Development in competitive immunoassay of a point-of-care testing for cotinine (COT) detection in urine.

We present a sensitive and selective lateral flow immunoassay (LFIA) for cotinine (COT), the primary metabolite of nicotine. COT is widely recognized as a superior biomarker to evaluate tobacco smoke exposure. The LFIA uses a competitive assay format where the COT-BSA capture competes with the target COT in urine samples for binding to the monoclonal antibody against COT (mAb-COT) conjugated with gold nanoparticles (mAb-COT-AuNPs). To improve the sensitivity and selectivity of the LFIA-COT, we focused on optimizing the diameter of AuNPs, the conjugation of mAb-COT, and the concentration of the COT-BSA capture. Our findings reveal that the utilization of 40 nm AuNPs in conjugation with a concentration of 4 mg mL-1 of mAb-COT demonstrated significantly greater efficacy compared to LFAs utilizing 20 nm AuNPs. Under the optimal conditions, the LFIA-COT demonstrated sensitive detection of COT at a level of 150 ng mL-1 within 15 min, as observed by the naked eye. It possesses a linear range of 25 to 200 ng mL-1 of COT, with the limit of detection (LOD) of 11.94 ng mL-1 in human urine samples when the color intensity is analyzed using ImageJ software. Our LFIA described here is simple and requires less time for COT detection. It can be used for the rapid and quantitative detection of COT in urine samples in clinical settings.

Development of a Prognostic Score for Cholangiocarcinoma Patients Using a Combination of Biochemical Parameters.

BACKGROUND/AIM: Prognosis of cholangiocarcinoma (CCA), especially of intrahepatic CCA (iCCA), is poor primarily due to difficulties in earlier diagnosis. Since the majority of iCCA patients are elders, their prognosis cannot be correctly predicted by pathological features and/or resection status alone. Consideration for comorbidity and/or risks of subclinical diseases at diagnosis is critically necessary for the prediction of prognosis of iCCA patients. This study aimed to develop a simple but reliable scoring system for prognosis of iCCA patients at the time of diagnosis. PATIENTS AND METHODS: Serum samples from 152 iCCA patients were collected, and four commonly used biochemical markers, serum aspartate aminotransferase, alkaline phosphatase, cystatin C and creatinine-based estimated glomerular filtration rate were measured. Then, the values of individual patients were scored as 0, 1, and 2 (low, medium, and high) by tertiles or clinically relevant cut-off points and summed to construct a prognostic score with a range between 0 to 8. RESULTS: Patients with high scores of 2-4 and 5-8 exhibited significantly shorter survival times compared to those with low scores of 0-1 (Chi-square: 15.75, p<0.001). Cox regression analysis suggested that the score could be an independent predictor for the survival of iCCA patients. The odds of advanced tumor stage in high score iCCA patients (2-4 and 5-8) were 12.310 (95%CI=2.241-67.605) and 23.964 (95%CI=3.296-174.216), respectively. This scoring system allowed further stratification of death rates per 100 person-years of iCCA patients. CONCLUSION: The ability of such a simple scoring system to discriminate risk might be helpful for iCCA patients to determine therapeutic programs at the time of diagnosis.

Unveiling the Potent Antiviral and Antioxidant Activities of an Aqueous Extract from Caesalpinia mimosoides Lamk: Cheminformatics and Molecular Docking Approaches.

Our group previously demonstrated that Caesalpinia mimosoides Lamk exhibits many profound biological properties, including anticancer, antibacterial, and antioxidant activities. However, its antiviral activity has not yet been investigated. Here, the aqueous extract of C. mimosoides was prepared from the aerial parts (leaves, stalks, and trunks) to see whether it exerts anti-influenza (H1N1) effects and to reduce the organic solvents consumed during extraction, making it a desirable approach for the large-scale production for medical uses. Our plant extract was quantified to contain 7 g of gallic acid (GA) per 100 g of a dry sample, as determined using HPLC analysis. It also exerts potent antioxidant activities comparable to those of authentic GA. According to untargeted metabolomics (UPLC-ESI(-)-QTOF-MS/MS) with the aid of cheminformatics tools (MetFrag (version 2.1), SIRIUS (version 5.8.3), CSI:FingerID (version 4.8), and CANOPUS), the major metabolite was best annotated as "gallic acid", phenolics (e.g., quinic acid, shikimic acid, and protocatechuic acid), sugar derivatives, and dicarboxylic acids were deduced from this plant species for the first time. The aqueous plant extract efficiently inhibited an influenza A (H1N1) virus infection of MDCK cells with an IC50 of 5.14 µg/mL. Of equal importance, hemolytic activity was absent for this plant extract, signifying its applicability as a safe antiviral agent. Molecular docking suggested that GA interacts with conserved residues (e.g., Arg152 and Asp151) located in the catalytic inner shell of the viral neuraminidase (NA), sharing the same pocket as those of anti-neuraminidase drugs, such as laninamivir and oseltamivir. Additionally, other metabolites were also found to potentially interact with the active site and the hydrophobic 430-cavity of the viral surface protein, suggesting a possibly synergistic effect of various phytochemicals. Therefore, the C. mimosoides aqueous extract may be a good candidate for coping with increasing influenza virus resistance to existing antivirals.

Conjugation of Peptides to Gold Nanoparticles.

Peptides and proteins have played an important role in many biological processes, functioning as enzymes, hormones, ligands, receptors, cell mediators, and structural components of cells. Being intrinsic molecules in signaling pathways, peptides allow for therapeutic intervention that closely mimic natural signaling cascades. However, the short chain of amino acids in free peptides is susceptible to proteolysis in vivo. Conjugation of peptides onto nanoparticles has been used as a strategy to extend peptide half-life through conferring steric hindrance and a high packing density that prevents proteolytic enzymes to degrade them. Here, we describe a method to conjugate the anticancer p53 peptides as our model peptide onto 12 nm gold nanoparticles (AuNPs) to form the AuNP-p53 peptide conjugate. Conjugation of the p53 short-chain peptide of 25 amino acids occurs through a combination of electrostatic interactions and covalent bonds between cysteine residues at the N-terminal of the peptide and the surface of the AuNPs. The AuNPs and AuNP-p53 are characterized by UV-Vis spectroscopy for its optical absorbance and zetasizer for their hydrodynamic diameter and zeta potential. The semiquantitative analysis of the amount of conjugated peptides on the AuNPs and peptide stability under trypsin treatment is performed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Anti-metastatic Effects of Cationic KT2 Peptide (a Lysine/Tryptophan-rich Peptide) on Human Melanoma A375.S2 Cells.

BACKGROUND/AIM: KT2 is a lysine/tryptophan-rich peptide modified from Crocodylus siamensis Leucrocin I. In this study, we examined the cell toxicity, cellular uptake, anti-migration and anti-invasion activities of KT2 in A375.S2 human melanoma cells. MATERIALS AND METHODS: A375.S2 cells were treated with KT2 peptide and then we performed MTT assay, study of cellular uptake by a confocal microscope, wound healing assay, transwell migration/invasion assay, and evaluation of the expression of metastasis-associated proteins. RESULTS: KT2 can be internalized through the plasma membrane and can slightly alter cell morphology, decrease the percentage of viable cells and inhibit cell migration and invasion of A375.S2 cells in a dose-dependent manner. This peptide suppressed MMP-2 activity, as measured by gelatine zymography assay. The protein level of MMP-2 was decreased by KT2. KT2 also down-regulated metastasis pathway-related molecules, including FAK, RhoA, ROCK1, GRB2, SOS-1, p-JNK, p-c-Jun, PI3K, p-AKT (Thr308), p-AKT (Ser473), p-p38, MMP-9, NF-kB, and uPA. CONCLUSION: These results indicate that KT2 inhibits the migration and invasion of human melanoma cells by decreasing MMP-2 and MMP-9 expression through inhibition of FAK, uPA, MAPK, PI3K/AKT NF-kB, and RhoA-ROCK signalling pathways. These findings suggest that KT2 deserves further investigation as an anti-metastatic agent for human melanoma.

Conjugation with gold nanoparticles improves the stability of the KT2 peptide and maintains its anticancer properties.

One of the major weaknesses of therapeutic peptides is their sensitivity to degradation by proteolytic enzymes in vivo. Gold nanoparticles (GNPs) are a good carrier for therapeutic peptides to improve their stability and cellular uptake in vitro and in vivo. We conjugated the anticancer KT2 peptide as an anticancer peptide model to PEGylated GNPs (GNPs-PEG) and investigated the peptide stability, cellular uptake and ability of the GNPs-KT2-PEG conjugates to induce MDA-MB-231 human breast cancer cell death. We found that 11 nm GNPs protected the conjugated KT2 peptide from trypsin proteolysis, keeping it stable up to 0.128% trypsin, which is higher than the serum trypsin concentration (range 0.0000285 ± 0.0000125%) reported by Lake-Bakaar, G. et al., 1979. GNPs significantly enhanced the cellular uptake of KT2 peptides after conjugation. Free KT2 peptides pretreated with trypsin were not able to kill MDA-MB-231 cells due to proteolysis, while GNPs-KT2-PEG was still able to exert effective cancer cell killing after trypsin treatment at levels comparable to GNPs-KT2-PEG without enzyme pretreatment. The outcome of this study highlights the utility of conjugated anticancer peptides on nanoparticles to improve peptide stability and retain anticancer ability.

The cationic cell-penetrating KT2 peptide promotes cell membrane defects and apoptosis with autophagy inhibition in human HCT 116 colon cancer cells.

The anticancer activity of cationic antimicrobial peptides (AMPs) has become more interesting because some AMPs have selective recognition against cancer cells. However, their antitumor properties and underlying mechanisms in cancer cells have not been clearly understood. In this study, we evaluated the effects of KT2 (lysine/tryptophan-rich AMP) on the cellular uptake and internalization mechanism, cell viability, surface charge of the cell membrane, membrane integrity, apoptotic cell death, and autophagy in human HCT 116 colon cancer cells. We found that KT2 interacted with the cell membrane of HCT 116 cells and was internalized into HCT 116 cells via clathrin-mediated and caveolae-mediated endocytosis mechanisms. The interaction of KT2 with cells caused cell membrane structure change, elevated membrane permeability, and KT2 also affected the lipid component. The results of atomic force microscopy showed cellular membrane defects of KT2-treated cells. The internalized KT2 induced nuclear condensation and apoptotic cell death. It elevated the apoptotic factor levels including those of cytochrome c and apoptosis-inducing factor. Furthermore, KT2 inhibited autophagy by the suppression of autophagy-related 5, autophagy-related 7, autophagy-related 16 like 1, and Beclin-1 proteins. In conclusion, these results revealed the cytotoxicity of cationic KT2 against HCT 116 cells and may help to clarify the interactions between cationic AMPs and cancer cells.

Berberine Inhibits Human Melanoma A375.S2 Cell Migration and Invasion via Affecting the FAK, uPA, and NF-κB Signaling Pathways and Inhibits PLX4032 Resistant A375.S2 Cell Migration In Vitro.

Many studies have demonstrated that berberine inhibited the cell migration and invasion in human cancer cell lines. However, the exact molecular mechanism of berberine inhibiting the cell migration and invasion of human melanoma A375.S2 and A375.S2/PLX (PLX4032 induced resistant A375.S2) skin cancer cells remains unknown. In this study, we investigated the anti-metastasis mechanisms of berberine in human melanoma cancer A375.S2 cells and A375.S2/PLX resistant cells in vitro. Berberine at low concentrations (0, 1, 1.5 and 2 µM) induced cell morphological changes and reduced the viable cell number and inhibited the mobility, migration, and invasion of A375.S2 cells that were assayed by wound healing and transwell filter. The gelatin zymography assay showed that berberine slightly inhibited MMP-9 activity in A375.S2 cells. Results from western blotting indicated that berberine inhibited the expression of MMP-1, MMP-13, E-cadherin, N-cadherin, RhoA, ROCK1, SOS-1, GRB2, Ras, p-ERK1/2, p-c-Jun, p-FAK, p-AKT, NF-κB, and uPA after 24 h of treatment, but increased the PKC and PI3K in A375.S2 cells. PLX4032 is an inhibitor of the BRAFV600E mutation and used for the treatment of cancer cells harboring activated BRAF mutations. Berberine decrease cell number and inhibited the cell mobility in the resistant A375.S2 (A375.S2/PLX, PLX4032 generated resistant A375.S2 cells). Based on these observations, we suggest that the potential of berberine as an anti-metastatic agent in melanoma that deserves to be investigated in more detail, including in vivo studies in future.

Antitumor Ability of KT2 Peptide Derived from Leukocyte Peptide of Crocodile Against Human HCT116 Colon Cancer Xenografts.

BACKGROUND/AIM: Many antimicrobial peptides have been shown to have anticancer activity against human cancer cell lines. Cationic KT2 peptide, derived from white blood cell extract of Crocodylus siamensis has antibacterial activity and antitumor activity against human cervical cancer cells, but there are no data on the effect of KT2 peptide on tumor growth in vivo. The anticancer activity of KT2 peptide on human colon cancer xenografts was investigated in nude mice. MATERIALS AND METHODS: Tumors in nude mice (BALB/c -nu/nu mice) were induced by subcutaneous injection with HCT116 cells. Twelve days after cancer cell xenograft, mice were treated by intratumoral injection with phosphate-buffered saline or KT2 peptide (25 and 50 mg/kg) once every 2 days for a total of four times and mice were sacrificed at 2 days after the last treatment. RESULTS: KT2 peptide treatment did not lead to significant difference in mouse body weight among groups, but reduced both tumor volume and weight of colon cancer xenografts. Moreover, KT2 peptide increased the expression of apoptotic proteins, such as BCL2-associated X (BAX), cleaved caspase-3, and poly (ADP-ribose) polymerase and reduced that of BCL2 apoptosis regulator in xenograft tumors. CONCLUSION: This finding suggests that KT2 peptide may inhibit tumor growth via apoptosis induction in this mouse model and supports the antitumor ability of KT2 peptide.

Antitumor activity of RT2 peptide derived from crocodile leukocyte peptide on human colon cancer xenografts in nude mice.

RT2, derived from the leukocyte peptide of Crocodylus siamensis, can kill human cervical cancer cells via apoptosis induction, but no evidence has shown in vivo. In this study, we investigated the antitumor effect of RT2 on human colon cancer xenografts in nude mice. Twenty-four mice were injected subcutaneously with human colon cancer HCT 116 cells. Eleven days after cancer cell implantation, the mice were treated with intratumoral injections of phosphate buffered saline (PBS) or RT2 (0.01, 0.1, and 1 mg/mouse) once every 2 days for a total of 5 times. The effect of a 10-day intratumoral injection of RT2 on body weight, biochemical, and hematological parameters in BALB/c mice showed no significant difference between the groups. Tumor volume showed a significant decrease only in the treatment group with RT2 (1 mg/mouse) at day 6 (P < .05), day 8 (P < .01), and day 10 (P < .01) after the first treatment. The protein expression levels of cleaved poly (ADP-ribose) polymerase (PARP), apoptosis-inducing factor (AIF), and the p53 tumor suppressor protein (p53) in xenograft tumors increased after treatment with RT2 (1 mg/mouse) compared to those in the PBS-injected group. Moreover, RT2 increased the expression of Endo G and Bcl-2 family proteins. Therefore, the peptide RT2 can inhibit tumor growth via the induction of apoptosis in an in vivo xenograft model.