Different Concentrations Of Doxorubicin Research Articles

GMI-based biosensor for the detection and quantification of doxorubicin anticancer drugs labeled to Fe3O4 superparamagnetic nanoparticles

We present the results of research on a Co-based giant magnetoimpedance (GMI) biosensor for Fe3O4-labelled doxorubicin (DOX) anticancer drugs. Co62Fe5Ni4Si15B14 ribbons were used as biosensing material for test solutions containing different concentrations of DOX. The plain ribbon, ribbon+Fe3O4 and ribbon+DOX2 samples exhibited low coercivity (Hc), high saturation magnetization (Ms) and low anisotropy (Hk). The GMI ratio (ΔZ/Z) increased from 33% to 38% with the increase of doxorubicin content in the ribbon. It was found that the ΔZ/Z ratio first increased rapidly up to a frequency of ∼2 MHz, and then decreased slowly as the frequency increased. While the magnetic field dependent ΔZ/Z has a single peak at 1 MHz, it has a double peak at 5 MHz. It was found that the peak height of ΔZ/Z in the ribbons with test solution of 20 ng/ml and 50 ng/ml increased by ∼3% and ∼6%, respectively, compared to the ribbon without test solution. Then the ΔZ/Z ratio decreased very rapidly with the increase of the applied external magnetic fields. The frequency-dependent GMI sensitivity (η) increased with increasing frequency and reached a maximum value at a critical frequency (∼1.6 MHz), after which it decreased. The detection sensitivity of the GMI biosensor (ξ) increased rapidly with increasing doxorubicin concentration up to ∼80 ng/ml and then remained almost unchanged. The results show that doxorubicin (DOX+Fe3O4), used as an anticancer drug, can be effectively detected even at low concentrations using the GMI based sensors fabricated in this study.

Effect of Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells in 3D Culture.

Chemotherapy is one of the most common strategies for cancer treatment, whereas drug resistance reduces the efficiency of chemotherapy and leads to treatment failure. The mechanism of emerging chemoresistance is complex and the effect of extracellular matrix (ECM) surrounding cells may contribute to drug resistance. Although it is well known that ECM plays an important role in orchestrating cell functions, it remains exclusive how ECM stiffness affects drug resistance. In this study, we prepared agarose hydrogels of different stiffnesses to investigate the effect of hydrogel stiffness on the chemoresistance of breast cancer cells to doxorubicin (DOX). Agarose hydrogels with a stiffness range of 1.5 kPa to 112.3 kPa were prepared and used to encapsulate breast cancer cells for a three-dimensional culture with different concentrations of DOX. The viability of the cells cultured in the hydrogels was dependent on both DOX concentration and hydrogel stiffness. Cell viability decreased with DOX concentration when the cells were cultured in the same stiffness hydrogels. When DOX concentration was the same, breast cancer cells showed higher viability in high-stiffness hydrogels than they did in low-stiffness hydrogels. Furthermore, the expression of P-glycoprotein mRNA in high-stiffness hydrogels was higher than that in low-stiffness hydrogels. The results suggested that hydrogel stiffness could affect the resistance of breast cancer cells to DOX by regulating the expression of chemoresistance-related genes.

Doxorubicin/poly(caprolactone)/polyethylene glycol nanoparticles prepared by electrospray

AbstractThe development of sustainable drug delivery systems is very important in the field of pharmaceutical research and has many advantages over the usual methods of drug administration, including improving effectiveness, reducing side effects, and increasing patient compliance. Among various techniques, electrospraying as a voltage‐driven process has emerged as a promising technology for the fabrication of drug‐loaded nanoparticles. In recent years, the application of electrospray approach for drug release has been widely investigated. However, optimization of electrospray process parameters and development of mathematical models to predict drug release kinetics from microparticles are still challenging. To address these issues, a combination of Central Composite Design (CCD) and Response Surface Methodology (RSM) has been used. This paper focuses on the application of CCD‐RSM for modeling and optimization of doxorubicin (DOX)‐poly(caprolactone) (PCL)‐polyethylene glycol (PEG) microparticles prepared using electrospray method. Doxorubicin, a widely used anticancer drug, exhibits potent cytotoxicity against a wide range of cancers. PCL‐PEG microparticles are promising as drug carriers due to their biocompatibility, controlled release properties, and ability to protect the encapsulated drug from degradation. The CCD‐RSM approach allows systematic investigation and optimization of several formulation and process variables, including polymer concentration, PCL‐PEG weight ratio, and applied voltage on drug release behavior. In addition, the effect of rheology and electrical conductivity of polymer fluid on the size of particles obtained from electrospraying was investigated. The effect of adding folic acid and doxorubicin in the optimized sample of nanoparticles and the effect of different concentrations of doxorubicin were investigated, and SEM, DLS, and FTIR techniques were used to characterize the microparticles, and the MMT test was performed to evaluate their cytotoxicity. The results showed that the optimized formulation is effective for sustained drug release over a long period for drug delivery applications.

Doxorubicin-induced senescence promotes resistance to cell death by modulating genes associated with apoptotic and necrotic pathways in prostate cancer DU145 CD133+/CD44+ cells

Cancer stem cells (CSCs) are the most important cause of cancer treatment failure. Traditional cancer treatments, such as chemotherapy and radiotherapy, damage healthy cells alongside malignant cells, leading to severe adverse effects. Therefore, inducing cellular senescence without triggering apoptosis, which further damages healthy cells, may be an alternative strategy. However, there is insufficient knowledge regarding senescence induction in CSCs that show resistance to treatment and stemness properties. The present study aims to elucidate the effects of senescence induction on proliferation, cell cycle, and apoptosis in prostate CSCs and non-CSCs. Prostate CSCs were isolated from DU145 cancer cells using the FACS method. Subsequently, senescence induction was performed in RWPE-1, DU145, prostate CSCs, and non-CSCs by using different concentrations of Doxorubicin (DOX). Cellular senescence was detected using the senescence markers SA-β-gal, Ki67, and senescence-associated heterochromatin foci (SAHF). The effects of senescence on cell cycle and apoptosis were evaluated using the Muse Cell Analyzer, and genes in signaling pathways associated with the apoptotic/necrotic pathway were analyzed by real-time PCR. Prostate CSCs were isolated with 95.6 ± 1.4% purity according to CD133+/CD44+ characteristics, and spheroid formation belonging to stem cells was observed. After DOX-induced senescence, we observed morphological changes, SA-β-gal positivity, SAHF, and the lack of Ki67 in senescent cells. Furthermore; we detected G2/M cell cycle arrest and downregulation of various apoptosis-related genes in senescent prostate CSCs. Our results showed that DOX is a potent inducer of senescence for prostate CSCs, inhibits proliferation by arresting the cell cycle, and senescent prostate CSCs develop resistance to apoptosis.

Metabolomic Signatures in Doxorubicin-Induced Metabolites Characterization, Metabolic Inhibition, and Signaling Pathway Mechanisms in Colon Cancer HCT116 Cells.

Doxorubicin (DOX) is a chemotherapeutic agent is used for various cancer cells. To characterize the chemical structural components and metabolic inhibition, we applied a DOX to HCT116 colon cancer cells using an independent metabolites profiling approach. Chemical metabolomics has been involved in the new drug delivery systems. Metabolomics profiling of DOX-applied HCT116 colon cancer cellular metabolisms is rare. We used 1H nuclear magnetic resonance (NMR) spectroscopy in this study to clarify how DOX exposure affected HCT116 colon cancer cells. Metabolomics profiling in HCT116 cells detects 50 metabolites. Tracking metabolites can reveal pathway activities. HCT116 colon cancer cells were evenly treated with different concentrations of DOX for 24 h. The endogenous metabolites were identified by comparison with healthy cells. We found that acetate, glucose, glutamate, glutamine, sn-glycero-3-phosphocholine, valine, methionine, and isoleucine were increased. Metabolic expression of alanine, choline, fumarate, taurine, o-phosphocholine, inosine, lysine, and phenylalanine was decreased in HCT116 cancer cells. The metabolic phenotypic expression is markedly altered during a high dose of DOX. It is the first time that there is a metabolite pool and phenotypic expression in colon cancer cells. Targeting the DOX-metabolite axis may be a novel strategy for improving the curative effect of DOX-based therapy for colon cancer cells. These methods facilitate the routine metabolomic analysis of cancer cells.

The Impairment of Cell Metabolism by Cardiovascular Toxicity of Doxorubicin Is Reversed by Bergamot Polyphenolic Fraction Treatment in Endothelial Cells.

The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted.

Effects of hsa-miR-28-5p on Adriamycin Sensitivity in Diffuse Large B-Cell Lymphoma.

Background Adriamycin (doxorubicin) is an important traditional drug that exhibits cytotoxicity in Diffuse Large B-cell Lymphoma (DLBCL). Doxorubicin affects the DLBCL cells at all stages of their cell cycle. Combined with our previous results, this study discovered that the overexpression of hsa-miR-28-5p inhibited the proliferation, promoted apoptosis, and triggered cell cycle arrest at the S-phase in DLBCL cells. However, the effect of (Homo sapiens, hsa)-microRNA (miR)-28-5p on doxorubicin sensitivity in DLBCL has not been investigated. This study aims to reveal the effects of hsa-miR-28-5p on doxorubicin sensitivity at the level of DLBCL cells. Methods To determine the optimal concentration of doxorubicin, different concentrations of doxorubicin were used to treat DLBCL cells. CCK-8 assay was used to detect the proliferation of DLBCL cells. The hsa-miR-28-5p-mimic NC and hsa-miR-28-5p mimic were transfected to doxorubicin-mediated DLBCL cells. Simultaneously, blank control groups were set up. The cells were cultured and transfected for 24 h. Next, each group was administered with different concentrations of doxorubicin and cultured again for 24 h to observe the effects of hsa-miR-28-5p on doxorubicin sensitivity at different times. The proliferation, early apoptosis, and late apoptosis in DLBCL cells were determined using soft agar colony-forming assay, mitochondrial membrane potential assay, and caspase-3 activity assay, respectively. The apoptosis and cell cycle were explored using Annexin V-PE/7-AAD and PI/RNase staining buffer, respectively. We speculated that PD-L1 might be involved in the effect of hsa-miR-28-5p on the sensitivity of adriamycin (doxorubicin) in the DLBCL cells. Hence, we performed immunohistochemistry (IHC) to determine PD-L1 expression within formalin-fixed paraffin-embedded (FFPE) samples from 52 DLBCL cases. Results The optimal concentration of doxorubicin targeting DLBCL cells was found to be 3.028 μmol/l. The effect of doxorubicin on DLBCL cells was time- and concentration-dependent. hsa-miR-28-5p mimic + doxorubicin remarkably decreased proliferation of DLBCL. DLBCL cell apoptosis rate was the highest in hsa-miR-28-5p mimic + doxorubicin group. Apart from that, hsa-miR-28-5p mimic plus doxorubicin had the best effect in promoting DLBCL cell apoptosis. After the intervention of hsa-miR-28-5p mimic + doxorubicin on DLBCL cells, the cell cycle was arrested in the S-phase and DNA synthesis was blocked. hsa-miR-28-5p mimic + doxorubicin could regulate the cycle of DLBCL cells. As a result, overexpression of hsa-miR-28-5p combined with doxorubicin is possibly involved in the development of DLBCL by affecting the proliferation, apoptosis, and cycle of DLBCL cells. PD-L1 showed an association with the prognosis of DLBCL patients. Combining with the literature, this suggested hsa-miR-28-5p may influence DLBCL occurrence and therapeutic effect by regulating the PD-L1 level. Conclusion The combination of hsa-miR-28-5p mimic and doxorubicin may be considered more effective in inhibiting growth, arresting the cell cycle, and promoting cell apoptosis of DLBCL cells compared to using doxorubicin alone. The effects of doxorubicin on DLBCL cells were found to be time- and concentration-dependent. The overexpression of hsa-miR-28-5p enhanced the effect of doxorubicin on DLBCL cells, which may be attributed to the regulation of PD-L1 levels.

Extracellular Vesicles Carrying miR-887-3p Promote Breast Cancer Cell Drug Resistance by Targeting BTBD7 and Activating the Notch1/Hes1 Signaling Pathway.

Objective Chemoresistance remains the primary reason threatening the prognosis of breast cancer (BC) patients. Extracellular vesicles (EVs) contribute to chemoresistance by carrying microRNAs (miRNAs). This study investigated the mechanism of miR-887-3p mediated by EVs in BC cell drug resistance. Methods MDA-MB-231-derived EVs were extracted and identified. BC cells were treated with different concentrations of doxorubicin, cisplatin, and fulvestrant, and the cell survival was evaluated. PKH26-labeled EVs were cocultured with BC cells, and the uptake of EVs was observed. miR-887-3p expression in BC cells and EVs was detected. After silencing miR-887-3p in MDA-MB-231 cells, BC cells were treated with EV-inhi to observe drug resistance. The target gene of miR-887-3p was predicted and verified. The levels of downstream Notch1/Hes1 pathway were detected. Xenograft experiment was conducted to evaluate the effect of EVs on the growth and drug resistance in vivo. Results MDA-MB-231-derived EVs enhanced the drug resistance of BC cells. EVs carried miR-887-3p into BC cells. miR-887-3p expression was elevated in BC cells and EVs. miR-887-3p inhibition reduced the drug resistance of BC cells. miR-887-3p targeted BTBD7. Overexpression of BTBD7 partially reversed the drug resistance of BC cells caused by EV treatment. EV treatment increased the level of Notch1/Hes1, and overexpression of BTBD7 decreased the level of Notch1/Hes1. In vivo experiments further validated the results of in vitro experiments. Conclusion EVs carrying miR-887-3p could target BTBD7 and activate the Notch1/Hes1 signaling pathway, thereby promoting BC cell drug resistance. This study may offer novel insights into BC treatment.

Synergistic anti-cancer effects of Nigella sativa seed oil and conventional cytotoxic agent against human breast cancer.

Breast cancer is the most commonly diagnosed invasive non-skin malignancy in women worldwide, and it is the leading cause of cancer-related deaths in them. Nigella sativa Linn. seed oil has been found to be effective in cancer treatment as well as having anti-cancer properties in some other types of cancers. The study looked into the synergistic cytotoxic effects of N.sativa Linn. seed oil and doxorubicin in the treatment of human breast cancer cells (MCF-7). Nigella sativa Linn. seed oil was used to evaluate its effect on human breast cancer cells, either alone or in conjunction with doxorubicin. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to examine cell proliferation and cell viability, while phase-contrast inverted microscopy was used to examine cellular morphology. Furthermore, the role of N.sativa seed oil in decreasing cell tumorigenicity features was highlighted by testing the cancer cell migration using the wound healing assay. Results showed that higher concentrations (50μg/mL) of N.sativa Linn. seed oil changed the breast cancer cell morphology and decreased the cell proliferation and viability. Breast cancer cells treated with black seed oil decreased cell movement after 24hours compared to the untreated cell in the wound healing assay. Whereas, only the higher concentration of doxorubicin (0.5-2.5μg/mL) reduced cell proliferation and cell viability. Moreover, the combination treatment of 50μg/mL of black seed oil with different concentrations of doxorubicin caused a significant cell proliferation reduction and decreased cell viability. The activity was seen optimum at lower concentration (0.1µg/mL) of doxorubicin. There was decreased cell proliferation and cell viability when N.sativa seed oil was used alone or in conjunction with doxorubicin in Breast cancer cells (MCF-7) revealing potential opportunities in the field of cancer treatment.

Ozone combined with doxorubicin exerts cytotoxic and anticancer effects on Luminal-A subtype human breast cancer cell line.

We aimed to examine the potential anticancer effects of ozone applied after chemotherapeutic treatment with different concentrations of doxorubicin in Luminal-A subtype of human breast cancer cell line (MCF-7) and compare the results with effects on L929 fibroblast cell line. Both cell lines were incubated with increasing doses of doxorubicin (1-50 μM) for 24 h at 37°C. Then, half of groups were incubated with 30 μg/mL ozone for 25 min as combination groups. Cell viability was analyzed by MTT assay, apoptosis by flow cytometry, and levels of tumor necrosis factor alpha, transforming growth factor beta, and matrix metalloproteinase-2 and MMP-9 by immunocytochemistry. Doxorubicin + ozone treatment enhanced viability of L929 (p<0.01) but reduced viability of MCF-7 compared to only doxorubicin-applied cells without ozone treatment (p<0.001). This combined treatment also enhanced apoptotic effect of doxorubicin on MCF-cells (p<0.001), but not on L929. It significantly increased all protein levels of L929 compared with those of other groups (p<0.05 for tumor necrosis factor alpha and MMP-2; p<0.01 for transforming growth factor beta and MMP-9). This treatment reversed the effect of doxorubicin on tumor necrosis factor alpha levels and considerably reduced MMP-2 and MMP-9 levels of MCF-7 compared with those of control group (p<0.01 and p<0.001, respectively). Ozone treatment potentiated the apoptotic and anticancer activities of doxorubicin in MCF-7 cells and showed repairing and healing effect on healthy fibroblast cells, which were damaged from cytotoxic effects of chemotherapeutic agent. MCF-7 cells may acquire sensitivity against the doxorubicin combined with ozone treatment through activating tumor necrosis factor alpha, MMP-2, and MMP-9 expressions.

Using mathematical modeling to estimate time-independent cancer chemotherapy efficacy parameters.

One of the primary cancer treatment modalities is chemotherapy. Unfortunately, traditional anti-cancer drugs are often not selective and cause damage to healthy cells, leading to serious side effects for patients. For this reason more targeted therapeutics and drug delivery methods are being developed. The effectiveness of new treatments is initially determined via in vitro cell viability assays, which determine the of the drug. However, these assays are known to result in estimates of that depend on the measurement time, possibly resulting in over- or under-estimation of the . Here, we test the possibility of using cell growth curves and fitting of mathematical models to determine the as well as the maximum efficacy of a drug ( ). We measured cell growth of MCF-7 and HeLa cells in the presence of different concentrations of doxorubicin and fit the data with a logistic growth model that incorporates the effect of the drug. This method leads to measurement time-independent estimates of and , but we find that is not identifiable. Further refinement of this methodology is needed to produce uniquely identifiable parameter estimates.

Cytotoxic and Radiosensitizing Effects of Folic Acid-Conjugated Gold Nanoparticles and Doxorubicin on Colorectal Cancer Cells.

Purpose: Radiotherapy is one of the most important therapeutic options used to treat cancers. Radiation effects can be improved using nanoparticles and chemotherapeutic drugs as radiosensitizing agents. The aim of the present study was to evaluate the effects of folic acid-conjugated gold nanoparticles (GNP-F) in combination with doxorubicin (DOX) and x-Ray irradiation in colorectal cancer (CRC) cell line (HT-29). Methods: The cell viability assay (WST-1) was performed to study the cytotoxic effects of different concentrations of DOX and GNP-F after 24 and 48 hours treatments. Then, the effects of the GNP-F, X-Ray irradiation, and DOX drug in single and combined treatments were examined after 24 and 48 hours treatment with effective doses. Likewise, the caspase 3 gene expression ratio and the caspase 3 activity were assessed after 48 h treatment. Moreover, the malondialdehyde (MDA) level was determined in treated and untreated cells. Results: When GNP-F (at a concentration of 70 μM) was combined with X-ray irradiation (2 Gy) and DOX drug, induced more cytotoxic effects compared to the control group. The results of cell viability assay showed that GNP-F + X-Ray in combination with a low concentration of DOX (0.25 × IC50) enhanced the cytotoxic effects of cells compared to related single treatments. Caspase 3 gene expression ratio and caspase 3 activity increased in double and triple combination treatments in comparison with the single groups. Moreover, the MDA level increased in triple combination compared to the single treatments. Conclusion: Our findings confirmed the potential anti-cancer effects of the GNP-F and DOX in combination with X-Ray irradiation in CRC cells.

The Synergistic Anticancer Traits of Graphene Oxide Plus Doxorubicin Against BT474 and MCF7 Breast Cancer Stem Cells In Vitro.

Breast cancer is among the leading causes of death due to cancers around the globe. Current therapeutic approaches towards healing of breast cancer have been associated with poor outcomes. Graphene and its derivatives have a two-dimensional flat structure, which is characterized by the ability to carry drugs and modify the surface, low cytotoxicity, and high biocompatibility. This study was performed on MCF7 and BT474 human breast cancer cells. Different concentrations of doxorubicin (DOX), graphene oxide (GO), and graphene oxide plus doxorubicin (GO-DOX) were subjected to both cell lines at specified intervals. At the end of the treatments, MTT test was applied to determine the viability of cells, and then flow cytometry, colony formation, and spheroid tests were implemented in both cell lines treated with DOX, GO, and GO-DOX components. We used DLS and TEM to confirm the GO properties. According to the MTT test results, 1 μL of DOX at 10mg/ml (equivalent to 0.1mg/ml) caused 50% survival of MCF7 cells at 24h. In both cell lines, an increase in apoptosis occurred after incubation with GO and DOX. Although a rate of mortality of MCF-7 cells was due to necrosis, the BT474 cell death was merely through the apoptosis. Furthermore, the results of the colony formation test outlined an enhancing inhibitory effect in the presence of GO-DOX as a comparison to the control. Additionally, spheroids formed following treatment with GO-DOX exhibited a significant decrease compared to their control group, with an increase in the number of spheroids in BT474 cells compared to those in the MCF-7. The decreasing effect of compounds against the migration and cell invasion potential was also observed, being higher in MCF7 than BT474 cells. The effects of cytotoxic GO were observed at higher concentrations.

The effect of bee bread (Perga) with chemotherapy on MDA-MB-231 cells.

Bee bread (BB) is a bee product like propolis and honey. It is the main food for larvae and bees producing royal jelly in the hive. It also known as Perga. As with other bee products, it is increasingly popular due to its antioxidant properties. The aim of this study was to examine the effects of BB on MDA-MB-231 breast cancer cells and the effects on these cells when administered together with Doxorubicin (DOX) and Cisplatin (CDDP), used in cancer treatment. The proliferation of the cells was determined by applying 5mg/mL BB together with different concentrations of DOX and CDDP. In addition to these studies, the effect of DOX+BB and CDDP+BB combinations on the migration of MDA-MB-231 cells was determined by the wound healing method. The expression levels of Bid and Bcl-2 were determined by RtqPCR. According to these studies, as expected, BB did not show a significant toxic effect on MDA-MB-231 cells at different concentrations. BB significantly suppressed the effect of DOX and CDDP on the proliferation of MDA-MB-231 cells. BB with DOX and CDDP suppressed the proapoptotic Bid gene while overexpressing the anti-apoptotic Bcl-2 gene, separately. Interestingly, BB blocked the migration of MDA-MB-231 cells by 50% even after 72h. As a result, BB significantly reduced the toxicity of DOX and CDDP on MDA-MB-231 cells. The most interesting result of the study is that BB prevented the migration of cancer cells.

Development and validation of an in-silico tool for the study of therapeutic agents in 3D cell cultures

Computational models constitute a fundamental asset for cancer research and drug R&D, as they provide controlled environments for testing of hypotheses and are characterized by the total knowledge of the system. These features are particularly useful for 3D cell culture models where a complex interaction among cells and their environments ensues.In this work, we present a programmable simulator capable of reproducing the behavior of cells cultured in 3D scaffolds and their response to pharmacological treatment. This system will be shown to be able to accurately describe the temporal evolution of the density of a population of MDA-MB-231 cells following their treatment with different concentrations of doxorubicin, together with a newly described drug-resistance mechanism and potential re-sensitization strategy. An extensive technical description of this model will be coupled to its experimental validation and to an analysis aimed at identifying which variables and behaviors account for differences in the response to treatment.Comprehensively, this work contributes to the growing field of integrated in-silico/in-vitro analysis of biological processes which has great potential for both the increase of our scientific knowledge and the development of novel, more effective treatments.

Abstract 13783: Disease Modeling Doxorubicin-induced Cardiotoxicity Using Human Induced Pluripotent Stem Cells-derived Cardiomyocytes From Healthy and Type 2 Diabetic Patients

Introduction: Doxorubicin-Induced Cardiotoxicity (DIC) is one of the most common clinical complications in cancer patients. Patients with diabetes have a higher incidence of breast cancer, and their risks of developing DIC is also higher as compared to healthy patients. The human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) have the unique potential to recapitulate the genetic background and have emerged as a powerful tool for disease modeling and drug screening. Hypothesis: We hypothesized that cardiomyocytes derived from T2 diabetic patients will recapitulate greater sensitivity to DIC as compared to CMs derived from non-diabetic individuals. Methods: The patient specific iPSCs were differentiated into CMs with modifications to enhance cardiomyocyte purity (Fig. 1A, n=4 cell lines in each group). After 30 days from the initial differentiation, CMs were exposed to different concentrations of doxorubicin (DOX) based on the pharmacokinetic characteristics of doxorubicin in humans. The DOX-induced stress on the CMs after 24h and 48h was investigated via the analysis of 1) Cellular LDH release 2) Cellular viability using Alamar Blue assay and 3) CM-markers using real-time PCR. Results: Following the treatment with DOX the (iPSC)-derived cardiomyocytes (CMs) from the diabetic patients were more sensitive as compared to control CMs by: 1) An increase in the LDH release, Fig. 1B, 2) Decrease in the cellular viability, Fig. 1C, and 3) Greater downregulation of cardiac specific structural genes including Troponin T and Troponin I mRNA expression. Conclusions: For the first time, we demonstrate the ability of hiPSC-CMs derived from diabetic individuals to recapitulate greater sensitivity to DOX in vitro , validating this platform for predicting DIC severity in vivo .

AML-369: Studying Drug Resistance: NB4 as an APL Model

Context The NB4 cell line has been used as an in vitro model of acute promyelocytic leukemia (APL) in many studies. Current treatment paradigms call for the administration of ATRA and ATO as differentiating agents in patients with newly diagnosed APL. For a subset of patients, resistance to conventional treatment remains a clinical issue and is poorly understood. Objective The purpose of this study was to evaluate whether resistance to ATRA and ATO can influence the susceptibility of APL cells to anthracycline therapy like doxorubicin using the NB4 cell line as an in vitro model. Design Wild type NB4 cells as well as NB4 cells resistant to both ATRA and ATO (NB4-RAA) were cultured in suspension in RPMI and 10% fetal calf serum. NB4-RAA were established in Dr. Jimenez's lab from the wild type NB4 parent cell line. Resistance was achieved after exposing NB4 cells to increasing concentrations of ATRA and ATO over a period of twelve months. NB4 and NB4-RAA were plated in multi-wells at 50,000 cells/mL and exposed to different concentrations of doxorubicin. Cell counts were taken at 24 and 48 hours after treatment. Results NB4 and NB4-RAA were exposed to 0 μg/mL (Control), 0.001 μg/mL, 0.01 μg/mL, 0.05 μg/mL, and 0.1 μg/mL. The viability of both cell lines decreased at similar levels at 24 h and 48 h when exposed to concentrations of doxorubicin higher than 0.01 μg/mL (p Conclusion These results indicate that resistance to ATRA and ATO in APL cells does not confer subsequent resistance to anthracyclines such as doxorubicin. As a result, doxorubicin targets a pathway that is distinct from that of ATRA & ATO. No difference in cell viability was found between naive NB4 and resistant NB4-RAA when exposed to the same concentrations of doxorubicin. Our results indicate the effectiveness of overcoming resistance to frontline APL therapy by using doxorubicin.

Impedance-based drug-resistance characterization of colon cancer cells through real-time cell culture monitoring

Interest in impedance-based cellular assays is rising due to their remarkable advantages, including label-free, low cost, non-invasive, non-destructive, quantitative and real-time monitoring. In order to test their potential in cancer treatment decision and early detection of chemoresistance, we devised a new custom-made impedance measuring system based on electric cell-substrate impedance sensing (ECIS), optimized for long term impedance measurements. This device was employed in a proof of concept cell culture impedance analysis for the characterization of chemo-resistant colon cancer cells. Doxorubicin-resistant HT-29 cells were used for this purpose and monitored for 140 h. Analysis of impedance-based curves reveal different trends from chemo-sensitive and chemo-resistant cells. An impedance-based cytoxicity assay with different concentrations of doxorubicin was also performed using ECIS. The obtained results confirm the feasibility of ECIS in the study of drug resistance and show promises for studies of time-dependent factors related to physiological and behavioral changes in cells during resistance acquisition. The methodology presented herein, allows the continuous monitoring of cells under normal culture conditions as well as upon drug exposure. The ECIS device used, sets the basis for high-throughput early detection of resistance to drugs, administered in the clinical practice to cancer patients, and for the screening of new drugs in vitro, on patient-derived cells.

Combination of Doxorubicin and Berberine Generated Synergistic Anticancer Effect on Breast Cancer Cells Through Down-regulation of Nanog and miRNA-21 Gene Expression

Background: Our purpose was to investigate the effect of berberine (BER) and doxorubicin (DOX) on the expression of stem cell markers Nanog and microRNA- 21 in MCF-7 cells. Methods: The study was an in vitro study employing the human breast cancer cell line MCF-7 that was divided into four groups: Group I: MCF-7 cell line maintained in drug-free environment as untreated control, Group II: MCF-7 cell line treated with different concentrations of DOX, Group III: MCF-7 cell line treated with various concentrations of BER. Group IV: MCF-7 cell line treated with different concentrations of combined DOX and BER. MTT assay determined the metabolic activity and viability of MCF-7 cells for all groups. We further extracted total RNA from MCF-7 cells, and RT-PCR assayed the expression of Nanog and miRNA-21. Results: The results revealed that DOX and/or BER decreased the percentage of viable MCF-7 monolayer and mammospheres breast cancer cells in a concentrationdependent manner. Moreover, the combination of DOX and BER generated synergistic anticancer effect on MCF-7 monolayer cells and mammospheres. In addition, DOX alone, BER alone, and their combination significantly reduced Nanog and miRNA- 21 gene expression in MCF-7 mammospheres compared with untreated mammospheres. Conclusions: BER may affect the viability of breast cancer cells through downregulation of Nanog and miRNA-21 gene expression, ultimately enhancing the sensitivity of breast cancer cell line to DOX. BER may be an effective chemotherapeutic agent against breast cancer where the combination of DOX and BER generates synergistic anticancer effects.

Parallelized Wireless Sensing System for Continuous Monitoring of Microtissue Spheroids.

Currently, the use of electrical readout methods for the investigation of microtissue spheroids in combination with lab automation tools is hindered by the cable connections that are required to interrogate the on-chip-integrated electrodes. To overcome this limitation, we developed a wireless sensor scheme, which can detect the size variation of microtissues during long-term culturing and drug exposure assays. The sensor system includes an interrogation board, which is composed of an inductor-capacitor (LC) readout circuit, and the tissue culture platform with integrated split-ring sensors. The magnetic coupling between the LC circuit and the sensors enables the interrogation of the on-chip sensors without any wire connection to the culture platform. By optimizing the sensor dimensions and the LC resonance frequencies, we were able to avoid cross talk between neighboring sensors. We integrated 12 tissue compartments on a standard microscopy slide with a sensor-to-sensor pitch of 9 mm, which is in accordance with standard 96-well plate dimensions. As a proof-of-concept experiment for the developed system, we monitored continuously and during more than four days the growth inhibition of colon cancer microtissue spheroids that had been exposed to different concentrations of doxorubicin, a chemotherapeutic compound. The stability of the measurements during long-term culturing and the compatibility of the sensor scheme with standard lab equipment offer great potential for automated electrical microtissue spheroid characterization.