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

<div><p>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 <i>in vivo</i>.</p></div

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

<p>(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)).</p

Mexiletine IC₅₀ curves for wild type and mutant Na_v1.7 channels.

<p>(A–D) Mexiletine IC₅₀ curves of wild type, I136V, I848T, and V1316A mutant Na_v1.7 channels, respectively. The IC₅₀ value of wild type channel for mexiletine is 1.77±0.78 mM. No significant difference in IC₅₀ values of I136V (2.03±0.38 mM) and V1316A (1.73±0.20 mM) mutant channels compared with wild type channel. I848T mutant has a significant (<i>P</i><0.05) lower IC₅₀ value (1.08±0.11 mM) compared with wild type channel. N for wild type is 6, I136V, 7, I848T, 7, V1316A, 8; <i>t</i>-test; data shown as means ± SEM.</p

Effects of IL-1β neutralization on the accumulation of MDSCs in lungs.

<p>(A) Immunofluorescent photos showed that Gr-1+ (green)/CD11b+ (red) cells indicate MDSCs and increased in lungs of DJ-1 KO mice. Treatment with anti-IL-1β neutralizing antibody antagonized the accumulation of MDSCs. DAPI was used to stain nuclei. (B) Bar chart showed the summarized results of MDSCs in lungs of WT and DJ-1 KO mice. Note that the neutralizing antibody significantly reduced the number of MDSCs accumulated in lungs. n = 9 for each group, * p < 0.05 compared with control IgG-treated WT mice, # p<0.05 compared with respective control IgG in WT and DJ-1 KO mice.</p

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

<p>(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.</p

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.

<p>(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<0.05).</p

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

<p>(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).</p

The Biophysical Basis Underlying Gating Changes in the p.V1316A Mutant Na_v1.7 Channel and the Molecular Pathogenesis of Inherited Erythromelalgia

<div><p>The Na_v1.7 channel critically contributes to the excitability of sensory neurons, and gain-of-function mutations of this channel have been shown to cause inherited erythromelalgia (IEM) with neuropathic pain. In this study, we report a case of a severe phenotype of IEM caused by p.V1316A mutation in the Na_v1.7 channel. Mechanistically, we first demonstrate that the Na_vβ4 peptide acts as a gating modifier rather than an open channel blocker competing with the inactivating peptide to give rise to resurgent currents in the Na_v1.7 channel. Moreover, there are two distinct open and two corresponding fast inactivated states in the genesis of resurgent Na⁺ currents. One is responsible for the resurgent route and practically existent only in the presence of Na_vβ4 peptide, whereas the other is responsible for the “silent” route of recovery from inactivation. In this regard, the p.V1316A mutation makes hyperpolarization shift in the activation curve, and depolarization shift in the inactivation curve, vividly uncoupling inactivation from activation. In terms of molecular gating operation, the most important changes caused by the p.V1316A mutation are both acceleration of the transition from the inactivated states to the activated states and deceleration of the reverse transition, resulting in much larger sustained as well as resurgent Na⁺ currents. In summary, the genesis of the resurgent currents in the Na_v1.7 channel is ascribable to the transient existence of a distinct and novel open state promoted by the Na_vβ4 peptide. In addition, S4–5 linker in domain III where V1316 is located seems to play a critical role in activation–inactivation coupling, chiefly via direct modulation of the transitional kinetics between the open and the inactivated states. The sustained and resurgent Na⁺ currents may therefore be correlatively enhanced by specific mutations involving this linker and relevant regions, and thus marked hyperexcitability in corresponding neural tissues as well as IEM symptomatology.</p></div

Local Immunosuppressive Microenvironment Enhances Migration of Melanoma Cells to Lungs in DJ-1 Knockout Mice

<div><p>DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1β expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×10⁴) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1β and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1β) neutralizing antibody to see whether IL-1β is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1β to explore underlying molecular mechanisms. Our results showed that IL-1β enhanced survival and colony formation of cultured melanoma cells, and that IL-1β levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1β correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1β neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1β plays an important role in promoting the cancer migration.</p></div

IFN-γ and I-TAC reduce the survival of dopaminergic neurons in primary midbrain neuron-glia mixed cultures but not in neuron-enriched cultures.

<p>(A) Midbrain neuron-glia mixed cultures were derived from E14 rat embryos and treated with recombinant IFN-γ protein (30 and 300 ng/ml) or I-TAC protein (1 and 10 ng/ml) on Day-7 cultures for 48 hours. LPS (500 ng/ml) was used as positive control. Note that IFN-γ and I-TAC reduced the survival of TH-positive neurons in neuron-glia mixed cultures (B) The neuron-enriched cultures were obtained from E14 rat embryos and treated with IFN-γ and I-TAC on Day-7 cultures for 48 hours. Note that IFN-γ and I-TAC did not affect survival of TH-positive neurons in neuron-enriched cultures. The dopaminergic neurons (TH-positive) were counted and normalized as percentage of TH-positive neurons in control cells. Data were presented as mean ± S.E.M. (n = 4–5 for each group) * p<0.05 as compared with control.</p