Iron Deposition and Ferroptosis in the Spleen in a Murine Model of Acute Radiation Syndrome

Total body radiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated the impact of radiation-induced iron release on the spleen of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed ~20-60-fold increased Fe3+ in the spleen 7-14 days post-irradiation, also associated with altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not prevent iron deposition in the spleen, and did not significantly modulate most iron-binding proteins. Spleen volumes were markedly decreased 7-14 days, correlating with high Fe3+. At these time points, caspase-3 was activated and we identified four markers of ferroptosis, iron-dependent programmed cell death. Interestingly, p21/Waf1, a marker of accelerated senescence, was not upregulated in vivo. Macrophage inflammation is an important effect of TBI. We investigated the effects of radiation and Fe3+ on the cultured J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, within 24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron binding proteins and triggers ferroptosis.

Effects of Xinwei granule on expression levels of cyclin D1 and its upstream genes in gastric intraepithelial neoplasia tissues

Purpose: To explore the effects of Xinwei granule (XWG) on low-grade gastric intraepithelial neoplasia (LGIN) and the underlying mechanisms. Methods: To establish LGIN model, Wistar rats were treated with N-methyl-N'-nitrosoguanidine for 3 months. LGIN model rats were randomly grouped into five groups (n = 15), viz, negative control (NC), normal saline (NS) group, Xinwei granule (XWG) group, Weifuchun tablet (WFCT) group, and vatacoenayme tablet (VT) group. Normal rats (n = 17) served as negative control. Histological evaluation of gastric mucosa was undertaken using hematoxylin and eosin staining. Quantitative realtime polymerase chain reaction (qRT-PCR), western blot, and immunohistochemical assays were performed to determine mRNA expressions, protein expression, and the distribution of cyclin D1, kruppel-like factor 4 (KLF4), and p21-WAF1-CIP1, respectively. Results: Compared with LGIN group, the body weight of the rats increased in XWG, WFCT, and VT groups. The pathological characteristics of LGIN group were alleviated by XWG, WFCT and VT treatments. The positive expression of cyclin D1 was enhanced in LGIN group, but reduced in XWG, WFCT and VT groups. The expression levels of KLF4 and p21-WAF1-CIP1, upstream regulators of cyclin D1 reduced in LGIN groups. However, administration of XWG, WFCT and VT strengthened the expressions of KLF4 and p21-WAF1-CIP1. More importantly, the protective effects of XWG against LGIN were superior to those of WFCT and VT. Conclusion: Xinwei granules alleviate LGIN in vivo by inhibiting cyclin D1 expression and enhancing KLF4 and p21-WAF1-CIP1 expression.

Effect of Cassiopea andromeda Venom on P15INK4b, P21 WAF1/CIP1, P53, DNA methyltransferase 1, and Bcl-2 Genes Expression, Apoptosis Induction, and Cell Growth Inhibition in Acute Promyelocytic Leukemia NB4 Cell line

Background: One of the acute hematologic malignancies is acute promyelocytic leukemia (APL) that resulted in translocation of chromosomes 15 and 17, t (15; 17), and cessation in the maturation of myeloid cell line, and ultimate aggregation of neoplastic promyelocytes. Regarding that appetence of using herbal and marine medicine studies is increasing, and on the other hand, the features of Cassiopea andromeda Venom remained unclear; this study was conducted to determine its effects on NB4 cells as a model for APL. Materials and Methods: In this experimental study, the cells were treated with C. andromeda Venom concentrations at different periods and times. Growth inhibition and toxic effects of C. andromeda Venom were evaluated through methyl thiazole tetrazolium salt reduction (MTT test). The flow cytometry analysis was carried out using 7AAD and Annexin V stains for evaluating this venom’s effect on apoptotic pathways. Besides, Real-Time polymerase chain reaction was performed to evaluate the relative gene expression. Results: C. andromeda Venom inhibited the growth of NB4 cells as correlated with concentration and time. Cell growth was inhibited by 49.1%, after 24 hours of treating NB4 cells with 1000µg/mL C. andromeda Venom. This venom increased the apoptotic process, which was then verified by 7AAD/AnnexinV staining. The fold change of p15INK4b, p21 WAF1/CIP1, P53, DNMT1, and Bcl-2 genes in the NB4 cell line were 144, 2.78, 1.75, 15.24, and 0.33, respectively, which meant that the expression level of p15INK4b, p21 WAF1/CIP1, P53, and DNMT1 were increased by 14400%, 278%, 175%, and 1524%, respectively and the expression of Bcl-2 was decreased by 67%. Conclusion: Considering the inhibitory property of C. andromeda Venom, the authors recommended it as a part of combinational medication for treating APL in animal trials and for other leukemias’ in vitro studies.

TFEB Modulates p21/WAF1/CIP1 during the DNA Damage Response

The MiT/TFE family of transcription factors (MITF, TFE3, and TFEB), which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents.