Heme Sequestration Effectively Suppresses the Development and Progression of Both Lung Adenocarcinoma and Squamous Cell Carcinoma.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two most common subtypes of lung cancer. Here, to identify new, targetable molecular properties of both subtypes, we monitored changes in the levels of heme- and oxidative phosphorylation (OXPHOS)-related proteins during lung tumorigenesis. Heme is a central molecule for oxidative metabolism and ATP generation via OXPHOS. Notably, both lung ADC and SCC tumors can be induced in the genetically engineered KLLuc mouse model harboring the G12D Kras mutation and a conditional Lkb1 knockout. We found that the levels of the rate-limiting heme synthesis enzyme ALAS1 and uptake protein SLC48A1, along with OXPHOS complex subunits, progressively increased as lung tumorigenesis advanced. Our data demonstrated that elevated levels of heme- and OXPHOS-related proteins were associated with both ADC and SCC. Importantly, treatment of KLLuc mice with a heme-sequestering protein, HeSP2, that inhibits heme uptake in tumor cells effectively arrested lung tumor progression, and both ADC and SCC tumors were strongly suppressed. Additionally, HeSP2 effectively suppressed the growth of both SCC and ADC tumor xenografts in NOD/SCID mice. Further analyses indicated that HeSP2 effectively diminished OXPHOS in both ADC and SCC, reduced angiogenesis, alleviated tumor hypoxia, and suppressed cell proliferation. These results show that the advancing of lung tumorigenesis requires progressive increase in cellular heme synthesis and uptake, leading to intensified OXPHOS activity and ATP generation and promoting aggressive tumorigenic functions. IMPLICATIONS: Heme sequestration is an effective strategy for the suppression of both ADC and SCC tumor initiation and development.

The promise of targeting heme and mitochondrial respiration in normalizing tumor microenvironment and potentiating immunotherapy.

Cancer immunotherapy shows durable treatment responses and therapeutic benefits compared to other cancer treatment modalities, but many cancer patients display primary and acquired resistance to immunotherapeutics. Immunosuppressive tumor microenvironment (TME) is a major barrier to cancer immunotherapy. Notably, cancer cells depend on high mitochondrial bioenergetics accompanied with the supply of heme for their growth, proliferation, progression, and metastasis. This excessive mitochondrial respiration increases tumor cells oxygen consumption, which triggers hypoxia and irregular blood vessels formation in various regions of TME, resulting in an immunosuppressive TME, evasion of anti-tumor immunity, and resistance to immunotherapeutic agents. In this review, we discuss the role of heme, heme catabolism, and mitochondrial respiration on mediating immunosuppressive TME by promoting hypoxia, angiogenesis, and leaky tumor vasculature. Moreover, we discuss the therapeutic prospects of targeting heme and mitochondrial respiration in alleviating tumor hypoxia, normalizing tumor vasculature, and TME to restore anti-tumor immunity and resensitize cancer cells to immunotherapy.

Heme Sequestration as an Effective Strategy for the Suppression of Tumor Growth and Progression.

Heme is an essential nutritional, metabolic, and signaling molecule in living organisms. Pathogenic microbes extract heme from hosts to obtain metallonutrient, while heme fuels mitochondrial respiration and ATP generation in lung tumor cells. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bind to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non-small cell lung cancer (NSCLC) cells, while their effects on nontumorigenic 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. IHC, along with data from Western blotting and functional assays, revealed that HeSPs reduced the levels of key proteins involved in heme uptake, as well as the consumption of major fuels for tumor 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.

Water-soluble polycarbodiimides and their cytotoxic and antifungal properties.

We have successfully synthesized water-soluble neutral and polyelectrolyte helical polycarbodiimides and studied their biological properties. These polymers were prepared by decorating carbodiimide backbones with nonionic, hydrophilic functional groups such as dimethylamine, piperazine, and morpholine. Additionally, the 3° amines present in these functional groups were quaternized using methyl iodide as the alkylating agent to produce their ionic analogs. Polycarbodiimides were chosen as the base polymer used because of their facile chemical modification, pH tolerance in terms of both their helical conformations and degradation behaviors, and tunable helical inversion barriers. Hydrophilic side groups, such as morpholine, dimethylamine, and piperazine, can be used to balance the amphiphilic architecture of the polycarbodiimides along with lipophilic groups, such as alkyl side chains. A chiral R or S BINOL Ti(IV) isopropoxide catalyst was used to control the handedness of the polycarbodiimide helices in these studies. These ionic and neutral polycarbodiimides were subsequently studied for potential antimicrobial and cytotoxic properties. Poly[N-methyl-N'-2-morpholinoethylcarbodiimide], as an example, exhibited significant antifungal properties against Candida albicans. Also, Poly[N-methyl-N'-2-morpholinoethylcarbodiimide] showed significant inhibition of biofilm formation. This suggests that the polymer is a promising candidate for antifungal biomedical applications. Measuring cytotoxicity against urinary bladder cancer cells, poly[N-[3-(dimethylamino)propyl)]-N'-[3-(morpholino)propyl]carbodiimide] (S-cat) and poly[N-[3-(dimethylamino)propyl)]-N'-[3-(morpholino)propyl]carbodiimide]-MeI (S-cat) showed significantly low IC50 values. The IC50 values of poly[N-[3-(dimethylamino)propyl)]-N'-[3-(morpholino)propyl]carbodiimide] (S-cat) and Poly[N-[3-(dimethylamino)propyl)]-N'-[3-(morpholino)propyl]carbodiimide]-MeI (S-cat) are 3.50 µM and 1.27 µM, respectively. The significantly low cancer cell growth inhibition concentration implies the highest cytotoxicity of the polymers, suggesting potential applications as cancer therapeutics. These results also showed that the functionalization and chirality of polycarbodiimides modulate their anticancer and antifungal activity.

Heme, A Metabolic Sensor, Directly Regulates the Activity of the KDM4 Histone Demethylase Family and Their Interactions with Partner Proteins.

The KDM4 histone demethylase subfamily is constituted of yeast JmjC domain-containing proteins, such as Gis1, and human Gis1 orthologues, such as KDM4A/B/C. KDM4 proteins have important functions in regulating chromatin structure and gene expression in response to metabolic and nutritional stimuli. Heme acts as a versatile signaling molecule to regulate important cellular functions in diverse organisms ranging from bacteria to humans. Here, using purified KDM4 proteins containing the JmjN/C domain, we showed that heme stimulates the histone demethylase activity of the JmjN/C domains of KDM4A and Cas well as full-length Gis1. Furthermore, we found that the C-terminal regions of KDM4 proteins, like that of Gis1, can confer heme regulation when fused to an unrelated transcriptional activator. Interestingly, biochemical pull-down of Gis1-interacting proteins followed by mass spectrometry identified 147 unique proteins associated with Gis1 under heme-sufficient and/or heme-deficient conditions. These 147 proteins included a significant number of heterocyclic compound-binding proteins, Ubl-conjugated proteins, metabolic enzymes/proteins, and acetylated proteins. These results suggested that KDM4s interact with diverse cellular proteins to form a complex network to sense metabolic and nutritional conditions like heme levels and respond by altering their interactions with other proteins and functional activities, such as histone demethylation.

Cytotoxicity and alkaline phosphatase activity evaluation of endosequence root repair material.

INTRODUCTION: The purpose of this in vitro study was to evaluate the cytotoxicity and alkaline phosphatase (ALP) activity of a new bioceramic root repair material, EndoSequence Root Repair Material (ESRRM; Brasseler USA, Savannah, GA), and to compare these characteristics with those of ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK) and Geristore (GR; Den-Mat LLC, Santa Maria, CA). METHODS: Human Saos-2 osteoblast-like cells were exposed to 1-, 3-, and 7-day elutes of the materials (100% and 50% strength) for 24 hours after which the bioactivity and ALP activity of the cells were evaluated using a methylthiazol sulfophenyl (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and para-Nitrophenylphosphate colorimetric assay, respectively. In the positive control group, Triton X-100 (Boehringer Mannheim Corp, Indianapolis, IN) was used to lyse the cells, representing 100% cytotoxicity, and in the negative control group cells received fresh culture medium only. Data were statistically analyzed using the unpaired t test and 1-way analysis of variance. RESULTS: The results revealed that the bioactivity of the cells as well as ALP activity were significantly decreased after exposure to ESRRM elutes in almost all time periods, both in 100% and 50% concentrations, with the exception of ALP activity of day 1 elutes of ESRRM at 50% concentration. MTA did not change the bioactivity or ALP activity of the cells. GR elutes of 100% concentration reduced the bioactivity on days 1 and 3, whereas GR elutes of 50% concentration affected the cells only on day 1. None of the GR elutes had any effect on ALP activity of the cells. CONCLUSIONS: It was concluded that ESRRM elutes of all time periods in general reduced the bioactivity and ALP activity of osteoblast-like cells. GR reduced bioactivity only, whereas MTA had no effect on the cells.