Tooth germ injury can lead to abnormal tooth development and even tooth loss, affecting various aspects of the stomatognathic system including form, function, and appearance. However, the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited. Therefore, it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model. Here, we constructed a Tg(dlx2b:Dendra2-NTR) transgenic line that labeled tooth germ specifically. Taking advantage of the NTR/Mtz system, the dlx2b⁺ tooth germ cells were depleted by Mtz effectively. The process of tooth germ repair was evaluated by antibody staining, in situ hybridization, EdU staining and alizarin red staining. The severely injured tooth germ was repaired in several days after Mtz treatment was stopped. In the early stage of tooth germ repair, the expression of phosphorylated 4E-BP1 was increased, indicating that mTORC1 is activated. Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ. Normally, mouse incisors were repaired after damage, but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress. Overall, we are the first to construct a stable and repeatable repair model of severe tooth germ injury, and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair, providing a potential target for clinical treatment of tooth germ injury.
Animals ; Mice ; Mechanistic Target of Rapamycin Complex 1/pharmacology* ; Signal Transduction ; Tooth/metabolism* ; Tooth Germ/metabolism* ; Odontogenesis

OBJECTIVETo investigate the roles of the mammalian target of rapamycin-1 and -2 (mTORC1 and TORC2) in the proliferation and apoptosis of prostate cancer 22RV1 cells.

METHODSAfter silencing mTORC1 and TORC2, we examined the proliferation and apoptosis of prostate cancer 22RV1 cells by methylthiazol tetrazolium (MTT) assay and flow cytometry, respectively, and detected the expressions of the androgen receptor (AR) and Akt phosphorylation in the prostate cancer 22RV1 cells by Western blot after transfecting Raptor-siRNA and Rictor-siRNA to the 22RV1 cells.

RESULTSMTT showed that the prostate cancer 22RV1 cells had no significant change in the growth rate after mTORC1 silence (P > 0.05), but their proliferation was markedly inhibited after mTORC2 silence (P < 0.01). Flow cytometry revealed a dramatic increase in the apoptosis of the 22RV1 cells after mTORC1 silence (P < 0.01), but no obvious change after mTORC2 silence (P > 0.05). Western blot exhibited that mTORC1 silence significantly increased the expression of AR and Akt phosphorylation (P < 0.05), while mTORC2 silence markedly decreased them (P < 0.05).

CONCLUSIONmTORC2 is not only required for the survival of prostate cancer 22RV1 cells, but also a promising therapeutic target of prostate cancer.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Male ; Mechanistic Target of Rapamycin Complex 1 ; Mechanistic Target of Rapamycin Complex 2 ; Multiprotein Complexes ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, Androgen ; metabolism ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism

Mammalian target of rapamycin (mTOR) controls cellular anabolism, and mTOR signaling is hyperactive in most cancer cells. As a result, inhibition of mTOR signaling benefits cancer patients. Rapamycin is a US Food and Drug Administration (FDA)-approved drug, a specific mTOR complex 1 (mTORC1) inhibitor, for the treatment of several different types of cancer. However, rapamycin is reported to inhibit cancer growth rather than induce apoptosis. Pyruvate dehydrogenase complex (PDHc) is the gatekeeper for mitochondrial pyruvate oxidation. PDHc inactivation has been observed in a number of cancer cells, and this alteration protects cancer cells from senescence and nicotinamide adenine dinucleotide (NAD^+‍) exhaustion. In this paper, we describe our finding that rapamycin treatment promotes pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) phosphorylation and leads to PDHc inactivation dependent on mTOR signaling inhibition in cells. This inactivation reduces the sensitivity of cancer cells' response to rapamycin. As a result, rebooting PDHc activity with dichloroacetic acid (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, promotes cancer cells' susceptibility to rapamycin treatment in vitro and in vivo.
Humans ; Sirolimus/pharmacology* ; Dichloroacetic Acid/pharmacology* ; Pyruvate Dehydrogenase Complex ; TOR Serine-Threonine Kinases ; Mechanistic Target of Rapamycin Complex 1 ; Neoplasms/drug therapy*

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a "master switch" for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-interacting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.
Antineoplastic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Furans ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Indoles ; pharmacology ; Mechanistic Target of Rapamycin Complex 1 ; Mechanistic Target of Rapamycin Complex 2 ; Morpholines ; pharmacology ; Multiprotein Complexes ; antagonists & inhibitors ; Naphthyridines ; pharmacology ; Neoplasms ; pathology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Purines ; pharmacology ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; Quinolines ; pharmacology ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors

BACKGROUND: Pulmonary microvascular endothelial cells (PMVECs) were not complex, and the endothelial barrier was destroyed in the pathogenesis progress of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF), which was secreted by bone marrow mesenchymal stem cells, could decrease endothelial apoptosis. We investigated whether mTOR/STAT3 signaling acted in HGF protective effects against oxidative stress and mitochondria-dependent apoptosis in lipopolysaccharide (LPS)-induced endothelial barrier dysfunction and ALI mice. METHODS: In our current study, we introduced LPS-induced PMEVCs with HGF treatment. To investigate the effects of mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 were, respectively, used to inhibit mTOR/STAT3 signaling. Moreover, lentivirus vector-mediated mTORC1 (Raptor) and mTORC2 (Rictor) gene knockdown modifications were introduced to evaluate mTORC1 and mTORC1 pathways. Calcium measurement, reactive oxygen species (ROS) production, mitochondrial membrane potential and protein, cell proliferation, apoptosis, and endothelial junction protein were detected to evaluate HGF effects. Moreover, we used the ALI mouse model to observe the mitochondria pathological changes with an electron microscope in vivo. RESULTS: Our study demonstrated that HGF protected the endothelium via the suppression of ROS production and intracellular calcium uptake, which lead to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection) and specific proteins (complex I), raised anti-apoptosis Messenger Ribonucleic Acid level (B-cell lymphoma 2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2 have the same protective effects in mitochondria damage and apoptosis. In in vivo experiments of ALI mouse, HGF also increased mitochondria structural integrity via the mTOR/STAT3 pathway. CONCLUSION In all, these reveal that mTOR/STAT3 signaling mediates the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis, and endothelial junction protein in ARDS, contributing to the pulmonary endothelial survival and barrier integrity.
Animals ; Apoptosis ; Calcium/metabolism* ; Endothelial Cells/metabolism* ; Endothelium/metabolism* ; Hepatocyte Growth Factor/metabolism* ; Lipopolysaccharides/pharmacology* ; Mammals/metabolism* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Mechanistic Target of Rapamycin Complex 2/metabolism* ; Mice ; Mitochondria/metabolism* ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Respiratory Distress Syndrome ; Sirolimus/pharmacology* ; TOR Serine-Threonine Kinases/metabolism*

Autophagy is a conserved and programmed catabolic process that degrades damaged proteins and organelles. But the underlying mechanism and functions of autophagy in the ischemia-reperfusion (IR)-induced injury are unknown. In this study, we employed simulated IR of N2a cells as an in vitro model of IR injury to the neurons and monitored autophagic processes. It was found that the levels of Beclin-1 (a key molecule of autophay complex, Beclin-1/class III PI3K) and LC-3II (an autophagy marker) were remarkably increased with time during the process of ischemia and the process of reperfusion after 90 min of ischemia, while the protein kinases p70S6K and mTOR which are involved in autophagy regulation showed delayed inactivation after reperfusion. Administration of 3-methyladenine (3MA), an inhibitor of class III PI3K, abolished autophagy during reperfusion, while employment of rapamycin, an inhibitor of mTORC1 (normally inducing autophagy), surprisingly weakened the induction of autophagy during reperfusion. Analyses of mitochondria function by relative cell viability demonstrated that autophagy inhibition by 3-MA attenuated the decline of mitochondria function during reperfusion. Our data demonstrated that there were two distinct dynamic patterns of autophagy during IR-induced N2a injury, Beclin-1/class III PI3K complex-dependent and mTORC1-dependent. Inhibition of over-autophagy improved cell survival. These suggest that targeting autophagy therapy will be a novel strategy to control IR-induced neuronal damage.
Adenine ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Cell Line, Tumor ; Cell Survival ; Mechanistic Target of Rapamycin Complex 1 ; Mice ; Mitochondria ; metabolism ; Multiprotein Complexes ; antagonists & inhibitors ; metabolism ; Neurons ; drug effects ; metabolism ; Neuroprotective Agents ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Reperfusion Injury ; metabolism ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism

Niclosamide, an oral antihelminthic drug, has been used to treat tapeworm infection for about 50 years. Niclosamide is also used as a molluscicide for water treatment in schistosomiasis control programs. Recently, several groups have independently discovered that niclosamide is also active against cancer cells, but its precise mechanism of antitumor action is not fully understood. Evidence supports that niclosamide targets multiple signaling pathways (NF-κB, Wnt/β-catenin, Notch, ROS, mTORC1, and Stat3), most of which are closely involved with cancer stem cells. The exciting advances in elucidating the antitumor activity and the molecular targets of this drug will be discussed. A method for synthesizing a phosphate pro-drug of niclosamide is provided. Given its potential antitumor activity, clinical trials for niclosamide and its derivatives are warranted for cancer treatment.
Animals ; Antineoplastic Agents ; pharmacokinetics ; pharmacology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Mechanistic Target of Rapamycin Complex 1 ; Multiprotein Complexes ; metabolism ; NF-kappa B ; metabolism ; Neoplasm Metastasis ; Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; drug effects ; Niclosamide ; pharmacokinetics ; pharmacology ; Reactive Oxygen Species ; metabolism ; Receptors, Notch ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; metabolism ; Wnt Signaling Pathway ; drug effects

OBJECTIVE: To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. METHODS: Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells. RESULTS: In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P < 0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P < 0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P < 0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P < 0.05), which was strongly suppressed by transfection with Numb-siRNA (P < 0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P < 0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P < 0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P < 0.05). CONCLUSION Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.
Animals ; Male ; Mice ; Amino Acids/pharmacology* ; AMP-Activated Protein Kinases/metabolism* ; Cisplatin/pharmacology* ; Epithelial Cells ; Low Density Lipoprotein Receptor-Related Protein-2/metabolism* ; Mammals/metabolism* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Membrane Proteins/metabolism* ; Mice, Inbred BALB C ; Nerve Tissue Proteins/metabolism* ; RNA, Small Interfering/metabolism* ; Signal Transduction ; Vacuolar Proton-Translocating ATPases/metabolism*