Fed-batch fermentation strategy for efficient welan gum production by Sphingomonas sp. FM01.

BACKGROUND: As a novel type of extracellular polysaccharide produced by Sphingomonas sp., welan gum has been widely applied in various fields because of its excellent properties. The study has improved the fermentation process. RESULTS: The initial sucrose concentration, temperature and stirring speed were set to 20 g L-1, 33 °C and 400 rpm, respectively, and 13.3 g L-1 sucrose was added at 24, 40 and 56 h. The temperature and stirring speed were then set at 28 °C and 600 rpm from 24 to 48 h and 28 °C and 600 rpm from 48 to 72 h, respectively. As a result, welan gum production, dry cell weight, sucrose conversion rate and viscosity were correspondingly increased to 38.60 g L-1, 5.47 g L-1, 0.64 g g-1 and 3779 mPa·s, respectively. In addition, the mechanism by which fermentation strategy promotes welan gum synthesis was investigated by transcriptome analysis. CONCLUSION: Improving respiration and ATP supply, reducing unnecessary protein synthesis, and alleviating competition between cell growth and welan gum synthesis contribute to promoting the fermentation performance of Sphingomonas sp., thus providing a practical strategy for efficient welan gum production. © 2024 Society of Chemical Industry.

In Vivo Reversal of P-Glycoprotein-Mediated Drug Resistance in a Breast Cancer Xenograft and in Leukemia Models Using a Novel, Potent, and Nontoxic Epicatechin EC31.

The modulation of P-glycoprotein (P-gp, ABCB1) can reverse multidrug resistance (MDR) and potentiate the efficacy of anticancer drugs. Tea polyphenols, such as epigallocatechin gallate (EGCG), have low P-gp-modulating activity, with an EC50 over 10 µM. In this study, we optimized a series of tea polyphenol derivatives and demonstrated that epicatechin EC31 was a potent and nontoxic P-gp inhibitor. Its EC50 for reversing paclitaxel, doxorubicin, and vincristine resistance in three P-gp-overexpressing cell lines ranged from 37 to 249 nM. Mechanistic studies revealed that EC31 restored intracellular drug accumulation by inhibiting P-gp-mediated drug efflux. It did not downregulate the plasma membrane P-gp level nor inhibit P-gp ATPase. It was not a transport substrate of P-gp. A pharmacokinetic study revealed that the intraperitoneal administration of 30 mg/kg of EC31 could achieve a plasma concentration above its in vitro EC50 (94 nM) for more than 18 h. It did not affect the pharmacokinetic profile of coadministered paclitaxel. In the xenograft model of the P-gp-overexpressing LCC6MDR cell line, EC31 reversed P-gp-mediated paclitaxel resistance and inhibited tumor growth by 27.4 to 36.1% (p < 0.001). Moreover, it also increased the intratumor paclitaxel level in the LCC6MDR xenograft by 6 fold (p < 0.001). In both murine leukemia P388ADR and human leukemia K562/P-gp mice models, the cotreatment of EC31 and doxorubicin significantly prolonged the survival of the mice (p < 0.001 and p < 0.01) as compared to the doxorubicin alone group, respectively. Our results suggested that EC31 was a promising candidate for further investigation on combination therapy for treating P-gp-overexpressing cancers.

Discovery of a Flavonoid FM04 as a Potent Inhibitor to Reverse P-Glycoprotein-Mediated Drug Resistance in Xenografts and Improve Oral Bioavailability of Paclitaxel.

Biotransformation of flavonoid dimer FD18 resulted in an active metabolite FM04. It was more druggable because of its improved physicochemical properties. FM04 (EC50 = 83 nM) was 1.8-fold more potent than FD18 in reversing P-glycoprotein (P-gp)-mediated paclitaxel (PTX) resistance in vitro. Similar to FD18, FM04 chemosensitized LCC6MDR cells towards multiple anticancer drugs by inhibiting the transport activity of P-gp and restoring intracellular drug levels. It stimulated the P-gp ATPase by 3.3-fold at 100 µM. Different from FD18, FM04 itself was not a transport substrate of P-gp and presumably, it cannot work as a competitive inhibitor. In the human melanoma MDA435/LCC6MDR xenograft, the co-administration of FM04 (28 mg/kg, I.P.) with PTX (12 mg/kg, I.V.) directly modulated P-gp-mediated PTX resistance and caused a 56% (*, p < 0.05) reduction in tumor volume without toxicity or animal death. When FM04 was administered orally at 45 mg/kg as a dual inhibitor of P-gp/CYP2C8 or 3A4 enzymes in the intestine, it increased the intestinal absorption of PTX from 0.2% to 14% in mice and caused about 57- to 66-fold improvement of AUC as compared to a single oral dose of PTX. Oral co-administration of FM04 (45 mg/kg) with PTX (40, 60 or 70 mg/kg) suppressed the human melanoma MDA435/LCC6 tumor growth with at least a 73% (***, p < 0.001) reduction in tumor volume without serious toxicity. Therefore, FM04 can be developed into a novel combination chemotherapy to treat cancer by directly targeting the P-gp overexpressed tumors or potentiating the oral bioavailability of P-gp substrate drugs.

A multi-objective optimization method for identification of module biomarkers for disease diagnosis.

Biomarker identification aims at finding a set of biological indicators that best discriminate biological samples of different phenotypes. In this paper, we take the module containing the significant disease-related genes and their interactions from biological networks as a module biomarker, and propose an evolutionary multi-objective optimization method to identify module biomarkers for disease diagnosis. To be specific, we take the classification accuracy on control and disease samples, the association with disease and the intra-link density in the module as the optimization objectives. To achieve the best performance, a novel population initiation strategy is tailored to generate dense-connected initial solutions, and a specific population update strategy is employed to direct the evolution towards the global optimums with abundant diversity. Experimental results show that our method outperforms the previous state-of-the-art disease diagnosis methods. Meantime, the detected biomarker module can reflect the basic and significant biological functions and has a great correlation with a disease phenotype.

Disruption of SND1-MTDH Interaction by a High Affinity Peptide Results in SND1 Degradation and Cytotoxicity to Breast Cancer Cells In Vitro and In Vivo.

Staphylococcal nuclease domain-containing protein 1 (SND1) is a multifunctional oncoprotein overexpressed in breast cancer. Binding of metadherin (MTDH) to SND1 results in the stabilization of SND1 and is important in the initiation and progression of breast cancer. Disruption of such interaction is a potential therapeutic for breast cancer. SN1/2 domain of SND1 was used as bait in a phage display screening to identify a 12-amino acid peptide 4-2. The activity of peptide 4-2 was evaluated by ELISA, coimmunoprecipitation, MTS, Western blot analysis, and xenograft mouse model. Peptide 4-2 could disrupt SND1-MTDH interaction. Cell penetrating derivative of peptide 4-2 (CPP-4-2) could penetrate and kill breast cancer cells by disrupting SND1-MTDH interaction and degrading SND1. Tryptophan 10 (W10) of peptide 4-2 was essential in mediating cytotoxicity, SND1 interaction, SND1-MTDH disruption, and SND1 degradation. CPP-4-2 could inhibit the growth of breast cancer in a xenograft mouse model. The SND1-interacting peptide 4-2 could kill breast cancer cells both in vitro and in vivo by interacting with SND1, disrupting SND1-MTDH interaction, and inducing SND1 degradation. W10 was an essential amino acid in the activity of peptide 4-2.

SALL4 promotes tumor progression in breast cancer by targeting EMT.

Sal-like protein 4 (SALL4) is overexpressed in breast cancer and might contribute to breast cancer progression, but the molecular mechanism remains unknown. Here, we found that within a group of 371 ethnic Chinese breast cancer patients, SALL4 was associated with lower grade (P = .002) and progesterone receptor positivity (P = .004) for overall cases; lower Ki67 (P = .045) and high vimentin (P = .007) for luminal cases. Patients with high SALL4 expression in lymph node metastasis showed a significantly worse survival than those with low expression. Knockout of SALL4 in a triple-negative breast cancer cell line MDA-MB-231-Red-FLuc-GFP led to suppressed ability in proliferation, clonogenic formation, migration, and mammosphere formation in vitro, tumorigenicity and lung colonization in vivo. On the other hand, overexpression of SALL4 enhanced migration and mammosphere formation in vitro and tumorigenicity in vivo. Mechanistically, there was a positive correlation between SALL4 expression and mesenchymal markers including Zinc finger E-box binding homeobox 1 (ZEB1), vimentin, Slug, and Snail in vivo. Chromatin immunoprecipitation experiment indicated that SALL4 can bind to the promoter region of vimentin (-778 to -550 bp). Taken together, we hypothesize that SALL4 promotes tumor progression in breast cancer by inducing the mesenchymal markers like vimentin through directly binding to its promoter. Increased SALL4 level in metastatic lymph node relative to the primary site is an important poor survival marker in breast cancer.

LC-MS/MS determination and comparative pharmacokinetics of strychnine, brucine and their metabolites in rat plasma after intragastric administration of each monomer and the total alkaloids from Semen Strychni.

A rapid, specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma. Plasma samples were pretreated via simple protein precipitation with methanol and ephedrine hydrochloride was used as internal standard. Chromatographic separation was carried out on an ZORBAX Eclipse XDB-C18 column (2.1×150mm, 3.5µm) by gradient elution with methanol and 10mM ammonium acetate (adjusted to pH 4.0 with formic acid). The quantification of the analytes was performed by mass spectrometry with TurboIonSpray ionization (ESI) inlet in the positive ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 0.510∼306.3ngmL(-1) for strychnine, brucine and 0.102∼306.0ngmL(-1) for strychnine N-oxide and brucine N-oxide, respectively. The intra- and inter-day precisions were less than 14.9%, and the accuracy ranged from 89.4 to 113% at three QC levels for the 4 analytes. The validated method was successfully applied to the pharmacokinetic study of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma after oral administration of each monomer and the total alkaloids from Semen Strychni. After single oral administration of the total alkaloids from Semen Strychni at 4 dose levels, Cmax, AUC0-t of strychnine and brucine increased and were proportional to the oral doses. In comparative pharmacokinetics studies, no significant difference was found between each monomer and the total strychnos alkaloids on the pharmacokinetic parameters such as Cmax and AUC. Mean Cmax and AUC of strychnine and brucine were slight increased in the monomer groups in comparison to the total strychnos alkaloids groups, which suggested that some other alkaloids in the Semen Strychni may decrease the absorption of strychnine and brucine in body.

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts.

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity have been evaluated. IC50 of nobiletin and hesperidin were 1.49 mM and 16.08 mM, respectively and their inhibition mechanisms are competitive type with inhibition constant (Ki) 2.82 mM and noncompetitive type with Ki 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC50 values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition of melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.48% at 31.25 microg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE gave efficacious antiproliferation effects on the B16 mouse melanoma cell with IC50 values 88.6 microM and 62.96 microg/mL, respectively, through the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.

Tyrosinase inhibitory effects and inhibition mechanisms of nobiletin and hesperidin from citrus peel crude extracts.

The inhibitory effects of nobiletin and hesperidin from citrus peel crude extracts on tyrosinase diphenolase activity are evaluated. IC50 of nobiletin and hesperidin is 1.49 mM and 16.08mM, respectively and their inhibition mechanism is competitive type with Ki = 2.82 mM and noncompetitive with Ki = 9.16 mM, respectively. Crude extracts from citrus peel (C. unshiu Marc.) were extracted with 95% ethanol and fractionated by petroleum ether (PCPE). The ethanol phase (ECPE) was further desorbed from macroporous adsorption resin (FGRE). Their IC50 values were 8.09 mg/mL, 7.53 mg/mL and 4.80 mg/mL, respectively. Their inhibition on melanogenesis in B16 mouse melanoma cells was also evaluated. FGRE showed a significant inhibition (42.5% at 31.25 microg/mL, p < 0.01) while hesperidin showed almost no inhibition. Nobiletin and PCPE give efficacious antiproliferation effects on B16 mouse melanoma cell with IC50 values 88.6 microM and 62.96 microg/mL, respectively, by the MTT test. Hesperidin and other crude extracts showed very low cytotoxity to the B16 cell.