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 outcomes of hepatocellular carcinoma (HCC). HCC complicated by cholestasis is more prone to recurrence and metastasis. Previous studies have shown that bile acids are involved in the pathogenesis of HCC, but the underlying mechanisms remain unclear. Glycinecholic acid (GCDC) is an important component of bile acids (BA). This study investigated the role of GCDC in HCC cell invasion through in vitro and in vivo experiments. The results showed that GCDC significantly enhanced the invasive ability of HCC cells; further research indicated that GCDC can induce autophagy activation in HCC cells and enhance their invasiveness. Interestingly, chloroquine (CQ) inhibition of autophagy reversed this phenomenon. Subsequently, this study analyzed the correlation between total bile acid (TBA) expression levels and clinicopathological features in HCC patients. Clinical studies found that elevated TBA levels in HCC tissues were associated with increased invasiveness and decreased survival in HCC patients. Mechanistic studies suggest that bile acids induce autophagy by targeting the AMPK/mTOR pathway in liver cancer cells. Therefore, our results suggest that bile acids may promote the invasion of hepatocellular carcinoma cells by activating autophagy, and bile acid levels may serve as a potentially useful prognostic indicator for hepatocellular carcinoma patients. [1] Autophagy is a major degradation process responsible for clearing cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During autophagy, cells form double-membrane vesicles called autophagosomes, which encapsulate degradable substances in the cytoplasm and eventually fuse with lysosomes. In this study, we used an Ac2F rat hepatocyte culture system to investigate the inhibitory effect of the synthetic compound MHY1485 on autophagy. Autophagy activity was assessed by measuring autophagy flux. Autophagosomes in Ac2F cells transfected with AdGFP-LC3 were observed using live-cell confocal microscopy. In addition, the activity of mTOR, a major regulator of autophagy, was assessed using Western blot and AutoDock 4.2 molecular docking simulation. The results showed that MHY1485 treatment inhibited basal autophagy flux, and this inhibitory effect was further confirmed under starvation (a strong physiological inducer of autophagy). After treatment with MHY1485, the levels of p62 and beclin-1 did not change significantly. Confocal microscopy images showed that the colocalization of autophagosomes and lysosomes was reduced, indicating that MHY1485 inhibited the fusion of lysosomes during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and the enlargement of autophagosomes in a dose- and time-dependent manner. In addition, MHY1485 can induce mTOR activation and its docking score in molecular docking simulation is higher than that of the known ATP-competitive mTOR inhibitor PP242. In summary, MHY1485 inhibits the autophagy process by inhibiting the fusion of autophagosomes and lysosomes, leading to the accumulation of LC3II protein and the enlargement of autophagosomes. MHY1485 can also induce mTOR activity, which provides another possible mechanism for MHY compounds to regulate autophagy. The significance of this study is that a novel autophagy inhibitor with mTOR activation activity has been discovered. [2] Recent studies have reported the important role of autophagy in follicle development. However, its underlying molecular mechanism is still unclear. This study investigated the effects of follicle-stimulating hormone (FSH) on mouse granulosa cells (MGCs). Results showed that FSH induces autophagy and is a key hormone regulating follicle development and granulosa cell (GC) proliferation. During granulosa cell autophagy, activation of the target of rapamycin (mTOR, a key regulator of autophagy) was inhibited. Furthermore, the mTOR agonist MHY1485 significantly inhibited the autophagy signaling pathway by activating mTOR. FSH treatment increased the expression of hypoxia-inducible factor 1α (HIF-1α). Blocking HIF-1α attenuated the autophagy signaling pathway. In vitro experiments showed that cobalt chloride (CoCl2)-induced hypoxia in the presence of FSH enhanced autophagy and affected the expression of beclin1 and BCL2/adenovirus E1B interacting protein 3 (Bnip3). Knockdown of beclin1 and Bnip3 inhibited the autophagy signaling pathway in granulosa cells. In addition, our in vivo studies showed that FSH-induced weight gain was significantly reduced after chloroquine effectively inhibited autophagy, which was related to incomplete PINK1-Parkin pathway-mediated mitophagy, cell cycle delay, and reduced cell proliferation rate. Furthermore, chloroquine treatment reduced the expression of inhibin α subunit but enhanced the expression of 3β-hydroxysteroid dehydrogenase. Blocking autophagy resulted in a significant reduction in the proportion of antral follicles and pre-ovulatory follicles after FSH stimulation. In summary, our results indicate that FSH induces the autophagy signaling pathway in MGCs through HIF-1α. In addition, our results also provide evidence that FSH-induced autophagy is associated with follicular 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.)