Acridine Orange/ethidium Bromide Research Articles

Evaluation of antimicrobial and antioxidant activity of cleome gynandra l. and assess its anticancer activity against in vitro cancer model

Cleome gynandra Linn. is the native herb distributed at tropical and sub-tropical temperature. In this study, the antioxidant, antimicrobial and anticancer properties of the ethanolic extract of Cleome gynandra leaves were investigated also the anti-cancer activity was assessed against A549 lung cancer cell lines. The antioxidant activities were evaluated by 1,1‑diphenyl‑2‑picrylhydrazyl free radical scavenging activity. The IC50 value of DPPH was ranged from 65.67 µg/ml for the leaf extract. The anti-microbial activity was showed against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and Salomonella typhi. The cytotoxic study was conducted and the results exhibited the strongest inhibitory activity against A549 lung cancer cell line as concentration dependent manner. The cytotoxic activity of the leaf extract was supported by the induction of apoptosis cell. The other assays to evaluate anticancer activity are DAPI (4,6 – diamminodino-2-phenylindole), PI (propidium iodide) and EtBr/AO (Ethidium bromide/acridine orange) staining methods. All the study results revealed the efficient anticancer activity of the ethanolic leaf extract of Cleome gynandra leaves. Thus, the herbal drug treatment might highly be recommended to treat effectively lung cancer cell line as an ideal choice or combinational therapy.

The Essential Oil from Oliveria decumbens Vent. (Apiaceae) as Inhibitor of Breast Cancer Cell (MCF-7) Growth.

Oliveria decumbens Vent. is an aromatic and medicinal plant traditionally used in Iran for the treatment of infections, gastrointestinal diseases, cancer, and inflammation. This research was aimed at investigating the pharmacological potential of O. decumbens essential oil (OEO) and its main compounds, focusing on OEO's cytotoxic effects on MCF-7 breast cancer cells. OEO was obtained by hydro-distillation, and the chemical constituents were identified using GC-MS. Thymol, carvacrol, γ-terpinene, and p-cymene were the main OEO constituents. When MCF-7 cells were treated with OEO, the expressions of genes related to apoptosis (BIM and Bcl-2), tumor suppression (PTEN), and cell growth inhibition (AURKA), were evaluated using real-time PCR. Moreover, molecular docking was used for studying in silico the interaction of OEO principal compounds with PTEN and AURKA. The expression of AURKA was significantly reduced since the OEO treatment enhanced the expression of PTEN. Through in silico molecular docking, it was revealed that thymol, carvacrol, p-cymene, and γ-terpinene can activate PTEN and thus inhibit AURKA. Additionally, the DNA fragmentation assay, acridine orange/ethidium bromide (AO/EB) double-staining assay, and real-time PCR highlighted the fact that the OEO treatment could activate apoptosis and inhibit cell proliferation. Therefore, OEO is a viable candidate to be employed in the pharmaceutical industry, specifically as a possible agent for cancer therapy.

Enhancement of cytotoxic and antioxidant activities of Digenea simplex chloroform extract using the nanosuspension technique

Digenea simplex (D. simplex), an Egyptian marine red macroalga, contains a diverse group of phytochemicals with unique bioactivities. At the same time, the synthesis of nanosuspension (NS) has received increasing interest to optimize the technological aspects of drugs. Thence, the main objective of this work was to use the chloroform extract (ChlE) of D. simplex to prepare its nanosuspension (ChlE-NS) formulation to increase its aqueous solubility, thereby improving its bioactivity. By using FTIR, GC/MS analysis, and phytochemical screening assays, the chemical profiling of ChlE was assessed. NS was prepared by the antisolvent precipitation technique using 1.5% w/v polyvinyl alcohol (PVA). A light microscope, FTIR, particle size distribution, polydispersity index (PDI), and zeta potential (ZP) measurements was used to characterize the prepared NS. Four cancer cell lines were used in the MTT experiment to investigate the anticancer potential of ChlE and ChlE-NS. An apoptotic mechanism was established using acridine orange/ethidium bromide (AO/EB) dual staining, DNA fragmentation, and increased caspase activity. ChlE and ChlE-NS were also evaluated as antioxidants using DPPH and ABTS free radical assays. The results showed that, when compared to ChlE, ChlE-NS had greater cytotoxic activity against the four cancer cell lines. However, results of antioxidant activity showed that ChlE-NS had an IC50 of 36.86 ± 0.09 and 63.5 ± 0.47%, while ChlE had values of 39.90 ± 0.08 and 86.5 ± 0.8% in DPPH and ABTS assays, respectively. Based on the results of this research, D. simplex ChlE-NS may be an effective strategy for enhancing ChlE's cytotoxic and antioxidant activities.Graphical abstract

Synthesis of Quinoline-2-Carboxylic Acid Aryl Ester and Its Apoptotic Action on PC3 Prostate Cancer Cell Line.

The aim of the present study is to synthesise an aryl ester compound from quinoline-2-carboxylic acid to evaluate its apoptotic, cell cycle blockade, and antiproliferative activity on the prostate cancer cell lines (PC3). Chromatographic and spectroscopic analysis was used to identify the synthesised carboxylic acid compound. The synthesised compound was treated with a PC3 cell line for 24h with control. The cells were treated at various concentration ranges of 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, and 1000µg/mL each. The cytotoxicity effect was studied by MTT assay, and their anticancer activity was further evaluated using cell cycle analysis, DNA fragmentation assay, acridine orange-ethidium bromide staining, and Western blot analysis. The end antiproliferative result showed that PC3 cell viability decreases in a concentration-dependent manner and the synthesised compound exhibited potent cytotoxicity against PC3 cells with an IC50 value of 26µg/mL at the concentration of 125µg. The increase in the number of apoptotic cells was observed after treating PC3 cells with the sample in double-staining methods. S phase of the cell cycle was significantly blocked by the test sample, and a typical ladder pattern of internucleosomal fragmentation was observed. A decrease in the live cells was observed with the sample in AO/ET-BR. A significant increase in the Bax expression and a decrease in Bcl-2 expression observed enhance the activity of caspases-7 and -9. The synthesised compound had shown to possess excellent cytotoxic effect through inducing apoptosis, especially causing cell cycle arrest at the S phase.

Synthesis, spectral characterization, protein binding and cytotoxic evaluation of new cobalt(II) and cobalt(III) complexes containing benzimidazolylterpyridine as ligand

Two new six-coordinate cobalt(II) [Co(bitpy)2]Cl2 (1) and cobalt(III) [Co(bitpy)2](ClO4)3 (2) complexes containing benzimidazolylterpyridine (bitpy) ligand have been synthesized and characterized using spectroscopic and electrochemical techniques. The g ‖ and g ⊥ values in the EPR spectrum for 1 were 2.215 and 2.012, respectively, and clearly authenticated the formation of stable octahedral low spin Co(II) meridional isomer complex. Similarly, 1H NMR spectrum of 2 revealed low spin and its diamagnetic nature. The interaction of the complexes with bovine serum albumin (BSA) has been investigated and revealed that the cobalt complexes quenched the intrinsic fluorescence by following the static quenching mechanism. MTT assay was used to check the anticancer potential of 2 against human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa). The IC50 values of 2 were lower than 7 µM in both the cancer cells and higher than 100 µM for non-cancerous cells (human embryonic kidney cells), indicating the specificity of the complex toward cancer cells. Furthermore, the morphological changes induced by 2 on the tested cell lines were monitored by AO-EB (acridine orange-ethidium bromide) and DAPI (4′,6-diamidino-2-phenylindole) staining methods.

Quality by design steered development of Niclosamide loaded liposomal thermogel for Melanoma: In vitro and Ex vivo evaluation

Melanoma is the most malignant form of skin cancer across the globe. Conventional therapies are currently ineffective which could be attributed to the rampant chemo-resistance, metastasis, inability to cross the skin barriers and accumulate within the tumor microenvironment. This advent brings in the principles of drug repurposing by repositioning Niclosamide (NIC), an anthelmintic drug for skin cancer. Incorporation into the liposomes facilitated enhanced melanoma cell uptake and apoptosis. Cytotoxicity studies revealed 1.756-fold enhancement in SK-MEL-28 cytotoxicity by NIC-loaded liposomes compared to free drug. Qualitative and quantitative cell internalization indicated greater drug uptake within the melanoma cells illustrating the efficacy of liposomes as efficient carrier systems. Nuclear staining showed blebbing and membrane shrinkage. Elevated ROS levels and apoptosis shown by DCFDA and acridine orange-ethidium bromide staining revealed greater melanoma cell death by liposomes compared to free drug. Incorporating NIC liposomes into the thermogel system restricted the liposomes as a depot onto the upper skin layers. Sustained zero order release up to 48 h with liposomes and 23.58-fold increase in viscosity led to the sol-to-gel transition at 33℃ was observed with liposomal thermogel. Ex vivo gel permeation studies revealed that C-6 loaded liposomes incorporated within the thermogel successfully formed a depot over the upper skin layer for 6 h to prevent transdermal delivery and systemic adverse effects. Thus, it could be concluded that NIC loaded liposomal thermogel system could be an efficacious therapeutic alternative for the management of melanoma.

Synthesis, Characterization, and Antiproliferative Effect of CuO-TiO2-Chitosan-Amygdalin Nanocomposites in Human Leukemic MOLT4 Cells.

The main aim of this study was to synthesize copper oxide- (CuO-) titanium oxide- (TiO2-) chitosan-amygdalin nanocomposites (CTCANc) and to characterize them physically and biologically (antimicrobial and anticancer activity using MOLT4 blood cancer cell line) to endorse their useful applications as potential drug candidates in anticancer avenues. CuO-TiO2-chitosan-amygdalin nanocomposites were synthesized according to standard, reported methods. Physical characterization of the nanocomposites was performed using methods like X-ray diffractometer (XRD), and morphological and ultrastructural analysis of nanocomposites were done using electron microscope scanning and transmission. FTIR was recorded using a Perkin-Elmer spectrometer, and photoluminescence (PL) spectra were done using the spectrometer. Further, antibacterial activities were assessed using standard bacterial cultures. To demonstrate the nanocomposite's anticancer effects, MTT assay, morphological analysis, apoptosis studies using acridine orange/ethidium bromide (AO/EtBr) dual staining, reactive oxygen species (ROS) analysis, and levels of antioxidant enzymes were analyzed using the MOLT4 blood cancer cell line. Synthesized nanocomposites were characterized using XRD and showed various peaks, respectively, for CuO-TiO2, amygdalin, and chitosan. MTT assay indicated an IC50 value of 38.41 μg/ml concentration of CTCANc. Hence, 30 and 40 μg/ml were used for the subsequent experiments. Morphological analysis, staining for apoptosis using AO/EtBr, mitochondrial membrane potential (MMP or ΔΨm) analysis, ROS analysis, and determination of the SOD, CAT, MDA, and GSH levels were performed. Observations like a significant loss of morphology, induction of apoptosis, elevated ROS, and decreased MMP were significant in 30 and 40 μg/ml nanocomposite-treated cells when compared to control cells. The bimetallic nanocomposites exhibited typical nanocomposites characteristics and significant antibacterial and anticancer effects. The study results endorse the antibacterial, anticancer activity of CuO-TiO2-chitosan-amygdalin nanocomposites and strongly suggest that further in-depth research using CuO-TiO2-chitosan-amygdalin nanocomposites could reveal their efficacy in the clinical scenario.

Ilimaquinone (Marine Sponge Metabolite) Induces Apoptosis in HCT-116 Human Colorectal Carcinoma Cells via Mitochondrial-Mediated Apoptosis Pathway.

Ilimaquinone (IQ), a metabolite found in marine sponges, has been reported to have a number of biological properties, including potential anticancer activity against colon cancer. However, no clear understanding of the precise mechanism involved is known. The aim of this study was to examine the molecular mechanism by which IQ acts on HCT-116 cells. The anticancer activity of IQ was investigated by means of a cell viability assay followed by the determination of induction of apoptosis by means of the use of acridine orange–ethidium bromide (AO/EB) staining, Annexin V/PI double staining, DNA fragmentation assays, and TUNEL assays. The mitochondrial membrane potential (ΔΨm) was detected using the JC-1 staining technique, and the apoptosis-associated proteins were analyzed using real-time qRT-PCR. A molecular docking study of IQ with apoptosis-associated proteins was also conducted in order to assess the interaction between IQ and them. Our results suggest that IQ significantly suppressed the viability of HCT-116 cells in a dose-dependent manner. Fluorescent microscopy, flow cytometry, DNA fragmentation and the TUNEL assay in treated cells demonstrated apoptotic death mode. As an additional confirmation of apoptosis, the increased level of caspase-3 and caspase-9 expression and the downregulation of Bcl-2 and mitochondrial dysfunction were observed in HCT-116 cells after treatment with IQ, which was accompanied by a decrease in mitochondrial membrane potential (ΔΨm). Overall, the results of our studies demonstrate that IQ could trigger mitochondria-mediated apoptosis as demonstrated by a decrease in ΔΨm, activation of caspase-9/-3, damage of DNA and a decrease in the proportion of Bcl-2 through the mitochondrial-mediated apoptosis pathway.

Di-(2-Ethylhexyl) Phthalate and Microplastics Induced Neuronal Apoptosis through the PI3K/AKT Pathway and Mitochondrial Dysfunction.

Di-(2-Ethylhexyl) phthalate (DEHP) and microplastics (MPs) have released widespread residues to the environment and possess the ability to cause damage to humans and animals. However, there are still gaps in the study of damage to neurons caused by DEHP and MPs in mice cerebra and whether they have combined toxic effects. To investigate the underlying mechanism of action, mice were fed 200 mg/kg DEHP and 10 mg/L MPs in vivo. In vitro, NS20Y (CBNumber: CB15474825) cells were treated with 25 μM DEHP and 775 mg/L MPs. Next, qRT-PCR and western blot analysis were performed to evaluate PI3K/AKT pathway genes, mitochondrial dynamics-related genes, apoptosis-related genes, and GSK-3β and its associated genes, mRNA, and protein expression. To determine pathological changes in the mice cerebra, hematoxylin and eosin (H&E) staining, transmission electron microscopy, and TUNEL staining were employed. To determine the levels of reactive oxygen species (ROS) and apoptosis cells in vitro, ROS staining, acridine orange/ethidium bromide (AO/EB) staining, and flow cytometry were performed. Our results demonstrated that DEHP and MPs caused changes in mitochondrial function, and GSK-3β and its associated gene expression in mice through the PI3K/AKT pathway, which eventually led to apoptosis of neurons. Moreover, our findings showed that DEHP and MPs have a combined toxic effect on mice cerebra. Our findings facilitate the understanding of the neurotoxic effects of DEHP and MPs on neurons in the cerebra of mice and help identify the important role of maintaining normal mitochondrial function in protecting cerebrum health.

Cross-interactions between Norepinephrine, Methicillin-Resistant Staphylococcus aureus and Human Osteoblast Cells in Culture Conditions

Objective: The role of norepinephrine (NE) on growth, adhesion and invasion of methicillin-resistant Staphylococcus aureus (MRSA) ATTC 43300 was examined in human osteoblast (HOB) cells. The effects of NE and/or MRSA on the viability and cell death pathways of HOB cells were also investigated. Furthermore, the alterations of bacterial response to oxidative stress (H2O2) were analyzed in the presence/ absence of NE. Materials and Methods: Bacterial growth was detected spectrophotometrically. The colony counting method was examined for adhesion-invasion. The alteration of HOB cell viability was determined by methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay. The death pathways of HOB cells were examined microscopically using acridine orange-ethidium bromide dual staining and dichlorofluorescein-diacetate (DCF-DA) dye. The bacterial response to H2O2 was investigated by agar dilution. Results: The growth of bacterium was not affected in the presence of NE. Bacterial adhesion was decreased by NE (p<0.0001) while high-level NE induced invasion (p=0.013). HOB cell viability was reduced by MRSA and/or NE (p<0.001). MRSA and co-existence of MRSA and NE caused necrosis more than apoptosis in HOB cells (p<0.05). NE did not alter the bacterial response to oxidative stress. Conclusion: Norepinephrine has different effects on the biological properties of both MRSA and HOB cells.

Pomolic acid inhibits proliferation of human lung carcinoma cells via induction of apoptosis and suppression of cell migration and invasion

Purpose: To investigate the anti-proliferative effect of pomolic acid on lung cancer cells (A549), and theunderlying mechanism.Methods: The viability of pomolic acid-treated A549 cells was determined by MTT and colony formation assays. Cell colony formation was monitored with acridine orange/ethidium bromide (AO/EB) staining. Protein expressions of Bax and Bcl-2 were assayed by western blotting.Results: Pomolic acid suppressed the growth of A549 cells, with an half-maximal inhibitory concentration of (IC50) of 10 μM (p < 0.05). However, the IC50 of pomolic acid for normal BEAS-2B cells was 80 μM. Pomolic acid also decreased colony formation of A549 cells. At 20 μM, the percentage of A549 colonies decreased to 14 % of control. The dose-dependent cytotoxicity of pomolic acid against A549 cells was mediated via induction of apoptosis and oxidative stress. Pomolic acid treatment enhanced the expression of Bax and decreased the expression of Bcl-2 in A549 cells. Moreover, pomolic acid inhibited the migration and invasion in A549 cells in a dose-dependent manner (p < 0.05).Conclusion: These results indicate the potent anticancer effect of pomolic acid against human lung cancer cells. Thus, pomolic acid has promising potential as a lead molecule for the development of chemotherapy.

5-Hydroxyisophthalic acid and neocuproine containing copper(II) complex as a promising cytotoxic agent: Structure elucidation, topology, Hirshfeld surface, DFT calculations, and molecular docking analysis

We aimed to synthesize a new copper(II) complex, namely [Cu(H2IPA)(NC)(Cl)]⋅H2O (1) (where, H3IPA = 5-hydroxy isophthalic acid and NC = 2,9-dimethyl-1,10-phenanthroline or Neocuproine) as a promising chemotherapeutic agent. Complex 1 was structurally characterized by single-crystal X-ray diffraction (SCXRD), UV-Visible, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and powder X-ray diffraction (PXRD) methods. Single crystal diffraction analysis reveals that Cu(II) ion exists in a distorted square pyramidal geometry, with the ligation of a chloride ion, two oxygen atoms, and two nitrogen atoms. Topological structural simplification of complex 1 shows ins as an underlying net. The prominent intermolecular interactions and their quantitative contributions in complex 1 were investigated using Hirshfeld Surface (HS) analysis. The DFT calculations were also performed to validate the experimental results (XRD, FTIR, and UV-Visible). In vitro cytotoxicity of complex 1 was performed against acute myeloid leukemia (THP-1), colorectal (SW480), and prostate cancer (PC-3) cell lines by utilizing MTT assay. The result shows that complex 1 has the ability to inhibit the growth of cancer cells at a lower concentration. The morphological changes that occur during apoptosis were carried out with acridine orange/ethidium bromide (AO/EB) staining methods on THP-1 and SW480 cancer cell lines. Additionally, comprehensive molecular docking studies were performed to understand the potential binding interactions of complex 1 with target protein and DNA double helix.

Galangin/β-Cyclodextrin Inclusion Complex as a Drug-Delivery System for Improved Solubility and Biocompatibility in Breast Cancer Treatment.

The purpose of this study was to evaluate the potential of a newly modified cyclodextrin derivative, water-soluble β-cyclodextrin–epichlorohydrin (β-CD), as an effective drug carrier to enhance the poor solubility and bioavailability of galangin (GAL), a poorly water-soluble model drug. In this regard, inclusion complexes of GAL/β-CDP were prepared. UV-VIS spectrophotometry, Fourier-transform infrared spectroscopy (FTIR), X-ray crystallography (XRD), zeta potential analysis, particle size analysis, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) were applied to characterize the synthesized GAL/β-CD. Michigan Cancer Foundation-7 (MCF-7; human breast cancer cells) and rat embryo fibroblast (REF; normal cells) were employed to examine the in vitro cytotoxic effects of GAL/β-CD using various parameters. The dye-based tests of MTT and crystal violet clearly exhibited that GAL/β-CD-treated cells had a reduced proliferation rate, an influence that was not found in the normal cell line. The cells’ death was found to follow apoptotic mechanisms, as revealed by the dye-based test of acridine orange/ethidium bromide (AO/EtBr), with the involvement of the mitochondria via caspase-3-mediated events, as manifested by the Rh 123 test. We also included a mouse model to examine possible in vivo toxic effects of GAL/β-CD. It appears that the inclusion complex does not have a significant influence on normal cells, as indicated by serum levels of kidney and liver enzymatic markers, as well as thymic and splenic mass indices. A similar conclusion was reached on the histological level, as manifested by the absence of pathological alterations in the liver, kidney, thymus, spleen, heart, and lung.

Radiosensitizing effect of dendrosomal nanoformulation of curcumin on cancer cells.

Curcumin was found to possess numerous pharmacological activities in clinical research, however, its biological effects together with radiation are yet to be addressed. The present study investigated whether the combined treatment of dendrosomal nanoformulation of curcumin (DNC) and gamma radiation can enhance the radiosensitivity of U87MG and MDA-MB-231 cell lines. U87MG and MDA-MB-231 cell lines were exposed to 2 Gray (Gy) and 10μM DNC determined by MTT assay, then subjected to clonogenic assay, cell cycle assay, and flow cytometric apoptosis analysis. Acridine Orange/Ethidium Bromide (AO/EB) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) stained cells were used to study morphologic changes. The expression evaluation of putative cell cycle genes, i.e., P53, P21, CCND1, and CCNB1 was carried out by RT-qPCR. Our findings indicated that the combined treatment with DNC and radiation might cooperatively augment the efficacy of ionizing radiation in the cancer cells and notably decrease the survival and viability of the cells in a time- and concentration-dependent manner. In addition to a synergistic effect deducted by sensitizer enhancement ratio (SER) assessment, co-treatment resulted in greater apoptotic cells than the individual treatments. Further experiments then indicated that DNC could effectively induce G2/M phase cell cycle arrest and apoptosis following irradiation. Conformably, there was a decrement of CCND1 and CCNB1 expression, and an increment of P53, P21 expression. The data implied that DNC as a radiosensitizer can enhance the lethal effect of ionizing radiation on cancer cells which could be a promising adjuvant therapy in clinical treatments.

β-Hydroxyisovaleryl-Shikonin Exerts an Antitumor Effect on Pancreatic Cancer Through the PI3K/AKT Signaling Pathway.

Pancreatic cancer (PC) is marked with a low survival rate and lack of recognized effective treatment strategy. We investigated the antitumor effect of β-hydroxyisovaleryl-shikonin (β-HIVS) on PC and the associated working mechanism. Cell toxicity was determined using Cell Counting Kit-8 (CCK-8) assay. Acridine Orange/Ethidium Bromide (AO/EB) double-fluorescent staining assay accompanied by flow cytometry was utilized to estimate cell apoptosis. Cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential were all evaluated using flow cytometry. Transwell and wound healing assays were performed to evaluate cell migration and invasion. Protein expression was analyzed by Western blots. A xenograft mouse model was employed to determine the antitumor effect of β-HIVS in vivo. PC cell viability gradually decreased with increasing β-HIVS while apoptosis was enhanced together with cell-cycle blockage in the G0–G1 phases. β-HIVS induced mitochondrial dysfunction, ROS production, and DNA damage and inhibited the invasive and migratory ability of PC cells. We further confirmed the suppression of EMT and PI3K/AKT pathways as underlying mechanisms. The mouse model treated with the increasing dose of β-HIVS displayed decreased tumor growth rate, along with increased apoptosis. Thus, β-HIVS exerts antitumor effects on PC through inducing apoptosis, ROS production, decreasing mitochondrial membrane potential, and suppressing signal pathways, such as PI3K/AKT. In summary, β-HIVS might be a promising strategy for PC treatment.

Effects of dihydroartemisinin combined with cisplatin on proliferation, apoptosis and migration of HepG2 cells

Cisplatin (DDP) is a potent and widely applied chemotherapeutic agent. However, its clinical efficacy for the treatment of liver cancer is limited by adverse effects and the development of resistance. Combinatorial therapy may alleviate these issues. Dihydroartemisinin (DHA) is a first-generation derivative of artemisinin. The effects of DDP on liver cancer when applied in combination with DHA have not previously been studied. Therefore, the present study aimed to investigate the effects of DHA combined with DDP on HepG2 cells and their potential underlying molecular mechanisms. HepG2 cells were treated with different concentrations of DHA and/or DDP. Cell Counting Kit-8 assay was used to assess the cell viability. Cell proliferation and apoptosis were quantified using flow cytometry, acridine orange/ethidium bromide (AO/EB) fluorescent dual staining and the colony formation assay. Cell migration was quantified using the Transwell and wound healing assays. The HepG2 cell protein expression levels of Fas, Fas-associated death domain (FADD), procaspase-3, cleaved caspase-3, pro-caspase-8, cleaved caspase-8, Bax, Bcl-2, E-cadherin and N-cadherin, were detected via western blotting. Gelatin zymography was used to assess the levels of MMP-9 secreted by HepG2 cells into the supernatant. Following combined DHA and DDP treatment, the percentage of apoptotic cells was significantly increased, whereas cell proliferation and migration were significantly reduced, compared with cells treated with DDP only. DHA and DPP in combination significantly inhibited the expression of MMP-9, significantly increased the protein expression levels of Fas, FADD, Bax and E-cadherin, significantly increased the ratio of cleaved caspase-3 and cleaved caspase-8 to their precursor proteins and significantly decreased the protein expression levels of Bcl-2 and N-cadherin. The findings of the present study suggested that, DHA may confer synergistic effects with DDP in potentially promoting apoptosis and inhibiting the epithelial-mesenchymal transition for the treatment of liver cancer.

Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle

Objective“Multi-targeting” drugs can prove fruitful to combat drug-resistance of multifactorial disease—cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. MethodsThuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. ResultsThuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways—apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood–brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. ConclusionRobust “multi-targeting” anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.

Cytotoxic Activity, Cell Cycle Inhibition, and Apoptosis-Inducing Potential of Athyrium hohenackerianum (Lady Fern) with Its Phytochemical Profiling.

In the present study, we investigated the cytotoxic effects of Athyrium hohenackerianum ethanolic extract (AHEE) on the proliferation of breast, lung, and colon cancer cells. The AHEE was tested for its effect on the progression of the cell cycle, followed by induction of apoptosis determination by flow cytometry. Real-time qRT-PCR was also utilized to observe the initiation of apoptosis. In addition, GC-MS was performed in order to identify the active phytochemicals present in the AHEE. A cytotoxic activity with an IC50 value of 123.90 μg/mL against HCT-116 colon cancer cells was exhibited by AHEE. Following propidium iodide staining, annexin-V/PI, and clonogenic assays, AHEE treatment results in cell arrest in the S phase, causing an increase in the early and late phases of apoptosis and displaying antiproliferative potential, respectively. The morphological alterations were further monitored using acridine orange/ethidium bromide (AO/EB) staining. When compared with the control cells, features of apoptotic cell death, including nuclear fragmentation, in the AHEE-treated cells were noticed. The apoptosis was also confirmed by detecting the increased expression of p53 and caspase-3 along with the downregulation of Bcl-2. GC-MS analysis revealed that trans-linalool oxide, loliolide, phytol, 4,8,12,16-tetramethylheptadecan-4-olide, and gamma-sitosterol were the major phytochemical constituents. Based on these findings, it can be suggested that AHEE causes cellular death via apoptosis, which should be further explored for the identification of active compounds responsible for these observed effects. Therefore, AHEE can be used in the pharmaceutical development of anticancer agents for cancer therapeutics.

Monosaccharide Linked Schiff Base Metal Complexes of Cu(II), Zn(II) and Mn(II): Exploring the Antiproliferative Activity and Cell Death Mechanism

AbstractHerein, a new type of Schiff base ligand, HSL2 (where HSL2=2,2,7,7‐tetramethyltetrahydro‐3aH‐bis[1,3]dioxolo[4,5‐b:4′,5′‐d]pyran‐5‐yl) methyl‐4‐(3,5‐dichloro‐2‐hydroxybenzylidene amino)benzoate) anchored with a monosaccharide unit have been synthesized and characterized by FT‐IR, 1H NMR, and ESI‐MS. The ligand are chelated to metal ions Zn(II) (1), Cu(II) (2), and Mn(II) (3) to from square planar/tetrahedral or octahedral complexes. The DFT studies are carried out to optimize the shapes of the compounds. Moreover, Cu(II) and Mn(II) complexes were also characterized by EPR spectroscopy. The cytotoxicity of the complexes was screened towards three different cell lines A549 (lung cancer), HeLa (cervical cancer), and HT29 (colorectal cancer). These complexes exhibited significant cytotoxicity against three aforementioned cell lines and IC50 values are found in the range of 19.77–23 μM on HeLa cells, 9.42–12.25 μM on A549 cells and 8.84–16 μM on HT29 cells. The cell death mechanism was also studied by employing DNA fragmentation, Hoechst 33342 nuclear staining, and Acridine orange/ Ethidium Bromide (AO/EB) assay. In addition, ROS generation was also evaluated with these metal complexes.

Preparation and pharmacokinetics of glycyrrhetinic acid and cell transmembrane peptides modified with liposomes for liver targeted-delivery

The article presents a hepatocellular carcinoma cell surface-specific ligand glycyrrhetinic acid (GA) and cell-penetrating peptide (TAT) with good cell membrane penetration to modify the anti-tumor drug pingyangmycin (PYM) liver delivery system, which achieve targeted delivery of drugs and improve anti-tumor efficiency. In this study, we synthesized the pingyangmycin liposome modified by glycyrrhetinic acid and cell penetrating peptide(GA-TAT-PYM-L) and evaluated the anti-tumor effect of GA-TAT-PYM-L in vitro. Using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromidecell proliferation method, GA-TAT-PYM-L had a stronger inhibitory effect on HepG2 cells than the free drug PYM at the same concentration. Acridine orange-ethidium bromide staining assays showed that GA-TAT-PYM-L had stronger apoptosis promotion effects on HepG2 cells in comparison to PYM. Pharmacokinetic studies indicated that, compared with PYM, GA-TAT-PYM-L enhanced mean residence time (MRT0–∞) and area under curve (AUC0–∞) by about 2.79-fold and 2.45-fold. The T 1/2 was prolonged to 140.23 ± 14.13 min. Tissue distribution results showed that the PYM concentrations in livers from the GA-TAT-PYM-L group were always higher than other tissues at each monitoring period after 5 min, indicating that GA-TAT-PYM-L can achieve liver targeting.