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Nick Wolpert: Design, Application and Testing of a Novel Scratch Device for Analyzing Cell Movement Towards Migration, Invasion and Wound Healing
Motility and gap closure rates are solely dependent on how consistent and reliable the method of generating a scratch is to ensure relevant and reliable data. Current methods of determining cell movement most commonly use a pipet tip to scratch the surface of the substrate upon which the cells are cultured. This method is fraught with inconsistencies that primarily involve varying widths and depths of the scratch. To determine if the newly created device generated reproducible and consistent results across different users, while maintaining a shallow learning curve, a prototype device was developed and tested against the most common method – a pipet tip scratch. The wound assay device was designed in AutoCAD and 3D printed using a Dremel 3D fused deposition modeling (FDM) printer. The wound assay entails growing the cells to ~90% confluence and creating a scratch through the cells (Cormier et al. 2015). The device is referred to as the Wound Wizard. It results in the patent application: U.S. Provisional Patent Application Serial No. 63471160 entitled “WOUND ASSAY DEVICE AND RELATED METHODS OF USE”, filed on June 5th, 2023, Inventors: Wolpert, Nicholas, and Lauren Gollahon.
Noshin Mubtasim: Differentiation in presence of Fatty Acids combination increase the lipid accumulation profile of 3T3-L1 MBX
The increasing prevalence of obesity has prompted intensive research into understanding its role in pathogenesis, and to design appropriate treatments. To determine the signals generated from the interaction of fat cells with a target organ, a reliable white adipocyte model in vitro is needed. Differentiated fibroblasts are the most extensively studied in vitro cell model of white adipocytes. However, it can be argued that differentiated fibroblasts minimally recapitulate the consequences of obesity. Here, we describe 3T3-L1 MBX cells as a culture model for studying obese adipocytes and their effects. Differentiation of 3T3-L1 MBX cells was at first optimized and then were maintained in the presence of a fatty acid cocktail combination to induce the obese condition. Lipid accumulation and the adipokine secretion profiles were analyzed. Results showed that fatty acid-maintained, differentiated 3T3-L1 MBX cells had significantly greater accumulation of lipids and significant changes in the adipokine secretions in comparison to differentiated 3T3-L1 MBX cells maintained in medium without fatty acids. To elucidate the molecular changes associated with adipogenesis and lipid accumulation profile of 3T3-L1 MBX cells, we have also explored the expression of some of the regulatory proteins related to the development and maintenance of adipocytes from the preadipocyte lineage.
Soni Khandelwal: Cytotoxicity of Selenium Immunoconjugates against Triple Negative Breast Cancer Cells
Within the subtypes of breast cancer, those identified as triple negative for expression of estrogen receptor alpha (ESR1), progesterone receptor (PR) and human epidermal growth factor 2 (Her2), account for 10-20% of breast cancers, yet result in 30% of global breast cancer-associated deaths. Thus, it is critical to develop more targeted and efficacious therapies that also demonstrate less side effects. Selenium, an essential dietary supplement is incorporated as selenocysteine (Sec) in vivo into human selenoproteins, some of which exist as anti-oxidant enzymes and are of import to human health. Studies have also shown that selenium compounds hinder cancer cell growth and induce apoptosis in cancer cell culture models. The focus of this study was to investigate whether selenium-antibody conjugates could be effective against triple negative breast cancer cell lines using clinically relevant, antibody therapies targeted for high expressing breast cancers and whether selenium cytotoxicity was attenuated in normal breast epithelial cells. To that end, the humanized monoclonal IgG1 antibodies, Bevacizumab and Trastuzumab were conjugated with redox selenium to form Selenobevacizumab and Selenotrastuzumab and tested against the triple negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231 as well as a normal, immortalized, human mammary epithelial cell line, HME50-5E. VEGF and HER2 protein expression were assessed by Western. Although expression levels of Her2 were low or absent all test cells, our results showed that Selenobevacizumab and Selenotrastuzumab produced superoxide (O2-) anions in the presence of glutathione and confirmed by dihydroethidium (DHE) assay. Interestingly, superoxide was not elevated within HME50-5E cells assessed by DHE. The cytotoxicity of selenite and the selenium immunoconjugates towards triple negative cells compared to HME-50E cells was performed in a time and dose dependent manner as measured by Trypan Blue exclusion, MTT and Annexin V assays. Selenobevacizumab and Selenotrastuzumab were shown to arrest the cancer cell growth but not the HME50-5E cells. These results suggest that selenium-induced toxicity may be effective in treating TNBC cells by exploiting different immunotherapeutic approaches potentially reducing the debilitating side effects associated with current TNBC anticancer drugs. Thus, clinically relevant, targeting antibody therapies may be repurposed for TNBC treatment by attachment of redox selenium.
Caroline Schuster: Combinatorial Effects of the Natural Products Arctigenin, Chlorogenic Acid, and Cinnamaldehyde Commit Oxidation
Assassination on Breast Cancer Cells
Major obstacles in current breast cancer treatment efficacy include the ability of breast cancer cells to develop resistance to chemotherapeutic drugs and the off-target cytotoxicity of these drugs on normal cells, leading to debilitating side effects. One major difference between cancer and normal cells is their metabolism, as cancer cells acquire glycolytic and mitochondrial metabolism alterations throughout tumorigenesis. In this study, we sought to exploit this metabolic difference by investigating alternative breast cancer treatment options based on the application of phytochemicals. Herein, we investigated three phytochemicals, namely cinnamaldehyde (CA), chlorogenic acid (CGA), and arctigenin (Arc), regarding their anti-breast-cancer properties. These phytochemicals were administered alone or in combination to MCF-7, MDA-MB-231, and HCC1419 breast cancer or normal MCF-10A and MCF-12F breast cells. Overall, our results indicated that the combination treatments showed stronger inhibitory effects on breast cancer cells versus single treatments. However, only treatments with CA (35 μM), CGA (250 μg/mL), and the combination of CA[GL1] + [M2] CGA (35 μM + 250 μg/mL) showed no significant cytotoxic effects on normal mammary epithelial cells, suggesting that Arc was the driver of normal cell cytotoxicity in all other treatments. CA + CGA and, to a lesser extent, CGA alone effectively induced breast cancer cell death accompanied by decreases in mitochondrial membrane potential, increased mitochondrial superoxide, reduced mitochondrial and glycolytic ATP production, and led to significant changes in cellular and mitochondrial morphology. Altogether, the combination of CA + CGA was determined as the best anti-breast-cancer treatment strategy due to its strong anti-breast-cancer effects without strong adverse effects on normal mammary epithelial cells. This study provides evidence that targeting the mitochondria may be an effective anticancer treatment, and that using phytochemicals or combinations thereof offers new approaches in treating breast cancer that significantly reduce off-target effects on normal cells.
Jaehyung Lee: Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells.
NIMA-related kinase 2 (NEK2), a serine-threonine protein kinase, plays a role in features of mitotic progression including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Kinase death NEK2 mutant expression or NEK2 depleted cells lead to failure of centrosome separation while NEK2 overexpression results in premature centrosome separation. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with NEK2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between NEK2 and TRF1 are far from clear. Here, we show that mitotic aberrations through NEK2 overexpression are likely to require TRF1. Our results demonstrate that NEK2 directly binds and phosphorylates TRF1 in vitro and in vivo through multiple sites on TRF1. NEK2 overexpression in breast cancer cells, MCF 7 and MDA-MB-231, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the occurrence of unaligned chromosomes by NEK2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1 depleted cells demonstrated ~ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA transfected cells. Therefore, we propose that TRF1 is required for overexpressed NEK2 to trigger abnormal mitosis and chromosomal instability. This is demonstrated in the following model:
From this work, we were selected for the cover in the December 2013 volume of Cell Cycle.
Zhi Pan: Investigating the role of the endoplasmic reticulum in calcium regulation with regards to paclitaxel treatment.
This work involves three separate but related projects measuring changes in ER-calcium release, paclitaxel time and treatment concentrations and the role of the anti-apoptotic protein Bcl2 in regulating calcium concentrations in the cell. This is important because of implications for PCD.
Project 1. Taxol™, an important chemotherapy agent, employs apoptosis to induce cell death in breast cancer treatment. Our previous study showed that Taxol™ directly releases calcium from the endoplasmic reticulum (ER), which can promote subsequent apoptosis in breast cancer cells. Besides its effect on the ER, Taxol™ also stabilizes microtubules, causing mitotic arrest. However, the relationship between these events and apoptosis remains unclear.. Our results showed that the ER calcium store is a key common target for paclitaxel and Bcl-2, since both can directly change ER calcium release independent of each other. This direct ER-calcium involvement, linking the actions of paclitaxel and Bcl-2 together, helps elucidate the underlying mechanisms of the paclitaxel - Bcl-2 relationship in a novel way, outside of the previously described, indirect ER calcium regulation - through the phosphorylation of Bcl-2 by paclitaxel. Furthermore, the dosage of paclitaxel is crucial for inducing significant ER-calcium release to overcome Bcl-2-mediated apoptotic resistance. Therefore, our findings not only helps clarify the controversy regarding the efficacy of paclitaxel in the presence of Bcl-2 expression, but may also assist oncologists in optimizing the usage of paclitaxel in breast cancer treatment. More research is needed to fully understand the complex relationship between paclitaxel, calcium and Bcl-2. Our results can be summarized in the following figure and graphic abstract.
Project 2: Taxol Directly Induces Endoplasmic Reticulum-Associated Calcium Changes That Promote Apoptosis in Breast Cancer Cells.
Calcium, a key regulator of cell survival, is also important in regulating apoptosis. Although the chemotherapeutic agent Taxol employs apoptosis to induce cell death, the exact mechanism of how it induces apoptosis and the role of calcium in this process remains unclear. The main intracellular calcium storehouse, the endoplasmic reticulum, was identified as a new important gateway in apoptosis, possibly providing a target for Taxol. The goal of this study was to investigate whether calcium changes associated with the endoplasmic reticulum, were directly or indirectly generated by Taxol at clinically relevant doses, and related to Taxol-induced apoptosis in breast cancer cells. Time-lapsed imaging techniques followed by an endoplasmic reticulum-targeted construct, cameleon D1ER, were used to monitor cytosol––endoplasmic reticulum calcium dynamics in MDA-MB-468 (Bcl-2 negative) and MCF 7 (Bcl-2 positive) breast carcinoma cells. Apoptosis levels were measured with Annexin V and Propidium Iodide (PI) using flow cytometry. Our findings demonstrate that endoplasmic reticulum calcium stores provide a direct target for Taxol action and are important for induction of apoptosis, independent of Bcl-2 status. Furthermore, our results show for the first time, that the role of calcium in Taxol-induced endoplasmic reticulum-mediated apoptosis is dependent on Taxol dosage.
Project 3: Paclitaxel induces apoptosis in breast cancer cells through different calcium - regulating mechanisms depending on external calcium conditions.
Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed.
Kyungwoo Lee: Telomere Biology and Cancer Stem Cells
ZSCAN4 Directly Interacts with Human Rap1 in Cancer Cells Regardless of Telomerase Status.
Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of the shelterin complex that form capping structures. Through the interaction of shelterin complex-associated proteins, telomere length maintenance is regulated Western, pull-down, siRNA and overexpression assays we demonstrate, for the first time, that Zscan4 directly associates with Rap1 (physical association protein). Furthermore, by generating truncated versions of Zscan4, we identified its zinc finger domain as the Rap1 binding site. Using bimolecular fluorescence complementation (BIFC), we further validate this functional interaction in human cancer cells. Our results indicate that Zscan4 functions as a mediator of telomere length through its direct interaction with Rap1, possibly regulating shelterin complex-controlled telomere elongation in both telomerase positive and alternative lengthening of telomere pathways. This direct interaction between Zscan4 and Rap1 may explain how Zscan4 rapidly increases telomere length, yielding important information about the role of these proteins in telomere biology.
The following figure demonstrates the BIFC results for a telomerase positive breast cancer cell line (MCF7).
From this work, Dr. Lee proposed two possible models of Zscan4 interaction, based on telomerase status and was given the honor of having his work featured in the Cover Art for that Issue (shown below).
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