Search results for: cytometry analysis

Authors: Teodora Mocan, Matea Cristian, Cornel Iancu, Flaviu A. Tabaran, Florin Zaharie, Bartos Dana, Lucian Mocan

Abstract:

Colon cancer (CC) a lethal human malignancy, is one of the most commonly diagnosed cancer. With its high increased mortality rate, as well as low survival rate combined with high resistance to chemotherapy CC, represents one of the most important global health issues. In the presented research, we have developed a distinct nanostructured colon carcinoma vaccine model based on a nano-biosystem composed of 39 nm gold nanoparticles conjugated to colon cancer epitopes. We prove by means of proteomic analysis, immunocytochemistry, flow cytometry and hyperspectral microscopy that our developed nanobioconjugate was able to contribute to an optimal prophylactic effect against CC by promoting major histocompatibility complex mediated (MHC) antigen presentation by dendritic cells. We may conclude that the proposed immunoprophylactic approach could be more effective than the current treatments of CC because it promotes recognition of the tumoral antigens by the immune system.

Keywords: anticancer vaccine, colon cancer, gold nanoparticles, tumor antigen

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Authors: Bhargavi Santebennur Dwarakanath, Praveen Vadakke Kamath, Savitha Janakiraman

Abstract:

Cervical cancer is one of the leading causes of mortality in women and is the second most common malignancy worldwide. Lovastatin, a non polar, anticholesterol drug which also exerts antitumour activity in vitro. In the present study, lovastatin from Aspergillus terreus (KM017963) was purified by adsoprtion chromatography and evaluated for its anticancer and anti-oxidant properties in human cervical cancer cell lines (HeLa). The growth inhibitory and proapoptotic effects of purified lovastatin on HeLa cell lines were investigated by determining its influence on cytotoxicity, Mitochondrial Membrane Potential (MMP), DNA fragmentation and antioxidant property (Hydroxy radical scavenging effect and the levels of total reduced glutathione). Flow cytometry analysis by propidium iodide staining confirmed the induction of apoptotic cell death and revealed cell cycle arrest at G0/G1 phase. Results of the study give leads for anticancer effects of lovastatin and its potential efficacy in the chemotherapy of cervical cancer.

Keywords: apoptosis, Aspergillus terreus, cervical cancer, lovastatin

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Authors: M. Gowri, E. K. Girija, V. Ganesh

Abstract:

Background and Significance: Among the dental infections, inflammation and infection of the root canal are common among all age groups. Currently, the management of root canal infections involves cleaning the canal with powerful irrigants followed by intracanal medicament application. Though these treatments have been in vogue for a long time, root canal failures do occur. Treatment for root canal infections is limited due to the anatomical complexity in terms of small micrometer volumes and poor penetration of drugs. Thus, infections of the root canal seem to be a challenge that demands development of new agents that can eradicate E. faecalis. Methodology: In the present study, we synthesized and screened silver-curcumin nanoparticle against E. faecalis. Morphological cell damage and antibiofilm activity of silver-curcumin nanoparticle on E. faecalis was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model. Results: Screening data showed that silver-curcumin nanoparticle was active against E. faecalis. silver-curcumin nanoparticle exerted time kill effect. Further, SEM images of E. faecalis showed that silver-curcumin nanoparticle caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed eradication of E. faecalis and reduction in colony forming unit (CFU) after 24 h treatment in the infected tooth samples in this model. Further, silver-curcumin nanoparticle was found to be hemocompatible, not cytotoxic to normal mammalian NIH 3T3 cells and non-mutagenic. Conclusion: The results of this study can pave the way for developing new antibacterial agents with well deciphered mechanisms of action and can be a promising antibacterial agent or medicament against root canal infection.

Keywords: ex vivo dentine model, inhibition of biofilm formation, root canal infection, silver-curcumin nanoparticle

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Authors: Lukasz Szydlowski, Jiri Ehlich, Igor Goryanin

Abstract:

We demonstrate a single chamber, 96-well-plated based Microbial Fuel Cell (MFC) with printed, electronic components. This invention is aimed at robust selection of electrogenic microbial community under specific conditions, e.g., electrode potential, pH, nutrient concentration, salt concentration that can be altered within the 96 well plate array. This invention enables robust selection of electrogenic microbial community under the homogeneous reactor, with multiple conditions that can be altered to allow comparative analysis. It can be used as a standalone technique or in conjunction with other selective processes, e.g., flow cytometry, microfluidic-based dielectrophoretic trapping. Mobile conductive elements, like carbon paper, carbon sponge, activated charcoal granules, metal mesh, can be inserted inside to increase the anode surface area in order to collect electrogenic microorganisms and to transfer them into new reactors or for other analytical works. An array of 96-well plate allows this device to be operated by automated pipetting stations.

Keywords: bioengineering, electrochemistry, electromicrobiology, microbial fuel cell

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Authors: M. Gowri, E. K. Girija, V. Ganesh

Abstract:

Background and Significance: Among the dental infections, inflammation and infection of the root canal are common among all age groups. Currently, the management of root canal infections involves cleaning the canal with powerful irrigants followed by intracanal medicament application. Though these treatments have been in vogue for a long time, root canal failures do occur. Treatment for root canal infections is limited due to the anatomical complexity in terms of small micrometer volumes and poor penetration of drugs. Thus, infections of the root canal seem to be a challenge that demands development of new agents that can eradicate C. albicans. Methodology: In the present study, we synthesized and screened silver-curcumin nanoparticle against Candida albicans. Detailed molecular studies were carried out with silver-curcumin nanoparticle on C. albicans pathogenicity. Morphological cell damage and antibiofilm activity of silver-curcumin nanoparticle on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model. Results: Screening data showed that silver-curcumin nanoparticle was active against C. albicans. Silver-curcumin nanoparticle exerted time kill effect and post antifungal effect. When used in combination with fluconazole or nystatin, silver-curcumin nanoparticle revealed a minimum inhibitory concentration (MIC) decrease for both drugs used. In-depth molecular studies with silver-curcumin nanoparticle on C. albicans showed that silver-curcumin nanoparticle inhibited yeast to hyphae (Y-H) conversion. Further, SEM images of C. albicans showed that silver-curcumin nanoparticle caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, the antifungal activity of silver-curcumin nanoparticle was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed eradication of C. albicans and reduction in colony forming unit (CFU) after 24 h treatment in the infected tooth samples in this model. Conclusion: The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.

Keywords: C. albicans, ex vivo dentine model, inhibition of biofilm formation, root canal infection, yeast to hyphae conversion inhibition

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Authors: Debasish Pradhan, Shaktiprasad Pradhan, Rakesh Kumar Pradhan, Gitanjali Tripathy

Abstract:

Suppressors of cytokine signalling (SOCS1), a newly indentified antiapoptotic molecule is a downstream effector of the receptor tyrosine kinase-Ras signalling pathway. The current study has uncovered that SOCS1 may have wide and imperative capacities, particularly because of its close correlation with malignant tumors. To investigate the impact of SOCS1 on MDR, we analyzed the expression of P-gp and SOCS1 by immunohistochemistry and found there was a positive correlation between them. At that point, we effectively interfered with RNA translation by the contamination of siRNA of SOCS1 into MCF7/ADM breast cancer cell lines through a lentivirus, and the expression of the target gene was significantly inhibited. After RNAi, the drug resistance was reduced altogether and the expression of MDR1 mRNA and P-gp in MCF7/ADM cell lines demonstrated a significant decrease. Likewise, the expression of P53 protein increased in a statistically significant manner (p ≤ 0.01) after RNAi exposure. Moreover, flow cytometry analysis uncovers that cell cycle and anti-apoptotic enhancing capacity of cells changed after RNAi treatment. These outcomes proposed SOCS1 may take part in breast cancer MDR by managing MDR1 and P53 expression, changing cell cycle and enhancing the anti-apoptotic ability.

Keywords: breast cancer, multidrug resistance, SOCS1 gene, MDR1 gene, RNA interference

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Authors: Ajay Kumar, Satwinderjeet Kaur

Abstract:

Onosma bracteata Wall. (Boraginaceae), is known to be a medicinal plant, useful in the treatment of body swellings, abdominal pain and urinary calculi, etc. The present study focused on the radical scavenging and cancer growth inhibitory properties of isolates from O. bracteata. Obea fraction demonstrated noticeable free radical scavenging ability along with antiproliferative activity in human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung cancer A549 cell lines using MTT assay with GI50 values of 88.56, 101.61 and 112.7 μg/ml, respectively. The scanning electron and confocal microscopy studies showed morphological alterations including nuclear condensation and formation of apoptotic bodies in osteosarcoma MG-63 cells. Obea fraction in osteosarcoma MG-63 cells augmented the reactive oxygen species (ROS) level and decreased the mitochondrial membrane potential. Flow cytometry analysis revealed the Obea treated cells to be arrested in the G0/G1 phase in a dose dependent manner supported by the observed increase in the early apoptotic cell population. Western blotting analysis showed that the expression of p-NF-kB, COX-2, p-Akt, and Bcl-xL decreased whereas, the expression of GSK-3β, p53, caspase-3 and caspase-9 proteins increased. The downregulation of Bcl-2, Cyclin E, CDK2 and mortalin gene expression and upregulation of p53 genes was unfolded in RT-qPCR studies. The presence of catechin, kaempferol, Onosmin A and epicatechin, as revealed in high-performance liquid chromatography (HPLC) studies, contributes towards the chemopreventive potential of O. bracteata which can be tapped for chemotherapeutic use.

Keywords: apoptosis, confocal microscopy, HPLC, mitochondria membrane potential, reactive oxygen species

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

Abstract:

For chemotherapy, a drug delivery system should be able to specifically target cancer cells and deliver the therapeutic dose without affecting normal cells. Folate receptors (FR) can be considered key targets since they are commonly over-expressed in cancer cells and they are the molecular marker used in this study. Here, cyclodextrin (CD) has being studied as a vehicle for delivering the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within the CD cavity. In this study, β-CD has been modified using folic acid so as to specifically target the FR molecular marker. Thus, the system studied here for drug delivery consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 9.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 10.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 8.5 for A549 and 132.6 µM ± 12.1 and 288.1 µM ± 16.3 for BEAS-2B. These results demonstrate that MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug.

Keywords: cyclodextrins, cancer treatment, drug delivery, folate receptors, reduced folate carriers

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Authors: Sahar Rakhshan, Simonetta Geninatti Crich, Diego Alberti, Rachele Stefania

Abstract:

The combination of imaging and therapeutic agents in the same smart nanoparticle is a promising option to perform a minimally invasive imaging guided therapy. In this study, Low density lipoproteins (LDL), one of the most attractive biodegradable and biocompatible nanoparticles, were used for the simultaneous delivery of Paclitaxel (PTX), a hydrophobic antitumour drug and an amphiphilic contrast agent, Gd-AAZTA-C17, in B16-F10 melanoma cell line. These cells overexpress LDL receptors, as assessed by Flow cytometry analysis. PTX and Gd-AAZTA-C17 loaded LDLs (LDL-PTX-Gd) have been prepared, characterized and their stability was assessed under 72 h incubation at 37 ◦C and compared to LDL loaded with Gd-AAZTA-C17 (LDL-Gd) and LDL-PTX. The cytotoxic effect of LDL-PTX-Gd was evaluated by MTT assay. The anti-tumour drug loaded into LDLs showed a significantly higher toxicity on B16-F10 cells with respect to the commercially available formulation Paclitaxel Kabi (PTX Kabi) used in clinical applications. It was possible to demonstrate a high uptake of LDL-Gd in B16-F10 cells. As a consequence of the high cell uptake, melanoma cells showed significantly high cytotoxic effect when incubated in the presence of PTX (LDL-PTX-Gd). Furthermore, B16-F10 have been used to perform Magnetic Resonance Imaging. By the analysis of the image signal intensity, it was possible to extrapolate the amount of internalized PTX indirectly by the decrease of relaxation times caused by Gd, proportional to its concentration. Finally, the treatment with PTX loaded LDL on B16-F10 tumour bearing mice resulted in a marked reduction of tumour growth compared to the administration of PTX Kabi alone. In conclusion, LDLs are selectively taken-up by tumour cells and can be successfully exploited for the selective delivery of Paclitaxel and imaging agents.

Keywords: low density lipoprotein, melanoma cell lines, MRI, paclitaxel, personalized medicine application, theragnostic System

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Authors: Ferreira RC, Simons HZ, Thompson WS, Cutler AJ, Dopico XC, Smyth DJ, Mashar M, Schuilenburg H, Walker NM, Dunger DB, Wallace C, Todd JA, Wicker LS, Pekalski ML

Abstract:

Type 1 diabetes is caused by autoimmune destruction of insulin-secreting beta cells in the pancreas. T cells are known to play an important role in this immune-mediated destruction; however, there is no general consensus regarding alterations in cytokine production or T cell subsets in peripheral blood of patients with type 1 diabetes. Using polychromatic flow cytometry of peripheral blood mononuclear cells (PBMCs), we assessed production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 by memory CD4 T effector (Teff) cells in 69 patients with type 1 diabetes and 61 healthy donors. We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). In a separate cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. Consistent with the increased production of IL-21, we also found a 14.9% increase in circulating Tfh cells in the patients with type 1 diabetes (95% CI 2.9, 26.9; p = 0.016). Analysis of IL-21 production by PBMCs from a subset of 46 of the 62 donors immunophenotyped for Tfh showed that frequency of Tfh cells was associated with the frequency of IL-21+ cells (r2 = 0.174, p = 0.004). These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.

Keywords: T follicular helper cell, IL-21, IL-17, type 1 diabetes

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Authors: Florian Billing, Soren Segan, Meike Jakobi, Elsa Arefaine, Aliki Jerch, Xin Xiong, Matthias Becker, Thomas Joos, Burkhard Schlosshauer, Ulrich Rothbauer, Nicole Schneiderhan-Marra, Hanna Hartmann, Christopher Shipp

Abstract:

The immune response plays a major role in implant biocompatibility, but an understanding of how to design biomaterials for specific immune responses is yet to be achieved. We aimed to better understand how changing certain material properties can drive immune responses. To this end, we tested immune response to experimental implant coatings that vary in specific characteristics. A layer-by-layer approach was employed to vary surface charge and wettability. Human-based in vitro models (THP-1 macrophages and primary peripheral blood mononuclear cells (PBMCS)) were used to assess immune responses using multiplex cytokine analysis, flow cytometry (CD molecule expression) and microscopy (cell morphology). We observed dramatic differences in immune response due to specific alterations in coating properties. For example altering the surface charge of coating A from anionic to cationic resulted in the substantial elevation of the pro-inflammatory molecules IL-1beta, IL-6, TNF-alpha and MIP-1beta, while the pro-wound healing factor VEGF was significantly down-regulated. We also observed changes in cell surface marker expression in relation to altered coating properties, such as CD16 on NK Cells and HLA-DR on monocytes. We furthermore observed changes in the morphology of THP-1 macrophages following cultivation on different coatings. A correlation between these morphological changes and the cytokine expression profile is ongoing. Targeted changes in biomaterial properties can produce vast differences in immune response. The properties of the coatings examined here may, therefore, be a method to direct specific biological responses in order to improve implant biocompatibility.

Keywords: biomaterials, coatings, immune system, implants

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Authors: O. S. Adeyemi, C. G. Whiteley

Abstract:

The present study determined the toxic potential of metal nanoparticles in cell culture system. Silver and gold nanoparticles were synthesized and characterized following established "green" protocols. The synthesized nanoparticles, in varying concentrations ranging from 0.1–100 µM were evaluated for toxicity in metastatic MDA-MB-231 cells. The nanoparticles promoted a generation of reactive oxygen species and reduced cell viability to less than 50% in the demonstration of cellular toxicity. The nanoparticles; gold and the silver-gold mixture had IC50 values of 56.65 and 18.44 µM respectively. The IC50 concentration for silver nanoparticles could not be determined. Furthermore, the probe of the cell death using flow cytometry and confocal microscopy revealed the partial involvement of apoptosis as well as necrosis. Our results revealed cellular toxicity caused by the nanoparticles but the mechanism remains yet undefined.

Keywords: cell death, nanomedicine, nanotoxicology, toxicity

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Authors: Otilia Ruxandra Vasile, Ecaterina Andronescu, Cristina Daniela Ghitulica, Bogdan Stefan Vasile, Roxana Trusca, Eugeniu Vasile, Alina Maria Holban, Carmen Mariana Chifiriuc, Florin Iordache, Horia Maniu

Abstract:

Engineered powders offer great promise in several applications, but little information is known about cytotoxicity effects. The aim of the current study was the synthesis and cytotoxicity examination of silver powders using pyrosol method at temperatures of 600°C, 650°C and 700°C, respectively sol-gel method and calcinations at 500°C, 600°C, 700°C and 800°C. We have chosen to synthesize and examine silver particles cytotoxicity due to its use in biological applications. The synthesized Ag powders were characterized from the structural, compositional and morphological point of view by using XRD, SEM, and TEM with SAED. In order to determine the influence of the synthesis route on Ag particles cytotoxicity, different sizes of micro and nanosilver synthesized powders were evaluated for their potential toxicity. For the study of their cytotoxicity, cell cycle and apoptosis have been done analysis through flow cytometry on human colon carcinoma cells and mesenchymal stem cells and through the MTT assay, while the viability and the morphological changes of the cells have been evaluated by using cloning studies. The results showed that the synthesized silver nanoparticles have displayed significant cytotoxicity effects on cell cultures. Our synthesized silver powders were found to present toxicity in a synthesis route and time-dependent manners for pyrosol synthesized nanoparticles; whereas a lower cytotoxicity has been measured after cells were treated with silver nanoparticles synthesized through sol-gel method.

Keywords: Ag, cytotoxicity, pyrosol method, sol-gel method

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Authors: Azzam H., Abousamra N. K., Wafa A. A., Hafez M. M., El-Gilany A. H.

Abstract:

Platelet activation occurs in peripheral blood of patients with rheumatic mitral stenosis (MS) and atrial fibrillation (AF) and could be related to abnormal thrombogenesis. The CD40/CD40 ligand (CD40L) which reflects platelet activation, mediate a central role in thrombotic diseases. However, the role of CD40/CD40L system in rheumatic MS with or without AF remains unclear. Expressions of CD40 on monocytes and CD40L on platelets were determined by whole blood flow cytometry and serum levels of soluble CD40L were measured by enzyme-linked immunosorbent assay in group 1 (19 patients with MS) and group 2 (20 patients with MS and AF) compared to group 3 (10 controls). Patients with groups 1 and 2 had a significant increase in expression of CD40 on monocytes (P1 and P2 = 0.000) and serum levels of sCD40L (P1 = 0.014 and P2 = 0.033, respectively), but nonsignificant increase in expression of CD40L on platelets (P1 = 0.109 and P2 = 0.060, respectively) as compared to controls. There were no significant difference in all the parameters in group 1 compared to group 2. Correlation analysis demonstrated that there was a significant direct relationship between the severity of MS and serum levels of sCD40L (r = -0.469, p = 0.043). In conclusion, rheumatic MS patients with or without AF had upregulation of the CD40/CD40L system as well as elevated sCD40L levels. The levels of sCD40L had a significantly direct relationship with the severity of MS and it was the stenotic mitral valve, not AF, that had a significant impact on platelet activation.

Keywords: CD40, CD40L, mitral stenosis, atrial fibrillation

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Authors: S. Subramaniam, J. S. A. Rao, P. Ramdas, K. R. Selvaduray, N. M. Han, M. K. Kutty, A. K. Radhakrishnan

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Immune system is a complex system where the immune cells have the capability to respond against a wide range of immune challenges including cancer progression. However, in the event of cancer development, tumour cells trigger immunosuppressive environment via activation of myeloid-derived suppressor cells and T regulatory (Treg) cells. The Treg cells are subset of CD4+ T lymphocytes, known to have crucial roles in regulating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. Dysregulation of these mechanisms could lead to cancer progression and immune suppression. Recently, there are many studies reporting on the effects of natural bioactive compounds on immune responses against cancer. It was known that tocotrienol-rich-fraction consisting 70% tocotrienols and 30% α-tocopherol is able to exhibit immunomodulatory as well as anti-cancer properties. Hence, this study was designed to evaluate the effects of gamma-tocotrienol (G-T3) supplementation on T-reg cells in a syngeneic mouse model of breast cancer. In this study, female BALB/c mice were divided into two groups and fed with either soy oil (vehicle) or gamma-tocotrienol (G-T3) for two weeks followed by inoculation with tumour cells. All the mice continued to receive the same supplementation until day 49. The results showed a significant reduction in tumour volume and weight in G-T3 fed mice compared to vehicle-fed mice. Lung and liver histology showed reduced evidence of metastasis in tumour-bearing G-T3 fed mice. Besides that, flow cytometry analysis revealed T-helper cell population was increased, and T-regulatory cell population was suppressed following G-T3 supplementation. Moreover, immunohistochemistry analysis showed that there was a marked decrease in the expression of FOXP3 in the G-T3 fed tumour bearing mice. In conclusion, the G-T3 supplementation showed good prognosis towards breast cancer by enhancing the immune response in tumour-bearing mice. Therefore, gamma-T3 can be used as immunotherapy agent for the treatment of breast cancer.

Keywords: breast cancer, gamma tocotrienol, immune suppression, supplement

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

Abstract:

Drug delivery systems are proposed for use in cancer treatment to specifically target cancer cells and deliver a therapeutic dose without affecting normal cells. For that purpose, the use of folate receptors (FR) can be considered a key strategy, since they are commonly over-expressed in cancer cells. In this study, cyclodextrins (CD) have being used as vehicles to target FR and deliver the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within their cavities. Here, β-CD has been modified using folic acid so as to specifically target the FR. Thus, this drug delivery system consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 15.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 16.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 10.5 for A549 and 132.6 µM ± 16.1 and 288.1 µM ± 26.3 for BEAS-2B. These results demonstrate that free MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug. The use of cell imaging by confocal microscopy has allowed visualisation of FR targeting in cancer cells, as well as the identification of the interlisation pathway of the drug. Hence, the cellular uptake and internalisation process of this drug delivery system is being addressed.

Keywords: cancer treatment, cyclodextrins, drug delivery, folate receptors, reduced folate carriers

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Authors: Laura Gantley, Vanessa M. Conn, Stuart Webb, Kirsty Kirk, Marta Gabryelska, Duncan Holds, Brett W. Stringer, Simon J. Conn

Abstract:

Trinucleotide repeat disorders comprise ~20 severe, inherited human neuromuscular and neurodegenerative disorders, which are a result of an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington’s disease, results from the expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Non-coding RNAs have been implicated in the initiation and progression of many diseases; thus, we focus on one circular RNA (circRNA) molecule arising from non-canonical splicing (back splicing) of HTT pre-mRNA. This circRNA and its mouse orthologue were transgenically overexpressed in human cells (SHSY-5Y and HEK293T) and mouse cells (Mb1), respectively. High-content imaging and flow cytometry demonstrated the overexpression of this circRNA reduces cell proliferation, reduces nuclear size independent of cellular size, and alters cell cycle progression. Analysis of protein by western blot and immunofluorescence demonstrated no change to HTT protein levels but altered nuclear-cytoplasmic distribution without impacting the expansion of the HTT repeat region. As these phenotypic and genotypic changes are found in Huntington’s disease patients, these results may suggest that this circRNA may play a functional role in the progression of Huntington’s disease.

Keywords: cell biology, circular RNAs, Huntington’s disease, molecular biology, neurodegenerative disorders

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Authors: Magdalena Rusady Goey, Gordon Strathdee, Neil Perkins

Abstract:

Background: Acute lymphoblastic leukaemia (ALL) is the most frequent type of childhood malignancy, accounting for 25% of all cases. TWIST2, a basic helix-loop-helix transcription factor, has been implicated in ALL development. Prior studies found that TWIST2 undergoes epigenetic silencing in more than 50% cases of ALL through promoter hypermethylation and suggested that re-expression of TWIST2 may inhibit cell growth/survival of leukaemia cell lines. TWIST2 has also been implicated as a regulator of NF-kappaB activity, which is constitutively active in leukaemia. Here, we use a lentiviral transductions system to confirm the importance of TWIST2 in controlling leukaemia cell growth and to investigate whether this is achieved through altered regulation of NF-kappaB activity. Method: Re-expression of TWIST2 in leukaemia cell lines was achieved using lentiviral-based transduction. The lentiviral vector also expresses enhanced green fluorescent protein (eGFP), allowing transduced cells to be tracked using flow cytometry. Analysis of apoptosis and cell proliferation were done using annexinV and VPD450 staining, respectively. Result and Discussion: TWIST2-expressing cells were rapidly depleted from a mixed population in ALL cell lines (NALM6 and Reh), indicating that TWIST2 inhibited cell growth/survival of ALL cells. In contrast, myeloid cell lines (HL60 and K562) were comparatively insensitive to TWIST2 re-expression. Analysis of apoptosis and cell proliferation found no significant induction of apoptosis, but did find a rapid induction of proliferation arrest in TWIST2-expressing Reh and NALM6 cells. Initial experiment with NF-kappaB inhibitor demonstrated that inhibition of NF-kappaB has similar impact on cell proliferation in the ALL cell lines, suggesting that TWITST2 may induce cell proliferation arrest through inhibition of NF-kappaB. Conclusion: The results of this study suggest that epigenetic inactivation of TWIST2 in primary ALL leads to increased proliferation, potentially by altering the regulation of NF-kappaB.

Keywords: leukaemia, acute lymphoblastic leukaemia, NF-kappaB, TWIST2, lentivirus

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Authors: Maria Giovanna Lupo, Marina Camera, Marcello Rattazzi, Nicola Ferri

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A complex interplay among chronic kidney disease, lipid metabolism and aortic calcification has been recognized starting from results of many clinical and experimental studies. Here we investigated the influence of kidney function on PCSK9 levels, both in uremic rats and in clinical observation study, and its potential direct action on cultured smooth muscle cells (SMCs) calcification. In a cohort of 594 subjects enrolled in a single centre, observational, cross-sectional and longitudinal study, a negative association between GFR and plasma PCSK9 was found. Atherosclerotic cardiovascular disease (ASCVD), as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats, induced aortic calcification and increased total cholesterol and PCSK9 levels in plasma, livers and kidneys. Immunohistochemical analysis confirmed PCSK9 expression in aortic SMCs. SMCs overexpressing PCSK9 (SMCsPCSK9), cultured for 7-days in a pro-calcification environment (2.0mM or 2.4mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to mocked SMCs. Under the same experimental conditions, the addition of exogenous recombinant PCSK9 did not increase the extracellular calcification of SMCs. By flow cytometry analysis we showed that SMCsPCSK9, in response to 2.4mM Pi, released higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALPL), compared to EVs from mocks SMCs. In conclusion, our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated to CKD condition. This effect appears to be mediated by a positive effect of endogenous PCSK9 on the release of EVs containing Ca2+ and ALP, which facilitate the deposition inorganic calcium phosphate crystals.

Keywords: PCSK9, calcification, extracellular vesicles, chronic kidney disease

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Authors: Nathalie Baeza-Kallee, Raphaël Bergès, Victoria Hein, Stéphanie Cabaret, Jeremy Garcia, Abigaëlle Gros, Emeline Tabouret, Aurélie Tchoghandjian, Carole Colin, Dominique Figarella-Branger

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Glioblastoma (GBM) contains cancer stem cells that are resistant to treatment. GBM cancer stem cell expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity, and tumorigenesis of GBM cancer stem cells. Our aim was to characterize the resulting effects of neuraminidase that remove A2B5 in order to target GBM cancer stem cells. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3, and GBM cancer stem cell lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography-mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size, and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM cancer stem cell lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM cancer stem cell lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.

Keywords: cancer stem cell, ganglioside, glioblastoma, targeted treatment

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Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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Authors: Liza Meutia Sari, Gus Permana Subita, Elza Ibrahim Auerkari

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Background: Many herbs have been discovered to be potential sources of anticancer drugs. Biji Pinang or areca nut (Areca catechu Linn.) has a high content of phenolics and flavonoids, and which is related to antioxidant activity. However, data on its effects on oral squamous cell carcinoma is not available. Objectives: Identification of the cytotoxicity and apoptosis activity in HSC-2 and HSC-3. Methods: The areca nut was extracted by ethanol 96%, MTS assay and apoptosis activity with flow cytometry. Results: The extract of areca nut showed higher toxicity on HSC-3 cell compared to HSC-2. The IC₅₀ of HSC-3 was 164.06 μg/ml vs. 629.50 μg/ml in HSC-2. There was an increase in late apoptosis percentage after 24 and 48 hours in HSC-2. There was a significant increase in early apoptosis percentage after 24 hours and late in 48 hours in HSC-3. Conclusion: The antioxidant activity of the extract of areca nut might be associated with the selective cytotoxicity on HSC-2 and HSC-3. Apoptosis is the major cell death mechanism involved. The areca nut may play an important role in anticancer herb medicine.

Keywords: areca nut, cytotoxicity, apoptosis, oral carcinoma

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Authors: Mona Vijayaran, Gurleen Oberoi, Sanjay Mishra

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Introduction: p190 BCR‐ABL1 in CML is often associated with monocytosis. In the case described here, monocytosis is associated with coexisting p210 BCR‐ABL and CMML clones. Mutation analysis using next‐generation sequence (NGS) in our case showed TET2 and SRSF2 mutations. Aims & Objectives: A 75-year male was evaluated for monocytosis and thrombocytopenia. CBC showed Hb-11.8g/dl, TLC-12,060/cmm, Monocytes-35%, Platelets-39,000/cmm. Materials & Methods: Bone marrow examination showed a hypercellular marrow with myeloid series showing sequential maturation up to neutrophils with 30% monocytes. Immunophenotyping by flow cytometry from bone marrow had 3% blasts. Making chronic myelomonocytic leukemia as the likely diagnosis. NGS for myeloid mutation panel had TET2 (48.9%) and SRSF2 (32.5%) mutations. This report further supported the diagnosis of CMML. To fulfil the WHO diagnostic criteria for CMML, a BCR ABL1 by RQ-PCR was sent. The report came positive for p210 (B3A2, B2A2) Major Transcript (M-BCR) % IS of 38.418. Result: The patient was counselled regarding the unique presentation of the presence of 2 clones- P210 CML and CMML. After discussion with an international faculty with vast experience in CMML. It was decided to start this elderly gentleman on Imatinib 200mg and not on azacytidine, as ASXL1 was not present; hence, his chances of progressing to AML would be less and on the other end, if CML is left untreated then chances of progression to blast phase would always be a possibility. After 3 months on Imatinib his platelet count improved to 80,000 to 90,000/cmm, but his monocytosis persists. His 3rd month BCR-ABL1 IS% is 0.004%. Conclusion: After searching the literature, there were no case reports of a coexisting CML p210 with CMML. This case might be the first case report. p190 BCR ABL1 is often associated with monocytosis. There are few case reports of p210 BCR ABL1 positivity in patients with monocytosis but none with coexisting CMML. This case highlights the need for extensively evaluating patients with monocytosis with next-generation sequencing for myeloid mutation panel and BCR-ABL1 by RT-PCR to correctly diagnose and treat them.

Keywords: CMML, NGS, p190 CML, Imatinib

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Authors: Eun Ho Kim

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Innovations have conjured up a mode of treating GBM cancer cells in the newly diagnosed patients in a period of 4.9 months at an improved median OS, which brings along only a few minor side effects in the phase III of the clinical trial. This mode has been termed the Alternating Electric Fields (AEF). The study at hand is aimed at determining whether the AEF treatment is beneficial in sensitizing the GBM cancer cells through the process of increasing the AEF –induced senescence. The methodology to obtain the findings for this research ranged across various components, such as obtaining and testing SA-β-gal staining, flow cytometry, Western blotting, morphology, and Positron Emission Tomography (PET) / Computed Tomography (CT), immunohistochemical staining and microarray. The number of cells that displayed a senescence-specific morphology and positive SA-ß-Gal activity gradually increased up to 5 days. These results suggest that p16, p21 and p27 are essential regulators of AEF -induced senescence via NF-κB activation. The results showed that the AEF treatment is functional in enhancing the AEF –induced senescence in the GBM cells via an apoptosis- independent mechanism. This research concludes that this mode of treatment is a trustworthy protocol that can be effectively employed to overcome the limitations of the conventional mode of treatment on GBM.

Keywords: alternating electric fields, senescence, glioblastoma, cell death

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Authors: Izabela Poprawa, Alina Chachulska-Zymelka, Lukasz Chajec, Grazyna Wilczek, Piotr Wilczek, Sebastian Student, Magdalena Rost-Roszkowska

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Heavy metals polluting the environment, especially soil, have a harmful effect on organisms, because they can damage the organ structure, disturb their function and cause developmental disorders. They can affect not only the somatic tissues but also the germinal tissues. In the natural environment, plants and animals are exposed to short- and long-term exposure to these stressors, which have a major influence on the functioning of these organisms. Numerous animals have been treated as the bioindicators of the environment. Therefore, studies on any alterations caused by, e.g., heavy metals are in the center of interests of not only environmental but also medical and biological science. Myriapods are invertebrates which are bioindicators of the environment. One of the species which lives in the upper layers of soil, particularly under stones and rocks is Lithobius forficatus (Chilopoda), commonly known as the brown centipede or stone centipede. It is a European species of the family Lithobiidae. This centipede living in the soil is exposed to, e.g., heavy metals such as cadmium, lead, arsenic. The main goal of our project was to analyze the impact of long-term exposure to cadmium on the structure of ovary with the emphasis on the course of oogenesis. As the material for analysis of cadmium exposure to ovaries, we chose the centipede species, L. forficatus. Animals were divided into two experimental groups: C – the control group, the animals cultured in laboratory conditions in a horticultural soil; Cd2 – the animals cultured in a horticultural soil supplemented with 80 mg/kg (dry weight) of CdCl2 for 45 days – long-term exposure. Animals were fed with Acheta and Chironomus larvae maintained in tap water. The analyzes were carried out using transmission electron microscopy (TEM), flow cytometry and laser scanning (confocal) microscopy. Here we present the results of long-term exposure to cadmium concentration in soil on the organ responsible for female germ cell formation. Analysis with the use of the transmission electron microscope showed changes in the ultrastructure of both somatic and germ cells in the ovary. Moreover, quantitative analysis revealed the decrease in the percentage of cells viability, the increase in the percentage of cells with depolarized mitochondria and increasing the number of early apoptotic cells. All these changes were statistically significant compared to the control. Additionally, an increase in the ADP/ATP index was recorded. However, changes were not statistically significant to the control. Acknowledgment: The study has been financed by the National Science Centre, Poland, grant no 2017/25/B/NZ4/00420.

Keywords: cadmium, centipede, ovary, ultrastructure

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Authors: Ratchaneewan Maneemaroj, Paveena Noisuwan, Chonlada Lakhonphon

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The smoking is one of the major risk factors in Non-Communicable Disease. Free radicals from cigarette smoke can cause oxidative stress. The oxidative insults can lead to red blood cell (RBC) senescence and are involved in the clearance of red blood cells. The objective of the present study is to assess the association between smoke, oxidative stress evaluated with serum Malondialdehyde (MDA) level and phosphatidylserine (PS) externalization (biomarker of RBC senescence) evaluated with annexin V binding. A total of sixty-four male volunteers aged 25-60 years old were recruited in this study. MDA was measured by colorimetric method. Annexin V binding was detected by flow cytometry. Our results show that there was a significant increase in MDA levels in cigarette smokers as compared to non-smokers (p < 0.001). However, there was no significant different between annexin V binding (% gate) in cigarette smokers and non-smokers (p = 0.978). These results provide evidence of free radical from smoking is associated with oxidative damage to erythrocytes. However, our results suggest that PS externalization is unlikely to have a role in RBC senescence pathway of stressed erythrocytes from cigarette smoke. The other biomarker of RBC senescence should be determined on cigarette smoker erythrocytes.

Keywords: malondialdehyde, phosphatidylserine, RBC senescence, annexin V

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Authors: Neda Menbari, Ramin Mehdiabadi

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Background: breast cancer remains a critical global health issue, constituting a leading cause of cancer-related mortality in women. MicroRNAs (miRs) are natural RNA molecules that play an important role in cellular processes and regulate post-transcriptional gene expression. MiR-216b-5p is a miR that acts as a tumor suppressor. The expression levels of FoxM1 and miR-216b-5p in malignant and control cells have been evaluated by quantitative polymerase chain reaction (qPCR) technique and flow cytometry. Results: the results of this study revealed a significant downregulation of miR-216b-5p in cancerous cells compared to the control MCF-10A cells (P=0.0004). Interestingly, the expression of miR-216b-5p exhibited an inverse relationship with key clinical indicators such as tumor size, grade, and lymph node invasion. Conclusion: The study's findings showed the prognostic value of miR-216b-5p levels in breast cancer, and its reduced expression correlates with unfavorable tumor characteristics. This research recommends performing more studies on the role of FoxM1 and miR-216b-5p in breast cancer pathology which potentially paving the way for targeted therapeutic interventions.

Keywords: breast cancer, gene expression, FOXM1, microRNA

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Authors: A. Vijayendra Chary, R. Hemalatha

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Background: Vitamin D is a potent modulator of the immune system and is involved in regulating cell proliferation and differentiation. Vitamin D deficiency has been linked to increased severity of asthma in children. Asthma has dramatically increased in past decades, particular in developing countries and affects up to 20% of the population. IgE and its receptors, CD23 (FcεRII) and CD 21, play an essential role in all allergic conditions. Methods: A case control study was conducted on asthma and age and sex matched control children. 25 hydroxyvitamin D3 was quantified by HPLC; CD23; and CD21 expression on B cells were performed by flow cytometry. Total Histamine, total IGE and IL-5 and IFN-γ cytokines were determined by ELISA in blood samples of bronchial asthma (n=45) and control children (n=45). Results: The mean ± SE of vitamin D was significantly (p<0.05) low in asthma children (13.6±0.54 ng/mL) than in controls (17.4 ± 0.37 ng/mL). The mean (%) ± SE of CD23 and CD21 expression on B cells were significantly (p<0.01) high in asthma (1.02±0.09; 1.67± 0.13), when compared to controls (0.24±0.01; 0.94±0.03) respectively. The mean± SE of Serum IgE and blood histamine levels in asthma children (354.52 ± 17.33 IU/mL; 53.27 ± 2.54 nM/mL) were increased (P<0.05) when compared to controls (183.12±17.62 IU/mL 39.34±4.16 nM/mL) respectively and IFN-γ (Th1 cytokine) was lower (P<0.01) (16.37±1.27 pg/mL) than in controls (43.34±6.21 pg/mL). Conclusion: Our study provides evidence that low vitamin D levels are associated with increased IgE receptors CD23 and CD21 on B cells. In addition, there was preferential activation of Th2 (IL-5) and suppression of Th1 (IFN-γ) cytokines in children with asthma.

Keywords: bronchial asthma, CD23, IgE, vitamin D

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Authors: Cóndor C. José, Rodrigues E. Camila, Noronha L. Irene, Dos Santos Fernando, Irigoyen M. Claudia, Andrade Lúcia

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Sepsis induces alterations in hemodynamics and autonomic nervous system (ASN). The autonomic activity can be calculated by measuring heart rate variability (HRV) that represents the complex interplay between ASN and cardiac pacemaker cells. Wharton’s jelly mesenchymal stem cells (WJ-MSCs) are known to express genes and secreted factors involved in neuroprotective and immunological effects, also to improve the survival in experimental septic animals. We hypothesized, that WJ-MSCs present an important role in the autonomic activity and in the hemodynamic effects in a cecal ligation and puncture (CLP) model of sepsis. Methods: We used flow cytometry to evaluate WJ-MSCs phenotypes. We divided Wistar rats into groups: sham (shamoperated); CLP; and CLP+MSC (106 WJ-MSCs, i.p., 6 h after CLP). At 24 h post-CLP, we recorded the systolic arterial pressure (SAP) and heart rate (HR) over 20 min. The spectral analysis of HR and SAP; also the spontaneous baroreflex sensitivity (measure by bradycardic and tachycardic responses) were evaluated after recording. The one-way ANOVA and the post hoc Student– Newman– Keuls tests (P< 0.05) were used to data comparison Results: WJ-MSCs were negative for CD3, CD34, CD45 and HLA-DR, whereas they were positive for CD73, CD90 and CD105. The CLP group showed a reduction in variance of overall variability and in high-frequency power of HR (heart parasympathetic activity); furthermore, there is a low-frequency reduction of SAP (blood vessels sympathetic activity). The treatment with WJ-MSCs improved the autonomic activity by increasing the high and lowfrequency power; and restore the baroreflex sensitive. Conclusions: WJ-MSCs attenuate the impairment of autonomic control of the heart and vessels and might therefore play a protective role in sepsis. (Supported by FAPESP).

Keywords: baroreflex response, heart rate variability, sepsis, wharton’s jelly-derived mesenchymal stem cells

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Authors: Kirti Hira, A. Sajeli Begum, S. Mahibalan, Poorna Chandra Rao

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Introduction: Cancer is one of the leading causes of death worldwide. Although existing therapy effectively kills cancer cells, they do affect normal growing cells leading to many undesirable side effects. Hence there is need to develop effective as well as safe drug molecules to combat cancer, which is possible through phyto-research. The currently available plant-derived blockbuster drugs are the example for this. In view of this, an investigation was done to identify cytotoxic lead molecules from Hedyotis umbellata (Family Rubiaceae), a widely distributed weed in India. Materials and Methods: The methanolic extract of the whole plant of H. umbellata (MHU), prepared through Soxhlet extraction method was further fractionated with diethyl ether and n-butanol, successively. MHU, ether fraction (EMHU) and butanol fraction (BMHU) were lyophilized and were tested for the cytotoxic effect using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay against non-small cell lung cancer (NSCLC) A549 cell lines. The potentially active EMHU was subjected to chromatographic purification using normal-phase silica columns, in order to isolate the responsible bioactive compounds. The isolated pure compounds were tested for their cytotoxic effect by MTT assay against A549 cells. Compound-3, which was found to be most active, was characterized using IR, 1H- and 13C-NMR and MS analysis. The study was further extended to decipher the mechanism of action of cytotoxicity of compound-3 against A549 cells through various in vitro cellular models. Cell cycle analysis was done using flow cytometry following PI (Propidium Iodide) staining. Protein analysis was done using Western blot technique. Results: Among MHU, EMHU, and BMHU, the non-polar fraction EMHU demonstrated a significant dose-dependent cytotoxic effect with IC50 of 67.7μg/ml. Chromatography of EMHU yielded seven compounds. MTT assay of isolated compounds explored compound-3 as potentially active one, which inhibited the growth of A549 cells with IC50value of 14.2μM. Further, compound-3 was identified as cedrelopsin, a coumarin derivative having molecular weight of 260. Results of in vitro mechanistic studies explained that cedrelopsin induced cell cycle arrest at G2/M phase and down-regulated the expression of G2/M regulatory proteins such as cyclin B1, cdc2, and cdc25C, dose dependently. This is the first report that explores the cytotoxic mechanism of cedrelopsin. Conclusion: Thus a potential small lead molecule, cedrelopsin isolated from H. umbellata, showing antiproliferative effect mediated by G2/M arrest in A549 cells was discovered. The effect of cedrelopsin against other cancer cell lines followed by in vivo studies can be performed in future to develop a new drug candidate.

Keywords: A549, cedrelopsin, G2/M phase, Hedyotis umbellata

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