Free heme exacerbates colonic injury induced by anti-cancer therapy.

Gastrointestinal inflammation and bleeding are commonly induced by cancer radiotherapy and chemotherapy but mechanisms are unclear. We demonstrated an increased number of infiltrating heme oxygenase-1 positive (HO-1+) macrophages (Mø, CD68+) and the levels of hemopexin (Hx) in human colonic biopsies from patients treated with radiation or chemoradiation versus non-irradiated controls or in the ischemic intestine compared to matched normal tissues. The presence of rectal bleeding in these patients was also correlated with higher HO-1+ cell infiltration. To functionally assess the role of free heme released in the gut, we employed myeloid-specific HO-1 knockout (LysM-Cre : Hmox1flfl), hemopexin knockout (Hx-/-) and control mice. Using LysM-Cre : Hmox1flfl conditional knockout (KO) mice, we showed that a deficiency of HO-1 in myeloid cells led to high levels of DNA damage and proliferation in colonic epithelial cells in response to phenylhydrazine (PHZ)-induced hemolysis. We found higher levels of free heme in plasma, epithelial DNA damage, inflammation, and low epithelial cell proliferation in Hx-/- mice after PHZ treatment compared to wild-type mice. Colonic damage was partially attenuated by recombinant Hx administration. Deficiency in Hx or Hmox1 did not alter the response to doxorubicin. Interestingly, the lack of Hx augmented abdominal radiation-mediated hemolysis and DNA damage in the colon. Mechanistically, we found an altered growth of human colonic epithelial cells (HCoEpiC) treated with heme, corresponding to an increase in Hmox1 mRNA levels and heme:G-quadruplex complexes-regulated genes such as c-MYC, CCNF, and HDAC6. Heme-treated HCoEpiC cells exhibited growth advantage in the absence or presence of doxorubicin, in contrast to poor survival of heme-stimulated RAW247.6 Mø. In summary, our data indicate that accumulation of heme in the colon following hemolysis and/or exposure to genotoxic stress amplifies DNA damage, abnormal proliferation of epithelial cells, and inflammation as a potential etiology for gastrointestinal syndrome (GIS).

DNA in fresh urine supernatant is not affected by additional centrifugation and is protected against deoxyribonuclease.

Urinary DNA is widely studied as a non-invasive marker for monitoring of kidneys after transplantation or the progression of urinary tract tumors. The quantity of urinary DNA especially of mitochondrial origin has been reported to mirror kidney damage in various renal diseases and their models. Processing of samples might affect urinary DNA concentrations but the details are not clear. Samples of urine were collected from fifteen healthy volunteers. DNA was extracted from the whole urine, but also from the supernatant after centrifugation at 1600 g and 16000 g. In addition, we have analyzed the DNA in the microparticles in the pellet after the last spin. DNA was measured using fluorometry and real time PCR targeting nuclear and mitochondrial sequences. Addition of deoxyribonuclease to aliquots of samples enabled the characterization of DNA protection. Centrifugation at 1600 g decreased the concentration of extracted DNA by 66% at least in samples with higher DNA in whole urine. Interestingly, the additional spin at 16000 g did not result in a significant decrease in DNA concentration in the supernatant despite detectable microparticle-associated DNA. Deoxyribonuclease decreases total and nuclear DNA by 26% and 31% in whole urine. The majority of urinary mitochondrial DNA seems to be protected against deoxyribonuclease. Our results indicate high variability in urinary DNA even in healthy probands. Extracellular urinary DNA is partially bound to cell debris or microparticles, but a considerable part is still in the supernatant and is protected against cleavage. Further research should identify the nature of the protection, especially for mitochondrial DNA. Better understanding of the biology of urinary DNA should help its clinical interpretation.

Labile Heme and Heme Oxygenase-1 Maintain Tumor-Permissive Niche for Endometriosis-Associated Ovarian Cancer.

Endometriosis, a painful gynecological condition accompanied by inflammation in women of reproductive age, is associated with an increased risk of ovarian cancer. We evaluated the role of peritoneal heme accumulated during menstrual cycling, as well as peritoneal and lesional macrophage phenotype, in promoting an oncogenic microenvironment. We quantified the heme-degrading enzyme, heme oxygenase-1 (HO-1, encoded by Hmox1) in normal peritoneum, endometriotic lesions and endometriosis-associated ovarian cancer (EAOC) of clear cell type (OCCC). HO-1 was expressed primarily in macrophages and increased in endometrioma and OCCC tissues relative to endometriosis and controls. Further, we compared cytokine expression profiles in peritoneal macrophages (PM) and peripheral blood mononuclear cells (PBMC) in women with endometriosis versus controls as a measure of a tumor-promoting environment in the peritoneum. We found elevated levels of HO-1 along with IL-10 and the pro-inflammatory cytokines (IL-1ß, IL-16, IFNγ) in PM but not in PBMC from endometriosis patients. Using LysM-Cre:Hmox1flfl conditional knockout mice, we show that a deficiency of HO-1 in macrophages led to the suppression of growth of ID8 ovarian tumors implanted into the peritoneum. The restriction of ID8 ovarian tumor growth was associated with an increased number of Mac3+ macrophage and B cells in LysM-Cre:Hmox1flfl mice compared to controls. Functional experiments in ovarian cancer cell lines show that HO-1 is induced by heme. Low levels of exogenous heme promoted ovarian cancer colony growth in soft agar. Higher doses of heme led to slower cancer cell colony growth in soft agar and the induction of HO-1. These data suggest that perturbation of heme metabolism within the endometriotic niche and in cancer cells themselves may be an important factor that influences tumor initiation and growth.

Measurement of labile and protein-bound heme in fixed prostate cancer cells and in cellular fractions.

Labile heme is present in the cells at very low concentrations, either unbound or loosely bound to molecules, and accessible for signaling as alarmin. Our recent work suggests that extracellular heme can be taken up and detected in the nuclei of cancer cells. Here, we describe the detailed protocol for detection of labile and total heme in prostate cancer cells and its measurement in subcellular compartments in vitro. The protocol can be adapted to be used for other cell types. For complete details on the use and execution of this protocol, please refer to Canesin et al. (2020).

Deoxyribonucleases and Their Applications in Biomedicine.

Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or enhancing inflammation. The cleavage of extracellular DNA is crucial for limiting the inflammatory response and maintaining homeostasis. Deoxyribonucleases (DNases) as enzymes that degrade DNA are hypothesized to play a key role in this process as a determinant of the variable concentration of extracellular DNA. DNases are divided into two families-DNase I and DNase II, according to their biochemical and biological properties as well as the tissue-specific production. Studies have shown that low DNase activity is both, a biomarker and a pathogenic factor in systemic lupus erythematosus. Interventional experiments proved that administration of exogenous DNase has beneficial effects in inflammatory diseases. Recombinant human DNase reduces mucus viscosity in lungs and is used for the treatment of patients with cystic fibrosis. This review summarizes the currently available published data about DNases, their activity as a potential biomarker and methods used for their assessment. An overview of the experiments with systemic administration of DNase is also included. Whether low-plasma DNase activity is involved in the etiopathogenesis of diseases remains unknown and needs to be elucidated.