Synchrotron FTIR microspectroscopy revealed apoptosis-induced biomolecular changes of cholangiocarcinoma cells treated with ursolic acid
Graphical abstract
Introduction
Cholangiocarcinoma (CCA) is a heterogeneous cancer that arises from malignant transformation of biliary epithelial cells [1]. The highest incidence worldwide of CCA (85 cases per 100,000 individuals per year) is found in the Northeast of Thailand, where the carcinogenic liver fluke Opisthorchis viverrini is endemic [2,3]. Chronic inflammation induced by O. viverrini infection could promote the malignant transformation of cholangiocytes, the induction of oxidative stress and thus CCA genesis [4]. CCA remains a disease which is difficult to diagnose in its early stages due to the late onset of symptoms, leading to it being less amenable to curative treatment and high mortality [5]. Complete resection is the only curative therapy for CCA but this can only be performed in less than one third of the patients [6,7]. After surgery the 5-year survival rate of CCA cases is still poor with recurrence rates of 60–90% [8,9]. Current chemotherapeutic agents used in the treatment of CCA still give mediocre results [10,11]. The combination of gemcitabine and cisplatin (GC) has been shown to improve the overall survival compared to gemcitabine alone and is considered a standard chemotherapy for patients with advanced CCA [12]. However, the GC regimen can cause adverse effects, e.g., nephrotoxicity and hematological toxicity. These limitations led us into the search for a novel therapeutic candidate from natural sources for CCA therapy.
Natural products have gained a considerable interest as anti-cancer agents due to their promising effectiveness and low side effects [13]. Ursolic acid (UA; also known as 3-β-3-hydroxy-urs-12-ene-28-oic-acid) is a natural pentacyclic triterpenoid derived from a wide variety of fruits and herbs, e.g., apple peels and holy basil leaves [14]. UA has been reported to exhibit a wide range of biological properties, including anti-inflammatory, anti-cancer and antioxidant activities [15]. Numerous studies have shown that UA exhibited in vitro anti-cancer effects in various kinds of cancer through multiple mechanisms, including preventing tumor angiogenesis and metastasis, inducing apoptosis and cell cycle arrest, and inhibiting cell proliferation [[16], [17], [18], [19]]. In addition, the in vivo administration of UA resulted in the inhibition of tumor growth in gallbladder cancer and hepatocellular carcinoma [19,20].
The ultimate goal of cancer therapy is to promote the death of cancer cells. The two main types of cell death are necrosis and apoptosis. Necrosis is considered as a passive, accidental cell death which is generally caused by non-physiological conditions, such as infection and injury [21]. Necrosis is morphologically characterized by rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and the spilling of the intracellular content [22]. In contrast, apoptosis is a form of programmed cell death which is a highly regulated process. Apoptosis is characterized by distinct morphological features which include plasma membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation and formation of membrane-bound apoptotic bodies [23]. Apoptosis also leads to structural changes within the cell membrane and this is clearly evident where there is translocation of phosphatidylserine (PS) from its inner to outer leaflets. Several chemotherapeutic agents, such as cisplatin, doxorubicin, resveratrol and epigallocatechin-3-gallate, are capable of inducing apoptosis in several types of cancer including CCA [[24], [25], [26], [27], [28], [29]]. However, the above-stated anti-cancer compounds, i.e., cisplatin, doxorubicin and resveratrol, have been demonstrated to promote necrosis depending on cancer cell types [[30], [31], [32]].
Biomolecular and morphological changes are potential biomarkers of apoptosis. At the molecular level, apoptosis is a highly conserved mechanism involving the activation of a family of cysteine proteases termed caspases (e.g., caspase-2, ‐3, ‐6, ‐7, ‐8, ‐9 and ‐10), upregulation of pro-apoptotic proteins (e.g., Bax and Bid) and downregulation of anti-apoptotic proteins (e.g., Bcl-2 and survivin/BIRC5) [33,34]. Conventional techniques used for detecting apoptosis are, for instance, Annexin V-FITC/propidium iodide (PI) staining, the TUNEL assay, and caspase activity assays [35]. The aforementioned techniques require time-consuming processes and the use of dyes/chemicals; therefore, a label-free and non-invasive alternative method is needed.
Fourier transform infrared (FTIR) spectroscopy is a well-established analytical technique used to probe different chemical bonds and functional groups present in molecules [36]. This technique has also been applied for the analysis of molecular composition in biological specimens [37,38]. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy, which combines microscopy and synchrotron light source, is a high-throughput and non-destructive technique for detecting changes in the functional groups of biomolecules belonging to cell or tissue components. Recently, SR-FTIR microspectroscopy has been used in cancer research to study cell response to drugs/phytochemicals, to detect apoptosis changes, and to discriminate between benign and malignant cells [[39], [40], [41], [42]]. The typical region for FTIR spectroscopy is from 4000 cm−1 to 400 cm−1 because the vast majority of molecules exhibit FTIR peaks in this range. SR-FTIR allows the characterization of the structure and conformation of biomolecules in biological samples due to vibrations of functional groups of lipids (3000–2800 cm−1), proteins (1700–1500 cm−1), nucleic acids (1300–1100 cm−1) and carbohydrates (1100–900 cm−1) [43].
Herein, the activity and molecular mechanism of UA in CCA were investigated for the first time. The effects of UA on growth inhibition and apoptosis induction through biomolecular changes in KKU-213 and KKU-055 CCA cell lines were assessed by the sulforhodamine B (SRB) method, Annexin V-FITC/PI staining, Western blot analysis, and caspase-3/7 activity assay. While SR-FTIR microspectroscopy was utilized as a label-free technique to effectively evaluate biomolecular alterations in CCA cells in response to UA treatment. These methods have enabled us to detect the effect of UA on biomolecules, notably lipids and proteins, in CCA, providing useful information on the anti-cancer activity and biomolecular changes that occurred to CCA cells following UA treatment.
Section snippets
Materials and reagents
Calcium fluoride (CaF2) optical windows for IR light were purchased from Crystran, Dorset, UK. Ursolic acid (UA; ≥90%), sulforhodamine B sodium salt (Technical grade, dye content ≥75%), acetic acid (Analytical grade, ≥99.7%), 37% formaldehyde (Formalin), dimethyl sulfoxide (DMSO; HPLC grade, ≥99.9%), Trizma base (Tris base; ≥99.9%), skimmed milk, sodium phosphate dibasic (≥99.95%) and potassium dihydrogen phosphate (≥99.5%) were obtained from Merck KGaA, Darmstadt, Germany. Tween-20 was
Ursolic acid exerted cytotoxic effects on CCA cells
The cytotoxicity of two CCA cell lines (KKU-213 and KKU-055) treated with UA and cisplatin (0–60 μM) for 24 and 48 h was evaluated using SRB assay. This method is based on the binding of dye to basic amino acids of cellular proteins, permitting measurement of total protein content which is proportional to the number of viable cells. The cytotoxic results showed that UA inhibited the proliferation of both CCA cell lines, in a dose- and time-dependent manner (Fig. 1A). After 24- and 48-h
Discussion
The findings of this study demonstrated that UA possessed anti-proliferative and apoptotic activities through biomolecular changes and caspase-3/7 activation in CCA cells. UA inhibited cell proliferation and caused morphological changes in KKU-213 and KKU-055 cell lines. The IC50 values of UA obtained in this study are in good agreement with those previously reported in gallbladder, melanoma, and breast cancer [18,19,48]. It should be noted that the KKU-055 cell line showed higher sensitivity
Conclusion
Promoting cancer cell apoptosis is an important goal of effective cancer therapy. Our study revealed, for the first time, that UA has the ability to inhibit cell proliferation and induce apoptosis of CCA cells through biomolecular changes, altered pro- and anti-apoptotic protein expression levels and caspase-3/7 activation. We also demonstrated that SR-FTIR microspectroscopy in combination with PCA can be used as a non-labeled tool to identify changes in lipid and protein contents induced by
Declaration of Competing Interest
The authors disclose no conflicts of interest.
Acknowledgements
This study was supported by the Khon Kaen University Research Fund (620009002) to C.S.; Research and Academic Services Affairs of Khon Kaen University (KKUS (2562)-009) to C.S.; the Cholangiocarcinoma Research Institute (CARI), Khon Kaen University, Thailand (CARI 012/2560) to C.S. and P.M.; and Faculty of Medicine, Khon Kaen University, Thailand (Grant number IN62319). The authors would like to thank Dr. Andrew J. Hunt for his assistance in the preparation of this manuscript.
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