Dual-Armed Oncolytic Myxoma Virus Encoding IFN-γ and CD47 Promotes Lymphocyte Infiltration and Tumor Suppression of Syngeneic Murine Melanoma.

Myxoma virus (MyxV) is a rabbit-specific poxvirus. However, its ability to selectively target tumor cells has established it as a safe and effective anticancer therapy. To strengthen its preclinical efficacy, transgenes that can prolong cancer cell infection and enhance anti-tumor effector functions are currently being investigated. We engineered MyxV armed with CD47, to turn on a 'do not eat me' signal within infected cells with actively replicating viruses, and with IFN-γ to further activate host immune anticancer responses. Tumor suppressive activities were significantly enhanced by the dual-armed MyxV_CD47/IFN-γ compared to parental MyxV or single-armed MyxV_CD47 or MyxV_IFN-γ. In addition, significant increases in IFN-γ+ CD8+T-cells and CD4+ T-cells populations within tumor-infiltrating lymphocytes (TIL) were observed after MyxV_CD47/IFN-γ treatment. Notably, all groups treated with MyxV showed a marked reduction in Foxp3+ CD4+ regulatory T-cells (Tregs) within TIL. We also show that MyxV infection induces PD-L1 up-regulation in cancer cells, and combinational treatment of MyxV with anti-mouse PD-L1 antibodies (αPD-L1) further controlled tumor burden and increased survival in the syngeneic melanoma model B16F10. Our data demonstrate that a CD47 and IFNγ dual-armed MyxV is an effective oncolytic viral immunotherapeutic. These findings strongly support further preclinical investigations to develop next-generation MyxV-based immunotherapy approaches.

Comparative analysis of metabolic variations, antioxidant potential and cytotoxic effects in different parts of Chelidonium majus L.

Metabolic variations, antioxidant potential and cytotoxic effects were investigated in the different plant parts like the leaf, stem, flower, pod, and root of C. majus L. using spectroscopic and chromatographic methods. Total phenolics and flavonoids were studied in the different parts of C. majus L., leaf showed higher flavonoid content (137.43 mg/g), while the pod showed the highest phenolic (23.67 mg/g) content, when compared with the stem, flower and root. In the ABTS antioxidant assay, the flower extract showed 57.94% effect, while the leaf, pod and root extract exhibited 39.10%, 36.08% and 28.88% activity, respectively. The pod and leaf extracts demonstrated the potential effect, exhibiting 45.46 and 41.61% activity, respectively, for the DPPH assay. Similar to the phosphomolybdenum assay, the flower revealed higher antioxidant activity (46.82%) than the other plant parts. The in vitro SRB assay facilitated evaluation of the cytotoxic effect against the HeLa and CaSki human cervical cancerous cells. The extract displayed dose-dependent inhibitory effect on both the cell lines. The highest cytotoxic effect was observed in the pod and flower extracts post 48 h of exposure at 1000 µg/mL. The results of C. majus L. offered new insights in the preliminary steps regarding the development of a high value product for phytomedicine applications though promising metabolic variations with antioxidant and anticancer potentials.

Cytotoxic and apoptotic potential of Phyllodium elegans extracts on human cancer cell lines.

The extract of Phyllodium (P.) elegans was investigated for its anti-cancer properties on brain astroglioma cells (U251-MG), colorectal carcinoma cells (HCT116), and malignant melanoma cells (A375). P. elegans methanolic extract (PeME) showed cytotoxicity on all three cancer cell lines tested. The cell viability assay revealed that PeME significantly reduced the viability of these cells. Clear apoptotic features such as cellular morphology, cell shrinkage, and augmentation of dead cells were observed. Flow cytometry and fluorescence staining techniques confirmed the apoptotic property of PeME. In vitro scratch invasion assay showed that cell migration rate was significantly reduced. Fluorescence microscopic studies using 4',6-diamidino-2-phenylindole staining showed early and late signs of apoptosis after PeME treatment. Upon PeME stimulation, activation of caspase-3/-9 and Mu-2-related death-inducing gene (MUDENG, MuD) was observed by western blot analysis. JC-1 staining analysis by flow cytometry showed that PeME depolarized the mitochondria membrane potential (MMP). Collectively, these findings, for the first time, point to the fact that PeME has anti-cancer properties against brain, colon, and skin cancer cell lines by depolarizing the MMP and activating apoptotic signaling through the activation of caspase-3/-9 as well as MuD. This is the first report reporting the anticancer activity of this specific plant extract.[Figure: see text].

Utilization of Dianthus superbus L and its bioactive compounds for antioxidant, anti-influenza and toxicological effects.

Dianthus superbus (DS) is a traditional medicinal herb well known for its medicinal and therapeutic potential and widely distributed in various Asian countries. The ethyl acetate (EA), butanol (Bu) and distilled water (DW) extracts of DS assessed for extraction of bioactive compounds and their biological activities. The chemical analysis was done using LC-MS/MS and antioxidant, anticancer and antiviral activities were determined. EA extracts showed strong anticancer activity with IC50 of 9.5, 13.8 and 69.9 µg/mL on SKOV, NCL-H1299 and Caski cancer cell lines, respectively. The Bu extracts exhibited strongest antiviral activity with respect to both influenza A and B viruses with IC50 values of 4.97 and 3.9 µg/mL, respectively. Also the metabolic profile for EA, Bu and DW extracts shows high variations and influence precisely the antioxidant, anticancer and antiviral properties. The quercetin 3- rutinoside and isorhamnetin 3- glucoside showed higher neuraminidase inhibition activity in dose dependent manner. Molecular docking study revealed that flavonol glycosides have higher binding activities towards influenza polymerase membrane glycoprotein. Correlation study showed that flavonol glycosides were linked to anti-influenza activity and cyclic peptides with anticancer activities. This study provides vital information for effective utilization of DS for medicinal, food and therapeutic purposes.

Cytotoxic effect of TDZ on human cervical cancer cells.

The present study investigates the anticancer activity of Thidiazuron (TDZ). Anticancer activity of TDZ was evaluated in cervical carcinoma cells (HeLa cells). Sulforhodamine-B (SRB) assay indicates that TDZ was about 100 times more toxic to the cancer cell than normal cells. TUNEL assay showed TDZ induced DNA damage in tumor cells. The loss of mitochondrial membrane potential (MMP) in cancer cells was observed following TDZ treatment. The Bax and bcl-2 gene expression ratio are highly responsible for the regulation of MMP balance, and these ratio was significantly altered following TDZ treatment. The p53 and caspase-3 expressions were increased in cancer cells following treatment. Caspase-3 activation is the key factor for apoptosis. Cytotoxicity of TDZ on HeLa cells was 100 times higher than normal kidney cell (MDCK cells). Moreover, the anticancer activity of TDZ was tested by DNA damage, mitochondrial dysfunction, some gene expression and caspase-3 inhibition in silico. TDZ detected has higher ability on early apoptosis of cancer cell through DNA damage. Additionally, cancer cellular MMP was significantly reduced under inoculation of TDZ. In silico assay confirmed that TDZ was able to bind with the active site of the capase-3 protein. Therefore, taking all these data together it is suggested that the TDZ may be a potential agent to act against cervical cancer cells.

ß-Alanine intercede metabolic recovery for amelioration of human cervical and renal tumors.

ß-Alanine is a non-essential amino acid and presents as a major component of various sports supplements. It is a non-proteogenic amino acid, formed in vivo by degradation of carnosine, anserine, balenine, and dihydrouracil. The present study was aimed at investigating the anti-tumor effects of ß-alanine in renal and cervical tumor cells. Sulforhodamine-B assay and flow cytometric analysis were used to measure cell viability. Lactate dehydrogenase (LDH) expression was analyzed using FITC-conjugated fluorescent antibody. The cellular adenosine triphosphate (ATP) content was measured using bioluminescence method. Cell migration was determined by the simple standard-scratch method. ß-Alanine reduced renal and cervical cell growth significantly. Percentage of inhibition of renal and cervical tumor cells was increased at higher concentration of ß-alanine. LDH expression and ATP content were significantly reduced in renal and cervical tumor cells in a dose-dependent manner. Renal and cervical tumor cell migration were significantly reduced following 10 and 100 mM of ß-alanine treatment. In our study, ß-alanine exerts no significant effect on normal MDCK cells except a marginal effect at the highest concentration (100 mM). In summary, our experimental data suggest that ß-alanine may be a potential anti-tumor agent exhibiting several anti-cancer effects in renal and cervical tumor cells.

Extreme effects of Seabuckthorn extracts on influenza viruses and human cancer cells and correlation between flavonol glycosides and biological activities of extracts.

Seabuckthorn is a medicinal plant that is used to prevent cold. It was tested for its metabolic content followed by activity against cancer and virus. The metabolic distribution of different polarity solvent extractions from the leaves was analyzed by LC-MS/MS. Flavonol glycoside contents in EA and Bu extracts were higher than MeOH and DW was observed. MeOH and EA extracts recorded high activity against influenza A/PR virus with IC50 of 7.2 µg/mL and 10.3 µg/mL compared with known drug Oseltamivir of 60.3 µg/mL. A similar trend showed in influenza A/Victoria virus. In case of influenza B viruses such as B/Lee and B/Maryland, EA extract (2.87 µg/mL and 4.5 µg/mL of IC50) emerged strongest among other extracts and Oseltamivir (103.73 µg/mL and 71.6 µg/mL). Each extract showed potent anticancer activities. Interestingly, Bu extract showed stronger anticancer activity against human cancer cells such as NCL-H1299, HeLa, SKOV and Caski (8.2 µg/mL, 8.6 µg/mL, 18.2 µg/mL and 9.2 µg/mL of IC50) respectively. Correlation study reveals that aglycones and flavonol mono-glycosides highly correlated with anti-influenza activities but not correlated with anticancer activities. Reversely, di-glycosides and tri-glycosides have a high correlation with cytotoxic effect with both normal and cancer cells. Therefore, this study provides significant information concerning Seabuckthorn for further medicinal drug development.

Investigation of role of aspartame on apoptosis process in HeLa cells -->.

Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. The study reports that consumption of aspartame containing product could lead to cancer. However, the effect of aspartame on apoptosis process in cancer is not yet understood clearly. HeLa cells were exposed to different concentrations (0.01-0.05 mg/ml) of aspartame for 48 h. Cytotoxicity of aspartame on cancer cells was determined by SRB assay. The result indicates no significant changes on cell viability. Aspartame suppresses apoptosis process in cancer cells by down-regulation of mRNA expression of tumor suppressor gene p53, and pro-apoptotic gene bax. It up-regulates anti-apoptotic gene bcl-2 mRNA expression. In addition, Ki 67 and PCNA mRNA, and protein expressions were determined. Taking all these together, we conclude that aspartame may be a potent substance to slow-down the apoptosis process in HeLa cells. Further works are ongoing to understand the biochemical and molecular mechanism of aspartame in cancer cells.

Anticancer studies of synthesized ZnO nanoparticles against human cervical carcinoma cells.

A metal oxide nanoparticle has been widely investigated for its potential use in the biomedical application. The present study investigates the cytotoxicity of ZnO nanoparticle in human cervical carcinoma cells. Cell viability was determined, and it showed the possible cytotoxic effect of ZnO nanoparticles. The characteristic apoptotic features such as rounding and loss of adherence were observed in the treated cells. Fluorescence and Confocal Laser Scanning Microscope (CLSM) studies have showed reduced nuclear volume and condensed cytoplasm. The mRNA expression of apoptotic gene p53 and caspase 3 was up-regulated following ZnO nanoparticle exposure, which confirms the occurrence of apoptosis at the transcriptional level. Reactive oxygen species (ROS) was increased in a dose-dependent manner, and initiate lipid peroxidation of the liposomal membrane, which in turn regulate several signaling pathways and influencing the cytokinetic movements of cells. ZnO nanoparticles showed a dynamic cytotoxic effect in cervical carcinoma cells. ZnO nanoparticle might induce the apoptosis through increased intracellular ROS level. Moreover, up-regulated apoptotic gene expression confirms the occurrence of apoptosis. Taking all these data together, it may be concluded that ZnO nanoparticle may exert cytotoxicity on HeLa cell through the apoptotic pathway, implies the probable utility of ZnO nanoparticle in the cancer treatment and therapy.

Therapeutic efficacy of natural dipeptide carnosine against human cervical carcinoma cells.

Natural substances have been attracted several researchers in the recent years, because of its potential antioxidant, anti-inflammatory and anti-cancer properties. We have investigated the effect of carnosine on cell viability, apoptosis, DNA damage, reactive oxygen species (ROS) and caspase 3 enzyme expression in human cervical carcinoma and Madin-Darby Kidney Cells (MDCK) cells. Carnosine inhibited cancer cell growth up to 23%. ROS level was increased up to 30 and 31% in MDCK and HeLa cells respectively. Tunnel assay showed 42 and 14% of positive apoptotic cells in cancer and normal cells respectively. The alteration in mitochondrial and nuclear morphology was determined. The extended lace-like network of normal mitochondria found in control cells. Carnosine treatment significantly altered the mitochondrial morphology of normal cervical carcinoma cell. Mitochondria were condensed clump structures in carnosine treated cancer cells. Carnosine reduced the number of colonies of cervical carcinoma cells. Caspase 3 expression was corresponded to the appearance of immunofluorescence in the cytoplasm. Caspase 3 expression was gradually increased in cervical carcinoma cells. In Silico, docking study was performed to recognize the binding activity of carnosine against a subunit of the caspase 3, and carnosine was able to bind to the drug binding pocket of caspase 3. The glide energy is -5.2 kcal/mol, suggesting the high binding affinity of carnosine to caspase 3. Taking all these data together, the natural dipeptide L-carnosine could be a suitable antiproliferative agent in cervical carcinoma cells. Copyright © 2016 John Wiley & Sons, Ltd.

In Vitro Therapeutic Potential of Tio2 Nanoparticles Against Human Cervical Carcinoma Cells.

Cellular and physiological responses to the degradation products of titanium implants are key indicators to determine the quality of biocompatibility of implant devices. The present study investigated titanium dioxide (TiO2) nanoparticle-induced cytotoxicity, apoptotic morphological modification, and apoptotic-related gene expressions in the human cervical carcinoma cells. TiO2 nanoparticle-induced cytotoxicity on cancer cells was determined by the sulphorhodamine-B assay. Apoptotic morphological modification such as nuclear fragmentation, rounding, cytoplasm shrinkage, loss of adhesion, and reduced cell volume were observed by an inverted, fluorescence, and confocal laser scanning microscope (CLSM). The DNA fragmentation study showed the occurrence of necrosis and apoptosis in nanoparticle-treated cells. The qPCR study showed the increased p53 and bax mRNA expression in the nanoparticle-treated cells compared to control. In addition, caspase 3 activity was increased in nanoparticle-treated cells, which indicates the increased auto-catalysis. Taking all these data together, it may suggest that TiO2 nanoparticle could inhibit the growth of HeLa cells.

Time and Concentration-Dependent Therapeutic Potential of Silver Nanoparticles in Cervical Carcinoma Cells.

Silver nanoparticles (AgNPs) have well-known anti-bacterial properties and have been widely used in daily life as various medical and general products. There is limited information available on the cytotoxicity of AgNPs. Therefore, the present study aimed to investigate the cytotoxicity of AgNPs in HeLa cells. Cytotoxicity and apoptosis have been observed in the AgNPs treated in the HeLa cells. Sulphorhodamine-B assay (SRB assay) showed the cytotoxic effect in the AgNP-treated HeLa cells. Inverted microscope, fluorescence microscope, and confocal laser scanning microscope (CLSM) analyses showed the apoptosis-induced morphological changes such as rounding in shape, nuclear fragmentation, cytoplasm reduction, loss of adhesion, and reduced cell volume. Necrosis and apoptosis were observed in the AgNP-treated HeLa cells by DNA fragmentation study. Mitochondria-derived reactive oxygen species (ROS) have increased in AgNP-treated HeLa cells. Up-regulation of messenger RNA (mRNA) expression of p53, bax, and caspase 3 were found in AgNP-treated HeLa cells. Caspase 3 enzyme activity was found to increase in AgNP-treated HeLa cells. The AgNPs showed the right cytotoxic effect in cervical carcinoma cells. Our results suggest that metal-based nanoparticles might be a potential candidate for the treatment of cervical cancer.

Cytotoxic effects of aspartame on human cervical carcinoma cells.

Aspartame is used as an artificial sweetener in more than 6000 food varieties. The present study aims to determine the effects of aspartame at various concentrations on the cell viability, morphology, ROS level and DNA of human cervical carcinoma cells over two time periods of exposure. The effects of aspartame on HeLa cell viability were investigated using the sulphorhodamine-B assay (SRB assay) and flow cytometry. Alkaline comet assay was carried out to determine the possible DNA damage induced by aspartame. Mitochondria-derived reactive oxygen species (ROS) were determined using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Fluorescence microscopy was used to determine the presence of apoptotic and necrotic cells following aspartame treatment. Cell viability was significantly altered following a higher concentration of aspartame exposure. Mitochondria-derived ROS increased at higher concentrations of aspartame exposure. Exposure to 10 mM and 20 mM of aspartame induced DNA fragmentation. Apoptotic and necrotic bodies were found in the range of 1-20 mM aspartame exposure. Exposure to high concentrations of aspartame may alter cell viability and morphology, and it may induce ROS generation and DNA damage in cervical carcinoma cells.

Metabolic variations, antioxidant potential, and antiviral activity of different extracts of Eugenia singampattiana (an endangered medicinal plant used by Kani tribals, Tamil Nadu, India) leaf.

Eugenia singampattiana is an endangered medicinal plant used by the Kani tribals of South India. The plant had been studied for its antioxidant, antitumor, antihyperlipidemic, and antidiabetic activity. But its primary and secondary metabolites profile and its antiviral properties were unknown, and so this study sought to identify this aspect in Eugenia singampattiana plant through different extraction methods along with their activities against porcine reproductive and respiratory syndrome virus (PRRSV). The GC-MS analysis revealed that 11 primary metabolites showed significant variations among the extracts. Except for fructose all other metabolites were high with water extract. Among 12 secondary metabolites showing variations, the levels of 4-hydroxy benzoic acid, caffeic acid, rutin, ferulic acid, coumaric acid, epigallocatechin gallate, quercetin, myricetin, and kaempferol were high with methanol extract. Since the flavonoid content of methanol extracts was high, the antioxidant potential, such as ABTS, and phosphomolybdenum activity increased. The plants antiviral activity against PRRSV was for the first time confirmed and the results revealed that methanol 25 µg and 75 to 100 µg in case of water extracts revealed antiviral activity.

Metabolic variation and antioxidant potential of Malus prunifolia (wild apple) compared with high flavon-3-ol containing fruits (apple, grapes) and beverage (black tea).

Secondary metabolic variation of wild apple (Malus prunifolia) was compared with fruits that contained high flavan-3-ol like grapes (GR), apple (App) and the beverage, black tea (BT). The polyphenol contents in wild apple was higher than in GR and App but less than BT. The identified phenolic acids (gallic, protocatechuic, chlorogenic, p-coumaric and ferulic acids) and flavonoids (quercetin and myricetin) indicate that wild apple was higher than that of App. Among all the samples, BT had highest antioxidant potential in terms of 2,2'-Azinobis (3-thylbenzothiazoline-6-sulfonic acid) diammonium salt (95.36%), metal chelating (45.36%) and phosphomolybdenum activity (95.8 mg/g) because of the high flavan-3-ol content. The gallic acid and epigallocatechin gallate were highly correlated with antioxidant potential and these metabolites levels are higher in wild apple than that of App. Wild apples being a non-commercial natural source, a detailed study of this plant will be helpful for the food additive and preservative industry.