Proteomic Analysis of Butyrate-Resistant Colorectal Cancer-Derived Exosomes Reveals Potential Resistance to Anti-Cancer Drugs.

BACKGROUND: Butyrate-resistant (BR) cells play an important role in acquiring chemoresistance in colorectal cancer (CRC). Our previous study demonstrated that BR CRC cells showed cross-resistance to chemotherapy drugs, including 5-fluorouracil and oxaliplatin, in both monolayer and spheroid cultures. The mechanisms underlying drug resistance were also elucidated. However, the link between parental (PT) and BR cells remains unclear. Extracellular vesicles (EVs) are key cell-cell communications that transport various molecules, including DNA, RNA, and proteins, between the donor and target cells. EVs contribute to drug resistance in cancers, such as melanoma and lung cancer. Recently, we focused on the correlation of proteomic profiles of EVs from different cell types. METHODS: In this study, we analyzed the proteomic profiles of EVs derived from PT and BR cells to investigate the mechanisms underlying the butyrate- and chemo-resistant phenotypes. EVs were isolated from PT and BR cells using ultracentrifugation. The characteristics of the EVs were evaluated using western blot and transmission electron microscopy. The EV proteomic data were further analyzed using liquid chromatography-mass spectrometry. RESULTS: We identified a unique protein expressed in BR cells related to the chemoresistant phenotype. Functional enrichment analysis showed that BR cells had higher protein catalytic activity, binding, and transcription activity. The STITCH database showed a greater correlation between protein-drug interactions in BR cells than in PT cells. Moreover, our findings support the hypothesis that EVs promote tumor progression and metastasis and affect the tumor microenvironment. CONCLUSIONS: Proteomic analysis of EVs from BR CRC cells reveals insights into drug resistance mechanisms, including protein-mediated carcinogenesis and reduced drug uptake, offering potential strategies to overcome resistance in clinical practice.

Non-coding RNAs in enzalutamide resistance of castration-resistant prostate cancer.

Enzalutamide (Enz) is a next-generation androgen receptor (AR) antagonist used to treat castration-resistant prostate cancer (CRPC). Unfortunately, the relapsing nature of CRPC results in the development of Enz resistance in many patients. Non-coding RNAs (ncRNAs) are RNA molecules that do not encode proteins, which include microRNAs (miRNA), long ncRNAs (lncRNAs), circular RNAs (circRNAs), and other ncRNAs with known and unknown functions. Recently, dysregulation of ncRNAs in CRPC, particularly their regulatory function in drug resistance, has attracted more and more attention. Herein, we introduce the roles of dysregulation of different ncRNAs subclasses in the development of CRPC progression and Enz resistance. Recently determined mechanisms of Enz resistance are discussed, focusing mainly on the role of AR-splice variant-7 (AR-V7), mutations, circRNAs and lncRNAs that act as miRNA sponges. Also, the contributions of epithelial-mesenchymal transition and glucose metabolism to Enz resistance are discussed. We summarize the different mechanisms of miRNAs, lncRNAs, and circRNAs in the progression of CRPC and Enz resistance, and highlight the prospect of future therapeutic strategies against Enz resistance.

Drug resistance phenotype analysis and mechanism investigation of extensively drug resistant Klebsiella pneumoniae / 中华检验医学杂志

Objective:This work aims to investigate the phenotype-characteristics of drug resistance and the possible mechanisms of extensively drug-resistance Klebsiella pneumoniae(XDRKP). Methods:Screened by the previous drug susceptibility results, 116 clinical Klebsiella pneumoniae isolates were collected from Shanxi Bethune Hospital from January 2018 to December 2020. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) rapid microbial identification system and VITEK-compact 2 were used. The modified carbapenem inactivation method (mCIM) combining with EDTA carbapenem inactivation method (eCIM) was used to identify the strains′ carbapenemase phenotypes, which were compared with subsequent qPCR results. The qPCR amplification combining with agarose gel electrophoresis were carried out to detect various drug-resistant related genes, including: carbapenemase genes: blaKPC, blaNDM, blaVIM, blaIMP, blaOXA; aminoglycosides resistance genes: ① 16S rRNA methylase genes: rmtA, rmtC, rmtD, rmtG, rmtH, armA, npmA, rmtB, rmtE, rmtF, ② variant of aminoglycosides acetyltransferase gene: aac(6′)-Ib-cr; quinolone resistance genes: DNA gyrase protection protein qnr family: qnrA, qnrB, qnrC, qnrD, qnrS, efflux pump protein gene: oqxAB, qepA, variant of aminoglycoside acetyltransferase gene: aac(6′)-Ib-cr; and tigecycline-resistant Tet protein genes: efflux pump protein gene: tet (A), tet (L), ribosome protection protein gene: tet (M), tigecycline modified enzyme gene: tet (X). Each isolate′s phenotype and resistance gene result were compared and analyzed correspondingly. Results:A total number of 116 XDRKP isolates were collected in 3 years, 115 of which are identified as carbapenem resistant. Both cephalosporins and quinolones resistant rate were 100%, while the resistant rate of aminoglycosides antibiotic gentamicin, tobramycin and amikacin was 95.69% (111/116), 94.83% (110/116), or 88.79% (103/116) respectively. Sulfonamide antibiotics and tigecycline showed a relatively lower resistant rate. Compared with PCR amplification results, mCIM combining with eCIM phenotype testing had a high conformity, up to 95.65% (110/115). Positive rate of each resistance related gene was: blaKPC 90.52% (105/116), blaNDM 10.34% (12/116), rmtB 81.90% (95/116), armA 2.59% (3/116), oqxAB 65.52% (76/116), qnrB 6.03% (7/116), qnrS 12.93% (15/116), aac(6′)-Ib-cr 7.76% (9/116), or tet(A) 21.55% (25/116), respectively. Other resistance related genes were not detected. Corresponding analysis between the resistant phenotypes and resistance related genes indicated that a total of 65 XDRKP didn′t have a matched pairs, i.e. bacteria′s resistance to specific antibiotic could not be interpreted by carrying some associated resistant genes.Conclusions:The wide distribution of resistant genes and multiple-antibiotic-inactivated trait of some genes(such as aac(6′)-Ib-cr and oqxAB) in XDRKP are potential causes of the generation of extensively drug resistant phenotype. Different XDRKP isolates may carry one or more resistant genes in responding to specific antibiotic. In addition, there are some bacteria with an unmatched phenotype-gene feature indicating that both resistance genes′ regulation and some other mechanisms also play a role in development of XDR.

Molecular mechanism of R-bicalutamide switching from androgen receptor antagonist to agonist induced by amino acid mutations using molecular dynamics simulations and free energy calculation.

R-bicalutamide, a first generation antiandrogen, was used to treat prostate cancer for decades. Although it is very effective at the beginning, resistance appears after 2-3 years of treatment. Mutation of androgen receptor (AR) is considered a main reason for drug resistance. It is reported that AR W741C, W741L, W741C_T877A, T877A, F876L, F876L_T877A and L701H mutations can convert R-bicalutamide from AR antagonist to agonist, but the switching mechanisms are not clear. In this study, molecular dynamics simulations and molecular mechanics generalized Born surface area (MM-GBSA) calculations were performed to analyze the interaction mechanisms between R-bicalutamide and wild type/mutant ARs. The results indicate that helix H12, which lies on the top of AR LBD like a cover, plays a vital role in R-bicalutamide binding. When interacting with AR, the B-ring of R-bicalutamide pushes H12 aside, distorting the coactivator binding site (AF2) resulting in the inactivation of transcription. Several residue mutations appear to enlarge the distance between the B-ring of R-bicalutamide and H12, reducing steric clash, which is conducive to a closed H12 conformation, leading to the formation of the coactivator binding site AF2 and increased transcription. Hydrogen bond and per-residue free energy decomposition analyses are also investigated to explore the interacting mechanisms, and M895 is found to be a key residue in the antagonist mechanism. The obtained molecular mechanisms will aid rational screening and design of novel AR antagonists, even to mutant AR.

Mechanism of resistance of Pseudomonas aeruginosa to carbapenems / 上海第二医科大学学报

Objective To investigate the mechanism of resistance of clinical isolates of Pseudomonas aeruginosa to carbapenems.Methods Fifty-eight strains of imipenem-and meropenem-resistant Pseudomonas aeruginosa were isolated.Modified K-B technique was adopted in the susceptibility test by adding ?-lactamases inhibitors and efflux pumps inhibitors to M-H agar plates.The refined three-dimensional extract test was used to detect ?-lactamases.The genes of metallo-enzyme IMP,VIM,as well as outer membrane porin D2(OprD2) were analyzed with polymerase chain reaction.Results Among the 58 strains of carbapenems-resistant Pseudomonas aeruginosa,53 produced over-expressed active efflux and continuously produced large amont of AmpC enzyme,15 of which were accompanied by the loss of OprD2,and 1 of which were accompanied by extended spectrum ?-lactamases(ESBLs).Among the 5 strains which neither produced over-expressed active efflux nor ?-lactamases,only 1 was found with OprD2 gene deletion.Metallo-enzyme was not detected in any of the 58 strains.Conclusion The mechanism of resistance of Pseudomonas aeruginosa to carbapenems was mainly the production of the over-expressed active efflux combined with the continuous production of large amount of AmpC enzyme.Sixteen of the strains were accompanied by the loss of OprD2 gene.

Resistance Analysis of Pseudomonas aeruginosa / 中华医院感染学杂志

OBJECTIVE To investigate the distribution and drug resistance of Pseudomonas aeruginosa.METHODS All specimens isolated and cultured from patients in our hospital were identified by using the automatic microorganism analyzer WalkAway-40,Dade Behring made in America,and bacteria′s drug susceptibility test and identifications were performed on strains using NC21 Microscan Panel.RESULTS From sixty-two strains of P.aeruginosa 36 strains were isolated of sputum.The resistance rate to the third generation of cephalosporins cefotaxime and ceftriaxone was 55.6% and 47.5%,respectively.The resistance rate to the other ?-lactamases antibiotics such as ceftazidime was 7.5%,piperacillin/tazobactam 11.0%,cefepime 14.8% and penicillin was 30.0%,the ratio of resistance for imipenem was 20.0%,the lowest one was amikacin(4.6%).CONCLUSIONS P.aeruginosa is one of the main bacteria in the lower respiratory tract infection.The drug-resistant mechanism of P.aeruginosa is very complex,including multidrug resistance characteristics,and it is originally resistant to several antibiotics.To avoid being produced ?-lactamases and result in resisting drug widely,the antibiotics should be selected according to low drug-resistant rate and taking into account sufficiently its drug resistance mechanism in the treatment of P.aeruginosa infection.