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1.
Langmuir ; 35(20): 6752-6761, 2019 05 21.
Article in English | MEDLINE | ID: mdl-31030514

ABSTRACT

There is a great demand worldwide for bone-related implant materials. The drawbacks of chronic infections and poor bone healing of current implant materials have limited their clinical applications. Functionalizing the implant surfaces with antibacterial and osteogenic films on implant materials provides new opportunities for fabricating novel implant materials. In the present study, an ultrathin (GO/Lys)8 film of several tens of nanometers was fabricated using a layer-by-layer (LBL) technique with alternative deposition of graphene oxide (GO) and lysozyme (Lys). The deposition of the (GO/Lys) n film exhibited a successive growth as supported by ellipsometry, UV-vis, and Fourier transform infrared data, and the physical properties (morphology, roughness, and stiffness) of this film were characterized with an atomic force microscope. The ultrathin films exhibited a great effect on bacterium sterilization of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and enhanced osteogenic differentiation efficiency, showing the potential application in bone implant coatings. We believe that this LBL assembling strategy will pave the way for fabricating dual-functional surfaces and guide the design of the implanted surfaces in the future.


Subject(s)
Anti-Bacterial Agents , Dental Pulp/metabolism , Escherichia coli/growth & development , Graphite , Membranes, Artificial , Osteogenesis/drug effects , Staphylococcus aureus/growth & development , Stem Cells/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Dental Pulp/cytology , Graphite/chemistry , Graphite/pharmacology , Humans , Muramidase/chemistry , Muramidase/pharmacology , Stem Cells/cytology
2.
EMBO Mol Med ; 9(11): 1491-1503, 2017 11.
Article in English | MEDLINE | ID: mdl-28899928

ABSTRACT

The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta-blockers, constipation by Ca2+ channel blockers, and dry cough by ACEI, lead to non-compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca2+-activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high-salt diet, N(G)-nitro-l-arginine intake, or angiotensin II delivery, showed decreased TRPV4-KCa2.3 interaction in ECs. Perturbation of the TRPV4-KCa2.3 interaction in mouse ECs by overexpressing full-length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small-molecule drug, JNc-440, which showed affinity for both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the impaired TRPV4-KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4-KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.


Subject(s)
Antihypertensive Agents/therapeutic use , Hypertension/drug therapy , Potassium Channels, Calcium-Activated/metabolism , TRPV Cation Channels/metabolism , Angiotensin II/genetics , Angiotensin II/metabolism , Animals , Antihypertensive Agents/chemistry , Blood Pressure , Cells, Cultured , Disease Models, Animal , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , Hypertension/metabolism , Hypertension/pathology , Mesenteric Arteries/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Mutagenesis , Nitroprusside/pharmacology , Plasmids/genetics , Plasmids/metabolism , Potassium Channels, Calcium-Activated/genetics , TRPV Cation Channels/deficiency , TRPV Cation Channels/genetics , Vasodilation/drug effects
3.
Clin Sci (Lond) ; 131(3): 227-237, 2017 02 01.
Article in English | MEDLINE | ID: mdl-27895148

ABSTRACT

Transient receptor potential channel 5 (TrpC5) is a member of the TrpC subgroup, and it forms a receptor-activated, non-selective Ca2+ channel. The architecture of the TrpC5 channel is poorly understood. In the present study, we report that TrpC5 is a key factor in regulating differentiation in colorectal cancer (CRC). Through a study of specimens from a large cohort of patients with CRC, we found that TrpC5 was highly expressed and its cellular level correlated with tumour grade. We showed further that up-regulated TrpC5 caused a robust rise in intracellular calcium concentration [Ca2+]i, increased Wnt5a expression and the nuclear translocation of ß-catenin, leading to a reduction in cancer differentiation and an increase in cancer cell stemness. Notably, patients with tumours that expressed high levels of TrpC5 showed significantly poorer disease-free and overall survival. Therefore, our findings suggest that TrpC5 is an independent adverse prognostic factor for death in CRC, reducing differentiation through the Ca2+/Wnt5a signalling pathway.


Subject(s)
Calcium Signaling , Carcinoma/metabolism , Colorectal Neoplasms/metabolism , TRPC Cation Channels/metabolism , Wnt-5a Protein/metabolism , Animals , Caco-2 Cells , Carcinoma/diagnosis , Carcinoma/pathology , Cell Differentiation , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/pathology , HT29 Cells , Humans , Mice, Inbred BALB C , Mice, Nude , Neoplasm Grading , Prognosis , beta Catenin/metabolism
4.
Biochem Pharmacol ; 118: 18-30, 2016 Oct 15.
Article in English | MEDLINE | ID: mdl-27520484

ABSTRACT

It is believed that tumor cells can give rise to endothelial cells and tumor endothelium has a neoplastic origin. Yet, the stimuli and underlying mechanism remain unclear. Here, we demonstrate that adriamycin or paclitaxel, first-line chemotherapy agent, induced breast cancer cells to generate morphological, phenotypical and functional features of endothelial cells in vitro. In xenografts models, challenges from adriamycin or paclitaxel induced cancer cells to generate the majority of microvessels. Importantly, in breast cancer specimens from patients with neoadjuvant anthracycline-based or taxane-based chemotherapy, tumor-derived endothelial microvessels, lined by EGFR-amplified or/and TP53+-CD31+ endothelial cells, was significantly higher in patients with progressive or stable disease (PD/SD) than in those with a partial or complete response (PR/CR). Further, exposure to the Notch signaling inhibitor and gene silencing studies showed that Notch signaling inhibition or silencing Nothc4/Dll3 decreased endothelial markers and function of tumor-derived endothelial cells under chemotherapy treatment, which may be through VEGFR3. Thus, our findings demonstrate that chemotherapy induces functional tumor-derived endothelial microvessels by mediating Notch signaling and VEGF signaling, and may provide new targets for anti-angiogenesis therapy in breast cancer.


Subject(s)
Antineoplastic Agents/adverse effects , Breast Neoplasms/drug therapy , Endothelium, Vascular/drug effects , Neoplasm Proteins/agonists , Neovascularization, Pathologic/chemically induced , Receptors, Notch/agonists , Signal Transduction/drug effects , Animals , Anthracyclines/adverse effects , Anthracyclines/therapeutic use , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/toxicity , Biomarkers/metabolism , Breast Neoplasms/blood supply , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Bridged-Ring Compounds/adverse effects , Bridged-Ring Compounds/therapeutic use , Doxorubicin/adverse effects , Doxorubicin/therapeutic use , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Humans , MCF-7 Cells , Mice, Nude , Microvessels/drug effects , Microvessels/metabolism , Microvessels/pathology , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Paclitaxel/adverse effects , Paclitaxel/therapeutic use , RNA Interference , Receptors, Notch/antagonists & inhibitors , Receptors, Notch/genetics , Receptors, Notch/metabolism , Specific Pathogen-Free Organisms , Taxoids/adverse effects , Taxoids/therapeutic use , Xenograft Model Antitumor Assays
5.
Acta Pharmacol Sin ; 37(9): 1199-207, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27397542

ABSTRACT

AIM: TRPV4-C1 heteromeric channels contribute to store-operated Ca(2+) entry in vascular endothelial cells. However, the negative regulation of these channels is not fully understood. This study was conducted to investigate the inhibitory effect of PKG1α on TRPV4-C1 heteromeric channels. METHODS: Immuno-fluorescence resonance energy transfer (FRET) was used to explore the spatial proximity of PKG1α and TRPC1. Phosphorylation of endogenous TRPC1 was tested by phosphorylation assay. [Ca(2+)]i transients and cation current in MAECs were assessed with Fura-2 fluorescence and whole-cell recording, respectively. In addition, rat mesenteric arteries segments were prepared, and vascular relaxation was examined with wire myography. RESULTS: In immuno-FRET experiments, after exposure of these cells to 8-Br-cGMP, more PKG1α was observed in the plasma membrane, and PKG1α and TRPC1 were observed to be in closer proximity. TAT-TRPC1(S172) and TAT-TRPC1(T313) peptide fragments, which contain the PKG targeted residues Ser172 and Thr313, respectively, were introduced into isolated endothelial cells to abrogate the translocation of PKG1α. Furthermore, a phosphorylation assay demonstrated that PKG directly phosphorylates TRPC1 at Ser172 and Thr313 in endothelial cells. In addition, PKG activator 8-Br-cGMP markedly reduced the magnitude of the 4αPDD-induced and 11,12-EET-induced [Ca(2+)]i transients, the cation current and vascular relaxation. CONCLUSION: This study uncovers a novel mechanism by which PKG negatively regulates endothelial heteromeric TRPV4-C1 channels through increasing the spatial proximity of TRPV4-C1 to PKG1α via translocation and through phosphorylating Ser172 and Thr313 of TRPC1.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Cyclic GMP-Dependent Protein Kinase Type I/metabolism , Endothelium, Vascular/metabolism , Mesenteric Arteries/metabolism , TRPC Cation Channels/metabolism , Animals , Cell Culture Techniques , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Endothelial Cells/metabolism , Fluorescence Resonance Energy Transfer , In Vitro Techniques , Membrane Potentials/drug effects , Mice, Knockout , Patch-Clamp Techniques , Peptide Fragments/pharmacology , Phosphorylation , Protein Transport , Rats, Sprague-Dawley , TRPC Cation Channels/genetics , Vasodilation
6.
Pharmacol Res ; 93: 36-42, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25579062

ABSTRACT

Chemotherapy targeting anti-angiogenesis in tumors may have insufficient efficacy, but little is known about the underlying mechanisms. Here, we showed that the Ca(2+)-permeable channel, TrpC5, is highly expressed in human breast cancer after long-term chemotherapy drug-treatment. It mediates downstream hypoxia-inducible factor 1α accumulation in the nucleus, and then activates the transcription of vascular endothelial growth factor which promotes tumor angiogenesis, leading to a poor chemotherapeutic outcome. We verified this mechanism at both the cellular and xenograft levels. Moreover, in samples from patients, high TrpC5 expression was correlated with enhanced tumor vasculature after chemotherapy. Taken together, our research demonstrated the essential role of TrpC5 in tumor angiogenesis when facing the challenge of chemotherapy and presents a new potential target for overcoming the high vasculature of human breast cancer after chemotherapy.


Subject(s)
Breast Neoplasms/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , TRPC Cation Channels/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Antibiotics, Antineoplastic/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Doxorubicin/pharmacology , Female , Humans , Mice, Nude , Neovascularization, Pathologic/metabolism , RNA, Small Interfering/administration & dosage , TRPC Cation Channels/genetics , Xenograft Model Antitumor Assays
7.
J Biol Chem ; 290(1): 448-56, 2015 Jan 02.
Article in English | MEDLINE | ID: mdl-25404731

ABSTRACT

5-Fluorouracil (5-Fu) is commonly used in the chemotherapy of colorectal cancer (CRC), but resistance to 5-Fu occurs in most cases, allowing cancer progression. Suppressing ABCB1 (ATP-binding cassette, subfamily B, member 1), which is a pump overproduced in cancer cells to export cytotoxic drugs, is an attractive strategy to overcome drug resistance. In the present study, transient receptor potential channel TrpC5 was found to be overproduced at the mRNA and protein levels together with ABCB1 in 5-Fu-resistant human CRC HCT-8 (HCT-8/5-Fu) and LoVo (LoVo/5-Fu) cells. More nuclear-stabilized ß-catenin accumulation was found in HCT-8/5-Fu and LoVo/5-Fu cells than in HCT-8 and LoVo cells. Suppressing TrpC5 expression with TrpC5-specific siRNA inhibited the canonical Wnt/ß-catenin signal pathway, reduced the induction of ABCB1, weakened the ABCB1 efflux pump, and caused a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu and LoVo/5-Fu cells. On the contrary, enforcing TrpC5 expression resulted in an activated Wnt/ß-catenin signal pathway and up-regulation of ABCB1. Taken together, we demonstrated an essential role of TrpC5 in ABCB1 induction and drug resistance in human CRC cells via promoting nuclear ß-catenin accumulation.


Subject(s)
Antineoplastic Agents/pharmacology , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic , TRPC Cation Channels/genetics , beta Catenin/genetics , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Cell Line, Tumor , Colon/drug effects , Colon/metabolism , Colon/pathology , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , TRPC Cation Channels/antagonists & inhibitors , TRPC Cation Channels/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism
8.
FEBS Lett ; 588(11): 2009-15, 2014 May 29.
Article in English | MEDLINE | ID: mdl-24786471

ABSTRACT

To investigate the role of microRNAs in the development of chemoresistance and related epithelial-mesenchymal transition (EMT), we examined the effect of miR-489 in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). MiR-489 was significantly suppressed in MCF-7/ADM cells compared with chemosensitive parental control MCF-7/WT cells. Forced-expression of miR-489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR-489 and is highly expressed in MCF-7/ADM cells. Forced expression of miR-489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR-489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.


Subject(s)
Breast Neoplasms/metabolism , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , MicroRNAs/physiology , 3' Untranslated Regions , Animals , Anthracyclines/pharmacology , Anthracyclines/therapeutic use , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Chemotherapy, Adjuvant , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Female , Gene Expression Regulation, Neoplastic , Humans , Inhibitory Concentration 50 , MCF-7 Cells , Mice , Mice, Nude , Neoadjuvant Therapy , RNA Interference , Smad3 Protein/genetics , Smad3 Protein/metabolism , Taxoids/pharmacology , Taxoids/therapeutic use , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
9.
Proc Natl Acad Sci U S A ; 111(17): 6389-94, 2014 Apr 29.
Article in English | MEDLINE | ID: mdl-24733904

ABSTRACT

A critical challenge for chemotherapy is the development of chemoresistance in breast cancer. However, the underlying mechanisms and validated predictors remain unclear. Extracellular vesicles (EVs) have gained attention as potential means for cancer cells to share intracellular contents. In adriamycin-resistant human breast cancer cells (MCF-7/ADM), we analyzed the role of transient receptor potential channel 5 (TrpC5) in EV formation and transfer as well as the diagnostic implications. Up-regulated TrpC5, accumulated in EVs, is responsible for EV formation and trapping of adriamycin (ADM) in EVs. EV-mediated intercellular transfer of TrpC5 allowed recipient cells to acquire TrpC5, consequently stimulating multidrug efflux transporter P-glycoprotein production through a Ca(2+)- and activated T-cells isoform c3-mediated mechanism and thus, conferring chemoresistance on nonresistant cells. TrpC5-containing circulating EVs were detected in nude mice bearing MCF-7/ADM tumor xenografts, and the level was lower after TrpC5-siRNA treatment. In breast cancer patients who underwent chemotherapy, TrpC5 expression in the tumor was significantly higher in patients with progressive or stable disease than in patients with a partial or complete response. TrpC5-containing circulating EVs were found in peripheral blood from patients who underwent chemotherapy but not patients without chemotherapy. Taken together, we found that TrpC5-containing circulating EVs may transfer chemoresistance property to nonchemoresistant recipient cells. It may be worthwhile to further explore the potential of using TrpC5-containing EVs as a diagnostic biomarker for chemoresistant breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cytoplasmic Vesicles/metabolism , Drug Resistance, Neoplasm , TRPC Cation Channels/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/ultrastructure , Cell Line, Tumor , Cytoplasmic Vesicles/drug effects , Cytoplasmic Vesicles/ultrastructure , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm/drug effects , Female , Humans , Mice , Mice, Nude , TRPC Cation Channels/blood , Treatment Outcome , Xenograft Model Antitumor Assays
10.
Biochem Biophys Res Commun ; 446(1): 85-90, 2014 Mar 28.
Article in English | MEDLINE | ID: mdl-24582564

ABSTRACT

Treatment of carcinoma commonly fails due to chemoresistance. Studies have shown that endothelial cells acquire resistance via the tumor microenvironment. Microvesicle (MV) shedding from the cell membrane to the microenvironment plays an important role in communication between cells. The aim of the present study was to determine whether MCF-7 adriamycin-resistant cells (MCF-7/ADM) shed MVs that alter the characteristics of human microvessel endothelial cells (HMECs). MVs from tumor cells transferred a Ca(2+)-permeable channel TrpC5 to HMECs, inducing the expression of P-glycoprotein (P-gp) by activation of the transcription factor NFATc3 (nuclear factor of activated T cells isoform c3). Expression of the mdr1 gene was blocked by the TrpC5-blocking antibody T5E3, and the production of P-gp in HMECs was reduced by blockade of TrpC5. Thus, we postulate that endothelial cells acquire the resistant protein upon exposure to TrpC5-containg MVs in the microenvironment, and express P-gp in the TrpC5-NFATc3 signal pathway.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , NFATC Transcription Factors/metabolism , TRPC Cation Channels/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/blood supply , Cell Communication , Cell-Derived Microparticles/metabolism , Cells, Cultured , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Endothelial Cells/metabolism , Female , Gene Expression , Heterografts , Humans , MCF-7 Cells , Mice , Mice, Nude , Signal Transduction , Tumor Microenvironment
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