| Purposes:In present, there is a trend that morbidity of lung cancer were increased. Non-small cell lung cancer (NSCLC) accounts for appoximately 75%-85% of all the lung cancers, and the five-year survival rate of patients with NSCLC was only 14%. The majority of the dignosed patients with NSCLC died of side effects of chemotherapy and radiotherapy. Therefore, there is an important target in searching for more safe, effective and low-toxical anti-cancer drugs from the natural compounds. Arenobufagin, one of the most important active ingredients of toad venom, is a type of C-24 steroid with a characteristic a-pyrone ring at C-17. The molecular formula was C24H32O6 and formula weight was 416.511. Previous reseach have shown that arenobufagin could inhibit human hepatoma HepG2 cell adhesion, migration, and invasion. Arenobufagin could induce human hepatoma HepG2 cell apoptosis. However, these types of C-24 steroid, including arenobufagin usually have narrow therapeutic index and high toxicity. Pharmacokinetics was that drugs are handled by body in four stages:Absorption, distribution, metabolism, and elimination (ADME). The pharmacological effect was related to the concentration of drug at the site of action. The pharmacological and toxicological effect was changed by biotransformed in body. Understanding ADME, which is beneficial to understanding the effect of pharmacology and toxicology. Purpose:To investigate arenobufagin and 1β-hydroxyl-arenobufagin induced apoptosis in A549 cells and their Pharmacokinetics. The main content of this study were as follows:Firstly the influence of arenobufagin on human lung adenocarcinoma A549 cell apoptosis. Secondly the biotransformation of arenobufagin was performed in rat. The metabolite of arenobufagin in rat was preliminary screened by LC-MS technology and Light Sight software. Then we investigated that the apoptotic mechanism of 1β-hydroxyl-arenobufagin and PI3K/Akt pathway involved. Finally, The Pharmacokinetics and tissue distribution of arenobufagin was conducted. A rapid, sensitive, and specific method for the pharmacokinetics and tissue distribution study of arenobufagin in rat plasma and tisssues by ultra fast liquid chromatography-tandem mass spectrometry was developed. The pharmacokinetics and tissue distribution of arenobufagin were clarifed.Methods:(1) MTT assay was performed to determine cell inhibition ration and IC50 value. The phase-contrast microscope and the Hoehest33342 staining were performed to observe the morphological changes of A549 cells apoptosis and apoptotic body. Annexin V/PI double stain and PI single stain were used to determined apoptosis rate and cell cycle.(2) ①Metabolism and Elimination:LC-MS/MS method was performed to determination of arenobufagin in rat bile, urine, and feces. The metabolites were identificated by Lightsight software and LC-MS technology. ②1β-hydroxyl-arenobufagin induced the A549 cells apoptotic mechanism and inhibited PI3K/Akt pathway:MTT assay was performed to determine cell inhibition ration and IC50 value. The phase-contrast microscope and the Hoehest33342 staining were performed to observe the morphological changes of A549 cells apoptosis and apoptotic body. Annexin V-PI double stain and PI single stain were used to determined apoptosis rate and cell cycle. PCR and Western Blot method were determined the expression of PI3K/Akt/mTOR pathway, Bax, Bcl-2, Caspase-3 and Caspase-9. Cell scratch assay and transwell chamber assay were used to determine changes in migration and invasion of A549 cells.(3) Quantitative determination of arenobufagin and 1β-hydroxyl-arenobufagin in rat plasma and tissues by ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) and applicantion to pharmacokinetics and tissue distribution study. The pharmacokinetics parameters were acquired and the tissue distribution was understood.Result:(1) Arenobufagin could induce apoptosis of A549 cells in a dose-dependent manner. The IC50 value was 431nM. The capacity of cells became smaller. The cell nucleus was high agglutination and pyknosis, the side cells were increased, and apoptotic bodies were appeared by the hoechst 33342 fluorescent staining assay, arenobufagin could induce A549 cells apoptosis in morphology. Arenobufagin treatment could increase the apoptotic G2/M fraction in dose-dependent manner by PI single stain assay. And arenobufagin could induce G2/M block. Annexin V and PI double stain assay was shown that arenobufagin induced cell apoptosis.(2) ①In elimination and metaabolism:The main parameters obtained after single dose of 4.0 mg/kg arenobufagin to rats were as follows:the value t1/2 of 1.66±0.07h,2.54±0.78h and 5.87±1.22h, excretion rate of 2.301%,19.67% and 1.726% respectively. The results showed that a total of 24 metabolites were found from the biosamples of rat plasma, urine, bile and feces, including phase I metabolites and phase Ⅱ metabolites. Metabolic reactions included oxidation, reduction, methylation, glucuronic acid conjugation and sulfate conjugation. Also there were some of arenobufagin detected in bile, urine, and feces. It maybe pass out of the body directly via urine, bile, and feces without any biotransformations. So liver is the main metabolic organ of arenobufagin, and then the metabolites and arenobufagin are eliminated via urine and bile.②1β-hydroxyl-arenobufagin induced the A549 cells apoptotic mechanism and inhibited PI3K/Akt pathway: 1 β-hydroxyl-arenobufagin could induce apoptosis of A549 cells in a dose-dependent manner. IC50 value was 208nM. The capacity of cells became smaller. The cell nucleus was high pyknosis and agglutination, side cells were increased, and apoptotic bodies were detected by Hoechst 33342 fluorescent staining assay. 1β-hydroxyl-arenobufagin could induce A549 cells apoptosis in morphology. 1β-hydroxyl-arenobufagin treatment increased the apoptotic G2/M fractions in a dose-dependent manner by PI single stain assay. And 1β-hydroxyl-arenobufagin could induce G2/M block. Annexin V and PI double stain assay was shown that 1β-hydroxyl-arenobufagin induced cell apoptosis owe to earlier apoptosis. 1β-hydroxyl-arenobufagin could down-regulate the protein and mRNA expression of PI3K/Akt/m-TOR pathway and up-regulation the rate of Bax/Bcl-2. The protein and mRNA expression of Caspase-3 and Caspase-9 were increased. The migration and invasion of A549 cells was inhibited.(3) The method was applied to study the pharmacokinetics of SD rats after administration of 2.0,4.0,8.0 mg/kg of arenobufagin. Mean peak plasma levels of arenobufagin (Cmax) of 1078±182.6ng/mL,1981±566.5ng/mL,4373±1354ng/mL and Tmax of 12.5±2.7min,11.7±2.6min, and 11.7±2.6 min were observed. The mean ti/2 value of 103.8±40.43min,113.0±47.02min and 110.3±21.17min was obtained. MRT0-t was calculated to be 45.6±6.8min,38.6±9.6min,42.7±10.5min. AUC0-t and AUC0-∞ were calculated to be 39.56±11.10mg·min/L,79.67±26.06mg·min/L, 166.7±57.96mg·min/L and 39.63±11.14mg·min/L,79.78±26.11mg·min/L, 166.9±57.80 mg·in/L, respectively. Mean peak plasma levels of 1β-hydroxyl-arenobufagin (Cmax) of 125.48±15.31 ng/mL,206.3±79.88 ng/mL, 468.1±135.7ng/mL and Tmax of 59.28±7.319min,47.5±6.124min,47.50±6.124min were observed. The mean t1/2 value of 113.8±37.48 min,106.3±39.42 min and 123.8±47.43min was obtained. MRT0-t were calculated to be 43.61±11.63min, 38.57±12.61min, and 47.63±14.72min. AUC0-t and AUCo-∞ were calculated to be 607.3±196.1μg·min/L,912.5±316.1μg·min/L,2389±743.8μg·min/mL and 611.9±203.5μg·min/L,927.8±343.5 μg·min/L,2412±809.1 μg-min/L, respectively. The result has found that AUC0-t and Cmax were linear over the three doses (2.0,4.0, and 8.0 mg/kg). Arenobufagin and 1 β-hydroxyl-arenobufagin has widely tissue distributed in rats after administration of 4.0mg/kg arenobufagin. Maximum concentrations of arenobufagin for tissues examined were seen at 5.0 or 15 min and 1β-hydroxyl-arenobufagin were at 45 or 60 min after administration. The tissue concentration of arenobufagin and 1 P-hydroxyl-arenobufagin wre highest in the liver, kidney, and lung followed by spleen, heart, and muscle after 45min. Arenobufagin and 1β-hydroxyl-arenobufagin was detected in brain and testis after administration. Arenobufagin and 1β-hydroxyl-arenobufagin can cross the blood-brain barrier and blood-testis barrierConclusion:Arenobufagin could inhibit A549 cells proliferation; induce A549 cells apoptosis, and G2/M block. In biotransformation:The total elimination rate was 23.43%. A total of 24 metabolites were detected, including phase I metabolites and phase Ⅱ metabolites. Liver is the main metabolic organ of arenobufagin, and then the metabolites are eliminated via bile and urine.1β-hydroxyl-arenobufagin is one of metabolite of arenobufagin.1β-hydroxyl-arenobufagin could also inhibit A549 cells proliferation, migration, and invasion.1β-hydroxyl-arenobufagin could induce A549 cells apoptosis and G2/M block. The mechanism may be as follows: ① down-regulate the protein and mRNA expression of PI3K/Akt/m-TOR pathway.②up-regulation protein and mRNA expression of Bax and down-regulate the protein and mRNA expression of Bcl-2, so that increased the rate of Bax/Bcl-2.③ increased protein and mRNA expression of Caspase-3 and Caspase-9. After study of pharmacokinetics and tissue distribution, the result has found that AUCo.t and Cmax were linear over the three doses (2.0,4.0, and 8.0 mg/kg). Arenobufagin and 1 β-hydroxyl-arenobufagin has widely tissue distributed in rats after administration of 4.0mg/kg arenobufagin. Arenobufagin and 1 P-hydroxyl-arenobufagin can cross the blood-brain barrier and blood-testis barrier. |
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