In vitro cytotoxic potential of Solanum nigrum against human cancer cell lines

Plants have natural products which use to possess antiproliferative potential against many cancers. In the present study, six isolated fractions (ethyl acetate, petroleum ether, chloroform, n-butanol, ethanol and aqueous) from Solanum nigrum were evaluated for their cytotoxic effect on different cell lines. Hepatic carcinoma cell line (HepG2), cervical cancer cell line (HeLa) and baby hamster kidney (BHK) used as normal non-cancerous cells were evaluated for cytotoxicity against isolated fractions. Cell viability assay was performed to evaluate the cytotoxicity of all fractions on different cell lines followed by the lactate dehydrogenase and vascular endothelial growth factor assays of most active fraction among all screened for cytotoxic analysis. HPLC analysis of most active fractions against cytotoxicity was performed to check the biological activity of compounds. Results displayed the potent cytotoxic activity of ethyl acetate fraction of S. nigrum against HepG2 cells with IC₅₀ value of 7.89 μg/ml. Other fractions exhibited potent anticancer activity against HepG2 cells followed by HeLa cells. Fractions in our study showed no cytotoxicity in BHK cells. Cytotoxic activity observed in our current study exposed high antiproliferative potential and activity of ethyl acetate fraction against HepG2 cells. The results demonstrated that S. nigrum fractions exhibited anticancer activity against hepatic and cervical cancer cell lines with non-toxic effect in normal cells. These results reveal significant potential of S. nigrum for the therapeutic of cancers across the globe in future.     

Anticancer effects of the engineered stem cells transduced with therapeutic genes via a selective tumor tropism caused by vascular endothelial growth factor toward HeLa cervical cancer cells

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/or human interferon-beta (IFN-β) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-β was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTECs. These GESTECs transduced with CD gene and IFN-β may provide a potential of a novel gene therapy for anticervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.     

Pomegranate peel induced biogenic synthesis of silver nanoparticles and their multifaceted potential against intracellular pathogen and cancer

In the field of nano-biotechnology, silver nanoparticles (AgNPs) share a status of high repute owing to their remarkable medicinal values. Biological synthesis of environment-friendly AgNPs using plant extracts has emerged as the beneficial alternative approach to chemical synthesis. In the current study, we have synthesized biogenic silver nanoparticles (PG-AgNPs) using the peel extract of Punica granatum as a reducing and stabilizing agent. The as-synthesized PG-AgNPs were characterized and evaluated for their antibacterial and anticancer potential. UV–Visible spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the formation of biogenic PG-AgNPs. The antibacterial potential was assessed against the biofilm of Listeria monocytogenes. The PG-AgNPs were efficacious against sessile bacteria and their biofilm as well. The as-synthesized nanoparticles at sub-MIC values showed dose-dependent inhibition of biofilm formation. Corroborating results were observed under crystal violet assay, Congo red staining, Confocal microscopy and SEM analysis. The anticancer ability of the nanoparticles was evaluated against MDA-MB-231 metastatic breast cancer cells. As evident from the MTT results, PG-AgNPs significantly reduced the cell viability in a dose-dependent manner. Exposure of MDA-MB-231 cells led to the accumulation of reactive oxygen species (ROS). Morphological changes and DNA fragmentation showed the strong positive effect of PG-AgNPs on the induction of apoptosis. Collectively, the as-synthesized PG-AgNPs evolved with synergistically emerged attributes that were effective against L. monocytogenes and also inhibited its biofilm formation; moreover, the system displayed lower cytotoxic manifestation towards mammalian cells. In addition, the PG-AgNPs embodies intriguing anticancer potential against metastatic breast cancer cells.     

Identification of alpha-enolase as a potential immunogenic molecule during allogeneic transplantation of human adipose-derived mesenchymal stromal cells

Background aimsGiven their low immunogenicity, immunoregulatory effects and multiple differentiation capacity, mesenchymal stromal cells (MSCs) have the potential to be used for “off-the-shelf” cell therapy to treat various diseases. However, the allorejection of MSCs indicates that they are not fully immune-privileged. In this study, the authors investigated the immunogenicity of human adipose-derived MSCs (Ad-MSCs) and identified potential immunogenic molecules.MethodsTo evaluate the immunogenicity of human Ad-MSCs in vivo, cells were transplanted into humanized mice (hu-mice), then T-cell infiltration and clearance of human Ad-MSCs were observed by immunofluorescence and bioluminescence imaging. One-way mixed lymphocyte reaction and flow cytometry were performed to evaluate the immunogenicity of human Ad-MSCs in vitro. High-throughput T-cell receptor (TCR) repertoire sequencing and mass spectrometry were applied to identified potential immunogenic molecules.ResultsThe authors observed that allogeneic Ad-MSCs recruited human T cells and caused faster clearance in hu-mice than non-humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The proliferation and activation of T cells were significantly enhanced during in vitro co-culture with human Ad-MSCs. In addition, the level of HLA-II expression on human Ad-MSCs was dramatically increased after co-culture with human peripheral blood mononuclear cells (PBMCs). High-throughput sequencing was applied to analyze the TCR repertoire of the Ad-MSC-recruited T cells to identify dominant TCR CDR3 sequences. Using synthesized TCR CDR3 peptides, the authors identified several potential immunogenic candidates, including alpha-enolase (ENO1). The ENO1 expression level of Ad-MSCs significantly increased after co-culture with PBMCs, whereas ENO1 inhibitor (ENOblock) treatment decreased the expression level of ENO1 and Ad-MSC-induced proliferation of T cells.ConclusionsThe authors’ findings improve the understanding of the immunogenicity of human Ad-MSCs and provide a theoretical basis for the safe clinical application of allogeneic MSC therapy.     

Comparative Evaluation of Nimesulide-Loaded Nanoparticles for Anticancer Activity Against Breast Cancer Cells

Recent clinical and epidemiological researches have declared that non-steroidal anti-inflammatory agents may display as antineoplastic agents and indicate pro-apoptotic and antiproliferative effects on cancer cells. The major purpose of this research was to develop a novel poly(ethyleneglycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nano-sized particles encapsulated with nimesulide (NMS), a selective COX-2 inhibitor, and to evaluate its anticancer activity against MCF-7 breast cancer cells. NMS-encapsulated PEG-b-PCL nanoparticles were fabricated using three different production techniques: (i) by emulsion-solvent evaporation using a high shear homogenizer, (ii) by emulsion-solvent evaporation using an ultrasonicator, and (iii) by nanoprecipitation. Nanoparticles were evaluated with respect to the entrapment efficiency, size characteristics, drug release rates, thermal behavior, cell viability assays, and apoptosis. The resulting nanoparticles were found to be spherical shapes with negative surface charges. The average diameter of all nanoparticles ranged between 148.5 and 307.2 nm. In vitro release profiles showed that all nanoparticles exhibited a biphasic release pattern. NMS-loaded PEG-b-PCL nanoparticles demonstrated significant anticancer activity against MCF-7 breast cancer cells in a dose-dependent manner, and the effects of nanoparticles on cell proliferation were significantly affected by the preparation techniques. The nanoparticles developed in this work displayed higher potential for the NMS delivery against breast cancer treatment for the future.     

Soursop leaves (Annona muricata L.) endophytic fungi anticancer activity against HeLa cells

Cervical cancer causes many deaths in females worldwide, including in Indonesia. Several studies have reported that soursop (Annona muricata L.) leaves can be used to treat cervical cancer. This study aims to determine the use of endophytic fungi of A. muricata leaves extract as an ingredient that inhibits cervical cancer. The isolated endophytic fungi from various soursop leave accessions were grown in culture media, then extracted using ethyl acetate. The extract was then tested against anti-yeast, cervical cancer cells, and on normal cells as control using the MTT method. Five isolated fungi were selected based on the greatest inhibition in one concentration, and the inhibitory concentration 50 (IC₅₀) value was determined. The soursop leaves endophytic fungi extracts showed cytotoxicity against cervical cancer cells by inhibiting the multiplication of HeLa cancer cells in vitro. The Sir-SM2 endophytic fungi crude ethyl acetate extract showed high cytotoxicity to cervical cancer cells (HeLa cells) but less harmful to the normal Chang cells; therefore can be a natural anticancer. Identification based on morphology shows that the isolated Sir-SM2 endophytic fungi belong to the Penicillium genus, and molecular identification based on Internal Transcribed Spacer shows high similarities with Penicillium crustosum.     

In vitro anticancer activity of extracted oil from Parrotiopsis jacquemontiana (Decne) Rehder

BackgroundParrotiopsis jacquemontiana, commonly referred to as “Beranj” in the local community, is widely used traditionally and has numerous health benefits. However, no studies have been conducted to investigate its anticancer potential, particularly its extracted oil.PurposeThe present study was put forth to appraise the anticancer potential of Parrotiopsis jacquemontiana extracted oil against liver (hcclm3 and hepg2) and breast cancer (mda-mb 231 and mcf-7) cell lines relative to normal cell lines (lo2 and mcf-10a) via MTT assay.MethodsFlow cytometry indicated the apoptotic effect whereas invasion and migration capabilities of oil against cancer cells were determined by Matrigel invasion chamber and wound-scratch assays.ResultsThe results of oil revealed a time and dose-dependent increase in cell proliferation inhibition, conferring to least IC50 shown against hcclm3 (144.9 ± 0.75 μg/ml) and mda-mb 231 (145.7 ± 0.32 μg/ml) cell line at 72 h, whereas no cytotoxic effect on normal cells was observed. In addition, the oil significantly (p < 0.001) suppressed the migration and invasion of hcclm3 and mda-mb 231 cells, showing noteworthy anti-metastatic potential. Furthermore, cell death was confirmed by Annexin‒V/PI staining where the maximum apoptotic percentage was calculated for oil (200 μg/ml) alongside mda-mb 231 conferring to 15.36 ± 1.22, 26.7 ± 1.2, and 36.43 ± 1.65 at 24, 48, and 72 h whereas 12.33 ± 1.05, 19.36 ± 1.62, and 29.3 ± 0.79 was recorded alongside hcclm3 at similar time intervals, respectively.ConclusionIn conclusion, the extracted oil exhibited strong anti-proliferative, anti-metastatic, and apoptotic effects and therefore may have potential applications in cancer treatment, however, further studies of oil regarding the action mechanisms and compounds involved in anticancer therapy are necessary.     

Cytotoxic activity of hirsutanone,a diarylheptanoid isolated from Alnus glutinosa leaves

BackgroundThe low efficacy of cancer therapy for the treatment of patients with advanced disease makes the development of new anticancer agents necessary. Because natural products are a significant source of anticancer drugs, it is important to explore cytotoxic activity of novel compounds from natural origin.PurposeThe aim of this work is to evaluate the cytotoxic capacity of hirsutanone, a diarylheptanoid isolated from Alnus glutinosa leaves. Hirsutanone cytotoxic way of action was also studied.Material and methodsThe cytotoxic ability of Alnus glutinosa leaves ethyl acetate extract was studied over HeLa and PC-3 cell lines, with the MTT colorimetric assay. Hirsutanone was isolated from this extract using chromatographic methods, and its structure elucidated by spectroscopic analysis. HT-29 cell viability after hirsutanone treatment was determined using SRB assay. In order to understand hirsutanone way of action, cytotoxicity was evaluated adding the diarylheptanoid and antioxidants. DNA topoisomerase II (topo II) poison activity, was also evaluated using purified topo II and a supercoiled form of DNA that bears specific topo II recognition and binding region; topo II poisons stabilize normally transient DNA-topo II cleavage complexes, and lead an increased yield of linear form as a consequence of a lack of double-strand breaks rejoining.ResultsThe diarylheptanoid hirsutanone was isolated from Alnus glutinosa (L.) Gaertn. (Betulaceae) leaves extract that showed cytotoxic activity against PC-3 and HeLa cell lines. Hirsutanone showed cytotoxic activity against HT-29 human colon carcinoma cells. Pre-treatment with the antioxidants NAC (N-acetylcysteine) and MnTMPyP (Mn(III)tetrakis-(1-methyl-4-pyridyl)porthyrin) reduced this activity, suggesting that reactive oxygen species (ROS) participate in hirsutanone-induced cancer cell death. Using human topo II and a DNA supercoiled form, hirsutanone was found to stabilize topo II-DNA cleavage complexes, acting as a topo II poison.ConclusionOur data suggest that, like curcumin, an induction of oxidative stress and topo II-mediated DNA damage may play a role in hirsutanone-induced cancer cell death. Since both compounds share similar structure and cytotoxic profile, and curcumin is in clinical trials for the treatment of cancer, our results warrant further studies to evaluate the anticancer potential of hirsutanone.     

流式细胞仪分选人外周血T淋巴细胞的方法建立与评价

目的:利用流式细胞仪同时分离外人周血单个核细胞中T淋巴细胞并检测其分离纯度及存活率。方法:本文采用流式细胞仪同时分选人外周血CD4~+、CD8~+T淋巴细胞为例,推而广之,采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞,采用流式细胞仪同时分选CD4~+、CD8~+T淋巴细胞,分离细胞再通过流式细胞仪回测其分离纯度并通过台盼蓝染色检测分离细胞的存活率。结果:采用此方法能有效人外周血细胞CD4~+、CD8~+T淋巴细胞,分选前CD4~+淋巴细胞纯度为(50.5±11.5)%、CD8~+T淋巴细胞纯度为纯度为(15.4±7.1)%;分选后CD4~+T淋巴细胞纯度为(94.3±1.3)%、CD8~+T淋巴细胞纯度为(93.6±1.6)%;分选后CD4~+T淋巴细胞存活率为(95.3±1.8)%,CD8~+T淋巴细胞存活率为(94.8±1.5)%,细胞的形态完整。结论:采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞后利用流式细胞仪分选的方法能够高效、快速的分离人外周血CD4~+、CD8~+T淋巴细胞,且存活率高,为进一步研究其功能提供了保证。采用不同的荧光抗体标记其他淋巴细胞亚群,也能高效、快速的分离出细胞。     

Biogenic synthesis of gold nanoparticles using Commiphora wightii and their cytotoxic effects on breast cancer cell line (MCF-7)

The current study aimed at developing gold nanoparticles (AuNPs) using the aqueous extract of the medicinal plant Commiphora wightii. The phytosynthesized gold nanoparticles (Cw@AuNPs) were evaluated for their anticancer activity against MCF-7 breast cancer cell model. The formation of AuNPs by Commiphora wightii leaf extract was confirmed by UV–vis spectra where their surface plasmon resonance was found at 533 nm. Further characterization of Cw@AuNPs was done by transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX) analysis, and fourier-transform infrared spectroscopy (FTIR) analysis. In vitro anticancer potential of thus obtained AuNPs was evaluated against MCF-7 and where the IC₅₀ was found to be 66.11 μg/mL Further, apoptotic studies were carried out using ethidium bromide dual staining, DNA fragmentation, comet assay, and flow cytometry studies. Results revealed that Cw@AuNPs at higher concentration significantly increased the apoptotic cells when compared to control cells. Cell cycle analysis of MCF-7 cells confirmed the cell cycle arrest at G2/M phase. These results demonstrate that the biosynthesized Cw@AuNPs appear to be promising for therapeutical applications against breast cancer.     

Zinc oxide nanoparticles from Cassia auriculata flowers showed the potent antimicrobial and in vitro anticancer activity against the osteosarcoma MG-63 cells

Osteosarcoma (OS) is a foremost mesenchymal bone neoplasm and it can occur at any age with survival rate is nearly 2–8 times lesser in elders than in teenagers. The clinical therapies for cancer treatment have gradually becoming outdated because of the developments of nano-medicine and multi-targeted drug-delivery. In this work, we green synthesized the zinc oxide nanoparticles from the Cassia auriculata flower (AS-ZnONPs) extract and evaluated its antimicrobial and in vitro anticancer potential against the OS MG-63 cells. The synthesized AS-ZnONPs were confirmed and characterized by using UV–vis spectroscopy, XRD, FE-SEM, and photoluminescence techniques. The antimicrobial activity of AS-ZnONPs was studied by disc diffusion technique. The viability of AS-ZnONPs treated MG-63 cells were examined by MTT assay. The apoptotic cells in the AS-ZnONPs treated MG-63 cells were assayed by dual staining. The MMP status of AS-ZnONPs treated cells were tested by Rh-123 staining. The cell adhesion assay was performed to detect the anticancer effects of AS-ZnONPs against MG-63 cells. The results of UV–vis spectroscopy, XRD, FE-SEM, and photoluminescence techniques proved the formation of AS-ZnONPs and it has the hexagonal wurtzite structures. AS-ZnONPs displayed the potent antimicrobial activity against the tested microbial strains. The AS-ZnONPs were appreciably inhibited the cell viability of MG-63 cells. The outcomes of fluorescence staining proved that AS-ZnONPs reduced the MMP and prompted the apoptosis in MG-63 cells. In conclusion, our discoveries demonstrated that the formulated AS-ZnONPs has the potent antimicrobial and in vitro anticancer activity against the MG-63 cells. The AS-ZnONPs could be potent chemotherapeutic agent in the future to treat the OS.     

Unveiling the anticancer and antimycobacterial potentials of bioengineered gold nanoparticles

Synthesis of gold nanoparticles was carried out using Pongammia pinnata (pongam) leaf extract and their anticancer and antimycobacterial activities were studied. Gold nanoparticle formation was confirmed by UV–vis, XRD and HR-TEM. The anticancer efficacies of the biogenic gold nanoparticles were analyzed using cytotoxicity, cell morphology analysis, oxidative DNA damage, apoptosis detection and toxicity studies. Biogenic gold nanoparticles inhibited breast cancer cell line (MCF-7) proliferation with an efficacy of IC₅₀ of 1.85 μg/mL. The antimycobacterial potential of the biogenic gold nanoparticles was screened against M. tuberculosis by Luciferase Reporter Phage (LRP) assay. The gold nanoparticles showed inhibition against sensitive M. tuberculosis with the minimum inhibitory concentration (MIC) of 10 μg/mL whereas no inhibition was found against the rifampicin resistant M. tuberculosis.     

NMK-BH2, a novel microtubule-depolymerising bis (indolyl)-hydrazide-hydrazone,induces apoptotic and autophagic cell death in cervical cancer cells by binding to tubulin at colchicine – site

BackgroundMicrotubules, the key components of the eukaryotic cytoskeleton and mitotic spindle, are one of the most sought-after targets for cancer chemotherapy, especially due to their indispensible role in mitosis. Cervical cancer is a prevalent malignancy among women of developing countries including India. In spite of the remarkable therapeutic advancement, the non-specificity of chemotherapeutic drugs adversely affect the patients' survival and well-being, thus, necessitating the quest for novel indole-based anti-microtubule agent against cervical cancer, with high degree of potency and selectivity.MethodsFor in vitro studies, we used MTT assay, confocal microscopy, fluorescence microscopy, flow cytometry and Western blot analysis. Study in cell free system was accomplished by spectrophotometry, fluorescence spectroscopy and TEM and computational analysis was done by AutodockTools 1.5.6.ResultsNMK-BH2 exhibited significant and selective anti-proliferative activity against cervical cancer HeLa cells (IC₅₀ = 1.5 μM) over normal cells. It perturbed the cytoskeletal and spindle microtubules of HeLa cells leading to mitotic block and cell death by apoptosis and autophagy. Furthermore, NMK-BH2 targeted the tubulin-microtubule system through fast and strong binding to the αβ-tubulin heterodimers at colchicine-site.ConclusionThis study identifies and characterises NMK-BH2 as a novel anti-microtubule agent and provides insights into its key anti-cancer mechanism through two different cell death pathways: apoptosis and autophagy, which are mutually independent.General significanceIt navigates the potential of the novel bis (indolyl)-hydrazide-hydrazone, NMK-BH2, to serve as lead for development of new generation microtubule-disrupting chemotherapeutic with improved efficacy and remarkable selectivity towards better cure of cervical cancer.     

Silver nanoparticles from <Emphasis Type="Italic">Dendropanax morbifera</Emphasis> Léveille inhibit cell migration,induce apoptosis,and increase generation of reactive oxygen species in A549 lung cancer cells

Green synthesized silver nanoparticles have significant potential in the pharmaceutical field because of their biological functions such as antioxidant and anticancer activities. Novel silver nanoparticles synthesized from Dendropanax morbifera Léveille leaves (D-AgNPs) exhibit antimicrobial activity and reduce the viability of cancer cells without affecting the viability of RAW 264.7 macrophage-like cells. In this study, we evaluated the anticancer effect of D-AgNPs by measuring the levels of reactive oxygen species (ROS) production and toxicity against A549 and HepG2 cell lines. The effect of D-AgNPs on cell migration, induction of apoptosis, and modification of gene and/or protein expression of cancer-related markers was determined using A549 cells. D-AgNPs exhibited cytotoxicity in A549 and HepG2 cell at different concentrations and enhanced the production of ROS in both cell lines. An increase in cell apoptosis and a reduction in cell migration in A549 cells were also observed after D-AgNP treatment. Furthermore, the effect of D-AgNPs in A549 cells was shown to be related to modification of the EGFR/p38 MAPK pathway. Our data provide the first evidence supporting the potential of D-AgNPs as a possible anticancer agent, particularly for the treatment of non-small cell lung carcinoma.     

The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells

BackgroundAbietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells.MethodsThe cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry.ResultsParvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-x_L did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation.ConclusionParvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.     

Peripheral blood derived mononuclear cells enhance osteoarthritic human chondrocyte migration

IntroductionA major problem in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into defects. Cartilage is essentially avascular and therefore its healing is not considered to involve mononuclear cells. Peripheral blood derived mononuclear cells (PBMC) offer a readily available autologous cell source for clinical use and therefore this study was designed to evaluate the effects of PBMCs on chondrocytes and cartilage.MethodsHuman primary chondrocytes and cartilage tissue explants were taken from patients undergoing total knee replacement (n = 17). Peripheral blood samples were obtained from healthy volunteers (n = 12) and mononuclear cells were isolated by density-gradient centrifugation. Cell migration and chemokinetic potential were measured using a scratch assay, xCELLigence and CyQuant assay. PCR array and quantitative PCR was used to evaluate mRNA expression of 87 cell motility and/or chondrogenic genes.ResultsThe chondrocyte migration rate was 2.6 times higher at 3 hour time point (p < 0.0001) and total number of migrating chondrocytes was 9.7 times higher (p < 0.0001) after three day indirect PBMC stimulus and 8.2 times higher (p < 0.0001) after three day direct co-culture with PBMCs. A cartilage explant model confirmed that PBMCs also exert a chemokinetic role on ex vivo tissue. PBMC stimulation was found to significantly upregulate the mRNA levels of 2 chondrogenic genes; collagen type II (COL2A1 600–fold, p < 0.0001) and SRY box 9 (SOX9 30–fold, p < 0.0001) and the mRNA levels of 7 genes central in cell motility and migration were differentially regulated by 24h PBMC stimulation.ConclusionThe results support the concept that PBMC treatment enhances chondrocyte migration without suppressing the chondrogenic phenotype possibly via mechanistic pathways involving MMP9 and IGF1. In the future, peripheral blood mononuclear cells could be used as an autologous point-ofcare treatment to attract native chondrocytes from the diseased tissue to aid in cartilage repair.     

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants

BackgroundThis study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids.MethodsA differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously.ResultsThe screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%.ConclusionThis study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.     

Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

Background

Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs).

Methods

PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.

Results

The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34⁺VEGFR2⁺CD133⁺ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.

Conclusion

Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34⁺VEGFR2⁺CD133⁺ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.