Doxycycline potentiates antitumor effect of 5-aminolevulinic acid-mediated photodynamic therapy in malignant peripheral nerve sheath tumor cells

Abstract

Neurofibromatosis type 1 (NF1) is one of the most common neurocutaneous disorders. Some NF1 patients develop benign large plexiform neurofibroma(s) at birth, which can then transform into a malignant peripheral nerve sheath tumor (MPNST). There is no curative treatment for this rapidly progressive and easily metastatic neurofibrosarcoma. Photodynamic therapy (PDT) has been developed as an anti-cancer treatment, and 5-aminolevulinic (ALA) mediated PDT (ALA-PDT) has been used to treat cutaneous skin and oral neoplasms. Doxycycline, a tetracycline derivative, can substantially reduce the tumor burden in human and animal models, in addition to its antimicrobial effects. The purpose of this study was to evaluate the effect and to investigate the mechanism of action of combined doxycycline and ALA-PDT treatment of MPNST cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the combination of ALA-PDT and doxycycline significantly reduce MPNST survival rate, compared to cells treated with each therapy alone. Isobologram analysis showed that the combined treatment had a synergistic effect. The increased cytotoxic activity could be seen by an increase in cellular protoporphyrin IX (PpIX) accumulation. Furthermore, we found that the higher retention of PpIX was mainly due to increasing ALA uptake, rather than activity changes of the enzymes porphobilinogen deaminase and ferrochelatase. The combined treatment inhibited tumor growth in different tumor cell lines, but not in normal human Schwann cells or fibroblasts. Similarly, a synergistic interaction was also found in cells treated with ALA-PDT combined with minocycline, but not tetracycline. In summary, doxycycline can potentiate the effect of ALA-PDT to kill tumor cells. This increased potency allows for a dose reduction of doxycycline and photodynamic radiation, reducing the occurrence of toxic side effects in vivo.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Cell viability of malignant peripheral nerve sheath tumor cell line, S462 under the treatment of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) alone or in combination with doxycycline.

(A), the percentage of S462 cell viability post the treatment of ALA-PDT. The cells were incubated with 1 mM ALA for 24 hours followed by light illumination. The MTT assay was performed to evaluate the cell viability at different light doses. (B), the percentage of S462 cell viability after the treatment of different concentration of doxycycline. (C) Left panel shows the percentage of cell viability under PDT alone and in combination with doxycycline (10- and 50- μg/mL) under the light dose of 8 J/cm2. The contour connecting every point (d1, d2) in the isobologram bows inwardly, which indicates a Loewe synergy between the treatment of ALA-PDT and doxycycline (right panel).

Fig 2. Cell viability and death mode under the treatment of ALA-PDT, doxycycline, and combined treatment ALA-PDT/doxycycline.

(A) The percentage of cell viability treated with ALA-PDT did not have a significant change after the pretreatment of benzyloxycarbony-Val-Ala-Asp-fluoromethylketone (co-treat + Z-VAD) or 3-methyladenine (co-treat + 3-MA) (left panel). The morphology and fluorescence staining of S462 cells were observed by bright field and fluorescent microscopy after ALA-PDT (right panel) (B) The survival rate of S462 cell was rescued when pre-treated with different concentrations (0, 2.5, 5.0, and 10.0 mM) of 3-MA (left panel). The right panel shows the results of fluorescent microscopical analysis. Abundant autophagosomes stained by MDC (bright punctate in cytoplasm) were found in the cytoplasm after treatment of doxycycline (doxycycline treatment); however, after co-treatment of 3-MA (doxycycline + 3-MA), the amount was decreased conspicuously. (C) The percentage of cell viability received ALA-PDT/doxycycline did not have a significant change under the pretreatment of Z-VAD (co-treat + Z-VAD) or 3-MA (co-treat + 3-MA) (left panel). The morphology of the cells post the co-treatment was observed by bright field and fluorescent microscopy (right panel of (C)).

Fig 3. Accumulation of protoporphyrin IX (PpIX), enzyme activities for porphobilinogen deaminase (PBGD) and ferrochelatase (FC), and uptake of 5-aminolevulinic acid (ALA) in cells co-treated with ALA and doxycycline.

(A) The relative fluorescence intensity of PpIX was evaluated against the time post either ALA (open square) or the combined treatment of ALA and doxycycline (filled square). The relatively enzyme activity of PBGD (B) and FC (C) after the combined treatment of ALA and different concentration of doxycycline. (D) The uptake of ALA was assessed against the time post either ALA alone (open square) or the combined treatment of ALA and doxycycline (filled square). (E and F) The relative uptake level of ALA (E) and the fluorescence intensity of PpIX (F) were measured in S462 cells after incubated with 1 mM of ALA and different concentrations of doxycycline for 24 hours (*, p

Fig 4. The effect of doxycycline on cellular uptake of methyl 5-aminolevulinic acid (me-ALA) and chlorin e6 (Ce6).

(A and B) Application of me-ALA leads to the uptake of ALA and accumulation of PpIX, similar to that found after ALA treatment. The duration of incubation was one or two hours for the uptake assay (A). For PpIX accumulation measurements, these four groups of cells were incubated for three and six hours (B). (C) The increased uptake of Ce6 was also found by doxycycline. *, p

Fig 5. Cell survival, uptake of ALA and accumulation of PpIX in tumor and normal cells.

(A), the percentage of cell viability post the treatment of ALA-PDT alone (PDT alone), doxycycline alone (Doxy alone), combined treatment (co-treatment) and sham operation (control) was evaluated in two malignant cell lines (the human malignant melanoma cell, A375 and lung adenocarcinoma CL1-0) and the mouse colon carcinoma, C26. The light dose for A375 cells is 9 J/cm2; 12 J/cm2 is used for CL1-0 and C26. The combined treatment significantly inhibited the growth of the five different cancer cells. (B), the percentage of cell viability in normal human Schwann cells (HSC), human fibroblast (Hs68) and mouse fibroblast (NIH3T3) cells after the same treatment as shown in (A). The light dose for NSC, Hs68, and NIH3T3 cells is 8 J/cm2. There were no significant changes in cell viability after the treatment of PDT (PDT alone), doxycycline (Doxy alone) or in combination (Co-treatment). The uptake of ALA (C) and accumulation of PpIX (D) after the treatment of ALA-PDT only (ALA alone), combined treatment ALA-PDT/doxycycline (ALA + Doxy), or sham operation (control) in both normal human fibroblast (Hs68) and four malignant cell lines (A375, CL1-10, and C26).

Fig 6. Synergistic effects of tetracycline and its derivatives combined with ALA-PDT treatment.

(A), Left panel shows the percentage of cell viability after the treatment of ALA-PDT only (ALA only), or in combination with tetracycline (ALA + Tetra), doxycycline (ALA + Doxy), or minocycline (ALA + Mino). Right panel shows the relative PpIX amounts after ALA only and the combined treatment. (B), isobologram analysis for the combined treatment of ALA-PDT/tetracycline (left panel), and ALA-PDT/minocycline (right panel). (C), the amounts of ALA uptake after the ALA only and the combined treatment.

Fig 7. ALA uptake in MPNST, S462 cells with combined treatment of ALA-PDT, doxycycline, and GABA.

Measurements of intracellular uptake of ALA were recorded by the time after the treatment of 1 mM of ALA (ALA only), 1 mM of ALA and 10 mM of γ–aminobutyric acid (ALA + GABA), 1 mM of ALA and 50 μg/mL of doxycycline (ALA + Doxy), and 1 mM of ALA, 50 μg/mL of doxycycline and 10 mM of γ–aminobutyric acid (ALA + Doxy + GABA).