sphingolipids ( sls ) are structural components of cell membranes , as well as important players in a variety of biological functions . ceramide and sphingosine have been associated with proapoptotic and tumor - suppressor functions , whereas sphingosine-1-phosphate ( s1p ) appears to have prosurvival and tumor - promoting effects     [ 2 , 3 ] . targeting sls has been used for new anticancer drug design     [ 4 , 5 ] . exposure of chemoresistant mcf-7 cells to short - chain ceramide analogs , for example , 4,6-dieneceramide , leads to the accumulation of endogenous ceramide , apoptosis , and inhibition of clonogenic survival     [ 68 ] . cationic ceramide conjugates with pyridinium salts ( ccps analogs ) were synthesized that target mitochondria and with improved physicochemical properties     . mitochondrial targeting is important for cancer treatment because cancer cells tend to have mitochondria with more negative mitochondrial membrane potential . these novel ceramide analogs ( c2-c16 ccps ) are potent anticancer agents , alone or in combination with standard chemotherapy     [ 9 , 1114 ] . exposure of mcf-7 cells to the short - chain homolog lcl29 ( c6-ccps ) targets mitochondria to trigger cell killing . similarly , lcl124 , the ( 2s,3s ) isomer of lcl29 , alone or in combination with gemcitabine , had potent anticancer activity against human head and neck squamous cell carcinomas in vitro and in vivo     . long - chain ceramide analog lcl30 ( c16-ccps ) was shown to target mitochondria and to trigger cell death in sw403 human colon carcinoma cells     . in addition , lcl30 was an effective anticancer agent in a colorectal cancer mouse model     . photodynamic therapy ( pdt ) is a treatment modality for effective abolishment of malignancies including head and neck cancers . in pdt , a light - absorbing agent ( photosensitizer ) is activated by highly - focused laser light to trigger oxidative stress and destruction of a cellular target     [ 16 , 17 ] . pdt alone , however , can be ineffective with some tumors     [ 18 , 19 ] . to overcome inefficiency of pdt itself to eradicate tumors , combined treatments become a necessary option . using lcl29     [ 9 , 20 ] in combination with photofrin - pdt , we have shown moderately improved in vivo response of mouse sccvii squamous carcinomas     .   here , we used for the first time lcl30 in combination with pdt to determine their effects on endogenous sls , apoptosis , and clonogenic survival in sccvii mouse squamous carcinoma cells ( sccvii cells ) . the model was chosen in order to use the results of this study in our future in vivo work in syngeneic mouse sccvii squamous carcinomas , a recognized mouse model for human head and neck cancers , since intact immune system is a key to pdt therapeutic success     [ 16 , 17 ] . for pdt , silicon phthalocyanine pc 4 , a photosensitizer with physicochemical properties superior to photofrin     [ 23 , 24 ] , sls that were analyzed by mass spectrometry ( ms ) are shown in figure 1 as part of , or relative to the de novo ceramide pathway . others     [ 2530 ] and we     [ 21 , 3135 ] have shown that the de novo ceramide pathway modulates response to anticancer drugs , including pdt . for example , we have shown in jurkat cells that silencing of ceramide - utilizing enzyme sphingomyelin synthase leads to enhanced ceramide and dihydroceramide accumulation with concomitant promotion of apoptosis     . combining pdt with the anticancer lcl30 is expected to promote tumor response to pdt and , therefore , improve clinical pdt . d - erythro-2-n-[16-(1-pyridinium)-hexadecanoyl]-sphingosine bromide ( lcl30 ) was synthesized in the lipidomics shared resource of the medical university of south carolina . sccvii cells were grown in rpmi medium ( invitrogen ) containing 10% fetal bovine serum ( hyclone ) 100  units / ml penicillin and 100  g / ml streptomycin . cells were maintained at 37c in a 5% co2 atmosphere and were treated in the growth medium . for pdt experiments , after overnight incubation with pc 4 at 37c , cells were irradiated with red light ( 2 milliwatts / cm ; max  ~  670  nm ) using a light - emitting diode array light source ( efos ) at the fluence of 200  mj / cm at room temperature . following pdt , cells were incubated at 37c for 2 hours . for the combination , lcl30 ( 1  m ) was added to pc 4-pretreated cell cultures 2 hours prior to irradiation . for cells treated with lcl30 alone , following treatments , cells were collected on ice and processed for various analyses . for ms analysis , cells were washed twice with cold phosphate - buffered saline ( pbs ) , resuspended in the mixture of ethyl acetate / methanol ( 1  :  1 , v / v ) , dried under nitrogen , and shipped overnight on dry ice to the lipidomics shared resource ( charleston , sc ) for further processing . following extraction , sls were separated by high - performance liquid chromatography , introduced to electrospray ionization source , and then analyzed by double ms using tsq 7000 triple quadrupole mass spectrometer ( thermo - fisher scientific ) as described previously     . as we showed previously , the lipophilic cationic dye jc-1 ( 5,5,6,6-tetrachloro-1,13,3-tetraethylbenzimidazolylcarbocyanine iodide ; molecular probes ) jc-1 forms aggregates in normal mitochondria with a high negative membrane potential that emit a red fluorescence ( 590  nm ) . in mitochondria with low membrane potential ( depolarized ) , the dye forms monomers in the cytosol that emit a green fluorescence ( 527  nm ) . following treatments , cells were collected , washed in pbs , resuspended in jc-1-containing medium ( 2  m final concentration ) , and incubated at 37c for 15 minutes . the samples were centrifuged , resuspended in growth medium , and analyzed by flow cytometry using bd lsr ii flow cytometer ( bd biosciences ) . as a positive control for mitochondrial depolarization and apoptosis , cells were treated with camptothecin ( 5  m ) overnight and processed for flow cytometry . as we showed previously , to detect apoptosis , the exposure of phosphatidylserine in the outer leaflet of the cell membrane was measured using annexin v , a protein which binds with high affinity to negatively charged phosphatidylserine in the presence of calcium . as apoptosis progresses , cell membrane integrity is lost , and this can be detected using dna - binding fluorescent dye propidium iodide ( pi , red fluorescence ) . by attaching annexin v to the fluorescein isothiocyanate ( fitc ; green fluorescence ) , one can discriminate between intact cells ( annexin v negative , pi negative ) , early apoptotic ( annexin v positive , pi negative ) , and late apoptotic or necrotic cells ( annexin v positive , pi positive ) . the kit was purchased from bd pharmingen and the flow cytometry protocol was followed , as described by the manufacturer . as described previously , the activity of the apoptotic marker devdase was determined in cytosol by an assay based on the enzyme 's ability to cleave the fluorogenic derivative 7-amino-4-methylcoumarin ( amc ; biomol ) of the tetrapeptide substrate n - acetyl - asp - glu - val - asp ( devd ) . the released fluorescence of the cleaved devd substrate was measured in f-2500 hitachi spectrofluorometer ( 380  nm excitation and 460  nm emission ) . the protocol that we described previously     [ 38 , 39 ] was modified using preplating approach , which allows keeping the treatments in the medium during colony formation . following addition of pc 4 or lcl30 , after eight days of growth at 37c , colonies ( 50 cells ) were stained with crystal violet ( 0.1% ) and counted . statistical differences were determined using student 's t - test ( p < .05 ) . we showed rapid uptake of lcl30 by mcf-7 cells . here , we determined uptake of lcl30 by sccvii cells using ms . following a four - hour incubation of cells with lcl30 ( 1  m ) , cellular levels of lcl30 were 260  4  pmol/10 cells , corresponding to approximately 5% of the concentration applied . after pdt ( 250  nm pc 4 + 200  mj / cm ) + lcl30 ( 1  m ) , cellular levels of lcl30 were increased by 29% ( figure 2 ) . we have already demonstrated that pdt has signature effects on the sl profile in cancer cell lines and in vivo     [ 21 , 32 , 39 ] . we   and others      have also shown that lcl30 triggers changes in endogenous ceramides and s1p in human cancer cell lines . in the present study , we used ms to determine the effects of the combination , as well as of the individual treatments , on the sl profile in sccvii cells . the effects of treatments on ceramides , individual and global , are shown in table 1 and figure 3(a ) . globally , there was 1.29- and 1.47-fold increase in ceramide levels after pdt and pdt / lcl30 , respectively . the differences between individual treatments , lcl30 or pdt , and the combination , as well as between individual treatments themselves , were significant ( p < .05 ) . the effects of treatments on the levels of dihydroceramides ( dhceramides ) , individual and global , are depicted in table 1 and figure 3(b ) . maximal increases were observed for c22- and c22:1-dhceramide after pdt and pdt / lcl30 , respectively . in contrast , pdt increased total dhceramides 1.48-fold , and the effect was maintained after the combination . the effects of treatments on other sls are shown in table 1 and figure 3(c ) . the levels of dihydrosphingosine ( dhsphingosine ) , a metabolic precursor of dhceramide , were reduced by 29 , 41 , and 75% after lcl30 , pdt , and pdt / lcl30 , respectively . thus , pdt / lcl30 was more effective than individual treatments in reducing dhsphingosine levels . pdt reduced s1p levels by 86% , and in combination with lcl30 , by 44% . we have shown that pdt combined with c16-ceramide enhances mitochondrial depolarization in jurkat cells undergoing apoptosis . here , we tested whether pdt / lcl30 triggers the collapse of mitochondrial potential in sccvii cells . as depicted in figure 4(a ) , neither individual treatments nor the combination had any effect on mitochondrial membrane potential . in contrast , overnight treatment with camptothecin ( 5  m ) led to the appearance of 63% cells with depolarized mitochondria . surprisingly , in the presence of pdt , with or without lcl30 , no apoptosis was detected in sccvii cells using either annexin v / pi staining ( figure 4(b ) ) or tunel assay ( not shown ) . however , overnight treatment with camptothecin ( 5  m ) led to the appearance of 9% and 75% cells that were annexin v ( + ) /pi (  ) and annexin v ( + ) /pi ( + ) , respectively . although lcl30 was reported not to induce apoptosis     , pdt is an effective apoptotic inducer . devdase was activated 1.47- and 1.95-fold after lcl30 and pdt , respectively ( figure 5 ) . after treatment of sccvii cells with the combination , a 5.68-fold increase in devdase activity was recorded . previously , we showed that enhanced ceramide accumulation is associated with increased activation of devdase after pdt     [ 32 , 35 ] . we also showed that devdase activation is accompanied by mitochondrial depolarization and/or apoptosis post - pdt     [ 3336 , 42 , 43 ] . the present study shows devdase activation in the absence of mitochondrial depolarization and apoptosis after pdt , with or without lcl30 . caspases , including caspase-3 , have been associated with nonapoptotic functions , for example , survival     [ 4446 ] . curiously , there was positive correlation between devdase activation and the levels of prosurvival s1p after lcl30 . in addition , there was negative correlation between the proapoptotic dhsphingosine      and devdase activation . the levels of proapoptotic sphingosine   were abolished after pdt or the combination with concomitant activation of devdase . the role of devdase activation in the absence of apoptosis in response to pdt , with or without lcl30 , remains to be established . to determine the effect of lcl30 on overall cell killing , clonogenic assay was employed . as shown in figure 6(a ) , treatment of sccvii cells with 0.1 , 1 and 5  m lcl30 led to 7.8 , 21.5 and 97.7% cell killing , respectively . the differences between each dose were significant ( p < .05 ) , supporting a dose - dependent cell killing after lcl30 .   to test for potential enhancement of cell killing by the combination , lds < when sccvii cells were treated with lcl30 ( 1  m ) and pdt ( 250  nm pc 4 + 200  mj / cm ) , 69.8% of the overall cell killing was observed ( figure 6(b ) ) . the combination led to cell killing that was greater than that by each treatment alone ( p < .05 ) . taken together , the present study demonstrates for the first time that ( i ) lcl30 is taken up by sccvii cells ; ( ii ) cellular total ceramides are enhanced after pdt / lcl30 ; ( iii ) the activity of devdase is upregulated in the absence of mitochondrial depolarization and apoptosis post - pdt / lcl30 ; ( iv ) total cell killing is augmented after the combination . our findings warrant the use of other apoptotic markers besides devdase to verify the triggering of apoptosis , and in vivo testing of pdt / lcl30 in view of translational potential of the combination .
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 .