GDNF induces RET-SRC-HER2-dependent growth in trastuzumab-sensitive but SRC-independent growth in resistant breast tumor cells.

We investigated the role of glial cell line-derived neurotrophic factor (GDNF) in compensating trastuzumab (TZMB)-induced apoptosis in HER2+ breast cancer (BC) cells using xenograft tumors. We generated BC xenografts in nude mice using samples from three patients selected based on their HER2 status and response to TZMB therapy. TZMB treatment resulted in shrinkage of the HER2+ TZMB-sensitive xenograft tumor but not the HER2- or HER2+ TZMB-resistant ones. GDNF neutralized TZMB activity and induced growth in all tumors. Three distinct cell lines were derived from these tumors and named, respectively, TZMB-sensitive (TSTC), HER2- (HNTC), and TZMB-resistant (TRTC). Over 50% of TRTC but 1% of TSTC cells expressed CD44, whereas 84% of TSTC were CD24+ compared to only 1% of TRTC, despite comparable levels of HER2 detected in both. TZMB induced profound morphological changes toward apoptosis in TSTC but not in TRTC or HNTC. However, GDNF significantly compensated TZMB-mediated TSTC cell loss and promoted growth by 37 and 50%, respectively, in TSTC and TRTC. Inhibition of SRC by Saracatinib (SARC) blocked GDNF function and accelerated TZMB-mediated cell death in TSTC, but GDNF continued promoting TRTC growth. These changes paralleled with expression levels of the key molecules involved in growth and apoptosis. Collectively, we found in our xenograft samples that firstly SRC mediates GDNF pro-survival functions by bridging RET-HER2 crosstalk in TZMB-responsive BC tumors. Secondly, SARC-TZMB interactions can synergistically eradicate such tumor cells; and thirdly, GDNF can support antibody resistance by acting independent from SRC in tumors with poor HER2 response to TZMB therapy.

Phase I study of safety and efficacy of allogeneic natural killer cell therapy in relapsed/refractory neuroblastomas post autologous hematopoietic stem cell transplantation.

Despite low incidence, neuroblastoma, an immunologically cold tumor, is the most common extracranial solid neoplasm in pediatrics. In relapsed/refractory cases, the benefits of autologous hematopoietic stem cell transplantation (auto-HSCT) and other therapies are limited. Natural killer (NK) cells apply cytotoxicity against tumor cells independently of antigen-presenting cells and the adaptive immune system. The primary endpoint of this trial was to assess the safety of the injection of allogenic, ex vivo-expanded and primed NK cells in relapsed/refractory neuroblastoma patients after auto-HSCT. The secondary endpoint included the efficacy of this intervention in controlling tumors. NK cells were isolated and primed ex vivo (by adding interleukin [IL]-2, IL-15, and IL-21) in a GMP-compliant CliniMACS system and administered to four patients with relapsed/refractory MYCN-positive neuroblastoma. NK cell injections (1 and 5 × 107 cells/kg in the first and second injections, respectively) were safe, and no acute or sub-acute adverse events were observed. During the follow-up period, one complete response (CR) and one partial response (PR) were observed, while two cases exhibited progressive disease (PD). In follow-up evaluations, two died due to disease progression, including the case with a PR. The patient with CR had regular growth at the 31-month follow-up, and another patient with PD is still alive and receiving chemotherapies 20 months after therapy. This therapy is an appealing and feasible approach for managing refractory neuroblastomas post-HSCT. Further studies are needed to explore its efficacy with higher doses and more frequent administrations for high-risk neuroblastomas and other immunologically cold tumors.Trial registration number: irct.behdasht.gov.ir (Iranian Registry of Clinical Trials, No. IRCT20201202049568N1).

Different Effects of Low-Level Laser Therapy on the Proliferation of HT29 Cells in Culture and Xenograft Models.

Introduction: Different kinds of treatments have been developed to fight cancers. Low-level laser therapy (LLLT), also known as photobiomodulation therapy (PBMT), is a low-power monochromatic and coherent light that has been used successfully for healing injuries and combating malignancies. However, there are concerns about the application of LLLT to cancers due to the increased proliferation of some cancer cells after LLLT. Methods: This study investigated the effects of 650 nm and 870 nm lasers on the proliferation of HT29 colorectal cancer cell lines in vitro and in vivo. Results: The results showed that the laser with a wavelength of 870 nm did not meaningfully alter the proliferation of cultured cells. However, cell proliferation was promoted when the laser was applied within a wavelength of 650 nm. Treatment of HT29-derived tumors in nude mice with the 650 nm laser resulted in the decline of the tumor progression rate compared to controls. This result was inconsistent with the proliferative effects of the laser on the cultured cells. Conclusion: Cell behavior in response to LLLT might be different between cell culture and xenograft models.

Mechanical and Thermal Properties of Functionally Graded Polyolefin Elastomer Foams.

In this work, uniform and graded polyolefin elastomer (POE) foams were prepared using a single-step technology based on a fixed chemical blowing agent (azodicarbonamide) concentration of 4 phr (parts per hundred rubber). The effect of molding temperature, including the average temperature (Tavg) and temperature difference (ΔT), on the foams' morphology, mechanical properties (tension, compression and hardness) and thermal conductivity was investigated. Two series of samples were produced by fixing Tavg with different ΔT or setting different ΔT, leading to different Tavg. The morphological analyses showed that two or three regions inside the foams were produced depending on the molding conditions, each region having different cellular structure in terms of cell size, cell density and cell geometry. The results obtained for the conditions tested showed a range of density (0.55-0.72 g/cm3), tensile modulus (0.44-0.70 MPa) and compression elastic modulus (0.35-0.71 MPa), with a thermal conductivity between 0.125 and 0.180 W/m.K. Based on the information provided, it can be concluded that the foam's properties can be easily controlled by the cellular structure and that graded samples are more interesting than uniform ones, especially for thermal insulation applications, such as packaging, construction, transportation, automotive and aerospace industries.

Magnonic contribution in thermal conductance of a nanostructure in the presence of magnon-phonon interaction.

Using Green's function method, we study thermal transport properties of magnons transmitting through a magnetic nanowire at a certain temperature. In a small part of the nanowire in the middle, we are supposed to have two types of local and nonlocal magnon-phonon interactions. The self-energies for this part due to the other parts of the wire are analytically derived. First, we calculate the phonon mode-dependent magnon transmission coefficient in the classical canonical ensemble. Then, by taking an average of the phonon modes, we obtain the total magnon transmission coefficient and use it for computing magnon thermal conductance. We use the model to investigate four configurations of magnetic nanowires composed of ferromagnetic and/or antiferromagnetic parts. The results show that, when the scattering region has an antiferromagnetic alignment, the magnons transfer in the structure more weakly than for ferromagnetic alignment. There is a phonon-assisted mechanism for tunneling of magnons which are transmitted through the gap or between magnon quasifrequencies of the scattering region. Generally, in the same values for local and nonlocal strengths of magnon-phonon interaction, the nonlocal one has a greater effect on the magnonic thermal transport properties. We found the fitting functions in order to relate the macroscopic quantities of the magnon transmission coefficient and thermal conductivity to microscopic parameters of the strengths of the local and nonlocal magnon-phonon interaction.

Osteogenic differentiation of follicular stem cells on nano-Saghez scaffold containing BMP2.

BACKGROUND: Bone tissue is one of the tissues that are capable of self-regeneration. However, bone self-regeneration is defeated in the case of broad lesion of bone structure. Isolated stem cells from wisdom tooth follicles can potentially differentiate into ectodermal and mesodermal cells. Saghez is a natural substance that has been extracted from Pistacia terebinthus with unique features, such as high temperature and mechanical stability, adhesive structure, biocompatibility, and anti-neoplastic properties. METHODS: In this study, Saghez-encapsulated BMP2 was applied as a scaffold for wisdom tooth follicle stem cell differentiation into the osteocyte. A total of three wisdom tooth follicles were obtained for stem cell isolation. For verification of differentiation of the isolated stem cells into osteocyte and adipocyte, Oil Red and Alizarin staining were applied, respectively. Moreover, mesenchymal stem cells were distinguished by profiling their cell surface markers, includingCD73, CD90, CD44, and CD105, by flow cytometry. Saghez scaffold loaded with BMP2 factor was prepared using sol-gel method. Four experimental groups were considered in this study: cells seeded on BMP2 encapsulated in Saghez scaffold, Saghez scaffold, osteogenic medium, and DMEM medium. RESULTS: Mechanical properties of Saghez scaffold, including tensile Young's modulus, ultimate tensile stress, compression Young's modulus, and complex shear modulus, were 19 MPa, 32 MPa, 0.42 MPa, and 0.9 MPa, respectively. The porosity of the scaffold was 70-140 µm, and the percentage of porosity was 75-98%. The results of flow cytometry studies indicated that CD44, CD73, CD90, and CD105 were positively expressed on the membrane of the tooth follicles' stem cell. The results indicated that the rate of differentiation of the follicle stem cells into osteocyte was the highest in the Saghez-BMP2 scaffold containing differentiation medium groups. These findings were verified by morphological studies, osteoblast and osteocalcin gene and protein expression investigations, and alkaline phosphatase activity measurement. The highest osteopontin and osteocalcin genes expression levels (1.7 and 1.9) were seen in positive control, followed by DMEM + differentiation factor (1.5 and 1.6), scaffold + BMP2 (1.2 and 1.4), DMEM + stem cell (1 and 1) and scaffold (0.4 and 0.5), and negative control respectively. CONCLUSION: This study provides a novel system for differentiation of the stem cell into osteocytes. The results of this study suggest that loaded BMP2 in Saghez scaffold possibly acts as an osteocyte differentiator factor.

Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis.

The aim of the present study was to assess the recombinant bonemorphogenetic protein 2 (RHBMP-2) with higher substantively and solubility for use in calcium phosphate scaffolds for better release in differentiation of mesenchymal stem cells to osteoblast cells. Using bioinformatics tools, two mutations (p. L10D and p. S12E) were chosen and applied in BMP2 CDS sequence to increase interaction with calcium derived composite. The new recombinant mutated sequence (BMP2mut ) was synthesized and then subcloned to expression vector pBV220. Experimental data regarded functional protein expression in E. coli. Since no modification was made in the active sites of proteins namely ß-sheets and α-helixes, not only was there any change in the specific activity occurred in the specific activity of the enzyme in comparison to its commercial counterpart, but also mesenchymal osteogenesis occurred more efficient on biphasic CaP scaffold model. As we hypothesized, use of negatively charged amino acids such as aspartate and glutamate in protein loops increased the interactions of BMP2-Ca2+ and resulted in its slower and more sustained released from CaP scaffolds compare to commercial RHBMP2. Our data suggested that new BMP2mut have greater osteoinductive capacity than RHBMP2 in the same time and dose than RHBMP2. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2799-2805, 2017.

Upregulation of glutathione peroxidase-1 expression and activity by glial cell line-derived neurotrophic factor promotes high-level protection of PC12 cells against 6-hydroxydopamine and hydrogen peroxide toxicities.

We examined the impact of strong co-presence and function of glutathione peroxidase-1 (GPX-1) and glial cell line-derived neurotrophic factor (GDNF) on protecting the rat dopaminergic pheochromocytoma cell line PC12 against 6-hydroxydopamine (6-OHDA) and hydrogen peroxide (H2O2) toxicities. Primarily, GPX-1 over-expression by PC12 cells infected with pLV-GPX1 lentivirus vectors significantly increased cell survival against 6-OHDA toxicity (p<0.01). Addition of conditioned medium collected from growing wild-type astrocytes (Control astro-CM) increased survival rate of pLV-GPX1 infectants by 10% compared to their un-treated counterparts (p<0.05) and 20% compared to their treated empty vector control (p<0.01). Treatment of pLV-GPX1 cells with astro-CM of GDNF-over-secreting astrocytes (Test astro-CM) significantly induced GPX-1 expression, peroxidase enzymatic activity, and intra-cellular glutathione (GSH) levels. These changes paralleled with protection of 90% of GDNF⁺/GPX1⁺ PC12 cells against toxicity, a rate that was 37% up from their un-infected un-treated (GDNF⁻/GPX1⁻) controls (p<0.001), and 12% up from pLV-GPX1 cells that received only Control astro-CM (GPX⁺/GDNF⁻) (p<0.01). GPX-1 over-expression per se suppressed intra-cellular H2O2 elevation upon 6-OHDA exposure, and addition of GDNF medium significantly accelerated this suppression (p<0.01). Substitution of 6-OHDA with H2O2 induced similar intra-cellular changes and comparable protection levels. In all cell groups, increased cell survival against either compound was further confirmed by increased live cell counts measured by double staining. Following depletion of intra-cellular GSH, only 46% of pLV-GPX1 cells survived 6-OHDA toxicity, whereas over 70% of them were saved upon GDNF treatment (p<0.001). Moreover, capase-3 activation was reduced in pLV-GPX1 cells and maximized by addition of GDNF. Comparison analyses established correlations between GPX-1-GDNF co-presence and both enhanced cell protection and diminished levels of activated caspase-3. Our data collectively indicate that GDNF is capable of inducing anti-oxidant activities of intra-cellular GPX-1 and that growth-promoting potential of GDNF and anti-oxidant properties of GPX-1 can, in concert, maximize survival of dopaminergic neurons.

Target points in trastuzumab resistance.

Epidermal growth factor (EGF) family of receptors is involved in cell growth and differentiation. The human EGF2 (HER2) lacks natural ligands, and correlation between HER2 levels and carcinogenesis makes the receptor an ideal candidate for targeted therapy in breast cancer. Trastuzumab is a humanized antibody applied against HER2-positive breast tumors in clinic. Metastatic tumors respond well to trastuzumab therapy for the first year, but development of antibody resistance helps the tumors to regrow allowing the disease to progress. Trastuzumab resistance is shaped via a range of intracellular signaling pathways that are interconnected and share in key effector molecules. Identification of a common node central to these resistance pathways could provide an ultimate solution for trastuzumab resistance in breast and other cancers.