Mechanism of Induction of P-gp Activity During MET Induced by DEX in Lung Cancer Cell Line.

Lung cancer metastasis often leads to a poor prognosis for patients. Mesenchymal-epithelial transition (MET) is one key process associated with metastasis. MET has also been linked to multidrug drug resistance (MDR). MDR arises from the overactivity of drug efflux transporters such as P-glycoprotein (P-gp) which operate at the cell plasma membrane, under the regulatory control of the scaffold proteins ezrin (Ezr), radixin (Rdx), and moesin (Msn), collectively known as ERM proteins. The current study was intended to clarify the functional changing of P-gp and the underlying mechanisms in the context of dexamethasone (DEX)-induced MET in lung cancer cells. We found that the mRNA and membrane protein expression of Ezr and P-gp was increased in response to DEX treatment. Moreover, the DEX-treated group exhibited an increase in Rho123 efflux, and it was reversed by treatment with the P-gp inhibitor verapamil or Ezr siRNA. The decrease in cell viability with paclitaxel (PTX) treatment was mitigated by pretreatment with DEX. The increased expression and activation of P-gp during the progression of lung cancer MET was regulated by Ezr. The regulatory mechanism of P-gp expression and activity may differ depending on the cell status.

Bacterial cellulose nanocrystal as drug delivery system for overcoming the biological barrier of cyano-phycocyanin: a biomedical application of microbial product.

Phycocyanin, produced by Spirulina platensis, has been reported as an anti-inflammatory, anti-hyperalgesia, antioxidant, anti-tumor, and anti-cancer agent. However, the ingestion of phycocyanin in the body is often hindered by its instability against gastric pH conditions. The nano-drug delivery system has developed as a promising platform for efficient drug delivery and improvement as well as drug efficacy. Bacterial cellulose nanocrystal (BCNC) has it superiority as DDS due to its inherent properties such as nanoscale dimension, large surface area, - biocompatibility, and non-toxic. To improve its mechanical properties, BCNC was crosslinked with glutaraldehyde and was analyzed as a potential candidate for DDS. The Fourier transform infrared analysis of the BCNC suggested that hydrolysis did not alter the chemical composition. The index of crystallinity of the BCNC was 18.31% higher than that of the original BC, suggesting that crystalline BC has been successfully isolated. The BCNC particle also showed a needle-like morphology which is 25 ± 10 nm in diameter and a mean length of 626 ± 172 nm. Crosslinked BCNC also had larger pores than the original BCNC along with higher thermal stability. Optimum phycocyanin adsorption on crosslinked BCNC reached 65.3% in 3 h. The release study shows that the crosslinked BCNC can protect the phycocyanin retardation by gastric fluid until phycocyanin reaches the targeted sites. This study provides an alternative potential DDS derived from natural bioresources with less expenses and better properties to promote the application of BCNC as functional nanomaterials in biomedical science.

Valorization of Salmo salar Skin Waste for the Synthesis of Angiotensin Converting Enzyme-1 (ACE1) Inhibitory Peptides.

One of potential inhibitors which is widely used for the clinical treatment of COVID-19 in comorbid patients is Angiostensin Converting Enzyme-1 (ACE1) inhibitor. A safer peptide-based ACE1 inhibitor derived from salmon skin collagen, that is considered as the by-product of the fish processing industry have been investigated in this study. The inhibitory activity against ACE1 was examined using in vitro and in silico methods. In vitro analysis includes the extraction of acid-soluble collagen, characterization using FTIR, Raman, UV-Vis, XRD, cytotoxicity assay, and determination of inhibition against ACE1. In silico method visualizes binding affinity, molecular interaction, and inhibition type of intact collagen and active peptides derived from collagen against ACE1 using molecular docking. The results of FTIR spectra detected amide functional groups (A, B, I, II, III) and imine proline/hydroxyproline, while the results of Raman displayed peak absorption of amide I, amide III, proline/hydroxyproline ring, phenylalanine, and protein backbone. Furthermore, UV-Vis spectra showed typical collagen absorption at 230 nm and based on XRD data, the chain types in the samples were α-helix. ACE1 inhibition activity was obtained in a concentration-dependent manner where the highest was 82.83% and 85.84% at concentrations of 1000, and 2000 µg/mL, respectively, and showed very low cytotoxicity at the concentration less than 1000 µg/mL. In silico study showed an interaction between ACE1 and collagen outside the active site with the affinity of - 213.89 kcal/mol. Furthermore, the active peptides of collagen displayed greater affinity compared to lisinopril, namely HF (His-Phe), WYT (Trp-Tyr-Thr), and WF (Trp-Phe) of - 11.52; - 10.22; - 9.58 kcal/mol, respectively. The salmon skin-derived collagen demonstrated ACE1 inhibition activity with a non-competitive inhibition mechanism. In contrast, the active peptides were predicted as potent competitive inhibitors against ACE1. This study indicated that valorization of fish by-product can lead to the production of a promising bioactive compound to treat COVID-19 patient with diabetic comorbid.

Transporter and metabolic enzyme-mediated intra-enteric circulation of SN-38, an active metabolite of irinotecan: A new concept.

SN-38, an active metabolite of irinotecan (CPT-11), is thought to circulate enterohepatically via organic anion-transporting polypeptides (OATPs), UDP-glucuronyl transferases (UGTs), multidrug resistance-related protein 2 (MRP2), and breast cancer resistance protein (BCRP). These transporters and enzymes are expressed in not only hepatocytes but also enterocytes. Therefore, we hypothesized that SN-38 circulates between the intestinal lumen and the enterocytes via these transporters and metabolic enzymes. To test this hypothesis, metabolic and transport studies of SN-38 and its glucuronide (SN-38G) were conducted in Caco-2 cells. The mRNA levels of UGTs, MRP2, BCRP, and OATP2B1 were confirmed in Caco-2 cells. SN-38 was converted to SN-38G in Caco-2 cells. The efflux of intracellularly generated SN-38G across the apical (digestive tract) membranes was significantly higher than the efflux across the basolateral (blood, portal vein) membranes of Caco-2 cells cultured on polycarbonate membranes. SN-38G efflux to the apical side was significantly reduced in the presence of MRP2 and BCRP inhibitors, suggesting that SN-38G is transported across the apical membrane by MRP2 and BCRP. Treatment of Caco-2 cells with OATP2B1 siRNA increased the SN-38 residue on the apical side, confirming that OATP2B1 is involved in the uptake of SN-38 into enterocytes. No SN-38 was detected on the basolateral side with or without siRNA treatment, suggesting that the enterohepatic circulation of SN-38 is limited, contrary to previous reports. These results suggest that SN-38 is absorbed into the enterocytes via OATP2B1, glucuronidated by UGTs to SN-38G, and excreted into the digestive tract lumen by MRP2 and BCRP. SN-38G can be deconjugated by ß-glucuronidase from intestinal bacteria in the digestive tract lumen to regenerate SN-38. We named this new concept of local drug circulation "intra-enteric circulation." This mechanism may allow SN-38 to circulate in the intestine and cause the development of delayed diarrhea, a serious side effect of CPT-11.

Synthesis, modification and application of fish skin gelatin-based hydrogel as sustainable and versatile bioresource of antidiabetic peptide.

Gelatin hydrogel is widely employed in various fields, however, commercially available gelatin hydrogels are mostly derived from mammalian which has many disadvantages due to the supply and ethical issues. In this study, the properties of hydrogels from fish-derived collagen fabricated with varying Glutaraldehyde (GA) determined. The antidiabetic properties of salmon gelatin (SG) and tilapia gelatin (TG) was also evaluated against α-glucosidase. Glutaraldehyde-crosslinked salmon gelatin and tilapia gelatin were used, and compared with different concentrations of GA by 0.05 %, 0.1 %, and 0.15 %. Water absorbency, swelling, porosity, pore size and water retention of the hydrogels were dependent on the degree of crosslinking. The synthesis of hydrogels was confirmed by FTIR study. Scanning electron microscope (SEM) observation showed that all hydrogels have a porous structure with irregular shapes and heterogeneous morphology. Performance tests showed that gelatin-GA 0.05 % mixture had the best performance. Antidiabetic bioactivity in vitro and in silico tests showed that the active peptides of SG and TG showed a high binding affinity to α-glucosidase enzyme. In conclusion, SG and TG cross-linked GA 0.05 % have the potential as an antidiabetic agent and as a useful option over mammalian-derived gelatin.

Differences in stress response between two altitudes assessed by salivary cortisol levels within circadian rhythms in long-distance runners.

There are conflicting reports regarding the efficacy of cortisol as a stress marker in altitude training due to the influence of the circadian rhythm. This study aimed to verify whether the automated measurement of salivary cortisol concentration via sequential sampling could detect the differences in exercise stress between two altitudes. We enrolled 12 elite female long-distance runners living near sea level. For the first higher-altitude camp, the runners lived at 1800 m and trained at 1700 m for 7 days. For the second lower-altitude camp, they lived at 1550 m and trained at 1300 m for 7 days. Their saliva was sequentially collected on the last 2 days during each camp which involved different intensity exercises in the morning and afternoon. The salivary cortisol concentrations were measured using electrochemiluminescence immunoassay. Before dinner, the basal salivary cortisol concentrations were significantly higher in the higher-altitude camp. The rate of change in the salivary cortisol concentration during the morning exercise was significantly higher in the higher-altitude camp than in lower-altitude camp (p = 0.028) despite the same exercise programs and intensities. Salivary cortisol level measurements during the athletes' circadian rhythms could detect the differences in acclimatization and exercise stress between two altitudes.

In silico proteolysis and molecular interaction of tilapia (Oreochromis niloticus) skin collagen-derived peptides for environmental remediation.

Fish skin collagen hydrolyzate has demonstrated the potent inhibition of dipeptidyl peptidase-IV (DPP-IV), one of the treatments for type-2 diabetes mellitus (type-2 DM), but the precise mechanism is still unclear. This study used in silico method to evaluate the potential of the active peptides from tilapia skin collagen (Oreochromis niloticus) for DPP-IV inhibitor. The methodology includes collagen hydrolysis using BIOPEP, which is the database of bioactive peptides; active peptide selection; toxicity, allergenicity, sensory analysis of active peptides; and binding of active peptides to DPP-IV compared with linagliptin. The result indicated that in silico enzymatic hydrolysis of collagen produced active peptides with better prediction of biological activity than intact collagen. There are 13 active peptides were predicted as non-toxic and non-allergenic, some of which have a bitter, salty, and undetectable taste. Docking simulations showed all active peptides interacted with DPP-IV through hydrogen bonds, van der Waals force, hydrophobic interaction, electrostatic force, π-sulfur, and unfavorable interaction, where WF (Trp-Phe), VW (Val-Trp), WY (Trp-Tyr), and WG (Trp-Gly) displayed higher binding affinities of 0.8; 0.5; 0.4; and 0.3 kcal/mol compared with linagliptin. In this study, we successfully demonstrated antidiabetic type-2 DM potential of the active peptides from tilapia skin collagen. The obtained data provided preliminary data for further research in the utilization of fish skin waste as a functional compound to treat the type-2 DM patients. Alternatively, this treatment can be synergistically combined with the available antidiabetic drugs to improve the insulin secretion of the type-2 DM patients.

Association Between Insulin Resistance and Cardinal Rheological Parameters in Young Healthy Japanese Individuals During 75g Oral Glucose Tolerance Test.

BACKGROUND: Insulin resistance is a well-known predictor and risk factor for type 2 diabetes mellitus (T2DM). Higher hematocrit induced by higher insulin resistance affects blood rheology. OBJECTIVE: This study intended to reveal the association between indices of insulin resistance and hemorheological parameters during a 75 g oral glucose tolerance test (75-g OGTT). METHODS: A total of 575 healthy young Japanese participants took 75-g OGTT. We then analyzed the association between insulin resistance indices and hematological parameters. RESULTS: The Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was significantly correlated with hematocrit (Ht), hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC), platelet count, lipid parameters and body mass index (BMI). The Matsuda index was negatively correlated with RBC count, WBC count, platelet count, total cholesterol (TC), low-density lipoprotein- cholesterol (LDL-C), triglyceride (TG), and positively correlated with high-density lipoprotein- cholesterol (HDL-C). The disposition index was negatively correlated with Hb, RBC count, LDL-C and BMI, and positively correlated with HDL-C. The Homeostasis Model Assessment of beta cell (HOMA-ß) was positively correlated with WBC count, platelet count, TC, LDL-C and TG. The insulinogenic index was positively correlated with WBC count, platelet count and TC. Multiple regression analysis revealed that HOMA-IR was independently associated with TG, and the Matsuda index was independently associated with TG, WBC count, and platelet count. The insulinogenic index was independently associated with WBC count. CONCLUSION: Cardinal rheological parameters reflected insulin resistance and release even in young healthy Japanese individuals within the physiological range of glycemic control.

Significance of serum branched-chain amino acid to tyrosine ratio measurement in athletes with high skeletal muscle mass.

BACKGROUND: Few nutritional markers reflect the hypermetabolic state of athletes with high levels of skeletal muscle. Although branched-chain amino acids (BCAA) play crucial roles in protein metabolism in skeletal muscle, the relationship between skeletal muscle mass and amino acid imbalances caused by the metabolism of BCAA and aromatic amino acids remains unclear. The aim of this study is to test the hypothesis that athletes with high levels of skeletal muscle mass have plasma amino acid imbalances, assessed by serum BCAA to tyrosine ratio (BTR) which can be measured conveniently. METHODS: The study enrolled 111 young Japanese men: 70 wrestling athletes and 41 controls. None of them were under any medications, extreme dietary restrictions or intense exercise regimens. Each participant's body composition, serum concentrations of albumin and rapid turnover proteins including transthyretin and transferrin, BTR, and thyroid function were assessed. RESULTS: Compared to the controls, the athletes had significantly higher skeletal muscle index (SMI) (p < 0.001), and lower serum albumin concentration (p < 0.001) and BTR (p < 0.001). Kruskal-Wallis tests showed that serum albumin concentration and BTR were significantly lower in the participants with higher SMI. Serum albumin concentration and BTR were inversely correlated with SMI by multiple regression analysis (logarithmic albumin, ß = - 0.358, p < 0.001; BTR, ß = - 0.299, p = 0.001). SMI was inversely and transthyretin was positively correlated with serum albumin (SMI, ß = - 0.554, p < 0.001; transthyretin, ß = 0.379, p < 0.001). Serum concentration of free 3,5,3'-triiodothyronine (FT3) was inversely correlated with BTR, and, along with SMI and albumin, was independent predictor of BTR (SMI, ß = - 0.321, p < 0.001; FT3, ß = - 0.253, p = 0.001; logarithmic albumin, ß = 0.261, p = 0.003). However, FT3 was not correlated with SMI or serum albumin. Serum concentrations of rapid turnover proteins were not correlated with BTR. CONCLUSIONS: Increased skeletal muscle mass enhances the circulating amino acid imbalances, and is independently facilitated by thyroid hormones. Serum BTR may be a useful biomarker to assess the hypermetabolic state of wrestling athletes with high levels of skeletal muscle.

Assessment of exercise-induced stress by automated measurement of salivary cortisol concentrations within the circadian rhythm in Japanese female long-distance runners.

BACKGROUND: Overtraining syndrome, caused by prolonged excessive stress, results in reduced performance and cortisol responsiveness in athletes. It is necessary to collect saliva samples sequentially within circadian rhythm for assessing exercise stress by measuring cortisol concentrations, and automated cortisol measurements using electrochemiluminescence immunoassay (ECLIA) may be useful for measuring a large number of saliva samples. In this study, we evaluated the appropriate use of cortisol-based exercise stress assessment within the circadian rhythm, which may diagnose and prevent overtraining syndrome in athletes. METHODS: We collected saliva and sera from 54 healthy participants and analyzed the correlation between salivary cortisol concentrations measured by ECLIA and enzyme-linked immunosorbent assay (ELISA) or serum cortisol analysis. We also collected saliva continuously from 12 female long-distance runners on 2 consecutive days involving different intensities and types of exercise early in the morning and in the afternoon and measured salivary cortisol concentrations using ECLIA. Each exercise intensity of runners was measured by running velocities, Borg Scale score, and rate of change in the pulse rate by exercise. RESULTS: ECLIA-based salivary cortisol concentrations correlated positively with those detected by ELISA (ρ = 0.924, p < 0.001) and serum cortisol (ρ = 0.591, p = 0.001). In long-distance runners, circadian rhythm of salivary cortisol, including the peak after waking and the decrease promptly thereafter, were detected on both days by continuous saliva sampling. The rates of change in salivary cortisol concentrations were significantly lower after an early morning exercise than after an afternoon exercise on both days (day 1, p = 0.002, and day 2, p = 0.003). In the early morning exercise, the rate of change in salivary cortisol concentration was significantly higher on day 1 than on day 2 (p = 0.034), similar to a significant difference in running velocities (p = 0.001). CONCLUSIONS: Our results suggest that automated ECLIA-based salivary cortisol measurements are able to detect the athletes' circadian rhythm and compare the exercise stress intensities at the same times on different days, even in the early morning, possibly leading to the prevention of overtraining syndrome.

Use of capillary electrophoresis with dual-opposite end injection for simultaneous analysis of small ions in saliva samples from wrestlers undergoing a weight training program.

Capillary electrophoresis-capacitively coupled contactless conductivity detection (CE-C4D), conducted using an in-house-developed polyvinyl alcohol (PVA)-coated capillary system, was applied for the simultaneous analysis of small anions and cations in saliva samples from wrestlers undergoing a weight training program. Use of the PVA capillary for CE provided good reproducible ion separation with minimization of the electroosmotic flow and suppression of protein adsorption onto the capillary wall. Four cations and eight anions were separated in 12min, using a background electrolyte of 20mM MES/20mM histidine and 18-crown-6 ether (pH 6) at 20kV. The relative standard deviations (n=5) of the migration times and peak areas were <1% and <8%, respectively. The detection limit at a signal-to-noise ratio of 3 ranged from 1.6 to 10µM. Using the optimized CE-C4D system, we investigated the correlations between the concentrations of salivary ions and cortisol, which is commonly used as a stress marker. Analysis of saliva samples from ten wrestlers, who were attempting rapid weight loss before a competition, showed the following trends: (1) all ion concentrations, except for Ca(2+), Na(+), and Cl(-), increased between the first and last days of weight loss; (2) Mg(2+) increased to 166% (from 0.50mM to 1.4mM) between the first and last days of weight loss, being the highest increase of all the ions; and (3) K(+), Mg(2+), NO3(-), and SCN(-) levels were strongly correlated (P<0.05) with cortisol. The CE-C4D rapidly produced useful data on saliva ion contents, with good ion recovery as determined by the standard addition method (89-110%).