<p>Supplementary Figure S2. Representative flow cytometry dot plots of apoptosis analysis in SP cells under exposure to combination of afatinib with topotecan or mitoxantrone were presented.</p>
A38 A major problem in cancer treatment involves the development of drug resistance to a variety of structurally unrelated anti-cancer drugs, also known as multidrug resistance (MDR). MDR can result from several diverse mechanisms; yet, the predominant one is the increased efflux of chemotherapeutics attributed to the overexpression of ATP-binding Cassette (ABC) drug transporters on the plasma membrane of tumor cells. ABC transporters utilize energy from ATP hydrolysis to efflux compounds out of the cell which in turn reduces the drug concentration actually exposed to the tumor. Thus, the tumor cells survive, and the treatment fails. Although over 12 of ABC transporters have been linked to MDR, little is known about the expression of these transporters during the early stages of MDR. We have recently found that ABC transporter mRNA expression patterns vary with single vs. multistep treatment with doxorubicin or etoposide in several cancer cell types. In this work, we report that a single-step selection with low doses of anti-cancer agents similar to concentrations reported in vivo can cause MDR and that this resistance is mediated by ABCG2. We established single-step-selected sublines of breast, ovarian and colon cancer cells (MCF-7, IGROV-1, and S-1) using 14 or 21 nM doxorubicin treatment for only 10 days followed by continuously culturing in drug-free medium. These cell lines showed overexpression of ABCG2 at the mRNA and protein levels after the single-step selection. RNAi analysis confirmed that ABCG2 confers drug resistance in these clones. Furthermore, ABCG2 up-regulation was facilitated by histone hyperacetylation due to weaker HDAC1-promoter association indicating that these epigenetic changes elicit changes in ABCG2 gene expression. Consequently, these studies suggest that the MDR phenotype can arise following low dose single-step selections and that ABCG2 mediates the early stages of MDR development. This is the first report of single-step, low dose selection causing overexpression of ABCG2 by epigenetic changes in various cancer cell lines suggesting that ABCG2 may be an important biomarker in the development of multidrug resistance in cancer in vitro. Further work evaluating the early stages of doxorubicin resistance in prostate cancer and melanoma cells is currently in progress.
<p>Supplementary Figure S2. Representative flow cytometry dot plots of apoptosis analysis in SP cells under exposure to combination of afatinib with topotecan or mitoxantrone were presented.</p>
Supplementary Figure 3 from Apatinib (YN968D1) Reverses Multidrug Resistance by Inhibiting the Efflux Function of Multiple ATP-Binding Cassette Transporters
<div>Abstract<p>ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in pharmacology and may play a role in stem cell biology and clinical drug resistance. To study the mechanism(s) regulating ABCG2 expression, we used ChIP to investigate the levels of acetylated histone H3, histone deacetylases (HDAC), histone acetyltransferases, and other transcription regulatory proteins associated with the <i>ABCG2</i> promoter. Following selection for drug resistance and the subsequent overexpression of <i>ABCG2</i>, an increase in acetylated histone H3 but a decrease in class I HDACs associated with the <i>ABCG2</i> promoter was observed. Permissive histone modifications, including an increase in histone H3 lysine 4 trimethylation (Me<sub>3</sub>-K4 H3) and histone H3 serine 10 phosphorylation (P-S10 H3), were observed accompanying development of the resistance phenotype. These changes mirrored those in some cell lines treated with a HDAC inhibitor, romidepsin. A repressive histone mark, trimethylated histone H3 lysine 9 (Me<sub>3</sub>-K9 H3), was found in untreated parental cells and cells that did not respond to HDAC inhibition with <i>ABCG2</i> up-regulation. Interestingly, although all five studied cell lines showed global histone acetylation and <i>MDR1</i> up-regulation upon HDAC inhibition, only those cells with removal of the repressive mark, and recruitment of RNA polymerase II and a chromatin remodeling factor Brg-1 from the <i>ABCG2</i> promoter, showed increased <i>ABCG2</i> expression. In the remaining cell lines, HDAC1 binding in association with the repressive Me3-K9 H3 mark apparently constrains the effect of HDAC inhibition on <i>ABCG2</i> expression. These studies begin to address the differential effect of HDAC inhibitors widely observed in gene expression studies. (Mol Cancer Res 2008;6(1):151–64)</p></div>
Abstract Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted.
Importance of the field: The ABCG2 efflux transporter is expressed in multiple tissues and plays an important role in the disposition of many statins. The functional 421C>A polymorphism in ABCG2 that reduces transporter activity has been found to be associated with increased systemic exposures to certain statins.
Circulating tumor cells (CTCs) are precursors of distant metastasis in a subset of cancer patients. A better understanding of CTCs heterogeneity and how these CTCs survive during hematogenous dissemination could lay the foundation for therapeutic prevention of cancer metastasis. It remains elusive how CTCs evade immune surveillance and elimination by immune cells. In this study, we unequivocally identified a subpopulation of CTCs shielded with extracellular vesicle (EVs)-derived CD45 (termed as CD45
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