The phthalocyanine photosensitizer Pc 4, HOSiPcOSi(CH3)₂(CH2)₃N(CH₃)₂, was supplied by Dr Malcolm E. Kenney (Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA). N-[9,10-³H]D-e-C16-ceramide was synthesized at the Lipidomics Shared Resource (Medical University of South Carolina, Charleston, SC, USA). RPMI medium and serum were from Life Technologies (Carlsbad, CA, USA) and Hyclone (Logan, UT, USA), respectively. The inhibitors zVAD-fmk and dasatinib (BMS-354825) were from MBL International (Woburn, MA, USA) and Selleck Chemicals (Houston, TX, USA), respectively.

SCCVII cells, initially derived from the spontaneous abdominal wall tumor of a C3H mouse (15), were grown in RPMI medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Cells were maintained at 37°C in a 5% CO₂ atmosphere and were treated in the growth medium. For PDT experiments, after overnight incubation with Pc 4 at 37°C, cells were irradiated with red light (2 mW/cm²; λ_max ∼670 nm) using a light-emitting diode array light source (EFOS, Mississauga, ON, Canada) at the fluence of 200 mJ/cm² at room temperature and then incubated at 37°C for indicated periods of time. For PDT + dasatinib, dasatinib was added to the cells 22 h prior to irradiation, unless indicated otherwise. After treatments, cells were collected on ice and processed for various analyses. For mass spectroscopy (MS) analysis, cells were washed twice with cold phosphate-buffered saline (Corning Life Sciences, New York, NY, USA), resuspended in the mixture of ethyl acetate/methanol (1:1, v/v; EMD Chemicals, Billercia, MA, USA), dried under nitrogen and shipped overnight on dry ice to the Lipidomics Shared Resource (Medical University of South Carolina, SC, USA) for further processing.

After extraction, sphingolipids were separated by high performance liquid chromatography, introduced to electrospray ionization source and then analyzed by double MS using TSQ Quantum Access Max triple stage quadrupole mass spectrometer (Thermo-Fisher Scientific, Pittsburg, PA, USA) as described previously (16).

Total RNA isolation was performed with RNeasy^® Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of the total RNA using iScript™ cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). The concentration and quality of total RNA preparations were evaluated spectrophotometrically. RT-PCR was performed on a Bio-Rad CFX96 detection system using Bio-Rad SsoFast Probes Supermix™ and TaqMan^® Gene Expression Assays (Life Technologies) with the primers for ceramide synthases 1, 2, 4, 5, 6, the housekeeping gene products RPL37A and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and the fluorophore probe FAM-490 (6-carboxyflurescein; all obtained from Life Technologies). Initial steps of RT-PCR were 30 sec at 85°C, followed by 40 cycles consisting of a 5 sec at 95°C, followed by 10 sec at 60°C. Determination of the relative normalized expression of corresponding ceramide synthase mRNAs against the expression of housekeeping gene-encoded proteins RPL37A and HGPRT was performed by ΔΔC_T provided by CFX96 manager software 3.0 from Bio-Rad.

Acid ceramidase activity was performed as described previously (17). Cells were lysed under acidic condition (pH 4.5). Equal amounts of N-[9,10-³H] D-e-C16-ceramide were mixed with 0.2% Triton X-100 and 0.4% cholate and dried down under nitrogen. The lipid film was dissolved by mixing and sonication in deionized water. After additions of acidic assay buffer (0.2 M acetic acid, 0.2 M sodium acetate and 0.5% Triton X-100, pH 4.5) and cell lysate, the reaction was carried at 37°C for 1 h and stopped by adding Dole’s alkaline solution. [³H]palmitic acid, a hydrolytic product of acid ceramidase, was extracted and processed to calculate the enzyme activity (17). Quantitation of radioactivity was performed using LS 6500 multipurpose scintillation counter (Beckman Coulter, Brea, CA, USA).

As described previously (18), DEVDase activity was determined in the cytosol by an assay based on the enzyme’s cleavage of a fluorogenic derivative of the tetrapeptide substrate N-acetyl-Asp-Glu-Val-Asp (DEVD; Enzo Life Sciences, Farmingdale, NY, USA). The peptide sequence is based on the cleavage site Asp²¹⁶ of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). The fluorescence of the cleaved DEVD substrate was measured using a spectrofluorometer (F-2500 Hitachi, New York, NY, USA; 380 nm excitation, 460 nm emission).

The lipophilic cationic dye JC-1 (5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethylbenzimidazolylcarbocyanine iodide; BD Biosciences, San Diego, CA, USA) was used to determine mitochondrial membrane potential by flow cytometry, as we described previously (11,19,20). After treatments, cells were harvested and processed for flow cytometry according to the manufacturer’s instructions (BD Biosciences). BD LSR II flow cytometer was used for analysis (BD Biosciences).

As previously described (11,20,21), to detect apoptosis, the exposure of phosphatidylserine in the outer leaflet of the cell membrane and cell membrane integrity loss were measured using Annexin V and DNA-binding propidium iodide fluorescent dyes (BD Biosciences), respectively. Early apoptotic (Annexin V⁺/propidium iodide⁻) were distinguished from late apoptotic or necrotic cells (Annexin V⁺/propidium iodide⁺). The kit was obtained from BD Biosciences and the flow cytometric protocol was followed, as described by the manufacturer.

Long-term cell viability was assessed using clonogenic assay according to the modified protocol that we described previously (20). Plating density was 250 cells/plate. Plating efficiency was 34% (n=16).

Protein content was determined by a modified Bradford assay (Bio-Rad) or, for acid ceramidase assay, by a bicinchoninic acid protein assay kit (Thermo-Fisher Scientific).

Data are shown as the mean ± SEM. Statistical analyses were performed by Student’s t-test. Significance was defined as a two-tailed p<0.05.

To test whether killing of SCCVII cells is increased by the combination of Pc 4-PDT with dasatinib, colony formation assay was used as the experimental end-point. The treatments were first used as single agents to determine whether they induce dose-dependent cell killing. Incubation of SCCVII cells with 100, 200 and 500 nM dasatinib led to 2, 28 and 53% cell killing, respectively (Fig. 2A). Similarly, PDT with 100, 250 or 500 nM Pc 4, at the light fluence of 200 mJ/cm², induced 16, 26 and 87% cell killing, respectively (Fig. 2A). Treatment of cells with the combination of PDT and dasatinib, each used at LD <30, led to 72% cell killing, which was significantly greater than that of each treatment alone (Fig. 2B). Because both agents are apoptotic inducers (11,12,22–26), the requirement of caspases in cell killing by each agent and the combination was assessed using the pan caspase inhibitor zVAD-fmk. As shown in Fig. 2B, zVAD-fmk substantially inhibited cell killing after dasatinib, but not after either PDT alone or the combination. Overall, the data demonstrate that each agent induces dose-dependent cell killing, the combination enhances cell killing, and that, unlike PDT alone or the combination, dasatinib induces zVAD-fmk-dependent cell killing.

Because zVAD-fmk inhibits effector caspases, including caspase-3 (27), we verified caspase-3 as a zVAD-fmk target in dasatinib-induced cell death using DEVDase assay. Caspase-3 activation began at 2 h and peaked at 24 h after dasatinib (not shown). As depicted in Fig. 3A, dasatinib induced activation of DEVDase was abolished by zVAD-fmk. The data suggest that zVAD-fmk-sensitive cell killing after dasatinib involves caspase-3.

To further assess induction of apoptosis after treatments, caspase-3 activation, mitochondrial depolarization and the appearance of Annexin V⁺ and propidium iodide+ cells were determined. PDT induced a dose-dependent activation of caspase-3 and the effect was potentiated after PDT + dasatinib (Fig. 3B). PDT alone induced mitochondrial depolarization and the effect was inhibited after the combination (Fig. 3C). Annexin V⁺ and/or propidium iodide+ cells remained at control levels after treatments (Fig. 3D). We validated that SCCVII cells display depolarized mitochondria and undergo apoptosis using camptothecin as a positive control (Fig. 3C and D). The results show that the combined treatment augments PDT or dasatinib-induced caspase-3 activation in the absence of appearance of other apoptotic markers.

Dasatinib upregulates expression levels of ceramide synthase genes 1, 2, 5 and 6 (13). We showed that knockdown of ceramide synthase 1 or 6 leads to apoptotic resistance to PDT (11,12). To test whether ceramide synthases are affected by treatments, mRNA levels of ceramide synthases 1, 2 4, 5 and 6 were measured using RT-PCR. As depicted in Fig. 4A, ceramide synthase 1 mRNA levels were upregulated after dasatinib and the effect was further increased after PDT + dasatinib. PDT alone did not significantly increase ceramide synthase 1 levels. None of the treatments had any effect on mRNA levels of ceramide synthase 2, 4, 5 and 6 (Fig. 4A). The data show that dasatinib-induced upregulation of ceramide synthase 1 mRNA levels is enhanced after the combination.

To assess whether enhanced upregulation of ceramide synthase 1 mRNA after PDT + dasatinib is associated with increased ceramide production, ceramide levels were measured using MS. Dasatinib alone induced a modest increase in C20:1- and C22-ceramide but had no effect on total ceramide levels (Table I and Fig. 4B). PDT alone increased the levels of all 12 individual ceramides that were measured, as well as total ceramides. PDT-induced increases in ceramide levels were not, for the most part, further changed after the combination, with the exception of attenuated levels of C26- and C26:1-ceramide after PDT + dasatinib compared to individual treatments (Table I and Fig. 4B).

The effects of treatments on other sphingolipids were also measured by MS and are shown in Table I. Unlike dasatinib, and irrespective of the presence of dasatinib, PDT increased the levels of C16-dihydroceramide, an intermediate from the de novo sphingolipid biosynthesis pathway. Moreover, unlike dasatinib, and irrespective of the presence of dasatinib, PDT induced an 87% decrease in sphingosine levels. PDT also induced a 62% decrease in S1P levels, and the effect was not changed after the combination. Overall, the data show that in SCCVII cells PDT induced substantial changes in the sphingolipid profile that were not modulated by the addition of dasatinib.

The substantial decrease in sphingosine levels after PDT could result from inhibition of ceramidase, a sphingosine-producing enzyme (Fig. 1). The inhibition of acid ceramidase has been implicated in radiosensitization of prostate cancer cells (28). We measured the activity of acid ceramidase in cell lysates from untreated and PDT-treated cells. As depicted in Fig. 4C, acid ceramidase activity was reduced by 52% post-PDT. Thus, PDT-induced decrease in sphingosine correlated with inhibition of acid ceramidase.