How Heme Oxygenase-1 Prevents Heme-Induced Cell Death.

Earlier observations indicate that free heme is selectively toxic to cells lacking heme oxygenase-1 (HO-1) but how this enzyme prevents heme toxicity remains unexplained. Here, using A549 (human lung cancer) and immortalized human bronchial epithelial cells incubated with exogenous heme, we find knock-down of HO-1 using siRNA does promote the accumulation of cell-associated heme and heme-induced cell death. However, it appears that the toxic effects of heme are exerted by "loose" (probably intralysosomal) iron because cytotoxic effects of heme are lessened by pre-incubation of HO-1 deficient cells with desferrioxamine (which localizes preferentially in the lysosomal compartment). Desferrioxamine also decreases lysosomal rupture promoted by intracellularly generated hydrogen peroxide. Supporting the importance of endogenous oxidant production, both chemical and siRNA inhibition of catalase activity predisposes HO-1 deficient cells to heme-mediated killing. Importantly, it appears that HO-1 deficiency somehow blocks the induction of ferritin; control cells exposed to heme show ~10-fold increases in ferritin heavy chain expression whereas in heme-exposed HO-1 deficient cells ferritin expression is unchanged. Finally, overexpression of ferritin H chain in HO-1 deficient cells completely prevents heme-induced cytotoxicity. Although two other products of HO-1 activity--CO and bilirubin--have been invoked to explain HO-1-mediated cytoprotection, we conclude that, at least in this experimental system, HO-1 activity triggers the induction of ferritin and the latter is actually responsible for the cytoprotective effects of HO-1 activity

Chemical and siRNA-mediated inhibition of catalase activity in A549 cells enhances the cytotoxic effects of heme.

<p><b>(A)</b> A549 cells were pre-treated with 5 mM 3-AT for 2 hours and then challenged with 100 μM heme for 36 hours. Cell viability was measured by alamar blue. <b>(B)</b> A549 cells were transfected with both 10 nM HO-1 and human catalase siRNAs for 24 hours and then challenged with 100 μM heme for 36 hours. Cell viability was measured by alamar blue. In both cases, data are means ± SEM of 3 independent experiments. *p≤0.05.</p

Heme exposure of HO-1KO A549 cells leads to lysosomal rupture and loss of viability which is prevented by pre-incubation with DFO.

<p>A549 cells were challenged with 100 μM heme for 24 hours and then stained with 5 μg/ml AO. <b>(A)</b> A549 cells deficient in intact lysosomes (‘pale’ cells) were measured by flow cytometry. <b>(B)</b> The summary of data shown in (A). Data are means ± SEM of 3 independent experiments. *p≤0.01. <b>(C)</b> A549 cells were pre-treated with 1 mM DFO for 2 hours and then challenged with 100 μM heme for 24 hours. Cell viability was measured using alamar blue. Data are means ± SEM of 3 independent experiments. *p≤0.05.</p

HO-1KO in A549 cells sensitizes the cells to heme-mediated cytotoxicity.

<p><b>(A)</b> A549 cells were transfected with the indicated HO-1 siRNA for 24 hours, then treated with or without 100 μM heme for 36 hours. Western blot shows substantial reduction in HO-1 mRNA expression in cells transfected with HO-1 siRNA. <b>(B)</b> Cells were exposed to 100 μM heme for 36 hours and viability was assessed with Alamar blue reduction. Data are means ± SEM of 6 independent experiments. *p≤0.01. <b>(C)</b> Human bronchial epithelial cells (HBEC) were transfected with the indicated HO-1 siRNA for 24 hours, and HO-1 expression was measured by western blot. <b>(D)</b> HBEC were transfected with the indicated HO-1 siRNA for 24 hours, then treated with or without 25 μM heme for 24 hours. Cell viability was measured by Alamar blue reduction.</p

HO-1KO A549 cells accumulate increased amounts of “loose” and total intracellular iron.

<p>The cells were transfected with HO-1 siRNA for 24 hours and then challenged with 100 μM heme for a further 24 hours. The cells were harvested by scraping and extracted with cold 10% perchloric acid. <b>(A)</b> Concentration of “loose” iron in the extract supernatants. <b>(B)</b> “Bound” intracellular iron concentrations. Note that in a nitric acid digest, “bound” iron includes not just ferritin bound iron but also heme iron. Data are means ± SEM of 3 independent experiments. *p≤0.01.</p

TLR4 knockdown suppresses heme accumulation in HO-1KO A549 cells.

<p>Following 24 hour transfections with both 10 nM TLR4 and HO-1 siRNAs, the cells were challenged with 100 μM heme for 6 hours, detached with 0.05% trypsin, washed twice and lysed using snap freezing and thawing. Heme was measured spectrophotometrically. Data are means ± SEM of 3 independent experiments. *p≤0.01.</p

HO-1KO blocks heme-mediated induction of ferritin and compensatory over-expression of ferritin H chain prevents heme-mediated cytotoxicity.

<p><b>(A)</b> A549 cells were transfected with 10 nM HO-1 siRNA and western blot indicates pronounced ferritin H chain induction in control cells and those transfected with scrambled siRNA but not HO-1 siRNA. <b>(B)</b> The summary of normalized ferritin H chain levels shown in (A). Quantification of ferritin H chain expression normalized to actin (n = 3 independent experiments). <b>(C)</b> Over-expression of ferritin H chain in HO-1KO A549 cells blocks heme-mediated cytotoxicity. A549 cells were transfected with 2 μg of FTH1 expressing pCMV6-ferritin for 24 hours (± HO-1 siRNA) and then challenged with100 μM heme for 36 hours. Cell viability was measured with alamar blue. Data are means ± SEM of 3 independent experiments. *p≤0.01.</p

siRNA-mediated knockdown of TLR4 suppresses heme-induced cytotoxicity in A549 cells.

<p><b>(A)</b> Western blot showing successful knockdown of TLR4 after 24 hour exposure to 10 nM TLR4 siRNA. <b>(B)</b> TLR4 knockdown prevents the cytotoxic effects of 36 hour exposure of A549 cells to 100 μM heme (viability assessed by alamar blue). Data are means ± SEM of 3 independent experiments.*p≤0.01.</p

Knockdown of TLR4 in A549 cells suppresses heme-induced intracellular ROS generation.

<p>Following 24 hour transfection with both HO-1 and TLR4 siRNAs the cells were challenged with 100 μM heme for a further 24 hours. The cells were then detached with 0.05% trypsin and stained with 10 μM DCFDA at 37°C for 30 minutes. ROS was measured using flow cytometry. Data are means ± SEM of 3 independent experiments. *p≤0.05.</p

Transcriptomic Profiling Identifies Differentially Expressed Genes in Palbociclib-Resistant ER+ MCF7 Breast Cancer Cells

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in estrogen receptor-positive (ER+) breast cancer remains a significant clinical challenge. Efforts to uncover the mechanisms underlying resistance are needed to establish clinically actionable targets effective against resistant tumors. In this study, we sought to identify differentially expressed genes (DEGs) associated with acquired resistance to palbociclib in ER+ breast cancer. We performed next-generation transcriptomic RNA sequencing (RNA-seq) and pathway analysis in ER+ MCF7 palbociclib-sensitive (MCF7/pS) and MCF7 palbociclib-resistant (MCF7/pR) cells. We identified 2183 up-regulated and 1548 down-regulated transcripts in MCF7/pR compared to MCF7/pS cells. Functional analysis of the DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several pathways associated with breast cancer, including &lsquo;cell cycle&rsquo;, &lsquo;DNA replication&rsquo;, &lsquo;DNA repair&rsquo; and &lsquo;autophagy&rsquo;. Additionally, Ingenuity Pathway Analysis (IPA) revealed that resistance to palbociclib is closely associated with deregulation of several key canonical and metabolic pathways. Further studies are needed to determine the utility of these DEGs and pathways as therapeutics targets against ER+ palbociclib-resistant breast cancer