Date Published: May 30, 2017
Publisher: Public Library of Science
Author(s): Ming-Jen Lee, Shih-Hsuan Hung, Mu-Ching Huang, Tsuimin Tsai, Chin-Tin Chen, Michael Hamblin.
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.
Photodynamic therapy (PDT), a new approach to cancer treatment, uses photosensitizers (PS) and light to generate reactive oxygen species, which can interact with biomolecules and subcellular organelles to interrupt key molecular functions. This photoreaction leads to cell death and destruction of tissues . 5-aminolevulinic acid (ALA) itself is not a photosensitizer; but exogenously applied ALA leads to the increased formation of protoporphyrin IX (PpIX, a photosensitizer) by a series of enzymes in the heme biosynthetic pathway . There are two rate-limiting steps in this pathway. The first is ALA production from succinyl CoA and glycine by ALA synthase, which is regulated by heme via a negative feedback mechanism. The other is the formation of heme via the addition of a ferrous iron to PpIX, which is controlled by the rate-limiting enzyme, ferrochelatase (FC). It has been shown that ALA-induced PpIX accumulation is greater in certain tumor cells due to the reduced activity of ferrochelatase (FC)  and higher porphobilinogen deaminase (PBGD) activity . In addition, cancer cells usually have high rate of glycolysis followed by lactic acid fermentation (Warburg effect), resulting in abnormalities of iron metabolism and errors in iron insertion which lead to the enhanced accumulation of PpIX in cancer cells .
Recently, multiple drug regimens have been shown to be more effective to manage or eradicate malignant tissues. In this report, we demonstrated that doxycycline and its derivative could selectively enhance the therapeutic efficacy of ALA-mediated PDT in a MPNST derived cell line, S462. Although treatment with either ALA-PDT or doxycycline alone was moderately effective to suppress the growth of S462 cells, the combined treatment revealed a synergistic cytotoxic effect, as determined by isobologram analysis. In addition, we found that the enhancement of ALA uptake and PpIX accumulation induced by doxycycline is more pronounced in cancer cells compared to normal cells. The combined treatment of topical ALA-PDT and doxycycline given orally was first tried in patients with acne conglobate . Another study using phenothiazine chloride-induced anti-microbial PDT treatment in periodontitis patients with uncontrolled diabetic mellitus showed enhanced effectiveness with the topical application of doxycycline .