Browsing by Author "Zhang, Li"
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Item A Holistic View of Cancer Bioenergetics: Mitochondrial Function and Respiration Play Fundamental Roles in the Development and Progression of Diverse Tumors(Springer) Alam, Md Maksudul; Lal, Sneha; FitzGerald, Keely E.; Zhang, Li; Alam, Md Maksudul; Lal, Sneha; FitzGerald, Keely E.; Zhang, LiSince Otto Warburg made the first observation that tumor cells exhibit altered metabolism and bioenergetics in the 1920s, many scientists have tried to further the understanding of tumor bioenergetics. Particularly, in the past decade, the application of the state-of the-art metabolomics and genomics technologies has revealed the remarkable plasticity of tumor metabolism and bioenergetics. Firstly, a wide array of tumor cells have been shown to be able to use not only glucose, but also glutamine for generating cellular energy, reducing power, and metabolic building blocks for biosynthesis. Secondly, many types of cancer cells generate most of their cellular energy via mitochondrial respiration and oxidative phosphorylation. Glutamine is the preferred substrate for oxidative phosphorylation in tumor cells. Thirdly, tumor cells exhibit remarkable versatility in using bioenergetics substrates. Notably, tumor cells can use metabolic substrates donated by stromal cells for cellular energy generation via oxidative phosphorylation. Further, it has been shown that mitochondrial transfer is a critical mechanism for tumor cells with defective mitochondria to restore oxidative phosphorylation. The restoration is necessary for tumor cells to gain tumorigenic and metastatic potential. It is also worth noting that heme is essential for the biogenesis and proper functioning of mitochondrial respiratory chain complexes. Hence, it is not surprising that recent experimental data showed that heme flux and function are elevated in non-small cell lung cancer (NSCLC) cells and that elevated heme function promotes intensified oxygen consumption, thereby fueling tumor cell proliferation and function. Finally, emerging evidence increasingly suggests that clonal evolution and tumor genetic heterogeneity contribute to bioenergetic versatility of tumor cells, as well as tumor recurrence and drug resistance. Although mutations are found only in several metabolic enzymes in tumors, diverse mutations in signaling pathways and networks can cause changes in the expression and activity of metabolic enzymes, which likely enable tumor cells to gain their bioenergetic versatility. A better understanding of tumor bioenergetics should provide a more holistic approach to investigate cancer biology and therapeutics. This review therefore attempts to comprehensively consider and summarize the experimental data supporting our latest view of cancer bioenergetics.Item Analysis of Heme Functions in Therapy Resistance and Tumorigenesis in Non-small Cell Lung Cancer(2021-08-01T05:00:00.000Z) Dey, Sanchareeka; Zhang, Li; Zhang, Chuanwei; Reitzer, Lawrence J.; Burr, John G.; Misra, JyotiLung cancer remains the leading cause of cancer-related death in the United States, 84% of them being non-small cell lung cancer (NSCLC). Early-stage treatment includes surgery and radiotherapy followed by periodic radiographic imaging for routine surveillance. These curative treatments have shown promise in some lung cancer patients while being ineffective for majority (30–60%) of the patients who are predicted to have advanced disease, particularly local or distant metastasis. This limit overall survival rates in these patients to less than 60%. Moreover, despite the advent of personalized therapy which includes various targeted therapies and immunotherapies, there has not been a significant improvement in the 5-year survival rate. Therefore, there is a pressing need to further optimize current strategies while continuing to explore novel strategies to improve therapeutic outcomes for patients with lung cancer, based on individual patient needs. Numerous studies are now focusing on the importance of mitochondrial respiration or oxidative phosphorylation (OXPHOS) in cancer progression. However, very little is known about its role and potential as a therapeutic target in non-small cell lung cancer (NSCLC). Several studies in our lab show that NSCLC cells display elevated levels of intracellular heme. This increased level of heme is either through de novo heme synthesis or heme uptake. Our lab has also demonstrated elevated mitochondrial respiration/oxidative phosphorylation (OXPHOS) in NSCLCs. Studies in our lab show that elevated expression of enzymes involved in heme biosynthesis, uptake, and degradation, as well as oxygen-utilizing hemoproteins in resistant cells post treatment with vascular disrupting agents. Limiting oxidative functions using Cyclopamine tartrate (CycT), inhibition of heme uptake with heme sequestering peptides (HSP2) and heme synthesis using succinyl acetone, have all shown promise in delaying growth and progression of NSCLC cells and tumor xenografts. Another feature of NSCLC tumor is its heterogeneity. NSCLCs exhibit widespread inter- and intra-tumoral heterogeneity as well as incidences of subtype transdifferentiation. This kind of plasticity enable them to develop drug resistance and pose great challenges for their treatment. In this study I aimed to understand the comparative dependence of the two major NSCLC subtypes, adenocarcinoma (ADC) and squamous cell carcinoma (SCC), on heme and OXPHOS, for their growth and progression. I observed that both ADC and SCC have similar demands for heme uptake and synthesis in cell culture. My results also suggest that OXPHOS activities are elevated in both ADC and SCC to support tumorigenic functions in culture. I used the Genetically Engineered Mouse Model (GEMM), KLLuc to study NSCLC tumor heterogeneity since tumors developed in these mice consists of both ADC and SCC phenotypes. My findings in vitro were corroborated in this model, using immunohistochemistry (IHC) and histology. Bioluminescence imaging and histology studies demonstrate that heme sequestering peptides successfully reduce tumor development and progression in KLLuc mice.Item Biochemical Studies of Regulatory Heme-Protein Interaction and Heme Regulation in Yeast(2018-08) Comer, Jonathan M.; 0000-0002-7156-5979 (Comer, JM); Zhang, LiHeme is a small biomolecule produced and used by living organisms from bacteria to humans. Heme consists of an organic porphyrin ring that coordinates an iron ion, Fe(II) or Fe(III), in its center. Heme can be in an oxidized or reduced state and is used extensively by organisms in a variety of redox reactions. As a cofactor in proteins of the electron transport chain, heme is crucial for cellular respiration and energy production. Heme is also the oxygen binding cofactor in globins such as hemoglobin and myoglobin, allowing organisms to distribute oxygen. Free heme in the cell is capable of producing reactive oxygen species, which are toxic to the cell. However, the degradation product of heme, bilirubin, is a powerful protectant against oxidation. In cytochromes, such as P450s and cytochrome c, heme is used in the synthesis and degradation pathways of sterols, lipids, and neurotransmitters. With such a central role in energy production, synthesis and degradation, oxidative stress, and oxygen transport, it makes sense that heme also acts as a very important signaling molecule regulating many genetic pathways such as oxidative stress response and carbon metabolism. The function of heme as a prosthetic group in proteins, such as cytochromes, is now well documented. Less is known, however, about its role as a regulator of metabolic and energetic pathways. This is due in part to some inherent difficulties in studying heme. Due to its slightly amphiphilic nature, heme is a “sticky” molecule that can easily bind non-specifically to proteins. In addition, heme tends to dimerize, oxidize, and aggregate in purely aqueous solutions; therefore, there are constraints on buffer composition and concentrations. Despite these difficulties, our knowledge of heme’s regulatory role continues to grow. This work describes the application of common molecular biology techniques to the unique situation of studying heme-protein regulation. Chapter 2 describes our findings of the heme regulation of Gis1, a yeast transcription factor and demethylase. Gis1 regulates the response to metabolic stress after the diauxic shift. Our lab previously identified Gis1 as a fast responder to hypoxia and re-oxygenation. Heme is closely related to oxygen utilization and signaling, and the Gis1 sequence contains two heme regulatory motifs (HRM), therefore we decided to study the regulation of Gis1 by heme. It was found that heme bound to at least two locations on Gis1, and that the zinc finger domain, which contains an HRM, promoted heme activation of Gis1 transcriptional and demethylase activities. The Jumonji domain, which contains the second heme binding site and an HRM, did not convey heme regulation of Gis1, but it did cause heme regulation of a different transcriptional activating domain. As a member of the JMJD2b/KDM4 family of demethylases, conserved in mammals, Gis1 represents a new class of heme signaling protein.Item Broadband Transient Absorption Study of Photoexcitations in Lead Halide Perovskites: Towards a Multiband Picture(2016-02-24) Alam, Md Maksudul; Sohoni, Sagar; Kalainayakan, Sarada Preeta; Garrossian, Massoud; Zhang, Li; 0000-0001-9242-0763 (Zhang, L); Alam, Md Maksudul; Sohoni, Sagar; Kalainayakan, Sarada Preeta; Zhang, LiBACKGROUND: Aberrant Hedgehog (Hh) signaling is associated with the development of many cancers including prostate cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, and basal cell carcinoma. The Hh signaling pathway has been one of the most intensely investigated targets for cancer therapy, and a number of compounds inhibiting Hh signaling are being tested clinically for treating many cancers. Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined. Cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. To find novel strategies for combating lung cancer, we decided to characterize the effect of cyclopamine tartrate (CycT), an improved analogue of cyclopamine, on lung cancer cells and its mechanism of action. METHODS: The effect of CycT on oxygen consumption and proliferation of non-small-cell lung cancer (NSCLC) cell lines was quantified by using an Oxygraph system and live cell counting, respectively. Apoptosis was detected by using Annexin V and Propidium Iodide staining. CycT’s impact on ROS generation, mitochondrial membrane potential, and mitochondrial morphology in NSCLC cells was monitored by using fluorometry and fluorescent microscopy. Western blotting and fluorescent microscopy were used to detect the levels and localization of Hh signaling targets, mitochondrial fission protein Drp1, and heme-related proteins in various NSCLC cells. RESULTS: Our findings identified a novel function of CycT, as well as another Hh inhibitor SANT1, to disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased ROS generation, mitochondrial membrane hyperpolarization, and mitochondrial fragmentation, thereby disrupting mitochondrial function in NSCLC cells. CONCLUSIONS: Together, our work demonstrates that CycT, and likely other Hh signaling inhibitors, can interrupt NSCLC cell function by promoting mitochondrial fission and fragmentation, mitochondrial membrane hyperpolarization, and ROS generation, thereby diminishing mitochondrial respiration, suppressing cell proliferation, and causing apoptosis. Our work provides novel mechanistic insights into the action of Hh inhibitors in cancer cells.Item Characterizing the Role of Heme in Alzheimer’s Disease Pathogenesis(2021-05-01T05:00:00.000Z) Vidal, Chantal; Zhang, Li; Pantano, Paul; Burr, John G.; Ploski, Jonathan E.; Delk, NikkiIn humans, heme accounts for 97% of functional iron. With a porphyrin ring and an iron ion, heme possesses structural and chemical features fitting for electron transfer, oxidation/reduction, and interaction with oxygen. Three oxidative phosphorylation (OXPHOS) complexes, II, III, and IV, require heme for proper functioning. Cells that require high levels of adenosine triphosphate (ATP) and OXPHOS require elevated levels of heme. Heme also serves as a powerful antioxidant for cells because it can be degraded to biliverdin and reduced to bilirubin. The reduction of biliverdin to bilirubin helps relieve reactive oxygen species (ROS) in cells. Neuronal cells are known to be high-energy demanding cells that depend on mitochondrial respiration to function. Specifically, in neurodegenerative diseases such as Alzheimer’s Disease (AD), mitochondrial dysfunction is one of the key characteristics associated with the progression of this disease. Oxidative stress has also been implicated in the pathogenesis of AD. Due to the role of heme in both of these cellular functions, it is important to understand how heme contributes to neuronal function and how it may play a pivotal role in developing this neurodegenerative disease. The objective of this research is to dissect the role of heme in the pathogenesis of AD. Utilizing immunocytochemistry and Western blot techniques, I have depicted the importance of heme in neuronal development. Heme uptake, synthesis, and degradation are significantly increased in developing and differentiating neurons. This increase in heme flux coincides with an increase in mitochondrial proteins. Using the APPPS1 mouse model and microarray expression data of human patients, I detected specific heme-related enzymes that are downregulated in AD. These alterations can lead to a decrease in the availability of heme for cellular functions. The decrease in heme availability can lead to disturbed mitochondrial function and an increase in oxidative stress. Furthermore, to glean whether heme flux alterations are early and potentially initiating causes of AD, I utilized patient-derived neurons generated from human-induced pluripotent stem cells (iPSCs). These studies revealed a distinct role of heme in the development of familial (FAD) and sporadic AD (SAD). SAD neurons have downregulated heme synthetic and degradation enzymes, while FAD neurons only had a slight yet significant reduction in the biliverdin reductase B (BLVRB) enzyme involved in heme degradation. Moreover, analysis of the tricarboxylic acid (TCA) cycle enzymes and intermediates revealed a significant alteration in enzymes and intermediates within both SAD and FAD neurons relative to gender-matched controls. This study revealed that although heme flux alterations are likely an early event in SAD pathogenesis, perturbations in the TCA cycle are probably a common characteristic of both SAD and FAD.Item Dissecting the Role of Heme in Regulating Yeast and Mammalian KDM4 Histone Demethylases(2020-08) Konduri, Purna Chaitanya; 0000-0003-0564-7770 (Konduri, PC); Zhang, LiHeme (Iron protoporphyrin IX) is an essential biological molecule and an important metallonutrient for organisms ranging from bacteria to humans. It is also a signaling molecule that modulates the activities of diverse regulatory proteins. Recent studies have identified a novel class of heme-regulated proteins- the JmjC domain-containing proteins, constituted by the yeast Gis1, an ortholog of the human KDM4 proteins. Heme regulates the transcriptional and demethylase of activities of Gis1. The KDM4 subfamily of proteins containing the JmjC domain, function as a-ketoglutarate- and Fe (II)-dependent histone demethylases. Histone lysyl demethylases (KDMs) play important roles in chromatin remodeling and gene regulation by catalyzing demethylation of the lysine residues on the N-terminal tails of histones. The objective of this research was to dissect the molecular events underlying the heme regulation of Gis1 and KDM4 proteins. The JmjN/C domains of Gis1 and KDM4A/B/C share high homology, and the C-terminal domains of KDM4s possess the PHD zinc fingers. Here, using various biochemical methods, we showed that heme binds to purified KDM4 A/B/C proteins containing only the JmjN/C domain. Interestingly, we identified that heme stimulates the histone demethylase activities of KDM4 A and C proteins, containing only the JmjN/C domain and full length Gis1, but not KDM4B. Additionally, the C-terminal regions of KDM4s like Gis1 could confer heme regulation when fused to an unrelated transcriptional activator. Furthermore, a biochemical pulldown study coupled with mass spectrometry identified 147 heme-regulated protein interactors of Gis1. These proteins include a significant number of heterocyclic compound binding proteins, Ubl conjugated proteins, metabolic enzymes and acetylated proteins. These results demonstrate a previously uncharacterized heme-regulated protein complex formation of Gis1 and likely other KDM4s to sense metabolic and nutritional conditions and transduce signals to downstream regulatory pathways.Item Electrochemical Performance of Polymer Derived Carbon Nanofibers and Tungsten Compound/Carbon Composites(2021-12-01T06:00:00.000Z) Garcia, Juan Alexandro; Ferraris, John P; Zhang, Li; Pantano, Paul; Balkus, Jr., Kenneth J; Yang, Duck JooCurrent energy demands and advancements in energy harvesting have driven research towards developing storage devices with high energy and power densities to better store and deliver charge. Battery devices fulfill many of these demands, but supercapacitor devices have garnered more favor due to their rapid charge rate capabilities and their vast cyclability. The different classifications of supercapcitors (electric double layer capacitors, pseudocapacitors, and hybrid supercapcitors) rely on either physical charge storage mechanisms which provide rapid delivery of charge, faradaic charge storage mechanisms which can be used to achieve how quantities of charge, or a combination of the two. While advancements in the field have been great, they are largely driven by trial-and-error approaches. To fully achieve the potential of supercapacitor devices, a better fundamental understanding of the aspects of the electrode materials and their influence over charge capabilities is needed. Chapter 1 introduces supercapacitor devices, and some of the materials that can be used to make them. It provides details on the production of activated carbon nanofibers (CNFs) derived from electrospun polyacrylonitrile (PAN) and tungsten compounds that were used in this work. Chapter 2 demonstrates the influence of miscibility in electrospun polymer blends on electrochemical performance on devices with ionic liquid electrolyte. In comparison to blends of PAN:polystyrene (PS) which have a miscibility parameter (MP) of 118, the studied blend of PAN:poly(styrene-co-acrylonitrile) has a miscibility parameter of 79. This resulted in a distinct morphology for phase separation of the blends, as well as a channel like morphology with interspersed domains. Devices from the PAN:SAN blend achieved a maximum power densities of approximately 17,500 W/kg when tested at 10 A/g in galvanostatic charge/discharge (GCD) and achieved a maximum energy density of 81 Wh/kg at 1750 W/kg when tested at 1 A/g. This device also boasted a capacitance retention of 82% after 3,500 cycles. Chapter 3 describes the preparation of hybrid electrode materials derived from polymer fibers with tungsten oxide nanoparticles and the influence of their degree of interaction on performance. A novel synthesis was described for the preparation of WO2.72, which normally is synthesized through a hydrothermal procedure in an autoclave, was prepared by in-situ synthesis of hybrid tungsten compound at CNFs with CO2 activation. This hybrid material (WO@CNF) was compared to pure WO2.72 and CNFs to determine the degree of interaction between the components in the hybrid and determine that interactions influence on the performance of the device. The WO@CNF material was found to have a low degree of interaction, but this still provided an improvement on the energy storage capabilities of the material over the physical mixture composite. Chapter 4 describes the modification of the synthesis used to make WO2.72 nanoparticles in carbon fibers to produce WN nanoparticles and the comparison of their electrochemical performance. Upon carbonization without activation, WN was found in the WO@CNF hybrid material. Metal nitrides possess favorable qualities for pseudocapacitors, and hybrid supercapacitors compared to metal oxides, and WN is not widely researched for these devices. Utilizing ammonia activation drove the synthesis towards WN to form WN@CNF hybrid materials. The WN@CNF produced carbons with a higher graphitic quality (Id:Ig ratio of 0.77 compared to 0.91 for WO@CNF), and achieved comparable power densities, but possessed lower energy densities.Item Enhanced heme function and mitochondrial respiration promote the progression of lung cancer cells(2013-05-21) Hooda, Jagmohan; Cadinu, Daniela; Alam, Md Maksudul; Shah, Ajit; Cao, Thai M.; Sullivan, Laura A.; Brekken, Rolf; Zhang, Li; Zhang, LiLung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer biology and therapeutics.Item Experimental Methods for Studying Cellular Heme Signaling(MDPI) Comer, Jonathan M.; Zhang, Li; 0000-0001-9242-0763 (Zhang, L); Comer, Jonathan M.; Zhang, LiThe study of heme is important to our understanding of cellular bioenergetics, especially in cancer cells. The function of heme as a prosthetic group in proteins such as cytochromes is now well- documented. Less is known, however, about its role as a regulator of metabolic and energetic pathways. This is due in part to some inherent difficulties in studying heme. Due to its slightly amphiphilic nature, heme is a "sticky" molecule which can easily bind non-specifically to proteins. In addition, heme tends to dimerize, oxidize, and aggregate in purely aqueous solutions; therefore, there are constraints on buffer composition and concentrations. Despite these difficulties, our knowledge of heme's regulatory role continues to grow. This review sums up the latest methods used to study reversible heme binding. Heme-regulated proteins will also be reviewed, as well as a system for imaging the cellular localization of heme.Item Heme Promotes Transcriptional and Demethylase Activities of Gis1, a Member of the Histone Demethylase JMJD2/KDM4 Family(Oxford University Press, 2018-10-22) Lal, Sneha; Comer, Jonathan M.; Konduri, Purna C.; Shah, Ajit; Wang, Tianyuan; Lewis, Anthony; Shoffner, Grant; Guo, Feng; Zhang, Li; Lal, Sneha; Comer, Jonathan M.; Konduri, Purna C.; Wang, Tianyuan; Lewis, Anthony; Zhang, LiThe yeast Gis1 protein is a transcriptional regulator belonging to the JMJD2/KDM4 subfamily of demethylases that contain a JmjC domain, which are highly conserved from yeast to humans. They have important functions in histone methylation, cellular signaling and tumorigenesis. Besides serving as a cofactor in many proteins, heme is known to directly regulate the activities of proteins ranging from transcriptional regulators to potassium channels. Here, we report a novel mechanism governing heme regulation of Gis1 transcriptional and histone demethylase activities. We found that two Gis1 modules, the JmjN + JmjC domain and the zinc finger (ZnF), can bind to heme specifically in vitro. In vivo functional analysis showed that the ZnF, not the JmjN + JmjC domain, promotes heme activation of transcriptional activity. Likewise, measurements of the demethylase activity of purified Gis1 proteins showed that full-length Gis1 and the JmjN + JmjC domain both possess demethylase activity. However, heme potentiates the demethylase activity of full-length Gis1, but not that of the JmjN + JmjC domain, which can confer heme activation of transcriptional activity in an unrelated protein. These results demonstrate that Gis1 represents a novel class of multi-functional heme sensing and signaling proteins, and that heme binding to the ZnF stimulates Gis1 demethylase and transcriptional activities.Item Heme Sequestration as an Effective Strategy for the Suppression of Tumor Growth(2021-08-01T05:00:00.000Z) Wang, Tianyuan; Zhang, Li; Lumata, Lloyd; Kim, Tae Hoon; Spiro, Stephen; Xuan, ZhenyuHeme is an essential prosthetic group in proteins and enzymes involved in oxygen utilization and metabolism. Heme also plays versatile and fascinating roles in regulating fundamental biological processes ranging from aerobic respiration to drug metabolism. Increasing experimental and epidemiological data have also shown that altered heme homeostasis accelerates the development and progression of common diseases, including various cancers, diabetes, vascular diseases, and Alzheimer's disease. The effects of heme on the pathogenesis of these diseases may be mediated via its action on various cellular signaling and regulatory proteins, as well as its function in cellular bioenergetics, specifically, oxidative phosphorylation (OXPHOS). Elevated heme levels in cancer cells intensify OXPHOS, leading to higher ATP generation and fueling tumorigenic functions. In contrast, lowered heme levels in neurons may reduce OXPHOS, leading to defects in bioenergetics and causing neurological deficits. Additionally, heme has been shown to modulate the activities of diverse cellular proteins influencing disease pathogenesis. These include tumor suppressor P53 protein, progesterone receptor membrane component 1 protein PGRMC1, cystathionine-βsynthase CBS, and the nuclear receptor subfamily member Rev-Erbα. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket of hemophores and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bound to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non-small lung cancer (NSCLC) cells, while their effects on non-tumorigenic cell lines representing normal lung cells were not significant. HeSPs strongly suppressed the growth of human NSCLC tumor xenografts in mice. HeSPs decreased oxygen consumption rates and ATP levels in tumor cells isolated from treated mice, while they did not affect liver and blood cell functions. Immunohistochemistry revealed that HeSPs reduced the levels of key enzymes and transporters involved in heme synthesis and uptake, as well as the uptake and metabolism of the main fuels for cancer cells, glucose and glutamine. Further, we found that HeSPs reduced the levels of angiogenic and vascular markers, as well as vessel density in tumor tissues. Together, these results demonstrate that HeSPs act via multiple mechanisms, including the inhibition of oxidative phosphorylation, to suppress tumor growth and progression. Evidently, heme sequestration can be a powerful strategy for suppressing lung tumors and likely drug-resistant tumors that rely on oxidative phosphorylation for survival.Item Heme, an Essential Nutrient from Dietary Proteins, Critically Impacts Diverse Physiological and Pathological Processes(MDPI AG, 2014-03-13) Hooda, Jagmohan; Shah, Ajit; Zhang, Li; Zhang, LiHeme constitutes 95% of functional iron in the human body, as well as two-thirds of the average person's iron intake in developed countries. Hence, a wide range of epidemiological studies have focused on examining the association of dietary heme intake, mainly from red meat, with the risks of common diseases. High heme intake is associated with increased risk of several cancers, including colorectal cancer, pancreatic cancer and lung cancer. Likewise, the evidence for increased risks of type-2 diabetes and coronary heart disease associated with high heme intake is compelling. Furthermore, recent comparative metabolic and molecular studies of lung cancer cells showed that cancer cells require increased intracellular heme biosynthesis and uptake to meet the increased demand for oxygen-utilizing hemoproteins. Increased levels of hemoproteins in turn lead to intensified oxygen consumption and cellular energy generation, thereby fueling cancer cell progression. Together, both epidemiological and molecular studies support the idea that heme positively impacts cancer progression. However, it is also worth noting that heme deficiency can cause serious diseases in humans, such as anemia, porphyrias, and Alzheimer's disease. This review attempts to summarize the latest literature in understanding the role of dietary heme intake and heme function in diverse diseases.Item Increased Heme Flux and Mitochondrial Resporation Enhance Tumorigenic Functions in Non-Small Cell Lung Cancer Cells(2019-12) Sohoni, Sagar; Zhang, LiLung cancer is the leading cause of cancer related deaths in United States, and about 85% of the cases are Non-small cell lung cancer (NSCLC). Many targeted therapies have been developed to treat lung cancer. Unfortunately, however statistical data over the past two decades suggest only a slight improvement in a patient’s survival rate after diagnosis. Clonal evolution and tumor heterogeneity are the major obstacles in designing effective targeted treatments against cancer. To create more comprehensive treatments, emerging therapies target bioenergetic pathways of cancer cells. Like normal cells, cancer cells can generate energy only through glycolysis and oxidative phosphorylation. Notably, a number of studies have shown that many types of cancer cells rely heavily on mitochondrial respiration. Tumors of human non-small cell lung cancer (NSCLC) are heterogeneous but exhibit elevated glycolysis and glucose oxidation relative to benign lung. Heme is a central molecule for oxidative metabolism and ATP generation via mitochondrial oxidative phosphorylation (OXPHOS). Here, we have found that non-small cell lung cancer cells show elevated levels of heme synthesis and uptake, mitochondrial heme, oxygen-utilizing hemoproteins, oxygen consumption, ATP generation, and key mitochondrial biogenesis regulators relative to non-tumorigenic cells. Likewise, proteins and enzymes relating to heme and mitochondrial functions are found to be upregulated in human NSCLC tissues relative to normal tissues. Hence, we believe altering heme availability can be a useful strategy in treatment of highly heterogenous cancers. In order to seqester extra-cellular heme we engineered heme-sequestering peptides (HSPs). We observed that treatment with HSPs significantly reduce heme uptake, intracellular heme levels, and tumorigenic functions of NSCLC cells. This effect of HSPs is specific to heme as addition of heme largely reversed the effect of HSPs on tumorigenic functions. Furthermore, we also observed that HSP2 significantly suppresses the growth of human NSCLC xenograft tumors in mice. HSP2-treated tumors showed lowered oxygen consumption rates and ATP levels. To further verify the importance of heme in promoting tumorigenicity, we generated NSCLC cell lines with increased heme synthesis or uptake, which result from overexpression of the rate-limiting heme synthesis enzyme ALAS1 or uptake protein SLC48A1, respectively. These cells exhibited enhanced migration and invasion and accelerated tumor growth in mice. Notably, tumors formed by cells with increased heme synthesis or uptake also have elevated oxygen consumption rates and ATP levels. Our data show that elevated heme flux and function underlie enhanced OXPHOS and tumorigenicity of NSCLC cells. Therefore, targeting heme flux and function offers a novel strategy for developing lung cancer therapy.Item Mitochondrial Respiration Promotes Non-Small Cell Lung Cancer Cell Proliferation and Function(2016-12) Alam, Md Maksudul; Zhang, LiLung cancer is the leading cause of cancer related death both in the USA and worldwide. More than 80% of the lung cancer cases are non-small cell lung cancer (NSCLC). Since Otto Warburg made the first observation that the rate of aerobic glycolisis is increased in tumor cells, substantial amount of research has been done on altered cancer cell metabolism and bioenergetics. Recently, the use of more advanced metabolomics and genomics technologies has revealed the remarkable plasticity of tumor metabolism and bioenergetics. Many types of tumor cells have been shown to generate most of the cellular energy via mitochondrial respiration and oxidative phosphorylation. In this dissertation project it has been shown that the rate of respiration is intensified in an array of NSCLCs compared to the normal lung cell line. We also found that the respiration is greatly reduced when NSCLCs are grown in glutamine deprived media, whereas the respiration is unaffected when the cells are grown in glucose deprived media. In this project we have also shown that Hedgehog (Hh) signaling inhibitors Cyclopamine tartrate (CycT) and SNAT1 disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased reactive oxygen species (ROS) generation, causes mitochondrial membrane hyperpolarization, and mitochondrial fragmentation, thereby disrupting mitochondrial function in NSCLC cells. Furthermore, using two isogenic cell lines representing matched pairs of normal and cancer cells, we have identified changes in protein levels accompanying transformation of normal lung epithelial cells to cancer cells. Surprisingly, a substantial number of proteins involved in actin cytoskeleton were preferentially downregualted in cancer cells. However, similar numbers of proteins in other organelles were either up or down regulated. The formation of stress fibers and focal adhesions were also markedly decreased in cancer cells. Protein network analysis showed that the altered proteins are highly connected.Item Molecular Mechanisms Underlying Aerobic Respiration and Heme Regulation in Yeast(2017-12) Lal, Sneha; Zhang, LiHeme is an important signaling molecule with diverse functions in living organisms ranging from regulating gene transcription to circadian rhythm. In yeast, heme serves as a secondary messenger of oxygen, as heme synthesis depends on the intracellular levels of oxygen. Yeast and higher eukaryotes have adapted to adverse conditions including low oxygen or hypoxia. To understand the molecular mechanisms underlying hypoxia tolerance on a genome-wide scale, protein localization was studied using green fluorescence protein (GFP) tagged library of yeast genes under normoxia and hypoxia by Henke and colleagues. This study identified more than 200 proteins that change their localization under hypoxia. Among them were proteins of the chromatin remodeling SWI/SNF complex. Six proteins of the SWI/SNF complex- Swi3, Snf5, Snf6, Snf11, Snf12 and Swp82 relocalized under hypoxia. One of the objectives of this dissertation was to study the mechanism of regulation of cellular bioenergetics and respiration by the SWI/SNF proteins. Measurement of oxygen consumption rate and promoter activity show that Swi3, not Swi2, regulates aerobic gene expression and oxygen consumption. The levels of mitochondrial respiratory chain complex proteins were found to be increased in Δswi3 cells as compared to the parent cells. Deletion of SWI3 also induced the expression of aerobic respiration genes under high heme conditions. Computational analysis of the promoters of the genes bound by the human homologs of Swi3, BAF155 and BAF170 also show that they modulate aerobic respiration genes. In the next part of the dissertation, we have studied the regulation of a transcription factor Gis1 which was initially identified as a stress response regulator. In the previous genome-wide study of protein sub-cellular localization under hypoxia, Gis1 was identified as one of the fast responders to hypoxia and reoxygenation. Here we have studied the regulation of the transcriptional activity of Gis1 by heme. Gis1 belongs to the JmjC family of histone demethylases. Gis1 protein has two heme regulatory motifs (HRM). We show that the DNA binding zinc finger domain of Gis1 promotes the heme activation of its transcriptional activity, although heme does not affect Gis1 binding to DNA. These results have identified a new class of heme signaling proteins.Item The Nuclear Localization of SWI/SNF Proteins Is Subjected to Oxygen Regulation(2012-08-29) Dastidar, Ranita Ghosh; Hooda, Jagmohan; Shah, Ajit; Cao, Thai M.; Robert Michael Henke; Zhang, Li; Zhang, LiBackground: Hypoxia is associated with many disease conditions in humans, such as cancer, stroke and traumatic injuries. Hypoxia elicits broad molecular and cellular changes in diverse eukaryotes. Our recent studies suggest that one likely mechanism mediating such broad changes is through changes in the cellular localization of important regulatory proteins. Particularly, we have found that over 120 nuclear proteins with important functions ranging from transcriptional regulation to RNA processing exhibit altered cellular locations under hypoxia. In this report, we describe further experiments to identify and evaluate the role of nuclear protein relocalization in mediating hypoxia responses in yeast.Results: To identify regulatory proteins that play a causal role in mediating hypoxia responses, we characterized the time courses of relocalization of hypoxia-altered nuclear proteins in response to hypoxia and reoxygenation. We found that 17 nuclear proteins relocalized in a significantly shorter time period in response to both hypoxia and reoxygenation. Particularly, several components of the SWI/SNF complex were fast responders, and analysis of gene expression data show that many targets of the SWI/SNF proteins are oxygen regulated. Furthermore, confocal fluorescent live cell imaging showed that over 95% of hypoxia-altered SWI/SNF proteins accumulated in the cytosol in hypoxic cells, while over 95% of the proteins were nuclear in normoxic cells, as expected.Conclusions: SWI/SNF proteins relocalize in response to hypoxia and reoxygenation in a quick manner, and their relocalization likely accounts for, in part or in whole, oxygen regulation of many SWI/SNF target genes.Item Role of Heme in Non-Small Cell Lung Cancer Growth and Tumor Vascular Oxygenation(2020-04-21) Ghosh, Poorva; Zhang, LiDespite a decline in deaths caused by lung cancer, it remains the leading cause of cancer-related death in the U.S. Of these, about 84% are cases of non-small cell lung cancer (NSCLC). Despite the advent of various targeted therapies and immunotherapies; the 5-year survival rate for lung cancer is very low. Therefore, alternative therapeutic strategies should be explored to improve therapeutic outcomes for patients of lung cancer. Numerous studies have demonstrated that elevated levels of mitochondrial respiration or oxidative phosphorylation (OXPHOS) are a key feature of NSCLC cells. Recent studies in our lab show that NSCLC cells demonstrate elevated levels of heme via increased heme uptake and synthesis, as well as elevated mitochondrial respiration/oxidative phosphorylation (OXPHOS) which fuels NSCLC growth and progression. Also, limiting heme via inhibition of heme synthesis with Cyclopamine tartrate (CycT) or inhibition of heme uptake with heme sequestering peptides (HSP2) delays the growth and progression of NSCLC tumors. The tumor microenvironment (TME) influences tumor heterogeneity, invasion, metastasis, and is a major contributor to cancer progression. Tumor vasculature and oxygenation are important aspects of TME; however, faulty tumor vascular oxygenation is a marker for poor prognosis in cancer. Given the central role of heme in oxygen transport, utilization, and storage, it is important to investigate the effect of limiting heme on tumor vascular oxygenation. This study aimed to gain insights into how limiting heme using heme targeting drugs (CycT or HSP2) inhibits tumor growth and progression as well as how this influences the vascularization status of NSCLC tumors. Here, we used subcutaneous/orthotopic lung tumor xenografts models of NSCLCs expressing luciferase in NOD-SCID mice to monitor tumor growth after treatment with CycT or HSP2 using bioluminescence imaging (BLI). Also, mice were subjected to multispectral optoacoustic tomography (MSOT) to visualize tumor vasculature, oxygenation, and hypoxia. MSOT data were acquired to calculate total hemoglobin concentrations (THb) and oxygenation (sO2). Tissue homogenates of resected tumors were used to determine oxygen consumption rates and ATP levels. Tumors/lung tissues were harvested, processed and paraffinembedded for immunohistochemistry (IHC) and histology experiments. BLI data, tumor volumes, masses, and hematoxylin and eosin (H&E) show that CycT or HSP2 treatment suppresses the growth of subcutaneous/lung orthotopic tumor xenografts. CycT or HSP2 treated tumors also show reduced oxygen consumption and ATP levels compared to control. MSOT data show that THb levels decrease when treated with CycT or HSP2. OE-MSOT (Oxygen enhanced MSOT) data show increased levels of sO2 MSOT in HSP2- or CycT-treated tumors compared to control tumors. IHC data for various hypoxia and angiogenesis markers confirm that limiting heme effectively normalizes tumor vascular oxygenation. This study demonstrates that targeting heme holds immense potential as a therapeutic strategy that can effectively inhibit angiogenesis, normalize tumor vasculature, and alleviate tumor hypoxia in NSCLC tumors thereby delaying lung tumor growth and progression.Item The Swi3 Protein Plays A Unique Role In Regulating Respiration In Eukaryotes(2016-06-30) Lal, Sneha; Alam, Md Maksudul; Hooda, Jagmohan; Shah, Ajit; Cao, Thai M.; Xuan, Zhenyu; Zhang, Li; Lal, Sneha; Alam, Md Maksudul; Hooda, Jagmohan; Shah, Ajit; Cao, Thai M.; Xuan, Zhenyu; Zhang, LiRecent experimental evidence increasingly shows that the dysregulation of cellular bioenergetics is associated with a wide array of common human diseases, including cancer, neurological diseases and diabetes. Respiration provides a vital source of cellular energy for most eukaryotic cells, particularly high energy demanding cells. However, the understanding of how respiration is globally regulated is very limited. Interestingly, recent evidence suggests that Swi3 is an important regulator of respiration genes in yeast. In this report, we performed an array of biochemical and genetic experiments and computational analysis to directly evaluate the function of Swi3 and its human homologues in regulating respiration. First, we showed, by computational analysis and measurements of oxygen consumption and promoter activities, that Swi3, not Swi2, regulates genes encoding functions involved in respiration and oxygen consumption. Biochemical analysis showed that the levels of mitochondrial respiratory chain complexes were substantially increased in Delta swi3 cells, compared with the parent cells. Additionally, our data showed that Swi3 strongly affects haem/oxygen-dependent activation of respiration gene promoters whereas Swi2 affects only the basal, haem-independent activities of these promoters. We found that increased expression of aerobic expression genes is correlated with increased oxygen consumption and growth rates in Delta swi3 cells in air. Furthermore, using computational analysis and RNAi knockdown, we showed that the mammalian Swi3 BAF155 and BAF170 regulate respiration in HeLa cells. Together, these experimental and computational data demonstrated that Swi3 and its mammalian homologues are key regulators in regulating respiration.Item Targeting Heme Function and Mitochondrial Respiration in Non-Small Cell Lung Cancer(2018-12) Kalainayakan, Sarada Preeta; 0000-0003-4332-6425 (Kalainayakan, SP); Zhang, LiEach year lung cancer causes more morbidities than cancers of colon, breast, and prostate combined. The American Cancer Society estimates that lung cancer will claim about 150,000 lives in 2018. Conventional and targeted therapies are reported to have reached the plateau in effectively improving the survival of lung cancer patients. Despite the advent of advanced therapies like immunotherapy, 5-year survival rates remain abysmal at 30% and 10% for Stage III and Stage IV respectively. Therefore, it is imperative to explore different strategies to effectively treat lung cancer and improve survival outcomes. There are two major histological types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for about 85% of the lung cancer cases. Several studies demonstrate that enhanced mitochondrial respiration or oxidative phosphorylation (OXPHOS) is a key feature of NSCLC. Therefore, targeting OXPHOS could be an effective strategy for intervention in NSCLC. Previous studies from our lab showed that hedgehog pathway antagonist, cyclopamine tartrate (CycT), significantly reduced OXPHOS and proliferation in NSCLC cell lines. However, in vitro models do not offer reliable evidence of therapeutic efficacy, thereby, necessitating studies on in vivo models. Previous studies from our lab demonstrated intensified heme uptake and synthesis in NSCLC cell lines compared to normal cell line. Since heme is a central factor in oxygen consumption, this study also probes the effect of CycT on heme metabolism. The objective of this study is two-fold: (i) to test the efficacy of targeting OXPHOS in NSCLC in vivo, and (ii) to investigate the therapeutic efficacy and the mechanism of action of CycT in growth and progression of NSCLC in vivo. We utilized subcutaneous and lung orthotopic xenografts of NSCLC cell lines with luciferase to track the growth and progression of NSCLC in immunodeficient mouse model, NOD/SCID (Non-obese diabetic/ severe combined immunodeficiency), via bioluminescence imaging. The lung tissues of the mice were probed using immunohistochemistry to discern the mechanisms of action. We found that CycT effectively hampered the growth and progression of subcutaneous and lung orthotopic xenografts of NSCLC cell lines. CycT significantly reduced proteins involved in heme metabolism and OXPHOS in addition to other pro-oncogenic hemoproteins and regulators of OXPHOS. In vitro studies demonstrated that the effects of CycT on heme metabolism and OXPHOS are independent of its antagonist properties on the hedgehog pathway. The significance of this study is that it shows that CycT acts via diminishing heme metabolism, hemoproteins involved in oxygen consumption, and oxygen consumption in NSCLC. This is the first study to demonstrate the effect of CycT on heme metabolism and OXPOS in vivo. This novel mechanism of action of CycT is independent of its previously known antagonistic effect on hedgehog signaling. This study demonstrates that CycT has the potential to be an effective therapeutic agent in treating NSCLC. This study provides compelling evidence to further assess the feasibility of using CycT in treating NSCLC.Item The Dielectric Properties of Two-dimensional Materials and Their Applications in Electronic Devices: a First-principles Study(2022-05-01T05:00:00.000Z) Rostami Osanloo, Mehrdad; Vandenberghe, William; Zhang, Li; Lv, Bing; Cho, Kyeongjae; Kim, Moon J.; Wallace, Robert M.Recent developments in the field of two-dimensional (2D) van der Waals (vdW) materials have captured great interest for their possible applications in the next generation of complementary Metal-Oxide Semiconductor (CMOS) technologies. Easy cleavage along the layer planes, naturally passivated surfaces, and intriguing anisotropic electrical, thermal, and optical properties make vdW materials ideal candidates to reach the ultimate scaling limit of transistors. Moreover, vdW transistors could add great functionality in the backend-of-line or for other novel applications like neuromorphic computing. In this doctoral dissertation, I will identify and study novel 2D vdW dielectric candidates to address some drawbacks of currently available non-van der Waals (non-vdW) dielectrics, e.g., HfO2 and Al2O3 . Although traditional three-dimensional (3D) dielectrics provide high-k solutions for silicon-based semiconductor technologies, they cannot be easily scaled and therefore deteriorate device performance in vdW channel transistors. Moreover, the covalent bonds in non-vdW dielectrics may destroy the naturally passivated bonds in a vdW channel material. To address this concern, we investigate 40 alternative 2D vdW dielectrics and evaluate their performance in conjugation with six Transition Metal Dichalcogenide (TMD) channels. We employ Density Functional Theory (DFT) and Density Functional Perturbation Theory (DFPT) to calculate the electronic and dielectric properties of a wide range of 2DvdW dielectrics. We perform highly accurate calculations to investigate the electronic band structure, thermodynamic properties, structural stability, and dielectric properties of 2D vdW structures. We perform precise calculations at high DFT and DFPT levels to obtain the bandgap, electron affinity, out-of-plane electron effective mass, as well as in-plane and out-of-plane dielectric constant. We calculate the band offset (conduction and valence band edge) of each material to evaluate their insulating properties for the potential application in n-MOS and p-MOS technologies. We compute the Equivalent Oxide Thickness (EOT) for each compound and use direct tunneling and thermionic emission equations to examine the performance of a device made by a 2D dielectric and a TMD channel. We eventually introduce the most promising candidates that can satisfy the low-power limit introduced by the 2020 International Roadmap for Devices and Systems (IRDS) by having an acceptable leakage current. We eventually compare our results with the industry’s most desirable dielectrics (SiO2 , and HfO2) and 2D hexagonal Boron Nitride (h-BN). We explicitly show how some materials in our list outperform the available high-k dielectrics. Given the growing interest in the 2D layered dielectric materials and their possible use in future scaled electronic devices, we believe that 2D vdW dielectric candidates identified in this PhD dissertation could pave the way for the future design of advanced n-MOS and p-MOS transistors.