Human drug efflux transporter ABCC5 confers acquired resistance to pemetrexed in breast cancer.

AIM: Pemetrexed, a new generation antifolate drug, has been approved for the treatment of locally advanced or metastatic breast cancer. However, factors affecting its efficacy and resistance have not been fully elucidated yet. ATP-binding cassette (ABC) transporters are predictors of prognosis as well as of adverse effects of several xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and can contribute to the optimization of breast cancer treatment regimen. METHODS: First, we measured the expression levels of ABC transporter family members in cell lines. Subsequently, we assessed the potential role of ABC transporters in conferring resistance to pemetrexed in primary breast cancer cells isolated from 34 breast cancer patients and the role of ABCC5 in mediating pemetrexed transport and apoptotic pathways in MCF-7 cells. Finally, the influence of ABCC5 expression on the therapeutic effect of pemetrexed was evaluated in an in vivo xenograft mouse model of breast cancer. RESULTS: The expression levels of ABCC2, ABCC4, ABCC5, and ABCG2 significantly increased in the pan-resistant cell line, and the ABCC5 level in the MCF-7-ADR cell line was 5.21 times higher than that in the control group. ABCC5 expression was inversely correlated with pemetrexed sensitivity (IC50, r = 0.741; p < 0.001) in breast cancer cells derived from 34 patients. Furthermore, we found that the expression level of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cells, with IC50 values of 0.06 and 0.20 µg/mL in ABCC5 knockout and over-expression cells, respectively. In the in vivo study, we observed that ABCC5 affected the sensitivity of pemetrexed in breast tumor-bearing mice, and the tumor volume was much larger in the ABCC5-overexpressing group than in the control group when compared with their own initial volumes (2.7-fold vs. 1.3-fold). CONCLUSIONS: Our results indicated that ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and it may serve as a target or biomarker for the optimization of pemetrexed regimen in breast cancer treatment.

[Accumulation of microbial necromass carbon and their contribution to soil organic carbon in different vegetation types on the Loess Plateau, Northwest China]. / é»„åœŸé«˜åŽŸä¸åŒæ¤è¢«ç±»åž‹åœŸå£¤å¾®ç”Ÿç‰©æ®‹ä½“ç¢³çš„ç§¯ç´¯åŠå ¶å¯¹æœ‰æœºç¢³çš„è´¡çŒ®.

Microbial necromass carbon (MNC) is an important contributor to soil organic carbon (SOC). Soil carbon storage has increased significantly since the return of farmland to forestland (grassland) on the Loess Plateau. However, the contribution of MNC to SOC accumulation in different vegetation types and the influence factors remain unclear. Herein, we used the biomarker (amino sugar) technique to determine the MNC content and analyzed the influencing factors in 0-5 cm and 5-20 cm soil layers of natural grassland, shrubland (Caragana microphylla), and forestland (Quercus liaodongensis) in the Loess Plateau. The results showed that: 1) the soil pH decreased significantly from grassland to shrubland and then to forestland within the same soil layer. However, the SOC, total nitrogen (TN), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) contents showed a reverse trend, with forestland displaying the highest values followed by shrubland and then grassland. The 0-5 cm had significantly higher values than the 5-20 cm depth. 2) The MNC contents varied 0.69-16.41 g·kg-1 in the two soil horizons of the three vegetation types. There were significant increases in the contents of bacterial necromass carbon (BNC), fungal necromass carbon (FNC), and MNC in the 0-5 cm soil from grassland, shrubland to forestland. The contents of MBC were 1.9 times higher in forestland than in shrubland, and 3.2 times higher in shrubland than in grassland. In the 5-20 cm soil layer, the contents of FNC and MBC were significantly higher in the forestland than in the shrubland and grassland. The FNC content was significantly higher than that of the BNC, ranging from 1.16 to 9.83 times greater than the BNC. 3) The contribution of MNC to SOC was 0.6 and 0.7 times higher in shrubland and forestland than in grassland, respectively, with FNC accounting for 15.2%-42.7%, and BNC accounting for 1.4%-7.4%. 4) pH, TN, MBC, and MBN were important factors that influenced MNC accumulation. In summary, the variation in vegetation type altered soil nutrients, microbial activity, and soil pH, resulting in forestland and shrubland being more beneficial to the formation and accumulation of MNC, which was dominated by fungi, compared to grassland.

Mechanism of polyethylene and biodegradable microplastic aging effects on soil organic carbon fractions in different land-use types.

Microplastics (MPs) are widely present in terrestrial ecosystems, but knowledge about the aging characteristics of MPs in different land-use types and their impact on soil organic carbon fractions is still limited. Polyethylene (PE) and biodegradable MPs (Poly propylene carbonate and Polybutylene adipate terephthalate synthetic material (PPC + PBAT, Bio)), at 0 %, 0.03 %, and 0.3 % (w/w) dosages, were added to grassland, farmland, and facility soils for eight-week incubation. The aging degree of MPs was explored by quantifying the carbonyl index (CI). Soil organic C fractions such as SOC, particulate organic carbon (POC), mineral-associated organic carbon (MAOC), and microbial-derived C were analyzed. MPs underwent rapid aging after incubation, and the CI value for 0.03 % PE-MPs increased from 0.05 to 0.27 (farmland) and 0.26 (facility) (p < 0.05). The aging degree of 0.03 % and 0.3 % Bio-MPs was most significant in grassland, with CI decreasing by 46.6 % and 69.0 %, respectively. The CI of MPs were negatively correlated with their dosage. The 0.03 % and 0.3 % PE-MPs decreased soil organic carbon (SOC) content by 7.4 % and 8.2 % in grassland, and 3.0 % and 6.0 % in the facility (p < 0.05). POC content of farmland and facility soil was negatively correlated with PE-MPs' CI (p < 0.05). The 0.03 % PE and Bio-MPs decreased fungal necromass C (FNC) by 0.40 and 0.05 g kg-1 in grassland and 0.48 and 0.21 g kg-1 in farmland. Besides, the dosage of MPs regulated FNC content through soil pH, nutrients, and extracellular enzyme activity, either directly or indirectly, ultimately affecting the soil C pool. Therefore, this study demonstrates that MPs strongly affect SOC dynamics by influencing soil microbial enzyme activity and fungal necromass.

LC-MS/MS Method for Determination of Hydroxychloroquine and Metabolites: Application in a Pharmacokinetic Study.

Hydroxychloroquine (HCQ) was originally used as an antimalarial and immunomodulation drug. We developed and validated a simple and sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantitation of HCQ and its three metabolites in rat blood, and reported their pharmacokinetic parameters. The chromatographic separation and detection of analytes were achieved within 4 min on ZORBAX SB-C8 (3.5 µm, 2.1 × 150 mm) column with gradient elution, and the flow rate was 0.25 mL/min. Simple protein precipitation was successfully applied for sample pretreatment. The HCQ displays a good linearity in the range of 2.0-5000.0 ng/mL, and the three metabolites also show good linearity ranging from 1.0 to 2500.0 ng/mL, with all correlation coefficients (R 2) better than 0.98. In conclusion, this rapid, sensitive method was successfully developed, validated, and then applied to a pharmacokinetic study of HCQ in rat model in high dose. The results of the pharmacokinetic study presented an average half-life time 21.14 ± 10.31 h (mean ± SD) of HCQ, which is much shorter in human compared to that in mice. For the three metabolites, longer half-life times (approximately 100 h) were shown in rat.

Pharmacokinetic study of the main components of Tanreqing capsules and Tanreqing injections in beagles by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Tanreqing capsules (TRQCs) and Tanreqing injections (TRQIs) are widely used in the treatment of respiratory diseases. In this study, a simple, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of the main components of Tanreqing, which include chlorogenic acid, ursodeoxycholic acid, chenodeoxycholic acid, and baicalin, in beagle dog plasma to compare their pharmacokinetic parameters. METHODS: Plasma samples were pretreated with protein precipitation. Chromatographic separation was performed on Waters Acquity UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column using a gradient elution with (A) 0.1% (v/v) formic acid aqueous solution and (B) acetonitrile. Six healthy beagles were divided into two groups, and a crossover, comparative pharmacokinetic study of TRQC (0.09 g/kg) and TRQI (0.5 mL/kg) after a single-dose administration or daily doses over 7 days was carried out. One group was administrated a single dose of TRQC and followed continuously for 7 days, whereas the other group was treated with TRQI in the same way. RESULTS: The calibration curves were linear over the ranges of 2.00-1000.00 ng/mL for baicalin, 10.00-5000.00 ng/mL for ursodeoxycholic acid, 1.00-500.00 ng/mLfor chenodeoxycholic acid and chlorogenic acid, respectively. The relative standard deviation of both intra-day and inter-day accuracy is less than 11.23%. The average extraction recovery of all compounds was greater than 82.21%. The major pharmacokinetic parameters of the four compounds were not significantly different between the two formulations (P > 0.05). CONCLUSIONS: The measured levels of the four major components of TRQCs and TRQIs were comparable in these dogs, providing a reference for the clinical application of TRQCs instead of TRQIs.

A Direct and Sensitive Method for Determination of 5-Fluorouracil in Colorectal Cancer Cells: Evaluating the Effect of Stromal Cell on Drug Resistance of Cancer Cells.

Fibroblasts in the stroma play a critical role in tumor evolution. In this study, we assessed the influence of colonic fibroblasts on colon cancer cells treated with 5-fluorouracil (5-FU), and mouse colon cancer cell lines MC38 and colonic fibroblasts NIH3T3 were used in this study. A sensitive and rapid UHPLC-MS/MS method for the quantitation of 5-FU from the cell and their medium has been successfully developed and validated. The cells were lysed with methanol, and the mixture was evaporated and then redissolved to extract intracellular 5-FU. The analysis was performed on UHPLC-MS/MS using an Atlantis T3-C18 column (3 µm, 2. 1 ∗ 100 mm) and gradient elution with acetonitrile and 0.1% formic acid in water. Method validation included the following parameters: the matrix effect range 88.82%-93.64% and the recovery range 93.52%-94.56%. The intraday and interday precision and accuracy were <11% and within ±6%, and the stability, specificity, carry-over, dilution effect, and linearity all conformed to the criteria. The method was applied to detect the concentration of 5-FU inside cells and cell culture medium. The preliminary results present that NIH3T3 could enhance the drug resistance of MC38 to 5-FU with a decreased intracellular concentration of 5-FU in MC38, which showed a positive relationship with NIH3T3 number.

A rapid and sensitive method for simultaneous determination of eight protein-bound uremic toxins in human serum by UHPLC-MS/MS: application in assessing peritoneal dialysis.

A simple, rapid, reliable and sensitive ultra-high performance liquid chromatography tandem spectrometry (UHPLC-MS/MS) method was established for determination of eight serum protein-bound uremic toxins (hippuric acid, indoxyl sulfate, indole-3-acetic acid, kynurenic acid, L-kynurenine, melatonin, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, 4-hydroxyhippuric acid) in serum from chronic kidney disease (CKD) dialysis patients. The chromatographic separation was achieved on an Atlantis T3 column (3 µm, 2.1 mm × 100 mm) using a gradient elution with acetonitrile (phase B) and 0.1% formic acid and 10 mmol/L ammonium acetate aqueous solution (phase A). The flow rate was 0.3 mL/min with analytical time of 5 min. The pretreatment procedure was developed with a simple protein precipitation and the hydrochlorothiazide was used as internal standard. The calibration ranges were set as 156.250-20000.000 ng/mL for indoxyl sulfate, hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid; 78.125-10000.000 ng/mL for L-kynurenine, indole-3-acetic acid and 4-hydroxyhippuricacid; 1.562-200.000 ng/mL for kynurenic acid; 0.078-10.000 ng/mL for melatonin. The UHPLC-MS/MS method for quantification of eight protein-bound uremic toxins was successfully developed and validated, and its clinical practicability was assessed on 81 serum samples from CKD patients.