Research On The Mechanism Of Autophagy In Cardiomyocytes And Non-small Cell Lung Cancer Cells Promoted By Sphingosylphosphorylcholine

Background and objectiveSphingosylphosphorylcholine (SPC), a bioactive sphingolipid, was found to be naturally occurred in blood plasma or produced by the intermediary metabolism of sphingomyelin in cells. Similar with other structurally related bioactive lipids, such as LPA and SIP, SPC works not only as the membrane component, but also acts as a signal transduction modulator. SPC was reported to modulate the development and progression of diverse diseases including cancer, cardiovascular diseases, Niemann-Pick disease (NPD), atopic dermatitis, and vasospasm. Thereby, as a bioactive sphingolipid, SPC has promising prospect for research.Myocardial Infarction (MI) is one of the leading causes of the mortality in developing country. The direct pathological cause is the deficiency of the nutrient and oxygen. Cardiac myocyte apoptosis is the major mechanism for heart failure. Since cardiomyocyte is terminally differentiated with limited proliferation ability, the loss of cells caused by ischemia may be detrimental to the heart. Autophagy is a conservative process which eliminates the long-lived proteins and injured organisms to maintaining the homeostasis of the cell. Stimulations within and out of the cells may evoke autophagy. Accumulated evidences suggest that autophagy play a pivotal role in determining the cell fate. Autophagy suppresses apoptosis to promote cell survival, while overdue autophagy causes cell death which is definated as autophagic cell death. It remained to be elucidated about the relationship and the specific molecular mechanism of autophagy and apoptosis.To answer those scientific questions mentioned above, this study investigated the effect of SPC on autophagy and apoptosis as well as their relationship in cardiomyocytes. We further explored the molecular mechanism involved in SPC regulated autophagy. Our research is beneficial for understanding the autophagy mechanism and the signal transduction of bioactive lipid SPC. In addition, this will also provide a novel target for ischemic heart disease therapy.With regarding to the fact that SPC induced autophagy in both human vascular endotherlium cells and cardiomyocytes, we speculated that SPC could be a popular autophagy inducer. It has been accepted that autophagy plays a complicated role in the initiation and progression of cancers. Through promoting the recycling of metabolism substrates or hampering inflammation, autophagy preserves cell viability and may cause the anti-chemotherapeutic effect. Nevertheless, autophagic cell death can also be employed as mechanism of drugs to kill those cancer cells. Since information about the autophagy and treatment of lung cancers is limited, we investigated the effect of SPC in non-small cell lung cancer cells. Our research not only confirmed the polularity effect of SPC as an autophagy inducer, but also supported an effective biomarker for timely diagnosis of the malignant lung cancer.Results1. Sphingosylphosphorylcholine protects cardiomyocyte against ischemic apoptosis via Lipid raft/PTEN/Aktl/mTOR mediated autophagy.1.1 SPC suppressed cardiomyocyte apoptosis.We investigated the influence of SPC on hypoxia induced apoptosis of Neonatal Rat Cardiac Myocytes (NRCMs) and the serum deprivation induced apoptosis of H9c2 cells, respectively. Hoechst 33258 staning showed that SPC inhibited the nuclear condensation and fragmentation in both NRCMs and H9c2 cells. ANXA5-PE/PI staining and flow cytometry analysis revealed that SPC suppressed the hypoxia induced apoptosis of NRCMs. Western blot showed that SPC decreased levels of the cleaved caspase3 and PARP1 in H9c2 cells. Besides, SPC reversed the rising of the intracellular ROS level caused by serum deprivation in H9c2 cells. Thus SPC was identified as an apoptosis inhibitor for cardiomyocytes.1.2 SPC promoted autophagy in cardiomyocytes.Autophagy was a bona fide mediator regulating cardiomyocyte apoptosis. Thus we detected the effect of SPC on autophagy. Western blot showed that SPC increased the level of LC3Ⅱ in H9c2 cells. We transfected H9c2 cells with the GFP tagged LC3 plasmid to detect the formation of the autophagosomes. Fluorescence microscopy revealed a much more obvious punctate pattern of GFP-LC3 in SPC-treated cells.To validating the effect of SPC on the autophagy flux, we detected the specific substrate of autophagy, p62. SPC promoted the degradation of p62. Pretreatment with Bafilomycin Al, an inhibitor for the flow of the autophagy, aggravated SPC induced accumulation of LC3 Ⅱ. These data suggested that SPC promoted the flow of autophagy in H9c2 cells.1.3 Autophagy evoked by SPC was protective.In order to confirm the protective role of autophagy induced by SPG,we pretreated H9c2 cells with 3MA before the addition of SPC. Western blot showed that 3MA inhibited SPC induced accumulation of LC3 Ⅱ.3MA treatment reversed the suppressing effect of SPC on the cleavage of Caspase 3 and Caspase 7. These data confirmed the pivotal role of autophagy in the cardioprotective effect of SPC.1.4 SPC induced autophagy through mTOR and JNK signal pathway.MTORC1 complex is a fundamental gatekeeper of autophagic activity. Thus we first detected the activity of it. Western blot showed that the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) were upregulated by SPC at early 5 min while downregulated at 12 h when the autophagy occurs. Thus we deduced that mTOR mediated SPC-induced autophagy in H9c2 cells in a complicated manner. Since autophagy were mainly regulated through mTOR and Beclinl/Vps34 complex. We next detected the effect of SPC on the Beclinl/Vps34 signal pathway. Western blot showed no variation of Beclinl, which suggested that SPC induced a Beclinl-independent autophagy.JNK and p38MAPK, belonging to MAPK signaling pathway, are two other important elements in regulating both autophagy and apoptosis. Western blot showed that SPC significantly increased phosphorylation of JNK, but did not affect p38MAPK activity. JNK suppression by its inhibitor SP600125 hampered SPC-induced autophagy. Hence, SPC could induce autophagy via phosphorylation of JNK.1.5 SPC acted as a switch of Akt activity.PI3K/Akt is widely known as the regulator of both mTOR and JNK. We then proceeded to study the influence of SPC on Akt. Western blot showed that SPC activated Akt at early 5min and inhibited the activation of it later at 12 h. Pretreatment of H9c2 cells with PI3K/Akt inhibitors LY 294002 and Wortmanin further promoted the conversion of LC3, demonstrating that restraining Akt/mTOR pathway promoted the autophagy induced by SPC. The further immunoprecipitation experiment identified that it was Akt 1 isoform mainly regulated by SPC.1.6 Lipid raft might participate in the regulation of SPC induced autophagy through PTEN/Akt pathway.Abundant evidences have shown that activity of Akt might be affected by the lipid raft. It was reported that lipid raft was involved in SPC caused contraction of rat vascular smooth muscle cells. Thus we determined whether Lipid raft participates in SPC-induced autophagy.β-cyclodextrin could deplete the cholesterol on plasma membranes, thus being employed as a popular used tool to destroy the membrane lipid raft. Western blot showed that pretreatment with β-cyclodextrin eliminated SPC induced accumulation of LC3Ⅱ inhibition of Akt phosphorylation as well as PTEN phosphorylation.2. Inhibition of autophagy promoted sphingosylphosphorylcholin induced cell death in non-small cell lung cancer cells2.1 SPC induced autophagy in non-small cell lung cancer cells.Since SPC induced autophagy in both human vascular endotherlium cells and cardiomyocytes, we speculated that SPC could be a popular autophagy inducer. We investigated the effect of SPC on autophagy in non-small cell lung cancer cells. Western blot showed that SPC induced the accumulation of LC3-Ⅱ in A549 and H157 lung cancer cells.3MA suppressed SPC induced accumulation of LC3-Ⅱ. Pepstatin A, inhibiting the lysosome enzyme cathepstin D activity thereby blocking the autophagy flow, promoted the SPC induced accumulation of LC3-Ⅱ. It could be concluded from the above-mentioned results that SPC promoted the autophagy flux in non-small cell lung cancer cells.2.2 SPC induced cell loss in non-small cell lung cancer cells.Autophagy plays an important role in cancer cell survival which could promote cell growth or cause cell death.The morphology changes and SRB assay showed that SPC inhibited A549 and H157 lung cancer cell growth at 5 μM and 10μM.2.3 SPC suppressed apoptosis in in non-small cell lung cancer cells.To determine which way was responsible for SPC-induced cell death,we detected apoptosis related proteins. Western blot showed that SPC inhibited the cleavage of PARP1, indicating that SPC inhibited apoptosis in non-small cell lung cancer cells.2.4 SPC induced necrosis in non-small cell lung cancer cells.We further investigated the necrosis of the cells. LDH assay suggested that SPC drastically promoted the LDHactivity. Thereby, SPC caused cell death through necrosis but not apoptosis.2.5 SPC induced autophagy to inhibit apoptosisTo verify the relationship between SPC induced autophagy and cell death, we suppressed autophagy with 3MA. SRB assay showed that cell death was not reduced but furtherly increased. We further detected the apoptosis and necrosis. Apoptosis but not LDH release was reverted with the treatment of 3MA. So we concluded that SPC induced autophagy only works as a negative regulator of apoptosis in non-small cell lung cancer cells.2.6 SPC induced autophagy through Akt/mTOR pathway.Since the Akt/mTOR signaling pathway was involved in SPC modulated autophagy in cardiomyocyte, we verified this mechanism in non-small cell lung cancer cells. Western blot showed that 10 uM SPC repressed the phosphorylation of p70S6K and 4EBP1 (two substrates of mTORCl). Akt phosphorylation was inhibited by SPC at both 5 and 10μM. These data showed that SPC induced autophagy through the Akt /mTORCl signal pathway.2.7 Involvement of p53 in SPC induced autophagy.p53 was down regulated during the process of SPC-promoted autophagy in human umbilical vein endothelial cells (HUVECs). Thus we determined whether p53 participated in SPC induced autophagy in cancer cells by using two lung cancer cell lines:A549 (p53 wide type) and H1299 (p53 deficient type) cells. SPC induced accumulation of LC3-II in p53 wide-type A549 cells. On the contrary, SPC did not induce accumulation of LC3-Ⅱ in p53 deficient H1299 cells. These indicated that p53 may also participate in the regulation of SPC induced autophagy. We further detected that SPC reduced the mitochondria membrane potential and ROS level.