Gallium maltolate shows synergism with cisplatin and activates nucleolar stress and ferroptosis in human breast carcinoma cells.

PURPOSE: Breast cancer is the most common cancer in women. Triple-negative breast cancer (TNBC) is an aggressive disease with poor outcomes. TNBC lacks effective targeted treatments, and the development of drug resistance limits the effectiveness of chemotherapy. It is crucial to identify new drugs that can enhance the efficacy of traditional chemotherapy to reduce drug resistance and side effects. METHODS: TNBC cell lines, MDA-MB-231 and Hs 578T, and a normal cell line, MCF-10 A, were included in this study. The cells were treated with gallium maltolate (GaM), and their transcriptome was analyzed. Ferroptosis and nucleolar stress markers were detected by qPCR, western blotting, fluorescence microscopy, and flow cytometry. The impairment of ribosome synthesis was evaluated by northern blotting and sucrose gradients. RESULTS: GaM triggered cell death via apoptosis and ferroptosis. In addition, GaM impaired translation and activated nucleolar stress. Cisplatin (DDP) is a chemotherapeutic agent for advanced breast cancer. While single treatment with GaM or DDP at low concentrations did not impact cell growth, co-administration enhanced cell death in TNBC but not in normal breast cells. The enhancement of ferroptosis and nucleolar stress could be observed in TNBC cell lines after co-treatment. CONCLUSIONS: These results suggest that GaM synergizes with cisplatin via activation of nucleolar stress and ferroptosis in human breast carcinoma cells. GaM is marginally toxic to normal cells but impairs the growth of TNBC cell lines. Thus, GaM has the potential to be used as a therapeutic agent against TNBC.

High-dose copper activates p53-independent apoptosis through the induction of nucleolar stress in human cell lines.

Copper is an essential micronutrient involved in many redox reactions in human cells. However, a high concentration of copper, intake from the environment or abnormal accumulation within cells because of genetic mutation, leads to cell toxicity. This is attributable to oxidative damage, altered gene expression, and functional impairment of the mitochondria. Copper stress also alters the morphology of the nucleolus, but the process has not been fully elucidated. In this study, cells were treated with copper sulfate at 3-9 ppm and examined if a high dose of copper would block ribosome biogenesis. With the incorrect distribution of nucleolar proteins nucleophosmin and fibrillarin to the nucleoplasm, ribosomal RNA (rRNA) processing was impaired; 34S rRNA from an abnormal A2 cut increased, and downstream pre-rRNAs decreased. The under-accumulation of 60S subunits was detected using sucrose gradients. From transcriptome analysis, ribosome synthesis-related genes were misregulated. Blockage in ribosome synthesis under copper-treatment induced nucleolar stress and triggered p53-independent apoptosis pathways. Thus, nucleolar stress is one cause of cell death under copper exposure.

Molecular profiling of afatinib-resistant non-small cell lung cancer cells in vivo derived from mice.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC patients with overexpressed or mutated epidermal growth factor receptor (EGFR) related to disease progression are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Acquired drug resistance after TKI treatments has been a major focus for development of NSCLC therapies. This study aimed to establish afatinib-resistant cell lines from which afatinib resistance-associated genes are identified and the underlying mechanisms of multiple-TKI resistance in NSCLC can be further investigated. Nude mice bearing subcutaneous NSCLC HCC827 tumors were administered with afatinib at different dose intensities (5-100 mg/kg). We established three HCC827 sublines resistant to afatinib (IC50 > 1 µM) with cross-resistance to gefitinib (IC50 > 5 µM). cDNA microarray revealed several of these sublines shared 27 up- and 13 down-regulated genes. The mRNA expression of selective novel genes - such as transmembrane 4 L six family member 19 (TM4SF19), suppressor of cytokine signaling 2 (SOCS2), and quinolinate phosphoribosyltransferase (QPRT) - are responsive to afatinib treatments only at high concentrations. Furthermore, c-MET amplification and activations of a subset of tyrosine kinase receptors were observed in all three resistant cells. PHA665752, a c-MET inhibitor, remarkably increased the sensitivity of these resistant cells to afatinib (IC50 = 12-123 nM). We established afatinib-resistant lung cancer cell lines and here report genes associated with afatinib resistance in human NSCLC. These cell lines and the identified genes serve as useful investigational tools, prognostic biomarkers of TKI therapies, and promising molecule targets for development of human NSCLC therapeutics.

ADP-ribosylation factor-like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation.

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)-like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394-1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.