Soyeon Jeong

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.

Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition

The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.

Docosahexaenoic acid prevents paraquat-induced reactive oxygen species production in dopaminergic neurons via enhancement of glutathione homeostasis

Omega-3 polyunsaturated fatty acid levels are reduced in the substantia nigra area in Parkinsons disease patients and animal models, implicating docosahexaenoic acid (DHA) as a potential treatment for preventing Parkinsons disease and suggesting the need for investigations into how DHA might protect against neurotoxin-induced dopaminergic neuron loss. The herbicide paraquat (PQ) induces dopaminergic neuron loss through the excessive production of reactive oxygen species (ROS). We found that treatment of dopaminergic SN4741 cells with PQ reduced cell viability in a dose-dependent manner, but pretreatment with DHA ameliorated the toxic effect of PQ. To determine the toxic mechanism of PQ, we measured intracellular ROS content in different organelles with specific dyes. As expected, all types of ROS were increased by PQ treatment, but DHA pretreatment selectively decreased cytosolic hydrogen peroxide content. Furthermore, DHA treatment-induced increases in glutathione reductase and glutamate cysteine ligase modifier subunit (GCLm) mRNA expression were positively correlated with glutathione (GSH) content. Consistent with this increase in GCLm mRNA levels, Western blot analysis revealed that DHA pretreatment increased nuclear factor-erythroid 2 related factor 2 (Nrf2) protein levels. These findings indicate that DHA prevents PQ-induced neuronal cell loss by enhancing Nrf2-regulated GSH homeostasis.

Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation

Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel–Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.

Mechanism of Anti-Invasive Action of Docosahexaenoic Acid in SW480 Human Colon Cancer Cell

Colon cancer is one of the most common malignancies in the western world and the second leading cause of cancer death in Korea. Epidemiology studies have shown a reduced incidence of colon cancer among populations consuming a large quantity of ω3-polyunsaturated fatty acids (ω3-PUFA) of ma- rine origin. Recently, it has been found that ω 3-PUFA has an antineoplastic effect in several cancers. This study was designed to investigate the mechanism of the anti-invasive effect of ω3-PUFA in colon cancer. ω3-PUFA, docosahexaenoic acids (DHA) and eicosapentaenoic acid ( EPA) treatment resulted in a dose-dependent inhibition of cell growth in SW480 human colon cancer cells. In contrast, arach- idonic acid (AA), a ω6-PUFA, exhibited no significant effect. This action likely involves apoptosis, giv- en that DHA treatment increased apoptotic cells in TUNEL assay. Moreover, invasiveness of SW480 cells was inhibited following treatment of DHA in a dose-dependent manner; in contrast, AA had no effect. The levels of MMP-9 and MMP-2 mRNA decreased after DHA pretreatment. MMP-9 and MMP-2 promoter activities were also inhibited by DHA treatment. The levels of NF-kB and p-IkB pro- tein were down-regulated by DHA pretreatment in a dose dependent manner. In addition, DHA in- hibited NF-kB promoter reporter activities. These findings suggest that ω3-PUFA may inhibit cancer cell invasion by inhibition of MMPs via reduction of NF-kB in colon cancer. In conclusion, ω3-PUFA could be used for chemoprevention and treatment of human colon cancer.

Abstract 2867: Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling in human cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Although Omega 3-polyunsatuarated fatty acids (Omega 3-PUFAs) induce cytotoxicity in several cancer cell lines, the exact mechanisms are not identified yet. In this study, we showed that autophagy is involved in the Omega 3-PUFAs-induced cytotoxicity in cancer cells. Autophagy is characterized by the sequestration of cytoplasmic material within autophagosomes for bulk degradation by lysosomes. We found that docosahexaenoic acid (DHA), an Omega 3-PUFA, induced not only apoptosis but also autophagy in cancer cells. Autophagy was detected after DHA exposure as indicated by induction of LC3 expression, and formation of autophagic vacuolization. We observed that the DHA-induced autophagy was accompanied by loss of p53. Inhibition of p53 by Pifithrin-α or microRNA-p53 significantly increased the DHA-induced autophagy, suggesting that the DHA-induced autophagy is mediated by downregulation of p53. Further experiments showed that the mechanism of the DHA-induced autophagy associated with p53 attenuation, involved an increase in the active form of AMPK which attenuated the mTOR activity as revealed by p27 sequestration. In addition, compelling evidence for the interplay between autophagy and apoptotic cell death induced by DHA is supported by the findings that autophagy inhibition partially decreased the DHA-induced apoptotic cell death and further autophagy induction by p53 inhibitor enhanced apoptosis in response to treatment with DHA in cancer cells. Our results demonstrate that autophagy may be related to the DHA-induced cytotoxicity in cancer cells. [This work was supported by basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (E00026 and R13-2007-020-01000-0]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2011-2867

Abstract 2110: Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells

Glioblastoma (GBM) is the most aggressive and deadliest brain malignancy in adults. Despite of surgical techniques, radiotherapy and chemotherapy, GBM has remained an invariably lethal tumor with a median survival less than 15 months. Although the anticancer mechanisms of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been reported in several cancers, it is still unclear in brain cancer. Here, our data show the anti-invasive and anti-metastatic effects of ω3-PUFAs in GBM. Invasiveness using transwell chamber was inhibited by docosahexaenoic acid (DHA) treatment in D54MG and GL261 cells. In zymography, MMP-2 activity was suppressed by DHA in a dose dependent manner. MMP-2 promoter activity was also decreased. DHA inhibited both p-STAT3 and β-catenin levels that contribute to invasion by stimulating pro-invasion factors such as MMP-2. Additionally, fat-1 (ω3-desaturease) stable D54MG cell was established and the effect of the high level of ω3-PUFAs was investigated endogenously. The invasiveness were significantly inhibited in the fat-1 stable cells compared to control cell. Moreover, the metastasis in vivo was significantly reduced when GL261 mouse glioma cells were injected through tail vein into the fat-1 transgenic mice. In immunohistochemistry, intensity of p-STAT-3 and β-catenin were significantly weak in metastatic lung tumor from Fat-1 mice compared to wild type mice. These results indicate that anti-invasive and anti-metastatic actions of ω3-PUFA may be correlated with inhibition of MMP-2 through decrease of STAT3 and β-catenin in GBM cells. Thus, these findings provide important preclinical evidence and molecular insight for utilization of ω-3 PUFAs for the chemoprevention and treatment of human GBM. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)]. Citation Format: Soyeon Kim, Soyeon Shin, Soyeon Jeong, Seung-Hyeon Han, Yoon-Seon Yoo, Prashanta Silwal, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Anti-invasive and antimetastatic actions of omega-3 polyunsaturated fatty acids in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2110. doi:10.1158/1538-7445.AM2017-2110

Abstract 1301: Anti-invasive and antimetastatic effects of ω3-polyunsaturated fatty acids through inhibition of NF-kB/matrix metalloproteinases in ovarian cancer cells

Ovarian cancer is leading cause of gene cological cancer death in the United States. It is reported that about 14,000 patients will die to ovarian cancer in United States, 2015. ω3- and ω6-PUFAs are considered as essential fatty acids because they cannot be synthesized in mammals. The ω3-desaturase (fat-1) converts ω6-PUFAs to ω3-PUFAs. Cancer cells and transgenic mice stably expressing fat-1 geneare useful models to study the anti-cancer effects of ω3-PUFAs. Here, we show anti-invasive and anti-metastatic action of ω3-PUFA in ovarian cancer.DHA significantly inhibited cell invasion and wound healing activity of human ovarian PA-1 cells. DHA also abolishedthe induction of matrix-metalloproteinases (MMPs) and cyclooxygenase (COX2) as well as the transactivity of NF-κB in PA-1 cells. When ID8 cells were subcutaneously implanted into fat-1 transgenic mice, the tumor size and volume were smallin comparison to wild-type mice. The growth inhibition of tumor was also confirmed with the increase in the level of TUNEL-positive cells. Additionally, when ID8 cells were injected via tail vein of fat1 mice, the lung metastasis was remarkably inhibited in fat-1 transgenic mice. Furthermore, the proliferation of fat-1-stably expressing PA-1 (f-PA-1) cells was more attenuated than that of cells expressing control vectors. The invasion and NF-κB/MMPs promoter activities were also decreased in f-PA-1 cells. These results suggest that ω3-PUFAs inhibit invasion and metastasis of ovarian cancer cells through inhibition of NF-κB/MMPs. Therefore, ω3-PUFAs may contribute an effective and safe therapeutic approach for the chemoprevention and treatment of human ovarian cancer. [This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932)] Citation Format: Soyeon Jeong, Kaipeng Jing, Soyeon Shin, Soyeon Kim, Young-Joo Jeon, Jun-Young Heo, Gi-Ryang Kweon, Seung-Kiel Park, Jong-Il Park, Kyu Lim. Anti-invasive and antimetastatic effects of ω3-polyunsaturated fatty acids through inhibition of NF-kB/matrix metalloproteinases in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1301.