Systematic analysis of potential targets of the curcumin analog pentagamavunon-1 (PGV-1) in overcoming resistance of glioblastoma cells to bevacizumab

BackgroundGlioblastoma is one of the most aggressive and deadliest malignant tumors. Acquired resistance decreases the effectiveness of bevacizumab in glioblastoma treatment and thus increases the mortality rate in patients with glioblastoma. In this study, the potential targets of pentagamavunone-1 (PGV-1), a curcumin analog, were explored as a complementary treatment to bevacizumab in glioblastoma therapy.MethodsTarget prediction, data collection, and analysis were conducted using the similarity ensemble approach (SEA), SwissTargetPrediction, STRING DB, and Gene Expression Omnibus (GEO) datasets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using Webgestalt and DAVID, respectively. Hub genes were selected based on the highest degree scores using the CytoHubba. Analysis of genetic alterations and gene expression as well as Kaplan–Meier survival analysis of selected genes were conducted with cBioportal and GEPIA. Immune infiltration correlations between selected genes and immune cells were analyzed with database TIMER 2.0.ResultsWe found 374 targets of PGV-1, 1139 differentially expressed genes (DEGs) from bevacizumab-resistant-glioblastoma cells. A Venn diagram analysis using these two sets of data resulted in 21 genes that were identified as potential targets of PGV-1 against bevacizumab resistance (PBR). PBR regulated the metabolism of xenobiotics by cytochrome P450. Seven potential therapeutic PBR, namely GSTM1, AKR1C3, AKR1C4, PTGS2, ADAM10, AKR1B1, and HSD17B110 were found to have genetic alterations in 1.2%–30% of patients with glioblastoma. Analysis using the GEPIA database showed that the mRNA expression of ADAM10, AKR1B1, and HSD17B10 was significantly upregulated in glioblastoma patients. Kaplan–Meier survival analysis showed that only patients with low mRNA expression of AKR1B1 had significantly better overall survival than the patients in the high mRNA group. We also found a correlation between PBR and immune cells and thus revealed the potential of PGV-1 as an immunotherapeutic agent via targeting of PBR.ConclusionThis study highlighted seven PBR, namely, GSTM1, AKR1C3, AKR1C4, PTGS2, ADAM10, AKR1B1, and HSD17B110. This study also emphasized the potential of PBR as a target for immunotherapy with PGV-1. Further validation of the results of this study is required for the development of PGV-1 as an adjunct to immunotherapy for glioblastoma to counteract bevacizumab resistance.     

Selective inhibition of interleukin-1 receptor-associated kinase 1 ameliorates lipopolysaccharide-induced sepsis in mice

Interleukin-1 receptor-associated kinases (IRAKs), particularly IRAK1 and IRAK4, are important in transducing signal from Toll-like receptor 4. We interrogated if a selective inhibition of IRAK1 could alleviate lipopolysaccharide (LPS)-induced sepsis. In this study, we tested the impact of a novel selective IRAK1 inhibitor Jh-X-119-01 on LPS-induced sepsis in mice. Survival at day 5 was 13.3% in control group where septic mice were treated by vehicle, while the values were 37.5% (p = 0.046, vs. control) and 56.3% (p = 0.003, vs. control) for 5 mg/kg and 10 mg/kg Jh-X-119-01-treated mice. Jh-X-119-01 alleviated lung injury and reduced production of TNFα and IFNγ in peritoneal macrophages. Jh-X-119-01 decreased phosphorylation of NF-κB and mRNA levels of IL-6 and TNFα in LPS-treated macrophages in vitro. Jh-X-119-01 selectively inhibited IRAK1 phosphorylation comparing with a non-selective IRAK1/4 inhibitor which simultaneously inhibited phosphorylation of IRAK1 and IRAK4. Both Jh-X-119-01 and IRAK1/4 inhibitor increased survival of septic mice, but Jh-X-119-01-treated mice had higher blood CD11b⁺ cell counts than IRAK1/4 inhibitor-treated ones [24 h: (1.18 ± 0.26) × 10⁶/ml vs. (0.79 ± 0.20) × 10⁶/ml, p = 0.001; 48 h: (1.00 ± 0.30) × 10⁶/ml vs. (0.67 ± 0.23) × 10⁶/ml, p = 0.042]. IRAK1/4 inhibitor induced more apoptosis of macrophages than Jh-X-119-01 did in vitro. IRAK1/4 inhibitor decreased protein levels of anti-apoptotic BCL-2 and MCL-1 in RAW 264.7 and THP-1 cells, an effect not seen in Jh-X-119-01-treated cells. In conclusion, Jh-X-119-01 selectively inhibited activation of IRAK1 and protected mice from LPS-induced sepsis. Jh-X-119-01 showed less toxicity on macrophages comparing with a non-selective IRAK1/4 inhibitor.     

The protective effects of silymarin nanoemulsion on 5-fluorouracil-induced gastrointestinal toxicity in rats

5-Fluorouracil (5FUra) is the third most popular chemotherapeutic component employed to treat solid tumors. In the present study, we aimed to appraise the silymarin (SM) and silymarin nanoemulsion (SMN) effect on 5FUra-induced gastrointestinal toxicity in adult male rats. A total of 30 male Wistar rats were divided into 6 groups including the control (Crl) group, and groups treated with SMN (5 mg.kg⁻¹), SM (5 mg.kg⁻¹), 5FUra + SMN (5 mg.kg⁻¹), and 5FUra + SM (5 mg.kg⁻¹) by IP injection for 14 days. And gastrointestinal toxicity was induced by a single intraperitoneal (IP) injection of 5FUra (100 mg.kg⁻¹) for the last group in the study. Treating rats with SM and SMN diminished elevating malondialdehyde (MDA) levels, and improved total antioxidant capacity (TAC) levels. Also, the intensity of mRNA expression of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) caused by 5FUra in the gastrointestinal tissue tract, and macroscopic oral ulcerations decreased, ass well as weight loss was prevented, particularly in the SMN group. Moreover, in the microscopic scope, there were significant improvements in the levels of hyperemia, hyaline, and inflammatory cell infiltration in the tongue, esophagus, and intestinal tissues in the FUra + SMN and FUra + SM groups compared to 5FUra. Hence, treatment with SM and SMN reduced oxidative stress, histopathological degeneration, and gene expression of inflammatory markers in the gastrointestinal tract. According to the results, treatment with SM and SMN markedly decreases the gastrointestinal toxicity caused by 5FUra.     

Blood-Brain Barrier Permeability of Asiaticoside,Madecassoside and Asiatic Acid in Porcine Brain Endothelial Cell Model

Neurotherapeutic potentials of Centella asiatica and its reputation to boost memory, prevent cognitive deficits and improve brain functions are widely acknowledged. The plant's bioactive compounds, i.e. asiaticoside, madecassoside and asiatic acid were reported to have central nervous system (CNS) actions, particularly in protecting the brain against neurodegenerative disorders. Hence, it is important for these compounds to cross the blood-brain barrier (BBB) to be clinically effective therapeutics. This study aimed to explore the capability of asiaticoside, madecassoside and asiatic acid to cross the BBB using in vitro BBB model from primary porcine brain endothelial cells (PBECs). Our findings showed that asiaticoside, madecassoside and asiatic acid are highly BBB permeable with apparent permeability (P_app) of 70.61 ± 6.60, 53.31 ± 12.55 and 50.94 ± 10.91 × 10^?6 cm/s respectively. No evidence of cytotoxicity and tight junction disruption of the PBECs were observed in the presence of these compounds. Asiatic acid showed cytoprotective effect towards the PBECs against oxidative stress. This study reported for the first time that Centella asiatica compounds demonstrated high capability to cross the BBB, comparable to central nervous system drugs, and therefore warrant further development as therapeutics for the treatment of neurodegenerative diseases.     

UM171 promotes expansion of autologous peripheral blood hematopoietic stem cells from poorly mobilizing lymphoma patients

BackgroundAutologous hematopoietic stem cell transplantation is an effective therapeutic strategy for lymphoma patients. However, some patients have to give up receiving transplantation because of failing to obtain sufficient CD34⁺ cells yields. Therefore, we ex vivo expanded HSCs of lymphoma patients using UM171 to solve the problem of HSCs deficiency.MethodsMobilized peripheral blood-derived CD34⁺ cells from lymphoma patients were cultured for 10 days with or without UM171. The fold of cell expansion and the immunophenotype of expanded cells were assessed by flow cytometry. RNA-seq experiment was performed to identify the mechanism by which UM171 promoted HSCs expansion.ResultsUM171 treatment increased the proportion of CD34⁺ (68.97 ± 6.91%), CD34⁺ CD38⁻ cells (44.10 ± 9.20%) and CD34⁺CD38⁻CD45RA⁻CD90⁺ LT-HSCs (3.05 ± 2.08%) compared to vehicle treatment (36.08 ± 11.14%, 18.30 ± 9.49% and 0.56 ± 0.45%, respectively). UM171 treatment led to an 85.08-fold increase in LT-HSC numbers relative to initial cells. Importantly, UM171 promoted expansion of LT-HSCs achieved 138.57-fold in patients with poor mobilization. RNA-seq data showed that UM171 upregulated expression of HSC-, mast cell-specific genes and non-canonical Wnt signaling related genes, and inhibited genes expression of erythroid, megakaryocyte and inflammatory mediated chemokine.ConclusionsOur study shows that UM171 can efficiently promote ex vivo expansion of HSCs from lymphoma patients, especially for poorly mobilizing patients. In terms of mechanism, UM171 upregulate HSC-specific genes expression and suppress erythroid and megakaryocytic differentiation, as well as activate non-classical Wnt signaling.     

Design of a Novel Nucleus-Targeted NLS-KALA-SA Nanocarrier to Delivery Poorly Water-Soluble Anti-Tumor Drug for Lung Cancer Treatment

In this study, we designed a novel nucleus-targeted nanocarrier (NLS-KALA-SA, NKSN) consisting of Kala peptide (KALA), nuclear localization signal (NLS) and stearic acid (SA) using Fmoc solid phase synthesis method. We chose Curcumin (CUR), Paclitaxel (PTX), Ginsenoside compound K(CK) as models of poorly water-soluble antitumor drugs, The drugs loaded NLS-KALA-SA nanoparticles (CUR/NKSN, PTX/NKSN, CK/NKSN) were obained by the dialysis method, their physicochemical properties were determined and antitumor activity were evaluated. The NLS-KALA-SA nanoparticles were spherical shaped with an average size of 76.4 ± 7.6 mm and a zeta potential of 43.7 ± 5.8 mV. The drug-loaded NLS-KALA-SA nanoparticles were above 86.1% and 17.1% in entrapment efficiency and drug loading capacity, and had sustained drug release behavior. Biodistribution and cellular uptake study exhibited that PTX/NKSN mainly distributed in tumor site of A549-bearing mice, and coumarin-6(C6) loaded NLS-KALA-SA nanoparticle (C6/NKSN) was predominantly accumulated in the nucleus of A549 cells. Western blot analysis indicated that PTX/NKSN could more remarkably inhibit Bcl-2 expression and enhance the expression of Bax and Caspase-3 as compared to the controls in A549 cells. Cell apoptosis and antitumor activity study showed that PXT/NKSN could more obviously induce apoptosis of A549 cells compared with free PXT, the PTX/NKSN administration was more effective than free PTX for lung cancer treatment and displayed mild toxicity in A549-bearing mice. The results demonstrates that the NLS-KALA-SA nanoparticles system could enhance the antitumor effects of the encapsulated drug and reduce tissue toxicity due to its long circulating properties and tumor targeting, which might provide a promising strategy for lung cancer treatment.     

Excess salt intake promotes M1 microglia polarization via a p38/MAPK/AR-dependent pathway after cerebral ischemia in mice

A high salt diet (HSD) is among the most important risk factors for many diseases. One mechanism by which HSD aggravates cerebral ischemic injury is independent of blood pressure changes. The direct role of HSD in inflammation after cerebral ischemia is unclear. In this research, after twenty-one days of being fed a high salt diet, permanent focal ischemia was induced in mice via operation. At 12 h and 1, 3 and 5 days postischemia, the effects of HSD on the lesion volume, microglia polarization, aldose reductase (AR) expression, and inflammatory processes were analyzed. We report that in mice, surplus dietary salt promotes inflammation and increases the activation of classical lipopolysaccharide (LPS)-induced microglia/macrophages (M1). This effect depends on the expression of the AR protein in activated microglia after permanent middle cerebral artery ligation (pMCAL) in HSD mice. The administration of either the AR inhibitor Epalrestat or a p38-neutralizing antibody blocked the polarization of microglia and alleviated stroke injury.In conclusion, HSD promotes polarization in pro-inflammatory M1 microglia by upregulating the expression of the AR protein via p38/MAPK, thereby exacerbating the development of ischemia stroke.     

Antioxidant and anti-inflammatory activities of lycopene against 5-fluorouracil-induced cytotoxicity in Caco2 cells

5-fluorouracil (5FU) is widely used to treat colorectal cancer (CC) and its main mechanisms of anticancer action are through generation of ROS which often result in inflammation. Here, we test the effect of Lycopene against 5FU in Caco2 cell line. Caco2 cells were exposed to 3 µg/ml of 5FU alone or with 60, 90, 120 µg/ml of lycopene. This was followed by assessment of cytotoxicity, oxidative stress, and gene expression of inflammatory genes. Our findings showed that Lycopene and 5FU co-exposure induced dose-dependent cytotoxic effect without compromising the membrane integrity based on the LDH assay. Lycopene also significantly enhanced 5FU-induced SOD activity and GSH level compared to control for all mixture concentrations (p < 0.01). Lycopene alone and combination with 5FU-induced expression of IL-1β, TNF-α, and IL-6. Furthermore, IFN-γ expression was significantly enhanced by only mixture of lycopene (90 µg/ml) and 5FU (p < 0.05). In conclusion, Lycopene supplementation with 5FU therapy resulted in improvement in antioxidant parameters such as catalase and GSH levels giving the cell capacity to cope with 5FU-mediated oxidative stress. Lycopene also enhanced IFN-γ expression in the presence of 5FU, which may activate antitumor effects further enhancing the cancer killing effect of 5FU.     

ACTH4-10 protects the ADR-injured podocytes by stimulating B lymphocytes to secrete interleukin-10

ObjectivesIn the present study, we aimed to assess whether adrenocorticotropic hormone (ACTH) could protect the podocytes from adriamycin (ADR)-induced injury by stimulating B lymphocytes to secrete the associated cytokines.MethodsProliferation assay was used to assess the proliferation and activity of podocytes. Enzyme-linked immunosorbent assay was used to examine the secretion of IL-10 and IL-4. TUNEL apoptosis detection kit was used to detect the apoptosis of podocytes. Real-time PCR and Western blotting analysis were used to examine the expressions of nephrin and podocin at the mRNA and protein levels.ResultsCompared with the normal control group, the podocyte proliferation of ADR group was significantly inhibited. However, compared with the ADR group, the podocyte proliferation of the supernatant (1 µg/L, 10 µg/L or 100 µg/L ACTH_4-10) + ADR groups was generally increased, and the pro-proliferative effect of the supernatant containing 10 µg/L ACTH_4-10 was the highest. Moreover, we found that after B lymphocytes were intervened by 10 µg/L ACTH4-10, the IL-10 level in the cell supernatant was significantly elevated (p < 0.05). When anti-IL-10R was added, the podocyte proliferation of the supernatant (10 µg/L ACTH_4-10) + ADR group was significantly inhibited. Furthermore, the supernatant of B cells stimulated with 10 µg/L ACTH_4-10 could better decrease the apoptosis rate of injured podocytes and increase the expressions of nephrin and podocin at the mRNA and protein levels by elevating the secretion of IL-10.ConclusionCompared with ACTH_4-10, the supernatant of B cells stimulated with ACTH_4-10 could better protect the podocytes from ADR-induced injury by elevating the secretion of IL-10.     

Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8)

Using X. laevis oocyte expression system, we investigated whether human Na⁺-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na⁺-dependent and electrogenic. The Na⁺-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na⁺-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na⁺-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.     

In vitro inhibitory effects of components from Salvia miltiorrhiza on catalytic activity of three human Arachidonic acid ω-hydroxylases

CYP4 enzymes are involved in the metabolism of xenobiotics and endogenous molecules. 20-Hydroxyeicosatetraenoic acid (20-HETE), the arachidonic acid (AA) ω-hydroxylation metabolite catalyzed by CYP4A/4F enzymes, is implicated in various biological functions. The goal of this investigation is to examine the inhibitory effects of components from Salvia miltiorrhiza(Danshen) on AA ω-hydroxylation using recombinant CYP4A11, CYP4F2, CYP4F3B, and microsomal systems. Tanshinone IIA had noncompetitive inhibition on CYP4F3B (K_i = 4.98 μM). Cryptotanshinone (K_i = 6.87 μM) and tanshinone I (K_i = 0.42 μM) had mixed-type inhibition on CYP4A11. Dihydrotanshinone I had mixed-type inhibition on CYP4A11 (K_i = 0.09 μM), and noncompetitive inhibition on CYP4F2 (K_i = 4.25 μM) and CYP4F3B (K_i = 3.08 μM). Salvianolic acid A had competitive inhibition on CYP4A11 (K_i = 19.37 μM), and noncompetitive inhibition on CYP4F2 (K_i = 15.28 μM) and CYP4F3B (K_i = 6.45 μM). Salvianolic acid C had noncompetitive inhibition on CYP4F2 (K_i = 5.70 μM) and CYP4F3B (K_i = 18.64 μM). In human kidney, human liver or rat heart microsomes, 20-HETE formation was significantly inhibited (P < 0.05) by dihydrotanshinone I (5 and 20 μM) and salvianolic acid A (20 and 50 μM). Given that low plasma concentrations of Danshen components after oral administration, Danshen preparations may not play pharmacological roles by inhibiting AA ω-hydroxylases; however, as Danshen components may reach high concentration in human intestine, drugs that have an important pre-systemic metabolism by these CYP4A/4F enzymes should avoid being co-administered with Danshen preparations.     

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.     

Quantitative investigation of hepatobiliary transport of [11C]telmisartan in humans by PET imaging

The pharmacokinetics of telmisartan are nonlinear within the clinical dose range. To identify the underlying mechanism of this nonlinearity, we conducted a PET study in healthy subjects using [¹¹C]telmisartan. Eight healthy male subjects were enrolled in a 2-way crossover study. PET imaging was performed after intravenous administration of [¹¹C]telmisartan with or without a 1-h oral predose of two 40 mg Micardis® tablets. About 60% of the injected [¹¹C]telmisartan accumulated in the liver within 10 min after injection. With predosing of 80 mg telmisartan, the systemic elimination of [¹¹C]telmisartan was slightly delayed, but the liver exposure started to decrease earlier and biliary excretion was greatly enhanced. Hepatic uptake clearance of the radioactivity was not changed by telmisartan predosing, whereas the biliary clearance of radioactivity from the liver was significantly increased. Thus, the alteration in the pharmacokinetics of the radioactivity could not be explained simply by the saturation of hepatic uptake. Therefore, other mechanisms, such as the saturation of intracellular binding of telmisartan and/or its glucuronide, and the glucuronidation of telmisartan by uridine 5′-diphospho-glucuronosyltransferases, should be considered. This is the first reported human PET study using [¹¹C]telmisartan, the results of which can assist understanding of the hepatobiliary transport of telmisartan in humans.     

An eco-friendly HPLC-UV method for the determination of risedronate in its bulk and tablet dosage form with application to content uniformity,dissolution and stability testing

Risedronate is a nitrogen-containing bisphosphonate for the treatment and prevention of postmenopausal osteoporosis. The current work aims to develop a novel green HPLC-UV method for the rapid analysis of risedronate sodium in bulk and tablet formulation. The analyzed samples were separated on Waters Atlantis dC18 (150 mm × 3.9 mm; 5 μm) column using a green mobile phase consisting of potassium phosphate buffer pH 2.9 and potassium edetate buffer pH 9.5 in a ratio of 1:2, the final pH was adjusted to 6.8 with phosphoric acid, the mobile phase was pumped at a rate of 1.0 mL/min, with column temperature set at 30 °C, eluted samples were detected at 263 nm and the chromatographic run time was 3.0 min. The method was found to be linear over the concentration range of 14–140 μg/mL with a correlation coefficient (r²) of 0.9994. Accuracy and precision were evaluated from three QC samples (LQC, MQC and HQC) together with the five calibrators where the percentage accuracy was found to be 101.84%. Processed quality control samples of risedronate sodium were tested for stability at different conditions, short term, long term and freeze- thaw stability. The current method was further extended to study the content uniformity of Actonel® tablets following United States Pharmacopoeia (USP) guidelines. The proposed method was fully validated as per ICH guidelines.     

Design of semisynthetic derivatives of the Amaryllidaceae alkaloid ambelline and exploration of their in vitro cytotoxic activities

Ambelline, an alkaloid from the Amaryllidaceae family with a crinane-type skeleton, has not yet demonstrated any outstanding biological activity. However, its analogues prepared by derivatization of the C-11 hydroxyl group show different interesting effects. Continuing our earlier work, twelve novel aromatic esters were developed (10, 14, 16, 17, 22–25, 30–33) and studied, together with previously synthesized derivatives (2–9, 11–13, 15, 18–21, 26–29) in terms of their cytotoxic activity. The cytotoxic potential was determined on a panel of nine human cancer cell lines and one noncancerous cell line to characterize their biological activity spectrum. To describe and foresee the structure–activity relationship for further research, substances synthesized and described in our previous work were also included in this cytotoxicity study. The most significant activity was associated with analogues having methyl (10), methoxy (14–17), or ethoxy (18) substitution on the phenyl condensed to ambelline. However, the 4-chloro-3-nitrobenzoyl derivative (32) showed the most promising IC₅₀ values, ranging from 0.6 ± 0.1 µM to 9.9 ± 0.2 µM. In vitro cytotoxicity studies indicated the most potent antiproliferative activity of 32 in a dose-dependent and time-dependent manner. Besides, 32 was found to be effective in decreasing viability and triggering apoptosis of MOLT-4 T-lymphoblastic leukemia cells.     

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor. Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell. Then, we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatin III. We found that baccatin III could be efficiently deacetylated into 10-DAB by DBAT with CoASH and K⁺ under alkaline condition. Thus, we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatin III. The fermentation conditions were optimized and the baccatin III titers reached 2, 3 and 4.6 g/L, respectively, in a 3-L bioreactor culture when 2, 3 and 6 g/L of 10-DAB were supplied. Our study provides an environment-friendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis. The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.     

13C-caffeine breath test identifies single nucleotide polymorphisms associated with caffeine metabolism

We performed a caffeine (N-3-methyl-¹³C) breath test (CafeBT) to determine whether it can be employed to identify caffeine metabolism-associated single nucleotide polymorphisms. The study included 130 healthy adults (mean age: 21.9 years). Saliva was collected using an Oragene®•DNA saliva collection kit. Breath samples were collected from the subjects. The subjects orally ingested 100 mg ¹³C-caffeine dissolved in distilled water. Subsequently, breath samples were collected in bags every 10 min for a total of 90 min. An analysis of ¹³CO₂ in the expired breath was performed by infrared spectroscopy, and the sum of Δ¹³CO₂ over 90 min (S^90m) was calculated. DNA from saliva samples was genotyped using TaqMan® SNP Genotyping for the following genes: cytochrome P4501A2: rs762551, rs2472297, aryl-hydrocarbon receptor (rs4410790), and adenosine A_2A receptor (rs5751876). All subjects had the genotype CC in rs2472297 alleles. No significant difference was observed in S^90m among the genotypes of rs762551 and rs5751876; however, a significant difference was found in S^90m among the genotypes of rs4410790 (C > T). Our findings suggest that the N-3 demethylation of caffeine is dependent on the rs4410790 allele and that CafeBT may be used to determine rs4410790 genotypes.