Intestinal Epithelial Co-Culture Sensitivity to Pro-Inflammatory Stimuli and Polyphenols Is Medium-Independent.

The complexification of in vitro models requires the compatibility of cells with the same medium. Since immune cells are the most sensitive to growth conditions, growing intestinal epithelial cells in their usual medium seems to be necessary. This work was aimed at comparing the sensitivity of these epithelial cells to pro-inflammatory stimuli but also to dietary polyphenols in both DMEM and RPMI-1640 media. Co-cultures of Caco-2 and HT29-MTX cells were grown for 21 days in the two media before their stimulation with a cocktail of TNF-α (20 ng/mL), IL-1ß (1 ng/mL), and IFN-γ (10 ng/mL) or with LPS (10 ng/mL) from E. coli (O111:B4). The role of catechins (15 µM), a dietary polyphenol, was evaluated after its incubation with the cells before their stimulation for 6 h. The RPMI-1640 medium did not alter the intensity of the inflammatory response observed with the cytokines. By contrast, LPS failed to stimulate the co-culture in inserts regardless of the medium used. Lastly, catechins were unable to prevent the pro-inflammatory response observed with the cytokines in the two media. The preservation of the response of this model of intestinal epithelium in RPMI-1640 medium is promising when considering its complexification to evaluate the complex cellular crosstalk leading to intestinal homeostasis.

Induction of cell death and modulation of Annexin A1 by phytoestrogens in human leukemic cell lines.

BACKGROUND: Phytoestrogens are polyphenolic plant compounds which are structurally similar to the endogenous mammalian estrogen, 17ß-estradiol. Annexin A1 (ANXA1) is an endogenous protein which inhibits cyclo-oxygenase 2 (COX-2) and phospholipase A2, signal transduction, DNA replication, cell transformation, and mediation of apoptosis. OBJECTIVE: This study aimed to determine the effects of selected phytoestrogens on annexin A1 (ANXA1) expression, mode of cell death and cell cycle arrest in different human leukemic cell lines. METHODS: Cells viability were examined by MTT assay and ANXA1 quantification via Enzyme-linked Immunosorbent Assay. Cell cycle and apoptosis were examined by flow cytometer and phagocytosis effect was evaluated using haematoxylin-eosin staining. RESULTS: Coumestrol significantly (p < 0.05) reduced the total level of ANXA1 in both K562 and U937 cells and genistein significantly (p < 0.05) reduced it in K562, Jurkat and U937 cells, meanwhile estradiol and daidzein induced similar reduction in U937 and Jurkat cells. Coumestrol and daidzein induced apoptosis in K562 and Jurkat cells, while genistein and estradiol induced apoptosis in all tested cells. Coumestrol and estradiol induced cell cycle arrest at G2/M phase in K562 and Jurkat cells with an addition of U937 cells for estradiol. Genistein induced cell cycle arrest at S phase for both K562 and Jurkat cells. However, daidzein induced cell cycle arrest at G0/G1 phase in K562, and G2/M phase of Jurkat cells. Coumestrol, genistein and estradiol induced phagocytosis in all tested cells but daidzein induced significant (p < 0.05) phagocytosis in K562 and Jurkat cells only. CONCLUSION: The selected phytoestrogens induced cell cycle arrest, apoptosis and phagocytosis and at the same time they reduced ANXA1 level in the tested cells. The IC50 value of phytoestrogens was undetectable at the concentrations tested, their ability to induce leukemic cells death may be related with their ability to reduce the levels of ANXA1. These findings can be used as a new approach in cancer treatment particularly in leukemia.

Suppressive effect of ethanol extract from mango (Mangifera indica L.) peel on IgE production in vitro and in vivo.

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

Killing Activity of Micafungin Against Candida albicans, C. dubliniensis and Candida africana in the Presence of Human Serum.

We compared killing activity of micafungin in time-kill experiments in RPMI-1640 with and without 50% serum against Candida albicans, Candida dubliniensis and Candida africana reference strains and clinical isolates. Killing rates (k values) were determined for each strain and concentration. In RPMI-1640 MIC ranges were 0.015-0.03, 0.015-0.03 and 0.015 mg/L against C. albicans, C. dubliniensis and C. africana, respectively. In 50% serum MIC values for the three species increased 16- to 64-fold. In RPMI-1640 micafungin was fungicidal against two of three C. albicans isolates at 16 and 32 mg/L within 14.54 h and fungistatic against all C. africana and C. dubliniensis. Fifty per cent serum significantly decreased the growth rate of C. africana, but not of the other two species; weak in vivo replication ability of C. africana was confirmed in murine model. In 50% serum micafungin at 0.25 and 1 mg/L did not inhibit any of the three species (k values were always negative). Micafungin killing rate in 50% serum at 4, 16 and 32 mg/L was significantly decreased for C. albicans, but increased for C. dubliniensis compared to RPMI-1640. Killing activity of micafungin against C. africana was comparable or higher in 50% serum than in RPMI-1640. Although micafungin is a highly protein-bound drug, it was equally effective against the species of the C. albicans complex in 50% serum at therapeutic trough concentration (4 mg/L). Both in vitro and in vivo data confirmed the low virulence of C. africana compared to the two sibling species.

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer.

BACKGROUND: Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling. METHODS: Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots. RESULTS: Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity. CONCLUSION: In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies. GENERAL SIGNIFICANCE: Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.

HPLC method for the determination of thymoquinone in growth cell medium.

BACKGROUND: Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. OBJECTIVE: This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. METHODS: The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. RESULTS: The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 µM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. CONCLUSION: The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

Increased susceptibility to azithromycin of Pseudomonas aeruginosa biofilms using RPMI 1640 testing media.

Azithromycin (AZM) is efficient for treatment of chronic Pseudomonas aeruginosa biofilm lung infections, despite of resistance in conventional susceptibility testing. It has been shown that planktonic P. aeruginosa are more susceptible to AZM when tested in RPMI 1640 medium. The aim of the study was to test the susceptibility to AZM of P. aeruginosa biofilms in LB vs RPMI 1640 media. We investigated the effect of AZM on planktonic and biofilms of (WT) P. aeruginosa (PAO1), the hypermutable (ΔmutS) and the antibiotic-resistant phenotype(ΔnfxB) mutants. The effect of AZM on young and mature biofilms was investigated in the modified Calgary Biofilm Device by estimation of the minimal biofilm inhibitory concentration (MBIC). The AZM MBIC90 in LB/RPMI1640 on young biofilms treated for 24 h was 16/4 µg/mL for PAO1, 32/8 µg/mL for ΔmutS, and 256/16 µg/mL for ΔnfxB, while in mature biofilms was 256/2 µg/mL for PAO1 and ΔmutS and 16/1 µg/mL for ΔnfxB. The effect of AZM was improved when the treatment was prolonged to 72 h, supporting the intracellular accumulation of AZM. An increased susceptibility of P. aeruginosa biofilms to AZM was observed in RPMI 1640 than in LB medium. Our results might improve susceptibility testing and dosing of AZM for treatment of biofilm infections.

Micronutrient availability alters Candida albicans growth and farnesol accumulation: implications for studies using RPMI-1640.

Science is challenging because we do not know what we do not know. Commercial chemicals are often marketed with >99% purity, but 0.5-1% impurity can impact results and cloud data interpretation. We recently developed an assay for farnesol and aromatic fusel alcohols from Candida albicans. During proof-of-concept experiments using RPMI-1640 growth media, the buffering compound was switched from MOPS obtained from Acros Organics to MOPS obtained from Sigma-Aldrich, both labeled 99% + purity. We observed a twofold decrease in growth, along with a three- to fivefold increase in farnesol production per cell upon the switch. ICP-MS showed that trace Mn(II) was present in Acros MOPS but absent in Sigma MOPS. Optimal growth was achieved by the addition of Mn(II), Zn(II), and Fe(II). We established upper and lower limits for Fe(II), Zn(II), Cu(II), and Mn(II) that allowed similar growth and then assessed 16 different mineral combinations in RPMI-1640 base media. The results show an increased production of farnesol and the aromatic fusel alcohols when Zn(II) is abundant, and a further increase in the aromatic fusel alcohols when both Fe(II) and Zn(II) are abundant. Finally, antifungal susceptibility testing displayed no significant difference between RPMI/MOPS with and without mineral supplementation. Supplemental Mn(II) was most needed for cell growth, while supplemental Zn(II) was most needed for the production of farnesol and the aromatic fusel alcohols. To avoid these artifacts due to metal contamination, we now use a modified RPMI supplemented with 1 mg/ L of Cu(II), Zn(II), Mn(II), and Fe(II). IMPORTANCE: The dimorphic fungus Candida albicans is a major opportunistic pathogen of humans. RPMI-1640 is a chemically defined growth medium commonly used with C. albicans. We identified over 32,000 publications with keywords RPMI and C. albicans. Additionally, Antifungal Susceptibility Testing (AFST) protocols in the United States (CLSI) and Europe (EUCAST) utilize RPMI as a base media to assess drug efficacy against clinical fungal isolates. RPMI contains many nutrients but no added trace metals. We found that the growth characteristics with RPMI were dependent on which MOPS buffer was chosen and the contamination of that buffer by trace levels of Mn(II) and Zn(II). Added Mn(II) was most needed for cell growth while added Zn(II) was most needed for secretion of farnesol and other signaling molecules.

Characteristics of Aqueous Chemical Species Generation in Plasma-Facing Liquid Systems Using Helium Jet Plasma.

Plasma-facing liquids (PFLs) facilitate the storage of reactive O and N species (RONS), including H2O2 and NO2 -, which remain in the PFL after plasma treatment, and they can continuously influence the target immersed in the liquid. However, their behaviors and levels of generation and extinction depend strongly on the plasma characteristics and liquid condition. Therefore, understanding the effects of the liquid type on the plasma discharge characteristics and the RONS generated via plasma discharge is necessary. We compared the RONS generation and storage trends of deionized H2O and a high-conductivity PFL, RPMI 1640, which is a well-known cell culture medium commonly used to culture mammalian cells. RPMI 1640 acted as an electrode and enhanced the plasma discharge power by supplying abundant radicals and RONS. The production of gaseous hydroxyl radicals and NO markedly increased, which facilitated H2O2 and NO2 - production in the PFL for the first 200 s, and then the increase in the RONS concentration stagnated. With respect to storage, as the components within RMPI 1640 exhibited high reaction constants for their reactions with H2O2, H2O2 elimination was completed in <30 min. Unlike H2O2, the concentration of NO2 - in the PFL was unchanged.

The microenvironment in antibiotic susceptibility testing.

Antibiotic susceptibility testing (AST) by agar diffusion has been repeatedly standardized and, in most cases, gives results which predict clinical success when antibiotic treatment is based on such results. The formation of the inhibition zone is due to a transition from planktonic to biofilm mode of growth. The kinetics of the interaction of antibiotics with bacteria is similar during AST by agar diffusion and during administration of antibiotics to the patients. However, the Mueller-Hinton agar (MHA) recommended for AST agar diffusion test is fundamentally different from the composition of the interstitial fluid in the human body where the infections take place and human cells do not thrive in MH media. Use of RPMI 1640 medium designed for growth of eucaryotic cells for AST of Pseudomonas aeruginosa against azithromycin results in lower minimal inhibitory concentration, compared to results obtained by MHA. The reason is that the RPMI 1640 medium increases uptake and reduces efflux of azithromycin compared to MHA. During treatment of cystic fibrosis patients with azithromycin, mutational resistance occur which is not detected by AST with MHA. Whether this is the case with other antibiotics and bacteria is not known but it is of clinical importance to be studied.

Identification and characterisation of ROS modulator 1 in Lampetra japonica.

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive molecules that oxidise targets in biological systems. ROS are also considered important immune regulators. In this study, we identified a homologue of reactive oxygen species modulator 1 (Romo1) in the Japanese lamprey (Lampetra japonica). The L japonica Romo1 (Lj-Romo1) gene shares high sequence homology with the Romo1 genes of jawed vertebrates. Real-time quantitative PCR demonstrated the wide distribution of Lj-Romo1 in lamprey tissues. Furthermore, after the lampreys were stimulated with lipopolysaccharide (LPS), the level of Lj-Romo1 mRNA was markedly up-regulated in the liver, gill, kidney, and intestine tissues. Lj-Romo1 was localised to the mitochondria and has the capacity to increase the ROS level in cells. The results obtained in the present study will help us to understand the roles of Romo1 in ROS production and innate immune responses in jawless vertebrates.

The inhibitory effect of photodynamic therapy on dual-species biofilms of Candida albicans and Candida krusei can be determined by Candida albicans/Candida krusei ratio.

Candida krusei and Candida albicans present the ability to form communities of microorganisms called biofilms. Biofilms can be composed of a single species or more and are an important virulence factor. The inhibition of C. albicans and C. krusei as well as of their dual-species biofilms by antimicrobial Photodynamic Therapy (aPDT) has been demonstrated. This study aimed to investigate the effect of aPDT, with TBO, on dual-species biofilms of C. albicans and C. krusei using different culture mediums, RPMI-1640 and Sabouraud-dextrose broth (SDB) to produce biofilms presenting different C. albicans/C. krusei ratio. Biofilms formed using RPMI-1640 presented a higher C. albicans/C. krusei ratio, however, biofilms formed using SDB presented a predominance of C. krusei. The metabolic activity of biofilms produced using RPMI-1640 was inhibited by aP (∼40%), while biofilms produced using SDB were not affected by aPDT. In addition, biofilm biomass was reduced in biofilms produced using RPMI-1640 and treated with aPDT (∼20%). The results demonstrated that aPDT reduces C. albicans development in dual-species biofilms with C. krusei. However, no effect could be observed on C. krusei, demonstrating that C. krusei, when present in the structure of dual-species biofilms can be resistant to aPDT.

The Influence of Medium Composition on EUCAST and Etest Antifungal Susceptibility Testing.

There is an ongoing effort to optimize and revise antifungal susceptibility testing (AFST) methods due to the rising number of fungal infections and drug-resistant fungi. The rising antifungal resistance within Candida and Aspergillus species, which are common contributors to invasive fungal infections (IFIs), is a cause for concern, prompting an expanding integration of in vitro AFST to guide clinical decisions. To improve the relevance of in vitro AFST results to therapy outcomes, influential factors should be taken into account. The tested medium is one of several factors that could affect the results of AFST. The present study evaluated the effect of two complex media (Sabouraud dextrose and Columbia) versus the standard defined medium (RPMI 1640) on the AFST results of amphotericin B, posaconazole, and voriconazole against Candida spp. and Aspergillus spp. representatives, utilizing the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest methods. Overall, Candida species exhibited higher variability in minimum inhibitory concentration (MIC) across different media (more than three log2 dilutions) comparing to Aspergillus spp., while quality control isolates showed consistency regardless of tested media, antifungals, and methods. When comparing tested methods, MIC variation was mostly detected using EUCAST than it was using Etest.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.

Modifications of antifungal sensibility testing as suggested by CLSI document M27-A4: proposal for using different culture medium and buffer.

A common strategy in antifungal susceptibility testing is the utilization of the standardized protocol based on the microbroth dilution assay approach as described by the Clinical Laboratory Standards Institute (CLSI) (M27-A4). One major problem for laboratories in resource-limited countries with this protocol arises from the use of expensive culture media like RPMI-1640 and 3-N-morpholinopropanesulfonic acid (MOPS) buffer. One approach of circumventing this problem in cases of economic need is the evaluation of alternative culture media and buffers. The overall goal of this work was to investigate the influence of modifications in the protocol M27-A4 on diagnostic reliability. We performed univariate analyses evaluating (1) 2 different culture media (YNB and modified SAB); (2) three different buffers (sodium bicarbonate, Tris-HCL, and phosphate), as well as the influence of inoculum concentration (102, 103, 104, 105 cells/mL), the influence of incubation time, and the influence of the assessment mode (visual, biological dye, and spectrophotometer). Our results suggested that (1) RPMI-1640 may be substituted by modified SAB and (2) MOPS buffer may be substituted by Tris-HCl buffer for defined analyses. By comparing the CLSI protocol and the alternative protocol proposed in the present study (modified SAB and Tris-HCl buffer) for the assessment of fluconazole susceptibility of eighteen yeasts (clinical isolates), similar results with both methodologies were recorded. We feel that this study should stimulate a discussion on the feasibility and evolution of the M27-A4 protocol in order to include pragmatic alternatives for resource-limited settings.

Functional metabolomics reveal the role of AHR/GPR35 mediated kynurenic acid gradient sensing in chemotherapy-induced intestinal damage.

Intestinal toxicity induced by chemotherapeutics has become an important reason for the interruption of therapy and withdrawal of approved agents. In this study, we demonstrated that chemotherapeutics-induced intestinal damage were commonly characterized by the sharp upregulation of tryptophan (Trp)-kynurenine (KYN)-kynurenic acid (KA) axis metabolism. Mechanistically, chemotherapy-induced intestinal damage triggered the formation of an interleukin-6 (IL-6)-indoleamine 2,3-dioxygenase 1 (IDO1)-aryl hydrocarbon receptor (AHR) positive feedback loop, which accelerated kynurenine pathway metabolism in gut. Besides, AHR and G protein-coupled receptor 35 (GPR35) negative feedback regulates intestinal damage and inflammation to maintain intestinal integrity and homeostasis through gradually sensing kynurenic acid level in gut and macrophage, respectively. Moreover, based on virtual screening and biological verification, vardenafil and linagliptin as GPR35 and AHR agonists respectively were discovered from 2388 approved drugs. Importantly, the results that vardenafil and linagliptin significantly alleviated chemotherapy-induced intestinal toxicity in vivo suggests that chemotherapeutics combined with the two could be a promising therapeutic strategy for cancer patients in clinic. This work highlights GPR35 and AHR as the guardian of kynurenine pathway metabolism and core component of defense responses against intestinal damage.

Identification of a novel PAK1 inhibitor to treat pancreatic cancer.

Pancreatic cancer is one of the most aggressive cancers with poor prognosis and a low 5-year survival rate. The family of P21-activated kinases (PAKs) appears to modulate many signaling pathways that contribute to pancreatic carcinogenesis. In this work, we demonstrated that PAK1 is a critical regulator in pancreatic cancer cell growth. PAK1-targeted inhibition is therefore a new potential therapeutic strategy for pancreatic cancer. Our small molecule screening identified a relatively specific PAK1-targeted inhibitor, CP734. Pharmacological and biochemical studies indicated that CP734 targets residue V342 of PAK1 to inhibit its ATPase activity. Further in vitro and in vivo studies elucidated that CP734 suppresses pancreatic tumor growth through depleting PAK1 kinase activity and its downstream signaling pathways. Little toxicity of CP734 was observed in murine models. Combined with gemcitabine or 5-fluorouracil, CP734 also showed synergistic effects on the anti-proliferation of pancreatic cancer cells. All these favorable results indicated that CP734 is a new potential therapeutic candidate for pancreatic cancer.

Culture of Malignant Pleural Mesothelioma Cells and the Effects of CDKN2B on Cancer Cell / 昆明医科大学学报

Objective To investigate the effects of different culture conditions(RPMI-1640,DMEM and DMEM/F12 medium)on the passage of MPM cells isolated from the tissues of Malignant pleural mesothelioma(MPM),and to study the effects of CDKN2B on the proliferation,invasion and apoptosis of MPM cells.Methods MPM cells were isolated from MPM tissues and cultured in RPMI-1640,DMEM and DMEM/F12 medium,respectively.Cell proliferation was examined by CCK-8,and the nuclei and chromosomes were observed by Wright-Giemsa staining.Fluorescence intensities of Calretinin,CD141,CK5,EMA and WT-1 were conducted by immunofluorescence assay.The mRNA and protein expression of CDKN2B were detected by RT-qPCR and Western blot,respectively.Transwell was used to detect cell invasion and flow cytometry was used to detect cell apoptosis.Results The established MPM cells showed good viability when passaged to the 10th generation in RPMI-1640,DMEM and DMEM/F12 cultures,and the MPM markers Calretinin,CD141,CK5,EMA and WT-1 were all expressed in the cells.The viability of MPM cells in RPMI-1640 culture medium was relatively stable.CDKN2B was downregulated in MPM cells(P<0.05),and overexpression of CDKN2B significantly suppressed the proliferation(P<0.05),invasion(P<0.05)and epithelial interstitial transformation of MPM cells(P<0.01),and promoted the apoptosis(P<0.01).Conclusion The established MPM cells were stably passaged in RPMI-1640 culture medium,and CDKN2B may be a potential target for the diagnosis and treatment of MPM.

Transformative hyaluronic acid-based active targeting supramolecular nanoplatform improves long circulation and enhances cellular uptake in cancer therapy.

Hyaluronic acid (HA) is a natural ligand of tumor-targeted drug delivery systems (DDS) due to the relevant CD44 receptor overexpressed on tumor cell membranes. However, other HA receptors (HARE and LYVE-1) are also overexpressing in the reticuloendothelial system (RES). Therefore, polyethylene glycol (PEG) modification of HA-based DDS is necessary to reduce RES capture. Unfortunately, pegylation remarkably inhibits tumor cellular uptake and endosomal escapement, significantly compromising the in vivo antitumor efficacy. Herein, we developed a Dox-loaded HA-based transformable supramolecular nanoplatform (Dox/HCVBP) to overcome this dilemma. Dox/HCVBP contains a tumor extracellular acidity-sensitive detachable PEG shell achieved by a benzoic imine linkage. The in vitro and in vivo investigations further demonstrated that Dox/HCVBP could be in a "stealth" state at blood stream for a long circulation time due to the buried HA ligands and the minimized nonspecific interaction by PEG shell. However, it could transform into a "recognition" state under the tumor acidic microenvironment for efficient tumor cellular uptake due to the direct exposure of active targeting ligand HA following PEG shell detachment. Such a transformative concept provides a promising strategy to resolve the dilemma of natural ligand-based DDS with conflicting two processes of tumor cellular uptake and in vivo nonspecific biodistribution.

Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. and spathulenol.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Psidium guineense Sw. are used in popular medicine for the treatment of inflammatory disease. However, there is no scientific evidence demonstrating this activity. AIM OF THE STUDY: To evaluate the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of P. guineense and spathulenol (a major constituent). The study was conducted in part to provide evidence supporting the ethnobotanical use of the leaves of this species. MATERIAL AND METHODS: The essential oil (EOPG) was extracted from the leaves of P. guineense by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major compound, spathulenol (PG-1), was isolated in a chromatographic column and characterized by nuclear magnetic resonance (NMR). EOPG and PG-1 were evaluated in vitro for antioxidant activity by DPPH, ABTS and MDA methods; anti-inflammatory potential was assessed using two models, including pleurisy and oedema, in mice. The impact of EOPG and PG-1 on cell proliferation was determined via spectrophotometric quantification of the cellular protein content using a sulforhodamine B assay, and anti-Mycobacterium tuberculosis activity was determined using the REMA method. RESULTS: A total of 38 components were identified from the EOPG, with the sesquiterpenic alcohol spathulenol (PG-1) (80.7%) being the major constituent. EOPG and PG-1 exhibited the highest antioxidant activities in the DPPH and MDA system compared with reference standard, with IC50 values ranging from 26.13 to 85.60µg/mL. Oral administration of EOPG and PG-1 showed significant inhibition in the Cg-induced mice paw oedema and pleurisy model. The EOPG (GI50 = 0.89µg/mL) and PG-1 (GI50 = 49.30µg/mL) were particularly effective against the ovarian cancer cell line. Both showed moderate antimycobacterial activity. CONCLUSION: For the first time, this study demonstrated the antioxidant, anti-inflammatory, antiproliferative and antimycobacterial properties of the essential oil of P. guineense (leaves were collected in Dourados-MS) and spathulenol, collaborating the etnhopharmacologycal use of this plant due to its an anti-inflammatory effect.