📖 17 mins.

Ronen Shavit¹, Maya Ilouze, Tali Feinberg, Yaacov Richard Lawrence, Yossi Tzur, Nir Peled

¹ Thoracic Cancer Research and Detection Center, Sheba Medical Center Tel Hashomer, Ramat-Gan, 52621, POB 244, Israel.
e-mail: [email protected]
URL: http://medicine.mytau.org/peled/

Y. R. Lawrence
Center for Translational Research in Radiation Oncology, Sheba Medical Center, Ramat-Gan, Israel
M. Ilouze : N. Peled
Thoracic Cancer Service, Davidoff Cancer Center, Rabin Medical Center, Petach Tikva, Israel

Correspondence: [email protected]

Accepted: 10 December 2014
Published: 7 January 2015

Abstract

Introduction: Lung cancer is the leading cause of cancer death. Radiation therapy plays a key role in its treatment. Ionizing radiation induces cell death through chromosomal aberrations, which trigger mitotic catastrophe and apoptosis. However, many lung cancer patients show resistance to radiation. Dichloroacetate (DCA) is a small molecule that can promote mitochondrial activation by increasing the influx of pyruvate. Here, we tested whether DCA may increase the sensitivity of non-small cell lung cancer (NSCLC) cells to radiation through this mechanism.

Methods: Two representative NSCLC cell lines (A549 and H1299) were tested for their sensitivity to radiation with and without pre-exposure to DCA. The treatment efficacy was evaluated using a clonogenic survival assay. An extracellular flux analyzer was used to assess the effect of DCA on cellular oxygen consumption as a surrogate marker for mitochondrial activity.

Results: We found that DCA increases the oxygen consumption rate in both A549 and H1299 cells by 60% (p = 0.0037) and 20% (p = 0.0039), respectively. Pre-exposure to DCA one hour before radiation increased the cytotoxic death rate 4-fold in A549 cells (55 to 13%, p = 0.004) and 2-fold in H1299 cells (35 to 17%, p = 0.28) respectively, compared to radiation alone.

Conclusion: Mitochondrial induction by DCA may serve as a radio-sensitizer in non-small cell lung cancer.

Keywords: NSCLC; DCA; Mitochondria; Radiation; Radio-sensitizer; Warburg effect

INTRODUCTION

Lung cancer is the leading cause of cancer-related deaths in the United States with an overall 5-year survival rate for all stages of ~17 % ^[1–4]. Radiation therapy (RT) plays an important role in the clinical management of lung cancer patients, particularly those with stage IIIB disease who are candidates for definitive chemo-radiotherapy. In addition, RT may be applied as a neo-adjuvant or adjuvant therapy in stage IIIA, or in an ablative manner when stereotactic body radiotherapy (SBRT) is applied. Radiation induced pneumonitis (RIP) is the limiting factor when treating patients with RT. In order to minimize RIP, oncologists aim to keep the V20 (i.e., the percentage of lung volume receiving a radiation dose of ≥20 Gy) below 22 % ^[5]. Radio-sensitizers may increase the cytotoxic efficacy of radiation, thereby potentially improving cure rates without increasing the V20.

RT kills cells by causing DNA double-strand breaks. Unrepaired DNA breaks result in chromosomal aberrations which, in turn, lead to “mitotic catastrophe” – a mode of cell death that results from the premature or inappropriate entry of cells into mitosis^[6,7]. Furthermore, radiation may directly affect cell membranes and organelles. Although these latter changes are as yet poorly understood, they may lead to changes in signal transduction, gene expression, protein stability, cellular redox states and cell cycle regulation, all of which may lead to apoptosis^[8]. Moreover, radiation may induce mitochondrial reactive oxygen species (ROS) production, accompanied by up-regulation of mitochondrial electron transport chain functions, thereby increasing mitochondrial membrane potential, mitochondrial respiration and mitochondrial ATP production ^[9,^10]. However, genetic alterations limit the capacity of cancer cells to undergo apoptosis, suggesting that hypoxic tumor microenvironments may provide a selective pressure towards an apoptosis-resistant cancer phenotype and, as a result, resistance to RT ^[11]. Because of this RT resistant phenotype and the significant side effects caused by RT itself, it is of a crucial importance to explore ways to radio-sensitize lung cancer cells in order to decrease radiation doses and to improve responses to therapy.

Many tumor cells exhibit elevated levels of glucose uptake and reduced levels of oxidative phosphorylation. This paradoxical hyper-glycolysis and lack of mitochondrial activity in the presence of oxygen is known as Warburg effect (depicted in Fig. 1) ^[12]. The unique metabolism of most solid tumors stems from remodeling mitochondrial functions to produce a glycolytic phenotype and a strong resistance to apoptosis ^[13]. There is a growing body of evidence indicating that mitochondria may be the primary targets for cancer therapeutics ^[14–17]. Cancer-specific remodeling can be reversed by a small molecule named dichloroacetate (DCA) ^[18], which inhibits pyruvate dehydrogenase kinase (PDK), thereby increasing the influx of pyruvate into the mitochondria and promoting its oxidation via mitochondrial activity over glycolysis in various cancer types (depicted in Fig. 1) ^[13,18]. An increase in mitochondrial activity causes an increase in both the amount and the extent of release of free radicals (ROS) in the cell, leading to an increase in cellular apoptotic activity^[13].

Figure 1. Schematic representation of the proposed effect of DCA on cancer cells exposed to radiation. The left panel depicts the Warburg effect: even in the presence of oxygen, cancer cell metabolism relies on glycolysis. This is accomplished by the intracellular expression of PDK which, in turn, inhibits the action of PDH. The result is inhibition of the influx of pyruvate to mitochondria and, thus, mitochondrial activity. In spite of DNA damage caused by radiation, cancer cells are unable to undergo efficient apoptosis since apoptotic modulators like reactive oxygen species (ROS) remain in the mitochondria. The right panel depicts the effect of DCA: DCA abolishes the inhibition of PDH by inhibiting PDK. Consequently, DCA increases the influx of pyruvate into the mitochondria and, thus, mitochondrial activity. In addition to DNA damage due to radiation, apoptotic modulators are released from the mitochondria and, hence, cancer cells undergo a higher rate of apoptosis

Here, we investigated the potential radio-sensitizing effect of DCA in NSCLC cells. Our results indicate that mitochondrial activation by DCA increases the sensitivity to radiation in both A549 and H1299 NSCLC-derived cells.

Materials and methods

Cell cultures
Two non-small cell lung cancer (NSCLC) cell lines were used in this study: A549, derived from a human adenocarcinoma and H1299, derived from a human large cell carcinoma. Both cell lines were purchased from the ATCC. The cells were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin/streptomycin and 1 % glutamate at 37 °C in a 5 % CO₂ humidified atmosphere.

Treatment conditions
Dichloroacetate (DCA, Sigma 34795) was added to culture media for 1 h prior to radiation at 10 and 20 mM in case of H1299 cells and 40 and 60 mM in case of A549 cells, according to the respective IC₅₀ values. Cells were radiated with a Kimtron Polaris irradiator at a dose rate of 0.9 Gy/min at room temperature.

Clonogenic survival assay
The clonogenic survival assay that we used has been previously described^[19]. Briefly, cells were detached with trypsin, suspended in complete culture media, counted and seeded in 3 ml dishes at a density of 200 cells per dish, and then left to attach and stabilize overnight. The next day, test groups were treated with DCA (or control) 1 h before radiation, at the indicated concentrations. Cells were radiated to doses of 0, 2, 4 and 6 Gy. Following incubation for 48 h, the culture media were removed and replaced with fresh culture media without DCA (all groups). Cells were incubated for another 10 days to allow colony formation. Next, colonies were fixed and stained with 0.5 % crystal violet in 96 % ethanol. Colonies containing at least 50 viable cells were counted (Fig. 2). Each experiment was performed in duplicate and the mean and standard deviation were calculated from 3 independent experiments.

Figure 2. Flow chart of the clonogenic survival assay

Synergy evaluation
In order to evaluate whether the treatment schedules show synergistic effects, plots of averages with standard deviations of survival rates relative to radiation doses were made. Survival percentages were calculated, both for groups treated and untreated with DCA, relative to those of unirradiated cells:

where:

Specifically, colony formation of both DCA-treated and DCA-untreated groups without radiation treatment were normalized to 100 %. Synergistic effects were determined by a two-way ANOVA (p value <0.05) [20].

Oxygen consumption assay
Oxygen consumption was used as a surrogate marker for mitochondrial activity. Measurements were carried out using an XF24 extracellular flux analyzer (Seahorse Biosciences). This device uses fluorescence-based optical sensors and custom multi-well plates to perform repeated oxygen consumption measurements of intact cells growing as monolayers. H1299 and A549 cells were seeded in Seahorse XF24 cell culture plates at 10,000 and 20,000 cells per well, respectively, in growth medium. Cells were incubated for 48 h at 37 °C in 5 % CO₂. Next, cells were washed and transferred to XF assay medium/PBS, and incubated for 60 min at 37 °C without CO₂ before starting the experiment. After establishing baseline oxygen consumption rates, 20 and 40 mM DCA were added to H1299 and A549 cells, respectively. Oxygen consumption measurements were continued for 25 min. Data were acquired from at least three replicate plates per cell line. Results are represented as the percent of the baseline respiration rate.

Statistical analysis
Statistical significance was evaluated using Student’s t-test. Synergy was evaluated by a two-way ANOVA^[20]. A value of p < 0.05 was considered significant.

Results

DCA radio-sensitizes NSCLC cells
Dichloroacetate (DCA) is known to increase mitochondrial activity ^[10,15] and has previously been shown to act as a radio-sensitizer in several cancer cell lines derived from prostate, colorectal and brain tumors ^[21, 22]. Here, its effect on two non-small cell lung cancer (NSCLC)-derived cell lines was investigated, i.e., A549 (adenocarcinoma) and H1299 (large cell carcinoma). The concentrations of DCA used for each cell line were in accordance with the respective IC₅₀ values (A549: 63 mM and H1299: 36 mM; Fig. 3a and b).

Figure 3. Clonogenic survival of NSCLC cells treated with increasing DCA consentrations. a A549 cells treated with 20, 40, 60 and 80 mM DCA. b H1299 cells treated with 10, 20, 30 and 50 mM DCA. c A549 cells treated with radiation doses of 2, 4, 6 Gy alone and combined with 40 and 60 mM DCA. d H1299 cells treated with radiation doses of 2, 4, 6 Gy alone and combined with 10 and 20 mM DCA. *p < 0.05 compared to control

We found that pre-radiation exposure with DCA increased cell death in both A549 and H1299 cell samples. The maximal effect in A549 cells was observed at 4 Gy, where DCA treatment (40 and 60 mM) decreased cell survival from ~55 to 40 % (p = 0.27) and 13 % (p = 0.004), respectively (Fig. 3c). In H1299 cells, the maximal effect was reached at 6 Gy. At this radiation dose, DCA treatment (10 and 20 mM) decreased cell survival from 35 % (radiation only) to 25 and 17 %, respectively (not statistically significant; Fig. 3d). Pre-exposure of A549 cells to DCA (60 mM) increased the cytotoxic effect of 4 Gy radiation by 4-fold compared to radiation alone. A two-way ANOVA analysis indicated a synergistic effect only in A549 cells.

DCA increases oxygen consumption in both A549 and H1299 cells
An enhanced cellular oxygen consumption rate (OCR) is an indication of increased mitochondrial activity ^[23]. We found that DCA increases the OCR in both A549 and H1299 cells by 60 % (p = 0.0037) and 20 %, (p = 0.0039), respectively, compared to the basal OCR (Fig. 4).

Figure 4. Oxygen consumption rate (OCR) in A549 and H1299 cells. DCA was added after ~16 min. a 20 mM DCA added to H1299 cells. b 40 mM DCA added to A549 cells. OCR changes were measured relative to basal states. DCA increased the oxygen consumption rate in A549 and H1299 cells by 60 % (p = 0.0037) and 20 % (p = 0.0039), respectively

Discussion

By testing the sensitivity to radiation with and without pre-exposure to dichloroacetate (DCA), we found that mitochondrial activation by DCA can radio-sensitize two distinct non-small cell lung cancer (NSCLC)-derived cell lines (i.e., A549 and H1299). In this proof of concept study, a wide range of DCA concentrations was used, aiming to tailor the dose to each cell’s IC₅₀ value. The synergistic effect of DCA was statistically significant in A549 cells, while only approaching significance in H1299 cells. Possibly, an even more robust effect can be obtained with higher DCA doses. We also showed that DCA increases oxygen consumption in both A549 and H1299 cells, which is a surrogate marker for increased mitochondrial activity.

Ionizing radiation up-regulates the mitochondrial electron transport chain function, and increases mitochondrial membrane potential and ATP production^[9]. Mitochondrial hyper-activation by DCA further increases the amount and the extent of the released free radicals (ROS) by the mitochondria itself ^[13]. These factors may explain the synergistic effect observed by pre-exposing the cells to DCA before starting radiation.

Atkinson et al. [15] showed that the inhibition of cytochrome c peroxidase and the release of cytochrome c from mitochondria mitigates radiation-induced cell death. Lee et al.^[24] observed a decrease in inhibition of ROS-mediated cell death by blocking the nuclear factor erythroid 2 (nrf2)-dependent anti-oxidant response, which increases the radio-sensitivity of H1299, A549 and H640 cells. These studies showed the ability to affect the cellular reaction to radiation by interfering with processes that are related to cytochrome c and ROS, thus underscoring our assumption that DCA radio-sensitizes cancer cells through mitochondrial activation that allows ROS release.

Cao et al.^[21] also reported that DCA acts as a radio-sensitizer in prostate cancer cells. Zwicker et al.^[22] found that DCA acts as a radio-sensitizer in vitro but not in vivo in WIDR (colorectal) and LN18 (glioma) cells. The lack of synergy in the in vivo WIDR xenograft model was explained by a buffer effect of the environment, which may have allowed the tumor cells to maintain their glycolytic metabolic program. Studies demonstrating the in vivo effect of DCA and radiation in NSCLC still remain to be performed.

RT is a common therapy for lung cancer patients. The considerable damage it causes to surrounding normal tissues, however, limits its application. During the last two decades pharmacologic inhibitors of signaling molecules that regulate apoptotic potential have shown promise as radio-sensitizers in vitro^[25,29]. Although DCA has been reported as a radio-sensitizer in prostate cancer^[21], its clinical use in NSCLC has so far not been proven. Here, we show the ability of DCA to decrease radiation doses in A549 and H1299 NSCLC cells without compromising the effect level. Furthermore, by showing that treatment with DCA increases the oxygen consumption of both A549 and H1299 cells, we have uncovered the potential of DCA to act as a mitochondrial activator. Therefore, we conclude that mitochondrial activation may play a role in the therapy of NSCLC when integrated with radiation treatment. Additionally, we hypothesize that induction of increased mitochondrial activity, i.e., increased release of free radicals and apoptosis by DCA, is the leading cause of DCA’s potential in radio-sensitizing NSCLC cells.

In summary, we show that in vitro mitochondrial induction by DCA significantly radio-sensitizes A549 NSCLC cells in a synergistic manner. This observation warrants further assessment in an in vivo setting.

Acknowledgments

The authors thank Dr. Shoshana Paglin (Sheba Medical Center) for guidance and fruitful discussions regarding this work.

This work was performed in partial fulfillment of the MD thesis requirements of the Sackler Faculty of Medicine, Tel Aviv University and was supported by Sheba Medical Center. (Mr. Ronen Shavit)

Conflict of interest

The authors declare that they have no conflict of interest.

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