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Isoliquiritigenin (ISL), a natural flavonoid derived from the root of liquorice, has been reported to possess anti-inflammatory and antioxidant activities. Previous studies have found that ISL plays a crucial role in anti-fibrosis of adipose tissue and renal tissue; however, its effect on pulmonary fibrogenesis has not been demonstrated. In this study, we aimed to explore the roles and the underlying mechanisms of ISL in TGF-β1-induced fibrogenesis using human lung fibroblast-derived MRC-5 cells. Cell proliferation and migration were determined by MTT and wound healing assay, respectively. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIA1) and fibronectin (FN), microtubule-associated protein light chain 3 (LC3) and related signaling molecules were detected by quantitative real-time PCR, western blot and immunofluorescence assay, correspondingly. EGFP-LC3 transfection was used for autophagy analysis. The results showed that ISL inhibited the TGF-β1-induced proliferation and migration, and down-regulated the expressions of α-SMA, COLIA1 and FN. ISL treatment led to up-regulation of LC3 in TGF-β1-treated MRC-5 cells, accompanied by significant decrease in the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In addition, the inhibitory effects of ISL on TGF-β1-induced fibrogenic features in MRC-5 cells were enhanced by pretreatment with autophagy activator Rapmycin and PI3K/AKT inhibitor LY294002 and reversed by autophagy inhibitor 3-methyladenine and PI3K/AKT activator IGF-1. Taken together, our results demonstrated that ISL could attenuate the fibrogenesis of TGF-β1-treated MRC-5 cells by activating autophagy via suppressing the PI3K/AKT/mTOR pathway. Therefore, ISL holds a great potential to be developed as a novel therapeutic agent for the treatment of pulmonary fibrosis.

Background FAM92A1–289(abbreviated FAM289) is recognized as one of the newly-discovered putative oncogenes. However, its role and molecular mechanisms in promoting cancer progression has not yet been elucidated. This study was performed to reveal its oncogenic functions and molecular mechanisms in human glioblastoma multiforme (GBM) cell models with knockdown or overexpression of FAM289 in vitro and in vivo. Methods To elucidate the molecular mechanisms underlying FAM289-mediated tumor progression, the protein-protein interaction between FAM289 and Galectin-1 was verified by co-immunoprecipitation, followed by an analysis of the expression and activity of Galectin-1-associated signaling molecules. Knockdown and overexpression of FAM289 in glioma cells were applied for investigating the effects of FAM289 on cell growth, migration and invasion. The determination of FAM289 expression was performed in specimens from various stages of human gliomas. Results FAM289-galectin-1 interaction and concomitant activation of the extracellular signal-regulated kinase (ERK) pathway participated in FAM289-mediated tumor-promoting function. Since the expression of DNA methyl transferase 1 (DNMT1) and DNA methyl transferase 3B (DNMT3B) was regulated by FAM289 in U251 and U87-MG glioma cells, Galectin-1 interaction with FAM289 may promote FAM289 protein into the cell nucleus and activate the ERK pathway, thereby upregulating DNMTs expression. Drug resistance tests indicated that FAM289-mediated TMZ resistance was through stem-like property acquisition by activating the ERK pathway. The correlation between FAM289, Galectin-1 expression and the clinical stage of gliomas was also verified in tissue samples from glioblastoma patients. Conclusions Our results suggest that high expression of FAM289 in GBM tissues correlated with poor prognosis. FAM289 contributes to tumor progression in malignant glioma by interacting with Galectin-1 thereby promoting FAM289 protein translocation into the cell nucleus. FAM289 in the nucleus activated the ERK pathway, up regulated DNMTs expression and induced stem-like property gene expression which affects drug resistance of glioma cells to TMZ. This study provided functional evidence for FAM289 to be developed as a therapeutic target for cancer treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1393-7) contains supplementary material, which is available to authorized users.

Background: Cytoplasmic activation/proliferation-associated protein-1 (caprin-1) is a newly discovered RNA-binding protein and is now recognized as one of the putative oncogenes. This study was performed to reveal its presence in human gliomas and its oncogenic functions in human glioblastoma-derived Denver brain tumor research group 05 (DBTRG-05MG) cells. Materials and Methods: Clinical glioma samples were cumulatively collected for the identification of caprin-1 using immunoblot analysis and immunofluorescence detection. DBTRG cells transfected with caprin-1-specific small interfering RNA (siRNA) were used to verify caprin-1's oncogenic function using a real-time cell analyzer (RTCA) and scratch assay. Results: Seven of eight collected glioma samples were identified as positive for caprin-1 expression. siRNA dose-responsive inhibition of cell proliferation was observed in DBTRG cells with RTCA, and cell migration rate was significantly reduced by siRNA transfection (P < 0.05). Conclusion: The present study identified the higher expression of caprin-1 in human glioblastoma-derived DBTRG cells. Its oncogenic functions, mainly enhanced cell proliferation and promoted cell migration capacity, were also verified in these cells. This study provided fundamentals for developing caprin-1 as a therapeutic target for the treatment of gliomas.

The treatment of glioblastoma has been a big challenge for decades in the oncological field mainly owing to its unique biological characteristics, such as high heterogeneity, diffusing invasiveness, and capacity to resist conventional therapies. The mRNA-based therapeutic modality holds many superior features, including easy manipulation, rapid and transient expression, and adaptive convertibility without mutagenesis, which are suitable for dealing with glioblastoma's complexity and variability. Synthetic anticancer mRNAs carried by various vehicles act as the ultimate attackers of the tumor across biological barriers. In this modality, specifically targeted glioblastoma treatment can be guaranteed by adding targeting molecules at certain levels. The choice of mRNA-bearing vehicle and administration method is a fully patient-tailored selection. This review covers the advantages and possible limitations of mRNA-based gene therapy, the in vitro synthesis of mRNA, the feasible methods for synthetic mRNA delivery and clinical therapeutic prospects of mRNA-based gene therapy for glioblastoma.

RNA-binding proteins serve an essential role in post-transcriptional gene regulation. Cytoplasmic activation/proliferation-associated protein-1 (caprin-1) is an RNA-binding protein that participates in the regulation of cell cycle control-associated genes. Caprin-1 acts alone or in combination with other RNA-binding proteins, such as RasGAP SH3-domain-binding protein 1 and fragile X mental retardation protein. In the tumorigenesis process, caprin-1 primarily functions by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, which contributes to radiation and chemotherapy resistance. Given its role in various clinical malignancies, caprin-1 holds the potential to be used as a biomarker and a target for the development of novel therapeutics. The present review describes this newly identified putative oncogenic protein and its possible impact on tumorigenesis.

BACKGROUND/AIM FAM92A1-289 is recognized as one of the newly-discovered putative oncogenes. This study was performed to reveal its oncogenic functions in human cervical carcinoma cells. MATERIALS AND METHODS The FAM92A1-289+ cell line was established with knock-in technique and selected by puromycin-resistance screening. Scratch assay, methylthiazol tetrazolium assay, colony forming assay and xenograft test were used to examine cell migration, cell proliferation, cell viability and tumor formation, respectively. RESULTS FAM92A1-289+ cells showed higher migration rate (p

Xiang-Jun Tang,1,* Kuan-Ming Huang,1,* Hui Gui,1,* Jun-Jie Wang,2 Jun-Ti Lu,1 Long-Jun Dai,1,3 Li Zhang,1 Gang Wang2 1Department of Neurosurgery, TaiHe Hospital, Hubei University of Medicine, Shiyan, 2Department of Pharmaceutics, Shanghai Eighth People’s Hospital, Jiangsu University, Shanghai, People’s Republic of China; 3Department of Surgery, University of British Columbia, Vancouver, BC, Canada *These authors contributed equally to this work Abstract: As one of the natural herbal flavonoids, myricetin has attracted much research interest, mainly owing to its remarkable anticancer properties and negligible side effects. It holds great potential to be developed as an ideal anticancer drug through improving its bioavailability. This study was performed to investigate the effects of Pluronic-based micelle encapsulation on myricetin-induced cytotoxicity and the mechanisms underlying its anticancer properties in human glioblastoma cells. Cell viability was assessed using a methylthiazol tetrazolium assay and a real-time cell analyzer. Immunoblotting and quantitative reverse transcriptase polymerase chain reaction techniques were used for determining the expression levels of related molecules in protein and mRNA. The results indicated that myricetin-induced cytotoxicity was highly potentiated by the encapsulation of myricetin. Mitochondrial apoptotic pathway was demonstrated to be involved in myricetin-induced glioblastoma cell death. The epidermal growth factor receptor (EGFR)/PI3K/Akt pathway located in the plasma membrane and cytosol and the RAS-ERK pathway located in mitochondria served as upstream and downstream targets, respectively, in myricetin-induced apoptosis. MiR-21 inhibitors interrupted the expression of EGFR, p-Akt, and K-Ras in the same fashion as myricetin-loaded mixed micelles (MYR-MCs) and miR-21 expression were dose-dependently inhibited by MYR-MCs, indicating the interaction of miR-21 with MYR-MCs. This study provided evidence supportive of further development of MYR-MC formulation for preferentially targeting mitochondria of glioblastoma cells. Keywords: myricetin, glioblastoma, EGFR, miR-21, mitochondrial apoptosis, mixed micelles, anticancer, drug delivery

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p

// Xiang-Jun Tang 1,* , Xu-Yong Sun 2,* , Kuan-Ming Huang 1,* , Li Zhang 1 , Zhuo-Shun Yang 1 , Dan-Dan Zou 1 , Bin Wang 1,3 , Garth L. Warnock 4 , Long-Jun Dai 1,4 and Jie Luo 1 1 Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, China 2 Guangxi Key Laboratory for Transplantation Medicine, Institute of Transplant Medicine, 303 Hospital of People’s Liberation Army, Nanning, China 3 The Biomedical Research Center, University of British Columbia, Vancouver, Canada 4 Department of Surgery, University of British Columbia, Vancouver, Canada * These authors have contributed equally to this work Correspondence to: Long-Jun Dai, email: // Jie Luo, email: // Keywords : immunotherapy, chimeric antigen receptor (CAR), exosomes, cancer therapy, extracellular vesicles Received : August 12, 2015 Accepted : October 06, 2015 Published : October 19, 2015 Abstract Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

Animal models are indispensable for the study of tumorigenesis and the development of anti-cancer drugs for human pancreatic cancer. In the present study, two orthotopic xenograft mouse models were developed. AsPC-1 human pancreatic cancer cells were stably labeled with red fluorescent protein (RFP) and injected subcutaneously into nude mice. For the orthotopic tumor mass model, the formed subcutaneous tumors were cut into blocks and implanted into the pancreas of nude mice via laparotomy. For the Matrigel™ tumor block model, solidified Matrigel containing RFP-labeled AsPC-1 cells was cut into blocks and implanted into the pancreas of nude mice. A subcutaneous tumor xenograft model was used as a control. Tumor growth and metastasis were assessed using an in vivo fluorescence imaging system. Thirty-six days after implantation, all mice from the two orthotopic xenograft models (n=20 per group) and 55% of the subcutaneous xenograft mice (n=20) developed tumors. The tumor growth rate was significantly higher in the orthotopic models than that in the subcutaneous model (P

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, in which the SMAD signaling pathway plays an important role. The aim of the present study was to identify differentially expressed microRNAs (miRNAs) during the progression of DN and to investigate a selected miRNA in relation to SMAD3/4 and its therapeutic efficacy. The miRNA microarray was used to identify differentially expressed miRNAs in db/db DN mice. Reverse transcription-quantitative polymerase chain reaction and immunoblot analyses were used to detect SMAD3/4 expression. The development of DN in the db/db mice was demonstrated by glucose dysregulation and typical morphological changes in the kidney. miRNA-346 (miR-346) was identified as one of the differentially expressed miRNAs. The expression of SMAD3/4 was significantly attenuated by miR-346 administration and the therapeutic effects of miR-346 were observed in the DN mouse models. miR-346 was identified as a negative regulator of SMAD3/4. SMAD3/4 was upregulated in the renal tissue of db/db mice. The administration of miR-346 attenuated the SMAD3/4 expression in renal tissue and ameliorated the renal function and glomerular histology in DN mice. This study paves the way for clinical studies of miR-346 in DN.

Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.

Biobanks have played a decisive role in all aspects of the field of cancer, including pathogenesis, diagnosis, prognosis and treatment. The significance of cancer biobanks is epitomized through the appropriate application of various “-omic” techniques (omics). The mutually motivated relationship between biobanks and omics has intensified the development of cancer research. Human cancer tissues that are maintained in intravital biobanks (or living tissue banks) retain native tumor microenvironment, tissue architecture, hormone responsiveness and cell-to-cell signalling properties. Intravital biobanks replicate the structural complexity and heterogeneity of human cancers, making them an ideal platform for preclinical studies. The application of omics with intravital biobanks renders them more active, which makes it possible for the cancer-related evaluations to be dynamically monitored on a real-time basis. Integrating intravital biobank and modern omics will provide a useful tool for the discovery and development of new drugs or novel therapeutic strategies. More importantly, intravital biobanks may play an essential role in the creation of meaningful patient-tailored therapies as for personalized medicine.

This study was performed to demonstrate the transportation of an engineered MSC-produced intracellular anticancer gene product between mesenchymal stem cell (MSC) and cancer cells. MSC-mediated anticancer strategy has held great promise owing to MSCs’ capacity of tumor-directed migration and the availability of specific anticancer genes. All anticancer genes that have been used in previous MSC-mediated anticancer studies were limited in functioning via extracellular mechanisms, mainly because of the restriction by cell membrane to macromolecules including proteins. In order to apply the majority of potent anticancer genes to the MSC-mediated anticancer system, a specifically designed expression vector which bears an intracellular anticancer gene, PTEN, is utilized to demonstrate the feasibility of the system in cancer therapies. A transacting activator of transcription (TAT) was introduced into an expression vector followed by a segment for PTEN-RFP fusion protein. A direct demonstration of PTEN-RFP transportation between MSC and cancer cells was obtained from direct co-cultures. A marked cancer cell death was observed in indirect co-cultures with conditioned media from PTEN-transfected MSCs. The demonstration of PTEN-engineered MSC-produced PTEN transportation indicates the feasibility of applying intracellular anticancer gene expression system in MSC-mediated strategies for cancer therapy.

AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers.

There is increasing evidence of therapeutic benefits from bone marrow (BM)-derived mesenchymal stromal cells (MSC) in various animal models with neurologic disorders. It is of great interest to apply the approach to clinical patients, i.e. to take the investigations from laboratory bench to the patient's bedside. This clinical trial was performed to assess the safety and feasibility of a combined procedure to deliver autologous MSC to patients with traumatic brain injury.MSC were isolated by BM aspiration and expanded in culture. Seven patients received autologous cell transplantation. A primary administration of 10(7)-10(9) cells was applied directly to the injured area during the cranial operation; a second dose of 10(8)-10(10) cells was infused intravenously. All patients were followed up regularly for 6 months.There was no immediate or delayed toxicity related to the cell administration within the 6-month follow-up period. Neurologic function was significantly improved at 6 months after cell therapy.The procedure used is safe and feasible at ordinary medical facilities without additional invasive procedures for the patient. The combined cell delivery procedure is expected to enhance the engraftment efficacy of transplanted cells at injured brain tissue, thereby promoting neurologic recover.

Effective use of all available donated organs is critical, in order to meet the increasing demand for transplants. The present study explored liver transplantation with livers that were donated following cardiac death (DCD). According to the guidelines established by The Red Cross Society of China, 42 DCD organs were procured. Selected donors were treated with extracorporeal membrane oxygenation (ECMO) prior to the organ retrieval. The present single-center study included 6 liver transplantations of DCD organs (5 liver transplants and 1 liver-kidney combined transplant). All 6 recipients had a successful recovery without significant complications. The serum alanine transaminase, total bilirubin and international normalized ratio returned to the normal levels within a short period of time following transplantation, and the liver function remained normal during the follow-up period, which lasted up to 24 months. The present report demonstrated the feasibility of orthotopic liver transplantation using DCD livers. The pre-conditioning DCD donors and optimization of the recipient's condition using ECMO, played a crucial role in ensuring the success of transplantation.

MicroRNAs (miRs) serve a regulatory function in oxidative radical-mediated inflammation and apoptosis during ischemia/reperfusion (IR) injury. Lipocalin 2 (Lcn-2), a target protein of miR-138, is widely involved in the systemic response to IR injury. The aim of the present study was to investigate the association between miR-138 and Lcn-2 in a rat model of cerebral ischemia/reperfusion (CIR) injury and to verify the interaction between miR-138 and Lcn-2 in a PC12 cell model of hypoxia/reoxygenation injury. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect the mRNA and protein expression levels of miR-138 and Lcn-2. Cell proliferation was determined by MTT assay. The results suggested that the expression of miR-138 was inversely correlated with the expression of Lcn-2 in the CIR rat model and the PC12 cells subjected to hypoxia and reoxygenation. The expression of Lcn-2 was inhibited by miR-138 mimics and enhanced by miR-138 inhibitors, thereby indicating that miR-138 functions as a negative regulator for Lcn-2 expression. This study provides an experimental basis for the further study of miR-138-based therapy for CIR injury.

Mesenchymal stem cells (MSCs) have been considered to have potential as ideal carriers for the delivery of anticancer agents since the capacity for tumor-oriented migration and integration was identified. In contrast to DNA-based vectors, mRNA synthesized in vitro may be readily transfected and is mutagenesis-free. The present study was performed in order to investigate the effects of phosphatase and tensin homolog (PTEN) mRNA-engineered MSCs on human glioma U251 cells under indirect co-culture conditions. PTEN-bearing mRNA was generated by in vitro transcription and was transfected into MSCs. The expression of PTEN in transfected MSCs was detected by immunoblotting, and the migration ability of MSCs following PTEN-bearing mRNA transfection was verified using Transwell co-cultures. The indirect co-culture was used to determine the effects of PTEN-engineered MSCs on the viability of U251 glioma cells by luminescence and fluorescence microscopy. The synthesized PTEN mRNA was expressed in MSCs, and the expression was highest at 24 h subsequent to transfection. An enhanced migration rate was observed in MSCs transfected with PTEN mRNA compared with non-transfected MSCs (P

Zhuo-Shun Yang,1,* Xiang-Jun Tang,2,* Xing-Rong Guo,1 Dan-Dan Zou,1 Xu-Yong Sun,3 Jing-Bo Feng,1 Jie Luo,1 Long-Jun Dai,1,4 Garth L Warnock4 1Hubei Key Laboratory of Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China; 2Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic of China; 3Guangxi Key Laboratory for Transplant Medicine, 303 Hospital of PLA, Nanning, People’s Republic of China; 4Department of Surgery, University of British Columbia, Vancouver, BC, Canada *These authors contributed equally to this work Background: Mesenchymal stem cells (MSCs) have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) engineering on MSCs’ capacity for cancer cell-oriented migration. Methods: MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system. Results: The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed. Conclusion: MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs’ tropism post-anticancer gene engineering. Keywords: gene therapy, mesenchymal stem cells, phosphatase and tensin homolog, cancer

TNF-related apoptosis-inducing ligand (TRAIL) is considered as a tumor cell-specific cytotoxic agent. Through the aid of mesenchymal stem cells (MSCs), TRAIL is capable of inducing apoptosis of tumor cells in tumor sites. The present study was performed to investigate the cytotoxic effects of TRAIL-engineered MSCs on glioblastoma cells (C6) in vitro.An expression vector of secreting form of TRAIL was used to engineer MSCs. The cytotoxic effects of TRAIL-transfected MSCs on C6 cells were invstigated using the MTT method and Hochest33258 staining after co-culture of the two cell types.TRAIL and control plasmid transfection of MSCs showed no significant effect on MSC's viability (p0.05). A significant inhibition of C6 cells was observed when they were co-cultured with TRAIL-engineered MSCs (63.7%±0.12, p0.05).Mesenchymal stem cells were very well tolerant to the transfection of TRAIL-bearing vectors. The cytotoxic effects of TRAIL-engineered MSCs on C6 cells indicates the therapeutic potential of this strategy for treatment of glioblastoma patients.

tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg2+ balance, we determined the effects of adenosine on cellular Mg2+ uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg2+ uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted to 0.22 ± 0.01 mM by being cultured in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2; next, changes in intracellular Mg2+ concentration ([Mg2+]i) were determined. [Mg2+]i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 137 ± 16 nM/s. Adenosine stimulates basal Mg2+ uptake by 41 ± 10%. The selective A1 purinoceptor agonist N 6-cyclopentyladenosine (CPA) increased intracellular Ca2+ and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg2+uptake. On the other hand, the selective A2 receptor agonist 2-[ p-(2-carbonyl-ethyl)-phenylethylamino]-5′- N-ethylcarboxamidoadenosine (CGS) stimulated Mg2+ entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg2+ uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A2 agonist-mediated Mg2+ entry. Aldosterone potentiated CGS-mediated Mg2+ entry, and elevation of extracellular Ca2+ diminished CGS-responsive cAMP formation and Mg2+ uptake. Accordingly, MDCT cells possess both A1 and A2 purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg2+ uptake in MDCT cells through separate A1 and A2purinoceptor pathways.

First published July 12, 2001; 10.1152/ajprenal.00349.2001.—Nucleotides have diverse effects on water and electrolyte reabsorption within the distal tubule of the nephron. As the distal tubule is important in control of renal Mg2+balance, we determined the effects of ATP on cellular Mg2+uptake in this segment. The effects of ATP on immortalized mouse distal convoluted tubule (MDCT) cells were studied by measuring Mg2+uptake with fluorescence techniques. The mean basal Mg2+uptake rate was 165 ± 6 nM/s. ATP inhibited basal Mg2+uptake and hormone-stimulated Mg2+entry by 40%. Both P2X (P2X1–P2X5 subtypes) and P2Y2 receptor subtypes were identified in MDCT cells using differential RT-PCR. Activation of both receptor subtypes with selective agonists increased intracellular Ca2+concentration, P2X purinoceptors by ionotropic-gated channels, and P2Y receptors via G protein-mediated intracellular Ca2+release. The more relatively selective P2X agonists [β,γ-methylene ATP (β,γ-Me-ATP) and 2′- and -3′- O-(4-benzoyl-benzoyl)-ATP] inhibited arginine vasopressin (AVP)- and parathyroid hormone (PTH)-mediated Mg2+uptake whereas agonists more selective for P2Y purinoceptors (UTP, ADP, and 2-methylthio-ATP) were without effect. Removal of extracellular Ca2+diminished β,γ-Me-ATP-mediated increase in intracellular Ca2+and inhibition of AVP-stimulated Mg2+entry. We conclude from this information that ATP inhibited Mg2+uptake in MDCT cells through P2X purinoceptors expressed in this distal convoluted tubule cell line.

The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1α,25-dihydroxyvitamin D [1,25(OH)2D3] on magnesium transport. In this study, we examined the effect of 1,25(OH)2D3on distal cellular magnesium uptake and the modulation of this response by extracellular Ca2+and Mg2+in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca2+and Mg2+concentrations to diminish peptide hormone-stimulated Mg2+uptake. Mg2+uptake rates were determined by microfluorescence in Mg2+-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)2D3for 16–24 h stimulated basal Mg2+uptake in a concentration-dependent manner from basal levels of 164 ± 5 to 210 ± 11 nM/s, representing a 28 ± 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)2D3-stimulated.Mg2+uptake (154 ± 18 nM/s), suggesting that 1,25(OH)2D3stimulates Mg2+uptake through gene activation and protein synthesis. Elevation of extracellular Ca2+inhibited 1,25(OH)2D3-stimulated Mg2+uptake (143 ± 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca2+of 1,25(OH)2D3-stimulated Mg2+uptake (202 ± 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca2+on 1,25(OH)2D3-responsive Mg2+entry. This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary, 1,25(OH)2D3stimulated Mg2+uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca2+, acting via the CaSR, inhibited 1,25(OH)2D3-stimulated Mg2+entry. These data indicate that 1,25(OH)2D3has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.

The distal tubule reabsorbs ∼10% of the filtered Mg2+, but this is 70–80% of that delivered from the loop of Henle. Because there is little Mg2+reabsorption beyond the distal tubule, this segment plays an important role in determining the final urinary excretion. The distal convoluted segment (DCT) is characterized by a negative luminal voltage and high intercellular resistance so that Mg2+reabsorption is transcellular and active. This review discusses recent evidence for selective and sensitive control of Mg2+transport in the DCT and emphasizes the importance of this control in normal and abnormal renal Mg2+conservation. Normally, Mg2+absorption is load dependent in the distal tubule, whether delivery is altered by increasing luminal Mg2+concentration or increasing the flow rate into the DCT. With the use of microfluorescent studies with an established mouse distal convoluted tubule (MDCT) cell line, it was shown that Mg2+uptake was concentration and voltage dependent. Peptide hormones such as parathyroid hormone, calcitonin, glucagon, and arginine vasopressin enhance Mg2+absorption in the distal tubule and stimulate Mg2+uptake into MDCT cells. Prostaglandin E2and isoproterenol increase Mg2+entry into MDCT cells. The current evidence indicates that cAMP-dependent protein kinase A, phospholipase C, and protein kinase C signaling pathways are involved in these responses. Steroid hormones have significant effects on distal Mg2+transport. Aldosterone does not alter basal Mg2+uptake but potentiates hormone-stimulated Mg2+entry in MDCT cells by increasing hormone-mediated cAMP formation. 1,25-Dihydroxyvitamin D3, on the other hand, stimulates basal Mg2+uptake. Elevation of plasma Mg2+or Ca2+inhibits hormone-stimulated cAMP accumulation and Mg2+uptake in MDCT cells through activation of extracellular Ca2+/Mg2+-sensing mechanisms. Mg2+restriction selectively increases Mg2+uptake with no effect on Ca2+absorption. This intrinsic cellular adaptation provides the sensitive and selective control of distal Mg2+transport. The distally acting diuretics amiloride and chlorothiazide stimulate Mg2+uptake in MDCT cells acting through changes in membrane voltage. A number of familial and acquired disorders have been described that emphasize the diversity of cellular controls affecting renal Mg2+balance. Although it is clear that many influences affect Mg2+transport within the DCT, the transport processes have not been identified.

β-Adrenergic agonists influence electrolyte reabsorption in the proximal tubule, loop of Henle, and distal tubule. Although isoproterenol enhances magnesium absorption in the thick ascending limb, it is unclear what effect, if any, β-adrenergic agonists have on tubular magnesium handling. The effects of isoproterenol were studied in immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+ uptake with fluorescence techniques. Intracellular free Mg2+ concentration ([Mg2+]i) was measured in single MDCT cells by using microfluorescence with mag-fura-2. To assess Mg2+uptake, MDCT cells were first Mg2+ depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg2+]i were determined. [Mg2+]i returned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 168 ± 11 nM/s. Isoproterenol stimulated Mg2+ entry in a concentration-dependent manner, with a maximal response of 252 ± 11 nM/s, at a concentration of 10−7 M, that represented a 50 ± 7% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. Isoproterenol-stimulated Mg2+ uptake was completely inhibited with RpcAMPS, a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially blocked by RO 31–822, a protein kinase C inhibitor. Accordingly, isoproterenol-mediated Mg2+ entry rates involve multiple intracellular signaling pathways. Aldosterone potentiated isoproterenol-stimulated Mg2+ uptake (326 ± 31 nM/s), whereas elevation of extracellular Ca2+ inhibited isoproterenol-mediated cAMP accumulation and Mg2+ uptake, 117 ± 37 nM/s. These studies demonstrate that isoproterenol stimulates Mg2+ uptake in a cell line of mouse distal convoluted tubules that is modulated by hormonal and extracellular influences.

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg2+uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca2+and Mg2+. The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg2+uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, illicited transient increases in intracellular Ca2+from basal levels of 102 ± 13 nM to 713 ± 125 nM, suggesting a receptor-mediated response. In order to determine Mg2+transport, MDCT cells were Mg2+-depleted by culturing in Mg2+-free media for 16 h and Mg2+uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg2+uptake, d([Mg2+]i)/dt, was 138 ± 24 nM/s in control MDCT cells. Gentamicin (50 µM) did not affect basal Mg2+uptake (105 ± 29 nM/s), but inhibited PTH stimulated Mg2+entry, decreasing it from 257 ± 36 nM/s to 108 ± 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 ± 2.5 to 23 ± 1 pmol/mg protein·5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg2+entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg2+absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of these antibiotics.Key words: intracellular Mg2+, Mg2+uptake, aminoglycosides, gentamicin, tobramycin, streptomycin, neomycin, parathyroid hormone, microfluorescence, cAMP measurements.

The clinical use of aminoglycosides often leads to renal magnesium wasting and hypomagnesemia. Of the nephron segments, both the thick ascending limb of Henle's loop and the distal tubule play significant roles in renal magnesium conservation but the distal convoluted tubule exerts the final control of urinary excretion. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. Peptide hormones, such as parathyroid hormone (PTH), glucagon, calcitonin, and arginine vasopressin (AVP) stimulate Mg 2+ uptake in MDCT cells that is modulated by extracellular polyvalent cations, Ca 2+ and Mg 2+ . The present studies determined the effect of aminoglycosides on parathyroid hormone (PTH)-mediated cAMP formation and Mg 2+ uptake in MDCT cells. Gentamicin, a prototypic aminoglycoside, illicited transient increases in intracellular Ca 2+ from basal levels of 102 ± 13 nM to 713 ± 125 nM, suggesting a receptor-mediated response. In order to determine Mg 2+ transport, MDCT cells were Mg 2+ -depleted by culturing in Mg 2+ -free media for 16 h and Mg 2+ uptake was measured by microfluorescence after placing the depleted cells in 1.0 mM MgCl2. The mean rate of Mg 2+ uptake, d((Mg 2+ )i)/dt, was 138 ± 24 nM/s in control MDCT cells. Gentamicin (50 μM) did not affect basal Mg 2+ uptake (105 ± 29 nM/s), but inhibited PTH stimulated Mg 2+ entry, decreasing it from 257 ± 36 nM/s to 108 ± 42 nM/s. This was associated with diminished PTH-stimulated cAMP formation, from 80 ±2 .5 to 23 ±1p mol/mgprotein·5 min. Other aminoglycosides such as tobramycin, streptomycin, and neomycin also inhibited PTH-stimulated Mg 2+ entry and cAMP formation. As these antibiotics are positively charged, the data suggest that aminoglycosides act through an extracellular polyvalent cation-sensing receptor present in distal convoluted tubule cells. We infer from these studies that aminoglycosides inhibit hormone-stimulated Mg 2+ absorption in the distal convoluted tubule that may contribute to the renal magnesium wasting frequently observed with the clinical use of

Insulin has been shown to be a magnesium-conserving hormone acting, in part, through stimulation of magnesium absorption within the thick ascending limb. Although the distal convoluted tubule possesses the most insulin receptors, it is unclear what, if any, actions insulin has in the distal tubule. The effects of insulin were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+uptake with fluorescence techniques using mag-fura 2. To assess Mg2+uptake, MDCT cells were first Mg2+depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in intracellular Mg2+concentration ([Mg2+]i) were measured with microfluorescence. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 164 ± 5 nM/s. Insulin stimulated Mg2+entry in a concentration-dependent manner with maximal response of 214 ± 12 nM/s, which represented a 30 ± 5% increase in the mean uptake rate above control values. This was associated with a 2.5-fold increase in insulin-mediated cAMP generation (52 ± 3 pmol ⋅ mg protein−1⋅ 5 min−1). Genistein, a tyrosine kinase inhibitor, diminished insulin-stimulated Mg2+uptake (169 ± 11 nM/s), but did not change insulin-mediated cAMP formation (47 ± 5 pmol ⋅ mg protein−1⋅ 5 min−1). PTH stimulates Mg2+entry, in part, through increases in cAMP formation. Insulin and PTH increase Mg2+uptake in an additive fashion. In conclusion, insulin mediates Mg2+entry, in part, by a genistein-sensitive mechanism and by modifying hormone-responsive transport. These studies demonstrate that insulin stimulates Mg2+uptake in MDCT cells and suggest that insulin acts in concert with other peptide and steroid hormones to control magnesium conservation in the distal convoluted tubule.

Prostaglandins have diverse effects on renal electrolyte reabsorption, inhibiting NaCl absorption in the thick ascending limb and modulating sodium and calcium transport in cortical collecting cells. It is unclear what effect, if any, prostaglandins have on tubular magnesium handling. The effects of prostaglandin E2(PGE2) were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+uptake with fluorescence techniques. Intracellular free Mg2+concentration ([Mg2+]i) was measured on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+uptake, MDCT cells were first Mg2+depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg2+]iwere determined. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 173 ± 8 nM/s. Indomethacin, 5 μM, diminished basal Mg2+uptake, suggesting that endogenous prostaglandins may stimulate Mg2+entry in control cells. PGE2stimulated Mg2+entry in a concentration-dependent manner with maximal response of 311 ± 12 nM/s, at a concentration of 10−7M, which represented an 80 ± 3% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. PGE2-stimulated Mg2+uptake was completely inhibited with the Rp diastereoisomer of adenosine 3′,5′-cyclic monophosphothionate (Rp-cAMPS), a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially by chelerythrine, a protein kinase C inhibitor. Accordingly, PGE2-mediated Mg2+entry rates involve multiple intracellular signaling pathways. These studies demonstrate that PGE2stimulates Mg2+uptake in a cell line of MDCT.

The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. MDCT cells possess an extracellular polyvalent cation-sensing mechanism responsive to Mg2+, Ca2+, and neomycin. The present studies determined the effect of Mg2+/Ca2+sensing on hormone-mediated cAMP formation and Mg2+ uptake in MDCT cells. MDCT cells were Mg2+ depleted by culturing in Mg2+-free media for 16 h, and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg2+ uptake was 164 ± 5 nM/s in control MDCT cells. Activation of Mg2+/Ca2+sensing with neomycin did not affect basal Mg2+ uptake (155 ± 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg2+ entry. In the present studies, the addition of extracellular Mg2+ or Ca2+ inhibited glucagon- and AVP-stimulated cAMP formation and Mg2+ uptake in concentration-dependent manner with half-maximal concentrations of ∼1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg2+ uptake in the presence of Mg2+/Ca2+sensing activation. We infer from these studies that Mg2+/Ca2+-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.

Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg2+transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg2+-depleted cells. Intracellular free Mg2+concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+depleted (0.22 ± 0.01 mM) by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg2+]iwas determined. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 164 ± 5 nM/s. Both glucagon and AVP stimulated Mg2+ uptake into MDCT cells, 196 ± 11 and 189 ± 6 nM/s, respectively, at concentrations of 3 × 10−7 M and 10−7 M, respectively. Enhanced Mg2+ uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg2+ entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10−4 M, enhanced Mg2+ uptake (225 ± 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg2+ uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg2+ uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.

Metabolic alkalosis leads to renal magnesium conservation, whereas metabolic acidosis is associated with urinary magnesium wasting. Micropuncture studies suggest that these actions affect magnesium transport in the distal tubule. The cellular mechanisms of acid-base changes were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined by microfluorescence using the Mg(2+)-responsive dye, mag-fura 2. Mg2+ transport was assessed as a function of change in [Mg2+]i with time following placement of Mg(2+)-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+, d([Mg2+]i)/dt, into Mg(2+)-depleted cells determined with a buffer solution of pH 7.4 was 178 +/- 21 nM/s. Mg2+ uptake at pH 8.0 was markedly increased 278 +/- 35 nM/s, whereas transport at pH 6.0 was significantly reduced to 121 +/- 15 nM/s. Mg2+ uptake at pH 7.4 was not stimulated with 20 or 40 mM bicarbonate, nor were the differences in Mg2+ uptake with pH associated with changes in membrane voltage. Mg2+ uptake was stimulated with membrane hyperpolarization at pH 6.0 but not at pH 8.0. Chlorothiazide (10(-4) M), which stimulates Mg2+ uptake by hyperpolarizing the membrane voltage, increased uptake at pH 6.0, 59 +/- 14%, but decreased it at alkaline pH of 8.0, -55 +/- 3%. Accordingly, MDCT cells become refractory to the stimulating effects of hyperpolarization at alkaline pH values. These studies show that protons may directly affect Mg2+ transport in MDCT cells.

Mesenchymal stem cells (MSCs) have attracted significant attention in cancer research as a result of their accessibility, tumor-oriented homing capacity, and the feasibility of auto-transplantation. This review provides a comprehensive overview of current challenges in pancreatic cancer therapy, and we propose a novel strategy for using MSCs as means of delivering anticancer genes to the site of pancreas. We aim to provide a practical platform for the development of MSC-based therapy for pancreatic cancer.

Biobanks have played a decisive role in all aspects of the field of cancer, including pathogenesis, diagnosis, prognosis and treatment. The significance of cancer biobanks is epitomized through the appropriate application of various "-omic" techniques (omics). The mutually motivated relationship between biobanks and omics has intensified the development of cancer research. Human cancer tissues that are maintained in intravital biobanks (or living tissue banks) retain native tumor microenvironment, tissue architecture, hormone responsiveness and cell-to-cell signalling properties. Intravital biobanks replicate the structural complexity and heterogeneity of human cancers, making them an ideal platform for preclinical studies. The application of omics with intravital biobanks renders them more active, which makes it possible for the cancer-related evaluations to be dynamically monitored on a real-time basis. Integrating intravital biobank and modern omics will provide a useful tool for the discovery and development of new drugs or novel therapeutic strategies. More importantly, intravital biobanks may play an essential role in the creation of meaningful patient-tailored therapies as for personalized medicine.

Cancer remains one of the leading causes of mortality and morbidity throughout the world. To a significant extent, current conventional cancer therapies are symptomatic and passive in nature. The major obstacle for the development of effective cancer therapy is believed to be the lack of sufficient specificity. Since the discovery of tumor-oriented homing capacity of mesenchymal stem cells (MSCs), the application of specific anticancer gene-engineered MSCs has held great potential for cancer therapies. The MSC-based multiple-targeted anticancer strategy is based on MSCs’ capacity of tumor-directed migration and incorporation and in situ expression of tumor-specific anticancer genes. Aimed at translating the benchwork to meaningful clinical applications, we will describe MSCs’ tumor tropism and their use as therapeutic vehicles, the multiple-targeted anticancer potential of engineered MSCs and a personalized strategy for cancer therapy.

We have provided functional and molecular evidence for the presence of Na+/Ca2+ exchange in isolated porcine cortical thick ascending limb (CTAL) cells. The present studies were designed to show that this exchange activity may be modulated by phosphorylative processes. Control of intracellular Ca2+ concentration ([Ca2+]i) was determined in isolated CTAL cells with microfluorescence. CTAL cells were pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) from 10 to 20 mM. These cells had normal basal [Ca2+]i (79 +/- 3 nM). Substitution of extracellular NaCl (50 mM) with KCl resulted in the rapid elevation of [Ca2+]i to maximal levels of 795 +/- 60 nM (n = 17). The increments of [Ca2+]i were associated with [Na+]i. We next determined the modulation of Na+/Ca2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca(2+)-calmodulin inhibitor, resulted in a shift of the [Na+]i dependence curve to the right. Pretreatment with okadaic acid, a phosphatase 1 and 2A inhibitor, increased the Na+/Ca2+ exchanger activity resulting in half-maximal [Ca2+]i increase near normal [Na+]i of 12 mM. Furthermore, in the presence of okadaic acid in normal CTAL cells, pretreatment with ouabain and the elevation of [Na+]i was not required to elicit increments in [Ca2+]i. These data indicate that Na+/Ca2+ exchange is present in CTAL cells and the exchange activity appears to be modulated, directly or indirectly, by phosphorylation events.

Intracellular Ca2+ concentration ([Ca2+]i) plays an important role in the signal transduction processes within cortical thick ascending limb (CTAL) cells. Control of [Ca2+]i was investigated in isolated CTAL cells with microfluorescent techniques. CTAL cells pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) had basal [Ca2+]i of 86 +/- 2 nM. Removal of extracellular Na (Nao+) or voltage depolarization with KCl (in the presence of Nao+) resulted in a rapid and reversible maximal elevation of [Ca2+]i (1,023 +/- 72 nM, n = 28), which was dependent on the presence of external Ca2+ (Cao2+). The rise in [Ca2+]i was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+]i with either removal of Nao+ or voltage depolarization were dependent on pretreatment with ouabain and increases in [Na+]i. The presence of a Na+/Ca2+ exchanger was, confirmed with hybridization techniques, and the isoform was identified by sequencing the alternative splicing site within the intracellular loop. A gene transcript that encodes a portion of the intracellular loop of the renal Na+/Ca2+ exchanger was amplified from cortical tissue and single CTAL cells by reverse transcription-polymerase chain reaction, using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained exons B and D, which is characteristic of one isoform (NACA3) of the renal Na+/Ca2+ exchanger. The results provide both functional and molecular evidence for a Na+/Ca2+ exchanger in thick ascending limb cells of the porcine kidney.

Mesenchymal stem cells (MSCs) have attracted great interest in cancer therapy owing to their tumor-oriented homing capacity and the feasibility of autologous transplantation. Currently, pancreatic cancer patients face a very poor prognosis, primarily due to the lack of therapeutic strategies with an effective degree of specificity. Anticancer gene-engineered MSCs specifically target tumor sites and can produce anticancer agents locally and constantly. This study was performed to characterize pancreas-derived MSCs and investigate the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-engineered MSCs on pancreatic cancer cells under different culture conditions. Pancreas-derived MSCs exhibited positive expression on CD44, CD73, CD95, CD105, negative on CD34 and differentiated into adipogenic and osteogenic cells. TRAIL expression was assessed by both enzyme-linked immunosorbent assay and western blot analysis. Different patterns of TRAIL receptor expression were observed on the pancreatic cancer cell lines, including PANC1, HP62, ASPC1, TRM6 and BXPC3. Cell viability was assessed using a real-time monitoring system. Pancreatic cancer cell death was proportionally related to conditioned media from MSC(nsTRAIL) and MSC(stTRAIL). The results suggest that MSCs exhibit intrinsic inhibition of pancreatic cancer cells and that this effect can be potentiated by TRAIL-transfection on death receptor-bearing cell types.

Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of β-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both β-glucosidase and linamarase and was thus characterized as a cyanogenic β-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic β-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca2+ ([Ca2+]i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca2+]i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca2+]i in a dose-dependent manner. [Ca2+]i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10−6M, and to 766±162 nmol/l with AVP, 10−7 M. The increment in [Ca2+]i by both hormones was by intracellular Ca2+ release and entry through plasma membrane Ca2+ channels. 8-Bromoadenosine-3′, 5′-cyclic monophosphate (8-BrcAMP), 10−4M, increased [Ca2+]i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 μmol), ([Ca2+]i changes: 86±4 to 390±29 nmol/l) and removal of bath Cao2+, ([Ca2+]ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca2+]i. Thapsigargin, 1.5 μmol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca2+ transients, and partially inhibited PTH-mediated Ca2+ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca2+ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca2+]i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca2+ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca2+ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca2+ and be modulated by different pathways which may have diverse effects on cTAL function.

Mesenchymal stem cells (MSCs) have attracted great interest in cancer therapy since the discovery of their tumor tropism. This study was performed to investigate the effects of TNF-related apoptosis-inducing ligand (TRAIL)-engineered MSCs on hepatocellular carcinoma (HCC) cells (HepG2) under different culture conditions.MSCs engineered with non-secreting TRAIL (MSC(TRAIL-GFP)) (GFP, green fluorescence protein) and secreting TRAIL (MSC(stTRAIL)) were used for the direct co-cultures, and conditioned media (CM) from corresponding cultures were applied to HepG2 as indirect co-cultures. Immunoblotting, ELISA and FACS analysis were used to detect the expression of TRAIL and TRAIL receptors. Cell death was assessed using live/dead assay.Death receptor (DR) 5 was identified on the HepG2 cells. The expression of TRAIL was confirmed in the cell lysates (MSC(TRAIL-GFP)MSC(stTRAIL)) and the conditioned media (MSC(stTRAIL)MSC(TRAIL-GFP)). Higher cell death was observed in high MSC/HepG2 ratio co-cultures. HepG2 cell death was proportionally related to CM from MSC(TRAIL-GFP) and MSC(stTRAIL).MSCs exhibit intrinsic inhibition of HepG2 which is potentiated by TRAIL-transfection.

Cancer remains one of the leading causes of mortality and morbidity throughout the world. To a significant extent, current conventional cancer therapies are symptomatic and passive in nature. The major obstacle to the development of effective cancer therapy is believed to be the absence of sufficient specificity. Since the discovery of the tumor-oriented homing capacity of mesenchymal stem cells (MSCs), the application of specific anticancer gene-engineered MSCs has held great potential for cancer therapies. The dual-targeted strategy is based on MSCs' capacity of tumor-directed migration and incorporation and in situ expression of tumor-specific anticancer genes. With the aim of translating bench work into meaningful clinical applications, we describe the tumor tropism of MSCs and their use as therapeutic vehicles, the dual-targeted anticancer potential of engineered MSCs and a putative personalized strategy with anticancer gene-engineered MSCs.