Search Results (26)

Molecular mechanisms responsible for the poor prognosis in patients with intrahepatic cholangiocarcinoma (CCA) are still unknown, but stem cell marker Cluster Differentiation 90 (CD90) has been reported to be associated with a more aggressive cancer phenotype. In this scenario, the TGF-β1 signaling pathway likely has a role as master gene regulator. Aim of the study is to investigate the role of CD90 in iCCA aggressiveness. The molecular profile of HuCCT1/CD90+ and HuCCT1/CD90− cells was obtained through transcriptomic analysis (NGS). Bioinformatic data were confirmed in both cell lines by qRT-PCR and Western blot. Cells were treated with Gemcitabine in monotherapy or in combination with Galunisertib, a selective inhibitor of TGF-βRI, in 2D and 3D models. HuCCT1/CD90+ cells are more proliferative, less migratory, and resistant to Gemcitabine treatment. HuCCT1/CD90+ cells also express lower levels of TGF-β1 compared to /CD90− cell lines. Finally, HuCCT1/CD90+ cells are resistant to Gemcitabine, while the combination of Gemcitabine and Galunisertib displays a synergistic effect on HuCCT1/CD90+ cell proliferation. These results underline that CD90-induced Gemcitabine resistance can be overcome by adding a TGFβ1 inhibitor such as Galunisertib, thereby moving further toward a precision medicine approach in patients with iCCA. Full article

Epicardial fat density (EFD) is implicated in cardiovascular diseases. This study aimed to assess the regional variability of epicardial fat density (EFD) using coronary computed tomography (CCT) and evaluate the feasibility of ROI-based measurements as an alternative to full segmentation. A retrospective analysis was conducted on 171 patients undergoing coronary CCT. EFD was measured on non-contrast scans acquired globally and in three predefined regions of interest (ROIs) for coronary calcium scoring: the aortic bulb, right posterolateral wall, and cardiac apex. Global EFD was quantified using semi-automated segmentation software (3D Slicer 5.6.2), while regional EFD values were manually determined. Statistical analyses were performed to compare global and regional EFD measurements. Global EFD averaged −83.92 ± 5.19 HU, while regional EFD showed significant variability. The aortic bulb had lower EFD values (−97.54 ± 12.80 HU) compared to the apex (−93.42 ± 18.94 HU) and right posterolateral wall (−94.99 ± 12.16 HU). Paired t-tests confirmed statistically significant differences between global and regional EFD values (p < 0.000). This study highlights significant regional variability in EFD across specific cardiac regions, suggesting that ROI-based assessments may not reliably reflect global EFD characteristics. Full article

In intrahepatic cholangiocarcinoma (iCCA), thrombospondin 1 (THBS1) and 2 (THBS2) are soluble mediators released in the tumor microenvironment (TME) that contribute to the metastatic spreading of iCCA cells via a lymphatic network by the trans-differentiation of vascular endothelial cells to a lymphatic-like phenotype. To study the direct role of THBS1 and THBS2 on the iCCA cells, well-established epithelial (HuCCT-1) and mesenchymal (CCLP1) iCCA cell lines were subjected to recombinant human THBS1 and THBS2 (rhTHBS1, rhTHBS2) for cellular function assays. Cell growth, cell adhesion, migration, and invasion were all enhanced in both CCLP1 and HuCCT-1 cells by the treatment with either rhTHBS1 or rhTHBS2, although they showed some variability in their intensity of speeding up cellular processes. rhTHBS2 was more intense in inducing invasiveness and in committing the HuCCT-1 cells to a mesenchymal-like phenotype and was therefore a stronger enhancer of the malignant behavior of iCCA cells compared to rhTHBS1. Our data extend the role of THBS1 and THBS2, which are not only able to hinder the vascular network and promote tumor-associated lymphangiogenesis but also exacerbate the malignant behavior of the iCCA cells. Full article

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial–mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression. Full article

The epicardial adipose tissue (EAT) is the visceral fat located between the myocardium and the pericardium. We aimed to perform a longitudinal evaluation of the epicardial adipose tissue using an advanced computer-assisted approach in a population of patients undergoing Cardiac CT (CCT) during statin administration, in relation to their body mass index (BMI). We retrospectively enrolled 95 patients [mean age 62 ± 10 years; 68 males (72%) and 27 females (28%)] undergoing CCT for suspected coronary artery disease during statin administration. CCT was performed at two subsequent time points. At the second CCT, EAT showed a mean density increase (−75.59 ± 7.0 HU vs. −78.18 ± 5.3 HU, p < 0.001) and a volume decrease (130 ± 54.3 cm³ vs.142.79 ± 56.9 cm³, p < 0.001). Concerning coronary artery EAT thickness, a reduction was found at the origin of the right coronary artery (13.26 ± 5.2 mm vs. 14.94 ± 5.8, p = 0.001) and interventricular artery (8.22 ± 3.7 mm vs. 9.13 ± 3.9 mm, p = 0.001). The quartile (Q) attenuation percentage (%) distribution of EAT changed at the second CCT. The EAT % distribution changed by the BMI in Q1 (p = 0.015), Q3 (p = 0.001) and Q4 (p = 0.043) at the second CCT, but the normal-BMI and overweight/obese patients showed a similar response to statin therapy in terms of quartile distribution changes. In conclusion, statins may determine significant changes in EAT quantitative and qualitative characteristics detected by CCT; the BMI influences the EAT composition, but statins determine a similar response in quartile distribution’s variation, irrespective of the BMI. Full article

Bile duct cancer, or cholangiocarcinoma, is a rare disease with limited treatment options that include surgery and cytotoxic chemotherapy. The high recurrence rate and poor prognosis of this type of cancer highlights the need to identify new and more effective therapeutic targets. In this study, we found that AXL, a receptor tyrosine kinase, is highly expressed in biliary cancer patients and significantly correlated with poor patient outcomes, including metastasis and low survival rates. We also demonstrated that targeting AXL inhibits tumor progression. In vitro studies with bile duct cancer cells (SNU1196 and HUCCT1) showed that genetic knockdown of AXL significantly reduced both tumor cell growth and invasion. In addition, in vivo studies using subcutaneous and orthotopic intrahepatic models demonstrated that genetic inhibition of AXL resulted in tumor-growth delay. To further examine the possible clinical translation of AXL inhibition in the clinic, we tested the efficacy of AVB-500, a soluble AXL receptor, in reducing AXL activation and tumor growth. AVB-500 was effective at inhibiting AXL activation and decreasing the growth and invasion of SNU1196 and HUCCT1 tumors which possess high AXL expression. Most importantly, AVB-500 was highly effective at decreasing tumor dissemination of bile duct tumor cells in the peritoneal cavity. This study strongly supports the idea of using the AXL receptor as a new therapeutic target to treat the growth and progression of biliary cancer. Full article

Metabolic syndrome (MetS) is a common condition closely associated with non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). Recent meta-analyses show that MetS can be prodromal to intrahepatic cholangiocarcinoma (iCCA) development, a liver tumor with features of biliary differentiation characterized by dense extracellular matrix (ECM) deposition. Since ECM remodeling is a key event in the vascular complications of MetS, we aimed at evaluating whether MetS patients with iCCA present qualitative and quantitative changes in the ECM able to incite biliary tumorigenesis. In 22 iCCAs with MetS undergoing surgical resection, we found a significantly increased deposition of osteopontin (OPN), tenascin C (TnC), and periostin (POSTN) compared to the matched peritumoral areas. Moreover, OPN deposition in MetS iCCAs was also significantly increased when compared to iCCA samples without MetS (non-MetS iCCAs, n = 44). OPN, TnC, and POSTN significantly stimulated cell motility and the cancer-stem-cell-like phenotype in HuCCT-1 (human iCCA cell line). In MetS iCCAs, fibrosis distribution and components differed quantitatively and qualitatively from non-MetS iCCAs. We therefore propose overexpression of OPN as a distinctive trait of MetS iCCA. Since OPN stimulates malignant properties of iCCA cells, it may provide an interesting predictive biomarker and a putative therapeutic target in MetS patients with iCCA. Full article

It is well established that Cholangiocarcioma (CCA) drug resistance plays a crucial role in the spread and survival of cancer cells. The major enzyme in the nicotinamide-adenine dinucleotide (NAD+)-mediated pathways, nicotinamide phosphoribosyltransferase (NAMPT), is essential for cancer cell survival and metastasis. Previous research has shown that the targeted NAMPT inhibitor FK866 reduces cancer cell viability and triggers cancer cell death; however, whether FK866 affects CCA cell survival has not been addressed before. We show herein that NAMPT is expressed in CCA cells, and FK866 suppresses the capacity of CCA cells to grow in a dose-dependent manner. Furthermore, by preventing NAMPT activity, FK866 significantly reduced the amount of NAD+ and adenosine 5′-triphosphate (ATP) in HuCCT1, KMCH, and EGI cells. The present study’s findings further show that FK866 causes changes in mitochondrial metabolism in CCA cells. Additionally, FK866 enhances the anticancer effects of cisplatin in vitro. Taken together, the results of the current study suggest that the NAMPT/NAD+ pathway may be a possible therapeutic target for CCA, and FK866 may be a useful medication targeting CCA in combination with cisplatin. Full article

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, has high mortality rates because of its limited treatment options and acquired resistance to chemotherapy. Sulforaphane (SFN), a naturally occurring organosulfur compound found in cruciferous vegetables, exhibits multiple therapeutic properties, such as histone deacetylase (HDAC) inhibition and anti-cancer effects. This study assessed the effects of the combination of SFN and gemcitabine (GEM) on human iCCA cell growth. HuCCT-1 and HuH28 cells, representing moderately differentiated and undifferentiated iCCA, respectively, were treated with SFN and/or GEM. SFN concentration dependently reduced total HDAC activity and promoted total histone H3 acetylation in both iCCA cell lines. SFN synergistically augmented the GEM-mediated attenuation of cell viability and proliferation by inducing G2/M cell cycle arrest and apoptosis in both cell lines, as indicated by the cleavage of caspase-3. SFN also inhibited cancer cell invasion and decreased the expression of pro-angiogenic markers (VEGFA, VEGFR2, HIF-1α, and eNOS) in both iCCA cell lines. Notably, SFN effectively inhibited the GEM-mediated induction of epithelial–mesenchymal transition (EMT). A xenograft assay demonstrated that SFN and GEM substantially attenuated human iCCA cell-derived tumor growth with decreased Ki67⁺ proliferative cells and increased TUNEL⁺ apoptotic cells. The anti-cancer effects of every single agent were markedly augmented by concomitant use. Consistent with the results of in vitro cell cycle analysis, G2/M arrest was indicated by increased p21 and p-Chk2 expression and decreased p-Cdc25C expression in the tumors of SFN- and GEM-treated mice. Moreover, treatment with SFN inhibited CD34-positive neovascularization with decreased VEGF expression and GEM-induced EMT in iCCA-derived xenografted tumors. In conclusion, these results suggest that combination therapy with SFN with GEM is a potential novel option for iCCA treatment. Full article

Cholangiocarcinoma (CCA) is a malignant neoplasm of the bile ducts, being the second most common type of cancer in the liver, and most patients are diagnosed at a late stage with poor prognosis. Targeted therapy aiming at receptors tyrosine kinases (RTKs) such as c-Met or EGFR have been developed but with unsatisfactory outcomes. In our recent report, we found several oncogenic molecules downstream of RTKs, including hydrogen peroxide clone-5 (Hic-5), Src, AKT and JNK, were elevated in tissues of a significant portion of metastatic CCAs. By inhibitor studies and a knockdown approach, these molecules were found to be within the same signal cascade responsible for the migration of HuCCT1 cells, a conventionally used CCA cell line. Herein, we also found Src inhibitor dasatinib and Hic-5 siRNA corporately suppressed HuCCT1 cell invasion. Moreover, dasatinib inhibited the progression of the HuCCT1 tumor on SCID mice skin coupled with decreasing the expression of Hic-5 and EGFR and the activities of Src, AKT and JNK. In addition, we found a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and several cytoskeletal molecules such as tubulin and cofilin were dramatically decreased after a long-term treatment of the HuCCT1 tumor with a high dose of dasatinib. Specifically, GAPDH was shown to be a downstream effector of the Hic-5/Src/AKT cascade involved in HuCCT1 cell migration. On the other hand, TFK1, another CCA cell line without Hic-5 expression, exhibited very low motility, whereas an ectopic Hic-5 expression enhanced the activation of Src and AKT and marginally increased TFK1 migration. In the future, it is tempting to investigate whether cotargeting Src, Hic-5 and/or GAPDH is efficient for preventing CCA progression in future clinical trials. Full article

Thymoquinone (TQ), a plant-based bioactive constituent derived from the volatile oil of Nigella sativa, has been shown to possess some anti-neoplastic activities. The present study aimed to investigate the mitochondria and apoptosis observed when TQ is applied against hepatocellular carcinoma (HepG2) and cholangiocarcinoma (HuCCT1) cells, two of the most common primary tumors of the liver. All cell lines were treated with increasing concentrations of TQ for varying durations. The anti-proliferative effect of TQ was measured using the methoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and resulted in dose- and time-dependent growth inhibition in both cell lines. Cell cycle, apoptosis, and assessment of mitochondria viability by morphology assessment and evaluation of the mitochondrial membrane potential were investigated. The present study confirms that TQ caused cell cycle arrest at different phases and induced apoptosis in both cell lines. A systematic review of rodent animal models was also carried out. Overall, our data seem to represent the most robust results, suggesting that TQ possesses promising therapeutic potential as an anti-tumor agent for the treatment of hepatocellular carcinoma and cholangiocarcinoma. Full article

Alpha-mangostin (AM), a significant component isolated from the pericarp of mangosteen (Garcinia mangostana L.), has been demonstrated as a potential compound for the treatment of cholangiocarcinoma (CCA). Due to its hydrophobic nature, however, its clinical uses may be limited by its low aqueous solubility, poor stability, and low bioavailability. The present study aimed to formulate and characterize the AM-loaded PLGA nanoparticles (AM-PLGA-NPs) and further evaluate the antiproliferative and proapoptotic activities, including the inhibitory activities on CCA cell (CL-6 and HuCCT-1) invasion and migration. The AM-PLGA-NPs were prepared using PLGA MW 7000–17,000 and 38,000–54,000 by the solvent displacement method. The methods used to evaluate these activities included a MTT assay, flow-cytometry, QCM ECMatrix cell migration, and cell invasion assays, respectively. The optimized AM-PLGA-NPs were characterized for physical (particle size and morphology, polydispersity index, and zeta potential) and pharmaceutical (encapsulation efficiency, loading efficiency, and drug release profile) parameters. AM-PLGA-NPs showed relatively potent and selective antiproliferative and proapoptotic activities in both CCA cell lines in a concentration- and time-dependent manner. The results revealed that the PLGA nanoparticles could be a suitable nanocarrier to encapsulate AM for its delivery to the CCA cells. Full article

Cholangiocarcinoma (CCA) is a rare and highly lethal disease with few effective treatment options. Cannabinoids, cannabidiol (CBD) and cannabigerol (CBG) are non-psychedelic components extracted from cannabis. These non-psychoactive compounds have shown anti-proliferative potential in other tumor models; however, the efficacy of CBD and CBG in CCA is unknown. Furthermore, two cell death pathways are implicated with CBD resulting in autophagic degeneration and CBG in apoptosis. HuCC-T1 cells, Mz-ChA-1 cells (CCA cell lines) and H69 cells (immortalized cholangiocytes), were treated with CBD and CBG for 24 to 48 h. The influence of these cannabinoids on proliferation was assessed via MTT assay. Apoptosis and cell cycle were evaluated via Annexin-V apoptosis assay and propidium iodide, respectively. The expression of proliferation biomarker Ki-67, apoptosis biomarker BAX, and autophagic flux biomarkers LC3b and LAMP1 were evaluated via immunofluorescence. Cell migration and invasion were evaluated via wound healing assay and trans-well migration invasion assays, respectively. The colony formation was evaluated via colony formation assay. In addition, the expression of autophagy gene LC3b and apoptosis genes BAX, Bcl-2, and cleaved caspase-3 were evaluated via Western blot. CBD and CBG are non-selective anti-proliferative agents yielding similar growth curves in CCA; both cannabinoids are effective, yet CBG is more active at lower doses. Low doses of CBD and CBG enhanced immortalized cholangiocyte activity. The reduction in proliferation begins immediately and occurs maximally within 24 h of treatment. Moreover, a significant increase in the late-stage apoptosis and a reduction in the number of cells in S stage of the cell cycle indicates both CBD and CBG treatment could promote apoptosis and inhibit mitosis in CCA cells. The fluorescent expression of BAX and LC3b was significantly enhanced with CBD treatment when compared to control. LAMP1 and LC3b colocalization could also be observed with CBD and CBG treatment indicating changes in autophagic flux. A significant inhibition of migration, invasion and colony formation ability was shown in both CBD and CBG treatment in CCA. Western blot showed an overall decrease in the ratio of anti-apoptotic protein Bcl-2 with respect to pro-apoptotic protein BAX with CBG treatment. Furthermore, CBD treatment enhanced the expression of Type II cell death (autophagic degeneration) protein LC3b, which was reduced in CBG-treated CCA cells. Meanwhile, CBG treatment upregulated Type I cell death (programmed apoptosis) protein cleaved caspase-3. CBD and CBG are effective anti-cancer agents against CCA, capable of inhibiting the classic hallmarks of cancer, with a divergent mechanism of action (Type II or Type I respectively) in inducing these effects. Full article

Overexpression of casein kinase 2 (CK2) has an oncogenic and pro-survival role in many cancers. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-angiogenic effects. Up to date, the anti-cancer effect and mechanism of CX-4945 on human cholangiocarcinoma (CCA) remain unclear. This study investigated whether CX-4945 inhibits growth and induces apoptosis of HuCCT-1 cells, a human CCA cell line. Of note, treatment with CX-4945 at 20 μM markedly reduced survival and induced apoptosis of HuCCT-1 cells, as evidenced by nuclear DNA fragmentation, PARP cleavage, activation of caspase-9/3, and up-regulation of DR-4. Although CX-4945 did not affect the phosphorylation and expression of CK2, it vastly inhibited the phosphorylation of CK2 substrates, supporting the drug’s efficacy in inhibiting CK2 and its downstream pathway. Importantly, knockdown of CK2 that partially suppressed the phosphorylation of CK2 substrates resulted in a significant reduction of HuCCT-1 cell survival. In addition, CX-4945 reduced the phosphorylation and expression of STAT-3 and STAT-5 in HuCCT-1 cells, and pharmacological inhibition or respective knockdown of these proteins resulted in significant growth suppression of HuCCT-1 cells. CX-4945 also had abilities to decrease Mcl-1 expression while increasing eIF-2α phosphorylation in HuCCT-1 cells. Furthermore, there was a time-differential negative regulation of HIF-1α expression by CX-4945 in HuCCT-1 cells, and knockdown of HIF-1α caused a significant reduction of the cell survival. In summary, these results demonstrated that CX-4945 has anti-growth, anti-angiogenic, and pro-apoptotic effects on HuCCT-1 cells, which are mediated through control of CK2, caspase-9/3, DR-4, STAT-3/5, Mcl-1, eIF-2α, and HIF-1α. Full article

The aim of this study is to prepare pH- and redox-sensitive nanoparticles for doxorubicin (DOX) delivery against DOX-resistant HuCC-T1 human cholangiocarcinoma (CCA) cells. For this purpose, L-histidine methyl ester (HIS) was attached to chitosan oligosaccharide (COS) via dithiodipropionic acid (abbreviated as ChitoHISss). DOX-incorporated nanoparticles of ChitoHISss conjugates were fabricated by a dialysis procedure. DOX-resistant HuCC-T1 cells were prepared by repetitive exposure of HuCC-T1 cells to DOX. ChitoHISss nanoparticles showed spherical morphology with a small diameter of less than 200 nm. The acid pH and glutathione (GSH) addition induced changes in the size distribution pattern of ChitoHISss nanoparticles from a narrow/monomodal distribution pattern to a wide/multimodal pattern and increased the fluorescence intensity of the nanoparticle solution. These results indicate that a physicochemical transition of nanoparticles can occur in an acidic pH or redox state. The more acidic the pH or the higher the GSH concentration the higher the drug release rate was, indicating that an acidic environment or higher redox states accelerated drug release from ChitoHISss nanoparticles. Whereas free DOX showed decreased anticancer activity at DOX-resistant HuCC-T1 cells, DOX-incorporated ChitoHISss nanoparticles showed dose-dependent anticancer activity. Intracellular delivery of DOX-incorporated ChitoHISss nanoparticles was relatively increased at an acidic pH and in the presence of GSH, indicating that DOX-incorporated ChitoHISss nanoparticles have superior acidic pH- and redox-sensitive behavior. In an in vivo tumor xenograft model, DOX-incorporated ChitoHISss nanoparticles were specifically delivered to tumor tissues and then efficiently inhibited tumor growth. We suggest that ChitoHISss nanoparticles are a promising candidate for treatment of CCA. Full article