Sodium dichloroacetate

Synergistic effect of dichloroacetate on talaporfin sodium-based photodynamic therapy on U251 human astrocytoma cells

Yo Shinodaa, Kohei Aoki, Ayaka Shinkai, Kumi Seki, Tsutomu Takahashi, Yayoi Tsuneoka, Jiro Akimoto, Yasuyuki Fujiwara
a Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan
b Department of Neurosurgery, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku, Tokyo, 160-0023, Japan

Background: Talaporfin sodium (TS) is an authorized photosensitizer for photodynamic therapy (PDT) against some tumors in Japan; however, the drawbacks of the drug include its high cost and side effects. Thus, reducing the dose of TS in each round of TS-PDT against tumors is important for reducing treatment costs and improving patients’ quality of life. Dichloroacetate (DCA) is approved for treating lactic acidosis and hereditary mi- tochondrial diseases, and it is known to enhance reactive oxygen species production and induce apoptosis in cancer cells. Therefore, DCA has the potential to enhance the effects of TS-PDT and permit the use of lower TS doses without reducing the anti-cancer effect.
Methods: U251 human astrocytoma cells were simultaneously incubated with TS and DCA using different concentrations, administration schedules, and treatment durations, followed by laser irradiation. Cell viability was determined using the CCK-8 assay.
Results: The combinational use of DCA and TS resulted in synergistically enhanced TS-PDT effects in U251 cells. The duration of DCA treatment before TS-PDT slightly enhanced the efficacy of TS-PDT. The intensity of laser irradiation was not associated with the synergistic effect of DCA on TS-PDT. In addition, the relationship be- tween the elapsed time after TS/DCA combination treatment and PDT ineffectiveness was identical to that of TS monotherapy.
Conclusions: DCA synergistically enhanced the anti-cancer effect of TS-PDT, illustrating its potential for drug repositioning in cancer therapy in combination with PDT.

1. Introduction
Photodynamic therapy (PDT) is a high selective therapeutic proce- dure used in the treatment of cancer [1–3]. PDT is performed using a photosensitizer, a small chemical compound that induces reactive oxygen species (ROS) production in response to light, and laser irra- diation to kill the ROS-exposed cancer cells. Talaporfin sodium (TS, NPe6, Laserphyrin®) is known as a second-generation photosensitizer that is clinically used in PDT for early-stage lung cancer, primary ma- lignant brain tumors, and locally remnant recurrent esophageal cancer in Japan [4–8]. Despite the efficacy of TS in PDT for these cancers, the drug is expensive and carries several side effects including photo- sensitivity, increased sputum production, and liver dysfunction [9]. Therefore, it is meaningful to reduce the effective dose of TS to over- come these problems, and the potential use of chemotherapeutic drugs in combination with TS represents a promising treatment strategy forcancer.
There have been several efforts to reduce the doses of photo- sensitizers via the simultaneous use of approved anti-cancer drugs, such as 5-aminolevulinic acid (5-ALA) with gefitinib [10], pheophorbide with doxorubicin [11], and photofrin with cisplatin [12]. In addition, another strategy for reducing the photosensitizer dose is the simulta- neous use of drugs that affect cancer-specific metabolic pathways. Previously described combinations include chlorin e6 plus 2-deox- yglucose or 3-bromopyruvate [13] and 5-ALA plus dichloroacetate (DCA) [14]. In most cancer cells, mitochondrial dysfunction results in a characteristic phenotype typified by a metabolic shift from mitochon- drial oxidative phosphorylation to aerobic glycolysis, which is called the Warburg effect [15,16]. Mitochondria are associated with both energy production and apoptosis in their host cells, and it appears that the glycolytic phenotype of cancer cells is indeed associated with a state of apoptosis resistance [17,18]. Thus, reversing the metabolic pathwayfrom aerobic glycolysis to oxidative phosphorylation to reduce the anti-apoptotic effects of mitochondria in cancer cells is a potential strategyfor enhancing the efficacy of PDT in cancer therapy. DCA is known as an inhibitor of pyruvate dehydrogenase kinase (PDK), which enhances mitochondrial oxidative phosphorylation by activating pyruvate dehy- drogenase (PDH), leading to the conversion of pyruvate to acetyl-CoA [18,19]. Activation of aerobic metabolism in mitochondria results in increased ROS production [20–22]. Therefore, DCA has the potential to enhance the effects on PDT by increasing ROS production. In addition to enhancing ROS production, DCA itself induces apoptosis in colorectalcancer [23], neuroblastoma [24], glioblastoma [25], and endometrial cancer [26] by modulating mitochondrial metabolism [27]. Moreover,DCA has already been clinically used in the treatment of lactic acidosis and hereditary mitochondrial diseases [28,29] with high tolerability and safety [30,31]. Therefore, DCA is a potential option for drug re-positioning if it can be used to enhance the effects of TS-PDT. In the present study, we evaluated whether the combined use ofDCA could reduce the effective dose of TS in cultured U251 human astrocytoma cells and identify a synergistic effect of DCA on the efficacy of TS-PDT.

2. Materials and methods
2.1. Cell culture
U251 cells (Riken Cell Bank, Ibaraki, Japan) were cultured as de- scribed previously with small modifications [32]. Briefly, cells werecultured at 1 × 104 cells/well in non-coated cell culture plastic 96-well plates (Nippon Genetics, Tokyo, Japan) in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS, FUJIFILM Wako Pure Chemical, Osaka, Japan) at 37 °C in a 5 % CO2 humidified atmosphere. All experimental protocols were evaluated and approved by the Regulations for Biolo- gical Research at Tokyo University of Pharmacy and Life Sciences and conducted in accordance with the approved protocols.

2.2. TS treatment and laser irradiation
The in vitro PDT experiment was performed as described previously with small modifications [33]. Twenty-four hours after plating, U251 cells were treated with several concentrations (0, 12.5, 25, 37.5, 50, or 62.5 μM) of TS (Meiji Seika Pharma, Tokyo, Japan) for 1 h at 37 °C in 5% CO2. Cells were washed once with phosphate-buffered saline (PBS),and fresh medium supplemented with 10 % FBS was added. The cells were immediately subjected to laser irradiation (wavelength: 664 nm, laser power 3.4 mW/cm2, total dose of laser irradiation: 1 J/cm2) using a ZH-L5011HJP semiconductor laser irradiator (Meiji Seika Pharma) and then incubated for 24 h at 37 °C in 5 % CO2. The viabilities of the cells were analyzed using Cell counting kit-8 (CCK-8, Dojindo Labora- tories, Tokyo, Japan) according to the manufacturer’s protocol, and absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Tokyo, Japan).

2.3. DCA treatment
Twenty-four hours after plating, U251 cells were treated with sev- eral concentrations (0, 10, 20, 40, or 80 mM) of sodium DCA (Sigma- Aldrich, St. Louis, MO, USA) and incubated for 24 h at 37 °C in 5 % CO2. The viabilities of the cells were analyzed using CCK-8 and a microplate reader.

2.4. Combination treatment of TS and DCA
Twenty-four hours after plating, U251 cells were treated with TS (0, 12.5, 25, 37.5, 50, or 62.5 μM) plus DCA (0, 10, or 40 mM) in fresh medium supplemented with 10 % FBS for 1 h at 37 °C in 5 % CO2. Cellswere washed once with PBS, and fresh medium supplemented with 10% FBS was added. The cells were immediately subjected to laser irra- diation (664 nm, 1 J/cm2) and subsequently incubated for 24 h at 37 °C in 5 % CO2. The viabilities of the cells were analyzed using CCK-8 and a microplate reader. Phase contrast imaging was performed using a DMi1inverted microscope (Leica Microsystems, Wetzlar, Germany).
To evaluate the effects of the duration of DCA treatment, U251 cells were treated with DCA (0, 10, or 40 mM) for 0, 2, 5, 11, or 23 h before TS (12.5 μM) treatment. Then, cells were treated with combinations ofTS (12.5 μM) and DCA (0, 10, or 40 mM) for 1 h, and after washing withPBS and fresh medium replacement, and laser irradiation (1 J/cm2), viability was assessed 24 h after irradiation as described previously. To evaluate the effects of laser intensity, U251 cells were treated withcombinations of TS (12.5 μM) and DCA (0, 10, or 40 mM) for 1 h. After washing with PBS wash and medium replacement, laser irradiation (1, 1.5, 2, or 2.5 J/cm2) and viability assays were performed as describedpreviously.
To evaluate the duration of the efficacy of TS/DCA combination treatment, U251 cells were treated with TS alone (25 μM) or the combination of TS (12.5 μM) and DCA (40 mM) for 1 h, followed by washing in PBS and fresh medium replacement. Then, cells were irradiated (1 J/cm2) for up to 23 h after each treatment, and viability assays were performed at 24 h after irradiation.

2.5. Statistical analysis
The data are expressed as the mean ± SEM. If not stated otherwise, differences between two datasets were assessed using Student’s t-test, and multiple datasets were assessed using one-way analysis of variance (ANOVA) or two-way ANOVA with a post hoc Tukey–Kramer test. All data were collected and analyzed using a double-blind approach.

3. Results
3.1. The anti-cancer effects of TS or DCA alone
We first tested the effects of TS or DCA alone on the viability of U251 cells. As we reported previously [32], TS exerted concentration- dependent anti-cancer effects on U251 cells (Fig. 1A). At 12.5 μM TS,small but significant anti-cancer activity was detected. Next, DCA wasapplied to U251 cells at different concentrations for 24 h. Treatment of U251 cells with 40–80 mM DCA for 24 h resulted in significantly re- duced viability, whereas no effect on viability was observed at lower concentrations (Fig. 1B). These data suggest that DCA itself has little anti-cancer activity at concentrations < 40 mM for 24 h treatment. Moreover, administration of 40 mM DCA for 1 h did not show any cytotoxicity (data not shown). Thus, DCA concentrations of 0–40 mM were selected for subsequent experiments. Similar experiments were carried out using rat meningioma cell line KMY-J (Supplemental Fig. 1). 3.2. Synergistic anti-cancer effects of the combination of TS and DCA Next, we assessed whether the combination of TS and DCA had synergistic anti-cancer effects at concentrations that were not cytotoxic when the drugs were used alone. The results illustrated that the com- bination treatment had concentration-dependent anti-cancer effects (Fig. 2). Interestingly, strong synergistic anti-cancer effects were ob-served when the TS concentration was 12.5 μM (Fig. 2C). At this con-centration, single-agent TS itself exhibited a small but significant anti- cancer effect (86.2 % viability, Fig. 1A). In addition, although single- agent DCA had little cytotoxic effects at 10 or 40 mM for 1 h treatment, this drug exhibited substantial anti-cancer activity when used in com- bination with TS. At TS concentrations of 25 and 37.5 μM, the combi-nation regimen exerted small but significant synergistic anti-cancereffects. Synergy was not observed at higher TS concentrations, in- dicating that the effect was saturable. Similar synergistic effect was obtained using KMY-J cell line (Supplemental Fig. 2). 3.3. Effects of the treatment schedule on the anti-cancer effects of the combination regimen In an attempt to enhance the synergistic effects of the TS/DCA combination treatment, we evaluated different durations of DCA treatment before TS administration and increased the irradiation in- tensity. Increasing the duration of DCA treatment significantly en- hanced the synergistic effect of the TS/DCA combination regimen, but the effect of the drug concentration on viability was not large (Fig. 3A). Increasing the laser power also significantly enhanced the anti-cancer effect, but the effects were small as previously observed in this study (Fig. 3B). 3.4. Duration of the efficacy of the TS/DCA combination regimen Finally, we examined the duration of the anti-cancer effects of the combination treatment and laser irradiation (Fig. 4). The results illu- strated that the combination regimen remained effective for the same duration as TS monotherapy. Therefore, the anti-cancer effect of theTS/DCA combination treatment was comparable to that of single-agent TS. 4. Discussion In the present study, we performed TS-PDT together with DCA in an effort to increase the cytotoxic effects of the therapy in U251 cells. This combinational treatment also showed a similar effect in rat meningioma cell line KMY-J so that this effect is thought to be not limited to U251 cells. As we expected, DCA synergistically enhanced the anti-cancer effect of TS-PDT. Moreover, the effects of the duration of DCA treatment before PDT and the intensity of laser irradiation as well as the duration of efficacy for the TS/DCA combination were verified in this in vitro preclinical study. In general, cancer cells tend to favor metabolism via aerobic gly- colysis rather than the mitochondrial tricarboxylic acid (TCA) cycle to obtain energy in the form of ATP [15]. This effect is mainly promoted by the hypoxia inducible factor 1 (HIF-1)-induced expression of several genes including PDK [34,35], which inactivates PDH via phosphoryla- tion [36]. PDH catalyzes the conversion of pyruvate to acetyl-CoA; thus, the overexpression of PDK inhibits the mitochondrial TCA cycle via PDH inactivation. DCA is a small synthetic drug that inhibits PDK [37], thereby enhancing mitochondrial TCA cycle activity [18,19]. In fact,DCA immediately activates PDH by inactivating PDK [38,39], thereby increasing mitochondrial acetyl-CoA concentrations [38,40]. The in- creased acetyl-CoA concentration promotes the TCA cycle and subse- quently enhances ROS production [22,39,41]. In addition to the en- hancement of ROS production, increased O2 consumption in tumor and fibrotic cells in response to DCA treatment has been reported [42,43]. These findings suggest that DCA both enhances ROS production in cancer cells but also stimulates PDT-induced ROS overproduction. Mitochondrial dysfunction in cancer cells is usually associated with changes in mitochondrial membrane potential [44,45]. DCA restores mitochondrial membrane potential [39,41,46], which is at least asso- ciated with the regulation of mitochondrial K+ channel expression [39]. In addition, it has been reported that DCA suppresses the ex- pression of HIF-1α and hexokinase-2 via PDK inhibition-mediated downregulation of protein kinase B/glycogen synthase kinase-3β sig- naling [46], which is also associated with the reversal of signs of mi-tochondrial dysfunction, including the impairment of mitochondrial membrane potential and aerobic glycolysis. These studies suggested that DCA promotes mitochondrial normalization in cancer cells. Taken together, these findings strongly support that DCA reverses apoptosis resistance in cancer cells [39,41,47]. Moreover, orally ad- ministered DCA easily penetrates the blood-brain barrier [22]. There- fore, DCA is considered a potential anti-tumor drug, including possibleeffects against brain tumors, in vitro and in vivo [22–26,39,41,48]. 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