‐illumination. DAPI staining shows membrane permeability due to PDP treatment at 2 μM BPD and 1 J/cm² (D (rhodamine 123), I (calcein)) compared to 2 μM BPD and 0 J/cm² (C (rhodamine 123), H (calcein)). Overlapping histograms show substrate retention in cell populations with DAPI staining (E (rhodamine 123), J (calcein)). Substrate accumulation was measured using a Sony ID7000 spectral analyzer and fluorescence was analyzed using FlowJo software. Data presented as histograms, with 2 μM BPD, 0 J/cm² in black and 1 J/cm² in blue and mean ± SD (n > 3), *p ≤ 0.05, **p ≤ 0.01, ordinary one‐way ANOVA.