RESUMO
Cholangiocarcinoma (CCA), an aggressive malignancy arising from the biliary epithelium, exhibits a high incidence in Thailand. CCA usually lacks specific symptoms and is typically diagnosed in its advanced stages, presenting significant treatment challenges. Current CCA therapeutic options, including surgery, chemotherapy, and radiation, have limited success rates and often cause side effects. Nature-derived compounds hold promise for reducing undesirable adverse effects and are an excellent source of anticancer drugs. Corosolic acid (CA), a triterpenoid found in Lagerstroemia speciosa L. leaves, exhibits anticancer properties; however, the effectiveness of CA against CCA and its molecular mechanisms remained unexplored. Herein, the anti-CCA and apoptosis-inducing effects of CA were investigated using various techniques, i.e., the MTT assay, flow cytometry with FITC-labeled Annexin V (Annexin V-FITC) and propidium iodide double staining, JC-1 staining, western blot analysis, caspase-3 activity assay, and molecular dynamics (MD) simulations. CA inhibited the proliferation of KKU-213A and KKU-213B CCA cells and triggered apoptosis through alterations in mitochondrial membrane potential (ΔΨm), and increases in the Bax/Bcl-2 expression ratio, cytochrome c release, and caspase-3 activity. As indicated by MD simulations, CA has the potential to bind to Bcl-2 through hydrogen bonds between amino acid residues R146 and N143. These findings underscore the potential of CA as a promising candidate for treatment of CCA.
RESUMO
Cholangiocarcinoma (CCA), an epithelial biliary tract malignancy, is a significant health concern in the Greater Mekong Subregion, particularly in northeastern Thailand. Prior to the development of advanced stages, CCA is typically asymptomatic, thereby limiting treatment options and chemotherapeutic effectiveness. Ursolic acid (UA), a triterpenoid derived from plants, was previously discovered to inhibit CCA cell growth through induction of apoptosis. Nevertheless, the therapeutic effectiveness of UA is limited by its poor solubility in water and low bioavailability; therefore, dimethyl sulfoxide (DMSO) is utilized as a solvent to treat UA with CCA cells. Enhancing cellular uptake and reducing toxicity, the utilization of polymeric nanoparticles (NPs) proves beneficial. In this study, UA-loaded PLGA nanoparticles (UA-PLGA NPs) were synthesized using nanoprecipitation and characterized through in silico formation analysis, average particle size, surface functional groups and ζ-potential measurements, electron microscopic imaging, drug loading efficiency and drug release studies, stability, hemo- and biocompatibility, cytotoxicity and cellular uptake assays. Molecular dynamics simulations validated the loading of UA into PLGA via hydrogen bonding. The synthesized UA-PLGA NPs had a spherical shape with an average size of 240 nm, a negative ζ-potential, good stability, great hemo- and bio-compatibility and an encapsulation efficiency of 98%. The NPs exhibited a characteristic of a simple diffusion-controlled Fickian process, as predicted by the Peppas-Sahlin drug release kinetic model. UA-PLGA NPs exhibited cytotoxic effects on KKU-213A and KKU-055 CCA cells even when dispersed in media without organic solvent, i.e., DMSO, highlighting the ability of PLGA NPs to overcome the poor water solubility of UA. Rhodamine 6G (R6G) was loaded into PLGA NPs using the same approach as UA-PLGA NPs, demonstrating effective delivery of the dye into CCA cells. These findings suggest that UA-PLGA NPs showed promise as a potential phytochemical delivery system for CCA treatment.
RESUMO
BACKGROUND: Ursolic acid (UA) is a natural triterpenoid which possesses anti-cancer activity. However, little is known regarding the activity and molecular mechanism of UA in cholangiocarcinoma (CCA). Thus, we investigated the effects of UA on growth inhibition and apoptosis induction through biomolecular changes in KKU-213 and KKU-055 CCA cell lines. METHODS: The anti-proliferative effect of UA against CCA cells was evaluated using SRB assay. Changes in biomolecules were assessed by SR-FTIR microspectroscopy combined with PCA and conventional methods (i.e., Annexin V-FITC/PI staining for lipid alteration and apoptosis induction; Western blot analysis and caspase-3/7 activity assay for apoptotic protein detection). RESULTS: UA suppressed the proliferation of CCA cells in a dose- and time-dependent manner. SR-FTIR data revealed a significant alteration in lipids attributable to changes in apoptotic cell membranes, confirmed by Annexin V-FITC/PI staining. SR-FTIR data showed that UA promoted changes in the protein secondary structure. Elevated expression of Bax and decreased expression of Bcl-2 and survivin/BIRC5 along with augmented caspase-3/7 activity supported alterations in apoptosis-related proteins. CONCLUSIONS: SR-FTIR microspectroscopy was successfully used as a label-free technique to monitor apoptosis-induced biomolecular changes in UA-treated CCA cells. UA exerted the cytotoxic and apoptotic activities in CCA cells through alterations in membrane lipids and apoptotic proteins. UA could be a potential anti-CCA candidate and a chemical starting point for the discovery of novel anti-cancer agents. SIGNIFICANCE: Our present study showed the first evidence that UA exhibited the anti-proliferative and pro-apoptotic activities toward CCA cells through changes in biomolecules, notably lipids and proteins.