Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer.

Oncogenic multidrug resistance is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (l-cycloserine) and CerS (fumonisin B1) in cell viability assays. Downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.

Sphingolipid pathway regulates innate immune responses at the fetomaternal interface during pregnancy.

For a successful pregnancy, the mother's immune system has to tolerate the semiallogeneic fetus. A deleterious immune attack is avoided by orchestration of cellular, hormonal, and enzymatic factors. However, the precise mechanisms underlying fetomaternal tolerance are not yet completely understood. In this study, we demonstrate that sphingolipid metabolism constitutes a novel signaling pathway that is indispensable for fetomaternal tolerance by regulating innate immune responses at the fetomaternal interface. Perturbation of the sphingolipid pathway by disruption of the sphingosine kinase gene (Sphk) during pregnancy caused unusually high expression of neutrophil chemoattractants, CXCL1 and CXCL2, in the decidua, leading to a massive infiltration of neutrophils into the fetomaternal interface with enhanced oxidative damage, resulting in early fetal death. Sphk-deficient mice also exhibited neutrophilia in the peripheral blood, enhanced generation of granulocytes in the bone marrow, and a decrease in the number of decidual natural killer cells. The blockage of neutrophil influx protected Sphk-deficient mice against pregnancy loss. Notably, a similar result was obtained in human decidual cells, in which Sphk deficiency dramatically increased the secretion of CXCL1 and IL-8. In conclusion, our findings suggest that the sphingolipid metabolic pathway plays a critical role in fetomaternal tolerance by regulating innate immunity at the fetomaternal interface both in mice and humans, and it could provide novel insight into the development of therapeutic strategies to treat idiopathic pregnancy loss in humans.

Targeting (cellular) lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-B13 ester prodrugs.

Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.

Combining anticancer agents photodynamic therapy and LCL85 leads to distinct changes in the sphingolipid profile, autophagy, caspase-3 activation in the absence of cell death, and long-term sensitization.

Two anticancer agents, LCL85 and photodynamic therapy (PDT) were combined to test whether the combination PDT/LCL85 evokes changes in the sphingolipid (SL) profile and promotes cell death. Treatment of SCCVII mouse squamous carcinoma cells using the silicone phthalocyanine Pc 4 for PDT induced increases in the prodeath global ceramides/dihydroceramides (DHceramides), and no changes in the prosurvival sphingosine-1-phosphate (S1P). In contrast, after LCL85, the levels of most ceramides and DHceramides were reduced, whereas the levels of S1P were increased. After PDT/LCL85 the levels of global ceramides and DHceramides, and of S1P, were restored to resting levels. PDT/LCL85 also enhanced the levels of C18-, C20-, and C20:1-ceramide, and C18-DHceramide. Treatment with PDT, with or without LCL85, led to substantial reductions in sphingosine levels. PDT/LCL85 induced enhanced autophagy and caspase-3 activation. None of the treatments affected short-term viability of cells. In contrast, long-term clonogenic survival was reduced not only after PDT or LCL85, but even more after PDT/LCL85. Overall, our data show that short-term exposure to PDT/LCL85 led to distinct signature effects on the SL profile, enhanced autophagy, and caspase-3 activation without cell death. Long-term exposure to PDT/LCL85 enhanced overall cell killing, supporting translational potential of PDT/LCL85.

Alterations of lipid metabolism provide serologic biomarkers for the detection of asymptomatic versus symptomatic COVID-19 patients.

COVID-19 pandemic exerts a health care emergency around the world. The illness severity is heterogeneous. It is mostly unknown why some individuals who are positive for SARS-CoV-2 antibodies stay asymptomatic while others show moderate to severe disease symptoms. Reliable biomarkers for early detection of the disease are urgently needed to attenuate the virus's spread and help make early treatment decisions. Bioactive sphingolipids play a crucial role in the regulation of viral infections and pro-inflammatory responses involved in the severity of COVID-19. However, any roles of sphingolipids in COVID-19 development or detection remain unknown. In this study, lipidomics measurement of serum sphingolipids demonstrated that reduced sphingosine levels are highly associated with the development of symptomatic COVID-19 in the majority (99.24%) SARS-CoV-2-infected patients compared to asymptomatic counterparts. The majority of asymptomatic individuals (73%) exhibited increased acid ceramidase (AC) in their serum, measured by Western blotting, consistent with elevated sphingosine levels compared to SARS-CoV-2 antibody negative controls. AC protein was also reduced in almost all of the symptomatic patients' serum, linked to reduced sphingosine levels, measured in longitudinal acute or convalescent COVID-19 samples. Thus, reduced sphingosine levels provide a sensitive and selective serologic biomarker for the early identification of asymptomatic versus symptomatic COVID-19 patients.

Blood sphingolipidomics in healthy humans: impact of sample collection methodology.

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C(14)-C(26) N-acyl part) of "normal" sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (C(n)-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C(16)-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C(16)-LacCer (10.0 µM) and the abundant HexCer was C(24)-HexCer (2.5 µM). The abundant Cer, C(24)-Cer (4.0 µM), was not influenced by fasting; however, levels of C(16)-C(20) Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL(3) was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.

Increased ceramide accumulation correlates with downregulation of the autophagy protein ATG-7 in MCF-7 cells sensitized to photodamage.

The purpose of this study was to determine the sphingolipid (SL) profile in cells defective in autophagy protein ATG-7 and overall cell death after photodynamic therapy (PDT) with the photosensitizer Pc 4. MCF-7 human breast cancer cells with downregulated ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to increased cell killing. PDT evoked an early (2h) greater global increase in ceramides in ATG-7 defective cells compared to Scr cells. The total increases in dihydroceramide (DHceramide) were significant at 2 and 24h in both cell types post-PDT. The levels of sphingosine-1-phosphate (S1P) and sphingosine were decreased below resting levels at both time points irrespective of the cell type. The data imply that ceramide might be a marker of ATG-7 deficiency in cells sensitized to PDT.

Sphingolipid analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Sphingolipid (SPL) metabolism (Fig. 1) serves a key role in the complex mechanisms regulating cellular stress responses to environment. Several SPL metabolites, especially ceramide (Cer), sphingosine (Sph) and sphingosinel-phosphate (S1P) act as key bioactive molecules governing cell growth and programmed cell death (Fig. 2). Perturbations in sphingolipids of one type may enhance or interfere with the action of another. To monitor changes in SPL composition therefore, reliable analytical methods are necessary. Here we present the liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for simultaneous qualitative and quantitative monitoring of SPL components (classes and molecular species) in biological material as an effective tool to study sphingolipid signaling events. The LC-MS/MS methodology is the only available technique that provides high specificity and sensitivity, along with a wealth of structural identification information.

Altered Levels of Serum Ceramide, Sphingosine and Sphingomyelin Are Associated with Colorectal Cancer: A Retrospective Pilot Study.

BACKGROUND/AIM: Because patients with cancer of apparently equivalent stage often have different outcomes, it is necessary to gather additional information to complement cancer staging. Dysregulated sphingolipid metabolism contributes to carcinogenesis. In this retrospective pilot study, we tested the hypothesis that changes in serum levels of sphingolipids are associated with stage IV colorectal cancer (CRC). PATIENTS AND METHODS: We used commercially available serum samples from healthy males and patients with CRC (adenocarcinoma of the large intestine, stage IV with metastases). Blood samples were obtained immediately prior to anesthesia/surgery. We measured sphingolipid levels in sera using mass spectrometry. RESULTS: In serum of patients with CRC, the levels of C16-, C18-, C18:1-, and C24:1-ceramide, as well as those of sphingosine, were significantly higher than those of controls. In contrast, the levels of C24-sphingomyelin were significantly lower than those of controls. A global test of association showed that ceramides and sphingomyelins but not hexosylceramides were significantly associated with stage IV CRC. CONCLUSION: Sphingolipids have a potential of serving as novel, non-invasive, inexpensive, and effective blood-based biomarkers to complement CRC staging for better prognosis and more personalized medicine.

Increased Plasma Levels of Select Deoxy-ceramide and Ceramide Species are Associated with Increased Odds of Diabetic Neuropathy in Type 1 Diabetes: A Pilot Study.

Plasma deoxy-sphingoid bases are elevated in type 2 diabetes patients and correlate with the stage of diabetic distal sensorimotor polyneuropathy; however, associations between deoxy-sphingolipids (DSL) and neuropathy in type 1 diabetes have not been examined. The primary aim of this exploratory pilot study was to assess the associations between multiple sphingolipid species including DSL and free amino acids and the presence of symptomatic neuropathy in a DCCT/EDIC type 1 diabetes subcohort. Using mass spectroscopy, plasma levels of DSL and free amino acids in DCCT/EDIC type 1 diabetes participants (n = 80), with and without symptoms of neuropathy, were investigated. Patient-determined neuropathy was based on 15-item self-administered questionnaire (Michigan Neuropathy Screening Instrument) developed to assess distal symmetrical peripheral neuropathy in diabetes. Patients who scored ≥4, or reported inability to sense their feet during walking or to distinguish hot from cold water while bathing were considered neuropathic. Plasma levels of ceramide, sphingomyelin, hexosyl- and lactosylceramide species, and amino acids were measured and analyzed relative to neuropathy status in the patient. Deoxy-C24-ceramide, C24- and C26-ceramide were higher in patients with neuropathy than those without neuropathy. Cysteine was higher in patients with neuropathy. No differences in other sphingolipids or amino acids were detected. The covariate-adjusted Odds Ratios of positive patient-reported neuropathy was associated with increased levels of deoxy-C24-, and deoxy-C24:1-ceramide; C22-, C24-, and C26-ceramide; and cysteine. Plasma deoxy-ceramide and ceramide species may have potential diagnostic and prognostic significance in diabetic neuropathy.

Fumonisin B1 Inhibits Endoplasmic Reticulum Stress Associated-apoptosis After FoscanPDT Combined with C6-Pyridinium Ceramide or Fenretinide.

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.

Enhanced apoptotic cancer cell killing after Foscan photodynamic therapy combined with fenretinide via de novo sphingolipid biosynthesis pathway.

We and others have shown that stresses, including photodynamic therapy (PDT), can disrupt the de novo sphingolipid biosynthesis pathway, leading to changes in the levels of sphingolipids, and subsequently, modulation of cell death. The de novo sphingolipid biosynthesis pathway includes a ceramide synthase-dependent reaction, giving rise to dihydroceramide, which is then converted in a desaturase-dependent reaction to ceramide. In this study we tested the hypothesis that combining Foscan-mediated PDT with desaturase inhibitor fenretinide (HPR) enhances cancer cell killing. We discovered that by subjecting SCC19 cells, a human head and neck squamous cell carcinoma cell line, to PDT+HPR resulted in enhanced accumulation of C16-dihydroceramide, not ceramide. Concomitantly, mitochondrial depolarization was enhanced by the combined treatment. Enhanced activation of caspase-3 after PDT+HPR was inhibited by FB. Enhanced clonogenic cell death after the combination was sensitive to FB, as well as Bcl2- and caspase inhibitors. Treatment of mouse SCCVII squamous cell carcinoma tumors with PDT+HPR resulted in improved long-term tumor cures. Overall, our data showed that combining PDT with HPR enhanced apoptotic cancer cell killing and antitumor efficacy of PDT. The data suggest the involvement of the de novo sphingolipid biosynthesis pathway in enhanced apoptotic cell killing after PDT+HPR, and identify the combination as a novel more effective anticancer treatment than either treatment alone.

C6-pyridinium ceramide sensitizes SCC17B human head and neck squamous cell carcinoma cells to photodynamic therapy.

Combining photodynamic therapy (PDT) with another anticancer treatment modality is an important strategy for improved efficacy. PDT with Pc4, a silicon phthalocyanine photosensitizer, was combined with C6-pyridinium ceramide (LCL29) to determine their potential to promote death of SCC17B human head and neck squamous cell carcinoma cells. PDT+LCL29-induced enhanced cell death was inhibited by zVAD-fmk, a pan-caspase inhibitor, and fumonisin B1 (FB), a ceramide synthase inhibitor. Quantitative confocal microscopy showed that combining PDT with LCL29 enhanced FB-sensitive ceramide accumulation in the mitochondria. Furthermore, PDT+LCL29 induced enhanced FB-sensitive redistribution of cytochrome c and caspase-3 activation. Overall, the data indicate that PDT+LCL29 enhanced cell death via FB-sensitive, mitochondrial ceramide accumulation and apoptosis.

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.

Plasma sphingolipids and lung cancer: a population-based, nested case-control study.

BACKGROUND: Sphingosine-1-phosphate (S1P) and ceramides are bioactive signaling sphingolipids that regulate pathways that are central to cancer pathogenesis. METHODS: A nested case-control study was implemented to test whether prediagnostic circulating concentrations of S1P and ceramides were associated with future lung cancer risk. In the community-based CLUE II cohort study in Washington County, Maryland, the study consisted of 100 incident lung cancer cases, each matched to two cancer-free controls on age, sex, race, and cigarette smoking status. Plasma stored at -70°C at the beginning of follow-up in 1989 was assayed for sphingolipids using liquid chromatography/tandem mass spectrometry methodology (LC/MS-MS). RESULTS: Compared with controls, geometric mean plasma concentrations of S1P and total ceramides were 2.9% (P = 0.10) and 5.1% (P = 0.02), respectively, greater in lung cancer cases. For S1P, the ORs and 95% confidence intervals (CI) for lung cancer risk were 2.7 (1.2-5.9), 2.7 (1.1-6.4), and 1.9 (0.8-4.5) for the second, third, and highest fourth, respectively, compared with the lowest fourth (overall P = 0.006). Compared with those with total ceramide concentrations in the lowest fourth, the ORs (and 95% CI) for lung cancer risk were 1.6 (0.7-3.3), 1.5 (0.7-3.4), and 2.1 (0.9-4.7) for the second, third, and highest fourth, respectively (P(trend) = 0.01). CONCLUSIONS: Higher concentrations of S1P and total ceramide in plasma were associated with increased future risk of lung cancer. IMPACT: These novel findings suggest that perturbation of sphingolipid metabolism and S1P generation may either contribute to the etiology of lung cancer or be a marker of latent lung cancer.

Dihydroceramide desaturase knockdown impacts sphingolipids and apoptosis after photodamage in human head and neck squamous carcinoma cells.

BACKGROUND: Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis. AIM: Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4. MATERIALS AND METHODS: Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed. RESULTS: Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced. CONCLUSION: Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT.

Increased killing of SCCVII squamous cell carcinoma cells after the combination of Pc 4 photodynamic therapy and dasatinib is associated with enhanced caspase-3 activity and ceramide synthase 1 upregulation.

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1- and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

siRNA-mediated down-regulation of ceramide synthase 1 leads to apoptotic resistance in human head and neck squamous carcinoma cells after photodynamic therapy.

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.

Ceramide synthase 6 knockdown suppresses apoptosis after photodynamic therapy in human head and neck squamous carcinoma cells.

BACKGROUND: The effectiveness of photodynamic therapy (PDT) for cancer treatment correlates with apoptosis. We observed that suppression of de novo-generated sphingolipids, e.g. ceramide, renders cells resistant to apoptosis post-PDT. Ceramide synthase 6 (CerS6) has been implicated in apoptosis after various stimuli. AIM: To investigate the involvement of down-regulation of CerS6 in apoptosis and its impact on 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 transfections and PDT treatment, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectroflurometry and flow cytometry for apoptotic marker detection, and trypan blue assay for cytotoxicity assessment, were used. RESULTS: CerS6 knockdown led to reduction in PDT-induced DEVDase activation, mitochondrial depolarization, apoptosis and cell death. CerS6 knockdown was associated with selective decreases in ceramides and dihydroceramides, markedly of C18-dihydroceramide, post-PDT. CONCLUSION: CerS6 might be a novel therapeutic target for regulating apoptotic resistance to PDT.

C16-Ceramide Analog Combined with Pc 4 Photodynamic Therapy Evokes Enhanced Total Ceramide Accumulation, Promotion of DEVDase Activation in the Absence of Apoptosis, and Augmented Overall Cell Killing.

Because of the failure of single modality approaches, combination therapy for cancer treatment is a promising alternative. Sphingolipid analogs, with or without anticancer drugs, can improve tumor response. C16-pyridinium ceramide analog LCL30, was used in combination with photodynamic therapy (PDT), an anticancer treatment modality, to test the hypothesis that the combined treatment will trigger changes in the sphingolipid profile and promote cell death. Using SCCVII mouse squamous carcinoma cells, and the silicone phthalocyanine Pc 4 for PDT, we showed that combining PDT with LCL30 (PDT/LCL30) was more effective than individual treatments in raising global ceramide levels, as well as in reducing dihydrosphingosine levels. Unlike LCL30, PDT, alone or combined, increased total dihydroceramide levels. Sphingosine levels were unaffected by LCL30, but were abolished after PDT or the combination. LCL30-triggered rise in sphingosine-1-phosphate was reversed post-PDT or the combination. DEVDase activation was evoked after PDT or LCL30, and was promoted post- PDT/LCL30. Neither mitochondrial depolarization nor apoptosis were observed after any of the treatments. Notably, treatment with the combination resulted in augmented overall cell killing. Our data demonstrate that treatment with PDT/LCL30 leads to enhanced global ceramide levels and DEVDase activation in the absence of apoptosis, and promotion of total cell killing.