Synthesis And Antitumor Mechanism Of NO - Donor Betulin Alcohol Derivatives

Objective:Betulin, an important pentacyclic triterpene, exhibits a wide range of pharmacological properties, including antitumor, anti HIV, antibacterial and anti-inflammatory. However, the clinical application of betulin is limited due to its low water solubility and weak effect. To attain NO-donor betulin of significant antitumor, a series of betulin derivatives have been synthesized. NO release in vivo of betulin derivatives was studied. The compounds were evaluated against apanel of two human carcinoma cell lines including Huh7 and MCF-7. And the most active derivative was subjected to mechanistic studies.Methods:Betulin was modified at the positions of C-3, C-28 or/and C-30 via esterification, nucleophilic substitution and radical substitution. NO release in vivo was determined by Griess assay. Cell viability was assessed using MTT assay and colony formation assay. Apoptotic rate was detected using Annexin V-FITC/PI kit. Mitochondrial membrane potential and cell cycle were analyzed by flow cytometry. Expression of cyclin, apoptosis-related proteins and PI3K/Akt signal pathway were investigated by Western blotting.Results:Seven new NO-donor betulin derivatives, including 3,28-di-[4-(2-nitrooxy ethoxy)-1,4-succinyl]-betulin,3,28-di-[4-(4-nitrooxy butoxy)-1,4-maleyol]-oxyl-betulin, 3,28-di-acetoxy-30-nitrooxy betulin,3,28-di-{4-(8-nitrooxy-2,6-dioxyabicyclo[3.3.0] octane-4-oxo)formyl-benzoyl oxyl}-betulin,3,28-di-(2-nitrooxy-acetoxy)-betulin, 3,28-di-(2-nitrooxy-propionyloxy)-betulin and 3,28-di-(2-nitrooxy-isobutyl acyloxy)-betulin, were synthesized successfully. Their structures were confirmed by IR, NMR. The compounds released different amount of NO in the presence of L-cysteine. The NO releasing from most compounds was low and achieved balanced quickly. In particular, 3,28-di-(2-nitroxy-acetoxy)-betulin showed potent antiprolifirative activity on Huh7 and MCF-7 cell lines in a dose-dependent manner.Annexin V-FITC and PI staining indicated that 3,28-di-(2-nitrooxy-acetoxy)-betulin induced Huh 7 cell apoptosis. By the regulation of Caspase family protein pathway. The expression of Caspase 3 and Caspase 9 decreased, and the expression of C-Caspase 3 and C-Caspase 9 increased significantly. In addition, 3,28-di-(2-nitrooxy-acetoxy)-betulin induced G2/M arrest in Huh 7. The loss of mitochondrial transmembrane potential was confirmed by JC-1 staining. The green fluorescence of JC-1 monomer increased from 3.48% to 23.4%, which showed that apoptosis was induced by mitochondrial pathway. Bcl-2 protein expression was found to decrease, while the Bax expression increased. The results of Western blotting showed that 3,28-di-(2-nitrooxy-acetoxy)-betulin decreased the levels of Akt, PI3K and p110α in a concentration-dependent manner.Conclusion:3,28-di-(2-nitrooxy-acetoxy)-betulin displayed high level of anti-hepatoma activity compared with betulin. The results of mechanistic studies revealed that 3,28-di-(2-nitrooxy-acetoxy)-betulin induced apoptosis in Huh7 cell lines by inhibiting PI3K/Akt signal pathway, decreasing mitochondrial membrane potential and the expression levels of Bcl-2 protein, promoting cytochrome c release. 3,28-di-(2-nitrooxy-acetoxy)-betulin, a potential anti-hepatoma agent, is worthy of further research.