Viability Curves Research Articles

Effects of 6PPD-Quinone on Human Liver Cell Lines as Revealed with Cell Viability Assay and Metabolomics Analysis.

N-(1,3-Dimethyl butyl)-N'-phenyl-phenylenediamine-quinone (6PPD-Q) is a derivative of the widely used rubber tire antioxidant 6PPD, which was first found to be acutely toxic to coho salmon. Subsequent studies showed that 6PPD-Q had species-specific acute toxicity in fishes and potential hepatotoxicity in mice. In addition, 6PPD-Q has been reported in human urine, demonstrating the potential widespread exposure of humans to this chemical. However, whether 6PPD-Q poses a higher risk to humans than its parent compound, 6PPD, and could cause adverse effects in humans is still unclear. In this study, we utilized two human liver cell models (the human proto-hepatocyte model L02 and the human hepatocellular carcinoma cell line HepG2) to investigate the potentially differential effects of these two chemicals. Cell viability curve analysis showed that 6PPD-Q had lower IC50 values than 6PPD for both liver cell lines, suggesting higher toxicity of 6PPD-Q to human liver cells than 6PPD. In addition, L02 cells are more sensitive to 6PPD-Q exposure, which might be derived from its weaker metabolic transformation of 6PPD-Q, since significantly lower levels of phase I and phase II metabolites were detected in 6PPD-Q-exposed L02 cell culture medium. Furthermore, pathway analysis showed that 6PPD-Q exposure induced changes in phenylalanine, tyrosine, and tryptophan biosynthesis and tyrosine metabolism pathways in L02 cells, which might be the mechanism underlying its liver cell toxicity. Gene expression analysis revealed that exposure to 6PPD-Q induced excessive ROS production in L02 cells. Our results further supported the higher risk of 6PPD-Q than 6PPD and provided insights for understanding the effects of 6PPD-Q on human health.

Daptomycin Liposomes Exhibit Enhanced Activity against Staphylococci Biofilms Compared to Free Drug.

The purpose of the present study was to investigate the anti-staphylococcal activity of liposomal daptomycin against four biofilm-producing S. aureus and S. epidermidis clinical strains, three of which are methicillin-resistant. Neutral and negatively charged daptomycin-loaded liposomes were prepared using three methods, namely, thin-film hydration (TFH), a dehydration-rehydration vesicle (DRV) method, and microfluidic mixing (MM); moreover, they were characterized for drug encapsulation (EE%), size distribution, zeta-potential, vesicle stability, drug release, and drug integrity. Interestingly, whilst drug loading in THF and DRV nanosized (by extrusion) vesicles was around 30-35, very low loading (~4%) was possible in MM vesicles, requiring further explanatory investigations. Liposomal encapsulation protected daptomycin from degradation and preserved its bioactivity. Biofilm mass (crystal violet, CV), biofilm viability (MTT), and growth curve (GC) assays evaluated the antimicrobial activity of neutral and negatively charged daptomycin-liposomes towards planktonic bacteria and biofilms. Neutral liposomes exhibited dramatically enhanced inhibition of bacterial growth (compared to the free drug) for all species studied, while negatively charged liposomes were totally inactive. Biofilm prevention and treatment studies revealed high antibiofilm activity of liposomal daptomycin. Neutral liposomes were more active for prevention and negative charge ones for treating established biofilms. Planktonic bacteria as well as the matured biofilms of low daptomycin-susceptible, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) strains were almost completely eradicated by liposomal-daptomycin, indicating the need for their further exploration as antimicrobial therapeutics.

PTTG1 induces pancreatic cancer cell proliferation and promotes aerobic glycolysis by regulating c-myc.

This study aimed to clarify the role of pituitary tumor-transforming gene 1 (PTTG1) in proliferation, migration, invasion, and aerobic glycolysis of pancreatic cancer cells, and evaluate the potential of PTTG1 as a therapeutic target. PTTG1 expression in pancreatic cancers was analyzed using the GEPIA databank. In the Panc1 cell with the PTTG1 knockdown or Mia-PaCa2 cells with PTTG1 overexpression, the cell proliferation was evaluated using cell viability curves and colony formation, and wound heal assay and transwell assay were performed to evaluate the migration and invasion, respectively. Furthermore, a western blot was performed to evaluate the expressions of PTTG1, proliferating cell nuclear antigen, E-cadherin, N-cadherin, and c-myc. Meanwhile, the glucose uptake, extracellular acidification rates (ECAR), and oxygen consumption rates (OCR) were analyzed. Our results showed that PTTG1 expression is upregulated in pancreatic cancer, which promoted cell proliferation. Low PTTG1 contributed to higher disease-free survival and overall survival. In Panc1 cell, PTTG1 knockdown resulted in reduced cell viability and colony formation. The migration and invasion abilities of the cells were also reduced in Panc1 with PTTG1 knockdown. Correspondingly, PTTG1 knockdown decreased c-myc expression, glucose uptake, ECAR, and OCR in Panc1 cells. In Mia-PaCa2 cells, PTTG1 overexpression promoted cell proliferation, aerobic glycolysis, and translocation of β-catenin to the nucleus by regulating c-myc. In conclusion, PTTG1 induces proliferation, migration, and invasion, and promotes aerobic glycolysis in pancreatic cancer cells via regulating c-myc, demonstrating the potential of PTTG1 as a therapeutic target.

Prognostic Utility of the Patient-Derived AML Cells' Ex Vivo Drug Sensitivity Results

Genomic mutational profiles substantially influence selection of the treatment as they impact drug responses. However, the expected response to a specific treatment based on the data from a large group of the patients with the similar mutational profile is not always equal to the actual response observed at an individual basis. In this study, we measured ex vivo sensitivity of the patient-derived AML cells to 21 different anti-cancer drugs and assessed their prognostic utility. The ex vivo drug sensitivity analysis was performed using the bone marrow (BM) aspirates of 45 out of 58 AML patients (78%), which contained sufficient number of live cancer cells. The isolated blast cells were incubated for 72 hours with various concentrations of the 21 drugs used to treat hematologic malignancies, and changes in viability were measured using the Alamar Blue assay. For each drug, we calculated the individual patient's drug sensitivity in terms of the three metrics-IC 50 (the drug concentration to inhibit cell population by 50%), area under the curve (AUC, area under the viability curve for a cell population over the tested drug concentration range), and E max (the fraction of viable cells at the highest drug concentrations tested). In the context of drug potency, smaller values of IC 50 AUC and E max correspond to high drug sensitivities. After sample acquisition, 14 out of 45 patients did not receive treatment while the remaining patients received the following treatments: Venetoclax plus hypomethylating agent (VEN+HMA) in either the newly diagnosed (ND) (n=9) or relapsed/refractory (R/R) setting (n=6), Anthracycline plus cytarabine(AraC) based induction (n=8) or reinduction (n=4), and others (n=4). Expectedly, the values of AUC, IC 50 and E max differed substantially across the cohort for all 21 drugs, in alignment with unequal responses and survival across the patients treated with the same regimen; the range of E max, for example, spanned 0 to 100% for 17 of the 21 drugs. We confirmed that the interpersonal differences in the drug sensitivity metrics were at least 8-fold greater than the variability observed across replicate experiments performed using the leukemia cell line HL60. Given the various disease setting and treatment modalities, we focused on the patients receiving the VEN+HMA (n=15) and Anthra + AraC based (re)induction (n=12) for further analysis. Upon evaluating the predictive utility of the drug sensitivity metrics, we found that the E max of VEN was the most effective in predicting survival after the administration of VEN+HMA, with an ROC-AUC of 0.84. The median E max of VEN was significantly higher (38% vs 17%) among the patients who died within 2 months of the first administration (n = 4) compared to those who survived (n = 11). When stratified into the high vs low risk subgroups based on the E max of VEN, overall survival of the low risk subgroup was significantly ( P=0.0025) superior with hazard ratio of 8.9 (95% CI, 0.7 - 108.9). On the other hand, the AUC of VEN exhibited the strongest utility in predicting the response (CR(i)) to VEN+HMA therapy, with an ROC-AUC of 0.72. While the E max or AUC values of the HMA drugs did not show any strong correlation to the treatment outcomes. We observed the similar tendency for other treatments including Anthracycline + AraC; E max of idarubicin (IDA) was significantly higher (21% vs 3+1%) for the patient who died (n = 1) within 100 days of the regimen administration than those who survived (n = 7). In contrast, E max of ARA was non-discriminant (21% vs 15+10%) although the values were in general much lower than those observed for the HMA drugs. ROC-AUC of survival and response prediction was 1.00 based on E max of IDA, however, a bigger cohort is needed to confirm the statistical significance of the observed utility. In conclusion, we report that our proprietary ex vivo drug sensitivity platform may have clinical utility in selecting the best-fitting therapy for AML patients in an individualized manner. Interestingly, different metrics representing the ex vivo drug sensitivity had disparate utility in prediction the clinical outcome. E max, which correlates to the proportion of the drug-resistant cancer cells, was particularly useful in discriminating overall survival in patients treated with VEN + HMA or Anthracycline + AraC regimens, while AUC showed promise in predicting response specifically in patients treated with VEN + HMA.

Enhancing the Antifungal Efficacy of Fluconazole with a Diterpene: Abietic Acid as a Promising Adjuvant to Combat Antifungal Resistance in Candida spp.

The increasing antifungal resistance rates against conventional drugs reveal the urgent need to search for new therapeutic alternatives. In this context, natural bioactive compounds have a critical role in antifungal drug development. Since evidence demonstrates that abietic acid, a diterpene found in Pinus species, has significant antimicrobial properties, this study aimed to evaluate the antifungal activity of abietic acid against Candida spp and its ability to potentiate the activity of fluconazole. Abietic acid was tested both individually and in combination with fluconazole against Candida albicans (CA INCQS 40006), Candida krusei (CK INCQS 40095), and Candida tropicalis (CT INCQS 40042). The microdilution method was used to determine the IC50 and the cell viability curve. Minimum Fungicidal Concentration (MFC) was determined by subculture in a solid medium. The plasma membrane permeability was measured using a fluorescent SYTOX Green probe. While the IC50 of the drugs alone ranged between 1065 and 3255 μg/mL, the IC50 resulting from the combination of abietic acid and fluconazole ranged between 7563 and 160.1 μg/mL. Whether used in combination with fluconazole or isolated, abietic acid exhibited Minimum Fungicidal Concentration (MFC) values exceeding 1024 μg/mL against Candida albicans, Candida krusei and Candida tropicalis. However, it was observed that the antifungal effect of fluconazole was enhanced when used in combination with abietic acid against Candida albicans and Candida tropicalis. These findings suggest that while abietic acid alone has limited inherent antifungal activity, it can enhance the effectiveness of fluconazole, thereby reducing antifungal resistance.

Dissecting the role of lactate metabolism LncRNAs in the progression and immune microenvironment of osteosarcoma

BackgroundThe process of lactate metabolism has been proved to play a critical role in the progression of various cancers and to influence the immune microenvironment, but its potential role in osteosarcoma remains unclear. MethodsWe have acquired transcriptomic and clinical data from 84 osteosarcoma samples and 70 normal bone samples from the TARGET and GTEx databases. We identified differentially expressed lactate metabolism-related LncRNAs (LRLs) in osteosarcoma and performed Cox regression and LASSO regression to establish LRLs prognostic signature (LRPS). The reliability of LRPS performance was examined by separate prognostic analysis, viability curves and receiver operating characteristic (ROC) curves. Furthermore, the effects of LRPS on the immune microenvironment of osteosarcoma were investigated, and the functions of the focal genes were experimentally validated. ResultA total of 856 differentially expressed LRLs were identified and 5 of them were selected to construct LRPS, which was a better prognostic predictor for osteosarcoma compared with other published prognostic signatures (AUC up to 0.947 and 0.839 in the training and test groups, respectively, with adj-p<0.05 for KM curves). We found that LRPS significantly affected the immune infiltration of osteosarcoma, while RP11-472M19.2 significantly promoted the metastasis of osteosarcoma, which was well validated experimentally. Encouragingly, a number of sensitive drugs were identified for LRPS and RP11-472M19.2 high-risk groups. ConclusionOur study shows that lactate metabolism plays a crucial role in the development of osteosarcoma and has been well validated experimentally, providing extremely important insights into the clinical treatment and in-depth research of osteosarcoma.

Formononetin acts synergistically with a JAK2 inhibitor to suppress growth in myeloproliferative neoplasm by inhibiting JAK/STAT3 signaling pathway

Purpose: To determine the effect of formononetin (FMNT) in the proliferation, drug resistance, and DNA damage of myeloproliferative neoplasm (MPN), and to evaluate the potential of FMNT as a therapeutic target.&#x0D; Methods: Cell viability curves and colony formation assays were used to characterize the proliferation of HEL or HEL/R cells. Flow cytometry was conducted to assess the apoptosis of HEL or HEL/R cells, while western blotting was performed to evaluate the expressions of DNA H2AX, DNA-PK, and Rad51, which are indicative of DNA damage, and the phosphorylation levels of JAK2 and STAT3 were determined.&#x0D; Results: Formononetin (FMNT) suppressed cell proliferation of HEL cells in a dose-dependent manner. It significantly reduced colony formation and promoted apoptosis of HEL cells (p &lt; 0.05). For HEL/R cells, FMNT treatment significantly reduced cell viability, and resistance, and promoted apoptosis. Moreover, FMNT elevated H2AX levels and significantly reduced the expressions of DNA-PK and Rad51 in TG101209 (TG)-induced JAK-TKI-resistant cells (p &lt; 0.05). Furthermore, FMNT decreased JAK2 and STAT3 phosphorylation in JAK- TKI-resistant cells.&#x0D; Conclusion: Treatment with FMNT represses proliferation, promotes apoptosis, weakens JAK-TKI resistance, and strengthens DNA damage in MPN cells by suppressing JAK/STAT3 pathway. This suggests that FMNT is a potential therapeutic candidate for the treatment of MPN.

Photon and Proton irradiation in Patient-derived, Three-Dimensional Soft Tissue Sarcoma Models

BackgroundDespite their heterogeneity, the current standard preoperative radiotherapy regimen for localized high-grade soft tissue sarcoma (STS) follows a one fits all approach for all STS subtypes. Sarcoma patient-derived three-dimensional cell culture models represent an innovative tool to overcome challenges in clinical research enabling reproducible subtype-specific research on STS. In this pilot study, we present our methodology and preliminary results using STS patient-derived 3D cell cultures that were exposed to different doses of photon and proton radiation. Our aim was: (i) to establish a reproducible method for irradiation of STS patient-derived 3D cell cultures and (ii) to explore the differences in tumor cell viability of two different STS subtypes exposed to increasing doses of photon and proton radiation at different time points.MethodsTwo patient-derived cell cultures of untreated localized high-grade STS (an undifferentiated pleomorphic sarcoma (UPS) and a pleomorphic liposarcoma (PLS)) were exposed to a single fraction of photon or proton irradiation using doses of 0 Gy (sham irradiation), 2 Gy, 4 Gy, 8 Gy and 16 Gy. Cell viability was measured and compared to sham irradiation at two different time points (four and eight days after irradiation).ResultsThe proportion of viable tumor cells four days after photon irradiation for UPS vs. PLS were significantly different with 85% vs. 65% (4 Gy), 80% vs. 50% (8 Gy) and 70% vs. 35% (16 Gy). Proton irradiation led to similar diverging viability curves between UPS vs. PLS four days after irradiation with 90% vs. 75% (4 Gy), 85% vs. 45% (8 Gy) and 80% vs. 35% (16 Gy). Photon and proton radiation displayed only minor differences in cell-killing properties within each cell culture (UPS and PLS). The cell-killing effect of radiation sustained at eight days after irradiation in both cell cultures.ConclusionsPronounced differences in radiosensitivity are evident among UPS and PLS 3D patient-derived sarcoma cell cultures which may reflect the clinical heterogeneity. Photon and proton radiation showed similar dose-dependent cell-killing effectiveness in both 3D cell cultures. Patient-derived 3D STS cell cultures may represent a valuable tool to enable translational studies towards individualized subtype-specific radiotherapy in patients with STS.

Apigenin-7-glucoside induces apoptosis and ROS accumulation in lung cancer cells, and inhibits PI3K/Akt/mTOR pathway

Purpose: To investigate how apigenin-7-glucoside (AGL) affects the proliferation, migration, invasion, and reactive oxygen species (ROS) accumulation in lung cancer cells, and to evaluate the potential of AGL as a therapeutic target.&#x0D; Methods: Human bronchial epithelial cells (BEAS-2B), human lung carcinoma (A549), and non-small cell lung cancer cells (H1975), were treated with 25, 50, 100, and 200 μM AGL. Cell viability curves and EdU assays were used to evaluate their proliferation, while apoptosis was assessed by flow cytometry. Cell migration and invasion were evaluated by Transwell assays, and western blot was performed to determine the expression level of cytochrome C and phosphorylation of PI3K, AKT, and mTOR. Furthermore, ELISA was used to quantify the level of malondialdehyde (MDA) and glutathione (GSH). Indirect immunofluorescent assay (IFA) was performed by staining with DCFH-DA to evaluate ROS level.&#x0D; Results: Proliferation of A549 and H1975 cells was suppressed by AGL in a dose-dependent manner. AGL significantly reduced proliferation, promoted cell apoptosis, and attenuated the migration and invasion of A549 or H1975 cells. It also elevated the levels of cytochrome C and MDA but reduced the production of GSH in A549 and H1975 cells. AGL enhanced the accumulation of ROS and weakened phosphorylation of AKT, PI3K, and mTOR in A549 and H1975 cells.&#x0D; Conclusion: AGL represses proliferation, promotes apoptosis, suppresses migration and invasion, and induces ROS accumulation in lung cancer cells by repressing PI3K/Akt/mTOR pathway, thus indicating the potential of AGL as a target in lung cancer treatment.

Inhibitory effects of flavonoids on glucose transporter 1 (GLUT1): From library screening to biological evaluation to structure-activity relationship

Glucose transporter 1 (GLUT1) is mainly responsible for glucose uptake and energy metabolism, especially in the aerobic glycolysis process of tumor cells, which is closely associated with the advancement of tumors. Numerous studies have demonstrated that the inhibition of GLUT1 can decrease the growth of tumor cells and enhance drug sensitivity, so GLUT1 is considered to be a promising therapeutic target for cancer treatment. Flavonoids are a group of phenolic secondary metabolites present in vegetables, fruits, and herbal products, some of which were reported to increase cancer cells' sensitivity to sorafenib by inhibiting GLUT1. Our objective was to screen potential inhibitors of GLUT1 from 98 flavonoids and assess the sensitizing effect of sorafenib on cancer cells. and illuminate the structure–activity relationships of flavonoids with GLUT1. Eight flavonoids, including apigenin, kaempferol, eupatilin, luteolin, hispidulin, isosinensetin, sinensetin, and nobiletin exhibited significant inhibition (>50%) on GLUT1 in GLUT1-HEK293T cells. Among them, sinensetin and nobiletin showed stronger sensitizing effects and caused a sharp downward shift of the cell viability curves in HepG2 cells, illustrating these two flavonoids might become sensitizers to enhance the efficacy of sorafenib by inhibiting GLUT1. Molecular docking analysis elucidated inhibitory effect of flavonoids on GLUT1 was related to conventional hydrogen bonds, but not Pi interactions. The pharmacophore model clarified the critical pharmacophores of flavonoids inhibitors are hydrophobic groups in 3’positions and hydrogen bond acceptors. Thus, our findings would provide useful information for optimizing flavonoid structure to design novel GLUT1 inhibitors and overcome drug resistance in cancer treatment.

Antifungal effect of the liposome encapsulation of the Trans- Caryophylene and its association with fluconazole

Trans-Caryophyllene (TC), a sesquiterpene, with proven biological activities, which in this work was tested alone, encapsulated in liposomes and associated with Fluconazole in vitro in an attempt to enhance the effect of the drug. Liposomes were characterized from vesicle size, polydispersity index, and Zeta potential, and imaging by scanning electron microscopy. Antifungal assays were performed against Candida albicans, Candida tropicalis and Candida krusei by microdilution to determine the IC50 values and the viability curve. The Minimum Fungicidal Concentration (MFC) was performed by subcultivation in solid medium and the inhibitory effect of the association of TC and Fluconazole and tests to verify morphological changes was performed in micro-cultivation chambers based on concentrations on microdilution plates. The corresponding IC50 data of the substances ranged from 34.4 to 65249 μg/mL, considerably high values compared to the control (Fluconazole). The MFC of all compounds showing fungistatic effect. The performance of the compounds on the cell viability curve was similar in all tested strains, as they showed no antifungal potential when compared to the control (FCZ), when associated with FCZ they showed no significant antifungal activity. The free and liposomal TC also managed to restrict 100% of the fungal dimorphism, in both concentrations, against C. albicans, and against C. tropicalis the isolated TC did not show a significant inhibitory effect; however, against the C. krusei strain inhibited 100% in filamentous growth in both concentrations, which is statistically relevant. The liposomes were homogeneous, with vesicles with diameters of 185.46 nm for the control and 143.8 nm for the liposomal TC, and a surface charge potential of - 42.6 mV. By scanning microscopy, the spherical shapes of the vesicles were verified.

An Insight into Symmetrical Cyanine Dyes as Promising Selective Antiproliferative Agents in Caco-2 Colorectal Cancer Cells.

Cancer remains one of the diseases with the highest worldwide incidence. Several cytotoxic approaches have been used over the years to overcome this public health threat, such as chemotherapy, radiotherapy, and photodynamic therapy (PDT). Cyanine dyes are a class of compounds that have been extensively studied as PDT sensitisers; nevertheless, their antiproliferative potential in the absence of a light source has been scarcely explored. Herein, the synthesis of eighteen symmetric mono-, tri-, and heptamethine cyanine dyes and their evaluation as potential anticancer agents is described. The influences of the heterocyclic nature, counterion, and methine chain length on the antiproliferative effects and selectivities were analysed, and relevant structure–activity relationship data were gathered. The impact of light on the cytotoxic activity of the most promising dye was also assessed and discussed. Most of the monomethine and trimethine cyanine dyes under study demonstrated a high antiproliferative effect on human tumour cell lines of colorectal (Caco-2), breast (MCF-7), and prostate (PC-3) cancer at the initial screening (10 µM). However, concentration–viability curves showed higher potency and selectivity for the Caco-2 cell line. A monomethine cyanine dye derived from benzoxazole was the most promising compound (IC50 for Caco-2 = 0.67 µM and a selectivity index of 20.9 for Caco-2 versus normal human dermal fibroblasts (NHDF)) and led to Caco-2 cell cycle arrest at the G0/G1 phase. Complementary in silico studies predicted good intestinal absorption and oral bioavailability for this cyanine dye.

Viability of Wildflower Seeds After Mesophilic Anaerobic Digestion in Lab-Scale Biogas Reactors.

The use of wildflower species as biogas feedstock carries the risk that their seeds survive anaerobic digestion (AD) and cause weed problems if spread with the digestate. Risk factors for seed survival in AD include low temperature, short exposure and hardseededness (HS). However, it is not possible to predict how AD will affect seed viability of previously unstudied species. In laboratory-scale reactors, we exposed seeds of eight species from a mixture of flowering wild plants intended as biogas feedstock and three reference species to AD at two mesophilic temperatures. Half of the species were HS, the other was non-HS (NHS). Viability was determined using a combination of tetrazolium and germination tests. Viability and germinability were modeled as functions of exposure time using a dose-response approach. Responses to AD varied considerably among species, and none of the considered influencing factors (time, temperature, HS) had a consistent effect. Seed lots of a species differed in inactivation times and seed-killing efficacy. The HS species Melilotus officinalis, Melilotus albus, and Malva sylvestris were particularly AD-resistant. They were the only ones that exhibited biphasic viability curves and tended to survive and germinate more at 42°C than at 35°C. Viability of the remaining species declined in a sigmoidal curve. Most NHS species were inactivated within a few days (Cichorium intybus, Daucus carota, Echium vulgare, and Verbascum thapsus), while HS species survived longer (Malva alcea). AD stimulated germination in the HS species A. theophrasti and its AD-resistance overlapped with that of the most resistant NHS species, C. album and tomato. In all seed lots, germinability was lost faster than viability, implying that mainly dormant seeds survived. After the maximum exposure time of 36 days, seeds of HS species and Chenopodium album were still viable. We concluded that viability responses to mesophilic AD were determined by the interplay of AD-conditions and species- and seed-lot-specific traits, of which HS was an important but only one factor. For the use of wildflowers as biogas feedstock, we recommended long retention times and special care with regard to HS species.

Functional Comparison of Human and Murine Equilibrative Nucleobase Transporter 1

BackgroundThiopurines are a key component for the immunosuppressive therapy of various leukemias, inflammatory bowels disease, and other autoimmune disorders. Thiopurine therapy discontinuation/interruption is associated with a higher risk of relapse, but is common due to the prevalence of severe and potentially fatal adverse events, such as hepatotoxicity.Drug transporter expression is a known factor for patient variability in drug response and toxicity. We have recently established that the SLC43A3‐encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which the thiopurine, 6‐mercaptopurine (6‐MP) enters cells. ENBT1 is known to be highly expressed in human hepatocytes, however, the relationship between ENBT1 and thiopurine‐induced hepatotoxicity has not been explored in the literature.To investigate this paradigm, our lab has proposed developing a novel SLC43A3 knockout mouse model. However, the functional differences between human and murine ENBT1, in mediating 6‐MP transport, has equally not been explored. Evidence that suggests ENBT1 is functionally similar between species, would be an essential foundation to assist bridging the gap between our proposed animal model and clinical studies within the literature.HypothesisWe hypothesize that hSLC43A3‐encoded hENBT1 and mslc43a3‐encoded mENBT1 are functionally similar and will have non‐significantly different 6‐MP transport kinetics and resulting cytotoxicity.MethodsThe ENBT1‐deficient human embryonic kidney 293 (HEK293) cell line was stably transfected with either hSLC43A3 or mslc43a3. [3H]Adenine and [3H]6‐MP were used in an oil‐stop centrifugation assay to assess ENBT1‐mediated transport activity in wildtype and transfected HEK293 cell lines. To determine cytotoxicity, wildtype and transfected HEK293 cell lines were incubated for 48 hours with a range of 6‐MP concentrations (78 nM – 1.28 mM) before being assessed via MTT cell viability assay.ResultsOil stop centrifugation assay of ENBT1‐mediated [3H]adenine and [3H]6‐MP transport revealed that hENBT1 and mENBT1 have similar transport kinetics (Km and Vmax), where resulting Michaelis‐Menten curves were not significantly different (unpaired T‐test: Adenine ‐ t16=0.32, p=0.76, n=5 &amp; 6‐MP ‐ t16=0.64, p=0.53, n=4). Adenine inhibition of ENBT1‐mediated [3H]6‐MP transport and 6‐MP inhibition of ENBT1‐mediated [3H]adenine transport also showed that hENBT1 and mENBT1 have concurring inhibition kinetics (Ki), inhibition curves were not significantly different (unpaired T‐test: Adenine: t17=0.29, p=0.77, n=5 &amp; 6‐MP: t14=0.18, p=0.86, n=4). Subsequent MTT cell viability assay showed that wildtype HEK293 are relatively 6‐MP resistant, while the transfected‐HEK293s are 6‐MP sensitive and have non‐significantly different cell viability curves (unpaired T‐test: t28=0.13, p=0.89, n=5).ConclusionOur results show that hENBT1 and mENBT1 are functionally similar in regards to ENBT1‐mediated adenine and 6‐MP transport in a stably transfected cell line. Additional work is necessary to investigate whether this is the case in human and murine hepatic cell lines/primary hepatocytes that have endogenous SLC43A3/ENBT1 expression and a larger range of xenobiotic metabolizing enzymes that may alter the equilibrium of 6‐MP within cells.

Mitochondrial coenzyme Q attenuates lipopolysaccharide-induced mitochondria-dependent apoptosis in type II alveolar epithelial cells via phosphatidylinositol 3-kinase/Akt pathway

To investigate the protective effect and potential mechanism of mitochondrial coenzyme Q (MitoQ) on mitochondria-dependent apoptosis in type II alveolar epithelial cells induced by lipopolysaccharide (LPS). The type II lung epithelial cell line (A549) were cultured with different concentrations of LPS in vitro, a cell model of acute lung injury (ALI) was reproduced, the optimal concentration of LPS was obtained according to the half maximal inhibitory concentration (IC50). The cells were pretreated with different concentrations of MitoQ to determine the best intervention concentration of MitoQ. The cells were divided into four groups: the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 10 mg/L of LPS for 24 hours; the cells in MitoQ+LPS group were pretreated with 1 μmol/L MitoQ for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours; and the cells in MitoQ+phosphatidylinositol 3-kinase (PI3K) selective inhibitor LY294002+LPS group were pretreated with 1 μmol/L MitoQ and 20 μmol/L LY294002 for 60 minutes, and then were co-cultured with 10 mg/L of LPS for 24 hours. Cell viability was measured using cell counting kit-8 (CCK-8). The cell apoptosis rate was determined by flow cytometry and TdT-mediated dUTP-nick end labeling (TUNEL) method. The protein expression levels of apoptosis protein Bax, anti-apoptotic protein Bcl-2 and PI3K-serine/threonine kinase (Akt) protein PI3K expression and Akt phosphorylation level were detected by Western blotting. According to the inhibition rate curve, the IC50 of LPS on A549 cells was 11.06 mg/L. Therefore, 10 mg/L was selected as the stimulating concentration of LPS. After stimulation with 10 mg/L LPS, the cell viability first increased and then decreased with the increase in MitoQ pretreatment concentration. According to the cell viability curve, 1 μmol/L was selected as the optimum concentration of MitoQ. Compared with LPS group, after pretreated with 1 μmol/L MitoQ, cell mitochondrial dependent apoptosis was significantly attenuated, which was characterized by the apoptosis rate was significantly decreased [flow cytometry: (8.73±0.25)% vs. (18.10±0.70)%, TUNEL: (12.30±0.82)% vs. (21.43±0.86)%, both P < 0.05], the expression of Bax was significantly down-regulated (Bax/β-actin: 0.58±0.03 vs. 1.06±0.10, P < 0.05) and Bcl-2 level was significantly up-regulated (Bcl-2/β-actin: 1.03±0.06 vs. 0.53±0.07, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly increased [PI3K protein (PI3K/β-actin): 1.20±0.02 vs. 0.96±0.04, phosphorylated Akt (p-Akt) protein (p-Akt/t-Akt): 1.22±0.08 vs. 0.92±0.04, both P < 0.05]. Pretreatment with LY294002 could inhibit the anti-apoptotic effect of MitoQ on cells, it was characterized by the apoptotic rate was significantly increased as compared with MitoQ+LPS group [flow cytometry: (14.50±0.57)% vs. (8.73±0.25)%, TUNEL: (16.50±0.53)% vs. (12.30±0.82)%, both P < 0.05], the expression of Bax was significantly up-regulated (Bax/β-actin: 0.95±0.03 vs. 0.58±0.03, P < 0.05) and Bcl-2 level was significantly down-regulated (Bcl-2/β-actin: 0.62±0.03 vs. 1.03±0.06, P < 0.05), meanwhile the expression of PI3K and Akt phosphorylation level were significantly decreased [PI3K protein (PI3K/β-actin): 0.90±0.05 vs. 1.20±0.02, p-Akt protein (p-Akt/t-Akt): 0.89±0.02 vs. 1.22±0.08, both P < 0.05]. MitoQ improved LPS induced mitochondria-dependent apoptosis of A549 cells by significantly activating PI3K/Akt signal pathway, which provided a new treatment for LPS induced ALI.

Repeated cell sorting ensures the homogeneity of ocular cell populations expressing a transgenic protein.

Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is constrained by the complex interplay among factors such as the temporal consistency of expression and compatibility with specific cell types, including ocular cells. Here, we report a more efficient way to express an enhanced green fluorescent protein (EGFP) in human corneal fibroblasts, corneal epithelial cells, and conjunctival epithelial cells through a lentiviral expression system. The relative transducing unit criterion for EGFP-expressing pseudovirions was first determined in HEK-293T cells. Homogeneous populations of EGFP-positive and EGFP-negative cells could be isolated by cell sorting. The half-maximal inhibitory concentration (IC50) value for puromycin was calculated according to viability curves for each cell type. The results revealed that cell types differed with respect to EGFP expression efficiency after transduction with the same amount of EGFP-encoding pseudovirions. Using a cell sorter, the homogeneity of EGFP-positive cells reached >95%. In the initial sorting stage, however, the efficiency of EGFP expression in the sorted cells was noticeably reduced after two rounds of sequential culture, but repeated sorting for up to four rounds yielded homogeneous EGFP-positive human corneal fibroblasts that could be maintained in continuous culture in vitro. The sorted EGFP-positive cells retained their proper morphology and cell type-specific protein expression patterns. Puromycin resistance was found to depend on cell type, indicating that the IC50 for puromycin must be determined for each cell type to ensure the isolation of homogeneous EGFP-positive cells. Taken together, repeated cell sorting is an efficient means of obtaining homogeneous populations of ocular cells expressing a transgenic protein during continuous culture without the potential confounding effects of antibiotics.

Evaluation of the antifungal activity of α, β, and δ-damascone and inclusion complexes in β-cyclodextrin against Candida spp.

Due to the increase in fungal resistance to existing drugs, a need exists to search for new antifungals. This study aimed to evaluate the antifungal activity of α, β, and δ-damascone and inclusion complexes with β-cyclodextrin against different Candida spp. The inclusion complex of β-damascone was prepared by the co-evaporation method using three molar proportions (1:1; 2:1; 3:1 (βDA-βCD)) and analyzed using Fourier transform infrared spectroscopy (FTIR). Standard Candida albicans (CA INCQS 40,006), Candida krusei (CK INCQS 40,095), and Candida tropicalis (CT INCQS 40,042) strains were used to evaluate antifungal activity. The substances were tested individually or in association with fluconazole (FCZ). The IC50 and cell viability curve constructions were performed using the microdilution method. The minimum fungicidal concentration (MFC) was determined by the subculture method in a solid medium. The α, β, and δ-DA isolated or in combination with fluconazole (FCZ) showed significant antifungal activity. β-damascone showed effective complexation in the three molar proportions assayed; however, none of the inclusion complexes was demonstrated clinically significant effects against the fungal tested. Then, all compounds have shown promising antifungal activities; however, in vivo assays are necessary to have therapeutical application in the future.

Particulate matter 2.5 induced hyperpigmentation in reconstructed human epidermis model (MelaKutis®).

Particulate matter 2.5 induced hyperpigmentation in reconstructed human epidermis model (MelaKutis®).

Preclinical Drug Response Metric Based on Cellular Response Phenotype Provides Better Pharmacogenomic Variables with Phenotype Relevance.

High-throughput screening of drug response in cultured cell lines is essential for studying therapeutic mechanisms and identifying molecular variants associated with sensitivity to drugs. Assessment of drug response is typically performed by constructing a dose-response curve of viability and summarizing it to a representative, such as IC50. However, this is limited by its dependency on the assay duration and lack of reflections regarding actual cellular response phenotypes. To address these limitations, we consider how each response-phenotype contributes to the overall growth behavior and propose an alternative method of drug response screening that takes into account the cellular response phenotype. In conventional drug response screening methods, the ranking of sensitivity depends on either the metric used to construct the dose-response curve or the representative factor used to summarize the curve. This ambiguity in conventional assessment methods is due to the fact that assessment methods are not consistent with the underlying principles of population dynamics. Instead, the suggested phenotype metrics provide all phenotypic rates of change that shape overall growth behavior at a given dose and better response classification, including the phenotypic mechanism of overall growth inhibition. This alternative high-throughput drug-response screening would improve preclinical pharmacogenomic analysis and the understanding of a therapeutic mechanism of action.

Utilizing Patient-Derived Epithelial Ovarian Cancer Tumor Organoids to Predict Carboplatin Resistance.

The development of patient-derived tumor organoids (TOs) from an epithelial ovarian cancer tumor obtained at the time of primary or interval debulking surgery has the potential to play an important role in precision medicine. Here, we utilized TOs to test front-line chemotherapy sensitivity and to investigate genomic drivers of carboplatin resistance. We developed six high-grade, serous epithelial ovarian cancer tumor organoid lines from tissue obtained during debulking surgery (two neoadjuvant-carboplatin-exposed and four chemo-naïve). Each organoid line was screened for sensitivity to carboplatin at four different doses (100, 10, 1, and 0.1 µM). Cell viability curves and resultant EC50 values were determined. One organoid line, UK1254, was predicted to be resistant to carboplatin based on its EC50 value (50.2 µM) being above clinically achievable Cmax. UK1254 had a significantly shorter PFS than the rest of the subjects (p = 0.0253) and was treated as a platinum-resistant recurrence. Subsequent gene expression analysis revealed extensively interconnected, differentially expressed pathways related to NF-kB, cellular differentiation (PRDM6 activation), and the linkage of B-cell receptor signaling to the PI3K–Akt signaling pathway (PI3KAP1 activation). This study demonstrates that patient-derived tumor organoids can be developed from patients at the time of primary or interval debulking surgery and may be used to predict clinical platinum sensitivity status or to investigate drivers of carboplatin resistance.