Abstract
Background/Aim: Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival. Materials and Methods: Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry. Results: Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids. Conclusion: Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies.
Although FoscanPDT alone was effective in treating early HNSCC, it was not as successful for recurrent HNSCC (1). Thus, there is a need to combine PDT with other anticancer agents for the purpose of achieving synergism of the anticancer benefits of each, with minimal toxicity. A good candidate for the combined treatment with FoscanPDT is HPR, an FDA-approved anticancer treatment, with minimal toxicity to normal cells (2, 3). We showed that in SCCVII squamous cell carcinoma tumors grown in syngeneic mice, FoscanPDT+HPR improved the therapeutic outcome (4). Using the same in vivo model we showed that combining FoscanPDT with LCL29, significantly enhanced tumor cures (5, 6). These agents affect the levels of sphingolipids, which can also act as anticancer agents. We and others showed that the de novo sphingolipid biosynthesis pathway is associated with apoptosis after exposure to stressors, including PDT and HPR, in HNSCC cells (7-11). The de novo sphingolipid biosynthesis pathway includes a ceramide synthase-dependent conversion of dihydrosphingosine to dihydroceramide, which is then converted, in a desaturase-dependent reaction, to ceramide (Figure 1). Desaturase 1, the ubiquitous isoform of desaturase, is inhibited by HPR (12). Dihydroceramide can be catabolized to dihydrosphingosine via ceramidase (13), and dihydrosphingosine can be phosphorylated by sphingosine kinase to dihydrosphingosine-1-phosphate (14). We showed that treatment of HNSCC cells with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide and caspase-3 activation (4). The ceramide synthase inhibitor FB (15) significantly reduced caspase-3 activation and the colony-forming ability of HNSCC cells after FoscanPDT±HPR (4).
Both FoscanPDT and HPR can induce cancer cell death via ER stress (16, 17). Cancer cells appear to be more susceptible to increased levels of ER stress than their normal counterparts (18). ER stress, triggered by stress and an imbalance between the load of unfolded or misfolded proteins inside the ER lumen and the capacity of the ER, results in the unfolded protein response, i.e. the build up of unfolded or misfolded proteins in the ER (19). The unfolded protein response involves the induction of three signaling pathways: the double-stranded RNA-activated protein kinase-like ER kinase (PERK)-eukaryotic translation initiation factor 2 alpha (eIF2α), activating transcription factor 6 (ATF6), and the inositol-requiring enzyme 1 (IRE1)-X-box binding protein 1 (XBP1) (20). These signaling pathways counteract stress by inducing the expression of ER chaperones, binding immunoglobulin protein (BiP), and other factors, that together promote the protein folding machinery (21). If the unfolded protein response fails to control the levels of unfolded and misfolded proteins in the ER, ER-induced apoptosis is triggered through activation of the pro-apoptotic factor CCAAT/enhancer-binding protein homologous protein (CHOP) (22).
In HNSCC cells the ER stress-mediated apoptosis involves the de novo sphingolipid biosynthesis pathway-associated ceramide synthase (11). It is not known, however, whether the ER stress-associated apoptosis is induced after combining FoscanPDT with HPR or LCL29, and whether sphingolipids modulate the pathway. In the present study we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival.
Materials and Methods
Materials. The photosensitizer Foscan (m-tetrahydroxyphenylchlorin, Biolitec AG, Edinburgh, UK) was dissolved (2 mg/ml) in a mixture of ethanol:polyethyleneglycol200:water (2/3/5, v/v). C6-pyridinium ceramide or LCL29 [D-erythro-2-N-[6’-1”-pyridinium-hexanoyl sphingosine bromide] was obtained from Avanti Polar Lipids (Alabaster, AL, USA). DMEM/F-12 medium was obtained from Thermo-Fisher Scientific (Waltham, MA, USA). Fetal bovine serum, HPR [N-(4-hydroxyphenyl) retinamide], L-histidine, and 4-phenylbutyrate were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Fumonisin B1 and zVAD-fmk were from Cayman Chemicals (Ann Arbor, MI, USA) and MBL International Corporation (Woburn, MA, USA), respectively. 4-mu8c, salubrinal and thapsigargin were all obtained from EMD Millipore (Billerica, MA, USA).
De novo sphingolipid biosynthesis pathway-associated metabolism of dihydrosphingolipids. CDase, Ceramidase; Cer, ceramide; CerS, ceramide synthase; DES1, desaturase 1; DHCer, dihydroceramide; DHS, dihydrosphingosine; DHS1P, dihydrosphingosine-1-phosphate; SK, sphingosine kinase.
Cell culture and treatments. SCC19 cells were kindly provided by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI, USA). Cells were grown in DMEM/F-12 medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Carlsbad, CA, USA) in a humidified incubator at 37°C and 5% CO2. For all experiments, incubation of cells was carried out in a humidified incubator at 37°C and 5% CO2. All treatments were added to the cells in the growth medium. After overnight incubation with Foscan, LCL29 (1 μM) or HPR (2.5 μM) were added immediately prior to irradiation. Cells were irradiated at room temperature with red light (power density: 2 mW/cm2; fluence: 400 mJ/cm2; λmax ~ 670 nm), using a light-emitting diode array light source (EFOS, Mississauga, ON, Canada), and incubated for the indicated times. The inhibitors were used at their non-toxic doses (LD <5) and were added 1 h prior to individual or combined treatments. FoscanPDT, LCL29 and HPR were used at their LD20s. With the exception of the clonogenic assay, in all other assays used in the present study, the total number of cells exposed to the treatments was in the range of 1-2×106. In the clonogenic assays using preplating and postplating protocols, the total number of cells was approximately 500 and 1×106, respectively. At the constant fluence of 400 mJ/cm2, the final concertation of Foscan of 0.06 μM and 0.15 μM in preplating and postplating protocols, respectively, was required to result in the LD20. Apparently, there was a correlation between the total number of cells exposed to FoscanPDT and the actual LD20. This was not the case for LCL29 or HPR alone.
Clonogenic assay. Cell survival was assessed using clonogenic assay according to the preplating and postplating protocols (23, 24). For the preplating protocol, cells were resuspended in the growth medium containing Foscan (0.06 μM) and aliquots were seeded in 6-well plates (Thermo-Fisher Scientific) in triplicate in sufficient numbers to give rise to approximately 50 colonies per well. After overnight incubation, cells were irradiated. Other treatments and inhibitors were added prior to irradiation, as described above. For the postplating protocol, cells were grown in T25 flasks (Thermo-Fisher Scientific) and treated with FoscanPDT (0.15 μM Foscan + 400 mJ/cm2)±LCL29 or HPR. Overnight incubation with Foscan was followed by addition of other treatments and irradiation. After treatments, cells were trypsinized and aliquots were plated in triplicate in sufficient numbers to give rise to approximately 50 colonies per well. For both protocols, after 10 days of incubation, colonies were stained with crystal violet (0.1%) and counted. Plating efficiency was 28% (n=42).
DEVDase (caspase-3) activity assay. As described previously (8), DEVDase activity was determined in the cytosol obtained from treated cells 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 fluorescence of the cleaved DEVD substrate was measured using a spectrofluorometer (F-2500 Hitachi, New York, NY, USA; 380 nm excitation, 460 nm emission).
RNA extraction and quantitative real-time polymerase chain reaction (RT-PCR). Total RNA was isolated from treated cells using RNeasy® Mini Kit (Qiagen, Valencia, CA, USA) according to 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 primer/probe sets for CHOP and BiP, the ribosomal protein L13A, and hypoxanthine-guanine phosphoribosyltransferase (all from Life Technologies). Initial steps of RT-PCR were 30 s 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 CHOP and BiP mRNAs against the internal control housekeeping genes, the ribosomal protein L13A and hypoxanthine-guanine phosphoribosyltransferase was performed by ΔΔCT provided by CFX96 manager software 3.0 from Bio-Rad.
Immunoblotting. After treatments, cells were collected, lysed in reducing Laemmli buffer, boiled and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and western immunoblotting (25). Equal protein loading was confirmed using antibody to actin. Antibodies to BiP, CHOP and actin were from Enzo-Life Sciences, Thermo-Fisher Scientific, and EMD Millipore, respectively. Following visualization of blots using ECL Plus chemifluorescence kit and STORM 860 imaging system (GE Healthcare, Piscataway, NJ, USA), they were quantified by ImageQuant 5.2 (GE Healthcare).
Electrospray ionization tandem mass spectrometry (MS) analysis. Lipids were extracted from treated cells, and sphingolipids were separated by high performance liquid chromatography, introduced to electrospray ionization source and then analyzed by tandem MS using Thermo Scientific Vanquish Ultra-High Performance Liquid Chromatography System and Thermo Scientific TSQ Quantum Access Max Triple Quadrupole Mass Spectrometer (both from Thermo-Fisher Scientific) [cf. (25)].
Protein determination. Protein content was determined by a modified Bradford assay (Bio-Rad).
Statistical analysis. Significant differences (p<0.05) were determined using Student's t-test.
Results and Discussion
Enhanced loss of clonogenicity after the combined treatments is dependent on oxidative- and ER- stress, apoptosis, and FB-sensitive sphingolipid production. To assess the role of oxidative and ER stress, apoptosis and FB-sensitive sphingolipid generation in cell survival after FoscanPDT combined with HPR or LCL29, we used the following pharmacologic inhibitors: the singlet oxygen quencher L-histidine (27), the ceramide synthase inhibitor FB, the pan-caspase inhibitor zVAD-fmk (28), and the ER stress-related agents. As shown in Figure 2, enhanced loss of clonogenicity was induced after FoscanPDT combined with LCL29 or HPR, and the effect was counteracted by L-histidine, FB and zVAD-fmk. The ER stress inducer thapsigargin (29) augmented enhanced loss of clonogenicity after FoscanPDT+HPR. Enhanced loss of clonogenicity after the combined treatments was counteracted by: 4-mu8c, an inhibitor of IRE1 (30), salubrinal, an inhibitor of dephosphorylation of eIF2α (31), and 4-phenylbutyrate, an inhibitor of CHOP (32). The data suggest that the combined treatments induced enhanced loss of clonogenicity that were dependent upon singlet oxygen, ER stress, apoptosis and FB-sensitive sphingolipid generation.
FB inhibited caspase-3 activation after FoscanPDT alone or in combination with LCL29. We showed that after FoscanPDT+HPR caspase-3 activation was enhanced. FB counteracted caspase-3 activation after FoscanPDT±HPR (4). Similarly, enhanced caspase-3 activation was induced after FoscanPDT+LCL29, and inhibited by FB (Figure 3). The data suggest that enhanced caspase-3 activation after FoscanPDT+LCL29 is dependent upon FB-sensitive sphingolipid generation.
The effect of FoscanPDT±LCL29±FB on BiP and CHOP mRNA expression. To assess the induction of ER stress after treatments, we determined the expression of the ER stress markers BiP and CHOP. As shown in Figure 4A, the ER stress prosurvival marker BiP was up-regulated at the mRNA level after FoscanPDT alone. BiP mRNA was down-regulated after LCL29 alone, and the effect was counteracted after combining LCL29 with FoscanPDT. FB had no significant effect on BiP mRNA after FoscanPDT±LCL29. The ER stress proapoptotic marker CHOP mRNA was up-regulated after FoscanPDT±LCL29 (Figure 4A). FB modesty (but insignificantly) attenuated enhanced CHOP mRNA expression after FoscanPDT+LCL29, and had no significant effect on CHOP mRNA levels after each treatment alone.
Enhanced loss of clonogenicity after combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production. L-histidine (L-hist; 1 mM), fumonisin B1 (FB; 10 μM), thapsigargin TG (1 nM), 4-mu8c (1 μM), salubrinal (Sal; 5 μM), 4-phenylbutyrate (PB; 2 mM), and zVAD-fmk (zVAD; 10 μM) were added 1 h prior to FoscanPDT (0.06 μM Foscan + 400 mJ/cm2), LCL29 (1 μM), HPR (2.5 μM) or the combined treatments. Colonies were stained with crystal violet (0.1%) and counted 10 days after treatments. The data are shown as the average±SEM (n=3-39). Significant differences are shown between: †treatment and untreated control; *combination and individual treatments; #(treatment+inhibitor) and treatment. Untreated controls had 0% loss of clonogenicity.
The effect of FoscanPDT±HPR±FB on BiP and CHOP mRNA expression. In contrast to FoscanPDT alone, there was no up-regulation of BiP mRNA after HPR alone (Figure 4B). FB had no significant effect on BiP mRNA levels after FoscanPDT or HPR alone. Enhanced up-regulation of BiP mRNA after FoscanPDT+HPR was inhibited by FB. CHOP mRNA levels were elevated after individual treatments and were unaffected by FB. Enhanced CHOP mRNA expression after FoscanPDT+HPR was attenuated by FB.
The effect of treatments on CHOP and BiP protein expression. As depicted in Figure 4C, elevated CHOP protein expression was induced after FoscanPDT alone or in combination with LCL29 or HPR. LCL29 or HPR alone did not induce CHOP protein expression. FB inhibited induced CHOP protein expression after FoscanPDT alone or in combination with LCL29 or HPR. BiP was up-regulated at the protein level only after the combinations, and FB attenuated the effect. Overall, the combined treatments induced enhanced BiP and CHOP expression that was inhibited by FB at the mRNA and/or protein level. The data imply that FB-sensitive sphingolipid generation modulates enhanced up-regulation of BiP and CHOP after the combined treatments. Our findings also indicate that the long-term effect of enhanced expression of the prosurvival BiP and the proapoptotic CHOP was tipping the balance in favor of enhanced loss of clonogenicity.
Enhanced accumulation of sphingolipids is induced after treatments. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide (4). Similar trend was shown only for C18:1-ceramide, not for any other ceramide (4). Here we show that combining FoscanPDT with HPR enhanced accumulation of dihydrosphingosine-1-phosphate (Table I). Moreover, the combination of FoscanPDT with LCL29 enhanced accumulation of the dihydrosphingolipids C16-dihydroceramide, dihydrosphingosine, and dihydrosphingosine-1-phosphate, as well as C18-ceramide (Figure 5). Similar trends were observed for other ceramides, i.e. C14-, C16-, and C26:1-ceramide, as well as sphingosine-1-phosphate (Table II).
Enhanced caspase-3 activation after FoscanPDT+LCL29 is inhibited by FB. FB (10 μM) was added 1 h prior to PDT (0.15 μM Foscan + 400 mJ/cm2), LCL29 (1 μM) or the combination. Twenty-four hours after treatments, cells were collected and processed for DEVDase assay. The enzyme activity is expressed in arbitrary units (a.u.) and the data are shown as the average±SEM (n=2-6). Significant differences are shown between: †treatment and untreated control (Con); *combination and individual treatments; #(treatment+FB) and treatment.
Taken together, the combined treatments enhanced the levels of dihydrosphingolipids and enhanced the ER stress and apoptosis. Because dihydroceramide can be converted into dihydrosphingosine and dihydrosphingosine-1-phosphate, this can lead to cell death or survival depending on dihydrosphingosine/dihydrosphingosine-1-phosphate balance (13). Similar reasoning could be applied to increases in the proapoptotic ceramide and the prosurvival sphingosine-1-phosphate after FoscanPDT+LCL29. The enhanced loss of clonogenicity after the combined treatments indicates that the dihydrosphingosine/dihydrosphingosine-1-phosphate and ceramide/sphingosine-1-phosphate balances were tipped in favor of this outcome.
In summary, our present study indicates that enhanced loss of clonogenicity after the combined treatments is dependent on oxidative- and ER- stress, apoptosis, and FB-sensitive sphingolipid production. The study also provides novel insights into modulation of the ER stress by FB-sensitive sphingolipid production after the combined treatments. These novel findings are expected to help develop more effective mechanism-based therapeutic strategies.
The effect of treatments without and with FB on BiP and CHOP expression. FB (10 μM) was added 1 h prior to PDT (0.15 μM Foscan + 400 mJ/cm2), LCL29 (1 μM), HPR (2.5 μM) or the combinations. After the treatments, the cells were incubated for the indicated times, collected, and processed for appropriate assays. (A and B): The effect of treatments±FB on BiP and CHOP mRNA levels. Twenty-four hours after the treatments, the cells were collected and processed for RT-PCR. The data were calculated as the relative normalized expression of BiP and CHOP mRNAs against the expression of housekeeping gene-encoding proteins the ribosomal protein L13A and hypoxanthine-guanine phosphoribosyltransferase, and are shown as the average±SEM (n=2-4). Significant differences are shown between: †treatment and untreated control (Con); *combination and individual treatments; #(treatment+FB) and treatment; acombination and LCL29. (C): The effect of treatments±FB on BiP and CHOP protein levels. Forty eight hours post-treatments, cells were collected and processed for PAGE/western immunoblotting. Equal protein loading was verified using anti-actin. Protein levels were quantified from the blots and are expressed in arbitrary units (a.u.).
FoscanPDT+HPR enhances accumulation of dihydrosphingosine-1-phosphate in SCC19 cells at 24 h.
FoscanPDT+LCL29 induces enhanced accumulation of dihydrosphingolipids and C18-ceramide. Cells were treated with PDT (0.15 μM Foscan + 400 mJ/cm2)±LCL29 (1 μM), incubated for 24 h, then collected and processed for MS. The levels of sphingolipids were calculated as pmoles/mg protein and are shown as the average±SEM (n=2-3). C16-DHCer, C16-dihydroceramide; DHS, dihydrosphingosine; DHS1P, dihydrosphingosine-1-phosphate; C18-Cer, C18-ceramide.
Effect of FoscanPDT±LCL29 on sphingolipid levels in SCC19 cells at 24 h.
Acknowledgements
This work was supported by: the Faculty Research Award Program Individual Investigator Award from the Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, USA (to DS). The MS-related work at the Lipidomics Shared Resource Facility (Medical University of South Carolina, Charleston, SC, USA) was supported by the following grants from the National Institutes of Health (NIH): P30 CA 138313, SC COBRE P20 RR017677, and C06 RR018823.
Footnotes
This article is freely accessible online.
- Received December 15, 2016.
- Revision received January 10, 2017.
- Accepted January 12, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.