Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

Brominated oxime nucleophiles are efficiently reactivating cholinesterases inhibited by nerve agents.

Six novel brominated bis-pyridinium oximes were designed and synthesized to increase their nucleophilicity and reactivation ability of phosphorylated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Their pKa was valuably found lower to parent non-halogenated oximes. Stability tests showed that novel brominated oximes were stable in water, but the stability of di-brominated oximes was decreased in buffer solution and their degradation products were prepared and characterized. The reactivation screening of brominated oximes was tested on AChE and BChE inhibited by organophosphorus surrogates. Two mono-brominated oximes reactivated AChE comparably to non-halogenated analogues, which was further confirmed by reactivation kinetics. The acute toxicity of two selected brominated oximes was similar to commercially available oxime reactivators and the most promising brominated oxime was tested in vivo on sarin- and VX-poisoned rats. This brominated oxime showed interesting CNS distribution and significant reactivation effectiveness in blood. The same oxime resulted with the best protective index for VX-poisoned rats.

Alisertib shows negligible potential for perpetrating pharmacokinetic drug-drug interactions on ABCB1, ABCG2 and cytochromes P450, but acts as dual-activity resistance modulator through the inhibition of ABCC1 transporter.

Alisertib (MLN8237), a novel Aurora A kinase inhibitor, is currently being clinically tested in late-phase trials for the therapy of various malignancies. In the present work, we describe alisertib's potential to perpetrate pharmacokinetic drug-drug interactions (DDIs) and/or to act as an antagonist of multidrug resistance (MDR). In accumulation assays, alisertib potently inhibited ABCC1 transporter, but not ABCB1 or ABCG2. The results of molecular modeling suggested a bifunctional mechanism for interaction on ABCC1. In addition, alisertib was characterized as a low- to moderate-affinity inhibitor of recombinant CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 isoenzymes, but without potential clinical relevance. Drug combination studies revealed the capability of alisertib to synergistically antagonize ABCC1-mediated resistance to daunorubicin. Although alisertib exhibited substrate characteristics toward ABCB1 transporter in monolayer transport assays, comparative proliferation studies showed lack of its MDR-victim behavior in cells overexpressing ABCB1 as well as ABCG2 and ABCC1. Lastly, alisertib did not affect the expression of ABCC1, ABCG2, ABCB1 transporters and CYP1A2, CYP3A4, CYP2B6 isozymes on mRNA level in various systemic and tumoral models. In conclusion, our study suggests that alisertib is a drug candidate with negligible potential for perpetrating systemic pharmacokinetic DDIs on ABCB1, ABCG2 and cytochromes P450. In addition, we introduce alisertib as an effective dual-activity chemosensitizer whose MDR-antagonistic capacities are not impaired by efflux or effect on MDR phenotype. Our in vitro findings provide important pieces of information for clinicians when introducing alisertib into the clinical area.

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates.

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.

Tepotinib Inhibits Several Drug Efflux Transporters and Biotransformation Enzymes: The Role in Drug-Drug Interactions and Targeting Cytostatic Resistance In Vitro and Ex Vivo.

Tepotinib is a novel tyrosine kinase inhibitor recently approved for the treatment of non-small cell lung cancer (NSCLC). In this study, we evaluated the tepotinib's potential to perpetrate pharmacokinetic drug interactions and modulate multidrug resistance (MDR). Accumulation studies showed that tepotinib potently inhibits ABCB1 and ABCG2 efflux transporters, which was confirmed by molecular docking. In addition, tepotinib inhibited several recombinant cytochrome P450 (CYP) isoforms with varying potency. In subsequent drug combination experiments, tepotinib synergistically reversed daunorubicin and mitoxantrone resistance in cells with ABCB1 and ABCG2 overexpression, respectively. Remarkably, MDR-modulatory properties were confirmed in ex vivo explants derived from NSCLC patients. Furthermore, we demonstrated that anticancer effect of tepotinib is not influenced by the presence of ABC transporters associated with MDR, although monolayer transport assays designated it as ABCB1 substrate. Finally, tested drug was observed to have negligible effect on the expression of clinically relevant drug efflux transporters and CYP enzymes. In conclusion, our findings provide complex overview on the tepotinib's drug interaction profile and suggest a promising novel therapeutic strategy for future clinical investigations.

The Evaluation of Glioblastoma Cell Dissociation and Its Influence on Its Behavior.

PURPOSE: Primary cell lines are a valuable tool for evaluation of tumor behavior or sensitivity to anticancer treatment and appropriate dissociation of cells could preserve genomic profile of the original tissue. The main aim of our study was to compare the influence of two methods of glioblastoma multiforme (GBM) cell derivation (mechanic-MD; enzymatic-ED) on basic biological properties of thus derived cells and correlate them to the ones obtained from stabilized GBM cell line A-172. METHODS: Cell proliferation and migration (xCELLigence Real-Time Cell Analysis), expression of microRNAs and protein markers (RT-PCR and Western blotting), morphology (phase contrast and fluorescent microscopy), and accumulation of temozolomide (TMZ) and its metabolite 5-aminoimidazole-4-carboxamide (AIC) inside the cells (LC-MS analysis) were carried out in five different samples of GBM (GBM1, GBM2, GBM32, GBM33, GBM34), with each of them processed by MD and ED types of isolations. The same analyses were done in the A-172 cell line too. RESULTS: Primary GBM cells obtained by ED or MD approaches significantly differ in biological behavior and properties of these cells. Unlike in primary MD GBM cells, higher proliferation, as well as migration, was observed in primary ED GBM cells, which were also associated with the acquired mesenchymal phenotype and higher sensitivity to TMZ. Finally, the same analyses of stabilized GBM cell line A-172 revealed several important differences in measured parameters. CONCLUSIONS: GBM cells obtained by MD and ED dissociation show considerable heterogeneity, but based on our results, MD approach should be the preferred method of primary GBM cell isolation.

The Effects of Selected Sesquiterpenes from Myrica rubra Essential Oil on the Efficacy of Doxorubicin in Sensitive and Resistant Cancer Cell Lines.

ß-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER) and valencene (VAL) are constituents of the essential oil of Myrica rubra (MEO), which has significant antiproliferative effect in various cancer cell lines. In the present study, we compared the antiproliferative effect of these sesquiterpenes alone and in combination with the cytostatic drug doxorubicin (DOX) in cancer cell lines with different sensitivity to DOX. Two ovarian cancer cell lines (sensitive A2780 and partly resistant SKOV3) and two lymphoblast cancer cell lines (sensitive CCRF/CEM and completely resistant CEM/ADR) were used. The observed effects varied among sesquiterpenes and also differed in individual cell lines, with only VAL being effective in all the cell lines. A strong synergism of DOX with NER was found in the A2780 cells, while DOX acted synergistically with HUM and CAO in the SKOV3 cells. In the CCRF/CEM cells, a synergism of DOX with CAO and NER was observed. In resistant CEM/ADR cells, sesquiterpenes did not increase DOX efficacy, although they significantly increased accumulation of DOX (up to 10-times) and rhodamine-123 (substrate of efflux transporter ABCB1) within cancer cells. In conclusion, the tested sesquiterpenes were able to improve DOX efficacy in the sensitive and partly resistant cancer cells, but not in cells completely resistant to DOX.

Essential Oil from Myrica rubra Leaves Potentiated Antiproliferative and Prooxidative Effect of Doxorubicin and its Accumulation in Intestinal Cancer Cells.

Essential oil from the leaves of Myrica rubra, a subtropical Asian fruit tree traditionally used in folk medicines, has a significant antiproliferative effect in several intestinal cancer cell lines. Doxorubicin belongs to the most important cytostatics used in cancer therapy. The present study was designed to evaluate the effects of defined essential oil from M. rubra leaves on efficacy, prooxidative effect, and accumulation of doxorubicin in cancer cell lines and in non-cancerous cells. For this purpose, intestinal adenocarcinoma CaCo2 cells were used. Human fibroblasts (periodontal ligament) and a primary culture of rat hepatocytes served as models of non-cancerous cells. The results showed that the sole essential oil from M. rubra has a strong prooxidative effect in cancer cells while it acts as a mild antioxidant in hepatocytes. Combined with doxorubicin, the essential oil enhanced the antiproliferative and prooxidative effects of doxorubicin in cancer cells. At higher concentrations, synergism of doxorubicin and essential oil from M. rubra was proved. In non-cancerous cells, the essential oil did not affect the toxicity of doxorubicin and the doxorubicin-mediated reactive oxygen species formation. The essential oil increased the intracellular concentration of doxorubicin and enhanced selectively the doxorubicin accumulation in nuclei of cancer cells. Taken together, essential oil from M. rubra leaves could be able to improve the doxorubicin efficacy in cancer cells due to an increased reactive oxygen species production, and the doxorubicin accumulation in nuclei of cancer cells.

The Influence of Sesquiterpenes from Myrica rubra on the Antiproliferative and Pro-Oxidative Effects of Doxorubicin and Its Accumulation in Cancer Cells.

The sesquiterpenes ß-caryophyllene, ß-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER), and valencene (VAL) are substantial components of the essential oil from Myrica rubra leaves which has exhibited significant antiproliferative effects in several intestinal cancer cell lines, with CaCo-2 cells being the most sensitive. The present study was designed to evaluate the effects of these sesquiterpenes on the efficacy and toxicity of the anticancer drug doxorubicin (DOX) in CaCo-2 cancer cells and in primary culture of rat hepatocytes. Our results showed that HUM, NER, VAL and CAO inhibited proliferation of CaCo-2 cancer cells but they did not affect the viability of hepatocytes. CAO, NER and VAL synergistically potentiated the efficacy of DOX in cancer cells killing. All sesquiterpenes exhibited the ability to selectively increase DOX accumulation in cancer cells and did not affect DOX concentration in hepatocytes. Additionally, CAO and VAL were able to increase the pro-oxidative effect of DOX in CaCo-2 cells. Moreover, CAO mildly ameliorated DOX toxicity in hepatocytes. Based on all results, CAO seems to be the most promising compound for further testing.

Anthracycline resistance mediated by reductive metabolism in cancer cells: the role of aldo-keto reductase 1C3.

Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantly contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2'-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment.

Purification and reconstitution of human membrane-bound DHRS7 (SDR34C1) from Sf9 cells.

Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.

7-Amino-3-phenyl-2-methyl-pyrazolopyrimidine derivatives inhibit human rhinovirus replication.

Small molecules that exhibit broad-spectrum enteroviral inhibitory activity by targeting viral replication proteins are highly desired in antiviral drug discovery studies. To discover new human rhinovirus (hRV) inhibitors, we performed a high-throughput screening of 100,000 compounds from the Korea Chemical Bank library. This search led to identification of two phosphatidylinositol-4-kinase IIIß (PI4KIIIß) inhibitors having the pyrazolo-pyrimidine core structure, which display moderate anti-rhinoviral activity along with mild cytotoxicity. The results of a study aimed at optimizing the activity of the hit compounds showed that the pyrazolo-pyrimidine derivative 6f exhibits the highest activity (EC50 = 0.044, 0.066, and 0.083 µM for hRV-B14, hRV-A16, and hRV-A21, respectively) and moderate toxicity (CC50 = 31.38 µM). Furthermore, 6f has broad-spectrum activities against various hRVs, coxsackieviruses and other enteroviruses, such as EV-A71, EV-D68. An assessment of kinase inhibition potencies demonstrated that 6f possesses a high and selective kinase inhibition activity against PI4KIIIß (IC50 value of 0.057 µM) and not against PI4KIIIα (>10 µM). Moreover, 6f exhibits modest hepatic stability (46.9 and 55.3 % remaining after 30 min in mouse and human liver microsomes, respectively). Finally, an in vivo study demonstrated that 6f possesses a desirable pharmacokinetic profile reflected in low systemic clearance (0.48 L∙h-1 kg-1) and modest oral bioavailability (52.4 %). Hence, 6f (KR-26549) appears to be an ideal lead for the development of new antiviral drugs.

BODIPY-labelled acetylcholinesterase reactivators can be encapsulated into ferritin nanovehicles for enhanced bioavailability in the CNS.

The BODIPY-labelled oxime reactivator was prepared and used to study its biodistribution into central nervous system. The newly synthesized oxime was found to be weak inhibitor of acetylcholinesterase and strong inhibitor of butyrylcholinesterase. Its reactivation ability for organophosphate inhibited acetylcholinesterase was found similar to a parent oxime. The BODIPY-labelled oxime was further encapsulated into recombinant human H-ferritin and evaluated in vitro and in vivo. The oxime or encapsulated oxime were found to be bioaccumulated primarily in liver and kidneys of mice, but some amount was distributed also to the brain, where it was detectable even after 24 h. The BODIPY-labelled oxime encapsulated to human H-ferritin showed better CNS bioaccumulation and tissue retention at 8 and 24 h time points compared to free oxime, although the fluorescence results might be biased due to BODIPY metabolites identified in tissue homogenates. Taken together, the study demonstrates the first utilization of recombinant ferritins for changing the unfavourable pharmacokinetics of oxime reactivators and brings promising results for follow-up studies.

Development of submicromolar 17ß-HSD10 inhibitors and their in vitro and in vivo evaluation.

17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is a multifunctional mitochondrial enzyme and putative drug target for the treatment of various pathologies including Alzheimer's disease or some types of hormone-dependent cancer. In this study, a series of new benzothiazolylurea-based inhibitors were developed based on the structure-activity relationship (SAR) study of previously published compounds and predictions of their physico-chemical properties. This led to the identification of several submicromolar inhibitors (IC50 ∼0.3 µM), the most potent compounds within the benzothiazolylurea class known to date. The positive interaction with 17ß-HSD10 was further confirmed by differential scanning fluorimetry and the best molecules were found to be cell penetrable. In addition, the best compounds weren't found to have additional effects for mitochondrial off-targets and cytotoxic or neurotoxic effects. The two most potent inhibitors 9 and 11 were selected for in vivo pharmacokinetic study after intravenous and peroral administration. Although the pharmacokinetic results were not fully conclusive, it seemed that compound 9 was bioavailable after peroral administration and could penetrate into the brain (brain-plasma ratio 0.56).

Frentizole derivatives with mTOR inhibiting and senomorphic properties.

Frentizole is immunosuppressive drug with low acute toxicity and lifespan-prolonging effect. Recently, frentizole´s potential to disrupt toxic amyloid ß (Aß) - Aß-binding alcohol dehydrogenase (ABAD) interaction in mitochondria in Alzheimer´s brains has been revealed. Another broadly studied drug with anti-aging and immunosuppressive properties is an mTOR inhibitor - rapamycin. Since we do not yet precisely know what is behind the lifespan-prolonging effect of rapamycin and frentizole, whether it is the ability to inhibit the mTOR signaling pathway, reduction in mitochondrial toxicity, immunosuppressive effect, or a combination of all of them, we have decided within our previous work to dock the entire in-house library of almost 240 Aß-ABAD modulators into the FKBP-rapamycin-binding (FRB) domain of mTOR in order to interlink mTOR-centric and mitochondrial free radical-centric theories of aging and thus to increase the chances of success. Based on the results of the docking study, molecular dynamic simulation and MM-PBSA calculations, we have selected nine frentizole-like compounds (1 - 9). Subsequently, we have determined their real physical-chemical properties (logP, logD, pKa and solubility in water and buffer), cytotoxic/cytostatic, mTOR inhibitory, and in vitro anti-senescence (senolytic and senomorphic) effects. Finally, the three best candidates (4, 8, and 9) have been forwarded for in vivo safety studies to assess their acute toxicity and pharmacokinetic properties. Based on obtained results, only compound 4 demonstrated the best results within in vitro testing, the ability to cross the blood-brain barrier and the lowest acute toxicity (LD50 in male mice 559 mg/kg; LD50 in female mice 575 mg/kg).

Role of N-Cadherin in Epithelial-to-Mesenchymal Transition and Chemosensitivity of Colon Carcinoma Cells.

(1) Background: N-cadherin expression, epithelial-to-mesenchymal transition (EMT) and aggressive biological phenotype of tumor cells are linked although the underlying mechanisms are not entirely clear. (2) Methods: In this study, we used two different in vitro cell models with varying N-cadherin expression (stabilized lines and primocultures) and investigated their select biological features including the degree of their chemoresistance both in vitro as well as in vivo. (3) Results: We report that although enforced N-cadherin expression changes select morphological and behavioral characteristics of exposed cells, it fails to successfully reprogram cells to the aggressive, chemoresistant phenotype both in vitro as well as in vivo as verified by implantation of those cells into athymic mice. Conversely, primocultures of patient-colonic cells with naturally high levels of N-cadherin expression show fully aggressive and chemoresistant phenotype pertinent to EMT (in vitro and in vivo), with a potential to develop new mutations and in the presence of dysregulated regulatory pathways as represented by investigated miRNA profiles. (4) Conclusions: The presented results bring new facts concerning the functional axis of N-cadherin expression and related biological features of colon cancer cells and highlight colon cancer primocultures as a useful model for such studies.

Discovery of small molecule mechanistic target of rapamycin inhibitors as anti-aging and anti-cancer therapeutics.

To date, the most studied drug in anti-aging research is the mTOR inhibitor - rapamycin. Despite its almost perfect anti-aging profile, rapamycin exerts one significant limitation - inappropriate physicochemical properties. Therefore, we have decided to utilize virtual high-throughput screening and fragment-based design in search of novel mTOR inhibiting scaffolds with suitable physicochemical parameters. Seven lead compounds were selected from the list of obtained hits that were commercially available (4, 5, and 7) or their synthesis was feasible (1, 2, 3, and 6) and evaluated in vitro and subsequently in vivo. Of all these substances, only compound 3 demonstrated a significant cytotoxic, senolytic, and senomorphic effect on normal and cancerous cells. Further, it has been confirmed that compound 3 is a direct mTORC1 inhibitor. Last but not least, compound 3 was found to exhibit anti-SASP activity concurrently being relatively safe within the test of in vivo tolerability. All these outstanding results highlight compound 3 as a scaffold worthy of further investigation.

Silencing of E-cadherin expression leads to increased chemosensitivity to irinotecan and oxaliplatin in colorectal cancer cell lines.

Colorectal carcinoma (CRC) is a leading malignant disease in most developed countries. In advanced stages it presents with metastatic dissemination and significant chemoresistance. Despite intensive studies, no convincing evidence has been published concerning the association of cadherins and epithelial-mesenchymal transition (EMT) as a direct cause of acquired chemoresistance in CRC. The present study was designed to investigate the role of E-cadherin in EMT and its associated chemosensitivity/chemoresistance in four immortalized CRC cell lines representing various stages of CRC development (i.e. HT29 and Caco-2-early, SW480 and SW620 late). The expression of E-cadherin gene CDH1 was downregulated by the specific siRNA. Cell proliferation and chemosensitivity to irinotecan (IT) and oxaliplatin (OPT) were detected using WST-1 and x-CELLigence Real Time analysis. Expression of selected EMT markers were tested and compared using RT-PCR and western blot analysis in both variants (E-cadherin silenced and non-silenced) of each cell line. We have discovered that downregulation of E-cadherin expression has a diverse effect on both cell proliferation as well as the expression of EMT markers in individual tested CRC cell lines, with Caco-2 cells being the most responsive. On the other hand, reduced E-cadherin expression resulted in increased sensitivity of all cell lines to IT and mostly to OPT which might be related to changes in intracellular metabolism of these drugs. These results suggest dichotomy of E-cadherin involvement in the phenotypic EMT spectrum of CRC and warrants further mechanistic studies.

Ensartinib (X-396) Effectively Modulates Pharmacokinetic Resistance Mediated by ABCB1 and ABCG2 Drug Efflux Transporters and CYP3A4 Biotransformation Enzyme.

Ensartinib (X-396) is a promising tyrosine kinase inhibitor currently undergoing advanced clinical evaluation for the treatment of non-small cell lung cancer. In this work, we investigate possible interactions of this promising drug candidate with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs), which play major roles in multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). Accumulation studies showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 transporters. Additionally, incubation experiments with recombinant CYPs showed that ensartinib significantly inhibits CYP3A4 and CYP2C9. Subsequent molecular docking studies confirmed these findings. Drug combination experiments demonstrated that ensartinib synergistically potentiates the antiproliferative effects of daunorubicin, mitoxantrone, and docetaxel in ABCB1, ABCG2, and CYP3A4-overexpressing cellular models, respectively. Advantageously, ensartinib's antitumor efficiency was not compromised by the presence of MDR-associated ABC transporters, although it acted as a substrate of ABCB1 in Madin-Darby Canine Kidney II (MDCKII) monolayer transport assays. Finally, we demonstrated that ensartinib had no significant effect on the mRNA-level expression of examined transporters and enzymes in physiological and lung tumor cellular models. In conclusion, ensartinib may perpetrate clinically relevant pharmacokinetic DDIs and modulate ABCB1-, ABCG2-, and CYP3A4-mediated MDR. The in vitro findings presented here will provide a valuable foundation for future in vivo investigations.

Carbonyl Reduction of Flubendazole in the Human Liver: Strict Stereospecificity, Sex Difference, Low Risk of Drug Interactions.

Flubendazole (FLU), an anthelmintic drug of benzimidazole type, is now considered a promising anti-cancer agent due to its tubulin binding ability and low system toxicity. The present study was aimed at determining more information about FLU reduction in human liver, because this information has been insufficient until now. Subcellular fractions from the liver of 12 human patients (6 male and 6 female patients) were used to study the stereospecificity, cellular localization, coenzyme preference, enzyme kinetics, and possible inter-individual or sex differences in FLU reduction. In addition, the risk of FLU interaction with other drugs was evaluated. Our study showed that FLU is predominantly reduced in cytosol, and the reduced nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme is preferred. The strict stereospecificity of FLU carbonyl reduction was proven, and carbonyl reductase 1 was identified as the main enzyme of FLU reduction in the human liver. A higher reduction of FLU and a higher level of carbonyl reductase 1 protein were found in male patients than in female patients, but overall inter-individual variability was relatively low. Hepatic intrinsic clearance of FLU is very low, and FLU had no effect on doxorubicin carbonyl reduction in the liver and in cancer cells. All these results fill the gaps in the knowledge of FLU metabolism in human.