3-Methyladenine (3-MA)

The slight preference for Vps34 prevention by 3-Methyladenine (3-MA) probably arises from a hydrophobic ring specific to Vps34, which encircles the 3-methyl group of this compound. [1] It has been reported to cause cancer cell death under both normal and starvation conditions, and could also suppress cell migration and invasion independently of its ability to inhibit autophagy, implying that it possesses functions other than autophagy suppression. This compound elicits caspase-dependent cell death that is independent of autophagy inhibition. Treatment with 5 mM of it reduces the percentage of glucose-starved HeLa cells displaying GFP-LC3 puncta to 23%. The levels of LC3-I are increasing and the levels of LC3-II are decreasing between 12 and 48 hours in cells that are treated with 3-MA. Conversion of LC3-I to LC3-II is suppressed by the compound. Treatment of HeLa cells with it at 2.5 mM or 5 mM for one day does not affect cell viability, whereas treatment with 10 mM for one day causes a 25.0% decrease in cell viability. Treatment of cells with 2.5, 5 or 10 mM for two days causes 11.5%, 38.0% and 79.4% decrease in viability, respectively. It decreases cell viability in a time- and dose-dependent manner, and significantly shortens the duration of nocodazole-induced-prometaphase arrest. [2] Suppression of autophagy by 3-MA inhibits SU11274-induced cell death. [3] Prolonged treatment with it (up to 9 hours) induces significant LC3 I to II conversion in wild type MEFs. Prolonged treatment with 3-MA, but not wortmannin, markedly increases GFP-LC3 punctuation/aggregation. Its induced LC3 conversion and free GFP liberation are ATG7-dependent. Treatment with it leads to evident increase of p62 protein level. The compound increases the p62 level even in Atg5−/− MEFs as well as in cells with DOX-mediated deletion of ATG5. It inhibits class I and class III PI3K in different temporal patterns. Its induced LC3 I to LC3 II conversion is dramatically compromised in Tsc2−/− cells compared with wild type cells. This compound disrupts the anti-autophagic function of mTOR complex 1. [4]

3-Methyladenine (3-MA) blocks autophagy through its effect on class III phosphatidylinositol 3-kinase (PI3K). Treatment with this compound does not alter the degree of hemorrhage compared with the subarachnoid hemorrhage (SAH) group. Its pretreatment significantly aggravates neurological symptoms when compared with the SAH + vehicle group. Autophagy is decreased when it is applied. Conversely, cleaved caspase-3 is markedly up-regulated in the SAH + 3-MA group. In line with the up-regulation of cleaved caspase-3 expression, the number of TUNEL-positive cells in the right cortex is significantly increased in the SAH + 3-MA group compared with the SAH + vehicle group. [5]

• Protein degradation assay

  HeLa cells are radiolabeled for 24 hours with 0.05 mCi/mL l-[U- ¹⁴C]valine. At the end of the labeling period, cells are rinsed three times with PBS. Cells are incubated for the designated times in either full medium or EBSS with or without the presence of 10 mM 3-Methyladenine (3-MA).

• Cell lines

  HeLa cell line

• Concentrations

  1-10 mM

• Incubation Time

  24, 48 or 72 hours

• Method

  After treatment with 3-Methyladenine (3-MA), cell (such as HeLa cell) viability is determined by a trypan blue exclusion assay. Briefly, both adherent and floating cells are collected and suspended in phosphate buffered saline (PBS, pH 7.4) at a final density of 1-2 × 10⁶/mL. An equal volume of 0.4% trypan blue solution (w/v, in PBS) is added to the cell suspension and mixed thoroughly. After incubation at room temperature for 3 min, cell counting is performed using a hemacytometer.

• Animal Models

  Adult male Sprague–Dawley rats weighing 300-350 g

• Dosages

  400 nM

• Administration

  Intracerebral ventricular