Effect of Radiotherapy on Activating the Pyroptotic Cell Death Pathway in Breast Cancer Patients: The Role of Serum GSDMD-CT, NLRP3 and IL-18.

BACKGROUND: Breast cancer (BC) is the most common form of cancer among women and the second leading cause of cancer-related death worldwide. Several malignancies can be successfully treated with radiation (RT), although radioresistance is still a major contributor to radiotherapy failure. Ionizing radiation (IR) induces pyroptosis in cancer cells. Pyroptosis is a designed method of death connected to routine immunity and directly related to the body ROS content. Objective for the study: The aim of this work was to investigate the role of serum GSDMD-CT, nucleotide-binding domain and leucine-rich-repeat-containing family pyrin 3 (NLRP3) and IL-18 as predictors of pyroptotic cell death mechanism induced by radiotherapy in breast cancer patients. SUBJECTS AND METHODS: The 70 female participants in this study were divided into two groups: Group (I): 40 breast cancer patients treated with radiotherapy. Group (II): a control group of 30 healthy volunteers with similar ages and sex. Patients with breast cancer received radiation, with a dose of 44 Gray administered over the course of 16 days in five daily fractions of 2.75 Gray each. Two blood samples were taken from breast cancer patients: one before radiotherapy and the other after radiotherapy. While one blood sample was taken from healthy controls. The levels of the circulating pyroptosis biomarkers IL-18, NLRP3, and GSDMD-CT were measured using the ELISA method. RESULTS: Our results showed that, there was a significant increase in serum pyroptosis markers GSDMD-CT, NLRP3 and IL-18 in BC Patients after RT when compared to before radiotherapy. CONCLUSION: Radiotherapy induced pyroptosis in breast cancer patients as a new cell death mechanism. GSDMD-CT, NLRP3 and IL-18 are biomarkers of pyroptosis that significantly increased post irradiation highlighting enhanced ROS and pyroptosis induction.

Studying ferroptosis and iron metabolism pre- and post-radiotherapy treatment in breast cancer patients.

BACKGROUND: Radiotherapy (RT) is an important part of the treatment of many tumors. Radiotherapy causes oxidative damage in all cellular compartments, including lipid membrane, on a random basis. Toxic lipid peroxidation accumulation has only lately been linked to a regulated type of cell death known as ferroptosis. Iron is required for ferroptosis sensitization in cells. AIM OF THE WORK: This work aimed to study ferroptosis and iron metabolism before and after RT in BC patients. SUBJECTS AND METHODS: Eighty participants were included divided into two main groups: group I: 40 BC patients treated with RT. Group II: 40 healthy volunteers' age and sex matched as control group. Venous blood samples were collected from BC patients (prior to and after RT) and healthy controls. Glutathione (GSH), malondialdehyde (MDA), serum iron levels and % of transferrin saturation were measured by colorimetric technique. Ferritin, ferroportin, and prostaglandin-endoperoxide synthase 2 (PTGS2) levels were assessed by ELISA. RESULTS: Serum ferroportin, reduced glutathione, and ferritin showed significant decrease after radiotherapy in comparison to before radiotherapy. However, there was significant increase in serum PTGS2, MDA, % of transferrin saturation and iron levels after radiotherapy in comparison to before radiotherapy. CONCLUSION: Radiotherapy induced ferroptosis in breast cancer patients as a new cell death mechanism and PTGS2 is a biomarker of ferroptosis. Iron modulation is a useful approach for the treatment of BC especially if combined with targeted therapy and immune-based therapy. Further studies are warranted to be translated into clinical compounds.

Assessment of thyroid gland hormones and ultrasonographic abnormalities in medical staff occupationally exposed to ionizing radiation.

BACKGROUND: Ionizing radiation (IR) is high-energy radiation that has the potential to displace electrons from atoms and break chemical bonds. It has the ability to introduce mutations, DNA strand breakage, and cell death. Being a radiosensitive organ, exposure of the thyroid gland to IR can lead to significant changes in its function. AIM OF THE WORK: Was to measure the levels of thyroid hormones panel and ultrasonography abnormalities in medical staff occupationally exposed to IR. SUBJECTS AND METHODS: A total of 120 subjects were divided into three main groups: Group I: radiation-exposed workers occupationally exposed to radioiodine (131I) (n = 40), Group II: radiation-exposed workers occupationally exposed to X-ray (n = 40), and Group III: non-exposed healthy professionals matched in age and sex with the previous groups (n = 40). Thyroid hormones panel including free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), anti-thyroperoxidase antibodies (anti-TPO), and thyroglobulin (Tg) were measured. Thyroid ultrasonography was performed. Oxidative stress markers such as malondialdehyde (MDA), hydrogen peroxide (H2O2), and total antioxidant capacity (TAC) were measured. RESULTS: Group I had significantly higher fT3 levels than the control group. fT3 levels were considerably higher, while TSH was substantially lower in group II participants than in the control group. Tg was markedly lower in radiation-exposed workers. However, anti-TPO levels in radiation-exposed workers were significantly higher than in the control group. MDA and H2O2 were substantially higher; TAC was significantly lower in radiation-exposed workers compared to the control group. According to ultrasonographic examination, thyroid volume and the percentage of thyroid nodules in all radiation workers were significantly higher than in the control group. CONCLUSION: Despite low exposure doses, occupational exposure to IR affects the thyroid hormones and links with a higher likelihood of developing thyroid immune diseases.

Assessment of Serum Hypoxia Biomarkers Pre- and Post-radiotherapy in Patients with Brain Tumors.

Hypoxia is a prevalent hallmark of many malignant neoplasms. The aim was to assess the serum hypoxia biomarkers HIF-1α, VEGF, osteopontin, erythropoietin, caveolin-1, GLUT-1, and LDH pre- and post-radiotherapy in patients with brain tumors. The study was conducted on 120 subjects were divided into two groups: group I: 40 healthy volunteers as control group. Group II: 80 brain tumor patients were subdivided into glioblastoma subgroup: 40 glioblastoma patients, meningioma subgroup: 40 malignant meningioma patients. Two venous blood samples were collected from every patient prior to and following RT and one sample from controls. Biomarkers were assayed by ELISA. In glioblastoma subgroup, HIF-1α, VEGF, and LDH were significantly increased after RT. On the contrary, these biomarkers were significantly decreased after RT in malignant meningioma subgroup. Osteopontin was significantly increased after RT in both subgroups. Regarding erythropoietin, it was significantly decreased in both subgroups when compared to before RT. Caveolin-1 showed a significant increase in glioblastoma subgroup after RT comparing to before RT. GLUT-1 was significantly increased after RT in both subgroups comparing to before RT. Association of significant elevation of hypoxia biomarkers either pre- or post-RT with aggressive tumor such as glioblastoma indicates that, they are markers of malignancy and may have a role in tumor development and progression.

Znhit1 and HIF-2α are correlated with cancer stem cell markers in breast cancer patients.

Epigenetic alterations have emerged as fundamental players in development and progression of breast cancer (BC). A hypoxic tumour microenvironment regulates the stemness phenotype in breast cancer stem cells (BCSCs). The aim of this study was to investigate Znhit1 and HIF-2α gene expression in breast cancer tissues as well as their relation to CSCs markers LGR5, ALDH1A1 and ß-catenin in tissue and serum of BC patients. The present study included 160 females divided into two groups, group I: 80 healthy females served as control group and group II: 80 breast cancer patients. Gene expression of tissue Znhit1 and HIF-2α was determined by qRT-PCR. Tissue and serum ALDH1A1, LGR5 and ß-catenin levels were determined by ELISA. We found that gene expression of Znhit1 was significantly downregulated in BC tissues. Moreover, it was significantly negatively correlated with clinical stage and ß-catenin levels in BC patients. Regarding HIF-2α, gene expression of HIF-2α was significantly upregulated in BC tissues. Moreover, it was significantly positively correlated with Her-2/neu expression and ß-catenin levels in BC patients. Based upon our results, Znhit1 and HIF-2α may serve as novel therapeutic targets for BC therapy. Additionally, each of serum ALDH1A1, LGR5 and ß-catenin may play a crucial role in non-invasive detection of BC with a high specificity and sensitivity.

Studying ferroptosis and pyroptosis as new cell death mechanisms induced by ionizing radiation in Ehrlich solid tumor-bearing mice.

OBJECTIVE: Our objective was to explore the effect of different fractionation schedule on ferroptosis and pyroptosis biomarkers as new cell death mechanisms induced by IR. MATERIALS AND METHODS: This study included 40 tumor bearing mice divided into: Group I: Includes 8 untreated tumor-bearing mice. Group II: Includes 8 tumor bearing mice exposed to single dose 6 Gy of IR. Group III: Includes 8 tumor bearing mice exposed to 12 Gy in 2 fractions (2 × 6 Gy) of IR. Group IV: Includes 8 tumor bearing mice exposed to 12 Gy in 3 fractions (3 × 4 Gy) of IR. Group V: Includes 8 tumor bearing mice exposed to 8 Gy in 4 fractions (4 × 2 Gy) of IR. IL-1ß, IL-18, and GSDMD-CT levels were assessed by ELISA. PTGS2 and ACSL4 expression were assessed by RT-PCR. RESULTS: (2 × 6 Gy) group showed the highest ACSL4 expression followed by (3 × 4 Gy), then (4 × 2 Gy) and finally 6 Gy. (2 × 6 Gy) group resulted in the highest PTGS2 expression followed by (3 × 4 Gy), then 6 Gy, and finally (4 × 2 Gy). MDA significantly increased at (2 × 6 Gy), (3 × 4 Gy), and 6 Gy groups and insignificantly increased at (4 × 2 Gy) group. Iron significantly increased at (2 × 6 Gy), (3 × 4 Gy), and (4 × 2 Gy) groups and insignificantly at 6 Gy. Glutathione was significantly decreased at (2 × 6 Gy), (3 × 4 Gy), and (4 × 2 Gy) groups and insignificantly at 6 Gy. GSDMD-CT, IL-1ß, and IL-18 levels significantly reduced in tumor tissues after exposure to IR at all doses. CONCLUSION: High dose per fraction regimens induce the expression of ferroptosis markers more than low dose per fraction regimen and single dose of IR. IR at all doses induces pyroptotic cell death.

Role of Resveratrol as Radiosensitizer by Targeting Cancer Stem Cells in Radioresistant Prostate Cancer Cells (PC-3).

AIM OF WORK: Here, we examined the role of resveratrol as a radiosensitizer by targeting cancer stem cells in radioresistant prostate cancer cells (PC-3) using stem cell markers CD44, CD49b and CD29, SOX2, OCT4, CXCR4, DCLK1 and EMT markers such as VIM and E-cadherin. MATERIAL AND METHODS: This study was an in vitro study involving PC-3 cell line which was dividing into four groups. Group I (CO): Control group composed of cells grown in the same medium without treatment with ionizing radiation or resveratrol. Group II (IR): Cells were treated with ionizing radiation alone. Group III (RV): Cells were treated with resveratrol alone. Group VI (IR&RV): The cells were treated with ionizing radiation and resveratrol in combination. The viability of cells was assessed by MTT assay. Genes of interest were measured by RT-PCR and the radiosensitizing efficacy of RV on proliferating cancer cells was determined by clonogenic assay. RESULTS: Ionizing radiation significantly reduced PC-3 viability, lowered stem cell markers and affected epithelial to mesenchymal transition (EMT) genes expression at all doses (2, 4, 6 and 8 Gray). Resveratrol significantly decreased PC-3 viability and lowered stem cell markers and EMT genes expression at concentrations 35, 70 and 140 µM. Combining resveratrol treatment with ionizing radiation leads to significant reduction in cell viability and stem cell markers genes which was noticed with increasing the radiation dose when compared to ionizing radiation alone treated group. CONCLUSION: Resveratrol has a radiosensitizing effect, that ability is triggered by reducing the expression of cancer stem cell markers and affecting EMT markers. Resveratrol showed to be a good candidate for further studies as anticancer drug in the treatment of human prostate cancer.

Radioprotective Effect of Thymoquinone in X-irradiated Rats.

BACKGROUND: Thymoquinone, has anti-inflammatory, anti-oxidant, and cardio protection properties. This study aimed to evaluate the radioprotective effect of thymoquinone in whole body X-irradiated rats. METHODS: This study conducted on 40 male adult Wistar albino rats randomized into the following groups: Group I: Control rats did not receive thymoquinone or ionizing radiation. Group II: Whole-body irradiated rats with 6 Gy of X-ray. Group III: Rats orally intubated with thymoquinone (10 mg/kg/day) for 7 days then subjected to whole-body irradiation with 6 Gy then supplemented with thymoquinone for another 7 days. Group IV: Rats orally intubated with thymoquinone (20 mg/kg/day) for 7 days then subjected to whole-body irradiation with 6 Gy then supplemented with thymoquinone (20 mg/kg/day) for another 7 days. LDH, CK-MB, ALT, AST, MDA, TAC, Catalase activity, GPX, GSR and GSH were measured. RESULTS: Lipid peroxidation biomarker in the blood of X-irradiated rats significantly increased and accompanied by decrease in the levels of GSH, GSR, GPX, catalase as well as TAC. Moreover, exposure to IR significantly increases cardiac and liver enzymes. However, administration of TQ to X-irradiated rats with either 10 mg/kg or 20 mg/kg have the same reform effects and significantly protects rats against adverse effects of IR. CONCLUSION: Exposure to X-ray leads to significant changes in cellular biochemical and morphological conditions. Administration of TQ before radiation treatment significantly decreases the adverse effects of IR. TQ can improve cardiac function, decrease myocardial enzyme levels and inhibit oxidative stress.

Repression of protocadherin 17 is correlated with elevated angiogenesis and hypoxia markers in female patients with breast cancer.

BACKGROUND: Altered cadherin expression plays a vital role in tumorigenesis, angiogenesis and tumor progression. However, the function of protocadherin 17 (PCDH17) in breast cancer remains unclear. OBJECTIVE: Our target is to explore PCDH17 gene expression in breast carcinoma tissues and its relation to serum angiopoietin-2 (Ang-2), carbonic anhydrase IX (CAIX) and % of circulating CD34+ cells in breast cancer patients (BCPs). METHODS: This study included Fifty female BCPs and 50 healthy females as control group. Cancerous and neighboring normal breast tissues were collected from BCPs as well as blood samples at diagnosis. PCDH17 gene expression was evaluated by RT-PCR. Serum Ang-2, CAIX levels were measured by ELISA and % CD34+ cells were assessed by flow cytometry. RESULTS: PCDH17 was downregulated in cancerous breast tissues and its repression was significantly correlated with advanced stage and larger tumor size. Low PCDH17 was significantly correlated with serum Ang-2, % CD34+ cells and serum CAIX levels. Serum CAIX, Ang-2 and % CD34+ cells levels were highly elevated in BCPs and significantly correlated with clinical stage. CONCLUSIONS: PCDH17 downregulation correlated significantly with increased angiogenic and hypoxia biomarkers. These results explore the role of PCDH17 as a tumor suppressor gene inhibiting tumor growth and proliferation.

Exposure to Radiation During Work Shifts and Working at Night Act as Occupational Stressors Alter Redox and Inflammatory Markers.

BACKGROUND: Studies of breast cancer etiology suggest evidence that night shift working and occupational exposure to ionizing radiation (IR) are defined risk factors for breast cancer development. There are few studies to clarify neuroendocrine and inflammatory status and the possible consequences particularly in occupational exposure. AIM OF THE STUDY: Our aim was to associate the redox and inflammatory biomarkers with either nightshift working or occupational radiation exposure, and to compare their levels between the two groups at Alexandria University Hospitals, Alexandria, Egypt. METHODS: We included 150 female nurses at Alexandria University Hospitals: 50 nightshift workers, 50 radiation workers, and 50 dayshift workers as a control group (neither work nightly nor radiation workers). In morning serum sample (7 am), we measured the concentrations of serum melatonin, Cortisol, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by ELISA; malondialdehyde (MDA) and total antioxidant capacity (TAC) levels colorimetrically, and C-reactive protein (C-RP) levels by turbidimetric method. RESULTS: Nightshift workers had significantly lower levels of melatonin and TAC, and higher levels of serum inflammatory markers and cortisol, than day shift control group of workers. Workers occupationally exposed to IR had significantly higher levels of serum melatonin, MDA and inflammatory markers, lower levels of serum cortisol, and lower TAC than day shift workers. CONCLUSION: Occupational exposure to IR and working nightly alter circulating redox and inflammatory biomarkers.

Study the relationship of endothelial damage / dysfunction due to occupational exposure to low dose ionizing radiation versus high dose exposure during radiotherapy.

BACKGROUND: Vascular injuries caused by irradiation include acute vasculitis with neutrophil invasion, endothelial cell (EC) swelling, capillary loss, and activation of coagulator mechanisms, along with local ischemia and fibrosis. The circulating endothelial cells (CECs), increase dramatically in diseases with vascular damage. AIM: The aim of this study is to provide data on the endothelial dysfunction due to occupational exposure to low dose ionizing radiation versus high dose exposure during radiotherapy (RT). PATIENTS AND METHODS: This study included 100 subjects divided into three main groups: Group I: High dose exposure group: 50 breast cancer patients treated with post-operative radiotherapy. Group II: Low dose exposure group: 25 hospital radiation workers. Group III: 25 healthy volunteers' age and sex matched as control group who had never worked in radiation-related jobs. TM levels measured by enzyme linked immunosorbent assay (ELISA). Circulating endothelial cells (CEC) enumerated in peripheral blood by flow cytometric analysis of their signature receptor CD146. RESULTS: % CD146+ cells and plasma TM were significantly increased in radiation workers and after exposure to radiotherapy treatment in breast cancer patients. When comparing patients group with radiation workers group, we found significant elevation in plasma TM in radiation workers while insignificant difference was found in % CD146+ cells. CONCLUSION: CECs and plasma TM both are increased in radiation workers and patients treated with radiotherapy. They may constitute valuable markers of endothelial injury. Workers exposed to low doses of ionizing radiation may develop significant endothelial dysfunction predisposes them to cardiovascular complications namely thrombosis, mostly due to oxidative stress among other causes.

Metformin and survival in diabetic patients with breast cancer.

BACKGROUND: Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of breast cancer. This drug is widely used in the treatment of type 2 diabetes, where it is often referred to as an 'insulin sensitizer' because it not only lowers blood glucose, but also reduces the hyperinsulinemia associated with insulin resistance. Metformin inhibits the growth of cancer cells in vitro and tumors in vivo. However, there are little clinical data to support this. OBJECTIVE: The aim of the study was to determine whether metformin use was associated with better clinical outcomes in diabetic patients with breast cancer receiving adjuvant chemotherapy. PATIENTS AND METHODS: In this retrospective study, we reviewed the records of 460 consecutive patients with pathologically proved stage I-III breast cancer. These patients received adjuvant chemotherapy during the period from January 2008 to December 2008. Patients were compared by groups that consisted of 25 diabetic patients taking metformin, 14 diabetic patients not taking metformin, and 400 nondiabetic patients. RESULTS: In Kaplan-Meier analysis, metformin use during adjuvant chemotherapy significantly improved survival outcomes in diabetic patients' disease-free survival and overall survival. In multivariate Cox regression, metformin usage was a predictive factor that decreased the risk for breast cancer mortality. CONCLUSION: There is an association between metformin use and better survival outcome in diabetic breast cancer patients who received metformin during adjuvant chemotherapy. The choice of antidiabetic pharmacotherapy may influence prognosis of this group.