Khalda Fadlalla

The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle.

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability, and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose-dependent manner, 12.5–50 μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 h. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that although long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M.

Variable NF-κB pathway responses in colon cancer cells treated with chemotherapeutic drugs.

BackgroundThe nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is activated in cells exposed to various stimuli, including those originating on the cell surface or in the nucleus. Activated NF-κB signaling is thought to enhance cell survival in response to these stimuli, which include chemotherapy and radiation. In the present effort, we determined which anticancer drugs preferentially activate NF-κB in colon cancer cells.MethodsNF-κB reporter cells were established and treated with 5-fluorouracil (5-FU, DNA/RNA damaging), oxaliplatin (DNA damaging), camptothecin (CTP, topoisomerase inhibitor), phleomycin (radiomimetic), or erlotinib (EGFR inhibitor). The activation of NF-κB was assessed by immunofluorescence for p65 translocation, luciferase assays, and downstream targets of NF-κB activation (cIAP2, and Bcl-XL) were evaluated by immunoblotting, by ELISA (CXCL8 and IL-6 in culture supernatants), or by gene expression analysis.ResultsColon cancer cells responded variably to different classes of therapeutic agents, and these agents initiated variable responses among different cell types. CPT activated NF-κB in SW480 colon cancer cells in a dose-dependent manner, but not in HCT116 cells that were either wild-type or deficient for p53. In SW480 colon cancer cells, NF-κB activation by CPT was accompanied by secretion of the cytokine CXCL8, but not by up-regulation of the anti-apoptotic genes, cIAP2 or Bcl-XL. On the contrary, treatment of HCT116 cells with CPT resulted in up-regulation of CXCR2, a receptor for CXCL8, without an increase in cytokine levels. In SW480 cells, NF-κB reporter activity, but not cytokine secretion, was inhibited by SM-7368, an NF-κB inhibitor.ConclusionThe results show that, in response to cancer therapeutic agents, NF-κB activation varies with the cellular make up and that drug-induced NF-κB activation may be functionally uncoupled from anti-apoptotic outcomes found for other stimuli. Some cancer cells in a heterogeneous tumor tissue may, under therapeutic pressure, release soluble factors that have paracrine activity on neighboring cells that express the cognate receptors.

3-(2-Bromoethyl)-indole inhibits the growth of cancer cells and NF-κB activation

Indole-3-carbinol (I3C) and diindolylmethane (DIM), found in cruciferous vegetables, have chemopreventive and anticancer properties. In the present study, 14 substituted indoles were tested for activity against SW480 colon cancer cells. Among these, 3-(2-bromoethyl)-indole, named BEI-9, showed the greatest inhibition. The effects of BEI-9 on cancer cells were analyzed by MTS and CellTiter-Glo assays for effects on cell viability, by microscopy for phenotypic changes, by scratch wound assays for effects on migration, by flow cytometry for changes in the cell cycle, by immunoblotting for cyclin D and A to assess effects on cell cycle regulation, and by NF-κB reporter assays for effects on basal and drug-induced NF-κB activation. BEI-9 inhibited the growth of SW480 and HCT116 colon cancer cells at concentrations of 12.5 and 5 µM, respectively. BEI-9 also inhibited cell motility as determined with scratch wound assays, and reduced the levels of cyclin D1 and A. Furthermore, in reporter cells, BEI-9 (0.8 µM) inhibited basal and induced NF-κB activation and increased cell death when combined with the cytokine TNFα or the drug camptothecin (CPT), both of which activate NF-κB. Preliminary experiments to identify a safe dose range for immunodeficient mice showed that BEI-9, administered intraperitoneally, was tolerable at doses below 10 mg/kg. Thus, BEI-9 and other indole derivatives may be useful in chemoprevention or as chemosensitizers. Since NF-κB activation is implicated in carcinogenesis and in reducing sensitivity to anticancer drugs, BEI-9 should be investigated in combination with drugs such as CPT, which activate NF-κB.

Bio-based calcium carbonate (CaCO3) nanoparticles for drug delivery applications

With the rapid increase in global population, potential water shortage, water quality has become a concern. In some extreme cases such as Arizona, we may have to switch from toilet to tap within the near future. However, our current monitoring methods for drinking water have limitations and do not provide fast and reliable results to deal with these challenges. By using intrinsic fluorescence, microbial contamination in drinking water can be monitored in real-time, continuously, without sample contact and at very low concentration (~50 cells/L). This technology uses intrinsic fluorescence to detect microorganisms. By introducing a pulse, microorganisms can be excited and as a result they will fluoresce. In addition, some of the cellular components (fluorophores) that fluoresce in these microorganisms can be used as indicators to distinguish viable cells, non-viable cells and spores. In our study, three different wavelengths were used to excite specific cellular components. We used UV, Red and Amber light. The fluorophores targeted were reduced pyridines nucleotides (RPNs) and flavins, cytochromes, and calcium dipicolinic acid (DPA) as indicators for viable cells, non-viable cells and spores respectively. The emissions collected allowed us to distinguish viable cells, nonviable cells and spores. By using this method, a wide range of microorganisms such as bacteria, protozoa, amoeba and other microorganisms of concern can be detected.Targeted delivery of a cytotoxic drug using a drug delivery system could maximise the efficacy of the drug and reduce the side effects. Calcium carbonate (CaCO3) nanoparticles are highly porous, biocompatible, biodegradable, and have pH-sensitive properties, which makes them good candidates for biological drug delivery systems. In this study, the nanoparticles were derived from egg shells and studied for their drug loading capacity and cytotoxicity, using human colon adenocarcinoma (SW480), and human dermal fibroblast (HDF) cells. The lactate dehydrogenase (LDH) assay indicated a 50% cytotoxicity for SW480 cells at a concentration of 0.3 mg/ml, but there was no cytotoxic effect observed in HDF cells at the same concentration. The anticancer drug 5-fluorouracil (5-FU) and natural compound indole-3-carbinol (I3C) were loaded into CaCO3 nanoparticles and release profiles were studied. These results showed that the drugs can be loaded in CaCO3 nanoparticles and released efficiently. The drug loaded nanoparticles were more cytotoxic compared to the as-received drugs alone. The 5FU-loaded nanoparticles in pH 1.0, 5.0, and 7.4 yielded a total loading and release of 86.98 µg, 77.83 µg, and 162.60 µg respectively. Depending on the pH of the solution, up to 162.60 µg of 5-FU was loaded and released from 50 milligrams of the particles, whereas, up to 563.47 micrograms of I3C was loaded and released from similarly loaded particles. These preliminary results indicate the potential of bio-based CaCO3 nanoparticles in therapeutic applications.T research focuses on developing an impedance measurement system by Electronic Design Automation (EDA) technology to reduce analog circuits and convert analog biological information into digital data for the ease of processing, analyzing, and storing. The impedance measurement system includes a field-programmable gate array (FPGA) platform, a function generator, and a personal computer. The FPGA’s internal circuit structure includes two analog-to-digital converters (ADC), a baud rate generator, a frequency divider, an up counter, a 2-to-1 multiplexor, a decoder, and a UART transmitter. In order to monitor biological properties and analyze impedance changes, the function generator produces alternating current (AC) signals of various frequencies with specified magnitudes for injecting current into the measured subject. To detect the impedance magnitude and phase, the computer receives the injected AC signal data and the induced signal data from the FPGA through the RS232 serial port. Then, discrete Fourier transform is conducted to obtain the impedance magnitudes and phases angle at various frequencies. A graphical user interface (GUI) in the computer is designed to serve as the gateway through which for the user to observe the measurement results and to view the relationships among various parameters such as frequency, impedance, and biochemical concentration. This electrochemical impedance spectroscopy (EIS) system will be used to measure composite solutions of human serum albumin (HSA) and phosphate buffered saline (PBS) at different concentrations with impedance biosensors. Ming-Lang Chang, J Biosens Bioelectron, 2:4 http://dx.doi.org/10.4172/2155-6210.S1.13

Abstract B11: Stromal response to prostate cancer therapeutics

Docetaxel (D) is a clinically used front-line chemotherapeutic agent for castration resistant advanced prostate cancer (Pca). However, its long-term benefits are limited, with most patients eventually progressing because of inherent or acquired drug resistance. The treatment of advanced Pca is a significant challenge and there are no effective treatments that stably suppress the disease. The mechanisms behind the resistance to Docetaxel are not fully understood. It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at different stages of cancer development or during chemotherapy are largely unidentified. The central goal is to identify the cellular and biochemical responses of stromal cells in tumor microenvironment with which prostate cancer cells interact. Specifically, the interaction of prostate cancer cells with stromal cells of the prostate or the bone microenvironment in the presence of clinically used cancer therapeutics will be examined. Key regulators of these interactions will also be identified. Such regulators include cytokines, growth factors, proteins, and their receptors. By identifying these regulators and their contribution to tumor drug response, novel therapeutics targeted to the microenvironment can be developed. Therefore, we hypothesize that the response of prostate stromal cells exposed to chemotherapeutic agents could modify drug response or therapeutic outcome of prostate cancer. Cellular proliferation was analyzed by MTT assay. The IC50 of Docetaxel for WPMY-1 (normal stromal), LNCaP (androgen dependent), DU-145 (androgen independent), and HS27A (bone stromal) showed inhibitory effects between 2-10nM. Cell cycle profiles were assessed by flow cytometry. The analysis showed that Docetaxel (2-50nM) induced prominent G2-M arrest in prostate and stromal cells within 24 hours. Gene expression profiling of prostate cancer and stromal cells lines were evaluated. To obtain the expression profiles, we conducted a human cytokines and chemokines array, which consisted of 84 specific genes. Analysis of the prostate cancer and stromal cells treated with Docetaxel revealed differential expression patterns. These results suggest Docetaxel exhibits strong anti-proliferative effects and analysis of the gene array suggest regulation of genes associated with, cell growth, survival, proliferation, metastasis, angiogenesis, and apoptosis. Note: This abstract was not presented at the conference. Citation Format: Dominique Nicole Gales, Khalda Fadlalla, Upender Manne, Clayton Yates, Temesgen Samuel. Stromal response to prostate cancer therapeutics. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B11.

Abstract B70: Differential activation of NF-kB signaling and CXCL8 secretion in colon cancer cells treated with chemotherapeutic drugs.

Understanding the determinants of individual patient responses to chemotherapeutic drugs is a key component of personalized cancer therapy. These determinants could be regarded as actual drug-response modifiers or as biomarkers for drug response. The NF-kB signaling pathway is linked with cancer initiation and progression. NF-kB signaling has also been suggested to contribute to drug resistance, but the determinants of drug response and resistance after NF-kB activation are not known. For colon cancer cells, we examined the activation of NF-kB activation and its target genes in response to chemotherapeutic drugs. Materials and Methods: NF-kB reporter stable cells were established by transducing p53-mutant SW480, p53-wild type HCT116, and p53-null HCT116 colon cancer cells with lentiviral constructs containing NF-kB transcriptional response elements (TREs) linked to the luciferase gene. Luciferase expression was quantified as relative luciferase units. To determine which anticancer drugs preferentially activate NF-kB, the reporter cells were treated with 5-fluorouracil (5-FU, DNA/RNA damaging), oxaliplatin (DNA damaging), camptothecin (topoisomerase inhibitor), phleomycin (radiomimetic), or erlotinib (EGFR inhibitor). In addition to luciferase expression, downstream targets of NF-kB activation were evaluated by Western blotting (cIAP2, and Bcl-xL) or ELISA (CXCL8 and IL-6 in culture supernatants). Results: There were differences in the degrees of activation of NF-kB in response to the drugs. Camptothecin activated NF-kB in SW480 cells, but not in HCT116 cells, which were sensitive to the drug. There was no activation even at drug concentrations that did not diminish viability of HCT116 cells. Phleomycin also activated NF-kB only in SW480 cells, whereas erlotinib activated the pathway only in HCT116 cells. 5-FU failed to activate the NF-kB pathway in any of the three cell lines. Western blotting for the NF-kB targets, cIAP2 and Bcl-xL, showed that activation of NF-kB in SW480 cells by camptothecin was not accompanied by increased expression of these two proteins, which are known mediators of the anti-apoptotic functions of NF-kB. However, there was a robust increase in the expression and release of the cytokine CXCL8 by SW480 cells treated with the drug. IL-6 was not detected in culture supernatants from the cells. Conclusion: CXCL8, but none the other target genes cIAP2, Bcl-xL nor IL-6, was up-regulated by camptothecin-activated NF-kB. Therefore, the NF-kB-CXCL8 signaling axis appears to be involved in modifying the drug response in colon cancer cells exposed to this topoisomerase inhibitor. Supported by the Morehouse School of Medicine/Tuskegee University and UAB Comprehensive Cancer Center Partnership grant (NIH U54CA18948-07). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B70. Citation Format: Temesgen Samuel, Khalda Fadlalla, Ramy Elgendy, Balananda-Dhurjati Kumar Putcha, James Posey, Upender Manne. Differential activation of NF-kB signaling and CXCL8 secretion in colon cancer cells treated with chemotherapeutic drugs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B70.

Abstract 4664: Co-treatment of cancer cells with DNA damaging drugs and quercetin suppresses cell growth independent of p21 and Bax induction

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Quercetin, one of the most abundant polyphenols, has been evaluated for its potential cancer preventive functions and for its anticancer activity in vitro and in vivo. Its interactions with chemotherapeutics, however, have not been established. Some reports indicate enhancement of drug activity by quercetin but others suggest precautions in co-administering antioxidants such as quercetin and chemotherapeutic drugs. Therefore, further investigations were needed to determine the conditions under which the co-treatment of cancer cells with drugs and quercetin could be clinically beneficial. This study was done to examine how quercetin modulates responses to the chemotherapeutic drugs, 5FU, camptothecin, and VP16, in colon and prostate cancer cells, and to determine the biochemical processes involved in the interactions. Survival of HCT116 colorectal cancer cells (p53 wild-type & null) was determined by assay of clonogenicity of cells exposed to selected concentrations of 5-FU, camptothecin, or VP16 in the presence or absence of quercetin. For cell cycle and biochemical experiments, cells were exposed for 24 hours to 10 µM 5FU, 1 µM camptothecin, or 10 µM VP-16 with or without 50 µM quercetin. The effects of the drugs on the cell cycle were measured by flow cytometry. The induction of p53 and its transcriptional targets, p21 and Bax, as well as the levels of cell cycle regulators, cyclin B1 and survivin, were determined by immunoblotting. Additionally, cell migration assays were used to evaluate the effect of combination treatments on the motility of RKO colorectal and PPC1 prostate cancer cells. Quercetin synergistically inhibited the clonogenicity of the wild-type HCT116 cells, but also inhibited effects on the cell cycle of all the drugs tested. In contrast, for p53-null cells, the combination of low concentrations of 5-FU with up to 6 µM quercetin promoted clonogenic survival. Exposure of wild-type cells to 50 µM quercetin reduced drug-induced up-regulation of p53, p21, and Bax. Combinations of quercetin and the drugs also reduced the levels of cyclin B1 and survivin. RKO and PPC1 cells exposed to the drugs had reduced migratory capacity. Although quercetin alone reduced their migratory capacity, combination treatments did not further reduce cell migration. In summary, quercetin in combination with 5-FU reduced the survival of p53-proficient HCT116 colorectal cancer cells independently of p21 and Bax, but combination of this flavonoid with low concentrations of 5-FU provided a survival advantage for p53-null cells. Further investigations are needed to determine the mechanisms and circumstances under which the combinations of bioactive dietary compounds and chemotherapeutic drugs are beneficial. Supported by grants from NCI/NIH (2U54-CA118948-06, and SC2CA138178). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4664. doi:1538-7445.AM2012-4664

Abstract 2005: Modulation of tumor suppressor gene DNA-methylation by quercetin and dietary indoles

Epidemiological and preclinical research provide strong evidence for the anticancer properties of bioactive dietary compounds abundantly found in fruits, vegetables and other fiber-rich food stuff. However, the mechanisms by which nutritional intake of such dietary compounds prevent oncogenesis is largely unknown. We hypothesize that synergistic activities of different dietary compounds through epigenetic mechanisms underlie some of the chemopreventive functions of the dietary compounds. We undertook a preliminary study to examine the effects of Quercetin, Indole-3-Carbinol (I3C) and diindolylmethane (DIM) on promoter methylation profiles of selected genes known to be suppressed by methylation in colorectal cancer. Quercetin is a polyphenolic flavonoid, while I3C and DIM are dietary indoles, DIM being a derivative of I3C. The genes queried in this study were p16INK4a, E-cadherin (CDH1), and IGFBP7. p16INK4a is a cell cycle regulator silenced in up to 40-50% of colorectal carcinomas. E-cadherin is a tumor suppressor and metastasis regulator protein the loss of which promotes wnt-signaling, which is dysregulated in the majority of colorectal cancers. IGFBP7 has recently been identified as a p53-responsive gene frequently silenced in colorectal and gastric cancers. In this study, RKO colorectal cancer cells were treated with the three dietary compounds, vehicle (DMSO), or the demethylating compound 5-azacytidine. DNA was harvested and bisulfite converted. Methylation of the gene promoters was examined by methylation-specific PCR using primer sets that amplify only methylated DNA. Gel electrophoresis and Real-time PCR were used to assess the relative abundance of methylated DNA in control and treated samples. Preliminary results show that the dietary compounds can reverse the methylated-promoter phenotype, with the indole compounds having higher potency compared to quercetin especially in demethylating the CDH1 gene promoter. Further experiments will elucidate the molecular mechanisms through which the compounds modulate gene methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2011-2005

Abstract 2490: The flavonoid quercetin interferes with the activity of anti-microtubule drugs

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Dietary compounds with anticancer properties such as polyphenols are intensively studied for their chemopreventive or therapeutic potentials. Quercetin is a polyphenolic flavonoid with anticancer properties and is abundantly available in fruits and green vegetables. In this study, we examined the effects of quercetin on the cell cycle, viability and proliferation of colorectal and prostate cancer cells, either singly or in combination with the microtubule targeting drugs taxol or nocodazole. Quercetin inhibited cell proliferation in a dose dependent manner in both HCT116 colorectal and PPC1 prostate cancer cells. However, unexpectedly, combination treatment of the cells with 50µM or lower quercetin and taxol or nocodazole neutralized the ability of both drugs to induce G2/M arrest and to decrease cell viability. BrdU incorporation was inhibited by quercetin or quercetin-drug combination treatment of cells, but the microtubule targeting of taxol or nocodazole was not inhibited. Moreover, colony formation by both wild type and p53-null HCT116 colorectal cancer cells was inhibited by the same dose of quercetin that neutralized G2/M arrest by Taxol and nocodazole, suggesting a p53-independent effect. We conclude that while quercetin by itself may reduce cell proliferation and survival, in the short-term, the interference of quercetin with cell cycle progression diminishes the ability of taxol and nocodazole to arrest cells at G2/M. Our study adds microtubule targeting drugs to the list of anticancer drugs the activity of which might be antagonized by flavonoids such as quercetin. Further studies are needed to examine the mechanisms of interactions between anticancer drugs and polyphenolic dietary components or supplements, and how these interactions may influence outcomes of cancer therapy, survivorship or recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2490.