Autophagy

Spautin-1 decreases the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2, which increases the apoptosis that imatinib mesylate (IM) induces in CmL cells. Spautin-1's pro-apoptotic action is associated with GSK3β activation, a significant PI3K/AKT downstream effector. In the CmL cell line K562, spautin-1 increases IM-induced cytotoxicity by lowering the IC50 from 1 μM to 0.5 μM [1]. Reduced autophagy inhibition is linked to the mechanism by which spautin-1 treats acute pancreatitis [2].

Spautin-1 ameliorates the pathophysiology of acute pancreatitis produced by cerulein or L-arginine. Spautin-1 pretreatment significantly reduced the increase in serum amylase and lipase levels, indicating trypsin activity. The increase in serum TNFα levels generated by cerulein was reduced in the presence of spautin-1. Spautin-1 medication can alleviate inflammatory damage such as edema, degeneration, coagulative necrosis and inflammatory cell infiltration produced by cerulein [2].

Cell proliferation assays by Cell Counting Kit-8 (CCK-8)[1]
Cell proliferation was evaluated using CCK-8. Cells (1×10~5/ml) were seeded into 96-well plates in triplicate and then treated with 125 to 4,000 nM IM alone or in combination with spautin-1 (10 μM). After 48 h of incubation, 10 μl of CCK-8 reagent was added to each well. Four hours later, the absorbance was read at 450 nm using a microplate reader. The background absorbance was measured in wells containing only dye solution and culture medium. Data presented are the values subtracting the background absorbance values from the total absorbance values. The mean of the triplicates were calculated.

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Fluorescence microscopy[1]
Apoptotic morphology was studied by staining the cells with Hoechst 33258 fluorescent stain. Cells (1×105/ml) were seeded into a 12-well plate with indicated concentration of IM (500 nM) for 12 h. Then spautin-1 (10 μM) or DMSO was added to K562 medium for further 36 h. After incubation, cells were stained with 20 mg/ml of Hoechst 33258 for 10 min and observed under a fluorescence microscope

In this study, mice models with acute pancreatitis, including cerulein- and L-arginine-induced models, were constructed as previously described. For the cerulein-induced model, four intraperitoneal injections of cerulein (50 μg/kg body weight) were given consecutively at hourly intervals. The L-arginine-induced model received hourly intraperitoneal injections of 1.4 g/kg (optimal dosage for this study) L-arginine three times.
Rats were randomly divided into six groups (n = 12 per group). The first three groups were designed for cerulein-induced model analysis: Group 1 (control); Group 2 (cerulein), cerulein-induced pancreatitis without spautin-1 treatment; and Group 3 (cerulein + spautin-1), cerulein-induced pancreatitis with spautin-1 treatment (2 mg/kg, given by intraperitoneal injection 30 min before the first cerulein injection). The next 3 groups related to the L-arginine-induced model analysis: Group 4 (control); Group 5 (L-arginine), L-arginine-induced pancreatitis without spautin-1 treatment; and Group 6 (L-arginine + spautin-1), L-Arginine-induced pancreatitis with spautin-1 treatment (2 mg/kg, given by intraperitoneal injection 30 min before the first L-arginine injection). To observe the changes in the inflammation process, some of the mice were randomly selected and killed for serum amylase analysis three, six and nine hours after the last injection of cerulein or L-arginine. The mice were sacrificed nine hours after the last injection of cerulein or L-arginine for the analysis of serum amylase, lipase and TNFα, Western blotting and histopathological change.[2]

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Four intraperitoneal injections of cerulein (50 μg/kg body weight) are given consecutively at hourly intervals; The L-arginine-induced model received hourly intraperitoneal injections of 1.4 g/kg (optimal dosage for this study) L-arginine three times

Mice models with acute pancreatitis, including cerulein- and L-arginine-induced models

[1]. Spautin-1, a novel autophagy inhibitor, enhances imatinib-induced apoptosis in chronic myeloid leukemia. Int J Oncol. 2014 May;44(5):1661-1668.

[2]. Spautin-1 Ameliorates Acute Pancreatitis via Inhibiting Impaired Autophagy and Alleviating Calcium Overload. Mol Med. 2016 Aug 18;22.

Imatinib mesylate (IM), a targeted competitive inhibitor of the BCR-ABL tyrosine kinase, has revolutionized the clinical treatment of chronic myeloid leukemia (CML). However, resistance and intolerance are still a challenge in the treatment of CML. Autophagy has been proposed to play a role in IM resistance. To investigate the anti-leukemic activity of specific and potent autophagy inhibitor-1 (spautin-1) in CML, we detected its synergistic effect with IM in K562 and CML cells. Our results showed that spautin-1 markedly inhibited IM-induced autophagy in CML cells by downregulating Beclin-1. Spautin-1 enhanced IM-induced CML cell apoptosis by reducing the expression of the anti-apoptotic proteins Mcl-1 and Bcl-2. We further demonstrated that the pro-apoptotic activity of spautin-1 was associated with activation of GSK3β, an important downstream effector of PI3K/AKT. The findings indicate that the autophagy inhibitor spautin-1 enhances IM-induced apoptosis by inactivating PI3K/AKT and activating downstream GSK3β, leading to downregulation of Mcl-1 and Bcl-2, which represents a promising approach to improve the efficacy of IM in the treatment of patients with CML.[1]

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Acute pancreatitis is characterized by zymogen pre-activation. Severe inflammation caused by zymogen activation can eventually lead to multiple organ dysfunctions, which contributes to the high mortality rate of severe acute pancreatitis. However, there is no specific treatment available for acute pancreatitis therapy. Here, we show that spautin-1, which effectively inhibits autophagy flux, ameliorated the pathogenesis of acute pancreatitis induced by cerulein or L-Arginine. CaMKII phosphorylation due to cytosolic calcium oeverload was revealed in this paper. It was also demonstrated that autophagic protein aggregates degradation blockade accompanying with impaired autophagy correlated positively to intra acinar cells digestive aymogen activation sitimulated by cerulein or L-Arginine. The role of spautin-1 in ameliorating acute pancreatitis was shown here to be associated with impaired autophagy inhibition and Ca2+ overload alleviation. We provided a promising therapy for acute pancreatitis here through targeting both impaired autophagy and increased cytosolic calcium.[2]

C15H11F2N3

271.26

271.092

C, 66.42; H, 4.09; F, 14.01; N, 15.49

1262888-28-7

51037431

Light yellow to khaki solid powder

1.4±0.1 g/cm3

419.6±40.0 °C at 760 mmHg

207.6±27.3 °C

0.0±1.0 mmHg at 25°C

1.672

3.4

1

5

3

20

308

0

AWIVHRPYFSSVOG-UHFFFAOYSA-N

InChI=1S/C15H11F2N3/c16-11-3-1-10(2-4-11)8-18-15-13-7-12(17)5-6-14(13)19-9-20-15/h1-7,9H,8H2,(H,18,19,20)

6-fluoro-N-[(4-fluorophenyl)methyl]quinazolin-4-amine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.22 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 25.0 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: ≥ 2.5 mg/mL (9.22 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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