Therapeutic Implications of Ceritinib in Cholangiocarcinoma beyond ALK Expression and Mutation.

Cholangiocarcinoma (CCA) is a difficult-to-treat cancer, with limited therapeutic options and surgery being the only curative treatment. Standard chemotherapy involves gemcitabine-based therapies combined with cisplatin, oxaliplatin, capecitabine, or 5-FU with a dismal prognosis for most patients. Receptor tyrosine kinases (RTKs) are aberrantly expressed in CCAs encompassing potential therapeutic opportunity. Hence, 112 RTK inhibitors were screened in KKU-M213 cells, and ceritinib, an approved targeted therapy for ALK-fusion gene driven cancers, was the most potent candidate. Ceritinib's cytotoxicity in CCA was assessed using MTT and clonogenic assays, along with immunofluorescence, western blot, and qRT-PCR techniques to analyze gene expression and signaling changes. Furthermore, the drug interaction relationship between ceritinib and cisplatin was determined using a ZIP synergy score. Additionally, spheroid and xenograft models were employed to investigate the efficacy of ceritinib in vivo. Our study revealed that ceritinib effectively killed CCA cells at clinically relevant plasma concentrations, irrespective of ALK expression or mutation status. Ceritinib modulated multiple signaling pathways leading to the inhibition of the PI3K/Akt/mTOR pathway and activated both apoptosis and autophagy. Additionally, ceritinib and cisplatin synergistically reduced CCA cell viability. Our data show ceritinib as an effective treatment of CCA, which could be potentially explored in the other cancer types without ALK mutations.

Preclinical evidence for anaplastic lymphoma kinase inhibitors as novel therapeutic treatments for cholangiocarcinoma.

Introduction: Bile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA. Methods: ALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells. Results: ALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis. Discussion: These results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

Low molecular weight sulfated galactan (LMSG) supplemented with octanoyl ester (Oct-LMSG) demonstrated superior wound healing activity compared to the unsupplemented LMSG in a fibroblast wound model. To test the hypothesis that the increased bioactivity of Oct-LMSG may depend on its penetration into the plasma membrane, its cellular uptake was investigated and collagen production in fibroblast cells was assessed for the first time. The cellular uptake of Oct-LMSG was examined using indirect immunofluorescence and a confocal laser scanning microscope. In addition, the degree of fibroblast activation associated with this uptake was evaluated. The results indicated increased LMSG internalization in fibroblasts treated with Oct-LMSG. Transmission electron micrographs revealed the ultrastructure of active protein production in fibroblasts upon treatment with Oct-LMSG. In addition, Oct-LMSG upregulated the expression of type I collagen mRNA and proteins, as well as related signaling molecules involved in collagen synthesis, including collagen type I α1 chain (Col1A1), Col1A2, phosphorylated (p)-Smad2/3 and p-Smad4. The current findings support the notion that the supplementation of LMSG with octanoyl enhanced its cellular uptake into fibroblasts and, as a result, regulated the expression of type I collagen in fibroblasts via the activation of the Smad signaling pathway. This study demonstrates the therapeutic potential of Oct-LMSG in promoting tissue regeneration.

Targeting FGFRs Using PD173074 as a Novel Therapeutic Strategy in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an architecturally complex tumour with high heterogeneity. Discovery at later stages makes treatment challenging. However, the lack of early detection methodologies and the asymptomatic nature of CCA make early diagnosis more difficult. Recent studies revealed the fusions in Fibroblast Growth Factor Receptors (FGFRs), a sub-family of RTKs, as promising targets for targeted therapy for CCA. Particularly, FGFR2 fusions have been of particular interest, as translocations have been found in approximately 13% of CCA patients. Pursuing this, Pemigatinib, a small-molecule inhibitor of FGFR, became the first targeted therapy drug to be granted accelerated approval by the FDA for treating CCA patients harbouring FGFR2 fusions who have failed first-line chemotherapy. However, despite the availability of Pemigatinib, a very limited group of patients benefit from this treatment. Moreover, as the underlying mechanism of FGFR signalling is poorly elucidated in CCA, therapeutic inhibitors designed to inhibit this pathway are prone to primary and acquired resistance, as witnessed amongst other Tyrosine Kinase Inhibitors (TKIs). While acknowledging the limited cohort that benefits from FGFR inhibitors, and the poorly elucidated mechanism of the FGFR pathway, we sought to characterise the potential of FGFR inhibitors in CCA patients without FGFR2 fusions. Here we demonstrate aberrant FGFR expression in CCA samples using bioinformatics and further confirm phosphorylated-FGFR expression in paraffinised CCA tissues using immunohistochemistry. Our results highlight p-FGFR as a biomarker to guide FGFR-targeted therapies. Furthermore, CCA cell lines with FGFR expression were sensitive to a selective pan-FGFR inhibitor, PD173074, suggesting that this drug can be used to suppress CCA cells irrespective of the FGFR2 fusions. Finally, the correlation analysis utilising publicly available cohorts suggested the possibility of crosstalk amongst the FGFR and EGFR family of receptors as they are significantly co-expressed. Accordingly, dual inhibition of FGFRs and EGFR by PD173074 and EGFR inhibitor erlotinib was synergistic in CCA. Hence, the findings from this study provide support for further clinical investigation of PD173074, as well as other FGFR inhibitors, to benefit a larger cohort of patients. Altogether, this study shows for the first time the potential of FGFRs and the importance of dual inhibition as a novel therapeutic strategy in CCA.

CP-673451, a Selective Platelet-Derived Growth Factor Receptor Tyrosine Kinase Inhibitor, Induces Apoptosis in Opisthorchis viverrini-Associated Cholangiocarcinoma via Nrf2 Suppression and Enhanced ROS.

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) play essential roles in promoting cholangiocarcinoma (CCA) cell survival by mediating paracrine crosstalk between tumor and cancer-associated fibroblasts (CAFs), indicating the potential of PDGFR as a target for CCA treatment. Clinical trials evaluating PDGFR inhibitors for CCA treatment have shown limited efficacy. Furthermore, little is known about the role of PDGF/PDGFR expression and the mechanism underlying PDGFR inhibitors in CCA related to Opisthorchis viverrini (OV). Therefore, we examined the effect of PDGFR inhibitors in OV-related CCA cells and investigated the molecular mechanism involved. We found that the PDGF and PDGFR mRNAs were overexpressed in CCA tissues compared to resection margins. Notably, PDGFR-α showed high expression in CCA cells, while PDGFR-ß was predominantly expressed in CAFs. The selective inhibitor CP-673451 induced CCA cell death by suppressing the PI3K/Akt/Nrf2 pathway, leading to a decreased expression of Nrf2-targeted antioxidant genes. Consequently, this led to an increase in ROS levels and the promotion of CCA apoptosis. CP-673451 is a promising PDGFR-targeted drug for CCA and supports the further clinical investigation of CP-673451 for CCA treatment, particularly in the context of OV-related cases.

Sulfated Galactans from Gracilaria fisheri with Supplementation of Octanoyl Promote Wound Healing Activity In Vitro and In Vivo.

Sulfated galactans (SG) isolated from Gracilaria fisheri is partially degraded (DSG), and subsequentially supplemented with octanoyl (DSGO) and sulfate (DSGS) groups. The molecular weights of DSG, DSGO, and DSGS are 7.87, 152.79, and 97.07 kDa, respectively. The modification is confirmed using FTIR and NMR, while in vitro wound healing activity is assessed using scratched wound fibroblasts. The results reveal that DSGO exhibits highest percentage of wound closure in scratched fibroblast L929 cells. Furthermore, DSGO is able to promote proliferation and accelerate migration of scratched fibroblasts, which correspond to the regulation of proteins and mRNA (Ki67, p-FAK, vimentin, and E-cadherin) determined by Western blotting and qPCR analysis. The superior wound healing activity of DSGO is also confirmed in excision wound of rats. The results demonstrate that DSGO significantly enhances the percentage of wound closure, re-epithelialization, and collagen arrangement, increases α-smoth muscle actin (α-SMA) and vimentin expression, and decreases that of tumor necrosis factor-α (TNF-α) at the wound site. The results suggest that degraded SG supplemented with medium-chain fatty acids of octanoyl group may pass through the membrane, subsequently activating the mediators associated with proliferation and migration of fibroblasts, which can potentially lead to the promotion of wound healing activity.

Depolymerized Fractions of Sulfated Galactans Extracted from Gracilaria fisheri and Their Antibacterial Activity against Vibrio parahaemolyticus and Vibrio harveyi.

Various seaweed sulfated polysaccharides have been explored for antimicrobial application. This study aimed to evaluate the antibacterial activity of the native Gracilaria fisheri sulfated galactans (NSG) and depolymerized fractions against the marine pathogenic bacteria Vibrio parahaemolyticus and Vibrio harveyi. NSG was hydrolyzed in different concentrations of H2O2 to generate sulfated galactans degraded fractions (SGF). The molecular weight, structural characteristics, and physicochemical parameters of both NSG and SGF were determined. The results revealed that the high molecular weight NSG (228.33 kDa) was significantly degraded to SGFs of 115.76, 3.79, and 3.19 kDa by hydrolysis with 0.4, 2, and 10% H2O2, respectively. The Fourier transformed spectroscopy (FTIR) and 1H- and 13C-Nuclear magnetic resonance (NMR) analyses demonstrated that the polysaccharide chain structure of SGFs was not affected by H2O2 degradation, but alterations were detected at the peak positions of some functional groups. In vitro study showed that SGFs significantly exerted a stronger antibacterial activity against V. parahaemolyticus and V. harveyi than NSG, which might be due to the low molecular weight and higher sulfation properties of SGF. SGF disrupted the bacterial cell membrane, resulting in leakage of intracellular biological components, and subsequently, cell death. Taken together, this study provides a basis for the exploitation and utilization of low-molecular-weight sulfated galactans from G. fisheri to prevent and control the shrimp pathogens.

Increased Sulfation in Gracilaria fisheri Sulfated Galactans Enhances Antioxidant and Antiurolithiatic Activities and Protects HK-2 Cell Death Induced by Sodium Oxalate.

Urolithiasis is a common urological disease characterized by the presence of a stone anywhere along the urinary tract. The major component of such stones is calcium oxalate, and reactive oxygen species act as an essential mediator of calcium oxalate crystallization. Previous studies have demonstrated the antioxidant and antiurolithiatic activities of sulfated polysaccharides. In this study, native sulfated galactans (N-SGs) with a molecular weight of 217.4 kDa from Gracilaria fisheri were modified to obtain lower molecular weight SG (L-SG) and also subjected to sulfation SG (S-SG). The in vitro antioxidant and antiurolithiatic activities of the modified substances and their ability to protect against sodium oxalate-induced renal tubular (HK-2) cell death were investigated. The results revealed that S-SG showed more pronounced antioxidant activities (DPPH and O2- scavenging activities) than those of other compounds. S-SG exhibited the highest antiurolithiatic activity in terms of nucleation and aggregation, as well as crystal morphology and size. Moreover, S-SG showed improved cell survival and increased anti-apoptotic BCL-2 protein in HK-2 cells treated with sodium oxalate. Our findings highlight the potential application of S-SG in the functional food and pharmaceutical industries.

Inhibition of serine/arginine-rich protein kinase-1 (SRPK1) prevents cholangiocarcinoma cells induced angiogenesis.

The serine/arginine-rich protein kinase-1 (SRPK1) is an enzyme that has an essential role in regulating numerous aspects of mRNA splicing. SRPK1 has been reported to be overexpressed in multiple cancers, suggesting it as a promising therapeutic target in oncology. No previous studies reported the role of SRPK1 in cholangiocarcinoma (CCA) cells. This study aimed to examine the expression of SRPK1 and the effects of SRPK1 inhibition on the viability and angiogenesis activity of CCA cells using a selective SRPK1 inhibitor, SPHINX31. Here, we demonstrate that SPHINX31 (0.3-10 µM) had no inhibitory effects on CCA cells' viability and proliferation. However, SPHINX31 decreased the mRNA expression of pro-angiogenic VEGF-A165a isoform. In addition, SPHINX31 attenuated SRSF1 phosphorylation and nuclear localization, and increased the ratio of VEGF-A165b/total VEGF-A proteins. Moreover, when HUVECs were grown in conditioned medium from SPHINX31-treated CCA cells, migration slowed, and tube formation decreased. The present study demonstrates that targeting SRPK1 in CCA cells effectively attenuates angiogenesis by suppressing pro-angiogenic VEGF-A isoform splicing. These findings suggest a potential therapeutic treatment using SRPK1 inhibitors for the inhibition of angiogenesis in cholangiocarcinoma.

Structural characterization, antioxidant activity, and protective effect against hydrogen peroxide-induced oxidative stress of chemically degraded Gracilaria fisheri sulfated galactans.

Sulfated polysaccharides (SPs) possess an extensive range of biological activities, such as the inhibition of oxidation, correlated with their molecular weight (MW) and chemical structure. In this study, we used the trifluoroacetic acid (TFA) controlled degradation method to degrade sulfated galactans (SG) isolated from Gracilaria fisheri and evaluated the antioxidant and protective effects of the low molecular weight SG (LMSG) against H2O2 on fibroblast cells for the first time. Degradation of native SG (NSG) with an initial MW of 217.45 kDa using different concentrations of TFA resulted in five degraded NSG with MW of 97.23, 62.26, 30.74, 2.63, and 2.59 kDa. The reduction in MW was positively correlated with TFA concentrations. Chemical structure analyses using FTIR and NMR indicated that the TFA degradation process did not significantly change the LMSG polysaccharide main chain but did change the functional groups. LMSG exhibited higher scavenging activities and enhanced the cellular activities of GSH, CAT, and SOD enzymes. Moreover, LMSG activated Nrf-2/ARE signaling and increased expression of antioxidant genes CAT and SOD, which corresponded to increased protective effects against H2O2-induced ROS generation in fibroblast cells. The study reveals modification of NSG by acid TFA degradation resulted in the creation of LMSG, which showed greater antioxidant activity.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding.

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded ß-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

Probing the Anti-Cancer Potency of Sulfated Galactans on Cholangiocarcinoma Cells Using Synchrotron FTIR Microspectroscopy, Molecular Docking, and In Vitro Studies.

Sulfated galactans (SG) isolated from red alga Gracilaria fisheri have been reported to inhibit the growth of cholangiocarcinoma (CCA) cells, which was similar to the epidermal growth factor receptor (EGFR)-targeted drug, cetuximab. Herein, we studied the anti-cancer potency of SG compared to cetuximab. Biological studies demonstrated SG and cetuximab had similar inhibition mechanisms in CCA cells by down-regulating EGFR/ERK pathway, and the combined treatment induced a greater inhibition effect. The molecular docking study revealed that SG binds to the dimerization domain of EGFR, and this was confirmed by dimerization assay, which showed that SG inhibited ligand-induced EGFR dimer formation. Synchrotron FTIR microspectroscopy was employed to examine alterations in cellular macromolecules after drug treatment. The SR-FTIR-MS elicited similar spectral signatures of SG and cetuximab, pointing towards the bands of RNA/DNA, lipids, and amide I vibrations, which were inconsistent with the changes of signaling proteins in CCA cells after drug treatment. Thus, this study demonstrates the underlined anti-cancer mechanism of SG by interfering with EGFR dimerization. In addition, we reveal that FTIR signature spectra offer a useful tool for screening anti-cancer drugs' effect.

Co-Clinical Trials: An Innovative Drug Development Platform for Cholangiocarcinoma.

Cholangiocarcinoma (CCA), a group of malignancies that originate from the biliary tract, is associated with a high mortality rate and a concerning increase in worldwide incidence. In Thailand, where the incidence of CCA is the highest, the socioeconomic burden is severe. Yet, treatment options are limited, with surgical resection being the only form of treatment with curative intent. The current standard-of-care remains adjuvant and palliative chemotherapy which is ineffective in most patients. The overall survival rate is dismal, even after surgical resection and the tumor heterogeneity further complicates treatment. Together, this makes CCA a significant burden in Southeast Asia. For effective management of CCA, treatment must be tailored to each patient, individually, for which an assortment of targeted therapies must be available. Despite the increasing numbers of clinical studies in CCA, targeted therapy drugs rarely get approved for clinical use. In this review, we discuss the shortcomings of the conventional clinical trial process and propose the implementation of a novel concept, co-clinical trials to expedite drug development for CCA patients. In co-clinical trials, the preclinical studies and clinical trials are conducted simultaneously, thus enabling real-time data integration to accurately stratify and customize treatment for patients, individually. Hence, co-clinical trials are expected to improve the outcomes of clinical trials and consequently, encourage the approval of targeted therapy drugs. The increased availability of targeted therapy drugs for treatment is expected to facilitate the application of precision medicine in CCA.

Effect of Combining EGFR Tyrosine Kinase Inhibitors and Cytotoxic Agents on Cholangiocarcinoma Cells.

PURPOSE: The potential of members of the epidermal growth factor receptor (ErbB) family as drug targets in cholangiocarcinoma (CCA) has not been extensively addressed. Although phase III clinical trials showed no survival benefits of erlotinib in patients with advanced CCA, the outcome of the standard-of-care chemotherapy treatment for CCA, gemcitabine/cisplatin, is discouraging so we determined the effect of other ErbB receptor inhibitors alone or in conjunction with chemotherapy in CCA cells. MATERIALS AND METHODS: ErbB receptor expression was determined in CCA patient tissues by immunohistochemistry and digital-droplet polymerase chain reaction, and in primary cells and cell lines by immunoblot. Effects on cell viability and cell cycle distribution of combination therapy using ErbB inhibitors with chemotherapeutic drugs was carried out in CCA cell lines. 3D culture of primary CCA cells was then adopted to evaluate the drug effect in a setting that more closely resembles in vivo cell environments. RESULTS: CCA tumors showed higher expression of all ErbB receptors compared with resection margins. Primary and CCA cell lines had variable expression of erbB receptors. CCA cell lines showed decreased cell viability when treated with chemotherapeutic drugs (gemcitabine and 5-fluorouracil) but also with ErbB inhibitors, particularly afatinib, and with a combination. Sequential treatment of gemcitabine with afatinib was particularly effective. Co-culture of CCA primary cells with cancer-associated fibroblasts decreased sensitivity to chemotherapies, but sensitized to afatinib. CONCLUSION: Afatinib is a potential epidermal growth factor receptor targeted drug for CCA treatment and sequential treatment schedule of gemcitabine and afatinib could be explored in CCA patients.

Dysregulation of microRNA in cholangiocarcinoma identified through a meta-analysis of microRNA profiling.

BACKGROUND: In the past decades, the potential of microRNA (miRNA) in cancer diagnostics and prognostics has gained a lot of interests. In this study, a meta-analysis was conducted upon the pooled miRNA microarray data of cholangiocarcinoma (CCA). AIM: To identify differentially expressed (DE) miRNAs and perform functional analyses in order to gain insights to understanding miRNA-target interactions involved in tumorigenesis pathways of CCA. METHODS: Raw data from 8 CCA miRNA microarray datasets, consisting of 443 samples in total, were integrated and statistically analyzed to identify DE miRNAs via comparison of levels of miRNA expression between CCA and normal bile duct samples using t-tests (P < 0.001). The 10-fold cross validation was performed in order to increase the robustness of the t-test results. RESULTS: Our data showed 70 up-regulated and 48 down-regulated miRNAs in CCA. Gene Ontology and pathway enrichment analyses revealed that mRNA targets of DE miRNAs were significantly involved in several biological processes. The most prominent dysregulated pathways included phosphatidylinositol-3 kinases/Akt, mitogen-activated protein kinase and Ras signaling pathways. CONCLUSION: DE miRNAs found in our meta-analysis revealed dysregulation in major cancer pathways involved in the development of CCA. These results indicated the necessity of understanding the miRNA-target interactions and the significance of dysregulated miRNAs in terms of diagnostics and prognostics of cancers.

Purification and Evaluation of N-benzyl Cinnamamide from Red Seaweed Gracilaria fisheri as an Inhibitor of Vibrio harveyi AI-2 Quorum Sensing.

Previously, we reported that the ethanol extract from red seaweed Gracilaria fisheri effectively decreased biofilm formation of Vibrio harveyi. In this study, the anti-biofilm active compounds in the ethanol extract were isolated and their structures identified. The anti-biofilm fractionation assay for minimum inhibitory concentration (MIC) produced two fractions which possessed maximal inhibitory activities toward the biofilm formation of V. harveyi strains 1114 and BAA 1116. Following chromatographic separation of the bioactive fractions, two pure compounds were isolated, and their structures were elucidated using FTIR, NMR, and HR-TOF-MS. The compounds were N-benzyl cinnamamide and α-resorcylic acid. The in vitro activity assay demonstrated that both compounds inhibited the biofilm formation of V. harveyi and possessed the anti-quorum sensing activity by interfering with the bioluminescence of the bacteria. However, the N-benzyl cinnamamide was more potent than α-resorcylic acid with a 10-fold lesser MIC. The present study reveals the beneficial property of the N-benzyl cinnamamide from the ethanol extract as a lead anti-microbial drug against V. harveyi.

Discovery of 4,6-O-Thenylidene-ß-d-glucopyranoside-(2″-acetamido, 3″-acetyl-di-S-5-fluorobenzothizole/5-fluorobenzoxazole)-4'-demethylepipodophyllotoxin as Potential Less Toxic Antitumor Candidate Drugs by Reducing DNA Damage and Less Inhibition of PI3K.

As an FDA-approved drug, teniposide, was utilized in cancer treatment but was accompanied by a strong side effect in long-term clinical trials. This work discovered potential candidate drugs with low toxicity by modifying the molecule structure of teniposide through a structure-guided drug design approach. The IC50 value of novel 4,6-O-thenylidene-ß-d-glucopyranoside-(2″-acetamido, 3″-acetyl-di-S-5-fluorobenzothizole/5-fluorobenzoxazole)-4'-demethylepipodophyllotoxin (compounds 15 and 16) was 120.4-125.1 µM, which was significantly improved by around 10 times more than teniposide (11.5-22.3 µM) against healthy human cells (i.e., HL-7702, H8, MRC-5, and HMEC). In vivo studies demonstrated compounds 15 and 16 significantly suppressed the tumor growth in the HepG2 cell xenograft model without exhibiting obvious toxicity (LD50 values of 208.45 and 167.52 mg/kg), which was lower than that of teniposide (LD50 = 46.12 mg/kg). Compounds 15 and 16 caused mild γH2AX phosphorylation for low DNA toxicity and less inhibition of PI3K/Akt. Compounds 15 and 16 might be potential antitumor drugs with low toxicity.

Bioencapsulation efficacy of sulfated galactans in adult Artemia salina for enhancing immunity in shrimp Litopenaeus vannamei.

Live food organisms like Artemia have been used for delivery of different substances such as nutrients, probiotics and immune-stimulants to aquatic animals. Previously, we reported that sulfated galactans (SG) from the red seaweed Gracilaria fisheri (G. fisheri) increased immune activity in shrimp. In the present study we further investigated the capacity and efficiency of bioencapsulation of SG in adult Artemia for delivery to tissues and potentially boosting the expression of immune genes in post larvae shrimp. SG were labelled with FITC (FITC-SG) for in vivo tracking in shrimp. Bioencapsulation of adult Artemia with FITC-SG (0-100 µg mL-1) was performed and the fluorescence intensity was detected in the gut lumen after enrichment periods of 30 min, 1 h, 2 h, 6 h and 24 h. The results showed the Artemia took up SG over time in a concentration-dependent manner. Shrimp were fed with the bioencapsulated Artemia (FITC-SG, 20 µg mL-1) and the shrimp were evaluated under a stereo-fluorescent microscope. At 24 h after administration, FITC-SG was located in gills and hepatopancreas and also bound with haemocytes. With daily SG administration, the genes IMD, IKKß were up-regulated (after 1 day) while genes dicer and proPO-I were up-regulated later (after 7 days). Moreover, continued monitoring of shrimp fed for 3 consecutive days only with SG at the dose of 0.5 mg g-1 BW showed increases in the expression of IMD, IKKß genes on day 1 and which gradually declined to normal levels on day 14, while the expression of dicer and proPO-I was increased on day 3 and remained high on day 14. These results demonstrate that bioencapsulation of SG in adult Artemia successfully delivers SG to shrimp tissues, which then bind with haemocytes and subsequently activate immune genes, and potentially increase immunity in shrimp. In addition, the present study suggests that a 3-consecutive-day regimen of SG supplemented in Artemia (0.5 mg g-1 BW) may boost and sustain the enhanced immune functions in post larvae shrimp.

Bioflocs substituted fishmeal feed stimulates immune response and protects shrimp from Vibrio parahaemolyticus infection.

Fishmeal is the main source of protein in the shrimp feed industry and is normally derived from trash fish. As such, the production of fishmeal has an adverse effect on the marine environment by taking away small and juvenile fish, leading to depletion of marine species. There is a need for alternative sources of protein which will substitute fishmeal in the aquaculture industry. This study evaluated the components and nutritional efficacy of bioflocs, which were used to substitute fishmeal protein. The effect of bioflocs diets on growth performance, survival rate, and immune response in shrimp compared to normal fishmeal feed were determined. Bioflocs were harvested from the shrimp ponds (C:N ratio >12:1) at Shrimp Village, Chaiya district, Surat Thani, Thailand. The total protein in bioflocs was about 48% and the total lipid was about 5% (dried weight) and the percentages of essential amino acids (EAA) and fatty acids (EFA) in bioflocs were similar to those of fishmeal feed. Shrimp fed with the different dietary bioflocs feed regimens [% to replace fishmeal; 0% (B0), 25% (B25), 50% (B50), 75% (B75), and 100% (B100)] for 42 days revealed that all growth parameters were almost similar to those of the control shrimp (shrimp fed with normal fishmeal, B0) including final body weight, weight gain, specific growth rate, and feed conversion ratio. Remarkably, the survival rates, the levels of immune parameters, and expression of immune genes (proPO-I, PEN-4 and dicer) were significantly higher in bioflocs fed shrimp, especially in B25 and B50 shrimp. Moreover, B25 and B50 bioflocs fed shrimp showed notably increased survival rates following Vibrio parahaemolyticus (V. parahaemolyticus) infection. In conclusion, the present study demonstrates that shrimp survival and immunity are enhanced by biofiocs substituted fishmeal. Significantly, the bioflocs diets activated the immune response to prevent V. parahaemolyticus infection.

Ethanolic extract of red seaweed Gracilaria fisheri and furanone eradicate Vibrio harveyi and Vibrio parahaemolyticus biofilms and ameliorate the bacterial infection in shrimp.

Bacteria respond to host immunity for their proliferation and survival by cell-cell communications such as biofilm formation, bioluminescence, and secreting virulence factors. In the biofilm form, bacteria are more resistant to various antimicrobial treatments and withstand the host's immune system. The approaches of deciphering biofilm formation for treating bacterial infections are therefore highly desirable. Recently, we have reported that the ethanolic extract of the red seaweed Gracilaria fisheri (G. fisheri) enhanced immune activities and inhibited growth of the luminescent bacteria Vibrio harveyi in shrimp. We undertook the present research study in order to evaluate and compare the effectiveness of the ethanolic extract from G. fisheri and furanone, a known biofilm inhibitor, in inhibiting the formation of clinically important Vibrio biofilms. The results showed that sub-lethal concentrations of both the ethanolic extracts (5, 10 and 100 µg ml-1) and furanone (5 µM) inhibited biofilm formation by V. harveyi and Vibrio parahaemolyticus and also light production (luminescence) in V. harveyi. It is known that V. harveyi mediated light production via autoinducer AI-2 pathway, we further determined whether the inhibitory effect of the extract was involved the AI-2 signaling. The bioluminescence assay was conducted in an AI-2 deletion mutant V. harveyi. Supplementation of the AI-2 containing media with the extract or furanone impaired the light production in the mutant V. harveyi suggesting that the extract interfered AI-2 mediated light production similar to furanone. In vivo challenge study showed that the low concentrations (Sub MICs) of the ethanolic extract and furanone decreased bacterial adhesion and colonization in the surfaces of stomach lumen, down-regulated expression of a virulence factor, and protected shrimp against mortality from V. harveyi and V. parahaemolyticus infection. In conclusion, the present results suggest a potential application of the low concentrations of the ethanolic extract of G. fisheri as an efficient approach for treating biofilm-associated Vibrio diseases in aquacultures.