The substrate ALA hydrochloride was purchased from Cosmo Oil Co., Ltd. (Tokyo, Japan). RPMI-1640 medium and antibiotic-antimycotic mixture (ABAM; penicillin-streptomycin-amphotericin B) were obtained from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS) was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were of analytical grade.

The human lung carcinoma cell line A549 was purchased from Riken BRC Cell Bank (Tsukuba, Ibaraki, Japan). Cells were grown in RPMI-1640 medium supplemented with 10% FBS and ABAM at 37°C in an incubator with a controlled humidified atmosphere composed of 5% CO₂.

The A549 cells (2×10⁴ cells) were incubated at 37°C in RPMI-1640 medium containing ALA, with or without 20 μM of the caspase inhibitor Z-VAD-FMK (Enzo Life Sciences, Farmingdale, NJ, USA), under 5% CO₂ for 72 h in the dark to avoid photocytotoxicity from the generated porphyrins. After 72 h, the number of living cells was determined by the trypan blue dye exclusion assay.

Confluent cultures were washed with ice-cold PBS (−) and then treated with lysis buffer A (50 mM Tris-HCl pH 7.4, 1 mM DTT, 1% v/v Triton X-100) containing a protease inhibitor cocktail (Nacalai Tesque). The preparation was homogenized by 10 passages through a 27-G needle. The homogenate was centrifuged at 1,000 × g for 10 min at 4°C, and the resulting supernatant was collected as the cell lysate. Protein concentration of the supernatant was determined by the Bradford assay (Bio-Rad Laboratories, Richmond, CA, USA) using bovine serum albumin as a standard.

SDS-PAGE and western blot analysis were performed as described previously (11), with some modifications. In brief, the cell lysate was mixed with SDS-PAGE sample buffer containing 10% v/v 2-mercaptoethanol (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), and proteins were electrophoretically separated on a 15% polyacrylamide gel. The gel was electroblotted onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membrane was incubated in blocking solution containing 5% w/v skim milk in TTBS (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% v/v Tween 20) at 4°C overnight. The specific antibodies used were: human anti-heme oxygenase-1 (HO-1) monoclonal antibody (1:200; Abcam, Cambridge, UK), human catalase monoclonal antibody, human manganese superoxide dismutase (MnSOD) polyclonal antibody, human caspase-3 monoclonal antibody and human COX IV-1 monoclonal antibody (all at 1:200 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA). Human GAPDH monoclonal antibody (1:1,000; American Research Products Inc., Waltham, MA, USA) was used as an internal standard. The secondary antibody used was anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (1:3,000; Cell Signaling Technology Inc., Danvers, MA, USA). HRP-dependent luminescence was developed using Western Lightning™ Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences Inc., Waltham, MA, USA) and detected using the lumino-image analyzer ImageQuant LAS-4000 mini (GE Healthcare Life Sciences UK, Little Chalfont, UK). Chemiluminescence intensity was quantified using an ImageQuant™ TL Analysis Toolbox (GE Healthcare Life Sciences).

Dissolved oxygen concentrations in cultured medium were determined using a 24-channel SDR SensorDish^® Reader (PreSens Precision Sensing GmbH, Regensburg, Germany), with some modifications (12). In brief, A549 cells were seeded on a 24-well OxoDish^® (5×10⁴ cells/well; PreSens Precision Sensing GmbH) and cultured at 37°C under 5% CO₂ for 16 h. Next, the incubation medium was replaced with 1,000 μl of fresh medium covered with 800 μl of liquid paraffin as a gas barrier. The cells were cultured at 37°C under atmosphere, and measurements of dissolved oxygen concentrations were performed for 24 h at 1-h intervals.

Since glycolysis causes lactic acidosis in vivo and a decrease in pH of culture medium in vitro, the impact of ALA on the glycolytic pathway was measured by monitoring culture medium pH using the 24-channel SDR SensorDish Reader (12). In brief, A549 cells were seeded on a 24-well HydroDish^® (5×10⁴ cells/well, PreSens Precision Sensing GmbH) and cultured at 37°C under 5% CO₂ for 16 h. Next, the incubation medium was replaced with 800 μl of fresh buffer-free RPMI-1640 medium without NaHCO₃, covered with 800 μl of liquid paraffin as a gas barrier. The cells were cultured at 37°C under atmosphere, and pH measurements were performed for 24 h at 1-h intervals.

Intracellular superoxide anion radical (O₂^•−) levels were detected using a Diogenes Cellular Luminescence Enhancement System (National Diagnostics, Somerville, NJ, USA) as described previously (13), with some modifications. The Diogenes kit is a supersensitive version of the traditional luminol assay that measures oxidation levels directly proportional to chemiluminescence. Cells seeded on a 96-well plate (3×10⁴ cells/well) were cultured overnight at 37°C under 5% CO₂. Next, the incubation medium was replaced with fresh medium containing ALA. The cells were further incubated in the dark for 2 or 4 h, and the medium was replaced with 100 μl of Diogenes reagent. Following incubation at 37°C for 10 min, the luminescence level of the reagent was measured using a luminescence microplate reader (LMax II; Molecular Devices, Sunnyvale, CA, USA).

The enzymatic assay was performed using a Caspase-3 Colorimetric Assay kit (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) as described previously (14), with some modifications. In brief, A549 cells (2×10⁶ cells) were lysed with 250 ml of lysis buffer on ice for 10 min. Following centrifugation (10,000 × g, 10 min, 4°C), the supernatant was collected for the caspase-3 assay. A total of 100 μg of protein was diluted in 50 μl of cell lysis buffer and mixed with 50 μl of 2X reaction buffer (containing 10 mM DTT) and 5 μl of substrate (10 mM Asp-Glu-Val-Asp-p-nitroanilide; DEVD-pNA). Following incubation at 37°C for 24 h, absorbance was read at 405 nm using the microtiter plate reader Multiskan^® FC (Thermo Fisher Scientific, San Jose, CA, USA).

The impact of ALA on the survival of cancer cells was tested using A549 cells. A significant decrease in cell survival was detected following a 72-h exposure to ALA (Fig. 1). Since all experiments were performed in complete darkness, cell death was not caused by photocytotoxicity of the porphyrins generated from ALA. By contrast, the caspase inhibitor Z-VAD-FMK prevented cell death, thereby supporting a mechanism of ALA-mediated apoptosis in cancer cells.

To investigate the effect of ALA on the Warburg effect, key elements of oxidative phosphorylation and glycolysis were monitored in A549 cells pre- and post-exposure to ALA. First, ALA induced an increase in COX protein expression (Fig. 2), supporting a stimulating effect on oxidative phosphorylation. This result is consistent with the result from our previous study, where COX expression in mouse liver was increased following ALA administration (8). In addition, oxygen consumption was significantly higher following ALA exposure (Fig. 3A). Furthermore, the oxygen consumption ratio induced by ALA was higher than the ratio induced by DCA, which is a common enhancer of oxidative phosphorylation. Taken together, these data suggest that the increase in the oxygen consumption level measured in A549 cells following ALA exposure requires the induction of key proteins supporting oxidative phosphorylation, including COX.

Since glycolysis causes acidosis, the impact of ALA on this pathway was determined by monitoring the pH of the A549 cell culture medium during a 24-h incubation period (Fig. 3B). In control cultures, the rate of pH decay was typical of cancer cells. The presence of ALA prevented any significant decrease in pH during the 24-h period, suggesting that the compound suppressed glycolysis in A549 cancer cells.

The impact of ALA on oxidative phosphorylation was additionally tested in A549 cells with respect to the generation of ROS, namely O₂^•−. Fig. 4 shows that ALA significantly increased O₂^•−generation over 4 h. It is well known that ROS generation stimulates the expression of free radical scavenging enzymes. Western blot analysis (Fig. 5) shows that ALA stimulates MnSOD, catalase and HO-1 protein expression. Altogether, these data show that ALA-mediated ROS generation was sufficient to recruit additional scavenger enzymes to protect cancer cells against oxidative stress-mediated apoptosis. Therefore, ALA significantly stimulated the oxidative phosphorylation pathway in A549 cancer cells.

Since ROS generation induces apoptosis in cancer cells (15), experiments were performed to test whether ALA induces apoptosis in A549 cells. Activated caspase-3 is a key player in the initiation of apoptosis. Fig. 6A shows that ALA induced an increase in the protein expression of activated (cleaved) caspase-3. The functional overexpression of active caspase-3 was demonstrated by enhanced cleavage of DEVD-pNA (Fig. 6B) in the presence of ALA. Therefore, ALA increases the caspase-3 activity level in cancer cells. Furthermore, the inhibitor of caspase-dependent apoptosis, Z-VAD-FMK, prevented ALA-mediated cell death (Fig. 7). Altogether, these data demonstrate that ALA induced caspase-dependent apoptosis in A549 cells.