Multidrug-resistant Cell Lines Research Articles

Abstract P2-15-03: Liposomal annamycin inhibition of lung localized breast cancer

Abstract Background and Rationale: Annamycin is an anthracycline antibiotic currently in clinical studies in AML patients. Our earlier studies showed that Annamycin is not cross-resistant with doxorubicin (DOX) and is a poor substrate for P-glycoprotein 1 (P-gp) [aka ATP-binding cassette sub-family B member 1 (ABCB1) or multidrug resistance protein 1 (MDR1)], a major mechanism of doxorubicin resistance in different types of cancer. Annamycin, in contrast to DOX, achieves relatively high levels of cellular accumulation, especially in multidrug resistant (MDR) cell lines, and induces significant DNA damage in MDR cancer cells. Clinically, Annamycin is administered in a liposomal formulation (L-Annamycin) the in vivo activity of which has been demonstrated in different tumor models. IND aimed preclinical studies revealed that in sharp contrast to DOX, Annamycin displays dramatically reduced to no cardiotoxicity. Subsequent pharmacokinetic and organ distribution studies revealed surprisingly high levels of Annamycin in lung tissue. Annamycin levels (AUC, 24 h) in lungs were over 10-fold greater than in plasma. In addition, when compared with DOX, lung levels of Annamycin were 6-fold higher than those achievable with DOX. Thus, studies aimed to assess efficacy of L-Annamycin in lung localized tumor models were initiated. Objective: The objective of this study was to assess the in vitro and in vivo activity of L-Annamycin in 4T1, a highly aggressive triple negative breast cancer (TNBC) lung colonization model. Methods: In vitro efficacy of Annamycin was investigated in a panel of TNBC cell lines including murine 4T1 and human MDA-MB 231, MDA-MB-468, BT20, and SUM149 cell lines. Uptake and subcellular distribution of Annamycin was assessed using fluorescent microscopy and FACS. Biodistribution of Annamycin was tested in naïve CD-1 mice after intravenous administration of the drug. Efficacy of L-Annamycin was tested in a syngeneic TNBC lung model, specifically, in immune-competent Balb/c mice injected intravenously with 4T1 cells expressing firefly luciferase. Results: Annamycin shows consistently high cytotoxicity across all tested cell lines with IC50s in the low nanomolar range. Uptake analysis performed on 4T1 cells indicated significantly higher uptake of the drug as compared to DOX. Annamycin shows distinct subcellular distribution with high cytosolic deposition, while DOX was mostly confined to the nucleus. In vivo efficacy studies in mice treated with L-Annamycin showed significantly increased survival when compared to vehicle treated animals. Delayed tumor progression was assessed by bioluminescent imaging and correlated with increased survival. Conclusion: Our in vitro studies demonstrate the high potency of L-Annamycin in vitro and in vivo. Its unique intracellular uptake and distribution might in part be responsible for L-Annamycin’s activity profile and lack of cardiotoxicity. The observed high in vivo efficacy of L-Annamycin in an aggressive TNBC lung metastasis model coupled with consistently high accumulation of L-Annamycin in lung parenchyma, may prove to open new therapeutic options for patients with tumors metastatic to the lung. Citation Format: Rafal Zielinski, Krzysztol Grela, Stanislaw Skora, Izabela Fokt, Matthew Sander, Adam Didluch, Waldemar Priebe. Liposomal annamycin inhibition of lung localized breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-15-03.

Further exploration of DVD-445 as a lead thioredoxin reductase (TrxR) inhibitor for cancer therapy: Optimization of potency and evaluation of anticancer potential

A series of analogs of the earlier reported lead compound DVD-445 (thioredoxin reductase inhibitor with anticancer activity) has been synthesized via a modified Ugi reaction and investigated. Seven most potent compounds (with IC50 below 5.00 μM against recombinant rTrxR1 enzyme) were examined for their effect on cell growth and viability, oxidative stress induction and P-glycoprotein (P-gp) inhibition in human glioblastoma cells cell line U87 and its corresponding multidrug resistant (MDR) cell line U87-TxR. Several of these frontrunner compounds were shown to be superior over DVD-445. Besides providing promising candidates for anticancer therapy, our study further validates the small electrophilic Ugi Michael acceptor (UMA) chemotype as efficacious inhibitor of thioredoxin reductase.

Enhanced antitumor potential induced by chloroacetate-loaded benzophenones acting as fused tubulin-pyruvate dehydrogenase kinase 1 (PDHK1) ligands.

The majority of cancers detected every year are treated with anti-cancer compounds. Unfortunately, many tumors become resistant to antineoplastic drugs. One option is to use cocktails of compounds acting on different targets to try to overcome the resistant cells. This type of approach can produce good results, but is often accompanied by a sharp increase of associated side effects. The strategy presented herein focuses on the use of a single compound acting on two different biological targets enhancing potency and lowering the toxicity of the chemotherapy. In this light, the approach presented in the current study involves the dual inhibition of human pyruvate dehydrogenase kinase-1 (PDHK1) and tubulin polymerization using mono-, di- and tri-chloroacetate-loaded benzophenones and benzothiophenones. Synthesized molecules were evaluated in vitro on tubulin polymerization and on pyruvate dehydrogenase kinase 1. The cell cycle distribution after treatment of DA1-3b leukemic cells with active compounds was tested. Twenty-two benzo(thio)phenones have been selected by the National Cancer Institute (USA) for evaluation of their anti-proliferative potential against NCI-60 cancer cell lines including multidrug-resistant tumor cell lines. Seventeen molecules proved to be very effective in combating the growth of tumor cells exhibiting inhibitory activities up to nanomolar range. The molecular docking of best antitumor molecules in the study was realized with GOLD in the tubulin and PDHK1 binding sites, and allowed to understand the positioning of active molecules. Chloroacetate-loaded benzo(thio)phenones are dual targeted tubulin- and pyruvate dehydrogenase kinase 1 (PDHK1)-binding antitumor agents and exhibited superior antitumor activity compared to non-chlorinated congeners particularly on leukemia, colon, melanoma and breast cancer cell lines.

Sitravatinib Sensitizes ABCB1- and ABCG2-Overexpressing Multidrug-Resistant Cancer Cells to Chemotherapeutic Drugs.

The development of multidrug resistance (MDR) in cancer patients driven by the overexpression of ATP-binding cassette (ABC) transporter ABCB1 or ABCG2 in cancer cells presents one of the most daunting therapeutic complications for clinical scientists to resolve. Despite many novel therapeutic strategies that have been tested over the years, there is still no approved treatment for multidrug-resistant cancers to date. We have recently adopted a drug repurposing approach to identify therapeutic agents that are clinically active and at the same time, capable of reversing multidrug resistance mediated by ABCB1 and ABCG2. In the present study, we investigated the effect of sitravatinib, a novel multitargeted receptor tyrosine kinase inhibitor, on human ABCB1 and ABCG2 in multidrug-resistant cancer cell lines. We discovered that at submicromolar concentrations, sitravatinib re-sensitizes ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic drugs. We found that sitravatinib blocks the drug efflux function of ABCB1 and ABCG2 in a concentration-dependent manner but does not significantly alter the protein expression of ABCB1 or ABCG2 in multidrug-resistant cancer cells. In conclusion, we reveal a potential drug repositioning treatment option for multidrug-resistant cancers by targeting ABCB1 and ABCG2 with sitravatinib and should be further investigated in future clinical trials.

Identification of chemoresistance-related mRNAs based on gemcitabine-resistant pancreatic cancer cell lines.

Gemcitabine (GEM) alone and GEM‐based chemotherapy are the preferred regimens for treating advanced unresectable and metastatic pancreatic cancer (PC). However, these treatments have limited efficacy due to acquired resistance of cancer cells to chemotherapy, the mechanisms of which are not fully understood. In this study, we established two stable multidrug‐resistant cell lines, BxPC‐3‐GR and CFPAC‐1‐GR, from their corresponding parental cells through exposure to GEM following a stepwise incremental dosing strategy. The GEM IC50 values of BxPC‐3‐GR and CFPAC‐1‐GR increased 112‐fold and 210‐fold, respectively, compared to parental cell lines. In vitro and in vivo experiments confirmed that both GEM‐resistant cell subgroups declined in proliferative capacity, but were more resistant to GEM. Unlike CFPAC‐1‐GR, BxPC‐3‐GR exhibited enhanced migratory and invasive properties compared with BxPC‐3 in vitro. We also compared differentially expressed mRNA profiles between parental and GEM‐resistant cells using transcriptome sequencing. RRM1, STIM1, and TRIM21 were significantly upregulated in both GEM‐resistant cell lines and confirmed to be associated with the degree of GEM resistance by quantitative reverse‐transcription polymerase chain reaction and western blot analysis. These three genes were more highly expressed in PC tissues and potentially regarded as prognostic biomarkers through database mining. Thus, our findings provide chemo‐resistant cell models to better understand the underlying mechanisms of chemoresistance, and to explore potential biomarkers for GEM response in PC patients.

New Chalcone Derivative Inhibits ABCB1 in Multidrug Resistant T-cell Lymphoma and Colon Adenocarcinoma Cells.

Development of new potential drugs to overcome multidrug resistance to chemotherapy is a big challenge for cancer treatment. Attention is also given to the natural compounds and their derivatives. The study aimed at evaluating the impact of a new chalcone derivative (1C) on multidrug resistant cell lines, focusing on P-glycoprotein (P-gp, ABCB1) inhibition, as well as 1C-doxorubicin interaction in vitro. Cytotoxic and antiproliferative effects of the 1C compound were assessed by thiazolyl blue tetrazolium bromide (MTT) method in mouse T-cell lymphoma and human colon adenocarcinoma cells expressing ABCB1. Alterations in ABCB1 activity were evaluated by rhodamine 123 accumulation assay using flow cytometry. Drug-drug interaction was studied using combination assay. Our results confirmed antiproliferative, cytotoxic, as well as ABCB1 inhibitory potential of 1C in both tested ABCB1-expressing cancer cell lines. Furthermore, 1C displayed synergistic interaction with doxorubicin. Our results suggest the 1C chalcone derivative as a promising compound against resistant lymphoma and colon cancer, which could be used in monotherapy or in combination with other chemotherapeutics.

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance

The use of taxanes has for decades been crucial for treatment of several cancers. A major limitation of these therapies is inherent or acquired drug resistance. A key to improved outcome of taxane-based therapies is to develop tools to predict and monitor drug efficacy and resistance in the clinical setting allowing for treatment and dose stratification for individual patients. To assess treatment efficacy up to the level of drug target engagement, we have established several formats of tubulin-specific Cellular Thermal Shift Assays (CETSAs). This technique was evaluated in breast and prostate cancer models and in a cohort of breast cancer patients. Here we show that taxanes induce significant CETSA shifts in cell lines as well as in animal models including patient-derived xenograft (PDX) models. Furthermore, isothermal dose response CETSA measurements allowed for drugs to be rapidly ranked according to their reported potency. Using multidrug resistant cancer cell lines and taxane-resistant PDX models we demonstrate that CETSA can identify taxane resistance up to the level of target engagement. An imaging-based CETSA format was also established, which in principle allows for taxane target engagement to be accessed in specific cell types in complex cell mixtures. Using a highly sensitive implementation of CETSA, we measured target engagement in fine needle aspirates from breast cancer patients, revealing a range of different sensitivities. Together, our data support that CETSA is a robust tool for assessing taxane target engagement in preclinical models and clinical material and therefore should be evaluated as a prognostic tool during taxane-based therapies.

Abstract C094: Dose and schedule-dependent efficacy of liposomal Annamycin in pre-clinical models of acute myeloid leukemia

Abstract Annamycin (Ann) is a clinically evaluated anthracycline whose antitumor activity is not cross-resistant with that of doxorubicin (DOX). Formulated in liposomes, L-Annamycin (L-Ann) is currently in clinical studies in acute myeloid leukemia (AML) patients. In contrast to DOX, high levels of cellular accumulation of Ann were observed in multidrug resistant (MDR) cell lines, and consequently there was a significant degree of DNA damage and apoptosis. In vivo activity of L-Ann and free Ann was confirmed in different tumor models. In addition, preclinical toxicology studies revealed that both L-Ann as well as free Ann displayed dramatically reduced or no cardiotoxicity, in sharp contrast to DOX. In the current study, we assessed the anti-AML efficacy of Ann using multiple in vitro and in vivo models. In vitro efficacy was assessed in a panel of human AML cell lines including THP-1, OCI-AML3 and MV4;11. The uptake and subcellular distribution of Ann was evaluated using flow cytometry and fluorescence microscopy. In vivo biodistribution of Ann was tested in naïve B6 mice after intravenous administration of the drug followed by fluorescence microscopy. Anti-tumor efficacy was extensively tested in syngeneic AML-Turq 2 model in immunocompetent B6 mice, and subsequently in human OCI-AML3 model in immunosuppressed NSG mice. In the murine syngeneic AML-Turq 2 model, AML cells were visualized based on the mTurquoise fluorescence. AML disease progression was monitored using whole body bioluminescence imaging or flow cytometry, respectively. We observed a high potency of Ann against established human AML cell lines, with the IC50 in low nanomolar range. Saturation experiments indicated high uptake of Ann by the cancer cells. Interestingly, in addition to the expected uptake of Ann in the nucleus, Ann was also localized in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. In vivo studies confirmed anti-tumor efficacy of the Annamycin in both human and murine AML models. Based on bioluminescence imaging, the liposomal formulation of the drug significantly delayed AML progression in the human OCL-AML3/NSG model at 4 mg/kg with once weekly dosing. Similarly, significant dose-dependent reduction of peripheral blood AML blasts was observed in the murine AML-Turq2 model, and this reduction was strongly correlated with prolongation of animals survival. The median survival of mice receiving four doses of L-Ann once a week at 4 mg/ml was 37 days while mice receiving vehicle lived only 14 days (p=0.0002). Different doses and administration schedules of L-Ann were tested in effort to maximize survival benefits. In summary L-Ann is effective in AML, demonstrating significant activity in both in vitro and in vivo mouse models with a distinct pattern of intracellular uptake and organ distribution using a once a week schedule. This suggests that L-Ann with this profile, including a lack of cardiotoxicity and activity against DOX resistant tumors, may be an advantageous approach in the treatment of AML. Citation Format: Rafal Zielinski, Tomasz Zal, Krzysztof Grela, Stanislaw Skora, Izabela Fokt, Matthew Sander, Magdalena Remiszewski, Edward Felix, Meenakshi Shanmugasundaram, Waldemar Priebe. Dose and schedule-dependent efficacy of liposomal Annamycin in pre-clinical models of acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C094. doi:10.1158/1535-7163.TARG-19-C094

Process optimization for green synthesis of gold nanoparticles mediated by extract of Hygrophila spinosa T. Anders and their biological applications

Plant extract-mediated green synthesis of gold nanoparticles (AuNPs) and their biomedical application is currently gaining huge interest in the field of nanotechnology . In the present study, AuNPs were synthesized using Hygrophila spinosa aqueous extract (HSAE). The formation of AuNPs was confirmed by observing color change and characteristic absorbance peak for surface plasmon resonance by UV–Vis spectroscopy. Different reaction parameters such as HSAE concentration, salt concentration, pH, reaction time and temperature were optimized for biosynthesis of AuNPs. The NPs were characterized for particle size, surface morphology , crystallinity , elemental composition and surface functionalities. Cytotoxicity of the AuNPs was studied against different cancer cell lines by MTT assay and antioxidant potential as total antioxidant capacity was also evaluated. The biosynthesized AuNPs were small in size and negatively charged. Phytochemical screening of HSAE, and FTIR spectrum of synthesized AuNPs demonstrated participation of various groups of chemical compounds as capping and stabilizing agents in biosynthesis of AuNPs. The green synthesized AuNPs exhibited significantly higher anticancer activity compared to HSAE against breast, ovarian, glioblastoma/brain and multi-drug resistant ovarian cancer cell lines. Antioxidant activity of the synthesized AuNPs was less than HSAE. The biosynthesized AuNPs could be studied further to access their potency in the treatment and imaging of various cancers. • Green synthesis of AuNPs has been carried out with Hygrophila spinosa aqueous extract (HSAE). • Concentration of HAuCl 4 solution, pH, reaction incubation temperature & time, and concentration of HSAE have been optimized for synthesis of AuNPs. • Characterization by UV–Vis, FTIR, SEM, TEM, EDS and XRD confirm that HSAE could synthesize AuNPs. • Synthesized AuNPs exhibited promising antioxidant and anticancer activities.

Biodistribution and Efficacy of L-Annamycin in a Novel Imageable AML Mouse Model

Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

Anticancer Activity of a Highly Potent Small Molecule Tubulin Polymerization Inhibitor, AB8939

We have identified the small chemical molecule AB8939 as being a structurally novel, synthesized tubulin inhibitor that can circumvent resistance mechanisms known to limit the effectiveness of existing tubulin inhibitors; e.g., P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance. A series of in vitro preclinical studies provide proof-of-concept that AB8939 has broad applicability as a potent anticancer drug, particularly in tumors of hematopoietic and lymphoid tissues, including acute myeloid leukemia (AML). Regarding mechanism of action, x-ray crystallography demonstrated that AB8939 binds to the colchicine-binding site on the beta-subunit of tubulin. Cell cycle arrest in the G2/M phase was evaluated using HCT116 cells (a human colorectal tumor), treated at various concentrations of AB8939 for 24 hours. It was seen that AB8939 produced a strong mitotic arrest at the sub-micromolar concentration range (90% of cells in G2/M phase at 10 nM), which was of comparable strength to that of established microtubule targeting agents, each at a concentration of 100 nM. Additional assays using cytarabine (Ara-C) resistant MOLM14 AML cells confirmed this activity, also demonstrating dose dependent (2 to 20 nM) G2/M phase cell cycle arrest in patient-derived AML blasts and that G2/M cell cycle arrest lead to cellular death by apoptosis at nanomolar concentrations. The effect of AB8939 (100 nM) on the integrity of the microtubule and actin networks was tested in 3T3NIH cells (murine embryonic fibroblast cell line). AB8939 induced a rapid (within 1 hour) and radical destabilization of the microtubule network but did not affect the actin network. Similarly, destabilization of the microtubule network was observed in human primary cardiomyocytes and primary human lung fibroblast cells treated for 24 hours at 10 to 1000 nM AB8939. Further in vitro analysis showed that AB8939 produces a direct and potent, dose-dependent depolymerization effect (50% inhibition of in vitro microtubule polymerization at around 1 µM, with 100% inhibition at >5 µM). The potential of AB8939 to overcome resistance to chemotherapeutic agents in Pgp-dependent multidrug-resistant cell lines was assessed using the drug-sensitive human sarcoma cell line MES-SA (parental) and its multidrug-resistant counterparts MES-SA/MX2 and MES-SA/Dx5 in a 6-day proliferation/survival assay. AB8939 efficiently inhibited each of these cells with an IC50 ≤10 nM. By comparison, the MES-SA/MX2 and MES-SA/Dx5 cell lines were highly resistant to the chemotherapeutic agents of doxorubicin and vincristine, as compared with the effect on parental cells (IC50 <1.5 - 2.0 µM versus 20 nM, respectively). Additional tests showed that AB8939 is a very poor substrate of Pgp efflux pump, comparable with combretastatin-4, and therefore has the potential to overcome multidrug resistance in cancer patients. The anti-proliferative activity of AB8939 in various hematopoietic tumors and solid tumors was evaluated using a colorimetric cell proliferation and viability assay. AB8939 produced good anti-tumor activity after 72 hours (IC50 of ≤50 nM) in 19 hematopoietic tumor cell lines tested, including AML (3 cell lines), B cell lymphoma (8 cell lines), T cell lymphoma (6 cell lines), and multiple myeloma (2 cell lines). AB8939 also showed good anti-tumor activity after 6 days (IC50 of ≤10 nM) in several solid tumor cell lines, including breast, colon, glioblastoma, head and neck, lung, kidney, melanoma neuroblastoma, ovary, pancreas and prostate cell lines. The therapeutic potential of AB8939 in refractory/resistant AML was investigated further on doxorubicin-resistant AML cell lines (HL60 and U937), doxorubicin being a commonly used AML induction drug and Pgp substrate. AB8939 produced a strong anti-proliferative effect in both cell lines whereas both were resistant to doxorubicin, thus demonstrating AB8939's potential to overcome refractory/resistant AML. Notably, HL60 and U937 are respectively MPO-positive and MPO-negative, indicating that unlike vinca alkaloids (e.g. vincristine or vinblastine) AB8939 it is not deactivated by this myeloid enzyme. These data show that AB8939 is a prolific and highly potent (nanomolar concentrations) Pgp-independent, next-generation microtubule-destabilizer drug for cancer therapy; in particular, difficult to treat hematopoietic tumors such as relapsed/refractory AML. Disclosures Humbert: AB Science: Employment. Goubard:AB Science: Employment. Mansfield:AB Science: Employment, Patents & Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Dubreuil:AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. AB8939 Study Group:AB Science: Consultancy, Employment.

OPTIMAL FERMENTATION CONDITIONS FOR ANTIBIOTIC PRODUCTION BY ENDOPHYTIC Streptomyces cavourensis YBQ59 ISOLATED FROM Cinnamomum cassia Presl

This study determined the optimal nutrient and environmental conditions for the production of antimicrobial and cytotoxic agents by Streptomyces cavourensis YBQ59 during the fermentation process. The antimicrobial and cytotoxic activities of eluted fractions based gradient solvents via silica gel column of chromatograph were also evaluated. S. cavourensis YBQ59 exhibited strong, board-spectrum antimicrobial activities under fermentation conditions as follows: MT6 medium with soluble starch and soybean powder as carbon and nitrogen sources, respectively, temperature 30oC, initial pH 7.0, 20% DO concentration and with 5% initial seed culture. The kinetic of fermentation showed that the antimicrobial and cytotoxic activities were highest at between 72h and 78h. The fraction 25/1 D-M (dichloromethane –methanol) exhibited the highest antimicrobial effect against methicillin-resistant Staphylococcus aureus ATCC 33591 and methicillin-resistant Staphylococcus epidermidis ATCC 35984 with minimum inhibitory concentrations of 9.4 µg/ml and 6.4 µg/ml, respectively. The fraction 10/1 D-M had strong cytotoxic effects towards multidrug-resistant A459 and H1299 lung carcinoma cell lines with the cell viability of 11.3% and 12.4%, respectively. In conclusion, S. cavourensis YBQ59 would be a potential producer of valuable bioactive compounds that may have a board application in pharmaceutical industry and agriculture (biocontrol, livestock, food safety and quality management)

LncRNA UCA1 Increases Proliferation and Multidrug Resistance of Retinoblastoma Cells Through Downregulating miR-513a-5p.

Chemoresistance is one of the major obstacles for cancer therapy. Abnormal expression of long noncoding RNAs (lncRNAs) was broadly implicated in chemoresistance of multiple cancers. This study was aimed to investigate the function of urothelial cancer associated 1 (UCA1) in multidrug resistance of retinoblastoma and its potential molecular mechanism. In this study, we observed that UCA1 was significantly upregulated in chemoresistant retinoblastoma tissues and multidrug resistant retinoblastoma cell lines and predicted an unfavorable overall survival. Functionally, knockdown of UCA1 remarkably inhibited proliferation and sensitized retinoblastoma cells to multiple chemotherapy drugs, including vincristine (VCR), carboplatin (CBP), cisplatin (DDP), VP-16 (etoposide), and 5-fluorouracil (5-Fu). Mechanistic studies demonstrated that UCA1 functioned as a miRNA sponge to increase stathmin 1 (STMN1) expression through sponging miR-513a-5p. In addition, silence of miR-513a-5p or STMN1 overexpression could partly reverse UCA1 knockdown-induced inhibitory effects on proliferation and multidrug resistance of retinoblastoma cells. Overall, this study is the first to demonstrate that UCA1 plays a critical role in retinoblastoma chemoresistance, and UCA1 may serve as a potential diagnostic biomarker and therapeutic target of retinoblastoma.

Enhancing the Therapeutic Efficacy of Daunorubicin and Mitoxantrone with Bavachinin, Candidone, and Tephrosin.

The capability of flavonoids in sensitizing cancer cells was demonstrated in numerous works to chemotherapy and converse multidrug resistance by modulating efflux pumps and apoptosis mechanisms. Three flavonoids, namely, bavachinin, tephrosin, and candidone, have been recently introduced to cancer treatment research presenting various activities, such as antibacterial, immunomodulatory, cell death, and anticancer. Less information exists regarding the therapeutic significance of these flavonoids in cancer treatment, especially in overcoming multidrug resistance (MDR). Here, we tempted to investigate the potency of these agents in reversing MDR by analyzing their effects as chemosensitizers on cell cytotoxicity, P-gp and ABCG2 protein expression levels, and their function on two multidrug-resistant cell lines, P-gp-overexpressing human gastric adenocarcinoma cell line (EPG85.257RDB) and ABCG2-overexpressing human epithelial breast cancer cell line (MCF7/MX). The inhibitory concentration of 10% (IC10) of bavachinin, tephrosin, and candidone in EPG85.257RDB cells was 1588.7 ± 202.2, 264.8 ± 86.15, and 1338.6 ± 114.11 nM, respectively. Moreover, these values in MCF7/MX cell were 2406.4 ± 257.63, 38.8 ± 4.28, and 27.9 ± 5.59 nM, respectively. Expression levels of ABCG2 and P-gp were not significantly downregulated by these flavonoids. Maximum levels of daunorubicin and mitoxantrone accumulations and minimum rates of drug efflux in both cell lines were detected 48 hrs posttreatment with tephrosin and bavachinin, respectively. Chemosensitization to mitoxantrone and daunorubicin treatments was, respectively, achieved in MCF7/MX and EPG85.257RDB cells in response to IC10 of bavachinin and tephrosin, independently. These effects did not follow time-dependent manner, and each flavonoid had its cell-dependent patterns. Overall, bavachinin, tephrosin, and candidone showed potency to sensitize MDR cells to daunorubicin and mitoxantrone and could be considered as attractive MDR modulators for cancer treatment. However, their action was time and cell specific.

A new compound Trichomicin exerts antitumor activity through STAT3 signaling inhibition

Trichomicin, a novel small-molecule compound isolated from the fungus Trichoderma harzianum and identified as new structure compound, exhibited antitumor activities in various human cancer cell lines and reversed drug resistance activity in the multidrug-resistant cancer cell line KBV. The underlying cellular and molecular mechanism was illuminated. Trichomicin can significantly induce cancer cell apoptosis and reduced IL-6 expression and phosphorylation of STAT3 were found in response to Trichomicin treatment. The blockade of IL-6 mediated JAK-STAT3 signaling pathway by Trichomicin was confirmed using reporter gene system. As a promising antitumor-activity compound, Trichomicin is presented in this study.

Isolation, Structure Elucidation, and Antiproliferative Activity of Butanolides and Lignan Glycosides from the Fruit of Hernandia nymphaeifolia.

Seven new butanolides, peltanolides A–G (1–7), and two lignan glucosides, peltasides A (8) and B (9), along with eleven known compounds, 10–20, were isolated from a crude CH3OH/CH2Cl2 (1:1) extract of the fruit of Hernandia nymphaeifolia (Hernandiaceae). The structures of 1–9 were characterized by extensive 1D and 2D NMR spectroscopic and HRMS analysis. The absolute configurations of newly isolated compounds 1–9 were determined from data obtained by optical rotation and electronic circular dichroism (ECD) exciton chirality methods. Butanolides and lignan glucosides have not been isolated previously from this genus. Several isolated compounds were evaluated for antiproliferative activity against human tumor cell lines. Lignans 15 and 16 were slightly active against chemosensitive tumor cell lines A549 and MCF-7, respectively. Furthermore, both compounds displayed significant activity (IC50 = 5 µM) against a P-glycoprotein overexpressing multidrug-resistant tumor cell line (KB-VIN) but were less active against its parent chemosensitive cell line (KB).

Leaf Extracts from Dillenia philippinensis Rolfe Exhibit Cytotoxic Activity to both Drug-Sensitive and Multidrug-Resistant Cancer Cells.

Background:Cancer is one of the leading causes of illness and death worldwide. Only palliative therapeutic options are available for many types of cancers, and most anticancer drugs in clinical use exhibit significant side effects. It is therefore important to develop new anticancer drugs that are more effective and less toxic. In this study, we evaluate the bioactivity of a Philippine endemic plant, “katmon” or Dillenia philippinensis, and its potential use in cancer therapy. Methods:The cytotoxicity of the crude leaf extract, partitions, and isocratic column chromatography fractions of Dillenia philippinensis was determined in vitro by MTT assay against drug-sensitive cancer cell lines MCF7 (human breast adenocarcinoma) and HCT 116 (human colorectal carcinoma), as well as against moderately multidrug resistant (MDR) cancer cell line HCT-15 (human colorectal carcinoma) and its highly MDR subline HCT-15/Dox. The selectivity of the extract to cancer cells was determined by computing for the selectivity index (SI) with respect to normal mouse embryonic fibroblasts (NIH/3T3) cell line. To check for a possible mechanism for overcoming cancer multiple drug resistance, Calcein-AM assay was performed to assess the activity of the extract against P-glycoprotein-activated efflux pump. Results: Dillenia philippinensis (DP1) fraction from the hexane partition exhibited cytotoxicity (IC50< 30 µg/ml) against MCF7, HCT 116, HCT-15, and HCT-15/Dox cells. DP1 also exhibited a moderate level of selectivity against cancer cells over normal cells as supported by the SI computed from the IC50 value obtained for the normal cell line. DP1 was able to inhibit P-glycoprotein (P-gp) activity in a dose-dependent manner, suggesting its possible role in targeting cancer cells with overexpressed P-gp. Conclusion:The present findings thus demonstrate the potential chemotherapeutic properties of D. philippinensis which can be promising for future drug development against cancer.

Reversal of drug resistance by JS-K and nitric oxide in ABCB1- and ABCG2-expressing multi-drug resistant human tumor cells

Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of ABC transporter proteins, including P-170 glycoprotein (P-gp), and breast cancer resistance protein (BCRP, ABCG2) have been implicated in this multi-drug resistance (MDR). These ABC transporters are ATP-dependent efflux proteins. We have recently shown that nitric oxide (NO) inhibits the ATPase activities of P-gp, resulting in a significant enhancement of drug accumulation and the reversal of multi-drug resistance in NCI/ADR-RES cells, a P-gp-overexpressing human MDR cell line. In this study, we used [O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)-piperazin-1 yl]-diazene-1-ium-1-2-diolate] (JS-K), a tumor-specific NO-donor to study the reversal of drug resistance in both P-gp- and BCRP-overexpressing human tumor cells. We report here that while JS-K was extremely effective in reversing adriamycin resistance in the P-gp-overexpressing tumor cells (NCI/ADR-RES); it was significantly resistant to BCRP-overexpressing (MCF-7/MX) tumor cells, suggesting that JS-K may be a substrate for BCRP. Using another NO-donor (DETNO), we show that NO directly inhibits the ATP activities of BCRP, inducing significant increases in the accumulations of both Hoechst 33342 dye and topotecan, substrates for BCRP. Furthermore, NO treatment significantly reversed topotecan and mitoxantrone resistance to MCF-7/MX tumor cells. Molecular docking studies indicated that while DETNO and JS-K bind to ATP binding site in both ABC proteins, binding score was significantly reduced, compared to the ATP binding. Our results indicate that appropriately designed NO donors may find success in reversing multidrug resistance in the clinic.

4-Carboxyphenylboronic acid-decorated, redox-sensitive rod-shaped nano-micelles fabricated through co-assembling strategy for active targeting and synergistic co-delivery of camptothecin and gemcitabine

To achieve redox-controlled and tumor active targeting synergistic self-delivery of camptothecin and gemcitabine, redox-sensitive rod-shaped nano-micelles are fabricated through co-assembling between camptothecin-disulfide bond-PEG2000-4-carboxyphenylboronic acid and camptothecin-disulfide bond-gemcitabine conjugate. Most of all, for multidrug resistant cancer cell line MCF-7/ADR which is more resistant against CPT, increasing content of CPT in the formulation is favorable for synergistic effect of CPT and GEM drug combination. Benefiting from simple co-assembling strategy, it is easy and convenient to adjust drug ratio of CPT/GEM to optimize the synergism of drug combination. In addition, nano-micelles fabricated from co-assembling are endowed with both high absolute drug concentration and enhanced colloidal stability, which is helpful to in vivo studies. Transmission electron microscopy observation confirmed the rod-shaped morphology, which is beneficial to cellular internalization, of co-assembled nano-micelles resulting from π-π stacking interactions of CPT moieties and appropriate hydrophilic and hydrophobic interactions during co-assembling. Taking advantages of the specific interactions between 4-carboxyphenylboronic acid and sialic acid, co-assembled nano-micelles exerted enhanced cellular internalization. Noteworthy, compared with cocktail mixture of free CPT and GEM, nano-micelles greatly alleviated drug reflux against MCF-7/ADR and 4T1 cells. The nano-micelles realized redox-controlled ratio-metric and synchronous delivery of CPT and GEM, thereby pronounced in vitro synergistic antiproliferative effect against MCF-7/ADR and 4T1cells. Furthermore, in vivo bio-distribution analysis indicated the preferential accumulation of nano-micelles at tumor site, which could increase therapeutic efficacy and decrease side effects of non-selective anticancer drugs. Taken together, the redox-sensitive CPBA decorated co-assembled nano-micelles provided a promising strategy for tumor active targeting and redox-controlled intracellular synergistic combinational delivery of chemotherapeutics.

New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations.

PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype. In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR). In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR). Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments. Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.