• Molecules and Cells
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• v.42 no.6
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• pp.448-459
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• 2019

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is a promising target for gastric cancer (GC) treatment; however the efficacy of PI3K/mTOR dual inhibitors in GC has not yet been maximized. Additionally, the effect of autophagy regulation by PI3K/mTOR dual inhibitors has not been clearly elucidated in GC treatment. We aimed to show that our newly developed PI3K/mTOR dual inhibitor, CMG002, when combined with an autophagy inhibitor, chloroquine (CQ), potently induces effective cancer cell death in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) cells, where both the PI3K/AKT/mTOR and autophagy pathways play important roles in disease pathogenesis. EBV- and mock-infected AGS and NUGC3 GC cell lines were treated with CMG002 +/- CQ. PI3K/AKT/mTOR signaling pathway mediators, cellular apoptosis and autophagy markers were confirmed by Western blot assay. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. CMG002 effectively blocked the PI3K/AKT/mTOR pathway by markedly decreasing phosphorylation of AKT and its downstream mediator S6. CMG002 induced G0/G1 cell cycle arrest and enhanced apoptotic cell death in AGS and NUGC3 cells, particularly EBV-infected cells compared with mock-infected cells, as confirmed by flow cytometric analyses and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays. The combination of CMG002 plus CQ synergistically increased apoptotic cell death in EBV-infected GC cell lines when compared with CMG002 alone (P < 0.05). Our results suggest that the new PI3K/mTOR dual inhibitor, CMG002, when used in combination with the autophagy inhibitor, CQ, provides enhanced therapeutic efficacy against EBVaGC.

• Development and Reproduction
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• v.4 no.1
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• pp.29-35
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• 2000

A phosphatidylinositol 3-kinase (PI3K) is a upstream component of insulin signaling by which protein synthesis can be stimulated in many systems. To elucidate involvement of PI3K and its downstream mammalian target of rapamycin (mTOR) in the insulin signaling in pleimplantation mouse embryos, 8-cell embryos were cultured to blastocysts in the presence or absence of insulin and／or inhibitor drugs. The number of blastomeres per blastocyst, protein synthesis, and protein phosphorylation were examined. There was significant difference in embryonic development to blastocyst stage and hatching was potentiated by the insulin supplementation. The increase in the mean celt numbers per blastocyst was apparent in the insulin culture. Wortmannin, a PI3K inhibitor and rapamycin, an inhibitor of mTOR abolished the stimulatory effect of insulin on morphological development mitosis and protein synthesis. In autoradiography, phosphoproteins pp22 and pp30 which undergo phosphorylation in response to insulin were identified. Taken together, it can be suggested that PI3K and mTOR engaged in insulin signaling in the mouse embryo 8-cell onward and mediate embryotropic offset of insulin.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.17 no.1
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• pp.24-30
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• 2017

Gorham-Stout disease is a rare disorder characterized by lymphovascular proliferation and destruction of osseous matrix. The etiology of this condition remains poorly understood. Chylothorax as a consequence of lymphatic leakage in thoracic cage may cause a severe life-threatening complication, accompanying respiratory difficulty. Currently, there is no standard management for this extremely rare condition. Here we describe a patient affected by Gorham-Stout disease successfully managed by the combined treatment of mTOR inhibitor and beta-blocker. A previously healthy 11-year-old female developed dyspnea and chest pain with a massive pleural effusion. The ligation of right thoracic duct and bilateral pleurodesis temporarily decreased her pleural effusion, which was aggravated repetitively and required frequent admission and tube thoracotomies. Along with bilateral pleural adhesiolysis with thoracotomy, the combined treatment of oral beta-blocker and mTOR inhibitor was commenced. After 1 month of oral medication, her pleural effusion was not increased and she was free of respiratory difficulty on room air without chest tubes. Over eleven months of treatment, no serious adverse reaction was noted and her condition has been stable with no further admission required.

• BMB Reports
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• v.49 no.1
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• pp.63-68
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• 2016

The serine/threonine kinase mTOR is essential for the phosphoinositide 3-kinases (PI3K) signaling pathway, and regulates the development and function of immune cells. Aberrant activation of mTOR signaling pathway is associated with many cancers including leukemia. Here, we report the contributions of mTOR signaling to growth of human leukemic cell lines and mouse T-cell acute leukemia (T-ALL) cells. Torin, an ATP-competitive mTOR inhibitor, was found to have both cytotoxic and cytostatic effects on U-937, THP-1, and RPMI-8226 cells, but not on Jurkat or K-562 cells. All cells were relatively resistant to rapamycin even with suppressed activity of mTOR complex 1. Growth of T-ALL cells induced by Notch1 was profoundly affected by torin partially due to increased expression of Bcl2l11 and Bbc3. Of note, activation of Akt or knockdown of FoxO1 mitigated the effect of mTOR inhibition on T-ALL cells. Our data provide insight on the effect of mTOR inhibitors on the survival and proliferation of leukemic cells, thus further improving our understanding on cell-context-dependent impacts of mTOR signaling. [BMB Reports 2016; 49(1): 63-68]

• Clinical and Experimental Pediatrics
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• v.54 no.6
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• pp.241-245
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• 2011

Tuberous sclerosis complex (TSC) is a genetic multisystem disorder that results from mutations in the TSC1 or TSC2 genes, and is associated with hamartomas in several organs, including subependymal giant cell tumors. The neurological manifestations of TSC are particularly challenging and include infantile spasms, intractable epilepsy, cognitive disabilities, and autism. The TSC1- and TSC2-encoded proteins modulate cell function via the mammalian target of rapamycin (mTOR) signaling cascade, and are key factors in the regulation of cell growth and proliferation. The mTOR pathway provides an intersection for an intricate network of protein cascades that respond to cellular nutrition, energy levels, and growth factor stimulation. In the brain, TSC1 and TSC2 have been implicated in cell body size, dendritic arborization, axonal outgrowth and targeting, neuronal migration, cortical lamination, and spine formation. The mTOR pathway represents a logical candidate for drug targeting, because mTOR regulates multiple cellular functions that may contribute to epileptogenesis, including protein synthesis, cell growth and proliferation, and synaptic plasticity. Antagonism of the mTOR pathway with rapamycin and related compounds may provide new therapeutic options for TSC patients.

• Journal of Life Science
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• v.32 no.6
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• pp.421-429
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• 2022

The expression of interleukin-1α (IL-1α) is elevated in monocytic cells, such as monocytes and macro-phages, within atherosclerotic arteries, yet the cellular molecules involved in cytokine upregulation remain unclear. Because peptidoglycan (PG), a major component of gram-positive bacterial cell walls, is detected within the inflammatory cell-rich regions of atheromatous plaques, it was investigated if PG contributes to IL-1α expression in monocytes/macrophages. Exposure of THP-1 monocytic cells to PG resulted in elevated levels of IL-1α gene transcripts and increased secretion of IL-1α protein. The transcription and secretion of IL-1α were abrogated by OxPAPC, an inhibitor of TLR2/4, but not by polymyxin B that inhibits lipopolysaccharide-induced TLR4 activation. To understand the molecular mechanisms of the inflammatory responses due to bacterial pathogen-associated molecular patterns (PAMPs) in diseased arteries, we attempted to determine the cellular factors involved in the PG-induced upregulation of IL-1α expression. Pharmacological inhibition of cell signaling pathways with LY294002 (a PI3K inhibitor), Akti IV (an inhibitor of Akt activation), rapamycin (an mTOR inhibitor), U0126 (a MEK inhibitor), SB202190 (a p38 MAPK inhibitor), SP6001250 (a JNK inhibitor), and DPI (a NOX inhibitor) also significantly attenuated the PG-mediated expression of IL-1α. These results suggest that PG induces the monocytic or macrophagic expression of IL-1α, thereby contributing to vascular inflammation, via multiple signaling molecules, including TLR2, PI3K/Akt/mTOR, and MAPKs.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.12
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• pp.1708-1716
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• 2016

The Akt/mammalian target of the rapamycin (mTOR) pathway is activated in the majority of human cancers. Activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy. In this study, we evaluated the apoptotic effect of ethanol extract of Artemisia annua L. through down-regulation of Akt signal pathways and the mitochondrial pathway in hepato-carcinoma cells (HepG2). A. annua extract is known as a medicinal herb that is effective against cancer. We evaluated anti-proliferative activity by MTT-based viability assay and apoptotic effect by Annexin-V/PI staining, mitochondrial membrane potential (MMP), and caspase-3/7 activity as determined by flow cytometry. A. annua treatment led to loss of MMP, resulting in cytochrome c-inducible activation of caspase-3/7. Treatment with A. annua extract reduced activities of Akt/mTOR/anti-apoptotic proteins (such as Bcl-2 and $Bcl-X_L$), leading to increased activation of tumor suppressor p53 and pro-apoptotic proteins (such as Bax and Bak). We applied LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR) to determine the relationship between signal transduction of proteins associated with apoptosis. LY294002 and rapamycin significantly reduced cell viability and increased apoptosis. These results indicate that Bcl-2 and caspase-3 are key regulators in A. annua extract-induced apoptosis in HepG2 cells and are controlled through the Akt/mTOR signaling pathway.

• Development and Reproduction
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• v.20 no.1
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• pp.1-10
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• 2016

Molecular targeting for the altered signaling pathways has been proven to be effective for the treatment of many types of human cancer, including colorectal cancer (CRC). The dual phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor BEZ235 has shown to exhibit potent antitumor activity against solid tumors. Autophagy is a cellular lysosomal catabolic process to maintain metabolic homeostasis, which has been known to be induced in response to many therapeutic agents in cancer cells. This process is negatively regulated by mTOR and often acts as prosurvival or prodeath mechanism following cancer therapeutics. The current study was designed to investigate the antiproliferation activity of BEZ235 and to evaluate the role of autophagy induced by BEZ235 using HCT15 CRC cells bearing ras oncogene mutation. We found that BEZ235 decreases cell viability, which was mostly dependent on $G_1$ arrest of cell cycle via suppression of cyclin A expression. BEZ235 affects PI3K/Akt/mTOR signaling pathway by increasing the phosphorylation of AKT at $Ser^{473}$ and RAS/RAF/MEK/ERK pathway by decreasing the phosphorylation of ERK at $Tyr^{204}$. BEZ235 also stimulated autophagy induction as evidenced by the increased expression of LC3-II and abundant acidic vesicular organelles (AVOs) in the cytoplasm. In addition, the combination of BEZ235 with autophagy inhibitor chloroquine, a known antagonist of autophagy, counteracted the antiproliferation effect of BEZ235. Thus, our study indicates that autophagy induced in response to BEZ235 treatment appears to act as cell death mechanism in HCT15 CRC cells.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.4
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• pp.365-374
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• 2021

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 μl) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

• Journal of Life Science
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• v.26 no.6
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• pp.663-672
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• 2016

The Cnidium monnieri (L.) Cusson is an annual plant distributed in China and Korea. The fruit of C. monnieri is used as a medicinal herb that is effective for the treatment of carbuncle and pain in female genitalia. However, the anti-cancer effects of CME have not yet been reported. In this study, we assessed the apoptotic effects and cell cycle arrest effects of ethanol extracts from C. monnieri on HCT116 colon cancer cells. The results of an MTT assay and LDH assay demonstrated a decrease in cell viability and the cytotoxic effects of CME. In addition, the number of apoptotic body and the apoptotic rate were increased in a dose-dependent manner through Hoechst 33342 staining and Annexin V-PI double staining. In addition, cell cycle arrest occurred at the G1 phase by CME. Protein kinase B (Akt) plays an important role in cancer cell survival, growth, and division. Akt down-regulates apoptosis-mediated proteins, such as mammalian target of rapamycin (mTOR), p53, and Glycogen Synthase kinase-3β (GSK-3β). CME could regulate the expression levels of p-Akt, p-mTOR, p-GSK-3β, Bcl-2 family members, caspase-3, and PARP. Furthermore, treatment with CME, LY294002 (PI3K/Akt inhibitor), BIO (GSK-3β inhibitor), and Rapamycin (mTOR inhibitor) showed that apoptotic effects occurred through the regulation of the AKT/mTOR/GSK-3β signaling pathway. Our results demonstrated CME could induce apoptosis and cell cycle arrest in HCT116 colon cancer cells.