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Enhanced killing of SCC17B human head and neck squamous cell carcinoma cells after photodynamic therapy plus fenretinide via the de novo sphingolipid biosynthesis pathway and apoptosis

Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.


Introduction
Photodynamic therapy (PDT), a clinically-approved cancer therapy, utilizes a photosensitizer, a light at a wavelength corresponding to the photosensitizer's absorbance, and molecular oxygen to produce reactive oxygen species in malignant cellular targets, and ultimately causing their destruction (1). Apoptosis is an important mechanism for eliminating tumors (2)(3)(4)(5)(6), and the effectiveness of PDT regimens correlates with tumor cell apoptosis (7). Because PDT itself is not always effective as a tumor treatment (8,9), PDT is combined with other anticancer agents for improved therapeutic benefit. 4HPR [N-(4-hydroxyphenyl) retinamide; fenretinide], a proapoptotic, clinically-relevant anticancer synthetic retinoid (10), is a potential candidate for combined treatment.
As others and we have shown, sphingolipids (SLs), e.g., ceramide, generated via the de novo SL biosynthetic pathway ( Fig. 1), have been implicated in apoptotic cell death after PDT and 4HPR in various malignant cell lines (11)(12)(13)(14). Ceramide synthase catalyzes a reaction in the de novo SL biosynthesis pathway, in which a fatty acyl group is added to dihydrosphingosine to form dihydroceramide. Ceramide is formed in the subsequent desaturase-dependent reaction, which can be inhibited by 4HPR (15). The ceramide synthase inhibitor fumonisin B1 (FB) renders cells resistant to apoptosis after PDT and 4HPR (14,16).
There have been no reports on combining PDT with 4HPR for improving the efficacy of PDT. The objective of the present study was to test the hypothesis that combining PDT with 4HPR enhances cell killing via apoptosis and the de novo SL biosynthesis pathway. We used SCC17B cells, a human head and neck squamous cell carcinoma cell line, representing a model that is potentially PDT-treatable in the clinic (17).
Cell culture and treatments. SCC17B cells were obtained from Dr Thomas Carey (University of Michigan, Ann Arbor, MI, USA). Cells were grown in DMEM/F-12 medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37˚C and 5% CO 2 . For all experiments, unless indicated otherwise, incubation of cells was carried out in a humidified incubator at 37˚C and 5% CO 2 . All treatments, as well as staining with Mitotracker Red CMXRos (see below) were added to cells in growth medium. After overnight incubation with Pc4 (20 nM), 4HPR (2.5 µM) was added immediately prior to irradiation. Cells were irradiated at room temperature with red light (2 mW/cm 2 ; λ max ~670 nm) using a light-emitting diode array light source (EFOS, Mississauga, ON, Canada) at the fluence of 200 mJ/cm 2 and incubated for 10 h. Phosphate-buffered saline (PBS) without calcium and magnesium was used for confocal microscopy. PBS containing calcium and magnesium was used for mass spectrometry (MS). Both types of PBS were purchased from Life Technologies.
Clonogenic assay. Cell survival was assessed using clonogenic assay according to the modified pre-plating protocol, as we have described (18). Cells were resuspended in growth medium containing Pc4 (20 nM) and seeded (250 cells/well) in a 6-well plate (Thermo Fisher Scientific). After overnight incubation, the cells were irradiated. 4HPR was added immediately prior to irradiation. The inhibitors FB, zVAD-fmk (zVAD) and ABT-199 (ABT) were added 1 h prior to PDT±4HPR. After 14 days of incubation, the medium was aspirated, the plates were stained with crystal violet (0.1% in 20% ethanol; Sigma-Aldrich) for 30 sec, rinsed with water and air-dried. Colonies (≥50 cells) were counted using eCount Colony Counter (VWR International, Radnor, PA, USA). Plating efficiency was 36% (n=16).
Quantitative confocal microscopy. Cells were grown on coverslips (Thermo Fisher Scientific) in 6-well plates (Thermo Fisher Scientific). To visualize mitochondria, treated cells were incubated with Mitotracker Red CMXRos (250 nM; Life Technologies) in growth medium for 30 min. After treatments, the coverslips were washed with cold PBS, and fixed by incubation for 15 min in 4% formaldehyde (Thermo Fisher Scientific) in PBS. After washing with PBS, cells were permeabilized with ice-cold acetone/methanol (1:1) for 10 min. After blocking with 3% goat serum and 3% fetal bovine serum in PBS for 1 h at 4˚C, cells were incubated at room temperature for 45 min with mouse monoclonal anti-dihydroceramide/ceramide antibodies  microscopy imaging was performed at the Microscopy, Imaging and Cytometry Resources Core at Wayne State University, School of Medicine.
Quantifications were carried out as follows: dihydroceramide/ceramide fluorescence associated with the ER and the mitochondria, as well as Bax fluorescence associated with the mitochondria, were all quantified with MetaXpress software (version 5 5.00.20; Molecular Devices, LLC; Sunnyvale, CA, USA) (19). Mitotracker or anti-KDEL antibodies were employed to demarcate the mitochondria or the ER. Dihydroceramide/ceramide or Bax-pixel intensities associated with these subcellular compartments were measured with the multiwavelength cell scoring module. The correct compartment identification was verified by visual inspection. All images were corrected for background contribution before quantification. For Bax associated with mitochondria a minimum of 488 regions were measured for each data point. For dihydroceramide/ceramide associated with the ER and mitochondria a minimum of 680 or 518 regions were measured for each data point, respectively. Significant differences (p<0.05) were determined using comparison of multiple samples by one-way ANOVA.
Electrospray ionization/double mass spectrometry (MS) analysis. After treatments, cells were collected on ice, washed twice with cold PBS, resuspended in a mixture of ethyl acetate/ methanol (1:1, v/v; EMD Millipore, Billerica, MA, USA), dried under nitrogen in the N-EVAP analytical evaporator (Organomation; Berlin, MA, USA), and shipped overnight on dry ice to the Lipidomics Shared Resource Facility (Medical University of South Carolina, Charleston, SC, USA) for further processing. After extraction, SLs were separated by high performance liquid chromatography, introduced to the electrospray ionization source and then analyzed by double MS using TSQ 7000 triple quadrupole mass spectrometer (Thermo-Fisher Scientific) as described previously (20). The pmoles of SLs were normalized per mg protein. Protein content was determined by a modified Bradford assay per manufacturer's instructions (Bio-Rad, Hercules, CA, USA).
Statistical analysis. Significant differences (p<0.05) were determined using Student's t-test or one-way ANOVA.

Results
Enhanced cell killing after PDT+4HPR is FB-, zVAD-fmkand ABT-199-sensitive. Clonogenic assay was used to test whether combining PDT with 4HPR sensitizes SCC17B cells to PDT. As shown in Table I, when PDT and 4HPR were used at LD20 each, i.e., the dose reducing survival by 20%, 63% of PDT+4HPR-treated cells were unable to form colonies. To determine whether apoptosis and ceramide synthase are necessary for enhanced cell killing after PDT+4HPR, we used the pancaspase inhibitor zVAD-fmk (zVAD), the Bcl2 inhibitor ABT-199 (ABT), and the ceramide synthase inhibitor FB (21)(22)(23). We have previously shown that FB and zVAD rendered cells resistant to PDT (16). In contrast, ABT sensitized cells to PDT (16). Apoptosis is a major pathway involved in the anticancer action of 4HPR (24) and the de novo SL biosynthesis pathway plays a role in 4HPR-induced apoptosis (14,25). As shown in Table I, all inhibitors were non-toxic (LD<5). FB and zVAD rendered the cells resistant not only to PDT and 4HPR alone, but also to PDT+4HPR. In contrast, ABT sensitized SCC17B cells to PDT±4HPR.

PDT+4HPR-enhanced Bax associated with mitochondria and cyt c redistribution is inhibited by FB.
The mitochondrial apoptosis pathway, including Bax translocation to mitochondria and cyt c redistribution/release, is induced by PDT and 4HPR (16,(26)(27)(28). Both of these processes are inhibited by FB after PDT (16). The question is whether the mitochondrial apoptosis pathway is affected by combining PDT with 4HPR, and whether the process is ceramide synthase-dependent. Using quantitative confocal microscopy we found that Bax associated with mitochondria and cyt c redistribution were induced after each individual treatment (Fig. 2). PDT+4HPR enhanced Bax associated with mitochondria and cyt c redistribution, and FB inhibited both processes.
PDT+4HPR enhances C16-dihydroceramide, not ceramide, accumulation. Both PDT and 4HPR regulate the de novo SL biosynthesis pathway (12)(13)(14)(15)(16)29). The question is what the effects of combining PDT with 4HPR on the cellular SL profile are. We used MS to address the question. As depicted in Fig. 3A, in contrast to 4HPR, PDT increased total ceramide accumulation. Combining PDT with 4HPR attenuated PDT-induced increase in total ceramide levels. The accumulation of individual ceramides, by and large, followed the same pattern (Table II). 4HPR did not significantly raise the levels of any individual ceramide. In contrast, 4HPR increased accumulation of C16-dihydroceramide, a de novo SL biosynthesis pathway metabolite by 445% above basal levels (Fig. 3B). PDT also increased the levels of C16-dihydroceramide by 138% beyond resting levels. Combining PDT with 4HPR enhanced accumulation of C16-dihydroceramide by 632%.

PDT+4HPR-induced enhanced ceramide accumulation in the ER is inhibited by FB.
Because the de novo SL biosynthesis pathway is localized to the ER, we tested the effect of combining PDT with 4HPR on dihydroceramide/ceramide accumulation in the ER. Using an antibody that recognizes dihydroceramide and ceramide (30) and the ER marker anti-KDEL for quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER (Fig. 4A and B). FB inhibited ER-associated ceramide accumulation after PDT±4HPR. We have shown that PDT-induced mitochondrial dihydroceramide/ceramide accumulation is FB-sensitive (16). However, it is unknown what impact on mitochondrial dihydroceramide/ ceramide accumulation combining PDT with 4HPR might have. Using the same anti-dihydroceramide/ceramide antibody and the mitochondrial marker Mitotracker for quantitative confocal microscopy, we found that PDT and 4HPR alone did induce mitochondrial dihydroceramide/ceramide accumulation (Fig. 4C). However, the effect was not enhanced after PDT+4HPR. FB inhibited mitochondrial ceramide accumulation after all the treatments.

Discussion
The present study is novel because, to our knowledge, no report exists on combining PDT with 4HPR. The results suggest that PDT+4HPR-induced enhanced killing of SCC17B cells depends on ceramide synthase, caspase activation and inhibition of Bcl2, and is associated with ceramide synthase-dependent mitochondrial apoptosis pathway. We have reported similar findings after PDT alone (16). In contrast, in breast cancer cells FB did not affect cell death after 4HPR (14). The discrepancy between the findings could be due to the use of different assays, i.e. clonogenic vs. short-term viability assay, respectively (31), or cell type-specificity of FB-induced resistance.
Our observation that 4HPR increased the levels of C16-dihydroceramide, the substrate of desaturase in the de novo SL biosynthesis pathway (Fig. 1), is consistent with the report showing 4HPR-induced inhibition of the enzyme (15). Our present findings that PDT-induced increased ceramides and C16-dihydroceramide in cells undergoing apoptosis are similar to our previous results in SCCVII mouse squamous carcinoma cells (18,32). Here we also demonstrate that combining PDT with 4HPR enhances C16-dihydroceramide levels, apoptosis and loss of clonogenicity. In addition, total ceramide levels, as well as most of the individual ceramides  remained above baseline levels after PDT+4HPR. These data support the notion that dihydroceramide, together with ceramide, has a pro-apoptotic/pro-death role. We demonstrate in the present report that PDT+4HPRinduced enhanced killing of SCC17B cells is associated with ER-localized and FB-sensitive de novo SL biosynthesis. This is in accordance with previous findings for PDT and 4HPR itself (15,16,29). Although each treatment alone increased mitochondrial dihydroceramide/ceramide accumulation in a FB-sensitive manner, combining PDT with 4HPR did not affect the process. The ER and mitochondria are connected by the mitochondria-associated membrane. Ceramide synthase activation has been shown in mitochondria-associated membrane after radiation (33). Reportedly, 4HPR can activate ceramide synthase (29). It remains to be established whether and at what subcellular site ceramide synthase is activated after PDT+4HPR.
We believe that our findings have important clinical implications. Combining PDT with another anticancer agent is advantageous because lower doses of individual treatments (LD20 ; Table I) are required for improved therapeutic benefit. This study also shows that combining PDT with 4HPR (C) Incubation with Mitotracker Red CMXRos was carried out prior to immunostaining with anti-ceramide antibodies. Nuclei were visualized with DAPI. All images were acquired by confocal microscopy with identical settings. MetaXpress software was used to quantify ceramide fluorescence located in the ER (B) and mitochondria (C). (B and C) Data are shown as the average ± SEM. The graphs depict ceramide fluorescence/ER area (B), or ceramide fluorescence/mitochondrial area (C). Results were normalized to the untreated control. Significant differences are shown between: † treatment and untreated control; # (treatment + inhibitor) and treatment; * combination and individual treatments. Con, untreated control. enhances cell killing via the de novo SL biosynthesis and mitochondrial apoptotic pathway. Thus, targeting these pathways could augment the therapeutic value of PDT.