Toxicity In Cancer Cells Research Articles

The Wonders of Silk Fibroin Biomaterials in the Treatment of Breast Cancer.

Breast cancer has continued to be a cause of increasing morbidity and mortality in women, being the most common cause of cancer-related deaths among them. Its management using chemotherapy is continually plagued with problems of systemic toxicity, lack of compliance by patients, and inadequate targeting of cancer cells. The future of breast cancer chemotherapy will likely involve the use of biocompatible high cell-targeting capacity drug-delivery vehicles like silk fibroin to ameliorate these problems. The utilization of silk fibroin nanoparticles to deliver cytotoxic drugs provides specificity, optimal entrapment, improved therapeutic index, and maximal breast cancer cell toxicity with minimal or no collateral damage to surrounding normal cells. The silk fibroin obtained from the cocoon of the Bombyx morii worm is processed and degummed to remove the sericin component; it is then made into nanoparticles utilizing the desolvation, ionic gelation, or electrospray method. It is then loaded with an appropriate chemotherapeutic drug (e.g., carboplatin), and characterization is performed using physico-chemical methods such as fourier-transform infrared spectroscopy, dynamic light scattering, or transmission electron microscopy. The nanoparticles are then tested for cytoxicity, and the induction of apoptosis on breast cancer cell lines MC-7 (Her2-) and MDA-MB-453 (Her2+). Toxicity and apoptosis are assessed using the MTT assay and ELISA methods, respectively. Silk fibroin has been demonstrated in various studies to be a very useful tool in specific active or passive drug delivery to target cancer cells, thus ensuring a maximum destruction and minimum damage to normal surrounding cells, which decreases systemic toxicity and enhances drug efficacy.

αv β3 mediated tumor imaging using 99m Tc labeled NAD/monosaccharide coated ferrihydrite nanoparticles.

This study describes the synthesis of highly water-soluble, non-toxic, and biocompatible nicotinamide adenine dinucleotide (NAD)/glucosamine (=Nga1Fh) and NAD/glucosamine/gluconic acid coated ferrihydrite nanoparticles (=Nga2Fh) and their possible uses to target tumors in living animals via 99m Tc and 125 I radioisotope labeling. The structural properties were investigated using DLS, zeta potential, TEM, FT-IR, XRD, and Raman spectroscopy. The cell toxicity in CT26 cancer cells and in vivo tumor targetability in U87MG and CT26 tumor-bearing mice was further evaluated using cRGDyK-tagged and cRGDfK-tagged ferrihydrite nanoparticles. The average diameters of the resulting Nga1Fh and Nga2Fh nanoparticles were <5 to 7 and <3nm, respectively. The Nga2Fh nanoparticles did not show cell toxicity until 0.1mg/mL. Using gamma camera imaging, 99m Tc-cRGDfK-Nga2Fh showed the highest tumor uptake in a U87MG tumor-bearing mouse when compared with that of 99m Tc-cRGDyK-Nga2Fh and 99m Tc-Nga2Fh. The image-based tumor-to-muscle ratio by time for 99m Tc-cRGDfK-Nga2Fh was 3.8±1.7, 4.2±2.0, 7±1.5, 13±2.0, 8±3.7, and 2±1.6 at 5 and 30minutes, 1, 2, 4, and 24hours, respectively. Although further studies are needed, the NAD/monosaccharide coated ferrihydrite nanoparticles could be presented as an interesting material for a drug delivery system.

The in vitro renal cell toxicity of some unconventional anticancer phenanthroline-based platinum(II) complexes

The cytotoxicity of platinum(II) complexes coordinated to a chiral diamine, 1S,2S-diaminocyclohexane or 1R,2R-diaminocyclohexane and 1,10-phenanthroline or 3,4,7,8-tetramethyl-1,10-phenanthroline has been investigated in the renal proximal tubule HK-2 cell line. All platinum(II) complexes exhibited lower cytotoxicity in HK-2 cells (IC50 1.7–25μM) than in A2780 ovarian cancer cells or cisplatin-resistant A2780cisR cells (IC50 0.2–2.1μM) (at 48h). PHENSS ([Pt(1,10-phenanthroline)(1S,2S-dach)]2+) induced apoptosis and necrosis in ovarian cancer cells at concentrations that are relatively cytostatic to renal cells. Cisplatin was similarly or more cytotoxic to renal cells than ovarian cancer cells. Similar trends were reflected with shorter term exposure (1.5h). PHENSS demonstrated a comparatively cytostatic mode of action in renal cell cultures than cisplatin, as demonstrated by lower toxicity at higher concentrations (90μM). PHENSS induced an elongated renal cell morphology, cytoskeletal stress fibre thickening, and increased β-galactosidase activity, but no detectable change in reactive oxygen species generation or cell cycle distribution. In contrast, cisplatin treatment was associated with increased oxidative stress, cellular enlargement, G2/M arrest and apoptosis. The cytotoxicity of all platinum(II) complexes in both renal and ovarian cell lines were reduced in the presence of organic cation transporter (OCT) inhibitors cimetidine, disopyramide and amantadine. PHENSS and analogues demonstrated low level genotoxicity in an in vitro micronuclei assay compared to cisplatin or etoposide. These findings highlight PHENSS and other phen-based unconventional platinum(II) complexes as promising anticancer agents with alternative modes of action that induce lower kidney cell toxicity and genotoxicity, while demonstrating greater cisplatin-resistant ovarian cancer cell toxicity.

Sodium butyrate promotes apoptosis in breast cancer cells through reactive oxygen species (ROS) formation and mitochondrial impairment

BackgroundSodium butyrate (NaBu) is a short-chain fatty acid which serves as a histon deacetylase inhibitor and has received considerable interest as a possible regulator of cancer cell death. The regulatory effect of NaBu on cancer cell growth or death has yet to be illustrated in many cancers including breast cancer. This study is aimed to elucidate the possible effect of NaBu on regulation of breast cancer growth and apoptosis.MethodsThe cytotoxic effect of NaBu on the growth of breast cancer cells (MCF-7 and MDA-MB-468) and normal breast cells (MCF-10A) was determined using MTT assay. Annexin-V-FITC staining and PI staining were performed to detect apoptosis and cell cycle distribution using Flow cytometry, the level of mitochondrial membrane potential (Δψm), Reactive oxygen species (ROS)formation and caspase activity were determined accordingly.ResultsBased on our data, NaBu induced a dose and time-dependent cell toxicity in breast cancer cells which was related to the cell cycle arrest and induction of apoptosis. The impact of NaBu on MCF-10A cell toxicity, cell cycle distribution and apoptosis was inconsiderable. NaBu-elicited apoptosis was accompanied by the elevated level of ROS, increased caspase activity and reduced mitochondrial membrane potential (Δψm) in MCF-7 and MDA-MB-468 cells and with no effect on the above mentioned factors in MCF-10A cells.ConclusionsOur study provided insight in to the role of NaBu on the regulation of breast cancer cell growth and lighten up the pro-apoptotic activity of NaBu.

115 - Disulfiram causes Selective Hypoxic Cancer Cell Toxicity and Radio-chemo-sensitization via Redox Cycling of Copper and O2•‒ mediated Oxidative Stress

Lung cancer is one of the most deadly cancers in the USA, and despite multiple modalities of treatment, the 5 year survival rate remains

Dual-Drug Containing Core-Shell Nanoparticles for Lung Cancer Therapy

Late-stage diagnosis of lung cancer occurs ~95% of the time due to late manifestation of its symptoms, necessitating rigorous treatment following diagnosis. Existing treatment methods are limited by lack of specificity, systemic toxicity, temporary remission, and radio-resistance in lung cancer cells. In this research, we have developed a folate receptor-targeting multifunctional dual drug-loaded nanoparticle (MDNP) containing a poly(N-isopropylacrylamide)-carboxymethyl chitosan shell and poly lactic-co-glycolic acid (PLGA) core for enhancing localized chemo-radiotherapy to effectively treat lung cancers. The formulation provided controlled releases of the encapsulated therapeutic compounds, NU7441 - a potent radiosensitizer, and gemcitabine - an FDA approved chemotherapeutic drug for lung cancer chemo-radiotherapy. The MDNPs showed biphasic NU7441 release and pH-dependent release of gemcitabine. These nanoparticles also demonstrated good stability, excellent hemocompatibility, outstanding in vitro cytocompatibility with alveolar Type I cells, and dose-dependent caveolae-mediated in vitro uptake by lung cancer cells. In addition, they could be encapsulated with superparamagnetic iron oxide (SPIO) nanoparticles and visualized by MRI in vivo. Preliminary in vivo results demonstrated the low toxicity of these particles and their use in chemo-radiotherapy to effectively reduce lung tumors. These results indicate that MDNPs can potentially be used as nano-vehicles to provide simultaneous chemotherapy and radiation sensitization for lung cancer treatment.

A Comparison of Floating-Electrode DBD and kINPen Jet: Plasma Parameters to Achieve Similar Growth Reduction in Colon Cancer Cells Under Standardized Conditions

A comparative study of two plasma sources (floating-electrode dielectric barrier discharge, DBD, Drexel University; atmospheric pressure argon plasma jet, kINPen, INP Greifswald) on cancer cell toxicity was performed. Cell culture protocols, cytotoxicity assays, and procedures for assessment of hydrogen peroxide (H2O2) were standardized between both labs. The inhibitory concentration 50 (IC50) and its corresponding H2O2 deposition was determined for both devices. For the DBD, IC50 and H2O2 generation were largely dependent on the total energy input but not pulsing frequency, treatment time, or total number of cells. DBD cytotoxicity could not be replicated by addition of H2O2 alone and was inhibited by larger amounts of liquid present during the treatment. Jet plasma toxicity depended on peroxide generation as well as total cell number and amount of liquid. Thus, the amount of liquid present during plasma treatment in vitro is key in attenuating short-lived species or other physical effects from plasmas. These in vitro results suggest a role of liquids in or on tissues during plasma treatment in a clinical setting. Additionally, we provide a platform for correlation between different plasma sources for a predefined cellular response.

Inhibition of aldehyde dehydrogenase 1 enhances the cytotoxic effect of retinaldehyde on A549 cancer cells.

We hypothesized that aldehyde dehydrogenase1 (ALDH1) protects cancer cells from retinaldehyde-induced cytotoxicity, and that targeting this enzyme would enhance the therapeutic effect of retinaldehyde. ALDEFLUOR™ assays showed high ALDH activity in A549 and H522 cancer cells and low activity in H1666 and T47D cancer cells. Immunoblots showed that expression of ALDH1A1 and ALDH1A3 was high in A549 and H522 cells, but low in H1666 cells. HPLC confirmed that N, N-diethylaminobenzaldehyde (DEAB) inhibits ALDH-mediated disposal of retinaldehyde in A549 cells and lysates. Treatment of A549 cells with retinaldehyde in the presence of DEAB augmented reactive oxygen species production and decreased glucose uptake and oxygen consumption. Importantly, DEAB substantially potentiated the ability of retinaldehyde to dose-dependently suppress the survival of A549 and H522 cells, whereas the added effect of DEAB was minor in H1666 and T47D cells. Gene silencing with specific siRNA revealed that ALDH1A1 contributed to protection of A549 cells against retinaldehyde toxicity. These results demonstrate that ALDH1 confers protection against retinaldehyde toxicity in cancer cells.

Alantolactone induces apoptosis, promotes STAT3 glutathionylation and enhances chemosensitivity of A549 lung adenocarcinoma cells to doxorubicin via oxidative stress

Alantolactone (ALT), a sesquiterpene lactone component of Inula helenium, has been reported to exert anticancer activity in various cancers. However, the cellular targets and underlying mechanism of anticancer activity of ALT in various cancers including lung cancer has not been fully defined. In the present study, we found that ALT effectively inhibits proliferation and triggers oxidative stress mediated-apoptosis in A549 lung adenocarcinoma cells by inducing ER stress and mitochondrial dysfunction. This ALT-mediated apoptosis was inhibited by NAC while diamide potentiated it. Moreover, ALT effectively suppressed both constitutive and inducible STAT3 activation, inhibited its translocation into nucleus and decreased its DNA binding activity. Further mechanistic study revealed that ALT abrogated STAT3 activation by promoting STAT3 glutathionylation. ROS scavenger NAC reverted ALT-mediated STAT3 glutathionylation and inhibition of STAT3 phosphorylation. Finally, ALT enhanced chemosensitivity of A549 cells to doxorubicin and reversed doxorubicin resistance in A549/DR cells by inhibiting STAT3 activation and P-glycoprotein expression and increasing intracellular accumulation of doxorubicin. Suppression of STAT3 activation by targeting ROS metabolism with ALT thus discloses a previously unrecognized mechanism underlying the biological activity of ALT. Taken together; ALT induces oxidative stress-dependent apoptosis, inhibits STAT3 activation and augments doxorubicin toxicity in A549 lung cancer cells. These findings provide an in-depth insight into the molecular mechanism of ALT in the treatment of lung cancer.

Hepatoprotective Role of Hydrangea macrophylla against Sodium Arsenite-Induced Mitochondrial-Dependent Oxidative Stress via the Inhibition of MAPK/Caspase-3 Pathways.

Sodium arsenite (NaAsO2) has been recognized as a worldwide health concern. Hydrangea macrophylla (HM) is used as traditional Chinese medicine possessing antioxidant activities. The study was performed to investigate the therapeutic role and underlying molecular mechanism of HM on NaAsO2-induced toxicity in human liver cancer (HepG2) cells and liver in mice. The hepatoprotective role of HM in HepG2 cells was assessed by using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), reactive oxygen species (ROS), and lactate dehydrogenase (LDH) assays. Histopathology, lipid peroxidation, serum biochemistry, quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses were performed to determine the protective role of HM against NaAsO2 intoxication in liver tissue. In this study, we found that co-treatment with HM significantly attenuated the NaAsO2-induced cell viability loss, intracellular ROS, and LDH release in HepG2 cells in a dose-dependent manner. Hepatic histopathology, lipid peroxidation, and the serum biochemical parameters alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were notably improved by HM. HM effectively downregulated the both gene and protein expression level of the mitogen-activated protein kinase (MAPK) cascade. Moreover, HM well-regulated the Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) ratio, remarkably suppressed the release of cytochrome c, and blocked the expression of the post-apoptotic transcription factor caspase-3. Therefore, our study provides new insights into the hepatoprotective role of HM through its reduction in apoptosis, which likely involves in the modulation of MAPK/caspase-3 signaling pathways.

Abstract 4066: The dihydroartemisinin oxime dimer (NSC735847) displays a selective toxicity in colon cancer cells which is potentially mediated by endoplasmic reticulum stress

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Common chemotherapeutic regimens for CRC include a combination of chemotherapeutic agents to produce synergistic drug activity and reduce adverse effects. However, adverse effects due to lack of selective toxicity is still a major problem with many chemotherapeutic agents. Novel drugs that display tumor selective toxicity are desperately needed. Several studies have shown that artemisinin monomers (Clinically used anti-malarial agent) possess antineoplastic activity with minimal toxicity. Interestingly, artemisinin dimers showed more potent antineoplastic activity compared to the monomers. However, few studies have fully characterized the activity of these molecules. In this study, we tested the antitumor activity of five chemically-synthesized dihydroartemisinin (DHA) dimers using the human colon cancer cell lines, HT29 and HCT116 and the non-tumorigenic colon cell line, FHC. Compared to other tested dimers, the DHA oxime dimer, NSC735847 showed pronounced selective toxicity in HT29 and HCT116 cells. Using MTS cell viability assays, NSC735847caused a preferential reduction in the viability of HT29 and HCT116 colon cancer cells compared to the non-tumorigenic FHC cells. In addition, NSC735847 significantly increased caspase 3/7 activity in HT29 and HCT116 cells but not in FHC cells which suggests that this compound causes selective apoptosis in these colon cancer cell lines. The combination of NSC735847 and the topoisomerase I inhibitor, irinotecan (commonly used chemotherapeutic agent for colorectal cancer) showed synergistic antitumor activity in HT29 cells. The combination of the two drugs caused a significant increase in cell death and caspase 3/7 activity which were greater than those caused by the individual drugs. Previous studies using other tumorigenic cell lines, suggested that NSC735847 induces oxidative and endoplasmic reticulum (ER) stress. To gain insight into the potential mechanisms of anti-colorectal cancer activity of NSC735847, we tested if this compound causes ER stress and/ or oxidative stress in HT29 cells and whether ER stress was required for NSC735847-induced apoptotic cell death. NSC735847 increased the expression of ER stress marker C/EBP homologous protein 10 (CHOP10) in HT29 cells and the use of ER stress inhibitor, salubrinal, significantly decreased NSC735847-induced cell death and apoptosis. Additionally, NSC735847 caused oxidative stress in HT29 cells which was inhibited by pretreatment of the cells with the antioxidant, trolox. The crosstalk between oxidative stress and ER stress in NSC735847-induced apoptosis in colon cancer cells is still under investigation. Our results suggest that NSC735847 causes ER stress in HT29 cells which plays a major role in drug-induced cell death and apoptosis in these cells. Citation Format: Ahmed E. Elhassanny, Eman Soliman, Paul McGuire, Mahmoud ElSohly, Waseem Gul, Rukiyah Van Dross. The dihydroartemisinin oxime dimer (NSC735847) displays a selective toxicity in colon cancer cells which is potentially mediated by endoplasmic reticulum stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2017-4066

Abstract 1069: Inhibition of the nuclear import receptor, KpnB1 synergizes with cisplatin toxicity in cervical cancer cells

Abstract Elevated expression of members of the nuclear transport protein family has been reported in multiple cancers and presents as novel anticancer therapeutic targets. Using a cervical cancer model system, we have previously shown that inhibition of the nuclear import receptor, KpnB1 by siRNA and a novel small molecule, Inhibitor of Nuclear Import (INI-43) resulted in cancer cell death via apoptosis. In this study, we investigated the cancer cell killing effects of KpnB1 inhibition in combination with Cisplatin (CDDP), a first-line chemotherapeutic agent used in the treatment of many cancers. KpnB1 siRNA and INI-43 treatment at sub-lethal concentrations enhanced cancer cells' sensitivity to CDDP. Our data shows that the combination treatment of INI-43 and CDDP significantly decreases CDDP IC50 compared to CDDP treatment alone. Increased PARP-cleavage was observed in combination treated cells and this correlated with increased γH2A.X, indicating increased apoptosis and DNA damage. Furthermore, INI-43 treatment reduced the nuclear import of NFκB, a stress-regulated response transcription factor known to be a KpnB1 cargo protein that promotes cancer cell survival. Decreased levels of downstream pro-survival and DNA-repair targets of NFκB were observed, including cyclinD1, c-Myc and XIAP, which correlated with increased DNA damage and apoptosis. Taken together, results suggest increased Cisplatin sensitivity of cervical cancer cells when nuclear import via KpnB1 is inhibited. Citation Format: Ru-pin Chi, Wei Wei, Michael Birrer, Virna D. Leaner. Inhibition of the nuclear import receptor, KpnB1 synergizes with cisplatin toxicity in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2017-1069

Abstract 5481: Role and regulation of Peroxiredoxin antioxidant proteins in K562 leukemia cells

Abstract Peroxiredoxins are a family of antioxidant proteins that have found to play a key role in many diseases, including cancer. A wide number of studies have found that Peroxiredoxin levels are elevated in several cancers, and reducing these levels can aid in inhibiting cell growth of cancer cells, suggesting that Peroxiredoxins confer resistance to reactive oxygen species (ROS)-induced cellular toxicity in cancer cells. Specific research has demonstrated that Peroxiredoxin levels are elevated in leukemia cells, and that targeting and inhibiting Peroxiredoxins in these cells can induce differentiation. Studies have also shown the use of doxorubicin as an effective chemotherapy treatment to significantly decrease cell proliferation and viability in leukemia. The purpose of this study is to examine Peroxiredoxin levels in K562 cells, a human chronic myelogenous leukemia cell line, and determine if high levels of Peroxiredoxins have the ability to protect cells from chemotherapy-induced death and possibly aid in chemoresistance. We first demonstrated that serum-deprived K562 cells exhibited significant growth inhibition over a 72 hour time period, but had no significant effect on viability. We also showed that treating K562 cells with 50 nM doxorubicin significantly decreased proliferation and viability. Corresponding changes in Peroxiredoxin protein levels are currently being analyzed to determine the effect of both growth-inhibition and chemotherapy-induced cytotoxicity on the Peroxiredoxin family in K562 cells, and siRNA experiments are being conducted to examine the protective role of Peroxiredoxins in these cells. Based on previous studies on MCF-7 cells conducted in our lab, we predict that Peroxiredoxin levels will be modified under both conditions, and that suppression of Prdxs by siRNA will increase chemotherapy susceptibility. Citation Format: Elizabeth Szabo, Shelley A. Phelan. Role and regulation of Peroxiredoxin antioxidant proteins in K562 leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5481. doi:10.1158/1538-7445.AM2017-5481

Abstract 2164: BLID is a novel drug-inducible apoptotic molecule: Identification of an integrative mechanism of chemosensitivity in breast cancer cells

Abstract The triple-negative breast cancer (TNBC) is an aggressive form of breast cancer frequently seen in African American women and BRCA1 mutation carriers. The TNBC tumors often relapse with distant metastases following standard chemotherapy. It is now evident that discoveries of new mechanisms and approaches that explain and target breast cancer biology are urgently needed for durable intervention of metastatic disease. Earlier, we have demonstrated that BLID, BH-3 Like motif containing Inducer of cell Death, is a strong prognostic factor in invasive breast cancer. Frequent lack of BLID has been associated with TNBC, African American ethnicity and younger women. Significant correlations exist between BLID negative breast cancer and declines in overall survival, local relapse-free survival and distant metastasis-free survival. Recently, BLID has been shown to inhibit breast cancer cell growth and metastasis. The purpose of this study was to investigate the role of BLID in response of breast cancer cells to chemotherapeutic drugs. In the dose response and time course studies, BLID mRNA expression was found to be induced by chemotherapeutic drugs. Expression of BLID cDNA nanocomplex (scLBLID) resulted in significant increase in chemosensitivity in SKBr3 and MDA-MB-231 cells, and a comparison of BLID with p53 showed that the chemosensitization effect of BLID was significantly greater than that of p53. Consistently, BLID knockdown led to reversal of drug-induced cytotoxicity. In the ChIP-PCR and ChIP-qPCR assays, drug treatment of breast cancer cells resulted in an increased binding of pro-apoptotic transcription factor FOXO3a to the BLID promoter, and the reversal of drug-induced BLID reporter activity was seen in presence of FOXO3a siRNA. Furthermore, siRNA silencing of FOXO3a was found to be associated with decrease in endogenous BLID mRNA expression. Remarkably, we found that expression of central tumor suppressor microRNA miR34a also resulted in increased BLID mRNA expression and drug toxicity in breast cancer cells. Because lack of BLID expression has been associated with poor prognosis in breast cancer patients, we reasoned that the silencing of BLID may reveal as yet unknown changes in gene expression that may drive breast cancer cell proliferation and therapy resistance. In this context, the mRNA array profiling studies showed that BLID knockdown in MDA-MB-231 cells was associated with increased expression of the oncogenic/anti-apoptotic molecules CYP1B1, BIRC3 and CSF1, and decreased expression of the anti-oncogenic/apoptotic molecules AKAP12, DFNA5 and CHRDL1. Our data suggest that chemotherapeutic drugs induce BLID expression via activation of FOXO3a, and the BLID signaling axis downstream of FOXO3a and miR34a is a novel integrative mechanism of breast cancer response to chemotherapy. SY and RH are equal contributors in this study. Citation Format: Sivaramakrishna Yadavalli, Rong Hu, Antonina Rait, Xin Li, Esther Chang, Robert Clarke, Usha Kasid. BLID is a novel drug-inducible apoptotic molecule: Identification of an integrative mechanism of chemosensitivity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2164. doi:10.1158/1538-7445.AM2017-2164

Abstract 4990: High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities

Abstract Monolayer, two-dimensional (2D) cell cultures have been a predominant in vitro model in anticancer drug discovery and high-throughput screening (HTS). However, 2D cultures of cancer cells lack numerous properties of in vivo tumors, such as tissue-like structure, cell-cell interactions and nutrient/oxygen gradients. Thus, in recent years there has been an increased interest in 3D cell cultures, such as multicellular tumors spheroids (MCTS), to address some of these limitations. Recently, we and others have applied MCTS for HTS and identified oxidative phosphorylation (OXPHOS) as a selective vulnerability of quiescent cancer cells persisting in hypoxic and nutrient-deprived milieu. However, prolonged continuous exposure to OXPHOS inhibitors is necessary for the cytotoxic effect. Thus, there is a need to identify processes that could be co-targeted for enhanced anticancer activity. Here, we present two distinct HTS approaches to identify combination partner molecules for OXPHOS inhibitors. Since we were interested in targeting non-dividing nutrient-deprived cancer cells, we used quiescent MCTS (Q-MCTS), as an in vitro model. Cells in Q-MCTS experience glucose concentrations and pH similar to those observed in deep tumor parenchyma in vivo. In our first screening approach, we have applied high-throughput gene-expression profiling to study drug effects in MCTS at a large scale. Using L1000 Gene Expression Profiling method, we generated a dataset of over 1000 drug-induced gene-expression profiles and found that co-targeting of OXPHOS and the mevalonate pathway results in selective synergistic toxicity in quiescent cancer cells. In the other approach, we screened a library of 1650 biologically active compounds, with or without addition of the FDA-approved anthelmintic agent nitazoxanide (an OXPHOS inhibitor with high drug repurposing potential). After the screen, we selected molecules that demonstrated pronounced synergy when combined with nitazoxanide, but not when used alone. Then, we validated the hits in an extensive dose-response combination experiments in Q-MCTS and chose 14 compounds that demonstrated strong synergistic interaction with nitazoxanide at broad range of concentrations. These included antifungal agents, kinase inhibitors and others. In summary, we here report on novel approaches, utilizing 3D cell cultures, to identify drug combinations targeting quiescent cancer cells. By high-throughput gene-expression profiling and large-scale combinatorial drug screens, we were able to identify drug combinations preferentially toxic to quiescent cells. This work also demonstrates how 3D cell cultures yield functional insights that are not accessible through standard 2D cultures. Citation Format: Wojciech Senkowski, Madiha Nazir, Malin Jarvius, Jenny Rubin, Johan Lengqvist, Mats G. Gustafsson, Peter Nygren, Kim Kultima, Rolf Larsson, Mårten Fryknäs. High-throughput drug combination screening in tumor spheroids identifies context-dependent synthetic lethalities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2017-4990

Antidiabetic, antioxidant and anti inflammatory properties of water and n-butanol soluble extracts from Saharian Anvillea radiata in high-fat-diet fed mice

Ethnopharmacological relevanceAccording to Saharian traditional medicine, Anvillea radiata Coss. & Dur. (Asteraceae) has been valued for treating a variety of ailments such as gastro-intestinal, liver and pulmonary diseases, and has gained awareness for its beneficial effect on postprandial hyperglycemia. However, to best of our knowledge, no detailed study of the antidiabetic curative effects of this plant has been conducted yet. Aim of the studyTo determine the hypoglycemic and antidiabetic effect of dietary supplementation with Anvillea radiata extracts on high-fat-diet (HFD)-induced obesity and insulin resistance in C57BL/6J mice in relation with antioxidant, anti-inflammatory, pancreatic beta-cells and skeletal muscle protection, and digestive enzyme inhibiting properties. Materials and methodsSix extracts (water soluble and organic) from aerial parts of the plant were analyzed phytochemically (total phenolic and flavonoid content) and screened for in vitro superoxide (by chemiluminescence) and hydroxyl radical (by electron paramagnetic resonance spin-trapping) scavenging, antioxidant (DPPH, TRAP and ORAC assays), xanthine oxidase, metal chelating, α-amylase and α-glucosidase inhibitory property, and protective effects on copper-induced lipoprotein oxidation. Then selected hydroalcoholic and aqueous extracts were assessed for toxicity in normal human lung fibroblasts and A549 cancer cells using FMCA and MTT assays. Two water-soluble extracts having the best overall properties were assessed for their (i) protective effect at 1−15µg/mL on metabolic activity of rat insulinoma-derived INS-1 cells exposed to hyperglycemic medium, and (ii) acute hypoglycemic effect on 16-weeks HFD-induced diabetic mice. Then diabetic mice were administered HFD supplemented by extracts (up to 150mg/kg/day) for 12 additional weeks using standard diet as control and the antidiabetic drug, metformin (150mg/kg), as positive control. Then the antidiabetic, anti-inflammatory and antioxidant activity of extracts were determined. ResultsOf the highly efficient polyphenolics-enriched hydroalcoholic and ethyl acetate extracts, the lyophilized aqueous (AQL) and butanol extracts were not toxic in cells (≤ 400µg/mL) or when given orally in normal mice (≤ 2000mg/kg), exerted a dose-dependent hypoglycemic action in diabetic mice, which was maximal at the dose of 150mg/kg. Upon administering this dose for 12 weeks, both extracts significantly ameliorated body weight control capacity, recovery of plasma glucose and insulin level, reduced oxidative stress in blood, myocardial and skeletal muscles, and improved hyperlipidemic and inflammatory status. Moreover, diabetes-related complications were optimally ameliorated by oral therapy based on halved doses (75mg/kg) of a mixture of AQL and metformin. ConclusionsCurrent investigation supports the traditional medicinal usage of Anvillea radiata and suggests that both readily accessible and low-cost bio-extracts have the potency to develop an antihyperglycemic, antihyperlipidemic and protective agent against beta-cells and muscle dysfunction at doses compatible with the common practices of indigenous people for the management of metabolic disorders.

Effect of Curcumin on SMCT-1 Expression and Dichloroacetate Toxicity in HCT116 Colon Cancer Cells

Background: Colorectal cancer is a common cause of cancer-related deaths Epigenetic regulation of the influx sodium dependent monocarboxylate transporter-1 )SMCT1), a tumor suppressor, was recognized in colorectal cancer. In this study, effects of Curcumin (Cur), on SMCT1 gene expression was determined. A low SMCT1 expression, HCT116, cell line was used to test an in vitro effect of Cur on epigenetic regulation of SMCT1 expression via DNA methylation and its function. It was hypothesized that Cur can induce SMCT1 expression in the cells via hypomethylation effect. Measurement of increase in SMCT1 function was performed using dichloroacetate (DCA), a cytotoxic substrate of SMCT1. Methods: The effect of 5′Azacytidine (Aza), a hypomethylating agent, and Cur on SMCT1 expression and function was determined. Cells were treated with Aza and various concentrations of Cur for 72 h. After that SMCT1 expression was determined by real time PCR and Western blotting. To evaluate the SMCT1 function, DCA was used in MTT assay. Results: After treatment with 40 µM Cur, SMCT1 mRNA was significantly increased (p < 0.05). This was correlated with SMCT1 protein expression. Cells treated with 40 µM of Cur showed significant increase of cytotoxicity at DCA concentrations of 25 (p < 0.001) and 12.5 mM (p <0.01), respectively. Conclusion: Cur was shown to significantly induce the SMCT1 mRNA and protein expression in HCT116 cells. The induction of the SMCT1 protein increased DCA cytotoxicity, presumably through an increase of DCA transport into the cells. The mechanism underlying of SMCT1 induction by Cur may result from not only hypomethylation but other epigenetics.

Glycinated fullerenes for tamoxifen intracellular delivery with improved anticancer activity and pharmacokinetics.

Glycine-tethered C60-fullerenes were conjugated with N-desmethyl tamoxifen and evaluated for drug delivery benefits. C60-fullerenes were functionalized with glycine, and N-desmethyl tamoxifen was conjugated, employing a linker and characterized for micromeritics, drug loading, drug release and evaluated for cancer cell toxicity, cellular uptake and pharmacokinetics. The nanoconjugate with a drug entrapment efficiency of 82.71 ± 6.23% and a drug loading of 66.01 ± 4.98% was hemocompatibile with appreciable MCF-7 cytotoxicity. The confocal results confirmed enhanced uptake of conjugate. Interestingly, pharmacokinetic outcomes of the conjugate were superior and the area under the curve was enhanced by approximately three-times, whereas the drug clearance was reduced by around five-times, after single intravenous injection. The conjugation assured improved availability of drug in a biological system for prolonged duration as well as in the interiors of target cells with a promise of enhanced efficacy and compatibility.

Cytotoxic effects of a novel maleimide derivative on epithelial and tumor cells

Novel N-triazolyl maleimide derivatives were synthesized by azide–alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.

D-penicillamine combined with inhibitors of hydroperoxide metabolism enhances lung and breast cancer cell responses to radiation and carboplatin via H2O2-mediated oxidative stress

D-penicillamine (DPEN), a copper chelator, has been used in the treatment of Wilson's disease, cystinuria, and rheumatoid arthritis. Recent evidence suggests that DPEN in combination with biologically relevant copper (Cu) concentrations generates H2O2 in cancer cell cultures, but the effects of this on cancer cell responses to ionizing radiation and chemotherapy are unknown. Increased steady-state levels of H2O2 were detected in MB231 breast and H1299 lung cancer cells following treatment with DPEN (100µM) and copper sulfate (15µM). Clonogenic survival demonstrated that DPEN-induced cancer cell toxicity was dependent on Cu and was significantly enhanced by depletion of glutathione [using buthionine sulfoximine (BSO)] as well as inhibition of thioredoxin reductase [using Auranofin (Au)] prior to exposure. Treatment with catalase inhibited DPEN toxicity confirming H2O2 as the toxic species. Furthermore, pretreating cancer cells with iron sucrose enhanced DPEN toxicity while treating with deferoxamine, an Fe chelator that inhibits redox cycling, inhibited DPEN toxicity. Importantly, DPEN also demonstrated selective toxicity in human breast and lung cancer cells, relative to normal untransformed human lung or mammary epithelial cells and enhanced cancer cell killing when combined with ionizing radiation or carboplatin. Consistent with the selective cancer cell toxicity, normal untransformed human lung epithelial cells had significantly lower labile iron pools than lung cancer cells. These results support the hypothesis that DPEN mediates selective cancer cell killing as well as radio-chemo-sensitization by a mechanism involving metal ion catalyzed H2O2-mediated oxidative stress and suggest that DPEN could be repurposed as an adjuvant in conventional cancer therapy.