Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells

Background Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells. Results Cell pellets were collected and membrane protein was isolated to determine GIRK protein expression. GIRK protein was also analyzed by immuno-precipitation. GIRK protein was over-expressed in cells by transfection of GIRK plasmids. Gene expression studies were done by real-time RT-PCR. GIRK protein expression was identified in breast cancer cell lines. Expression of GIRK1 at the indicated molecular weight (MW) (62 kDa) was seen in cell lines MDA-MB-453 and ZR-75-1. In addition, GIRK1 expression was seen at a lower MW (40–42 kDa) in MDA-MB-361, MDA-MB-468, MCF-7, ZR-75-1, and MDA-MB-453 cell lines. To prove the lower MW protein was GIRK1, MDA-MB-453 cells were immuno-precipitated. GIRK2 expression was seen in MDA-MB-468, MCF-7, and ZR-75-1 and was variable in MDA-MB-453, while GIRK4 protein expression was seen in all six cell lines tested. This is the first report indicating GIRK protein expression in breast cancer cells. To determine functionality, MDA-MB-453 cells were stimulated with ethanol. Decreased GIRK1 protein expression levels were seen after treatment with 0.12% ethanol in MDA-MB-453 breast cancer cells. Serum-free media decreased GIRK protein expression, possibly due to lack of estrogen in the media. Transfection of GIRK1 or GIRK4 plasmids increased GIRK1 protein expression and decreased gene expression in MDA-MB-453 breast cancer cells. Conclusion Our data indicates that functional GIRK channels exist in breast cancer cells that are involved in cellular signaling

G-Protein Inwardly Rectifying Potassium Channel 1 (GIRK1) Knockdown Decreases Beta-Adrenergic, MAP Kinase and Akt Signaling in the MDA-MB-453 Breast Cancer Cell Line

Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA) studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, β2-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, β2 mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling

Expression of G-protein inwardly rectifying potassium channels (GIRKs) in lung cancer cell lines

BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with β(2)-adrenergic antagonist ICI 118,551 (100 μM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and β-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results

Functionalized gold nanorod nanocomposite system to modulate differentiation of human mesenchymal stem cells into neural-like progenitors

A 2D multifunctional nanocomposite system of gold nanorods (AuNRs) was developed. Gold nanorods were functionalized via polyethylene glycol with a terminal amine, and, were characterized using transmission and scanning electron microscopy, ultra violet-visible and X-ray photoelectron spectroscopy, and Zeta-potential. The system was cytocompatible to and maintained the integrity of Schwann cells. The neurogenic potential of adipose tissue – derived human mesenchymal stem cells (hMSCs) was evaluated in vitro. The expression pattern and localization of Vimentin confirmed the mesenchymal origin of cells and tracked morphological changes during differentiation. The expression patterns of S100β and glial fibrillary acidic protein (GFAP), were used as indicator for neural differentiation. Results suggested that this process was enhanced when the cells were seeded on the AuNRs compared to the tissue-culture surface. The present study indicates that the design and the surface properties of the AuNRs enhances neural differentiation of hMSCs and hence, would be beneficial for neural tissue engineering scaffolds

Lifshitz spacetimes from AdS null and cosmological solutions

We describe solutions of 10-dimensional supergravity comprising null deformations of $AdS_5\times S^5$ with a scalar field, which have $z=2$ Lifshitz symmetries. The bulk Lifshitz geometry in 3+1-dimensions arises by dimensional reduction of these solutions. The dual field theory in this case is a deformation of the N=4 super Yang-Mills theory. We discuss the holographic 2-point function of operators dual to bulk scalars. We further describe time-dependent (cosmological) solutions which have anisotropic Lifshitz scaling symmetries. We also discuss deformations of $AdS\times X$ in 11-dimensional supergravity, which are somewhat similar to the solutions above. In some cases here, we expect the field theory duals to be deformations of the Chern-Simons theories on M2-branes stacked at singularities.Comment: Latex, 29pgs, v3. references, minor clarifications (subsection on Lifshitz geometry seen by scalar probes) added, to appear in JHE

Etched 3D-Printed Polycaprolactone Constructs Functionalized with Reduced Graphene Oxide for Enhanced Attachment of Dental Pulp-Derived Stem Cells

A core challenge in the field of tissue engineering is the ability to establish pipeline workflows for the design and characterization of scaffold technologies with clinically translatable attributes. The parallel development of biomaterials and stem cell populations represents a self-sufficient and streamlined approach for establishing such a pipeline. In the current study, rat dental pulp stem cell (rDPSC) populations were established to assess functionalized polycaprolactone (PCL) constructs. Initial optimization and characterization of rDPSC extraction and culture conditions confirmed that cell populations were readily expandable and demonstrated surface markers associated with multi-potency. Subset populations were transduced to express DsRed fluorescent protein as a mechanism of tracking both cells and cell-derived extracellular matrix content on complex scaffold architecture. Thermoplastic constructs included reduced graphene oxide (rGO) as an additive to promote cellular attachment and were further modified by surface etching a weak acetic acid solution to roughen surface topographical features, which was observed to dramatically improve cell surface coverage in vitro. Based on these data, the modified rGO-functionalized PCL constructs represent a versatile platform for bone tissue engineering, capable of being applied as a standalone matrix or in conjunction with bio-active payloads such as DPSCs or other bio-inks

A Novel Model for Acute Peripheral Nerve Injury in the Horse and Evaluation of the Effect of Mesenchymal Stromal Cells Applied in situ on Nerve Regeneration: a Preliminary Study

Transplantation of mesenchymal stromal cells (MSCs) to sites of experimentally-created nerve injury in laboratory animals has shown promising results in restoring nerve function. This approach for nerve regeneration has not been reported in horses. In this study, we first evaluated the in vitro ability of equine, bone marrow-derived MSCs (EBM-MSCs) to trans-differentiate into Schwann-like cells (SLCs) and subsequently tested the MSCs, in vivo for their potential to regenerate a transected nerve after implantation. The EBM-MSCs from 3 equine donors were differentiated into SCLs for 7 days, in vitro, in the presence of specialized differentiation medium and evaluated for morphological characteristics, by using confocal microscopy, and for protein characteristics, by using selected Schwann-cell markers (GFAP and S100b). The EBM-MSCs were then implanted into the fascia surrounding the ramus communicans of one fore limb of 3 healthy horses after a portion of this nerve was excised. The excised portion of the nerve was examined histologically at the time of transection, and stumps of the nerve were examined histologically at day 45 after transplantation. The EBM-MSCs from all donors demonstrated morphological and protein characteristics of those of Schwann cells 7 days after differentiation. Nerves implanted with EBM-MSCs after nerve transection did not show evidence of nerve regeneration at day 45. Examination of peripheral nerves collected 45 days after injury and stem cell treatment revealed no histological differences between nerves treated with MSCs and those treated with isotonic saline solution (controls). The optimal delivery of MSCs and the model suitable to study the efficacy of MSCs in nerve regeneration should be investigated

3D-Printing Graphene Scaffolds for Bone Tissue Engineering

Graphene-based materials have recently gained attention for regenerating various tissue defects including bone, nerve, cartilage, and muscle. Even though the potential of graphene-based biomaterials has been realized in tissue engineering, there are significantly many more studies reporting in vitro and in vivo data in bone tissue engineering. Graphene constructs have mainly been studied as two-dimensional (2D) substrates when biological organs are within a three-dimensional (3D) environment. Therefore, developing 3D graphene scaffolds is the next clinical standard, yet most have been fabricated as foams which limit control of consistent morphology and porosity. To overcome this issue, 3D-printing technology is revolutionizing tissue engineering, due to its speed, accuracy, reproducibility, and overall ability to personalize treatment whereby scaffolds are printed to the exact dimensions of a tissue defect. Even though various 3D-printing techniques are available, practical applications of 3D-printed graphene scaffolds are still limited. This can be attributed to variations associated with fabrication of graphene derivatives, leading to variations in cell response. This review summarizes selected works describing the different fabrication techniques for 3D scaffolds, the novelty of graphene materials, and the use of 3D-printed scaffolds of graphene-based nanoparticles for bone tissue engineering