mTORC1; mTORC2; mTORC1; Autophagy

MHY1485 (10 μM; 4 hours) demonstrates that GCDC-induced autophagic activity is inhibited by upregulating p-mTOR expression and downregulating LC3 and p62 expression in HCC cells[1]. MHY1485 (5 μM; 6 hours) increases the LC3I/LC3I ratio in a dose- and time-dependent manner because it ostensibly inhibits LC3II degradation in rat liver Ac2F cells[2]. MHY1485 (0.5-2 μM; 6 hours) increases the phosphorylation of mTOR at ser2448 and upregulates the level of phosphorylation of 4E-BP1 in a dose-dependently manner in Ac2F cells[2].

MHY1485 (intraperitoneal injection; 10 mg/kg, 2 days) inhibits the follicle-stimulating hormone (FSH)-induced autophagy signaling. While p-mTOR and p-S6K1 expression levels rise, the expression of LC3 does not differ noticeably from that of the control group[3].
In addition, the effect of the mTOR activator, MHY1485, (10 mg/kg, 2 days) before FSH treatment was investigated. The results suggested that MHY1485 blocked the autophagy signaling induced by FSH. p-mTOR and p-S6K1 expression levels were maintained at a high level in the presence of MHY1485 (Figure 2d, bottom, Figure 2f), whereas LC3 expression showed no marked change compared to that in the control group (Figure 2d, top, Figure 2e). These findings demonstrated that FSH induces MGCs autophagy through the AKT-mTOR signaling pathway and initiates a dynamic process occurring within 12 h post-treatment [2].

Western blot analysis is used to find changes in the levels of total protein and phosphorylated forms of mTOR and 4E-BP1, which are indicators of mTOR activity. MHY1485 in varying concentrations is applied to Ac2F cells for 1 hour while rapamycin (5 mM) is used as a positive control. Cells are harvested after being washed in cold PBS. RIPA buffer, which contains 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM DTT, 0.1 mM NaF, 1 mM PMSF, and 1 mg/mL each of pepstatin, leupeptin, and aprotinin, is used to make cell lysates. Bicarbonate of acid (BCA) analysis is used to measure protein concentration. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels are used to separate proteins in exactly the same amounts. The gels are then electroblotted for 2 hours at 60-75 V to transfer them onto a polyvinylidene difluoride membrane. The membranes are then incubated with primary antibodies after being blocked in a solution of 5% nonfat milk in Tris-buffered saline (TBS) with 0.5% Tween-20. For determining molecular weight, pre-stained protein markers are employed.

Western Blotting [2]
Cells were washed with cold PBS and harvested. Cell lysates were prepared using RIPA buffer containing 50 mM Tris-HCl (pH), 150 mM NaCl, 1% NP-40, 1 mM DTT, 0.1 mM NaF, 1 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Protein concentration was determined by the bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as a standard. Equal amounts of protein were separated on 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The gels were subsequently transferred onto a polyvinylidene difluoride membrane by electroblotting for 2 h at 60–75 V. The membranes were blocked in a 5% nonfat milk solution in Tris-buffered saline (TBS) with 0.5% Tween-20, and incubated with primary antibodies as indicated. Pre-stained protein markers were used for molecular-weight determination.

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Staining of Autophagosomes with GFP-LC3 and Confocal Microscopy [2]
Approximately 1×10?5 cells were seeded in coverglass-bottom-dish, incubated overnight, and then transfected with the adenovirus encoding green fluorescent protein-microtubule-associated protein 1 light chain 3 at a concentration of 1,000 virus particles/cell in DMEM. After incubation for 24 h, cells were treated with compounds or starved. For visualization of lysosomes, cells were incubated with LysoTracker® at a concentration of 60 nM for 1 h. Confocal images were obtained with FV10i FLUOVIEW Confocal Microscope.

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Cells were exposed to the drug at the indicated concentration for 24 hours.

4-week-old female ICR mice[3]
10 mg/kg, 2 days
Intraperitoneal injection

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For activator and inhibitor experiments, MHY1485 (10 mg/kg, 2 days) and chloroquine (20 mg/kg, 5 days) were injected before FSH administration. HIF-1α inhibitor, Px-478, and AMPK inhibitor, compound C, were injected before FSH treatment and the experiment protocol is described in Supplementary Figure S2. [3]

[1]. Glycochenodeoxycholate promotes hepatocellular carcinoma invasion and migration by AMPK/mTOR dependent autophagy activation. Cancer Lett. 2019 Jul 10;454:215-223.

[2]. Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion. PLoS One. 2012;7(8):e43418.

[3]. Administration of follicle-stimulating hormone induces autophagy via upregulation of HIF-1α in mouse granulosa cells.Cell Death Dis. 2017 Aug 17;8(8):e3001.

Metastasis and recurrence severely impact the treatment effect of hepatocellular carcinoma (HCC). HCC complicated with cholestasis is more prone to recurrence and metastasis. Previous studies have implicated pathogenesis of HCC by bile acid; however, the underlying mechanism is unknown yet. Glycochenodeoxycholate (GCDC) is one of most important component of bile acid (BA). In the present study, the role of GCDC in HCC cells invasion was detected by in vitro and in vivo assays. GCDC was found to significantly enhance the invasive potential of HCC cells; Further studies showed that GCDC could induce autophagy activation and higher invasive capability in HCC cells. Interestingly, inhibition of autophagy by chloroquine (CQ) reversed this phenomenon. Subsequently, the correlation between TBA expression level and clinicopathological characteristics was analyzed in HCC patients. Clinically, high TBA level in HCC tissue was found to be associated with more invasive and poor survival in HCC patients. Mechanistic study showed that bile acid induced autophagy by targeting the AMPK/mTOR pathway in HCC cells. Therefore, our results suggest that bile acid may promote HCC invasion via activation of autophagy and the level of bile acid may serve as a potential useful indicator for prognosis of HCC patients. [1]

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Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time-dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel inhibitor of autophagy with an mTOR activating effect. [2]

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Recent studies reported the important role of autophagy in follicular development. However, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of follicle-stimulating hormone (FSH) on mouse granulosa cells (MGCs). Results indicated that autophagy was induced by FSH, which is known to be the dominant hormone regulating follicular development and granulosa cell (GC) proliferation. The activation of mammalian target of rapamycin (mTOR), a master regulator of autophagy, was inhibited during the process of MGC autophagy. Moreover, MHY1485 (an agonist of mTOR) significantly suppressed autophagy signaling by activating mTOR. The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was increased after FSH treatment. Blocking hypoxia-inducible factor 1-alpha attenuated autophagy signaling. In vitro, CoCl2-induced hypoxia enhanced cell autophagy and affected the expression of beclin1 and BCL2/adenovirus E1B interacting protein 3 (Bnip3) in the presence of FSH. Knockdown of beclin1 and Bnip3 suppressed autophagy signaling in MGCs. Furthermore, our in vivo study demonstrated that the FSH-induced increase in weight was significantly reduced after effectively inhibiting autophagy with chloroquine, which was correlated with incomplete mitophagy process through the PINK1-Parkin pathway, delayed cell cycle, and reduced cell proliferation rate. In addition, chloroquine treatment decreased inhibin alpha subunit, but enhanced the expression of 3 beta-hydroxysteroid dehydrogenase. Blocking autophagy resulted in a significantly lower percentage of antral and preovulatory follicles after FSH stimulation. In conclusion, our results indicate that FSH induces autophagy signaling in MGCs via HIF-1α. In addition, our results provide evidence that autophagy induced by FSH is related to follicle development and atresia. [3]

C17H21N7O4

387.39314

387.165

C, 52.71; H, 5.46; N, 25.31; O, 16.52

326914-06-1

326914-06-1

2834965

Off-white to yellow solid powder

1.4±0.1 g/cm3

643.3±65.0 °C at 760 mmHg

259°C

342.9±34.3 °C

0.0±1.9 mmHg at 25°C

1.652

-0.98

1

10

4

28

476

0

O=[N+](C1=CC=C(NC2=NC(N3CCOCC3)=NC(N4CCOCC4)=N2)C=C1)[O-]

MSSXBKQZZINCRI-UHFFFAOYSA-N

InChI=1S/C17H21N7O4/c25-24(26)14-3-1-13(2-4-14)18-15-19-16(22-5-9-27-10-6-22)21-17(20-15)23-7-11-28-12-8-23/h1-4H,5-12H2,(H,18,19,20,21)

4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine

MHY-1485; MHY 1485; mhy1485; 326914-06-1; 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine; 4,6-dimorpholin-4-yl-N-(4-nitrophenyl)-1,3,5-triazin-2-amine; MHY 1485; 4,6-bis(morpholin-4-yl)-N-(4-nitrophenyl)-1,3,5-triazin-2-amine; MFCD00489974; MHY1485

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~33 mg/mL (85.2 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 0.77 mg/mL (1.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.77 mg/mL (1.99 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5 mg/mL (12.91 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: 1 mg/mL (2.58 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)