Sphingolipid Metabolites Affect The Inhibition Of Retinoids On Cancer Growth

Backgrounds:Sphingolipids are the essential components of plasma membrane of eukaryotic cells and their metabolites usually work as important biological signaling molecules. Sphingolipid metabolites, specifically ceramide (Cer) and sphingosine-1-phosphate (S1P), associate closely to cancer growth and progression. Cer, its function as a tumor-suppressor lipid, has antiproliferative activity and apoptotic responses in various cancer cells. Conversely, S1P induces responses that, on aggregate, render SIP a tumor-promoting lipid. The cellular balance between Cer and SIP is an important determinant of cell fate.Retinoids, a group of structural and functional analogs of vitamin A, regulate several essential biological processes including cell growth, differentiation, and apoptosis. The effects of retinoids are mainly mediated by nuclear retinoid receptors, which include retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RAR and RXR proteins act as ligand-dependent transcription factors, which can modulate the transcriptional activity of retinoid receptor target genes by binding as RAR/RXR heterodimeric complexes to specific RAR or RXR response elements. Loss of retinoid receptors expression happens frequently in the development of carcinogenesis and induction of resistance to retinoids. In addition to modulating gene transcription in nucleus, RXRa also has attractive effects in cytoplasm among which translocation of RXRa/TR3heterodimer from the nucleus to the cytoplasm that has been proved to be an important apoptosis pathway.In this study, we aimed to invesgigate whether the induction of apoptosis by Cer is mediated by RXRa's translocation. We also studied the mechanism of resistance to retinoids in cancer cells mediated by SIP. These studies will help us to further understand the mechanisms of sphingolipid metabolites on the signaling pathways of retinoic acid and the function of retinoid receptors in cancer cells.Methods:1. Cer-induced translocation of RXRα from nucleus into cytoplasmThe redistribution of RXRα in SMMC-7721and H460cells induced by Cer was detected by assays of immunohistochemistry and western blotting. A GFP-RXRα expression vector was transfected into SMMC-7721cells to further explore the effect of Cer on RXRa subcellular localization. Luciferase reporter gene assay was employed to investigate the antagonization of Cer on9-cis-RA-induced activation of gene with a retinoic acid responsive element (RARE) in293A cells. The difference of growth inhibition induced by Cer in normal SMMC-7721and SMMC-7721/RXRa was determined by MTT assay. Western blotting was also used to detect the levels of RXRα and cleaved-PARP in SMMC-7721and H460cells exposure with Cer.2. S1P induces cancer cells resistant to retinoidsThe proliferation of SMMC-7721and PLC/PRF/5cells by SIP was estimated by MTT assy. Flow cytometry assay was used to analyze the distribution of cell-cycle phases of HT-29cells. The effects of S1P on ATRA (all-trans retinoic acid)-induced RARp expression in HT-29cells were valued by the assays of western blotting and Real-time PCR. Luciferase reporter gene assay was used to investigate whether SIP antagonize ATRA-induced activation of gene containing RARE in HT-29cells.The coherent distribution of Sphingosine kinase2(Sphk2) and RXRa in HT-29cells was detected by immunofluorescence and transient transfection, and the binding activity was observed by co-immunoprecipitation. In HT-29cells tranfected with HA-Sphk2and vehicle vector, western blotting and semi-quantative PCR were conducted to explore whether over-expressed Sphk2could inhibit RARβ expression induced by ATRA or LGD1069(RXR agonist). Luciferase reporter gene assay was used to investigate the antagonization of Sphk2on activation of RXR/RAR by ATRA or9-cis-RA in HT-29or293A cells.Semi-quantative PCR was used to verify the modulation of SIP on ATRA-induced RARβ expression and whether this modulation could be relieved by proteasome inhibitor MG132. The effects of SIP on modification and degradation of RXRa and RARP by ATRA or LGD1069were explored by the assay of western blotting.Results:1. Cer induced RXRa translocation from nucleus into cytoplasmIn culture medium containing0.5%serum,12h after treatment with10μM Cer, H460and SMMC-7721cells exhibited apoptosis features accompany with RXRa nuclear export. Immunoblotting affirmed the translocation of RXRa induced by Cer in SMMC-7721cells. Cer also induced migration of transfected GFP-RXRa in SMMC-7721cells after10h of treatment with Cer.2. Cer induced apoptosis not relying on RXRa's migration into cytoplasmReporter gene assay showed that Cer had no effect on the activation of RXR/RAR by9-cis-RA. In SMMC-7721/RXRa cells over-expressed RXRa, Cer didn't cause an increased growth inhibition compared with normal SMMC-7721cells. During the apoptosis process induced by Cer in SMMC-7721cells, cleaved-PARP increased as earlier as3h, but RXRa began to degrade at9h and decreased by14.2%at12h when RXRa's translocation was obvious. The degradation of RXRa induced by Cer was more obvious in H460cells.3. S1P antagonized the growth inhibitory effect of ATRA and LGD1069MTT results showed that by comparing with10μM ATRA treatment alone, combination of0.01and0.1μM of SIP with ATRA increased HT-29cell the proliferation of cells was increased by15.1%and26.2%, respectively, in HT-29cells and14.0%and15.2%, respectively, in H460cells, when ATRA combined with0.01and0.1μM of SIP. Flow cytometry analysis suggested that ATRA (5μM) arrested cancer cells in G1phase. However, concurrent treatment with ATRA and SIP (0.1μM) completely prevented the effect of Gl arrest from ATRA treatment.4. S1P suppressed ATRA-induced RARβ expressionWestern blotting results showed that the level of RARβ expression in HT-29cells was significantly increased by ATRA (1μM). But the increase of RARβ expression was substantially and dose-dependently suppressed by SIP. This suppression effect also happened in293A and SMMC-7721cells. Real-time PCR results demonstrated that the antagonizing effect of SIP on ATRA-induced RARβ expression appeared at3h and became more obvious as the time went on. The assay of reporter gene showed that RARE activity in ATRA-treated cells was decreased by7.8%,19.4%or37.3%when combination use with0.001,0.01, or0.1μM of S1P.5. Sphk2binded to RXRa and suppressed the activation of RXR/RAR by ATRA and LGD1069By using the assays of immunofluorescence and transient transfection, we found that both HA-Sphk2and GFP-RXRα were stayed in nucleus of HT-29cells and then migrated into cytoplasm in the presence of the stimuli of TPA. This phenomenon also happened to endogenous Sphk2and RXRa. RXRa binded to Sphk2, but not Sphk1were certified by co-immunoprecipitation. The results of RT-PCR and Western blotting showed that in HT-29cells transfected with Sphk2, ATRA and LGD1069lost the ability to induce the expression of RARβ. Luciferase activity assay results indicated that SphK2down-regulated the transcription of RARE activated by ATRA and9-cis-RA.6. The proteasomal degradation pathway was involved in S1P's modulation of ATRA-induced RARβ expressionRT-PCR results showed that in the presence of SIP, the induction of RARβ by ATRA was only41.8%of that with ATRA treatment alone. However, when the cells were concurrently treated with ATRA, SIP and MG132, the expression of RARβ transcript was markedly increased by92.3%of that with ATRA treatment alone. The expression of COX-2showed that the SIP's modulation of ATRA-induced RARβ expression could be relieved by proteasome inhibitor MG132.7. S1P enhanced the ligand-dependent degradation of retinoid receptorsWestern blotting showed that LGD1069decreased RXRα expression and a further decrease of RXRa expression by47.3%was detected after treatment with a combination of LGD1069and SIP. In contrast with the decrease of RXRa by LGD1069plus S1P, a significant increase of RXRα by91.7%was observed following exposure to a combination of S1P and ATRA, indicating that the loss of RARβ expression blocked the degradation of RXR a by ATRA. When HT-29cells were exposed to ATRA and MG132, a higher-molecular-weight band appeared in the gel and became more obvious when S1P was applied; indicating that S1P might enhance ATRA induced modification of RARβ. Consistent with the modified RARβ band, when acetylated protein was detected, a new band between51and55kDa was observed in HT-29cells when exposed to ATRA and MG132and the bands were became more obvious when the cells were exposed concurrently to ATRA, SIP, and MG132. We hypothesize that the ligand-dependent acetylation might involve the modulation or degradation of RARβ induced by S1P.Conclusions:The nuclear export of RXRa by Cer did not accounted for the initiation of apoptosis but the degradation of RXRa. The enhancement of ligand-dependent degradation of retinoid receptors by SIP suppressed retinoids-induced gene expression which caused the induction of retinoid resistance in tumor cells. Sphk2could suppress the activation of RXR/RAR by their agonist.