Xanthohumol hinders invasion and cell cycle progression in cancer cells through targeting MMP2, MMP9, FAK and P53 genes in three-dimensional breast and lung cancer cells culture.

BACKGROUND: Despite recent advances in the treatment of lung and breast cancer, the mortality with these two types of cancer is high. Xanthohumol (XN) is known as a bioactive compound that shows an anticancer effect on cancer cells. Here, we intended to investigate the anticancer effects of XN on the breast and lung cancer cell lines, using the three-dimensional (3D) cell culture. METHODS: XN was isolated from Humulus lupulus using Preparative-Thin Layer Chromatography (P-TLC) method and its authenticity was documented through Fourier Transform Infrared spectroscopy (FT-IR) and Hydrogen Nuclear Magnetic Resonance (H-NMR) methods. The spheroids of the breast (MCF-7) and lung (A549) cancer cell lines were prepared by the Hanging Drop (HD) method. Subsequently, the IC50s of XN were determined using the MTT assay in 2D and 3D cultures. Apoptosis was evaluated by Annexin V/PI flow cytometry and NFκB1/2, BAX, BCL2, and SURVIVIN expressions. Cell cycle progression was determined by P21, and P53 expressions as well as PI flow cytometry assays. Multidrug resistance was investigated through examining the expression of MDR1 and ABCG2. The invasion was examined by MMP2, MMP9, and FAK expression and F-actin labeling with Phalloidin-iFluor. RESULTS: While the IC50s for the XN treatment were 1.9 µM and 4.74 µM in 2D cultures, these values were 12.37 µM and 31.17 µM in 3D cultures of MCF-7 and A549 cells, respectively. XN induced apoptosis in MCF-7 and A549 cell lines. Furthermore, XN treatment reduced cell cycle progression, multidrug resistance, and invasion at the molecular and/or cellular levels. CONCLUSIONS: According to our results of XN treatment in 3D conditions, this bioactive compound can be introduced as an adjuvant anti-cancer agent for breast and lung cancer.

Cold atmospheric plasma induced genotoxicity and cytotoxicity in esophageal cancer cells.

In this paper, we studied the functional effects of cold atmospheric plasma (CAP) on the esophageal cancer cell line (KYSE-30) by direct and indirect treatment and fibroblast cell lines as normal cells. KYSE-30 cells were treated with CAP at different time points of 60, 90, 120 and, 240 s for direct exposure and 90, 180, 240 and, 360 s for indirect exposure. Cell viability was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis induction in the treated cells was measured by Annexin-V/PI using flow cytometry. The expression of apoptotic related genes (BAX/BCL-2) was analyzed by real-time polymerase chain reaction. Moreover, the genotoxicity was analyzed by comet assay. Cell viability results showed that direct CAP treatment has a markedly cytotoxic impact on the reduction of KYSE-30 cells at 60 s (p = 0.000), while indirect exposure was less impactful (p > 0.05). The results of the Annexin-V/PI staining confirmed this analysis. Subsequently, the genotoxicity study of the direct CAP treatment demonstrated a longer tail-DNA length and caused increase in DNA damage in the cells (p < 0.00001) as well as shift BAX/BCL-2 toward apoptosis. The concentration of H2O2 and NO2- in direct CAP treatment was significantly higher than indirect (p > 0.05). Treatment with direct CAP showed genotoxicity in cancer cells. Collectively, our results pave a deeper understanding of CAP functions and the way for further investigations in the field of esophageal cancer treatment.

Functional biological pacemaker generation by T-Box18 protein expression via stem cell and viral delivery approaches in a murine model of complete heart block.

Despite recent advances in the treatment of cardiac arrhythmia, the available options are still limited and associated with some complications. Induction of biological pacemakers via Tbx18 gene insertion in the heart tissue has been suggested as a promising therapeutic strategy for cardiac arrhythmia. Following a previous in vitro study reporting the production of Tbx18-expressing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), we aimed to investigate the efficacy of these engineered cells to generate pacemaker rhythms in a murine model of complete heart block. We also attempted to generate a functional pacemaker by Tbx18 overexpression in native cardiac cells of rat heart. The hiPSC-derived pacemaker cells were produced by lentiviral delivery of Tbx18 gene to stem cells during a small molecule-based differentiation process. In the present study, 16 male albino Wistar rats were randomly assigned to Tbx18-lentivirus (n = 4) and Tbx18-pacemaker cells (n = 4) administered via injection into the left ventricular anterolateral wall. The control rats received GFP-lentiviruses (n = 4) and GFP-pacemaker cells (n = 4). Fourteen days after the injection, the rats were sacrificed and analyzed by electrocardiography (ECG) recording using a Langendorff-perfused heart model following complete heart block induced by hypokalemia and crashing. Immunofluorescence staining was used to investigate the expression of Tbx18, HCN4 and connexin 43 (Cx43) proteins in Tbx18-delivered cells of heart tissues. The heart rate was significantly reduced after complete heart block in all of the experimental rats (P < 0.05). Heart beating in the Tbx18-transduced hearts was slower compared with rats receiving Tbx18-pacemaker cells (P = 0.04). The duration of ventricular fibrillation (VF) was higher in the lentiviral Tbx18 group compared with the GFP-injected controls (P = 0.02) and the Tbx18-pacemaker cell group (P = 0.02). The ECG recording data showed spontaneous pacemaker rhythms in both intervention groups with signal propagation in Tbx18-transduced ventricles. Immunostaining results confirmed the overexpression of HCN4 and downregulation of Cx43 as a result of the expression of the Tbx18 gene and spontaneously contracting myocyte formation. We confirmed the formation of a functional pacemaker after introduction of Tbx18 via cell and gene therapy strategies. Although the pacemaker activity was better in gene-received hearts since there were longer VF duration and signal propagation from the injection site, more data should be gathered from the long-term activity of such pacemakers in different hosts.

Cell laden hydrogel construct on-a-chip for mimicry of cardiac tissue in-vitro study.

Since the leading cause of death are cardiac diseases, engineered heart tissue (EHT) is one of the most appealing topics defined in tissue engineering and regenerative medicine fields. The importance of EHT is not only for heart regeneration but also for in vitro developing of cardiology. Cardiomyocytes could grow and commit more naturally in their microenvironment rather than traditional cultivation. Thus, this research tried to develop a set up on-a-chip to produce EHT based on chitosan hydrogel. Micro-bioreactor was hydrodynamically designed and simulated by COMSOL and produced via soft lithography process. Chitosan hydrogel was also prepared, adjusted, and assessed by XRD, FTIR and also its degradation rate and swelling ratio were determined. Finally, hydrogels in which mice cardiac progenitor cells (CPC) were loaded were injected into the micro-device chambers and cultured. Each EHT in every chamber was evaluated separately. Prepared EHTs showed promising results that expanded in them CPCs and work as an integrated syncytium. High cell density culture was the main accomplishment of this study.

Design and evaluation of a skin-on-a-chip pumpless microfluidic device.

The development of microfluidic culture technology facilitates the progress of study of cell and tissue biology. This technology expands the understanding of pathological and physiological changes. A skin chip, as in vitro model, consisting of normal skin tissue with epidermis and dermis layer (full thickness) was developed. Polydimethylsiloxane microchannels with a fed-batched controlled perfusion feeding system were used to create a full-thick ex-vivo human skin on-chip model. The design of a novel skin-on-a-chip model was reported, in which the microchannel structures mimic the architecture of the realistic vascular network as nutrients transporter to the skin layers. Viabilities of full-thick skin samples cultured on the microbioreactor and traditional tissue culture plate revealed that a precise controlled condition provided by the microfluidic enhanced tissue viability at least for seven days. Several advantages in skin sample features under micro-scale-controlled conditions were found such as skin mechanical strength, water adsorption, skin morphology, gene expression, and biopsy longevity. This model can provide an in vitro environment for localizing drug delivery and transdermal drug diffusion studies. The skin on the chip can be a valuable in vitro model for representing the interaction between drugs and skin tissue and a realistic platform for evaluating skin reaction to pharmaceutical materials and cosmetic products.

Mini Bioreactor Can Support In Vitro Spermatogenesis of Mouse Testicular Tissue.

Objective: It was in the early 20th century when the quest for in vitro spermatogenesis started. In vitro spermatogenesis is critical for male cancer patients undergoing gonadotoxic treatment. Dynamic culture system creates in vivo-like conditions. In this study, it was intended to evaluate the progression of spermatogenesis after testicular tissue culture in mini-perfusion bioreactor. Materials and Methods: In this experimental study, 12 six-day postpartum neonatal mouse testes were removed and fragmented, placed on an agarose gel in parallel to bioreactor culture, and incubated for 8 weeks. Histological, molecular and immunohistochemical evaluations were carried out after 8 weeks. Results: Histological analysis suggested successful maintenance of spermatogenesis in tissues grown in the bioreactor but not on agarose gel, possibly because the central region did not receive sufficient oxygen and nutrients, which led to necrotic or degenerative changes. Molecular analysis indicated that Plzf, Tekt1 and Tnp1 were expressed and that their expression did not differ significantly between the bioreactor and agarose gel. Immunohistochemical evaluation of testis fragments showed that PLZF, SCP3 and ACRBP proteins were expressed in spermatogonial cells, spermatocytes and spermatozoa. PLZF expression after 8 weeks was significantly lower (P<0.05) in tissues incubated on agarose gel than in the bioreactor, but there was no significant difference between SCP3 and ACRBP expression among the bioreactor and agarose gel culture systems. Conclusion: This three-dimensional (3D) dynamic culture system can provide somewhat similar conditions to the physiological environment of the testis. Our findings suggest that the perfusion bioreactor supports induction of spermatogenesis for generation of haploid cells. Further studies will be needed to address the fertility of the sperm generated in the bioreactor system..

SARS-CoV-2 antibody response after BBIBP-CorV (Sinopharm) vaccination in cancer patients: A case-control study.

Background: Long-term safety and efficacy of BBIBP-CorV vaccine especially in individuals with chronic diseases, like cancer, is under investigation. In the present prospective study, we aimed to evaluate severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody response with BBIBP-CorV vaccine in Iranian cancer patients. Methods: All the patients registered to receive BBIBP-CorV (Sinopharm) vaccine were divided into two groups of with (cases = 107) and without (controls = 45) history of cancer. Serum levels of SARS-CoV anti-spike recombinant receptor binding domain (anti-sRBD) and anti-nucleocapsid (anti-N) IgG serum levels were measured on days 0 (phase 0), 28-32 (phase I), and 56-64 (phase II) of vaccination. The data were analyzed using SPSS, version 22. Results: Totally, 152 individuals (67.1% females) with the mean age of 46.71 ± 15.36 years were included. Solid cancers included 87.8% of the cancer cases (46.7% gynecological and 31.8% gastrointestinal cancer). At Phases I and II, positive anti-sRBD IgG and anti-N IgG were significantly lower among the cases in total analysis. Side effects were not significantly different between the cases and controls. The lowest positive anti-sRBD IgG test was observed among the cancer patients who were simultaneously receiving chemotherapy (35.3%). Anti-sRBD IgG and anti-N IgG serum levels significantly increased at phases I and II in total analysis and in each group. In addition, serum anti-sRBD IgG increased during the three phases and it was significantly higher in the control group. Conclusion: Full vaccination of COVID-19 by BBIBP-CorV in immunocompromised patients such as cancer patients is safe and effective and could induce antibody response but in lower levels compared to healthy people. Probable causes to have minor antibody response found in males, older ages, individuals with BMI ≥ 25, those without past history of COVID-19 and with hematologic cancers. No significant side effects after vaccination were seen.

Bioartificial injectable cartilage implants from demineralized bone matrix/PVA and related studies in rabbit animal model.

Functional cartilage tissue engineering needs a substantial, easy to handle scaffold with proper mechanical strength to repair defected area in articular cartilage. In this study, we report the development and characterization of demineralized bone matrix (DBM) in with a poly vinyl alcohol (PVA) to have a proper homogenous injectable scaffold. Injectabiliy of the biodegradable scaffolds, degradation rate, swelling ratio compression and tensile mechanical properties, and viability and proliferation of bone marrow mesenchymal stem cells (BM-MSCs) followed by differentiation of them In-vitro and In-vivo seeded within the scaffold were studied. It demonstrated that the PVA 20% could increase significantly (p < 0.05) the biodegradability of DBM after 720 hours.DBM with 20% of PVA scaffold has significantly higher (p < 0.05) compression and tensile mechanical strength and viscosity. SEM images showed a multilayer of cells on DBM scaffold incorporated with PVA 20%.BM-MSCs on scaffolds, DBM+PVA 20% had a significant growth rate (p < 0.0001) compare to 2D and low concentration of PVA after 21 days of culture. Viability of cells was significantly higher (p < 0.05) on DBM+PVA scaffold compare to DBM. DBM+PVA 20% enhanced cell viability (P < 0.05) compare to DBM scaffold. The PVA presence enhanced chondrogenesis differentiation at the cellular and molecular levels, as evidenced by increased COL II (P < 0.05) and SOX2 upregulation of Chondrogensis-specific genes (p < 0.001). Hyline-like cartilage covered the defect which was confirmed by microscopy and histology assessments. Having considered percentages of PVA with a constant amount of DBM, injectability, compressive mechanical properties, homogeneity of the scaffold, and providing sufficient surface area (12.25 cm2/ml) for cell attachment; 0.35 g/ml of DBM in 20% PVA (w/v) has applicable properties within the ranges of studies which can be proposed for the injectable engineered articular cartilage.

Cartilage tissue engineering using injectable functionalized Demineralized Bone Matrix scaffold with glucosamine in PVA carrier, cultured in microbioreactor prior to study in rabbit model.

Using 3D model of injectable scaffolds for cartilage tissue engineering is one of the challenges that should be addressed to avoid invasive surgery for treatment. For this purpose, chondrocytes on Demineralized Bone Matrix (DBM) scaffolds functionalized with glucosamine in 20% polyvinyl alcohol (PVA) as a carrier was applied to the micro-bioreactor in-vitro, then the study was continued on in-vivo stage. Scaffold biocompatibility tests were performed and the mechanical and physicochemical properties were studied showing the fact that DBM was functionalized by Glucosamine, scaffold degradation rate was 53% after 720 h and swelling ratio was 2.5 times after 16 h, injectable scaffold demonstrated better mechanical characteristics (P < 0.05) than other concentrations of PVA. Consequently, in-vitro tests, including live-dead imaging resulting in 99% viability after 14 days (P < 0.001), DAPI staining and scanning electron microscope imaging were performed to determine the number and viability of the cells on the scaffold, showing a cells proliferation property of this group compared with the control after 14 days (P < 0.0001), then relative gene expression was evaluated and protein expression was assessed. The overall chondrogenic gene expression improved (P < 0.05) compared to the control (2D culture). Subsequently, the scaffold were loaded with chondrocytes and injected into the cartilage lesion part After 24 weeks of surgery, MRI and immunocytochemistry were performed. Then all outputs proved that the scaffold plus cell group had a significantly higher topological score (P < 0.0001) than other groups compared to normal cartilage. Finally, studies have shown that transplantation of chondrocytes in DBM, polyvinyl alcohol and glucosamine scaffold through one surgical stage improves cartilage lesion and it can be considered as a breakthrough in tissue engineering.

Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

OBJECTIVE: Testing novel biomaterials for the three dimensional (3D) culture of ovarian follicles may ultimately lead to a culture model which can support the integrity of follicles during in vitro culture (IVC). The present study reports the first application of a chitosan (CS) hydrogel in culturing mouse preantral follicles. MATERIALS AND METHODS: In this interventional experiment study, CS hydrogels with the concentrations of 0.5, 1, and 1.5% were first tested for fourier transform infrared spectroscopy (FT-IR), Compressive Strength, viscosity, degradation, swelling ratio, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and live/dead assay. Thereafter, mouse ovarian follicles were encapsulated in optimum concentration of CS (1%) and compared with those in alginate hydrogel. The follicular morphology, quality of matured oocyte and steroid secretion in both CS and alginate were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of folliculogenesis, endocrine, and apoptotic related genes was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and compared with day that in 0. RESULTS: The rates of survival, and diameter of the follicles, secretion of estradiol, normal appearance of meiotic spindle and chromosome alignment were all higher in CS group compared with those in alginate group (P≤0.05). The expression of Cyp19a1 and Lhcgr in CS group was significantly higher than that of the alginate group (P≤0.05). CONCLUSION: The results showed that CS is a permissive hydrogel and has a beneficial effect on encapsulation of ovarian follicle and its further development during 3D culture.

Simultaneous Delivery of Wharton's Jelly Mesenchymal Stem Cells and Insulin-Like Growth Factor-1 in Acute Myocardial Infarction.

Wharton's jelly mesenchymal stem cells (HWJMSCs) hold promise for myocardial regeneration, but optimal treatment regimen (preferably with a growth factor) is required to maximize functional benefits. The aim of this study was to explore the cardioprotective and angiogenesis effects of HWJMSCs combined with insulin-like growth factor-1 (IGF-1) in the treatment of acute myocardial infarction. The hydrogel consisted of Polyethylene glycol (PEG) and hyaluronic acid was prepared and characterized with regards to rheology, morphology, swelling, degradation, and release behaviors. To examine in-vivo effects, the hydrogels containing HWJMSCs either alone (Cells/hydrogel group) or with IGF-1 (Cells/hydrogel/IGF-1 group) were intra-myocardially injected into a rabbit myocardial infarction model. In-vivo efficacy was evaluated histological, immunohistochemical, echocardiography, scanning electron microscopy, and SPECT analyses. Eight weeks after infusion, the Cells/hydrogel and Cells/hydrogel/IGF-1 groups exhibited significantly increased left ventricular ejection fraction by echocardiography. Percent of ejection fraction was respectively 18.5% and 40% greater than control (P < 0.01). Vascular density (CD31 positive cells) of both treatment groups were more than the control group and this superiority was more remarkable in Cells/hydrogel/IGF-1 group. Cells/hydrogel/IGF-1 group showed the least defect size in SPECT analysis. Combinatory therapy with HWJMSCs and IGF-1 may additionally improve cardiac function and promote angiogenesis.

Effects of Endothelial and Mesenchymal Stem Cells on Improving Myocardial Function in a Sheep Animal Model.

Background: Myocardial infarction is the main cause of death worldwide. Angiogenesis, a promising new therapy for the treatment of diffuse coronary artery disease, shows a poor response to conventional revascularization techniques. This study focused on improving myocardial function using endothelial cells (ECs) and mesenchymal stem cells (MSCs) in a sheep animal model. Methods: Acute myocardial infarction was induced in 18 sheep (12 treated cases and 6 controls). Autologous MSCs and ECs were injected in the infarcted area and the border zone. Two months after transplantation, echocardiography, electron microscopy, and immunohistochemistry were performed. Results: Echocardiography in both MSC and EC groups revealed a significant improvement in the ejection fraction compared with the control group (p value < 0.05). Vascular density, estimated by antibodies against the von Willebrand factor and smooth muscle actin, increased in both study groups. The pattern of vascularity in the MSC and EC groups was diffused. The electron microscopic evaluation of the infracted areas revealed cardiomyocytes in variable stages of development in the border zone in both EC and MSC groups. Conclusion: Both ECs and MSCs were able to promote angiogenesis and improve cardiac function. Presumably, MSCs differentiate into ECs and cause angiogenesis as it occurs for ECs.

Regenerating Heart Using a Novel Compound and Human Wharton Jelly Mesenchymal Stem Cells.

BACKGROUND: Myocardial infarction is a major problem in health system and most conventional therapy is not led to restoration of the health. Stem cell therapy is a method to regenerate the heart but today appropriate cell source and scaffold selection as extracellular matrix to achieve the best effect is disputing. AIM OF THE STUDY: In this study a combination of human Wharton jelly mesenchymal stem cells (HWJMSCs) with a novel compound consisting polyethylene glycol (PEG), hyaluronic acid and chitosan is presented to heart regeneration. METHODS: After proliferation and expansion of HWJMSCs, these cells were mixed with scaffold and injected into the infarcted rabbit myocardium. After two months cardiac function and infarcted area were evaluated. Immunohistochemistry performed for vessel count and demonstrating of differentiation ability into cardiomyocytes. To confirm this ability PCR was done. Scanning electron microscope was used to evaluate angiogenesis. RESULTS: Improving cardiac function was higher in cell/scaffold group than the others and it was confirmed by SPECT results which showed least defect size in the myocardium. There were a lot of neoangiogenesis in the target group and also cardiomyogenesis observed in cell/scaffold group. PCR results confirmed the presence of differentiated cardiomyocytes and SEM showed well developed vessel in this group. CONCLUSIONS: Comparing macroscopic and microscopic results between all groups revealed that HWJMSC in combination with this scaffold led to brilliant results regarding cardiac function, angiogenesis and cardiogenesis. It is recommended using these cells and materials for cardiac tissue engineering and regeneration therapy.

Skeletal muscle regeneration via engineered tissue culture over electrospun nanofibrous chitosan/PVA scaffold.

Skeletal muscle tissue shows a remarkable potential in regeneration of injured tissue. However, in some of chronic and volumetric muscle damages, the native tissue is incapable to repair and remodeling the trauma. In the same condition, stem-cell therapy increased regeneration in situations of deficient muscle repair, but the major problem seems to be the lack of ability to attachment and survive of injected cells on the exact location. In this study, chitosan/poly(vinyl alcohol) nanofibrous scaffold was studied to promote cell attachment and provide mechanical support during regeneration. Scaffold was characterized using scanning electron microscope, X-ray diffraction, and tensile test. Degradation and swelling behavior of scaffold were studied for 20 days. The cell-scaffold interaction was characterized by MTT assay for 10 days and in vivo biocompatibility of scaffold in a rabbit model was evaluated. Results showed that cells had a good viability, adhesion, growth, and spread on the scaffold, which make this mat a desirable engineered muscular graft. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1720-1727, 2016.