Anti-Angiogenic Effects of Betulinic Acid Administered in Nanoemulsion Formulation Using Chorioallantoic Membrane Assay

Toxicology Department, University of Medicine and Pharmacy Victor Babes Timisoara, Eftimie Murgu Square, No. 2, Timisoara, 300041, Romania.
Journal of Biomedical Nanotechnology (Impact Factor: 5.34). 04/2011; 7(2):317-24. DOI: 10.1166/jbn.2011.1297
Source: PubMed


Betulinic acid (3beta, hydroxy-lup-20(29)-en-28-oic acid, BA), a pentacyclic triterpenoid, is derived from a widely distributed natural anticancer compound betulin. It has selective anticancer activity against several tumor cells, and recently it was shown that it also possess anti-angiogenic effects. The objective of this study was to formulate betulinic acid, a poorly aqueous-soluble compound, in flax-seed oil containing nanoemulsion formulation for enhanced delivery efficiency and to effectively inhibit the tumor angiogenic process. The nanoemulsion was prepared using high pressure homogenization method with a Microfludizer processor. The betulinic acid nanoemulsion was studied for the effect on the angiogenic process by performing the in vivo chick embryo chorioallantoic membrane (CAM) assay. The sample volume of 1 microl and 5 microl of the blank and BA nanoemulsions were applied directly on the CAM. The preliminary results from macroscopic, morphological and immunohistochemical evaluations have shown that morphological change was produced in the CAM mesenchyme with a negative impact on the normal growth of the capillaries. Betulinic acid does possess anti-angiogenic activity in a dose dependent manner, and the nanoemulsion formulation maintained this effect.

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    • "Considering the reduced CAIX levels, the CAI-promoted internalization and degradation of CAIX has been discussed[39]. In further studies, BA treatment resulted in HIF-1α inhibition or anti-angiogenic activity131415. Shin et al. reported a STAT3-induced HIF-1α inhibition because BA treatment inhibited hypoxia-mediated STAT3 phosphorylation. Nevertheless, the mechanisms of the BA-induced inhibition of HIF-1α remain unclear[14]. "
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    ABSTRACT: Betulinic acid (BA), a natural compound of birch bark, is cytotoxic for many tumors. Recently, a betulinyl sulfamate was described that inhibits carbonic anhydrases (CA), such as CAIX, an attractive target for tumor-selective therapy strategies in hypoxic cancer cells. Data on combined CAIX inhibition with radiotherapy are rare. In the human breast cancer cell lines MDA-MB231 and MCF7, the effects of BA and betulinyl sulfamates on cellular and radiobiological behavior under normoxia and hypoxia were evaluated. The two most effective betulinyl sulfamates CAI 1 and CAI 3 demonstrated a 1.8-2.8-fold higher cytotoxicity than BA under normoxia in breast cancer cells, with IC50 values between 11.1 and 18.1 µM. BA exhibits its strongest cytotoxicity with IC50 values of 8.2 and 16.4 µM under hypoxia. All three substances show a dose-dependent increase in apoptosis, inhibition of migration, and inhibition of hypoxia-induced gene expression. In combination with irradiation, betulinyl sulfamates act as radiosensitizers, with DMF10 values of 1.47 (CAI 1) and 1.75 (CAI 3) under hypoxia in MDA-MB231 cells. BA showed additive effects in combination with irradiation. Taken together; our results suggest that BA and betulinyl sulfamates seem to be attractive substances to combine with radiotherapy; particularly for hypoxic breast cancer.
    Preview · Article · Nov 2015 · International Journal of Molecular Sciences
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    • "Previous studies have reported various formulation approaches for BA, such as complexation with β-cyclodextrin, nanocarriers and polymeric nanoparticles. However, no studies have demonstrated the increased oral bioavailability of BA [17], [28], [29] and systematic development as well as PK/PD evaluation of BBR containing oral formulations have not been demonstrated yet. "
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    ABSTRACT: The poor bioavailability of Berberine (BBR) and Betulinic acid (BA) limits the development of these promising anticancer agents for clinical use. In the current study, BBR and BA in spray dried (SD) mucoadhesive microparticle formulations were prepared. A patented dual channel spray gun technology established in our laboratory was used for both formulations. Gastrointestinal (GI) permeability studies were carried out using Caco-2 cell monolayer grown in in-vitro system. The oral bioavailability and pharmacokinetic profile of SD formulations were studied in Sprague Dawley rats. A549 orthotopic and H1650 metastatic NSCLC models were utilized for the anticancer evaluations. Pharmacokinetic studies demonstrated that BBR and BA SD formulations resulted in 3.46 and 3.90 fold respectively, significant increase in plasma Cmax concentrations. AUC levels were increased by 6.98 and 7.41 fold in BBR and BA SD formulations, respectively. Compared to untreated controls groups, 49.8 & 53.4% decrease in the tumor volumes was observed in SD formulation groups of BBR and BA, respectively. Molecular studies done on excised tumor (A549) tissue suggested that BBR in SD form resulted in a significant decrease in the survivin, Bcl-2, cyclin D1, MMP-9, HIF-1α, VEGF and CD31 expressions. Cleaved caspase 3, p53 and TUNEL expressions were increased in SD formulations. The RT-PCR analysis on H1650 tumor tissue suggested that p38, Phospho-JNK, Bax, BAD, cleaved caspase 3&8 mRNA expressions were significantly increased in BA SD formulations. Chronic administration of BBR and BA SD formulations did not show any toxicity. Due to significant increase in oral bioavailability and superior anticancer effects, our results suggest that spray drying is a superior alternative formulation approach for oral delivery of BBR and BA.
    Full-text · Article · Mar 2014 · PLoS ONE
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    ABSTRACT: Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells. The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression. BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10. These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.
    Full-text · Article · Aug 2011 · BMC Cancer
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