Seung-Kiel Park

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.

The Omega-3 Polyunsaturated Fatty Acid DHA Induces Simultaneous Apoptosis and Autophagy via Mitochondrial ROS-Mediated Akt-mTOR Signaling in Prostate Cancer Cells Expressing Mutant p53

Docosahexaenoic acid (DHA) induces autophagy-associated apoptotic cell death in wild-type p53 cancer cells via regulation of p53. The present study investigated the effects of DHA on PC3 and DU145 prostate cancer cell lines harboring mutant p53. Results show that, in addition to apoptosis, DHA increased the expression levels of lipidated form LC3B and potently stimulated the autophagic flux, suggesting that DHA induces both autophagy and apoptosis in cancer cells expressing mutant p53. DHA led to the generation of mitochondrial reactive oxygen species (ROS), as shown by the mitochondrial ROS-specific probe mitoSOX. Similarly, pretreatment with the antioxidant N-acetyl-cysteine (NAC) markedly inhibited both the autophagy and the apoptosis triggered by DHA, indicating that mitochondrial ROS mediate the cytotoxicity of DHA in mutant p53 cells. Further, DHA reduced the levels of phospho-Akt and phospho-mTOR in a concentration-dependent manner, while NAC almost completely blocked that effect. Collectively, these findings present a novel mechanism of ROS-regulated apoptosis and autophagy that involves Akt-mTOR signaling in prostate cancer cells with mutant p53 exposed to DHA.

Docosahexaenoic acid-induced apoptosis is mediated by activation of mitogen-activated protein kinases in human cancer cells

BackgroundThe role of omega-3 polyunsaturated fatty acids (ω3-PUFAs) in cancer prevention has been demonstrated; however, the exact molecular mechanisms underlying the anticancer activity of ω3-PUFAs are not fully understood. Here, we investigated the relationship between the anticancer action of a specific ω3-PUFA docosahexaenoic acid (DHA), and the conventional mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38 whose dysregulation has been implicated in human cancers.MethodsMTT assays were carried out to determine cell viability of cancer cell lines (PA-1, H1299, D54MG and SiHa) from different origins. Apoptosis was confirmed by TUNEL staining, DNA fragmentation analysis and caspase activity assays. Activities of the conventional MAPKs were monitored by their phosphorylation levels using immunoblotting and immunocytochemistry analysis. Reactive oxygen species (ROS) production was measured by flow cytometry and microscopy using fluorescent probes for general ROS and mitochondrial superoxide.ResultsDHA treatment decreased cell viability and induced apoptotic cell death in all four studied cell lines. DHA-induced apoptosis was coupled to the activation of the conventional MAPKs, and knockdown of ERK/JNK/p38 by small interfering RNAs reduced the apoptosis induced by DHA, indicating that the pro-apoptotic effect of DHA is mediated by MAPKs activation. Further study revealed that the DHA-induced MAPKs activation and apoptosis was associated with mitochondrial ROS overproduction and malfunction, and that ROS inhibition remarkably reversed these effects of DHA.ConclusionTogether, these results indicate that DHA-induced MAPKs activation is dependent on its capacity to provoke mitochondrial ROS generation, and accounts for its cytotoxic effect in human cancer cells.

Vimentin phosphorylation by Cdc2 in Schwann cell controls axon growth via β1-integrin activation

Although preconditioning injury on the peripheral nerve induces axonal regenerative capacity in neurons, it is not known whether similar lesion effects occur in glial cells. Here we demonstrate that Schwann cells are activated by peripheral nerve preinjury and primed to mediate axon regeneration. Cdc2, which was induced from Schwann cells after sciatic nerve injury, phosphorylated vimentin almost exclusively in the distal nerve area. Phospho‐vimentin‐positive Schwann cells showed increased migration activity and were in close contact with process outgrowth of co‐cultured neurons. Vimentin phosphorylation by Cdc2 was involved in β1‐integrin activation leading to FAK phoshorylation and associated with Erk1/2 activation in Schwann cells. Neurite outgrowth of dorsal root ganglion neurons was increased by co‐culture with activated Schwann cells, in which phospho‐vimentin signaling was transmitted into β1‐integrin activation. Then neurite outgrowth was suppressed by genetic depletion of phospho‐vimentin and β1 integrin as well as inhibition of vimentin phosphorylation by Cdc2 inhibitor purvalanol A. The sciatic nerve graft harboring activated Schwann cells into the spinal cord induced Schwann cell migration beyond the graft‐host barrier and facilitated regeneration of spinal axons, which was inhibited by purvalanol A pretreatment of the graft. This is the first report to our knowledge demonstrating that activation of phospho‐vimentin linked to β1‐integrin pathway may mediate transcellular signaling to promote axon growth.—Chang, I. A., Oh, M.‐J., Kim, M. H., Park, S.‐K., Kim, B. G., Namgung, U. Vimentin phosphorylation by Cdc2 in Schwann cell controls axon growth via β1‐integrin activation. FASEB J. 26, 2401‐2413 (2012). www.fasebj.org

Growth-promoting activity of Hominis Placenta extract on regenerating sciatic nerve

AbstractAim:Extract of Hominis Placenta (HP) has been used in oriental medicine as an agent for improving physiological function. The present study was conducted to investigate whether HP treatment in an experimental sciatic nerve injury animal model produces growth-promoting effects on regenerating peripheral nerve fibers after injury.Methods:After HP was injected into a sciatic nerve injury site, changes in protein levels were analyzed in the regenerating nerve area by Western blotting and immunofluorescence staining analyses. For quantitative assessment of axonal regeneration, a retrograde tracing technique was used to identify the neuronal cell bodies corresponding to regenerating axons, and the extent of neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons prepared from animals that had experienced a sciatic nerve crush injury 7 d before neuron collection was analyzed.Results:Induction levels of axonal growth-associated protein (GAP-43) in the injured sciatic nerves were elevated by HP treatment. HP treatment also upregulated cell division cycle 2 (Cdc2) protein levels in the distal stump of the injured sciatic nerve. Induced Cdc2 protein was detected in Schwann cells, suggesting that Cdc2 kinase activity maybe involved in the growth-promoting activity of regenerating axons via Schwann cell proliferation. Cell body measurement by retrograde tracing indicated that HP treatment produced significant increases in regenerating motor axons. Finally, HP treatment of cultured DRG sensory neurons significantly increased neurite arborization and elongation.Conclusion:HP promotes the regeneration of injured sciatic axons by upregulating the synthesis of regeneration-related protein factors such as GAP-43 and Cdc2.

Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC

BackgroundRET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although it has been established that RET/PTC kinase plays a crucial role in intracellular signaling pathways that regulate cellular transformation, growth, and proliferation in thyroid epithelial cells, the upstream signaling that leads to the activation of RET/PTC is largely unknown. Based on the observation of high levels of PLD expression in human papillary thyroid cancer tissues, we investigated whether PLD plays a role in the regulating the RET/PTC-induced STAT3 activation.MethodsCancer tissue samples were obtained from papillary thyroid cancer patients (n = 6). The expression level of PLD was examined using immunohistochemistry and western blotting. Direct interaction between RET/PTC and PLD was analyzed by co-immunoprecipitation assay. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of n-butanol. The transcriptional activity of STAT3 was assessed by m67 luciferase reporter assay.ResultsIn human papillary thyroid cancer, the expression levels of PLD2 protein were higher than those in the corresponding paired normal tissues. PLD and RET/PTC could be co-immunoprecipitated from cells where each protein was over-expressed. In addition, the activation of PLD by pervanadate triggered phosphorylation of tyrosine 705 residue on STAT-3, and its phosphorylation was dramatically higher in TPC-1 cells (from papillary carcinoma) that have an endogenous RET/PTC1 than in ARO cells (from anaplastic carcinoma) without alteration of total STAT-3 expression. Moreover, the RET/PTC-mediated transcriptional activation of STAT-3 was synergistically increased by over-expression of PLD, whereas the PLD activity as a lipid hydrolyzing enzyme was not affected by RET/PTC.ConclusionThese findings led us to suggest that the PLD synergistically functions to activate the STAT3 signaling by interacting directly with the thyroid oncogenic kinase RET/PTC.

Shikonin induces apoptosis of lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling antagonized by p300

Shikonin derivatives exert powerful cytotoxic effects including induction of apoptosis. Here, we demonstrate the cytotoxic efficacy of shikonin in vivo in xenograft models, which did not affect body weight as well as its reduction of cell viability in vitro using several non-small cell lung cancer (NSCLC) cell lines. We found that inhibition of AKT by shikonin activated the forkhead box (FOX)O3a/early growth response protein (EGR)1 signaling cascade and enhanced the expression of the target gene Bim, leading to apoptosis in lung cancer cells. Overexpression of wild-type or a constitutively active mutant of FOXO3a enhanced shikonin-induced Bim expression. The NAD+-dependent histone deacetylase sirtuin (SIRT)1 amplified the pro-apoptotic effect by deacetylating FOXO3a, which induced EGR1 binding to the Bim promoter and activated Bim expression. Meanwhile, PI3K/AKT activity was enhanced, whereas that of FOXO3a was reduced and p300 was upregulated by treatment with a sublethal dose of shikonin. FOXO3a acetylation was enhanced by p300 overexpression, while shikonin-induced Bim expression was suppressed by p300 overexpression, which promoted cell survival. FOXO3a acetylation was increased by p300 overexpression and treatment with SIRT1 inhibitor, improving cell survival. In addition, shikonin-induced FOXO3a nuclear localization was blocked by AKT activation and SIRT1 inhibition, which blocked Bim expression and conferred resistance to the cytotoxic effects of shikonin. The EGR1 increase induced by shikonin was restored by pretreatment with SIRT1 inhibitor. These results suggest that shikonin induces apoptosis in some lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling, and that AKT and p300 negatively regulate this process via Bim upregulation.

Adenine suppresses IgE-mediated mast cell activation

Nucleobase adenine is produced by dividing human lymphoblasts mainly from polyamine synthesis and inhibits immunological functions of lymphocytes. We investigated the anti-allergic effect of adenine on IgE-mediated mast cell activation in vitro and passive cutaneous anaphylaxis (PCA) in mice. Intraperitoneal injection of adenine to IgE-sensitized mice attenuated IgE-mediated PCA reaction in a dose dependent manner, resulting in a median effective concentration of 4.21 mg/kg. In mast cell cultures, only adenine among cytosine, adenine, adenosine, ADP and ATP dose-dependently suppressed FcɛRI (a high affinity receptor for IgE)-mediated degranulation with a median inhibitory concentration of 1.6mM. It also blocked the production of LTB4, an inflammatory lipid mediator, and inflammatory cytokines TNF-α and IL-4. In addition, adenine blocked thapsigargin-induced degranulation which is FcɛRI-independent but shares FcɛRI-dependent signaling events. Adenine inhibited the phosphorylation of signaling molecules important to FcɛRI-mediated allergic reactions such as Syk, PLCγ2, Gab2, Akt, and mitogen activated protein kinases ERK and JNK. From this result, we report for the first time that adenine inhibits PCA in mice and allergic reaction by inhibiting FcɛRI-mediated signaling events in mast cells. Therefore, adenine may be useful for the treatment of mast cell-mediated allergic diseases. Also, the upregulation of adenine production may provide another mechanism for suppressing mast cell activity especially at inflammatory sites.

Association of castration-dependent early induction of c-myc expression with a cell proliferation of the ventral prostate gland in rat

The protooncogene c-myc is known to be associated with both cell proliferation and apoptosis. The possible cellular affects of castration on the ventral prostate gland of rat as well as the relationship to a castration induced c-myc expression were examined. Levels of c-myc mRNA in the ventral prostate gland peaked at 6 h (early induction) and 48 h (late induction) after castration, respectively. Castration-induced DNA fragmentation was not observed at an early induction of c-myc mRNA. DNA fragmentation appeared to be testosterone-dependent. On the other hand, cellular DNA synthesis measured by [3H]thymidine uptake in the ventral prostate gland was increased to maximum at 6 h after castration. These results suggest that an early induction of c-myc mRNA in ventral prostate gland after castration is closely associated with cell proliferation of the gland.

ATF is important to late S phase-dependent regulation of DNA topoisomerase IIα gene expression in HeLa cells

DNA topoisomerase IIalpha (Topo IIalpha) is regulated in late S phase-dependent manner. To identify late S phase-dependent cis-acting elements of Topo IIalpha gene, we have investigated the synchronized HeLa cells with chloramphenicol acetyltransferase and DNase I footprinting assays. The level of Topo IIalpha mRNA increased after release from aphidicolin block and reached a maximum in 8h (late S phase) in HeLa cells, and Topo II unknotting activity was also in parallel with the level of Topo IIalpha mRNA. The late S phase-regulatory element was found to be located in the region containing ATF-binding element between -290 and -90bp and the region was required for a maximal stimulation during late S phase. DNase I footprinting assay showed that ATF-binding element and novel cis-acting element (Topo IIalpha-specific sequence) were the principal protein-binding sites and the proteins interacting with these elements were induced during late S phase. One DNA-protein complex was formed by DNA mobility shift assay when ATF-binding site was incubated with nuclear extract prepared from late S phase cells, but no protein bound in non-S phase cells. Taken together, these results suggest that ATF may be essential transacting factor for maximal expression of Topo IIalpha gene during late S phase in HeLa cells.