Abstract
Background: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We previously observed that the knockdown of ceramide synthase 6, an enzyme from the de novo sphingolipid biosynthesis pathway, is associated with marked reduction in C18-dihydroceramide and makes cells resistant to apoptosis post-PDT. Down-regulation of ceramide synthase 1 (CERS1) can also render cells resistant to anticancer drugs. Aim: To explore the impact of CERS1 knockdown on apoptosis and the sphingolipid profile, post-PDT, with the silicone phthalocyanine Pc 4, in a human head and neck squamous carcinoma cell line. Materials and Methods: Besides siRNA transfection and PDT treatment, the following methods were used: immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation. Results: CERS1 knockdown led to inhibition of PDT-induced caspase 3-like (DEVDase) activation, of apoptosis and cell death. CERS1 knockdown was associated with global and selective decreases in ceramides and dihydroceramides, in particular C18-, C18:1- and C20-ceramide post-PDT. Conclusion: Our novel findings are consistent with the notion that CERS1 regulates apoptotic resistance to PDT, partly via C18- and C20-ceramide, and that CERS1 is a molecular target for controlling resistance to PDT.
- Apoptosis
- ceramide
- ceramide synthase 1
- dihydroceramide
- PDT
- sphingolipids
- head and neck squamous carcinomas cells UM-SCC-22A
Production of the sphingolipid ceramide is associated with apoptosis (1-3). Ceramide can be generated via the de novo sphingolipid biosynthesis pathway (Figure 1), in which ceramide synthase (CERS) acylates dihydrosphingosine to give rise to dihydroceramide, which is then converted to ceramide. Six mammalian CERSs have been characterized with different yet overlapping fatty acyl CoA specificity (4-8). CERSs have been implicated in different biological functions (1, 2, 9). CERS1 is involved in C18-ceramide synthesis (10). The enzyme has been implicated in sensitization of cells to chemotherapeutic agents (11, 12). siRNA-mediated down-regulation of CERS1 inhibits imatinib-induced cell death (13). Unlike in non-squamous tumors, in head and neck cancer, reduced CERS1 expression correlates with lower C18-ceramide levels compared to normal tissue (14). Overexpression of CERS1 in head and neck squamous carcinoma cells is associated with the generation of C18-ceramide and promotion of apoptosis. In contrast, CERS1 knockdown renders the cells resistant to anticancer agents (12).
The de novo sphingolipid biosynthesis pathway modulates apoptosis after photodynamic therapy (PDT) (15-17). PDT utilizes a light-absorbing photosensitizer, visible light and oxygen to generate reactive oxygen species that can destroy malignant cellular targets by apoptosis (18). The efficacy of PDT correlates with tumor cell apoptosis (19). We have demonstrated that de novo sphingolipids affect apoptosis after PDT with the silicone phthalocyanine photosensitizer Pc 4 (15-17). We have recently shown that the knockdown of ceramide synthase 6 (CERS6) is associated with marked reduction in C18-dihydroceramide and renders cells resistant to apoptosis post-PDT (20). However, the role of CERS1 in PDT-induced apoptosis is unclear. In the current study, we explored the effects of CERS1 knockdown on apoptosis and the sphingolipid profile post-Pc 4-PDT in UM-SCC-22A, a human head and neck squamous carcinoma cell line.
Materials and Methods
Materials. Pc 4, HOSiPcOSi(CH3)2(CH2)3N(CH3)2, was kindly supplied by Dr. Malcolm E. Kenney (Case Western Reserve University, Cleveland, OH, USA). Dulbecco's modified Eagle's medium (DMEM) and serum were from Invitrogen Life Sciences (Grand Island, NY, USA) and Hyclone (Logan, UT, USA), respectively. UM-SCC-22A, a human head and neck squamous carcinoma cell line from the hypopharynx (21, 22), was kindly supplied by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI, USA).
Cell culture. UM-SCC-22A cells were grown in DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C in an incubator with 5% CO2 atmosphere, and were treated in the growth medium.
siRNA transfection and PDT treatment. The siRNA targeting the sequence AAG GTC CTG TAT GCC ACC AGT of human CERS1 was from Qiagen (Valencia, CA, USA) (12, 23). For control siRNA, AllStars Negative Control siRNA from Qiagen was used. UM-SCC-22A cells were transfected with double-strand siRNAs using Oligofectamine from Invitrogen Life Sciences, according to the manufacturer's instructions. To optimize the concentration of siCERS1, preliminary dose-response experiments (10-40 nM siCERS1) were carried out. As described previously (20), cells (1×106-2.5×106) were transfected with 25 nM of each siRNA. Twenty-four hours after transfection, cells were collected and seeded in fresh growth medium. Following overnight exposure to Pc 4 (250 and 500 nM), cells were irradiated with red light (2 mW/cm2; λmax ~670 nm) using a light-emitting diode array (EFOS; Mississauga, ON, Canada) at a fluence of 200 mJ/cm2 at room temperature. Following PDT, cells were incubated at 37°C for 2 or 24 h, were collected on ice and were processed for various analyses. For mass spectrometric analysis, cells were washed twice with cold phosphate-buffered saline, resuspended in a mixture of ethyl acetate/methanol (1:1, v/v), dried under nitrogen, and shipped overnight on dry ice to the Lipidomics Shared Resource (Medical University of South Carolina, Charleston, SC, USA) for further processing.
Sphingolipid analysis by quantitative high-performance liquid chromatography (HPLC)/mass spectrometry (MS). Following extraction, sphingolipids were separated by HPLC, introduced to an electrospray ionization source and were then analyzed by double MS using TSQ 7000 triple quadrupole mass spectrometer from Thermo-Fisher Scientific (Waltham, MA, USA), as described previously (24).
Immunoblotting. Following PDT, cells were collected, lysed in reducing Laemmli buffer, boiled and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western immunoblotting, as reported previously (16, 25). Equal protein loading was confirmed using anti-pan-actin and anti-HSP90. The following antibodies were obtained from Novus Biologicals (Littleton, CO, USA): anti-CERS1 and anti-CERS6 (mouse polyclonal each); anti-CERS2 (mouse monoclonal); anti-CERS5 (rabbit polyclonal). These antibodies were originally made by Abnova (Taipei City, Taiwan, ROC). Mouse monoclonal anti-HSP90 was from BD Biosciences (San Diego, CA, USA). Mouse monoclonal anti-pan-actin was from Neo Markers (Kalamazoo, MI, USA). Following visualization of blots using ECL Plus chemifluorescence kit and a STORM 860 imaging system (GE Healthcare, Piscataway, NJ, USA), they were quantified by ImageQuant 5.2 (GE Healthcare).
Caspase-3-like (DEVDase) activity assay. As described previously (20), following PDT, cell harvesting and cell lysis, DEVDase activity, was measured using a fluorogenic derivative of the tetrapeptide substrate acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) from Enzo Life Sciences (Farmingdale, NY, USA) and an F-2500 spectrofluorometer (Hitachi; Dallas, TX, USA) (380 nm excitation, 460 nm emission).
Mitochondrial membrane depolarization measurement. The lipophilic cationic dye JC-1 (5,5’,6,6’-tetrachloro-1,1’3,3’-tetraethylbenzimidazolylcarbocyanine iodide) was used to determine mitochondrial membrane potential by flow cytometry, as we described previously (17, 20, 26). Following PDT, cells were harvested and processed for flow cytometry according to the manufacturer's instructions (BD Biosciences, San Diego, CA, USA). ABD LSR II flow cytometer was used for analysis (BD Biosciences).
Apoptosis detection. As we showed previously (20, 26, 27), in order 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 (PI) fluorescent dyes, respectively. Early apoptotic (annexin V+/PI−) cells were distinguished from late apoptotic or necrotic cells (annexin V+/PI+). The kit was obtained from BD Biosciences and the flow cytometric protocol was followed, as described by the manufacturer.
Trypan blue dye exclusion assay. After PDT, cells were harvested, resuspended in cell growth medium, and diluted 1:1 with 0.4% trypan blue stain from Sigma-Aldrich (St. Louis, MO, USA). Stained and unstained cells were counted using a hemocytometer. Trypan blue-positive cells were designated as dead cells.
Statistical analysis. Following data collection, the mean value and the standard error of the untreated and each treated group were calculated for at least n=3. Data were analyzed for statistically significant differences (p≤0.05) between groups using the Student's t-test of unequal variance.
Results
Effect of CERS1 knockdown and PDT on ceramide synthase expression. To verify the down-regulation of CERS1 by RNA interference in UM-SCC-22A cells, protein expression levels were determined using immunoblotting. As shown in Figure 2A and B, CERS1 protein levels were reduced by 46% after CERS1 knockdown. Following PDT (500 nM Pc 4 + 200 mJ/cm2), the CERS1 protein was down-regulated in both siControl- and siCERS1-transfected cells.
To test for potential off-target effects, expression levels of CERS2, -5, and -6 were determined using immunoblotting. As shown in Figure 2C, expression of these CERSs was not affected by CERS1 knockdown. The effect of PDT on the expression of other CERSs was similar in both siControl- and siCERS1-transfected cells.
CERS1 knockdown suppressed apoptosis and cell death without affecting the mitochondrial depolarization after PDT. The loss of mitochondrial membrane potential accompanies PDT-evoked apoptosis (17, 28). CERS6 knockdown suppresses PDT-induced mitochondrial depolarization (20). We asked whether CERS1 affects apoptosis at the mitochondrial level after PDT. We found that PDT-induced mitochondrial depolarization was not modulated by CERS1 knockdown (data not shown). The data suggest that CERS1 is not involved in mitochondrial apoptosis after PDT.
CERS6 knockdown inhibits PDT-induced apoptosis (20). The involvement of CERS1 in PDT-induced apoptosis is unknown. We first examined the effect of CERS1 knockdown on DEVDase activity after PDT. We found that at 2 h after PDT, DEVDase activation was significantly inhibited by CERS1 knockdown (Figure 3A). These findings support the involvement of CERS1 in the activation of DEVDase post-PDT.
We investigated the role of CERS1 in apoptosis after PDT using the apoptotic marker annexin V. Flow cytometric data revealed that CERS1 knockdown led to significant reductions in the appearance of annexin V+ and annexin V+/PI+ cells at 24 h after PDT (Figure 3B). These findings suggest that CERS1 knockdown yields cells resistant to early and late apoptosis post-PDT.
To assess the effect of CERS1 knockdown on short-term cell viability post-PDT, trypan blue dye exclusion assay was used. We found that following PDT, the appearance of trypan blue-positive cells was attenuated by CERS1 knockdown at 24 h (Figure 3C). Thus, CERS1 knockdown, similarly to CERS6 knockdown (20), suppressed cytotoxicity after PDT.
Effect of CERS1 knockdown on the sphingolipid profile. CERS6 knockdown is associated with changes in the sphingolipid profile both at rest and after PDT (20). To determine the effect of CERS1 knockdown on the sphingolipid profile, resting levels of sphingolipids were measured by MS. CERS1 knockdown significantly increased the levels of dihydrosphingosine, a CERS substrate. Out of all ceramides and dihydroceramides tested, the only significant change evoked by CERS1 knockdown was an increase in the levels of C16-ceramide and C16-dihydroceramide (Table I).
We then explored the effect of PDT on the sphingolipid profile in cells transfected with siControl- or siCERS1. The data presented here were obtained at 2 h post-PDT. CERS1 knockdown significantly suppressed global accumulation of ceramides and dihydroceramides after PDT (Figure 4A).
Among all ceramides and dihydroceramides tested, PDT induced the greatest increase in C18-dihydroceramide (Figure 4B). The 60-fold increase in C18-dihydroceramide after PDT was substantially reduced by CERS1 knockdown. CERS1 knockdown also significantly reduced the accumulation of the following dihydroceramides after PDT: C14-, C16-, C18:1-, C22-, C22:1-, C24-, and C26:1-dihydroceramide. Furthermore, CERS1 knockdown significantly suppressed PDT-induced accumulation of the following ceramides: C18-, C18:1-, and C20-ceramide (Figure 4B). The effect of CERS1 knockdown on accumulation of other ceramides and dihydroceramides did not correlate with reduced functional responses (not shown). Taken together, CERS1 knockdown was associated with selective decreases in individual ceramides and dihydroceramides, as well as with global decreases in ceramides and dihydroceramides after PDT.
Discussion
The key findings of our present study are that CERS1 knockdown is associated with global and selective decreases in ceramides and dihydroceramides, in particular C18-, C18:1- and C20-ceramide, leading to suppression of apoptosis post-PDT. Our present data strongly support a role of CERS1 and sphingolipids in apoptotic resistance to PDT. Similarly, down-regulation of CERS1 inhibits imatinib-induced cell death (13) and renders head and neck squamous carcinoma cells resistant to anticancer agents (12). We have recently shown that CERS6 knockdown supresses apoptosis after PDT (20). Compared to CERS6 knockdown, CERS1 knockdown was unable to modulate mitochondrial depolarization and to suppress apoptosis post-PDT to the same extent. Regardless, we have demonstrated that CERS1 knockdown, similar to CERS6 knockdown, leads to apoptotic resistance in UM-SCC-22A cells post-PDT.
Mammalian CERS1 has been shown to utilize C18-CoA preferentially (5, 10), and to a lesser extent, C18:1- and C20-CoA (5, 10, 12, 29). However, specificity of CERS1 for these substrates was not observed following CERS1 knockdown. In the absence of differences in expressions of other CERSs between cells transfected with control- or CERS1-siRNA, the observed lack of specificity for C18-ceramide by CERS1 knockdown suggests relaxation of substrate specificity of the enzyme (9). Alternatively, low levels of CERS1 expression and C18-ceramide in head and neck squamous carcinoma cells (14) might counteract the ability of CERS1 knockdown to lower C18-ceramide levels any further. Nevertheless, a significant increase in the levels of dihydrosphingosine, a CERS1 substrate, is consistent with the idea of CERS1 knockown.
The involvement of CERS1 in PDT-induced changes in the sphingolipid profile is supported by the following data: C18-, C18:1- and C20-ceramide were the only ceramides that were significantly reduced by the CERS1 knockdown; the levels of C18- and C18:1-dihydroceramide were also substantially suppressed. However, specificity of CERS1 for PDT-induced changes in the sphingolipid profile was only partial, since CERS1 knockdown evoked significant decreases in global ceramides and dihydroceramides. Moreover, the levels of C14-, C16-, C22-, C22:1, C24- and C26:1-dihydroceramide were also reduced by CERS1 knockdown post-PDT. In the absence of differences in expressions of other CERSs between cells transfected with control- or CERS1-siRNA, post-PDT observed changes in global ceramides, global and individual dihydroceramides by CERS1 knockdown suggest partial substrate specificity of the enzyme after PDT (9).
In summary, our present study indicates that CERS1 controls apoptotic susceptibility to PDT in part via the de novo production of C18- and C20-ceramide. These novel findings support the idea that alterations in CERS1 expression might be utilized therapeutically to control resistance to PDT.
Acknowledgements
This work was supported by U.S. Public Health Service Grant R01 CA77475 from the National Cancer Institute, National Institutes of Health (DS) and the Veterans Administration Merit Awards from RR&D and BLRD programs (TIG). The MS-related work was performed by the Lipidomics Shared Resource (Medical University of South Carolina), supported by NCI grants: IPO1CA097132 and P30 CA 138313 and NIH/NCRR SC COBRE Grant P20 RR017677. Laboratory space for the Lipidomics Shared Resource was supported by the NIH, grant C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources. We thank Drs. Besim Ogretmen and Can Emre Senkal for helpful discussions over the manuscript.
Footnotes
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↵* These Authors contributed equally in this study.
- Received May 2, 2012.
- Revision received May 28, 2012.
- Accepted May 29, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved