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Osteoporosis is a skeletal disease that is both systemic and silent characterized by an unbalanced activity of bone remodeling leading to bone loss. Rising evidences demonstrate that thyroid stimulating hormone (TSH) has an important role in the regulation on the metabolism of bone. However, TSH regulation on human osteoblast essential transcriptional factors has not been identified. Current study examined the role of TSH on human osteoblastic Runx2 expression and their functional genes by in vitro and in slico analysis. Human osteoblast like (HOS and SaoS-2) cells were cultured with DMEM and treated with hTSH at the concentration of 0.01 ng/mL and 10 ng/mL. After treatment, osteoblastic Runx2 and IGF-1R beta expression were studied using RT-PCR and western blot analysis. TSH treatment induced osteoblastic essential transcriptional factor, Runx2 in HOS and SaOS2 cells on 48 h duration and elevated the expression of IGF-IR β gene and Protein in SaoS-2 cells. TSH also promotes Runx2 responsive genes such as ALP, Collagen and osteocalcin in SaOS2 cells on day 2 to day 14 of 10 ng/mL of treatment and favors’ matrix mineralization matrix in these cells. In addition, TSH facilitated human osteoblastic cells to mineralize their matrix confirmed by day 21 of alizarin red calcium staining. In silico study was performed to check CREB and ELK1 interaction with Runx2. Results of in silico analysis showed that TSH mediated signalling molecules such as CREB and ELK1 showed interaction with Runx2 which involve in osteobalstic gene expression and differentiation. Present findings confirm that TSH promotes Runx2 expression, osteoblastic responsive genes and bone matrix formation.

Exosomes derived from mesenchymal stem cells (MSCs) have been evaluated for their potential to be used as drug delivery vehicles. Synthetically personalized exosome mimetics (EMs) could be the alternative vesicles for drug delivery. In this study, we aimed to isolate EMs from human MSCs. Cells were mixed with paclitaxel (PTX) and PTX-loaded EMs (PTX-MSC-EMs) were isolated and evaluated for their anticancer effects against breast cancer. EMs were isolated from human bone marrow-derived MSCs. MSCs (4 × 106 cells/mL) were mixed with or without PTX at different concentrations in phosphate-buffered saline (PBS) and serially extruded through 10-, 5-, and 1-μm polycarbonate membrane filters using a mini-extruder. MSCs were centrifuged to remove debris and the supernatant was filtered through a 0.22-μm filter, followed by ultracentrifugation to isolate EMs and drug-loaded EMs. EMs without encapsulated drug (MSC-EMs) and those with encapsulated PTX (PTX-MSC-EMs) were characterized by western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The anticancer effects of MSC-EMs and PTX-MSC-EMs were assessed with breast cancer (MDA-MB-231) cells both in vitro and in vivo using optical imaging. EMs were isolated by the extrusion method and ultracentrifugation. The isolated vesicles were positive for membrane markers (ALIX and CD63) and negative for golgi (GM130) and endoplasmic (calnexin) marker proteins. NTA revealed the size of MSC-EM to be around 149 nm, while TEM confirmed its morphology. PTX-MSC-EMs significantly (p < 0.05) decreased the viability of MDA-MB-231 cells in vitro at increasing concentrations of EM. The in vivo tumor growth was significantly inhibited by PTX-MSC-EMs as compared to control and/or MSC-EMs. Thus, MSC-EMs were successfully isolated using simple procedures and drug-loaded MSC-EMs were shown to be therapeutically efficient for the treatment of breast cancer both in vitro and in vivo. MSC-EMs may be used as drug delivery vehicles for breast cancers.

Exosomes are natural nano-sized membrane vesicles that have garnered recent interest owing to their potential as drug delivery vehicles. Though exosomes are effective drug carriers, their production and in vivo biodistribution are still not completely elucidated. We analyzed the production of exosome mimetics (EMs) from red blood cells (RBCs) and the radio-labeling of the RBC-EMs for in vivo imaging. Engineered EMs from RBCs were produced in large-scale by a one-step extrusion method, and further purified by density-gradient centrifugation. RBC-EMs were labeled with technetium-99m (99mTc). For non-invasive imaging, 99mTc (free) or 99mTc-RBC-EMs were injected in mice, and their biodistribution was analyzed by gamma camera imaging. Animals were sacrificed, and organs were collected for further biodistribution analysis. RBC-EMs have similar characteristics as the RBC exosomes but have a 130-fold higher production yield in terms of particle numbers. Radiochemical purity of 99mTc-RBC-EMs was almost 100% till 2 h reduced to 97% at 3 h. Radio-labeling did not affect the size and morphology of RBC-EMs. In contrast to free 99mTc, in vivo imaging of 99mTc-RBC-EMs in mice showed higher uptake in the liver and spleen, and no uptake in the thyroid. Ex vivo imaging confirmed the in vivo findings. Furthermore, fluorescent imaging confirmed the nuclear imaging findings. Immunofluorescent imaging revealed that the hepatic uptake of RBC-EMs was significantly mediated by kupffer cells (resident hepatic macrophages). Our results demonstrate a simple yet large-scale production method for a novel type of RBC-EMs, which can be effectively labeled with 99mTc, and feasibly monitored in vivo by nuclear imaging. The RBC-EMs may be used as in vivo drug delivery vehicles.

ObjectiveGlioblastoma is a highly aggressive primary brain tumor that is resistant to radiotherapy and chemotherapy. Natural killer (NK) cells have been used to treat incurable cancers. Recent studies have investigated the effectiveness of NK-cell-derived exosomes (NK-Exo) for treating incurable cancers such as melanoma, leukemia, and neuroblastoma; however, NK-Exo have not been used to treat glioblastoma. In the present study, we investigated the antitumor effects of NK-Exo against aggressive glioblastoma both in vitro and in vivo and determined the tumor-targeting ability of NK-Exo by performing fluorescence imaging.MethodsU87/MG cells were transfected with the enhanced firefly luciferase (effluc) and thy1.1 genes; thy1.1-positive cells were selected using microbeads. U87/MG/F cells were assessed by reverse transcription polymerase chain reaction (RT-PCR), western blotting, and luciferase-activity assays. NK-Exo were isolated by ultracentrifugation, purified by density gradient centrifugation, and characterized by transmission electron microscopy, dynamic light scattering (DLS), nanoparticle-tracking analysis (NTA), and western blotting. Cytokine levels in NK-Exo were compared to those in NK cells and NK-cell medium by performing an enzyme-linked immunosorbent assay (ELISA). NK-Exo-induced apoptosis of cancer cells was confirmed by flow cytometry and western blotting. In vivo therapeutic effects and specificity of NK-Exo against glioblastoma were assessed in a xenograft mouse model by fluorescence imaging. Xenograft mice were treated with NK-Exo, which was administered seven times through the tail vein. Tumor growth was monitored by bioluminescence imaging (BLI), and tumor volume was measured by ultrasound imaging. The mice were intraperitoneally injected with dextran sulfate 2 h before NK-Exo injection to decrease the liver uptake and increase the tumor specificity of NK-Exo.ResultsRT-PCR and western blotting confirmed the gene and protein expression of effluc in U87/MG/F cells, with the bioluminescence activity of U87/MG/F cells increasing with an increase in cell number. NTA and DLS results indicated that the size of NK-Exo was ~100 nm, and the western blot results confirmed that NK-Exo expressed exosome markers CD63 and Alix. We confirmed the in vitro cytotoxic effects of NK-Exo on U87/MG/F cells by performing BLI, and the killing effect on U87/MG and U87MG/F cells was measured by CCK-8 and MTT assays (p

ObjectiveNatural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells.MethodsHuman NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h.ResultsNK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells to injured and inflamed areas. Noninvasive cell tracking methods could be useful for monitoring cell fate. Therefore, in this study, we evaluated the efficacy of an intravenous infusion of genetically engineered mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4) to home to the tumor, by optical imaging. We constructed a retroviral vector containing CXCR with dual reporter genes, eGFP and Fluc2, under the control of an EF1α promoter (pBABE-EF1α-CXCR4-eGFP-IRES-Fluc2). We also developed an eGFP-Fluc2 construct in the Retro-X retroviral vector (Retro-X-eGFP-Fluc2). MSCs were transduced with retroviruses to generate CXCR4-overexpressing MSCs (MSC-CXCR4/Fluc2) and MSCs (MSC/Fluc2). CXCR4 mRNA and protein expression was confirmed by RT-PCR and Western blotting, respectively, and it was higher in MSC-CXCR4/Fluc2 than in naive MSCs. eGFP expression was confirmed by confocal microscopy. The transfected MSC-CXCR4/Fluc2 cells showed higher migratory capacity than naive MSCs observed in Transwell migration assay. The in vivo migration of CXCR4-overexpressing MSCs to MDAMB231/Rluc tumor model by BLI imaging was also confirmed. Intravenous delivery of genetically modified MSCs overexpressing CXCR4 with a Fluc2 reporter gene may be a useful, noninvasive BLI imaging tool for tracking cell fate.

Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies. For example, use a synthetic "tetracycline-on" switch system to control the expression of the therapeutic gene thymidine kinase, which converts prodrugs to active drugs. The aim of this study was to develop therapeutic MSCs, harboring an inducible suicide gene, and to validate therapeutic gene expression using optical molecular imaging of ATC. We designed the Tet-On system using a retroviral vector expressing herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Mouse bone marrow-derived mesenchymal stromal cells (BM-MSC) were transduced using this system with (MSC-Tet-TK/Fluc2) or without (MSC-TK/Fluc) the Tet-On system. Transduced cells were screened and characterized. Engineered MSCs were co-cultured with ATC (CAL62/Rluc) cells in the presence of the prodrug ganciclovir (GCV) and stimulated with DOX. The efficiency of cell killing monitored by assessing Rluc (CAL62/Rluc) and Fluc (MSC-Tet-TK/Fluc and MSC-TK/Fluc) activities using IVIS imaging. Fluc activity increased in MSC-Tet-TK/Fluc cells in a dose dependent manner following DOX treatment (R2 = 0.95), whereas no signal was observed in untreated cells. eGFP could also be visualized after induction with DOX, and the HSV1-TK protein could be detected by western blotting. In MSC-TK/Fluc cells, the Fluc activity increased with increasing cell number (R2 = 0.98), and eGFP could be visualized by fluorescence microscopy. The Fluc activity and cell viability of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells decreased significantly following GCV treatment. A bystander effect of the therapeutic cells confirmed in co-cultures of CAL62 cells, an anaplastic thyroid cancer cell line, with either MSC-Tet-TK/Fluc cells or MSC-TK/Fluc cells. The Rluc activity in MSC-Tet-TK/Fluc co-cultures, derived from the CAL62/Rluc cells, decreased significantly with GCV treatment of DOX treated cultures, whereas no significant changes were observed in untreated cultures. In addition, the Fluc activity of MSC-Tet-TK/Fluc cells also decreased significantly with DOX treatment whereas no signal was present in untreated cultures. A bystander effect also be demonstrated in co-cultures with MSC-TK/Fluc cells and CAL62/Rluc; both the Rluc activity and the Fluc activity were significantly decreased following GCV treatment. We have successfully developed a Tet-On system of gene-directed enzyme/prodrug delivery using MSCs. We confirmed the therapeutic bystander effect in CAL62/Rluc cells with respect to MSC-Tet-TK/Fluc and MSC-TK/Fluc cells after GCV treatment with and without DOX. Our results confirm the therapeutic efficiency of a suicide gene, with or without the Tet-On system, for ATC therapy. In addition, our findings provide an innovative therapeutic approach for using the Tet-On system to eradicate tumors by simple, repeated administration of MSC-Tet-TK/Fluc cells with DOX and GCV.

Inhibition of apoptosis leads to activation of cell survival factors (e.g., AKT) causes continuous cell proliferation in cancer. Apoptosis, the major form of cellular suicide, is central to various physiological processes and the maintenance of homeostasis in multicellular organisms. A number of discoveries have clarified the molecular mechanism of apoptosis, thus clarifying the link between apoptosis and cell survival factors, which has a therapeutic outcome. Induction of apoptosis and inhibition of cell survival by anticancer agents has been shown to correlate with tumor response. Cellular damage induces growth arrest and tumor suppression by inducing apoptosis, necrosis and senescence; the mechanism of cell death depends on the magnitude of DNA damage following exposure to various anticancer agents. Apoptosis is mainly regulated by cell survival and proliferating signaling molecules. As a new therapeutic strategy, alternative types of cell death might be exploited to control and eradicate cancer cells. This review discusses the signaling of apoptosis and cell survival, as well as the potential contribution of marine bioactive compounds, suggesting that new therapeutic strategies might follow.

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(−) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(−) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.

The current practice of direct seeding in East‐African high‐altitude rice farming systems is constrained by water availability early in the season and low temperatures later in the season at the crop's critical reproductive stage. Thus, productivity is restricted as only short‐duration varieties can be grown due to the risk of crop failure. To fully exploit the yield potential of such rainfed systems, the best combination of crop establishment methods and climatic 'best fit' genotypes is required. In this study, nine rice genotypes were evaluated under direct seeding and transplanting in the 2016 and 2017 cropping seasons with the aim of identifying genotype by environment by management combinations best fitting the high‐altitude, rainfed rice production systems. On average across all genotypes, transplanting had a positive yield effect of 18% in 2016 and 23% in 2017. Regarding the phenological development, individual phenophases were not significantly affected by transplanting relative to direct seeding; however, vegetative development stages in transplanted rice tended to be about 15% longer than when direct seeded. Even though transplanting led to extended vegetative growth, the time in the nursery allowed the plants to escape the cold spell late in the season. The results from the current study provide options to adapt cropping calendars by combining genetic resources with targeted crop management, thus improving and stabilizing yields of rainfed lowland rice farming systems at high altitude. [ABSTRACT FROM AUTHOR]