Transferrin Receptor and Cellular Iron are Potential Molecular Targets for Diagnosis and Treatment of Lung Cancer

Abstract

Lung cancer is one of the leading causes of cancer death. Most current treatments have debilitating side effects with poor selectivity and pharmacodynamic properties. To develop more effective and safer anticancer drugs, we synthesized trioxane (DMR) and dioxazinane (HSM), both novel Artemisinin (ART) analogs. These analogs induced apoptosis in cancer but not normal lung cells and was reactive oxygen species (ROS) dependent. We also showed that cancer cells have higher transferrin receptor (TfR) expression compared to normal cells. We hypothesize high levels of TfR expression and [iron]i are responsible for the cancer specific effects of analogs. To study this, we confirmed iron’s role in ART analog‐induced apoptosis. We also knocked out TfR in cancer cells and overexpressed TfR in normal cells.Confluent normal (BEAS2B) and cancer (A549) human lung cells were treated (18 H) with 10μM of DMR, HSM ±Deferoxamine (DFO, iron chelator; 10 μM). [Iron]i was assessed in cell lysates using a colorimetric assay (Biovision, CA). Cell death was assessed by i) staining with FITC‐Annexin V (AV, apoptosis), propidium iodide (PI, cell death), followed by imaging, and quantification using flow cytometry and/or microscopy (Image J) and ii) measuring Lactate dehydrogenase (LDH) release. Cells were transfected with CRISPR/Cas9 overexpression (OE) or knockout (KO) plasmid (Santa Cruz, TX) containing Green Fluorescent Protein (GFP). Co‐transfection with HDR plasmid allowed for puromycin selection. Transfection efficiency was assessed using RT‐PCR, Western blot and GFP expression.A549 cells had 7‐fold more [iron]i than BEAS2B cells. This was essential for the apoptotic effects, as chelating iron with DFO prevented ART analog‐induced cell death (% AV+ A549 cells, 18 H: HSM: 54±4; HSM+DFO: 1±0.5; DMR: 32±3; DMR+DFO: 0.3±0.1, n≥3). KO of TfR in A549 cells yielded a low transfection efficiency (10%, Image J). Western blot of cell lysates did not show a significant reduction in TfR protein expression. Co‐transfection with HDR plasmid followed by puromycin selection increased transfection efficiency. However, there was increased cell death in TfR KO cells in culture (72 H, PI+ cells; mean pixel intensity (mpi), control: 15±0.2; GFP: 19±2, TfR KO: 49±42; n=3). Overexpression of TfR in BEAS2B cells yielded a higher transfection efficiency (~25%; Image J and RT‐PCR). Similar to TfR KO in A549 cells, after 72 H, there was a marked increase in cell death in TfR OE BEAS2B (PI+ cells (mpi), control: 18±2; GFP: 13±4, TfR OE: 83±20; n=3). TfR overexpression in normal lung cells possibly resulted in iron overload and ferroptosis.Our findings demonstrate that abnormal TfR expression and the associated changes in iron uptake induces death in normal and cancer cells, highlighting its importance in cell survival and proliferation. Understanding the role of TfR and iron in carcinogenesis will help develop potent therapeutic drugs to treat cancer, a disease that accounts for ~ 9 million deaths annually. Further, synthesizing novel analogs such as DMR‐tagged transferrin will provide specific and efficient drug delivery to cancer cells.Support or Funding InformationAPS‐STRIDE (Grant #1 R25 HL 115473‐01); NSSRP BenU Funds