Acridine Orange And Ethidium Bromide Research Articles

Construction of anticancer drug incorporated aptamer-functionalized cationic β-lactoglobulin: induction of cell cycle arrest and apoptosis in colorectal cancer

Nanoscale drug delivery systems that are both multifunctional and targeted have been developed using proteins as a basis, thanks to their attractive biomacromolecule properties. A novel nanocarrier, aptamer (AS1411)-conjugated β-lactoglobulin/poly-l-lysine (BLG/Ap/PL) nanoparticles, was developed in this study. To this unique formulation, the as-prepared nanocarrier blends the distinctive features of an aptamer as a chemotherapeutic targeting agent with those of protein nanocarriers. By loading cabazitaxel (CTX) onto the nanocarriers, the therapeutic potential of BLG/Ap/PL could be demonstrated. The CTX-loaded BLG/Ap/PL (CTX@BLG/Ap/PL) showed a regulated drug release profile in an acidic milieu, which could improve therapeutic efficacy in cancer cells and have a high drug encapsulation efficacy of up to 93%. However, compared to free CTX, CTX@BLG/Ap/PL killed colorectal HCT116 cancer cells with a higher efficacy at 24 and 48 h. Further investigation confirms the apoptosis by acridine orange and ethidium bromide (AO/EB), and DAPI staining confirms the morphological changes, chromatin condensation, and membrane blebbing in the treated cell through flow cytometry displayed the release of higher percentages of apoptosis. Cell cycle analysis revealed that CTX@BLG/Ap/PL induced sub-G1 and G2/M phase (apoptosis) at 24 and 48 h. Annexin V/propidium iodide (PI) flow cytometry analysis confirmed that CTX@BLG/Ap/PL induces apoptosis in HCT116 cells. Overall, this study proved that CTX@BLG/Ap/PL had several advantages over free chemotherapeutic drugs and showed promise as a solution to the clinical problems associated with targeted antitumor drug delivery systems.

Green synthesis of silver chloride nanoparticles derived from Artemisia sieberi extract (AgClNPs-ASE) induces cell cycle arrest and triggers apoptosis in human breast (MDA-MB-231) cancer cells

In recent years, metallic nanoparticles have attracted tremendous attention in nanomedicine. Here, we report green synthesis and determination of silver chloride nanoparticles synthesized using Artemisia sieberi extract (AgClNPs-ASE) and its anticancer properties against human breast cancer cells. Numerous techniques, such as X-ray diffraction (XRD), UV-visible spectra, transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive spectra (SEM–EDS), and dynamic light scattering (DLS) analysis determined the morphology characteristics and size of the obtained nanoparticles. Cytotoxic properties of AgClNPs-ASE were carried out toward human breast cancer MDA-MB-231 cells and nontumorigenic HEK293 cells. AgClNPs-ASE revealed better efficacy toward breast cancer cells and was relatively less cytotoxic to nontumorigenic cells. Induction of apoptosis was further revealed by Hoechst 33258 dye and dual acridine orange and ethidium bromide (AO/EB) staining and in cells along with upregulation of caspase-8 and 9 proteins. AgClNPs-ASE could trigger the apoptosis by upregulation of early (1.93%) and late (58.86%) apoptosis followed by fluorescein isothiocyanate (FITC)‐Annexin‐V/PI staining and enhanced cell cycle population (sub-G1). AgClNPs-ASE significantly increased NO production in cancer cells. Moreover, qRT-PCR study showed that AgClNPs-ASE treated cells induce increased expression of the LncRNA GAS5 gene. These findings suggest the remarkable apoptosis in cancer cells triggered by AgClNPs-ASE in vitro.

Marine seaweed endophytic fungi-derived active metabolites promote reactive oxygen species-induced cell cycle arrest and apoptosis in human breast cancer cells.

Endophytic fungi have an abundant sources rich source of rich bioactive molecules with pivotal pharmacological properties. Several studies have found that endophytic fungi-derived bioactive secondary metabolites have antiproliferative, anti-oxidant, and anti-inflammatory properties, but the molecular mechanism by which they induce cell cycle arrest and apoptosis pathways is unknown. This study aimed to determine the molecular mechanism underlying the anticancer property of the endophytic fungi derived active secondary metabolites on human breast cancer cells. In this study, we identified four endophytic fungi from marine seaweeds and partially screened its phytochemical properties by Chromatography-Mass Spectrometry (GC-MS) analysis. Moreover, the molecular mechanism underlying the anticancer property of these active secondary metabolites (FA, FB, FC and FE) on human breast cancer cells were examined on MCF-7 cells by TT assay, Apoptotic assay by Acridine orang/Ethidium Bromide (Dual Staining), DNA Fragmentation by DAPI Staining, reactive oxygen species (ROS) determination by DCFH-DA assay, Cell cycle analysis was conducted Flow cytometry and the apoptotic signalling pathway was evaluated by westernblot analysis. Doxorubicin was used as a positive control drug for this experiment. The GC-MS analysis of ethyl acetate extract of endophytic fungi from the marine macro-algae revealed the different functional groups and bioactive secondary metabolites. From the library, we observed the FC (76%), FB (75%), FA (73%) and FE (71%) have high level of antioxidant activity which was assessed by DPPH scavenging assay. Further, we evaluated the cytotoxic potentials of these secondary metabolites on human breast cancer MCF-7 cells for 24h and the IC50 value were calculated (FA:28.62 ± 0.3µg/ml, FB:49.81 ± 2.5µg/ml, FC:139.42 ± µg/ml and FE:22.47 ± 0.5µg/ul) along with positive control Doxorubicin 15.64 ± 0.8µg/ml respectively by MTT assay. The molecular mechanism by which the four active compound induced apoptosis via reactive oxygen species (ROS) and cell cycle arrest in MCF-7 cells was determined H2DCFDA staining, DAPI staining, Acridine orange and ethidium bromide (AO/EtBr) dual staining, flowcytometry analysis with PI staining and apoptotic key regulatory proteins expression levels measured by westernblot analysis. Our findings, revealed the anticancer potential of endophytic fungi from marine seaweed as a valuable source of bioactive compounds with anticancer properties and underscore the significance of exploring marine-derived endophytic fungi as a promising avenue for the development of novel anticancer agents. Further investigations are necessary to isolate and characterize specific bioactive compounds responsible for these effects and to validate their therapeutic potential in preclinical and clinical settings.

Fabrication of etoposide-loaded folic-acid-clocked mesoporous nanoparticles: Investigation of lung cancer proliferation and induction of apoptosis and ferroptosis

Etoposide (EPT) offers significant benefits in the tumor therapy domain. EPT may cause ferroptosis in lung cancer cells; however, its limited bioavailability and low water solubility inhibit its potential applications. Therefore, we investigated the properties of EPT-loaded folic acid clocked with mesoporous silica nanoparticles (MSNs) (FA-MSN@EPT) that target lung tumor cells. We assessed the capacities of MSN@EPT and FA-MSN@EPT to induce ferroptosis in lung cancer cells by measuring ferroptosis-related ferritin (Fe2+), malondialdehyde, and glutathione levels. Enhanced ferroptosis, inhibition of A549 and H1299 cell proliferation, migration, invasion, and considerable improvements in the EPT bioavailability were noted when the fabricated MSN@EPT and FA-MSN@EPT were utilized. Furthermore, the apoptotic mode of cell death was investigated by acridine orange and ethidium bromide (AO-EB) and 4′,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry using annexin V-FITC and propidium iodide staining. This opens new possibilities for the clinical treatment of various tumors and offers a possible therapeutic option for lung cancer.

Red propolis extract associated to platelet-rich plasma and stromal cells with focus in cell therapy and functional tissue regeneration.

The use of platelet-rich plasma (PRP) and adipose-derived stromal cells (ADSC) have been investigated as a form of wound healing enhancement. The objective of this work was to evaluate the association of red propolis (RP) and PRP as inducers of ADSC for application in tissue regeneration. Adipose tissue post-collection and post-cryopreservation was isolated with type II collagenase, characterized by flow cytometry, and differentiated into osteogenic, chondrogenic and adipose cell. The viability of ADSC was evaluated when exposed to different concentrations of RP using the MTT and trypan blue assay. Acridine orange and ethidium bromide (AO/EB) was performed to evaluate cell death events. Horizontal migration methods were investigated in ADSC using autologous and homologous PRP associated with RP (PRP/RP). All assays were processed in triplicate. Flow cytometry and cellular differentiation showed that type II collagenase was effective for isolating ADSC post-collection and post-cryopreservation. RP extracts at concentrations of up to 50 μg.mL-1 presented no cytotoxic effects. Association of PRP and RP at 25 and 50 μg.ml-1 influenced ADSC migration, with total closure on the seventh day after exposition. The results here presented could stimulate proliferation of ADSC cells that may contribute directly or indirectly to the reconstructive process of tissue regeneration.

Precise Construction of Dual-Promising Anticancer Drugs Associated with Gold Nanomaterials on Glioma Cancer Cells.

Multiple chemodrugs with nanotechnology have proven to be an effective cancer treatment technique. When taken combined, cabazitaxel (CTX) and cisplatin (PT) have more excellent cytotoxic effects than drugs used alone in the chemotherapy of several different cancers. However, several severe side effects are associated with using these chemotherapy drugs in cancer patients. Gold nanomaterials (AuNMs) are promising as drug carriers because of their small diameter, easy surface modifications, good biocompatibility, and strong cell penetration. This work aimed to determine the CTX and PT encapsulated with AuNMs against human glioma U87 cancer cells. The fabrication of the AuNMs achieved a negative surface charge, polydispersity index, and the mean sizes. The combined cytotoxic effect of CTX and PT bound to AuNMs was greater than that of either drug alone when tested on U87 cells. The half inhibitory concentration (IC50) values for free PT were 54.7 μg/mL (at 24 h) and 4.8 g μg/mL (at 72 h). Results acquired from the MTT assay show cell growth decreases time- and concentration-dependent AuNMs, free CTX, free PT, and AuNMs@CTX/PT-induced cytotoxicity and, ultimately, the cell death of U87 cells via apoptosis. The biochemical apoptosis staining techniques investigated the cells' morphological changes of the cells (acridine orange and ethidium bromide (AO-EB) and nuclear staining (DAPI) techniques). The AO-EB and nuclear staining results reveal that the NPs effectively killed cancer cells. Furthermore, the flow cytometry analysis examined the mode of cell death. Therefore, AuNMs@CTX/PT has excellent potential in the cancer therapy of different cancer cells.

Corilagin Induces ROS-mediated Apoptosis and Triggers Cell-Cycle Arrest at G0/G1 Stage in Osteosarcoma Cells

Background Osteosarcoma (OS) is an extremely aggressive primary bone cancer (BC) malignancy. A variety of malignancies can develop in the bones, including BC. Primary BCs are tumors that start in the bone. Bones can also become affected by tumors that start in the body’s organs or other tissues. Objectives Several reports suggested that corilagin (CL) exerts anticancer properties on various kinds of tumor cells; however, its molecular action on OS cells remains undefined. Materials and Methods The CL activity of OS cells’ cytotoxicity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, and cell-cycle distribution was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA), Rh-123, acridine orange and ethidium bromide (AO/EB), 4′,6-diamidino-2-phenylindole (DAPI), and flow cytometry analysis. Results Results revealed that CL could suppress OS cell proliferation via enhanced intracellular ROS, and MMP loss, and triggered apoptosis in a dose-dependent has an inferior manner. Our findings demonstrated that CL alleviates U2OS and MG-63 cell proliferation by the ROS-mediated apoptosis, which triggers G0/G1 cell-cycle arrest. Conclusion Thus, CL might be a protective therapeutic agent against BC.

Piezoeletric cold atmospheric plasma induces apoptosis and autophagy in human hepatocellular carcinoma cells through blocking glycolysis and AKT/mTOR/HIF-1α pathway

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the fourth leading cause of cancer-related death worldwide. Advanced or metastatic HCC is currently managed using systemic drug therapy with unsatisfactory patient survival. Cold atmospheric plasma has emerged as a promising, physicochemical, and broad-spectrum oncotherapy. In this preclinical study, we investigated the anti-neoplastic functions and mechanism of piezoelectric direct discharge technology-based CAP, Piezo-CAP, on HCC in vitro and in vivo. Various HCC cells lines, such as SMMC7721, HepG2 and LM3, were used as in vitro cancer model for the phenotypic and mechanistic studies. Specifically, the cell counting Kit-8 and colony formation assay, flow cytometry, Transwell assay, Western blot, reactive oxygen species (ROS) assay, and glutathione to oxidized glutathione ratio (GSH/GSSG) assay were used to demonstrate plasma-induced changes in HCC cell proliferation, cell cycle progression, migration and invasion, epithelial-to-mesenchymal transition, intracellular ROS, and antioxidant capacity, respectively. In addition, the Acridine orange and ethidium bromide (AO/EB) staining and transmission electron microscopy were performed for cellular and subcellular assessment of HCC cell apoptosis. The Ad-mCherry-RFP-LC3B fluorescent double-labeled lentiviral system was used to detect autophagic flux. On the other hand, RNA-sequencing, quantitative real-time PCR, and Western blot were used to demonstrate plasma-induced metabolic and molecular disruption of tumor glycolysis and oncogenic proliferation, respectively. In vivo experiments using a human cell-line-derived xenograft model and immunohistochemistry (IHC) were utilized to investigate the mechanism. Piezo-CAP exerted anti-neoplastic functions through inhibiting cell proliferation, migration and invasion, and promote cell apoptosis and autophagy. Treatment of Piezo-CAP could suppress proliferation and induce autophagy of HCC cells through simultaneously disrupts cancer survival pathways of redox deregulation, glycolytic pathway, and PI3K/AKT/mTOR/HIF1α pathway signaling. Moreover, upon translation of these in vitro results into the tissue level, Piezo-CAP significantly suppressed in situ tumor growth. These findings collectively suggest that Piezo-CAP-induced apoptosis and autophagy of HCC cells though a multitargeted blockade of major cancer survival pathways of deregulated redox balance, glycolysis, and PI3K/AKT/mTOR/HIF-1α signaling.

Effective regeneration of rat sciatic nerve using nanofibrous scaffolds containing rat ADSCs with controlled release of rhNGF and melatonin molecules for the treatment of peripheral injury model

Different therapeutic strategies have been designed and developed for the repair and regeneration of peripheral nerve injury (PNI) tissue as a result of advancements in tissue engineering and regenerative medicine. Due to its versatility, controlled delivery and administration of multifunctional therapeutic agents can be regarded of as an effective strategy in treating nerve injury. In this study, melatonin (Mel) molecules and recombinant human nerve growth factor (rhNGF) were loaded on the surface and in the core of polycaprolactone/chitosan (PCL/CS) blended nanofibrous scaffold. To simulate the in vivo microenvironment, a dual-delivery three-dimensional (3-D) nanofibrous matrix was developed and the in vitro neural development of stem cell differentiation process was systematically examined. The microscopic technique with acridine orange and ethidium bromide (AO/EB) fluorescence staining method was used to establish the adipose-derived stem cells (ADSCs) differentiation and cell–cell communications, which demonstrated that the effective differentiation of the ADSCs with nanofibrous matrix. As investigated observations, ADSCs differentiation was further evident through cell migration assay and gene expression analysis. According to the biocompatibility analysis, the nanofibrous matrix did not trigger any adverse immunological reactions. Based on these characteristics, a 5-week in vivo investigation examined the potential of the developed nanofibrous matrix in the regeneration of sciatic nerve of rats. Additionally, compared to the negative control group, the electrophysiological and walking track analyses demonstrated improved sciatic nerve regeneration. This study demonstrates the nanofibrous matrix's ability to regenerate peripheral nerves.

Benzo(ghi)perylene (BgP) a black tattoo ingredient induced skin toxicity via direct and indirect mode of DNA damage under UVA irradiation

Tattooing is a very common fashion trend across all the ages and gender of the society worldwide. Although skin inflammatory diseases are very frequent among tattoo users because of the active chemical ingredients used in tattoo ink, yet no ingredient-specific toxicity study has been performed. Benzo(ghi)perylene (BgP) is one of the PAHs and an important ingredient of black tattoo ink that shows strong absorption in UVA and UVB radiation of sunlight. Therefore, understanding the hazardous potential of BgP especially under UVA exposure is important for the safety of skin of tattoo users by considering the fact that penetration of UVA is in the dermis region where tattoo ingredients reside. To evaluate the hazardous potential of BgP on human skin under UVA exposure, different experimental tools i.e., in-chemico, in-silico and in-vitro were utilized. Our results illustrated that BgP photosensitized under UVA (1.5 mW/cm2) irradiation shows a degradation pattern till 4 h exposure. Photosensitized BgP reduced significant cell viability (%) at 1 μg/ml concentration. However, the pretreatment of singlet and hydroxyl radical quenchers, restoration of cell viability observed, confirmed the role of type-I and type-II photodynamic reactions in phototoxicity of BgP. Further, intracellular uptake of BgP in HaCaT cells was estimated and confirmed by UHPLC analysis. Molecular docking of BgP with DNA and formation of γ-H2AX foci demonstrated the DNA intercalation and double-stranded DNA damaging potential of BgP. Furthermore, acridine orange and ethidium bromide (AO/EB) dual staining showed apoptotic cell death via photosensitized BgP under UVA irradiation. The above findings suggest that BgP reached the human skin cell and induced dermal toxicity via direct and indirect mode of DNA damage under UVA exposure finally promoting the skin cell death. Thus, BgP-containing tattoo ink may be hazardous and may induce skin damage and diseases, especially in presence of UVA radiation of sunlight. To minimize the risk of skin diseases from synthetic ingredients in tattoo ink, the study highlights the importance of developing eco-friendly and skin-friendly tattoo ingredients by companies.

Methanol extract of Pergularia daemia (Forssk.) Chiov. leaves induce apoptosis in triple-negative breast cancer through intrinsic pathway

Most traditional anticancer medications are ineffective in treating triple negative breast cancer (TNBC), which has enhanced resistance to chemotherapeutic agents. Herbs continue to be a key element in the discovery of anticancer medications. In the present study, we assessed the cytotoxicity of methanol extract of leaves of Pergularia daemia (MLPD) in MDA-MB-231 triple-negative breast cancer (TNBC) cell line. The cytotoxicity of MLPD was assessed using MTT assay which revealed significant concentration-dependent cytotoxicity with an IC50 value of 35.95±3.57 µg/mL. Morphological evaluation of MDA-MB-231 cells treated with MLPD showed cytotoxic changes like vacuole formation, altered morphology, and reduction in the number of cells with apoptotic bodies formation. Staining techniques, such as acridine orange and ethidium bromide (AO/EB) showed early apoptosis characterized by yellow-green fluorescence and crescent-shaped nucleus. Hoechst 33258 staining of MDA-MB-231 cells treated with MLPD showed nuclear marginalization indicative of nuclear apoptotic changes. To assess the mitochondrial membrane potential (MMP), we did JC-1 staining and the results revealed green fluorescence indicative of mitochondrial dependent apoptotic pathway. Comet assay confirmed significant DNA damaging property in MDA-MB-231 cells treated with MLPD. Western blotting analysis showed significant downregulation of Bcl-2 by 0.52±0.03 folds with no significant change in caspase-8 expression. Hence, the present study demonstrated that MLPD possessed potent cytotoxic effect against MDA-MB-231 TNBC cells through mitochondria mediated intrinsic pathway of apoptosis.

Cirsilineol Inhibits the Proliferation of Human Prostate Cancer Cells by Inducing Reactive Oxygen Species (ROS)-Mediated Apoptosis

Cirsilineol has been reported to exhibit anticancer effects against several human cancer cell lines. The present study was designed to evaluate the anticancer effects of cirsilineol against the human DU-145 prostate cancer cells. The results showed that cirsilineol suppressed the proliferation of DU-145 cancer cells in a dose-dependent manner with minimal cytotoxic effects against the normal cells. The IC50 of cirsilineol was found to be 7 μM and 110 μM against prostate cancer DU-145 and normal HPrEC prostate cells, respectively. Acridine orange and ethidium bromide (AO/EB) staining showed that cirsilineol induced apoptosis in DU-145 prostate cancer cells. The Annexin V/PI staining further confirmed the induction of apoptosis in DU-145 cells. The western blot analysis showed that cirsilineol suppressed the expression of Bax and upregulated the expression of Bcl-2 in prostate cancer DU-145 cells. Moreover, cirsilineol caused a dose-dependent increase in reactive oxygen species (ROS) levels in prostate cancer. Wound healing and Transwell assays showed that cirsilineol inhibits migration and invasion of DU-145 prostate cancer cells. Summing up, the results suggest that cirsilineol suppresses the proliferation of prostate cancer cells and may prove to be a beneficial lead molecule for the development of chemotherapy for prostate cancer.

Tailored silver nanoparticles capped with gallic acid and its potential toxicity via ROS mediated pathway against osteosarcoma cells

Chemotherapeutic agents used to treat cancer cells does not kill cancer cells only but healthy cells as well. Silver nanoparticles (SNP) broad toxicity towards the cancer cells have attracted people to find the alternative anticancer agent. Therefore, in this work we biosynthesize SNP from E. coli and coated with gallic acid. Characterized process carried out through UV–visible spectrophotometer, Fourier transform infrared spectroscopy (FTIR), Thermic gravimetric analysis, scanning electron microscopy (SEM) Energy dispersive X- rays (EDX) and Transmission electron microscopy (TEM) showed particles are well dispersed, well stable, and particle size ranges from 11.44 to 34.97 nm. The gallic acid coated silver nanoparticles (gSNP) showed excellent toxicity at 20 µg/ml IC50 towards SAOS-2 osteosarcoma cancer while less toxicity towards normal 3T3 cells. Further analysis confirms the cell cycle inhibition takes place at S and G2/M phase while acridine orange and ethidium bromide (AO/EB) staining confirms the apoptosis, showed membrane blebbing, chromatin condensation and morphological changes in the treated cell through flow cytometry showed the release of higher percentages of apoptotic bodies. The gSNPs induces the production of ROS in the treated cancer cell through 2′,7′-Dichlorofluorescin diacetate method. The gSNP also showed the upregulation of p53 and caspase 9 proteins through flow cytometry. This study showed that gSNP could become a promising candidate in medicine and pharmaceutical applications for targeted drug delivery against cancer therapy.

Anti-proliferative and Anti-migratory Activities of Diphyllin on Human Colorectal Cancer Cells

Published on:June 2022 Journal of Young Pharmacists, 2022; 14(2):198-202 Original Article | doi:10.5530/jyp.2022.14.37 Authors: Sagar Puli, Ramasamy Raveendran* Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Dhanvantari Nagar, Puducherry, INDIA. Abstract: Background: Colorectal cancer (CRC) is the third leading cause of death, probably because of its invasive and metastatic potential. Agents that can mitigate the growth and prevent migration may benefit CRC. Diphyllin and its analogues belong to glycosylated compounds previously reported for their in vitro cytotoxicity against many viruses, candida, and human malignant cells. This study aims to evaluate the anti-proliferative and antimigratory activities of diphyllin on human CRC cells. Methods: The MTT assay was performed to determine the 50% inhibitory concentrations (IC50) in HT-29, SW-480, and HCT-15 CRC cells. The acridine orange and ethidium bromide (AO/EB) dual staining was used to ascertain the type of cell death. HT-29 cells were subjected to short-term 5-fluorouracil+oxaliplatin (5-FU+Ox) to eliminate the most sensitive cells. The remaining surviving cells were further treated with diphyllin alone or combined with 5-FU. The combination effect of diphyllin with 5-FU in 5-FU+Ox surviving HT-29 cells was determined by flow cytometry-based Annexin V apoptosis assay. The wound-healing assay was used to detect the anti-migratory activity of diphyllin on 5-FU+Ox surviving HT-29 CRC cells. Results: The IC50 values of diphyllin were found to be 2.9±0.38, 1.3±0.28, and 3.9±0.65 μg/mL against HT-29, SW-480, and HCT-15, respectively. Microscopic examination manifested apoptotic changes like chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies in diphyllin treated CRC cells. Flow cytometry analysis revealed that diphyllin enhances the apoptosis in 5-FU+Ox surviving HT-29 cells. Wound-healing assay displayed the inhibitory activity of diphyllin in 5-FU+Ox surviving HT-29 cells. Conclusion: Diphyllin shows anti-proliferative activity by inducing apoptosis in HT-29, SW-480, HCT-15 cells and 5-FU+Ox surviving HT-29 cells and impairs their migration. Key words: Anticancer, Apoptosis, Cytotoxicity, Flow cytometry, Woundhealing.

Role and mechanism of Cortex Moutan components in inhibiting production of toxic advanced glycation end products (AGEs)

Advanced glycation end products(AGEs) can lead to many diseases such as diabetes and its complications. In this study, an in vitro non-enzymatic glycosylation reaction model-bovine serum albumin/methylglyoxal(BSA/MGO) reaction system was constructed and incubated with Cortex Moutan extract. High performance liquid chromatography(HPLC) and ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) were used to detect and identify the active components that inhibited the formation of AGEs in the co-incubation solution of Cortex Moutan extract and MGO, and differential components such as salvianan, paeoniside, benzoylpaeoniflorin, mudanpioside J, galloyloxypaeoniflorin, benzoyloxy-paeoniflorin, 5-hydroxy-3 s-hydroxymethyl-6-methyl-2,3-dihydro benzofuran, and galloylpaeoniflorin were screened out, which were inferred to be the potential active components of Cortex Moutan extract to capture MGO. In addition, BSA-glucose reaction system was performed to investigate the influence of different concentrations of Cortex Moutan extract(decoction concentrations: 40, 80, 120, 160, and 200 mg·mL~(-1)) on inhibiting the production of AGEs in vitro. The inhibitory effects of Cortex Moutan extract and the differential components galloylpaeoniflorin and benzoyl paeoniflorin on the production of AGEs in human umbilical vein endothelial cells(HUVECs) induced by high glucose was further evaluated. Cell apoptosis was observed by acridine orange and ethidium bromide(AO/EB) double fluorescence staining. The results showed that Cortex Moutan Cortex extract and its differential components had certain inhibitory effects on the formation of AGEs, and could reduce cell apoptosis. This study provided reference for the treatment of diabetic vascular complications by Cortex Moutan inhibiting the toxic AGEs.

Transcriptome and biochemical analyses of rainbow trout (Oncorhynchus mykiss) RTG-2 gonadal cells in response to BDE-47 stress indicates effects on cell proliferation.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a biotoxin of polybrominated diphenyl ether (PBDEs) frequently detected in the environment. Apoptosis and cell cycle arrest are important toxic phenomena of xenobiotics that inhibit cell proliferation. In this study, we investigated the effects of BDE-47 (5μM, 10μM, 20μM, 40μM) on cell viability, morphology, cell cycle and apoptosis. BDE-47 significantly decreased cell viability, and morphological alterations were observed. The significant increase in cells at G1 phase indicated the occurrence of G1 phase cell cycle arrest in RTG-2 cells. An acridine orange and ethidium bromide (AO/EB) staining assay was employed and revealed the induction of apoptosis in RTG-2 cells. The results indicated that BDE-47 exposure inhibits cell proliferation. Transcriptome analysis was applied for further evidence. A total of 1300 differentially expressed genes (DEGs) were identified in RTG-2 cells, among which 26 DEGs were associated with the cell cycle and apoptosis. Western blotting and qPCR analyses also showed the expression of cell cycle- and apoptosis-related proteins and genes. Mapping the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, p53, Tumor necrosis factor (TNF), Mitogen-activated protein kinase (MAPK), phosphatidylinositide 3-kinase-AKT (PI3K-AKT), and reaction oxygen species (ROS)-mediated signaling pathways were determined to be the major pathways involved in modulating the cell cycle and apoptosis. Since we demonstrated simultaneous ROS overproduction during BDE-47 exposure in a previous study, we speculated a possible explanation for the observation: BDE-47-induced ROS overproduction was the initiating signal, which activated cell cycle arrest and apoptosis and finally inhibited cell proliferation.

CLEISTANTHIN B SHOWS A POTENT CYTOTOXIC ACTIVITY AGAINST COLORECTAL CANCER CELLS

Objective: The aim was to find out the cleistanthin B sensitive cancer cell type among a panel of cancer cell lines. Methods: The 50% inhibitory concentrations (IC50) of cleistanthin B against different cancer cells were determined by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. The cell death caused by cleistanthin B in colorectal cancer (CRC) cells was evaluated by acridine orange and ethidium bromide (AO-EB) dual staining. Using short exposure, we generated the 5-fluorouracil</AQ1> and oxaliplatin (5-FU+Ox) surviving cells from the parental HT-29 CRC cell lines. These surviving CRC cells were further treated with cleistanthin B either alone or combined with 5-FU. Annexin V apoptosis assay was used to determine the combined effect of cleistanthin B with 5-FU against HT-29 cells. Results: The IC50 values of cleistanthin B were found to be 3.6±0.55, 5.2±0.51, 8.6±1.02, 10.5±1.50, 18.3±3.71, 25.8±5.50, and 26.7±5.90 μg/mL against HT-29, SW-480, HCT-15, HELA, MDA-MB-231, A549, and DU145, respectively. The IC50 value of cleistanthin B against L132 cells was >100 μg/mL. The cleistanthin B treated HT-29, SW-480, and HCT-15 CRC cells showed apoptotic changes such as chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies in the AO-EB dual staining method. Flow cytometry analysis revealed that cleistanthin B enhances the 5-FU induced apoptosis against 5-FU+Ox surviving HT-29 CRC cells. Conclusion: Cleistanthin B is relatively more potent against CRC cells than other cancer cells, and it induces apoptosis mediated cell death in CRC cells. Cleistanthin B enhances the anticancer activity of 5-FU against HT-29 CRC cells.

Cytotoxic Effect of Lippia nodiflora Leaf Extract against the Prostate Cancer Cell Line

Prostate cancer is the second most common cause of cancer deaths for men. Lippia. Nodiflora (L. nodiflora) has been used as a natural remedy for various diseases, because of its antioxidant, anti-inflammatory, anti-bacterial, and anti-tumor effect. This study was to investigate the cytotoxic effect of L. nodiflora ethanolic leaf extract in prostate cancer cell lines (PC-3). The growth inhibitory effect of L. nodiflora ethanolic leaf extract was assessed by MTT assay. The cell morphological changes in L. nodiflora leaf extract-treated cells were observed using an inverted phase-contrast microscope. Apoptosis induction by L. nodiflora was determined by AO/EtBr (acridine orange and ethidium bromide) dual staining. MTT test results showed dose-dependent cell growth inhibition in PC-3 cells treated with L. nodiflora leaf extract (10-120 µg/mL). The IC-50 dose was observed at 40µg/ml. Morphological changes such as reduction in the number of cells, cell shrinkage, and cytoplasmic membrane blebbing were observed in the treated cells. Induction of apoptosis by L. nodiflora (40µg/ml) treated cells showed an increased number of early apoptotic and late apoptotic cells. The above data indicate that L. nodiflora inhibits cell proliferation and induces apoptosis in prostate cancer cells. Therefore, it can be concluded that L. nodiflora exhibits anti-cancer activity, and thus it raises new hope for its use in anti-cancer therapy.

Cytotoxic cobalt (III) Schiff base complexes: in vitro anti-proliferative, oxidative stress and gene expression studies in human breast and lung cancer cells.

Increasing cancer drug chemo-resistance, especially in the treatment of breast and lung cancers, alarms the immediate need of newer and effective anticancer drugs. Until now, chemotherapeutics based on metal complexes are considered the most effective treatment modality. In the present study, we have evaluated the cytotoxic effect of two cobalt (III) Schiff base complexes based on the leads from complex combinatorial chemistry. Cobalt (III) Schiff base complexes (Complex 3=Co(Ph-acacen)(HA)2](ClO4) and Complex 4= [Co(Ph-acacen)(DA)2](ClO4)] (Ph-acacen, 1-phenylbutane-1,3-dione; DA, dodecyl amine; HA, heptylamine) were evaluated against human breast cancer cell MCF-7 and lung cancer cell A549 using MTT cell viability assay, cellular morphological changes studied by Acridine Orange and Ethidium Bromide (AO/EB), Dual fluorescent staining, Hoechst staining 33248, Comet assay, Annexin V-Cy3 and 6 CFDA assay, JC-1 staining, Reactive oxygen species (ROS) assay, Immunofluorescence assay, and Real-time reverse transcription-polymerase chain reaction (RT-qPCR). Treatment of cobalt (III) Schiff base complexes (Complex 3 & 4) affected the viability of the cancer cells. The cell death induced by the complexes was predominantly apoptosis, but necrosis also occurred to a certain extent. Complex 4 produced better cytotoxic effect than complex 3, and MCF-7 cell was more responsive than A549. In that order, the complexes were more selective to cancer cell than normal cell, and more effective in overall performance than the standard drug cisplatin. Therefore, we conclude that cobalt (III) Schiff base complexes, especially complex 4, have the potential to be developed as effective drugs for treatment of cancers in general, and breast and lung cancers in particular.

Antineoplastic effectiveness of silver nanoparticles synthesized from Onopordum acanthium L. extract (AgNPs-OAL) toward MDA-MB231 breast cancer cells

The present research was done to investigate the anticancer properties of silver nanoparticles (AgNPs) fabricated using bioactive extract of Onopordum acanthium L. (AgNPs-OAL) against breast cancer cells MDA_MB231 in vitro. The determination studies of AgNPs-OAL were confirmed by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) analysis. Interestingly, the FESEM image observed the spherical shape of AgNPs-OAL with the range of 1-100 nm. As AgNP-OAL exhibited significant cytotoxicity properties on breast cancer MDA_MB231 cells with IC50 values of 66.04µg/mL, while lowing toxicity toward normal human embryonic kidney 293 (HEK293) cells with IC50 values of 101.04µg/mL was evaluated. Further, up-regulation of apoptotic Bax and CAD gene expressions were confirmed by quantitative real-time reverse transcription-PCR (qRT-PCR) technique results. Moreover, enhanced cell cycle population (sub-G1), annexin V/PI staining, acridine orange and ethidium bromide (AO/EB) staining, Hoescht 33,258 dye, and generation of reactive oxygen species were observed in AgNP-OAL-treated MDA_MB231 cancer cells. The green-synthesized AgNP-OAL has promising anticancer efficiency that can trigger apoptosis pathways in the MDA_MB231 breast cancer cells.