Various Concentrations Of Doxorubicin Research Articles

A mathematical model for predicting the spatiotemporal response of breast cancer cells treated with doxorubicin

ABSTRACT Tumor heterogeneity contributes significantly to chemoresistance, a leading cause of treatment failure. To better personalize therapies, it is essential to develop tools capable of identifying and predicting intra- and inter-tumor heterogeneities. Biology-inspired mathematical models are capable of attacking this problem, but tumor heterogeneity is often overlooked in in-vivo modeling studies, while phenotypic considerations capturing spatial dynamics are not typically included in in-vitro modeling studies. We present a data assimilation-prediction pipeline with a two-phenotype model that includes a spatiotemporal component to characterize and predict the evolution of in-vitro breast cancer cells and their heterogeneous response to chemotherapy. Our model assumes that the cells can be divided into two subpopulations: surviving cells unaffected by the treatment, and irreversibly damaged cells undergoing treatment-induced death. MCF7 breast cancer cells were previously cultivated in wells for up to 1000 hours, treated with various concentrations of doxorubicin and imaged with time-resolved microscopy to record spatiotemporally-resolved cell count data. Images were used to generate cell density maps. Treatment response predictions were initialized by a training set and updated by weekly measurements. Our mathematical model successfully calibrated the spatiotemporal cell growth dynamics, achieving median [range] concordance correlation coefficients of > .99 [.88, >.99] and .73 [.58, .85] across the whole well and individual pixels, respectively. Our proposed data assimilation-prediction approach achieved values of .97 [.44, >.99] and .69 [.35, .79] for the whole well and individual pixels, respectively. Thus, our model can capture and predict the spatiotemporal dynamics of MCF7 cells treated with doxorubicin in an in-vitro setting.

Synergistic cytotoxic effects of an extremely low-frequency electromagnetic field with doxorubicin on MCF-7 cell line

Breast cancer is one of the leading causes of cancer deaths in women worldwide. Magnetic fields have shown anti-tumor effects in vitro and in vivo as a non-invasive therapy method that can affect cellular metabolism remotely. Doxorubicin (DOX) is one of the most commonly used drugs for treating breast cancer patients. It can be assumed that combining chemotherapy and magnetotherapy is one of the most effective treatments for breast cancer. This study aimed to investigate the potential cytotoxic effect of DOX at low concentrations in combination with extremely low-frequency electromagnetic fields (ELF–EMF; 50 Hz; 20 mT). The breast cancer cell line MCF-7 was examined for oxidative stress, cell cycle, and apoptosis. MCF-7 cells were treated with various concentrations of DOX as an apoptosis-inducing agent and ELF–EMF. Cytotoxicity was examined using the MTT colorimetric assay at 12, 24, and 48 h. Consequently, concentration- and time-dependent cytotoxicity was observed in MCF-7 cells for DOX within 24 h. The MTT assay results used showed that a 2 μM concentration of DOX reduced cell viability to 50% compared with control, and as well, the combination of ELF–EMF and DOX reduced cell viability to 50% compared with control at > 0.25 μM doses for 24 h. In MCF-7 cells, combining 0.25 μM DOX with ELF–EMF resulted in increased ROS levels and DOX-induced apoptosis. Flow cytometry analysis, on the other hand, revealed enhanced arrest of MCF-7 cells in the G0-G1 phase of the cell cycle, as well as inducing apoptotic cell death in MCF-7 cells, implying that the synergistic effects of 0.25 μM DOX and ELF–EMF may represent a novel and effective agent against breast cancer.

Hyperthermia Improves Doxorubicin-Based Chemotherapy by Activating Mitochondrial Apoptosis in Bladder Cancer

Purpose: Although intravesical chemotherapy has several antitumoral benefits, it can also have severe side effects. The development of novel therapeutic approaches for bladder cancer (BC) is thus warranted. Hyperthermia (HT) is a widely applicable adjuvant therapy in various cancers. Therefore, this study investigated the effect of HT on improving the chemosensitivity of BC. Materials and Methods: The BC cell lines 5637 and T24 were cultured and treated with HT (43°C) for 24 h. Then, cell viability and survival were assessed using resazurin reagent and colony formation assay, respectively. Western blot assay was used to analyze the levels of Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) protein expression. Mitochondria degradation was observed by MitoTracker Green staining. Results: In BC cells, HT co-administered with various concentrations of doxorubicin significantly inhibited cell viability and survival. Moreover, HT combined with doxorubicin promoted mitochondrial apoptosis, which caused Bax upregulation and Bcl-2 downregulation. Levels of cleaved caspase-3 and PARP protein expression were also elevated after co-treatment. Conclusion: Taken together, HT improved the chemosensitivity of BC cells to doxorubicin. HT combined with chemotherapy further activated mitochondrial apoptosis in BC cells. The findings suggested that HT may serve as a potential adjunctive treatment for BC that is ready to be applied clinically.

Chemosensitizing effect and mechanism of imperatorin on the anti-tumor activity of doxorubicin in tumor cells and transplantation tumor model.

Multidrug resistance of tumors has been a severe obstacle to the success of cancer chemotherapy. The study wants to investigate the reversal effects of imperatorin (IMP) on doxorubicin (DOX) resistance in K562/DOX leukemia cells, A2780/Taxol cells and in NOD/SCID mice, to explore the possible molecular mechanisms. K562/DOX and A2780/Taxol cells were treated with various concentrations of DOX and Taol with or without different concentrations of IMP, respectively. K562/DOX xenograft model was used to assess anti-tumor effect of IMP combined with DOX. MTT assay, Rhodamine 123 efflux assay, RT-PCR, and Western blot analysis were determined in vivo and in vitro. Results showed that IMP significantly enhanced the cytotoxicity of DOX and Taxol toward corresponding resistance cells. In vivo results illustrated both the tumor volume and tumor weight were significantly decreased after 2-week treatment with IMP combined with DOX compared to the DOX alone group. Western blotting and RT-PCR analyses indicated that IMP downregulated the expression of P-gp in K562/DOX xenograft tumors in NOD/SCID mice. We also evaluated glycolysis and glutamine metabolism in K562/DOX cells by measuring glucose consumption and lactate production. The results revealed that IMP could significantly reduce the glucose consumption and lactate production of K562/DOX cells. Furthermore, IMP could also remarkably repress the glutamine consumption, α-KG and ATP production of K562/DOX cells. Thus, IMP may sensitize K562/DOX cells to DOX and enhance the anti-tumor effect of DOX in K562/DOX xenograft tumors in NOD/SCID mice. IMP may be an adjuvant therapy to mitigate the multidrug resistance in leukemia chemotherapy.

Numerical learning of deep features from drug-exposed cell images to calculate IC50 without staining

To facilitate rapid determination of cellular viability caused by the inhibitory effect of drugs, numerical deep learning algorithms was used for unlabeled cell culture images captured by a light microscope as input. In this study, A549, HEK293, and NCI-H1975 cells were cultured, each of which have different molecular shapes and levels of drug responsiveness to doxorubicin (DOX). The microscopic images of these cells following exposure to various concentrations of DOX were trained with the measured value of cell viability using a colorimetric cell proliferation assay. Convolutional neural network (CNN) models for the study cells were constructed using augmented image data; the predicted cell viability using CNN models was compared to the cell viability measured by colorimetric assay. The linear relationship coefficient (r2) between measured and predicted cell viability was determined as 0.94–0.95 for the three cell types. In addition, the measured and predicted IC50 values were not statistically different. When drug responsiveness was estimated using allogenic models that were trained with a different cell type, the correlation coefficient decreased to 0.004085–0.8643. Our models could be applied to label-free cells to conduct rapid and large-scale tests while minimizing cost and labor, such as high-throughput screening for drug responsiveness.

Antimalarial Effect of Doxorubicin on Plasmodium Falciparum: An in Vitro Study in FCR-3 Strain

Plasmodium falciparum is the most common species of Plasmodium that causes malaria in Southeast Asia. Artemisinin, a drug with the mechanism of action by inducing oxidative stress in infected red blood cells (RBC) is currently used as the main therapy for malaria, after resistance to chloroquine has been found. However, evidence of artemisinin resistance was discovered in several regions in Southeast Asia. Therefore, a research is required to prove the existence of other drugs that have anti-malaria effects. A drug candidate, doxorubicin also can induce the formation of oxidative stress inside the cells. This study aims to determine the activity of doxorubicin to inhibit the development of P. falciparum in vitro. Red blood cell (RBC) infected with P. falciparum were treated with various concentrations of doxorubicin. Giemsa technique was applied to detect P. falciparum inside RBC. After 48 hours of incubation, the culture was observed to measure the number and the confluence of RBC and P. falciparum in the medium. This study revealed that doxorubicin reduced the number of RBC infected with P. falciparum lysis. The effective dose of doxorubicin-inhibit RBC cell lysis is 0.4 μM, which only reduces 81% RBC cell lysis compared to the control group that reduces 95% RBC cell lysis. At this concentration also found a decrease in the number of P. falciparum cells in the medium. The results proved that doxorubicin has an inhibitory effect on the development of P. falciparum and can decrease the lysis of RBC due to P. falciparum infection. This findings provide an insight that doxorubicin is a potential candidate for antimalarial drugs.

Enhancing in vitro cytotoxicity of doxorubicin against MCF-7 breast cancer cells in the presence of water-soluble β-cyclodextrin polymer as a nanocarrier agent

This study was aimed to evaluate the impact of water-soluble β-cyclodextrin polymer (β-CDP), as a nanocarrier, on the therapeutic efficacy of the commercial doxorubicin (DOX). The β-CDP was synthesized by cross-linking of β-CD using epichlorohydrin. The chemical structure of the cross-linked β-CDP was verified by NMR and thermogravimetric analysis. The solubility of β-CDP in water was around 880 g in 1000 mL. Next, the DOX/β-CDP was formulated by the addition of the polymer into the various concentrations of DOX in a weight ratio of 1:10 and 1:20 (DOX: β-CDP). The formation of the inclusion complex of DOX with β-CDP was studied by UV–Vis spectroscopy. The UV–Vis spectra of DOX/β-CDP formulations show the appearance of the absorption peak at 480 nm and a significant decrease in the absorbance intensity of DOX signal compared to DOX solution. This verified the formation of the DOX/β-CDP complex and also the presence of free DOX in the DOX/β-CDP formulation. The hydrodynamic size of DOX/β-CDP formulation was obtained in the range of nanometer, and the complexion of DOX by β-CDP leads to a negative shift in the zeta potential. In vitro release profile shows a burst release followed by sustained release of DOX from DOX/β-CDP formulation. This is due to the presence of both free and encapsulated DOX. The cytotoxic effect of DOX solution and DOX/β-CDP formulation was evaluated by MTT assay against breast cancer (MCF-7) cell line. The anticancer activity of the DOX/β-CDP was higher than the DOX.

Real-time physiological sensor-based liver-on-chip device for monitoring drug toxicity

Organ-on-chip models, known as microphysiological systems, are created to mimic the anatomy and physiology of a human organ at the micro-level. Besides being pivotal in the reverse engineering of human organs and pathogenesis studies, they serve as an alternative to animal testing and the development of pharmaceutics. Monitoring the extracellular stromal environment is the basis for gaining in-depth knowledge of the pathophysiology of cell culture. Hence, it is extensively employed as an essential tool in the fields of organ-on-chip and in vitro toxicology. In this study, we explore the vitality of a microfluidic system for the automated, online detection of drug-induced physical changes in cellular viability by continual monitoring of a microfluidic 2D monolayer cell culture. Trans-epithelial electrical resistance (TEER) values and pH changes of the immortal HepG2 cell line were measured continuously using microfluidic-based electrical and photoelectric sensors. A chip-embedded transparent, flat, non-toxic sensor and in-house 3D manufactured portable digital microscope supersedes the conventional manual, expensive confocal microscopic assays, and off-line operated isolated sensor systems. The cytotoxicity was induced by various concentrations of doxorubicin, epirubicin and lapatinib, and the acute metabolic and physical response of cells was examined by detecting the variations in TEER, pH and other biological markers. Thus, our liver-on-chip device provides real-time online data on drug-induced liver injury in vitro.

Adrenomedullin protects rat dorsal root ganglion neurons against doxorubicin-induced toxicity by ameliorating oxidative stress.

Objective(s):Despite effective anticancer effects, the use of doxorubicin (DOX) is hindered due to its cardio and neurotoxicity. The neuroprotective effect of adrenomedullin (AM) was shown in several studies. The present study aimed to evaluate the possible protective effects of AM against DOX-induced toxicity in dorsal root ganglia (DRGs) neurons. Materials and Methods:Rat embryonic DRG neurons were isolated and cultured. The effect of various concentrations of DOX (0.0 to 100 µM) in the absence or presence of AM (3.125 -100 nM) on cell death, apoptosis, oxidative stress, expression of tumor necrosis-α (TNF-α), interleukin1- β (IL-1β), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP) 3 and 13, and SRY-related protein 9 (SOX9) were examined. Results:Based on MTT assay data, DOX decreased the viability of DRG neurons in a dose and time-dependent manner (IC50=6.88 µm) while dose-dependently, AM protected DRG neurons against DOX-induced cell death. Furthermore, results of annexin V apoptosis assay revealed the protective effects of AM (25 nm) against DOX (6.88 µM)-induced apoptosis and necrosis of DRG neurons. Also, AM significantly ameliorated DOX-induced oxidative stress in DRG neurons. Real-time PCR results showed a significant increase in the expression of TNF-α, IL-1β, iNOS, MMP 3, and MMP 13, and a decrease in the expression of SOX9 following treatment with DOX. Treatment with AM (25 nM) significantly reversed the effects of DOX on the above-mentioned genes expression.Conclusion:Our findings suggest that AM can be considered a novel ameliorating drug against DOX-induced neurotoxicity.

Attenuation of Doxorubicin Induced Genotoxicity in HepG2 Cells: Effect of Melatonin Loading Chitosan-Tripolyphosphate Nanoparticles on Oxidative stress

Doxorubicin is a chemotherapy medication applied in the treatment of numerous cancers. Like other topoisomerase II inhibitors, doxorubicin has been shown genotoxic effects in both in vitro and in vivo models. Melatonin acts as a potent antioxidant. In additional, anti-inflammatory, anti-apoptotic, cytoprotective and genoprotective effects of melatonin have been reported in previous studies. The aim of present study was to determine protective role of melatonin nanoparticles against doxorubicin induced-genotoxicity. HepG2 cells were treated with various concentrations of doxorubicin, melatonin and nano melatonin in both pre- and cotreatment conditions and then analyzed via comet assay. Besides the intracellular reactive oxygen species and glutathione levels have been assessed. The results of current study show that doxorubicin induced a clear genotoxic effect in HepG2 cells. Melatonin and its nanoparticles decreased the genotoxic effects of doxorubicin significantly in both types of experiment states that exhibited by comet assay. Furthermore, both forms of melatonin decreased the intracellular reactive oxygen species generation and increased the intracellular glutathione contents in HepG2 cells. However, nano melatonin was more effective in attenuating of oxidative stress and DNA damage induced by doxorubicin.

Hsp90 Inhibitor; NVP-AUY922 in Combination with Doxorubicin Induces Apoptosis and Downregulates VEGF in MCF-7 Breast Cancer Cell Line.

Objective:Breast cancer is one of the most prevalent malignancies and leading causes of females’ mortality worldwide. Because of resistance to various treatment options, new treatments based on molecular targeting has introduced as noticeable strategies in cancer treatment. In this regard, heat shock protein 90 (Hsp90) inhibitors are proposed as effective anticancer drugs. The goal of the study was to utilize a combination of the doxorubicin (DOX) and NVP-AUY 922 on the MCF-7 breast cancer model to investigate the possible cytotoxic mechanisms. Methods:MCF-7 breast cancer cell line was prepared and treated with various concentrations of DOX and NVP-AUY922 in single-drug treatments. We investigated the growth-inhibitory pattern by MTT assay after continuous exposure to NVP-AUY922 and DOX in order to determine dose-response. Then the combinatorial effects were evaluated in concentrations of 0.5 × IC50, 0.2 × IC50, 1 × IC50 and, 2 × IC50 of each drugs. Based on MTT results of double combinations, low effective doses were selected for Real-time PCR [caspase3 and vascular endothelial growth factor (VEGF)] and caspase 3 enzyme activity. Results:A dose-dependent inhibitory effects were presented with increasing the doses of both drugs in single treatments. The upregulation of caspase 3 and downregulation of VEGF mRNA were observed in double combinations of NVP-AUY922 and DOX versus single treatments. Also, in these combinations in low doses of examined drugs (0.5 × IC50, 0.2 × IC50), higher caspase 3 activity were presented in comparison to single treatments (p<0.05). Conclusions:Our findings indicate an effective action of NVP-AUY922 in combined with DOX in this cell line. These results can predict the treatment outcome in this model.

Abstract B106: Engineering cryogels to modulate oxygen tension in reconstructed tumor microenvironments

Abstract Introduction: Hypoxia, defined as low oxygen tension, is a characteristic feature of solid tumors and a hallmark of aggressive cancers. The rapid growth of tumors often results in the development of a hypoxic microenvironment, leading to tumor cell growth and invasion, resistance to apoptosis, and multidrug resistance. Multicellular tumor spheroids (MCTS) have been used to model solid tumors and emulate key aspects of tumor biology such as hypoxia. However, challenges with MCTS formation and reproducibility, inadequate biomechanical cues provided to cells, and uncontrolled oxygen depletion among other limitations led to nonphysiologic tumor cell responses. As a result, predicting clinical responses to anticancer drugs with MCTS remains a major challenge in cancer treatment. To model solid tumors more accurately, our team has recently developed an innovative approach using a cryogel scaffold inducing rapid oxygen depletion while enabling cell rearrangement into 3D spherical cellular aggregates. Our main objectives were: (1) to engineer hypoxia-inducing cryogels (HIC) to generate cellular hypoxia; (2) to provide a biophysical support enabling tumor cell attachment, proliferation, and remodeling; and (3) to evaluate acquired resistance of hypoxic B16-F10 melanoma cells to a common anticancer drug. Methods: HIC were fabricated via a cryogelation process using methacrylated hyaluronic acid (HAGM). RGD peptide, catalase, and glucose oxidase (GO) were incorporated into the gel to promote cell attachment and viability while depleting oxygen. Oxygen depletion was monitored using needle-type oxygen microprobes in the presence or absence of glucose. B16-F10 cell viability was evaluated by confocal imaging. Cellular hypoxia was monitored using a fluorescent hypoxyprobe. Finally, alamarblue assay was performed to evaluate drug resistance of B16-F10 cells to various concentrations of doxorubicin (DOX). Results: We demonstrated that HICs induce a controlled and sustained depletion of oxygen leading to hypoxia (~1% O2) up to 48h. Hypoxia induction is dependent on both GO and glucose contents. Unlike regular cryogels (control), HIC promoted cell remodeling into MCTS-like structure with high cell viability (95 ± 2%), while inducing a substantial level of cellular hypoxia (94 ± 3%) after 24h. Finally, HIC significantly induced melanoma resistance to DOX. With our highest DOX concentration (2μM), 100% and 77% of drug-resistant B16-F10 cells in HIC remained alive after 24h and 48h treatment, respectively. In comparison, all B16-F10 cells died within 48h in our control condition (blank cryogels). Conclusions: We have engineered advanced cryogels, namely HIC, capable of inducing hypoxia while promoting tumor cell remodeling and anticancer drug resistance. This suggests that tumor-laden HIC may mimic key aspects of aggressive tumor microenvironments, making them a promising platform for drug screening and potentially improving preclinical drug discovery and testing. Citation Format: Thibault Colombani, James Sinoimeri, Sidi A. Bencherif. Engineering cryogels to modulate oxygen tension in reconstructed tumor microenvironments [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B106.

L-Lysine increased the cytotoxicity effect of doxorubicin in MDA-MB-231 and MDA-MB-468 breast cancer cell lines

Abstract Background Breast cancer is the first cause of death among Women. Chemoresistance is believed to be responsible for most of the mortalities, thus using new therapeutic strategies are needed. Autophagy has been shown as a critical player in cancer cells response to chemotherapeutic drugs such as doxorubicin. However, autophagy effect on cancer cell can be viewed as a pro-death or a pro-survival role. Thus, increase or decrease of this process in cells relative to its baseline can induce cancer cell death. Thus, we investigated if L-Lysine can increase doxorubicin effectiveness in combination therapy through autophagy modulation. Methods MDA-MB-231 and MDA-MB-468 were used as two breast cancer cell lines. Cells were treated with various concentrations of doxorubicin and L-Lysine. Cell viability was assessed using MTT assay 24 and 48h after treatment. ROS and NO level was examined and the expression of various autophagy and apoptosis related proteins/genes were evaluated with western blotting and real-time PCR respectively. Results L-Lysine increased cytotoxicity effect of doxorubicin in both MDA-MB-231 and MDA-MB-468 by autophagy enhancement. L-Lysine reduced cell viability and increased apoptosis in combination with doxorubicin. Real-time PCR analysis indicated that the combination of these two drugs resulted in increased autophagy as well as apoptosis-related genes. Moreover, reactive oxygen species (ROS) and nitric oxide (NO) production increased 30min after treatment with both drugs. However, the amount of these molecules decreased after 24h which can be results of autophagy induction. These results were further confirmed at protein levels of LC-3 and mTOR by western blotting. Conclusion Our findings indicated that L-Lysine can reduce the resistance to doxorubicin in triple negative breast cancer cells, MDA-MB-231 and MDA-MB-468. Therefore combination therapy with L-Lysine and doxorubicin could be consider an effective strategy in breast cancer therapy.

Quantifying Anticancer Drug Doxorubicin Binding to DNA using Optical Tweezers

Doxorubicin is a successful anticancer drug approved for use in the 1970s and is considered to be one of the most effective cancer treatment methods today. Although Doxorubicin has positive survival statistics it has very negative side effects in many cases. Bleeding from the soles of the palms and feet, along with excruciating pain is often exhibited through the administration of this drug. Based on the preliminary findings utilizing optical tweezers we anticipate that this study will provide critical information about the drug binding mechanism. Single molecule biophysics techniques have provided useful insight into the DNA-binding mechanisms of small molecules. For this reason, in this study we quantify the binding kinetics and affinity of Doxorubicin to DNA using optical tweezers. The optical tweezers allow us to trap a single DNA molecule and manipulate it in the presence of various concentrations of Doxorubicin. Preliminary results suggest that DNA melting facilitates the intercalation process in the nanomolar range, implying a higher binding strength than the previously reported micromolar range.

Carbonic anhydrase IX enhances tumor cell proliferation and tumor progression in osteosarcoma.

PurposeWe investigated the effect of carbonic anhydrase IX (CA IX) inhibitor under hypoxia and normoxia in SaOS2 human osteosarcoma cell line. We also evaluated the expression of CA IX in 27 patients diagnosed with osteosarcoma.Materials and methodsCA IX expression in SaOS2 cells cultured under different oxygen tensions was analyzed by Western blotting. To evaluate the effect of CA IX inhibitor, MTS cell viability assay was performed after cells were treated with various concentrations of doxorubicin with or without a CA IX inhibitor. Finally, CA IX expression in patient-derived osteosarcoma samples was evaluated by immunohistochemistry.ResultsTreatment with CA IX inhibitor significantly suppressed cell proliferation and migration under hypoxic conditions. CA IX expression was observed in 81% of 27 patients. The 5-year survival rates in patients with high and low stain scores were 43.8% and 81.8%, respectively.ConclusionCA IX inhibitors have the potential to suppress cell proliferation, migration, and chemoresistance.

Downregulation of hepatic lipase is associated with decreased CD133 expression and clone formation in HepG2 cells.

The drug resistance of cancer remains a major obstacle to successful chemotherapy. New strategies for improving chemotherapeutic efficacy are urgently required. Recent studies have indicated that LIPC plays a role in promoting the liver metastasis of colorectal cancer. In the present study, we aimed to investigate the effects of LIPC on theproliferation and clone formation of colorectal cancer-derived cells, and chemoresistance in hepatoblastoma-derived HepG2 cells. The activity and expression of LIPC were determined in the cell lines by RT-qPCR and western blot analysis. HepG2 cells in which LIPC was knocked down by LIPC short hairpin RNA(shRNA) and control cells [shRNA control(shCON)] were established and analyzed for cell proliferation and colony formation rates. FACS analysis was used to explore the association between LIPC and the tumor-derived cell biomarker, CD133, and the percentages of CD133-positive cells were assessed by FACS. Additionally, shLIPC- and shCON-transfected cells were treated with various concentrations of doxorubicin and 5-floxuridine(5-FU), and cell viability was determined by MTT assay. mRNA levels in the shLIPC- and shCON-transfected cells were compared by cDNA microarray and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. The results revealed that the HepG2 cells exhibited a relatively higher LIPC activity and expression levels compared to the other colon cancer cell lines. The downregulation of LIPC in the HepG2 cells was associated with the decreased expression of CD133, decreased cell proliferation and colony formation, as well as increased resistance to chemotherapy. KEGG analysis of the cDNA microarray data revealed increased levels in the cell adhesion molecule(CAM) pathway, including CLDN10 and CLDN1, indicating that CAMs may play a role in LIPC-mediated tumor progression. The present findings indicate a potential role of LIPC as a promising therapeutic target in cancer.

Epidermal growth factor (EGF) fragment-guided anticancer theranostic particles for pH-responsive release of doxorubicin

EGF fragment (EGFfr) and doxorubicin were chemically co-decorated on single magnetic nanoparticles (MNPs) for concomitant cancer targeting and treatment. Magnetic nanoparticles were prepared by the precipitation of ferric chloride hydrates in an ammonia solution and subsequent surface-functionalization with amines. The terminal thiol group of the EGF fragment was first conjugated to surface amines of the MNPs using a heterofunctional crosslinker, and doxorubicin was sequentially conjugated to the MNPs via a hydrazone linker, where the degree of subsitution of the surface amines to EGFfr was varied from 1% to 40%. The decorated doxorubicin showed clear pH-dependency in the release profile, and doxorubicin showed fast release at pH 5.0 in comparison to pH 7.4. The EGF-decorated MNPs were tested for differential cellular uptakes against EGF overexpressing cells (A549), and the uptake levels gradually increased to 10% and saturated, which was quantified by ICP-OES. Internalized doxorubicin was also visualized by confocal microscopy, and A549 cells with EGF-decorated MNPs with EGF decoration showed higher fluorescence intensity of doxorubicin than those with non-decorated MNPs. Anti-cancer activity of the MNPs was compared at various concentrations of doxorubicin and EGFfr. Decoration of EGFfr significantly increased the anti-cancer activity of doxorubicin-incorporated MNPs in A549 cells; however, EGFfr alone did not affect the viability of the cells. Thus, we concluded that MNPs with optimized EGFfr and doxorubicin ratios showed higher targeting and drug payload against EGF receptor overexpressing cancer cells.

M-DNA/Transition Metal Dichalcogenide Hybrid Structure-based Bio-FET sensor with Ultra-high Sensitivity.

Here, we report a high performance biosensor based on (i) a Cu2+-DNA/MoS2 hybrid structure and (ii) a field effect transistor, which we refer to as a bio-FET, presenting a high sensitivity of 1.7 × 103 A/A. This high sensitivity was achieved by using a DNA nanostructure with copper ions (Cu2+) that induced a positive polarity in the DNA (receptor). This strategy improved the detecting ability for doxorubicin-like molecules (target) that have a negative polarity. Very short distance between the biomolecules and the sensor surface was obtained without using a dielectric layer, contributing to the high sensitivity. We first investigated the effect of doxorubicin on DNA/MoS2 and Cu2+-DNA/MoS2 nanostructures using Raman spectroscopy and Kelvin force probe microscopy. Then, we analyzed the sensing mechanism and performance in DNA/MoS2- and Cu2+-DNA/MoS2-based bio-FETs by electrical measurements (ID-VG at various VD) for various concentrations of doxorubicin. Finally, successful operation of the Cu2+-DNA/MoS2 bio-FET was demonstrated for six cycles (each cycle consisted of four steps: 2 preparation steps, a sensing step, and an erasing step) with different doxorubicin concentrations. The bio-FET showed excellent reusability, which has not been achieved previously in 2D biosensors.

Abstract LB-223: Intratumoral accumulation/entrapment of doxorubicin by tumor vascular infarction with retargeted tissue factor tTF-NGR

Abstract Truncated tissue factor (tTF) retargeted to tumor vasculature by C-terminal GNGRAHA peptide (tTF-NGR) is able to induce tumor vessel infarction. Here, we describe the identification of the optimum therapeutic regimen for the use of tTF-NGR (or a random-PEGylated derivative of tTF-NGR) in combination with the chemotherapeutic agent doxorubicin in vitro and in vivo. First, we treated human HT1080 xenografts subcutaneously transplanted in athymic CD-1 mice with doxorubicin (5 mg/kg body weight (bw), i.v.) and excised the tumors at different times after injection. Fluorescence spectrometric analyses of the autofluorescence of doxorubicin revealed that intratumoral maximum of doxorubicin accumulation was reached approximately 6 hours after injection. While tTF-NGR given before doxorubicin inhibits the uptake of the latter into HT1080 human fibrosarcoma xenografts in vivo, the reverse sequence does not influence the uptake of doxorubicin in tumor tissue, but significantly inhibits the late wash-out phase entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with various concentrations of doxorubicin in vitro increases concentrations of phosphatidylserine (PS) in the outer layer of the lipid bilayer induced by pro-apoptotic stress. In Factor X activation kinetics, both HUVEC and HT1080 cells showed a significantly higher procoagulatory efficacy when used with equimolar concentrations of tTF-NGR upon doxorubicin incubation and higher PS externalization than identical numbers of control cells with low PS externalization. For proof of a causal relation between doxorubicin-induced PS externalization and procoagulant behaviour of the cells, we could show that preincubation with an phosphatidylserine-inhibiting antibody completely abolished this elevated procoagulatory state of the cells. Thus, we hypothesized that both drugs by the reciprocal mechanisms described mutually increase their therapeutic anti-tumor efficacy in vivo. Therapeutic experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in CD1 nude mice revealed a significantly increased tumor growth inhibition applying suboptimal doses of doxorubicin and tTF-NGR in sequence over single drug treatment. In conclusion, combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by a new mechanism, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in endothelial and tumor cells. Citation Format: Janine Stucke-Ring, Julian Timo Ronnacker, Caroline Brand, Verena Mantke, Carsten Höltke, Christoph Schliemann, Torsten Kessler, Rolf M. Mesters, Christian Schwöppe, Wolfgang E. Berdel. Intratumoral accumulation/entrapment of doxorubicin by tumor vascular infarction with retargeted tissue factor tTF-NGR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-223.

Doxorubicin activates the Notch signaling pathway in osteosarcoma.

Notch signaling is critical in various biological processes, including cell proliferation, differentiation and apoptosis. Furthermore, accumulating evidence indicated that aberrant Notch signaling has a tumor-promoting function in osteosarcoma. However, the effect of the conventional chemotherapeutic agent, doxorubicin, on Notch signaling remains unclear. In the present study, osteosarcoma cells were treated with various concentrations of doxorubicin and the effect on Notch signaling was analyzed. A cytostatic dose of doxorubicin (<0.5 µM) was identified to significantly activate the Notch signaling pathway in a dose-dependent manner (P<0.01), as demonstrated by the elevated expression levels of Notch target genes. However, a toxic dose of doxorubicin (≥0.5 µM) significantly inhibited the Notch signaling pathway (P<0.01). These results indicated a significant correlation between doxorubicin administration and the Notch signaling pathway. Therefore, the present study supports further investigation into Notch and osteosarcoma chemoresistance.