3-Methyladenine
(Synonyms: 3-甲基腺嘌呤; 3-MA) 目录号 : GC10710
3-甲基腺嘌呤是一种经典的自噬抑制剂。
Cas No.:5142-23-4
Sample solution is provided at 25 µL, 10mM.
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| Cell experiment [1]: | |
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Cell lines |
Rat renal interstitial fibroblasts (NRK-49F) cells |
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Preparation Method |
Rat renal interstitial fibroblasts (NRK-49F) were cultured in DMEM with F12 containing 10% FBS, 1%penicillin and streptomycin in an atmosphere of 5%CO2, and 95% air at 37◦C. Cells were starved for 24h with DMEM containing 0.5% FBS. |
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Reaction Conditions |
Cells were exposed to uric acid (800 μM) for 36h in the presence or absence of 3-Methyladenine (0–10 mM). |
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Applications |
3-Methyladenine could abolish uric acid-induced α-SMA and collagen I expression. Uric acid also triggered a significant up-regulation of LC3II/I and Beclin-1. 3-Methyladenine dose-dependently suppressed these responses. In addition, treatment with 3-MA decreased TGF-βRI expression levels and the ratio of p-Smad3/Smad3 in a dose-dependent manner. |
| Animal experiment [2]: | |
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Animal models |
male Sprague-Dawley (SD) rats (2-months old; weight 200±20 g) |
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Preparation Method |
Mice were maintained on a 12h light/dark cycle and provided access to food and water ad libitum. All rats were pretrained to swim in the absence of a load for one week. The saline-treatment group was intravenously injected with 500 μL sterile saline by using 261/2 gauge needle via tail vein;3-MA-treatment group was intravenously injected with 500 μL 3-Methyladenine by using a 261/2 gauge needle via the tail vein. |
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Dosage form |
15 mg/kg |
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Applications |
3-Methyladenine could decrease the distortion of myocardium fibers as well as significantly decrease the width of cardiomyocytes of mice. In addition, 3-Methyladenine treatment obviously reduced autophagosome formation in the myocardium. 3-Methyladenine could also prevent the reduction of Bcl-2/Bax in the left ventricle of OE rats. |
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References: [1]. Bao J, et al. Pharmacological inhibition of autophagy by 3-MA attenuates hyperuricemic nephropathy. Clin Sci (Lond). 2018 Nov 2;132(21):2299-2322. [2]. Liu H, et al. Autophagy inhibitor 3-methyladenine alleviates overload-exercise- induced cardiac injury in rats. Acta Pharmacol Sin. 2017 Jul;38(7):990-997. |
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3-Methyladenine is a classic autophagy inhibitor. It inhibits phosphatidylinositol 3-kinase (PI3K), which is located upstream of the IGF/PI3K/mTOR/ULK pathway.[1] 3-Methyladenine is capable to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. In addition, 3-Methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis.[2]
In vitro study indicated that the inhibition of autophagy by 3-Methyladenine abolished uric acid-induced differentiation of renal fibroblasts to myofibroblasts and activation of transforming growth factor-β1 (TGF-β1), epidermal growth factor receptor (EGFR), and Wnt signaling pathways in cultured renal interstitial fibroblasts. Moreover, 3-Methyladenine was effective in attenuating renal deposition of extracellular matrix (ECM) proteins and expression of α-smooth muscle actin (α-SMA) and reducing renal epithelial cells arrested at the G2/M phase of cell cycle.[3]
In vivo study demonstrated that the administration of 3-Methyladenine inhibited Wnt/β-catenin and Notch/Jagged-1 signaling pathways as well as suppresses EGFR/ERK1/2 signaling pathway. Furthermore, 3-MA treatment remarkably inhibited the infiltration of macrophages and lymphocytes as well as release of multiple profibrogenic cytokines/chemokines in the injured kidney.[3]
3-甲基腺嘌呤是一种经典的自噬抑制剂。它抑制磷脂酰肌醇3-激酶（PI3K），该激酶位于IGF/PI3K/mTOR/ULK通路的上游[1]。 3-甲基腺嘌呤能够在一系列本体不相关的人类细胞系中诱导细胞存活率持续而突然地下降。此外， 3-甲基腺嘌呤引起的细胞毒性并非由AKT/mTOR轴的抑制驱动[2]。
实验室研究表明，使用3-甲基腺嘌呤可以抑制自噬作用，从而阻止尿酸诱导肾间质成纤维细胞向肌成纤维细胞分化和转化生长因子β1（TGF-β1）、表皮生长因子受体（EGFR）以及Wnt信号通路的激活。此外，3-甲基腺嘌呤还能有效减轻肾脏中胞外基质（ECM）蛋白沉积和α平滑肌动力蛋白（α-SMA）的表达，并降低停留在G2/M期的肾上皮细胞数量。
动物实验表明，给予3-甲基腺嘌呤可以抑制Wnt/β-catenin和Notch/Jagged-1信号通路，并且还能抑制EGFR/ERK1/2信号通路。此外，3-MA治疗显著抑制了肾损伤中巨噬细胞和淋巴细胞的浸润以及多种促纤维化的细胞因子/趋化因子的释放。
References:
[1]. Yang F, et al. Rapamycin and 3-Methyladenine Influence the Apoptosis, Senescence, and Adipogenesis of Human Adipose-Derived Stem Cells by Promoting and Inhibiting Autophagy: An In Vitro and In Vivo Study. Aesthetic Plast Surg. 2021 Jun;45(3):1294-1309.
[2].Chicote J, et al. Cell Death Triggered by the Autophagy Inhibitory Drug 3-Methyladenine in Growing Conditions Proceeds With DNA Damage. Front Pharmacol. 2020 Oct 15;11:580343.
[3].Bao J, et al. Pharmacological inhibition of autophagy by 3-MA attenuates hyperuricemic nephropathy. Clin Sci (Lond). 2018 Nov 2;132(21):2299-2322.
| Cas No. | 5142-23-4 | SDF | |
| 别名 | 3-甲基腺嘌呤; 3-MA | ||
| 化学名 | 3-methylpurin-6-amine | ||
| Canonical SMILES | CN1C=NC(=C2C1=NC=N2)N | ||
| 分子式 | C6H7N5 | 分子量 | 149.15 |
| 溶解度 | ≥ 7.45mg/mL in DMSO, ≥ 5mg/mL in Water | 储存条件 | Store at 2-8°C,Solutions of 3-MA are best fresh-prepared |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 6.7047 mL | 33.5233 mL | 67.0466 mL |
| 5 mM | 1.3409 mL | 6.7047 mL | 13.4093 mL |
| 10 mM | 0.6705 mL | 3.3523 mL | 6.7047 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
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工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Ferrostatin-1 and 3-Methyladenine Ameliorate Ferroptosis in OVA-Induced Asthma Model and in IL-13-Challenged BEAS-2B Cells
Ferroptosis was reported to be involved in the occurrence and development of asthma. However, the potential mechanism underlying the role of ferroptosis in asthma remains unclear. In this study, we established the mouse asthma model following the ovalbumin (OVA) method in C57BL/6 mice and the cell model with IL-13 induction in bronchial epithelial cells (BEAS-2B cells). Treatment of ferrostatin-1 (Ferr-1) and 3-methyladenine (3-MA) decreased iron deposition in IL-13-induced BEAS-2B cells and lung tissues of asthma mice, opposite to that in bronchoalveolar lavage fluid (BALF). Meanwhile, excessive lipid peroxidation asthma model in vivo and in vitro was alleviated by Ferr-1 or 3-MA treatment. In addition, Ferr-1 and 3-MA inhibited the expression of LC-3 in these cells and lung tissues of mice. Moreover, Ferr-1 and 3-MA also suppressed the production of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and oxidative stress factors (ROS and MDA), while promoting the level of SOD, in vivo and in vitro. Furthermore, application of Ferr-1 exhibited a greater inhibitory effect on iron release and lipid peroxidation in IL-13-induced BEAS-2B cells and asthma mice than 3-MA, accompanied with a weaker effect on ferritinophagy than 3-MA. Collectively, Ferr-1 and 3-MA ameliorated asthma in vivo and in vitro through inhibiting ferroptosis, providing a new strategy for the clinical treatment of asthma.
Inhibition of autophagy by 3-MA promotes hypoxia-induced apoptosis in human colorectal cancer cells
Objective: Cell autophagy reduces the sensitivity of cancer cells to therapeutic reagents in various types of human cancer. Therefore, the aim of our study was to use human colorectal cancer HCT116 cells to explore whether inhibition of autophagy by 3-Methyladenine (3-MA, an autophagy inhibitor) is able to enhance hypoxia-induced apoptosis in vitro.
Materials and methods: HCT116 cells were treated with 3-MA, hypoxia, or 3-MA plus hypoxia, and the autophagy, apoptosis and proliferation of the HCT116 cells were investigated. Western blot analysis was used to detect autophagy specificity protein microtubule-associated protein light chain 3 (LC3) expression. Effects on apoptosis were evaluated by using flow cytometry (JC-1 staining to measure mitochondrial membrane potential) and annexin V-propidium iodide (PI) staining.
Results: The results showed that the treatment of HCT116 cells in vitro with hypoxia alone increased autophagy as well as apoptosis, whereas combination treatment with 3-MA and hypoxia markedly inhibited hypoxia-induced autophagy, but increased hypoxia-induced cell apoptosis.
Conclusions: Autophagy might play a role as a self-defense mechanism in hypoxia-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.
Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase
A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.
Inhibition of autophagy by 3-methyladenine restricts murine cytomegalovirus replication
Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine, or rapamycin to block or stimulate autophagy. In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (hpi), but was increased at 48 hpi In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition, caspase-3 cleavage was increased at 48 hpi in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10, and 14 dpi. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication.
3-Methyladenine-enhanced susceptibility to sorafenib in hepatocellular carcinoma cells by inhibiting autophagy
As an effective targeted therapy for advanced hepatocellular carcinoma (HCC), sorafenib resistance has been frequently reported in recent years, with the activation of autophagy by cancer cells under drug stress being one of the crucial reasons. Sorafenib treatment could enhance autophagy in HCC cells and autophagy is also considered as an important mechanisms of drug resistance. Therefore, the inhibition of autophagy is a potential way to improve the sensitivity and eliminate drug resistance to restore their efficacy. To determine whether autophagy is involved in sorafenib resistance and investigate its role in the regulation of HepG2 cells' (an HCC cell line) chemosensitivity to sorafenib, we simultaneously treated HepG2 with sorafenib and 3-Methyladenine (3-MA) (a common autophagy inhibitor). First, by performing cell counting kit 8 cell viability assay, Hoechst 33342 apoptosis staining, and Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis kit detection, we found that both sorafenib and 3-MA effectively inhibitted the proliferative activity of HepG2 cells and induced their apoptosis to a certain extent. This effect was significantly enhanced after these two drugs were combined, which was also confirmed by the increased expression of apoptosis-related proteins. Subsequently, by using AAV-GFP-LC3 transfection methods and transmission electron microscopy, we found that both the number and activity of autophagosomes in HepG2 cells in sorafenib and 3-MA group were significantly reduced, suggesting that autophagy activity was inhibited, and this result was consistent with the expression results of autophagy-related proteins. Therefore, we conclude that 3-MA may attenuate the acquired drug resistance of sorafenib by counteracting its induction of autophagy activity, thus enhancing its sensitivity to advanced HCC therapy.