An attractive strategy to overcome multidrug resistance in cancer chemotherapy is to suppress P-glycoprotein (P-gp), which is a pump overproduced in cancer cells to remove cytotoxic drugs from cells. In the present study, a Ca 2+ -permeable channel TRPC5 was found to be overproduced together with P-gp in adriamycin-resistant breast cancer cell line MCF-7/ADM. Suppressing TRPC5 activity/expression reduced the P-gp induction and caused a remarkable reversal of adriamycin resistance in MCF-7/ADM. In an athymic nude mouse model of adriamycin-resistant human breast tumor, suppressing TRPC5 decreased the growth of tumor xenografts. Nuclear factor of activated T cells isoform c3 (NFATc3) was the transcriptional factor that links the TRPC5 activity to P-gp production. Together, we demonstrated an essential role of TRPC5-NFATc3-P-gp signaling cascade in P-gp induction in drug-resistant cancer cells.
Beclin 1, a protein essential for autophagy, regulates autophagy by interacting with Vps34 and other cofactors to form the Beclin 1 complex. Modifications of Beclin 1 may lead to the induction, inhibition or fine-tuning of the autophagic response under a variety of conditions. Here we show that Beclin 1 is acetylated by p300 and deacetylated by SIRT1 at lysine residues 430 and 437. In addition, the phosphorylation of Beclin 1 at S409 by CK1 is required for the subsequent p300 binding and Beclin 1 acetylation. Beclin 1 acetylation inhibits autophagosome maturation and endocytic trafficking by promoting the recruitment of Rubicon. In tumour xenografts, the expression of 2KR mutant Beclin 1 (substitution of K430 and K437 to arginines) leads to enhanced autophagosome maturation and tumour growth suppression. Therefore, our study identifies an acetylation-dependent regulatory mechanism governing Beclin 1 function in autophagosome maturation and tumour growth.
15However, in some other pathological conditions, including congestive heart failure, hypercholesterolemia, ischemiareperfusion, and restenosis after coronary angioplasty, the NO-mediated relaxant response is impaired, whereas the EDHFmediated relaxant response is increased as a compensation. 15Abstract-The small conductance and intermediate conductance Ca 2+-activated K + channels are known to be involved in the endothelium-dependent hyperpolarization. Ca 2+ entry into endothelial cells stimulates these channels, causing membrane hyperpolarization in endothelial cells and underlying smooth muscle cells. In the present study, with the use of coimmunoprecipitation and double immunolabeling methods, we demonstrated a physical interaction of transient receptor potential vanilloid 4 (TRPV4) with K Ca 2.3 in rat mesenteric artery endothelial cells. Acetylcholine and 4α-PDD mainly acted through TRPV4-K Ca 2.3 pathway to induce smooth muscle hyperpolarization and vascular relaxation. K Ca 3.1 was also involved in the process but at a much lesser degree than that of K Ca 2.3. Stimulating TRPV4-K Ca 2.3 signaling pathway also increased local blood flow in mesenteric beds and reduced systemic blood pressure in anesthetized rats. In streptozotocin-induced diabetic rats, the expression levels of TRPV4 and K Ca 2.3 were reduced, which could be an underlying reason for the dysfunction of endothelium-dependent hyperpolarization in these animals. These results demonstrated an important physiological and pathological role of TRPV4-K Ca 2. However, the molecular mechanism of altered EDHF responses in these disease states is poorly understood.In the present study, we identified a previously unknown physical association between TRPV4 and K Ca 2.3 and uncovered the functional role of this TRPV4-K Ca 2.3 signaling pathway in smooth muscle hyperpolarization and relaxation. We also found that reduced expression levels of TRPV4 and K Ca 2.3 could be an underlying mechanism for EDHF dysfunction in diabetic rats. Materials and MethodsSee the Methods section in the online-only Data Supplement for details. Cell Preparation and CultureAll animal experiments were conducted in accordance with the regulation of the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Primary mesenteric artery endothelial cells (MAECs) were isolated from male Sprague-Dawley rats, cultured for 3 to 5 days, and used for experiments without passage. Immunostaining, Immunoprecipitation, and ImmunoblotsDouble immunolabeling was performed in rat MAECs using anti-TRPV4 antibody together with either anti-K Ca 2.3 or anti-K Ca 3.1 antibody. Artery sections were stained with either anti-TRPV4 or anti-K Ca 2.3 antibody. Immunoprecipitation and immunoblots were performed following the protocol described elsewhere with slight modifications. . The membrane potentials of MAECs were also measured using perforated wholecell patch clamp with an EPC-9 amplifier. High-impedance sharp microelectrodes were used to measure smooth muscle cell membran...
AimsGenistein, an isoflavone derivative found in soy, is known as a promising treatment for rheumatoid arthritis (RA). However, the detailed molecular mechanism of genistein in suppression of proinflammatory cytokine production remains ambiguous. The aim of this work was to evaluate the signal pathway by which genistein modulates inflammatory cytokine expression.Materials and methodsMH7A cells were stimulated with tumor necrosis factor (TNF)-α and incubated with genistein, and interleukin (IL)-1β, IL-6, and IL-8 production was measured by enzyme-linked immunosorbent assay. Nuclear translocation of nuclear factor (NF)-κB was measured by a confocal fluorescence microscopy. The intracellular accumulation of reactive oxygen species (ROS) was monitored using the fluorescent probe 5-6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Signal-transduction protein expression was measured by Western blot.ResultsGenistein decreased the secretion of IL-1β, IL-6, and IL-8 from TNF-α-stimulated MH7A cells in a dose-dependent manner. Genistein prevented TNF-α-induced NF-κB translocation as well as phosphorylation of IκB kinase-α/β and IκBα, and also suppressed TNF-α-induced AMPK inhibition. The production of IL-1β, IL-6, and IL-8 induced by TNF-α was decreased by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting that inhibition of Akt activation might inhibit IL-1β, IL-6, and IL-8 production induced by TNF-α. In addition, we also found that pretreatment with the adenosine monophosphate-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside obviously inhibited TNF-α-induced proinflammatory cytokine production. These observations suggest that the inhibitory effect of genistein on TNF-α-induced proinflammatory cytokine production is dependent on AMPK activation.ConclusionThese findings indicate that genistein suppressed TNF-α-induced inflammation by inhibiting the ROS/Akt/NF-κB pathway and promoting AMPK activation in MH7A cells.
Adriamycin is a first-line chemotherapy agent against cancer, but the development of resistance has become a major problem. Although autophagy is considered to be an adaptive survival response in response to chemotherapy and may be associated with chemoresistance, its inducer and the underlying molecular mechanisms remain unclear. Here, we demonstrate that adriamycin up-regulates the both levels of TRPC5 and autophagy, and the increase in autophagy is mediated by TRPC5 in breast cancer cells. Blockade of TRPC5 or autophagy increased the sensitivity to chemotherapy in vitro and in vivo. Notably, we revealed a positive correlation between TRPC5 and the autophagy-associated protein LC3 in paired patients with or without anthracycline-taxane-based chemotherapy. Furthermore, pharmacological inhibition and gene-silencing showed that the cytoprotective autophagy mediated by TRPC5 during adriamycin treatment is dependent on the CaMKKβ/AMPKα/mTOR pathway. Moreover, adriamycin-resistant MCF-7/ADM cells maintained a high basal level of autophagy, and silencing of TRPC5 and inhibition of autophagy counteracted the resistance to adriamycin. Thus, our results revealed a novel role of TRPC5 as an inducer of autophagy, and this suggests a novel mechanism of drug resistance in chemotherapy for breast cancer.
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