Reactive Oxygen Species-mediated Apoptosis Research Articles

Tamarix articulata Inhibits Cell Proliferation, Promotes Cell Death Mechanisms and Triggers G0/G1 Cell Cycle Arrest in Hepatocellular Carcinoma Cells.

SUMMARYResearch backgroundFrom ancient times plants have been used for medicinal purposes against various ailments. In the modern era, plants are a major source of drugs and are an appealing drug candidate for the anticancer therapeutics against various molecular targets. Here we tested methanolic extract of dry leaves of Tamarix articulata for anticancer activity against a panel of hepatocellular carcinoma cells.Experimental approachCell viability of hepatocellular carcinoma cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after a dose-dependent treatment with the extract of T. articulata. Phase-contrast microscopy and 4՛,6-diamidino-2-phenylindole (DAPI) staining served to analyse cellular and nuclear morphology. Immunoblotting was performed to determine the expression of proteins associated with autophagy, apoptosis and cell cycle. However, flow cytometry was used for the quantification of apoptotic cells and the analysis of cells in different phases of the cycle after the treatment with various doses of T. articulata. Additionally, acridine orange staining and 2՛,7՛-dichlorofluorescein diacetate (DCFH-DA) dye were used to analyse the quantification of autophagosomes and reactive oxygen species.Results and conclusionOur results demonstrate that T. articulata methanolic extract exhibits promising antiproliferative activity with IC50 values (271.1±4.4), (298.3±7.1) and (336.7±6.1) µg/mL against hepatocellular carcinoma HepG2, Huh7D12 and Hep3B cell lines, respectively. Mechanistically, we found that T. articulata methanolic extract induces cell death by activating apoptosis and autophagy pathways. First, T. articulata methanolic extract promoted autophagy, which was confirmed by acridine orange staining. The immunoblotting analysis further confirmed that the extract at higher doses consistently induced the conversion of LC3I to LC3II form with a gradual decrease in the expression of autophagy substrate protein p62. Second, T. articulata methanolic extract promoted reactive oxygen species production in hepatocellular carcinoma cells and activated reactive oxygen species-mediated apoptosis. Flow cytometry and immunoblotting analysis showed that the plant methanolic extract induced dose-dependent apoptosis and activated proapoptotic proteins caspase-3 and PARP1. Additionally, the extract triggered the arrest of the G0/G1 phase of the cell cycle and upregulated the protein expression of p27/Kip and p21/Cip, with a decrease in cyclin D1 expression in hepatocellular carcinoma cells.Novelty and scientific contributionThe current study demonstrates that T. articulata methanolic extract exhibits promising anticancer potential to kill tumour cells by programmed cell death type I and II mechanisms and could be explored for potential drug candidate molecules to curtail cancer in the future.

Helenalin Facilitates Reactive Oxygen Species-Mediated Apoptosis and Cell Cycle Arrest by Targeting Thioredoxin Reductase-1 in Human Prostate Cancer Cells.

BackgroundHelenalin is a pseudoguaianolide natural product with anti-cancer activities. This study investigated the underlying mechanism of the anti-prostate cancer effects of helenalin in vitro.Material/MethodsCCK-8 assay was performed to detect the optimal concentrations of helenalin in DU145 and PC-3 cells. After exposure to helenalin and/or reactive oxygen species (ROS) inhibitor, ROS production was assessed by DCFH-DA staining. Thioredoxin reductase-1 (TrxR1) expression was detected by RT-qPCR and western blot. Moreover, apoptosis and cell cycle were evaluated by flow cytometry. Following TrxR1 knockdown or overexpression, TrxR1 expression, ROS generation, apoptosis, cell cycle, migration, and invasion were examined in cells co-treated with helenalin.ResultsHelenalin distinctly repressed the viability of prostate cancer cells in a concentration-dependent manner. We chose 8 μM and 4 μM as the optimal concentrations of helenalin for DU145 and PC-3 cells, respectively. Helenalin treatment markedly triggered ROS production and lowered TrxR1 expression, which was ameliorated by ROS inhibitor. Exposure to helenalin facilitated apoptosis as well as G0/G1 cell cycle arrest, which was reversed by ROS inhibitor. Helenalin relieved the inhibitory effect of TrxR1 on ROS production. Furthermore, helenalin ameliorated the decrease in apoptosis rate and the shortening of G0/G1 phase as well as the increase in migration and invasion induced by TrxR1 overexpression.ConclusionsOur findings revealed that helenalin accelerated ROS-mediated apoptosis and cell cycle arrest via targeting TrxR1 in human prostate cancer cells.

Application of ZnO/CNT@Fe3O4 nanocomposite in amplifying the anti-leukemic effects of Imatinib: a novel strategy to adjuvant therapy in chronic myeloid leukemia

The advent of tyrosine kinase inhibitors in the therapeutic protocols of chronic myeloid leukemia (CML) was a revolution in the treatment strategies that guaranteed the achievement of complete remission for patients. However, due to different mutations bypassing the efficacy of Imatinib, novel and more effective treatments are indeed required for the treatment of CML. Our study declared that the combination of synthesized ZnO/CNT@Fe3O4 nanocomposite with Imatinib decreased survival of CML-derived K562 cells, probably through inducing reactive oxygen species-mediated apoptosis. We also found improved cytotoxicity in the presence of a well known autophagy inhibitor, indicating that the apoptotic effect of this treatment is enhanced via autophagy suppression. Investigating the molecular mechanisms for the growth-suppressive effect of ZnO/CNT@Fe3O4-plus-Imatinib suggested that up-regulation of SIRT1 ceased cell cycle progression by increasing the expression of p21 and p27 cyclin-dependent kinase inhibitors. Notably, we reported here for the first time that either direct or indirect suppression of c-Myc results in enhanced anti-leukemic efficacy, suggesting that overexpression of c-Myc plays a contributory role in attenuating the efficacy of ZnO/CNT@Fe3O4—Imatinib in K562 cells. Given the promising effect of ZnO/CNT@Fe3O4 in potentiating the anti-cancer effects of Imatinib in K562 cells, our study suggested that nanocomposite could be used as a tool for combined-strategy treatment. However, further in vivo experiments are needed to provide clues for the safety and efficacy of this nanocomposite.

Linalool Induces Reactive Oxygen Species Mediated Apoptosis in Human Oral Squamous Carcinoma Cells

Development of oral cancer is associated with excessive cell proliferation, deregulation in cellular differentiation, insufficient apoptosis and genomic instability. Linalool is a natural monoterpene, contained in edible plants, considered to have potent antioxidant nature. In this context, it should be necessitate in broadening the understanding of the anticancer potential of linalool in experimental oral cancer model, which could be beneficial in anticancer therapy. Hence, this study is intended to assess the anticancer quality of linalool in keratin-forming tumor cell line. 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay was conducted to find out the half maximal inhibitory concentration of linalool in keratinforming tumor cell line. Linalool induced the loss of cell viability or cell death by a clear dose and time dependent manner. The half maximal inhibitory concentration on keratin-forming tumor cell line cells is 13, 24 and 45 μM for 24, 48 and 72 h respectively. Morphological changes, increased level of intracellular level of reactive oxygen species and loss of mitochondrial transmembrane potential of keratin-forming tumor cell line cells were evidently visualized on staining. Early growth 1 phase arrest in the cell cycle of keratin-forming tumor cell line cells was confirmed on dose and time dependent manner when compared with control during linalool treatment. Linalool treatment in various concentrations in vitro in keratinforming tumor cell line cells was able to reduce cell viability as compared to its counterparts. This study showed that linalool was a potent growth inhibitor of keratin-forming tumor cell line. In near future in vivo screening for its anticancer activity will be warranted and a linkage map will be developed between in vivo and in vitro studies.

β-sitosterol induces reactive oxygen species-mediated apoptosis in human hepatocellular carcinoma cell line.

Objective:It is of interest to investigate the anti-proliferative effect of β-sitosterol (BS) on human hepatocellular carcinoma (HepG2) cell line.Materials and Methods:β-sitosterol treatments (0.6 and 1.2 mM/ml) were done in HepG2 and after 24 hr, cell viability was evaluated by MTT assay. Reactive oxygen species (ROS) accumulating potential of BS was assessed by dichloro-dihydro-fluorescein diacetate staining. Morphology related to apoptosis was investigated by acridine orange and ethidium bromide dual staining. Cytochrome c and caspase 3 expressions were evaluated by immunofluorescence and western blot analyses.Results:β-sitosterol induced cytotoxicity (p<0.001) and intracellular ROS in HepG2 cells in a dose-dependent manner. BS treatments accumulated induced intracellular ROS accumulation which led to membrane damage and mitochondrial toxicity. At the molecular level, BS treatments induced cytochrome c release from mitochondria and enhanced the protein expressions (p<0.05 vs 0.6 mM/ml and p<0.001 vs 1.2 mM/ml) of both caspase 3 and cleaved caspase 3. Conclusion:β-sitosterol induced ROS accumulation which plays a critical role in apoptosis via the intrinsic pathway in HepG2 cells. The present investigation paves the way for further in vivo studies.

Curcumin reduced gold nanoparticles synergistically induces ROS mediated apoptosis in MCF-7 cancer cells

Curcumin nanotechnologies have increased the therapeutic efficacy of curcumin by enhancing its bio-availability. We have previously reported on the synthesis of stable, uniform and biocompatible gold nanoparticles using curcumin (cAuNPs) alone as the reducing and stabilizing agent. Here, we provide evidence for the synergistic effect produced by both the gold core and curcumin against breast cancer cells. Spectroscopic and electron microscopic observations show that cAuNPs in biological medium get coated by proteins which aids in their endocytosis. Inside the cells, cAuNPs triggers the generation of reactive oxygen species (ROS) which then depletes mitochondrial membrane potential (MMP). Consequently, the pro-apoptotic protein Bax is released that activates PARP cleavage and DNA fragmentation. The final stages of apoptosis including chromatin condensation and loss of filopodia are visualized through microscopy. These findings provide insights into the mechanism of action of cAuNPs in inducing apoptosis and also suggest their promising candidature as a chemotherapeutic drug.

Eu(III)-coordination polymer: inhibitory activity on cervical cancer via inducing ROS mediated apoptosis

Under the hydrothermal reaction condition, we prepared a new 3D Eu(III)-based coordination polymer (CP) [Eu(H2-DHBDC)0.5(DHBDC)0.5 (H2O)2] n ·H2O (1, H4-DHBDC = 2,5-dihydroxy-1,4-terephthalic acid). Furthermore, we assessed the inhibitory influence on cervical cancer cells of this compound. First, for testing the anti-viability of the compound against cervical cancer cells, the cell counting kit-8 (CCK-8) assay was carried out. Then, reactive oxygen species (ROS) detection kit was used to determine the ROS accumulation in the cervical cancer cells. Finally, after the Annexin V-FITC/PI apoptosis assay was finished, it was used for apoptotic cervical cancer cells after treated with compound. From the results of the software of pose scoring and molecular docking, we can see that the compound may bind to p91 by the functional side-chain of the ligand, which provided potential regulatory mechanisms for the compound.

Combination of tetrandrine and cisplatin synergises cytotoxicity and apoptosis in triple negative breast cancer

Abstract Background Breast cancer is one of the leading causes of cancer deaths among women worldwide. Cisplatin is a broad-spectrum anticancer drug used in the treatment of breast cancer. Recent research showed the potent anticancer effect of tetrandrine against different cancers. Objective Present study is aimed at determining the synergistic cytotoxic effect of tetrandrine and cisplatin against breast cancer under in vitro. Methods Cytotoxic effect of tetrandrine and cisplatin on MDA-MB-231 cells was performed using MTT - (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide). Synergistic, antagonistic and additive effects of tetrandrine and cisplatin were calculated using Compusyn software. Clonogenic assay was performed in 12 wells plate. The generation of Reactive Oxygen Species (ROS) was measured using H2DCF-DA. Cell cycle assay was carried out using flow cytometry. The caspase-3, -8 and -9 activities in the cells were measured based on the hydrolysis of peptide substrates by their respective enzymes to release p-nitroaniline (pNA). Results The study demonstrated synergistic cytotoxic effect on MDA-MB-231 cells in a dose dependent manner. The cells undergoing apoptosis and necrosis were evidenced by microscopic observation and colony formation and cell cycle assays. The study also revealed elevated levels of ROS and, increased activities of caspase-3 and -8 which also indicate the apoptosis of cells. Conclusion The study showed a promising synergistic cytotoxic effect of tetrandrine and cisplatin against MDA-MB-231 cells with cell cycle arrest and intracellular ROS mediated apoptosis. Hence, the study suggests the possible synergistic use of tetrandrine and cisplatin to treat triple negative breast cancer.

Treatment of Human Lens Epithelium with High Levels of Nanoceria Leads to Reactive Oxygen Species Mediated Apoptosis.

Nanoceria (cerium oxide nanoparticles) have been shown to protect human lens epithelial cells (HLECs) from oxidative stress when used at low concentrations. However, there is a lack of understanding about the mechanism of the cytotoxic and genotoxic effects of nanoceria when used at higher concentrations. Here, we investigated the impact of 24-hour exposure to nanoceria in HLECs. Nanoceria’s effects on basal reactive oxygen species (ROS), mitochondrial morphology, membrane potential, ATP, genotoxicity, caspase activation and apoptotic hallmarks were investigated. Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX) studies on isolated mitochondria revealed significant uptake and localization of nanoceria in the mitochondria. At high nanoceria concentrations (400 µg mL−1), intracellular levels of ROS were increased and the HLECs exhibited classical hallmarks of apoptosis. These findings concur with the cells maintaining normal ATP levels necessary to execute the apoptotic process. These results highlight the need for nanoceria dose-effect studies on a range of cells and tissues to identify therapeutic concentrations in vitro or in vivo.

Anti-proliferative and cytotoxic effect of Iranian snake (Vipera raddei kurdistanica) venom on human breast cancer cells via reactive oxygen species-mediated apoptosis

Background and purpose:Breast cancer is the most commonly occurring cancer in women around the world. Despite new advances in cancer therapy, breast cancer remains a disease with high morbidity and mortality. Snake venom is a poisonous mixture of different molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins, and peptides. Previous studies demonstrated that some snake venoms showed in vitro anti-cancer effects. In this study, the effects of the Iranian snake (Vipera raddei kurdistanica) venom on breast cancer cells were investigated.Experimental approach:The effect of increasing concentrations of snake venom on breast cell viability was assessed by trypan blue, MTT, and lactate dehydrogenase measurements. Apoptosis was detected and quantified by fluorescent staining and DNA fragmentation assay. The expression level of some apoptotic-related genes was investigated using real-time polymerase chain reaction (RT-PCR). The Western blotting method was also used to detect the protein expression profiles in the cells.Findings / Results:After treatment for 24, 48, 72, and 96 h, the cell viability was significantly reduced in a time- and dose-dependent manner (P < 0.05). The venom effect on normal breast cells was significantly smaller than cancer cells (P > 0.05). Apoptosis was significantly increased (P < 0.05). The RT-PCR and western blot data confirmed the increase of apoptosis in cells treated with venom.Conclusion and implications:These data suggested that the vipera raddei kurdistanica venom had a cytotoxic property via activation of apoptosis in breast cancer cells.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Pseudomonas aeruginosa synthesized silver nanoparticles inhibit cell proliferation and induce ROS mediated apoptosis in thyroid cancer cell line (TPC1)

We used cell-free culture filtrate of Pseudomonas aeruginosa as a reducing mediator of AgNO3 to silvernanoparticles (AgNPs) and possibly used as a potential anticancer agent against thyroid cancer cells (TPC1). The bio-generation of AgNPs was firmly established by taking a UV spectrum at 380–500 nm wavelength. The Fourier transform spectrum analysis reveals the association of alcohol, phenol and aromatic functional groups with P. aeruginosa synthesized AgNPs (Ps-AgNPs). By observing under transmission electron microscopy (TEM), the size and structure of the Ps-AgNPs were characterized as the size was 30–70 nm and spherical in shape. The concentration-dependent cytotoxicity of Ps-AgNPs on TPC1 cells was observed and IC50 value was calculated. The alteration of oxidative and antioxidant biomarkers in Ps-AgNPs treated cells were observed. The induced apoptosis was determined by staining the Ps-AgNPs treated cells with DCFH-DA, Rh-123 dye, Acridine Orange (AO) and ethidium bromide (EtBr). Increased level of intracellular reactive oxygen species (ROS) generation and decreased level of mitochondrial membrane potential was observed in Ps-AgNPs treated TPC1 cells. Moreover, the apoptotic morphological changes were explored, which indicates increased apoptosis by inducing cell membrane damage in Ps-AgNPs treated cells. This biogenic approach will enable an effective and significant improvement in nano-oncotherapy.

Repurposing an old drug for new use: Niclosamide in psoriasis-like skin inflammation.

Drug discovery is an onerous, extremely expensive, and time-consuming process. Instead, drug repurposing is an attractive strategy for exploiting novel indications for a drug beyond its original use. The untapped potential of drug repurposing compensates the barriers associated with the drug discovery pipeline. Psoriasis is an autoimmune skin disease, where hyperproliferation of keratinocytes and exaggerated immune responses are the important hallmarks of the disease. Extensive in vitro and preclinical research has demonstrated that niclosamide was found to exert potent anticancer and anti-inflammatory properties by targeting STAT3, p65 NF-κB, and NFATc-1 signaling paradigm with minimal host toxicity. From the disease perspective, the static intracellular molecular network in both cancer and psoriasis share overlapping pathological features in terms of hyperproliferation and chronic inflammation, which is mediated by the aforementioned signaling cascade. The plausible mechanistic relevance has prompted us to investigate the implementation of niclosamide for repositioning in psoriasis. Our in vitro and in vivo findings suggest that niclosamide inhibits keratinocytes hyperproliferation by reactive oxygen species-mediated apoptosis through the loss of mitochondrial membrane potential, cell cycle arrest at Sub G1 phase, and DNA fragmentation. Furthermore, niclosamide treatment resulted in abrogation of lipopolysaccharide-induced inflammatory cytokine levels in murine macrophages. Additionally, our results provided a preclinical rationale in imiquimod (IMQ)-induced BALB/c mouse model, where niclosamide diligently mitigated the IMQ-induced epidermal hyperplasia and inflammation by downregulating STAT3, p65 NF-κB, and NFATc-1 transcription factors along with Akt, Ki-67, and ICAM-1 protein expression.

Kaempferitrin Cause Cell Cycle Arrest at G2/M Phase and Reactive Oxygen Species Mediated Apoptosis in Human Colon Cancer HT-29 Cells

Kaempferitrin (Kaempferol-3,7-O-bisα-L-rhamnoside) belongs to a group of metabolites known as flavonoids. Kaempferitrin has been proven to have a wide range of important pharmacological activities, such as antioxidant, anti-inflammatory, anti-angiogenic and anti-carcinogenic activity. Current technologies dealing with drug discovery could provide the data necessary to consider kaempferitrin and its analogs as safe drugs for use in humans. The MTT test showed that colon cancer HT-29 cell viability was reduced dose-dependently with an increased kaempferitrin concentration. The fifty percent inhibition concentration (IC50) of kaempferitrin was found to be 30.0 μM, which can cause programmed cell death in HT-29 cells and enhance the levels of reactive oxygen species (ROS). After kaempferitrin treatment, apoptosis was observed in a dose-dependent manner 7.5, 15.0, and 30.0 µM, respectively. Excessive intracellular reactive oxygen species damaged cellular structures and led to cell death. The relation of ROS with kaempferitrin-induced apoptosis in HT-29 cells was analyzed by flow cytometry.

Carbohydrate Modifications of Neoandrographolide for Improved Reactive Oxygen Species-Mediated Apoptosis through Mitochondrial Pathway in Colon Cancer.

Modifications at the carbohydrate moiety of neoandrographolide, isolated from the medicinal plant Andrographis paniculata, result in more potent and less toxic derivatives, namely, 4′,6′-benzylidene neoandrographolide (2b) and 4′6′-p-methoxybenzylidene neoandrographolide (2c). These showed improved cytotoxicity against SW-620, PC-3, and A549 cancer cell lines. Nuclear morphology studies were conducted on compound 2b by 4′,6-diamidino-2-phenylidole staining and detection of intracellular reactive oxygen species (ROS) accumulation. It showed an increase in the generation of cellular and mitochondrial ROS level. The probable relation of B-cell lymphoma-2 (Bcl-2, an apoptosis inhibitor) to B-cell lymphoma-2-associated X protein (Bax, an apoptosis promoter) ratio with caspase-3 (apoptosis coordination enzyme) in the colon cancer cell line SW-620 was investigated, and it was discovered that upon 2b treatment, the expression of caspase-3 Bax increased remarkably. However, in 2b-treated cells, the expression of Bcl-2 was downregulated as compared to untreated cells.

Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy.

Osteosarcoma is the most frequently diagnosed primary malignant bone sarcoma in children and adolescents. Recent studies have shown that cancer stem cells (CSCs), a cluster of tumor cells with the ability to self-renew, play an essential role in tumor recurrence and metastasis. Thus, it is necessary to develop therapeutic strategies specifically targeting CSCs. Metformin, the first-line drug for type 2 diabetes, exhibits antineoplastic activities in various kinds of tumors. New evidence has suggested that metformin may target CSCs and prevent their recurrence. However, the underlying specific mechanisms remain unclear. In this study, we found that metformin significantly suppressed the self-renewal ability of osteosarcoma stem cells (OSCs) and induced G0/G1 phase arrest by blocking the activity of cyclin-dependent kinases. Furthermore, metformin induced apoptosis through a mitochondria-dependent pathway, leading to the collapse of the mitochondrial transmembrane potential and the production of reactive oxygen species (ROS). Importantly, metformin acted directly on the mitochondria, which resulted in decreased ATP synthesis. This change allowed access to the downstream AMPK kinase, and the activation of AMPK led to the reversal of the mTOR pathway, triggering autophagy. Particularly, metformin-mediated autophagy disturbed the homeostasis of stemness and pluripotency in the OSCs. Additionally, our mouse xenograft model confirmed the potential therapeutic use of metformin in targeting OSCs. In conclusion, our findings suggest that metformin suppresses the self-renewal ability and tumorigenicity of OSCs via ROS-mediated apoptosis and autophagy.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38− progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

Reactive Oxygen Species-Mediated Apoptosis and Cytotoxicity of Newly Synthesized Pyridazin-3-Ones In P815 (Murin Mastocytoma) Cell Line.

In cancer cells, the intracellular antioxidant capacity and the redox homeostasis are mainly maintained by the glutathione- and thioredoxin-dependent systems which are considered as promising targets for anticancer drugs. Pyridazinones constitute an interesting source of heterocyclic compounds for drug discovery. The present investigation focused on studying the in-vitro antitumor activity of newly synthesized Pyridazin-3(2h)-ones derivatives against P815 (Murin mastocytoma) cell line. The in-vitro cytotoxic activities were investigated toward the P815 cell line using tetrazolium-based MTT assay. Lipid peroxidation and the specific activities of antioxidant enzymes were also determined. The newly compounds had a selective dose-dependent cytotoxic effect without affecting normal cells (PBMCs). Apoptosis was further confirmed through the characteristic apoptotic morphological changes and DNA fragmentation. Two compounds (6F: and 7H: ) were highly cytotoxic and were submitted to extend biological testing to determine the likely mechanisms of their cytotoxicity. Results showed that these molecules may induce cytotoxicity via disturbing the redox homeostasis. Importantly, the anticancer activity of 6F: and 7H: could be due to the intracellular reactive oxygen species hypergeneration through significant loss of glutathione reductase and thioredoxin reductase activities. This eventually leads to oxidative stress-mediated P815 cell apoptosis. Furthermore, the co-administration of 6F: or 7H: with Methotrexate exhibited a synergistic cytotoxic effect. considering their significant anticancer activity and chemosensitivity, 6F: and 7H: may improve the therapeutic efficacy of the current treatment for cancer.

Thymoquinone loaded mesoporous silica nanoparticles retard cell invasion and enhance in vitro cytotoxicity due to ROS mediated apoptosis in HeLa and MCF-7 cell lines.

Thymoquinone (TQ) loaded monodispersed mesoporous silica nanoparticles (TQ-MSNPs) with size of 188 ± 3 nm were prepared and characterized using DLS, TEM and FTIR. These TQ-MSNPs overcome the limitations of free TQ like hydrophobicity, low aqueous and photo stability and thus enhance its anticancer activity. In vitro release kinetics showed biphasic drug release where up to 50% was released in first 8 h and subsequently 98% released after 48 h. Enhanced cytotoxicity of TQ-MSNPs was observed against MCF-7 and HeLa cell lines as compared to free TQ. DAPI and Annexin V-FITC/PI staining confirmed the induction of apoptosis in cancer cells following treatment with TQ-MSNPs. Also, TQ-MSNPs exhibited enhanced anti-invasion properties against both cell lines as very low concentration of loaded TQ imparts similar benefits as free TQ. Both TQ and TQ-MSNPs exerted their cytotoxicity via reactive oxygen species (ROS) generation, as addition of an antioxidant NAC attenuated their killing activity.

High levels of EGFR prevent sulforaphane-induced reactive oxygen species-mediated apoptosis in non-small-cell lung cancer cells

BackgroundSulforaphane (SFN) has been shown to induce the production of reactive oxygen species (ROS) and inhibit epidermal growth factor receptor (EGFR)–mediated signaling in non-small-cell lung cancer (NSCLC). NSCLC cells harboring constitutively active EGFR mutations are more sensitive to SFN treatment than cells with wild-type EGFR, but whether NSCLC cells with high levels of EGFR expression are more resistant or sensitive to SFN treatment is not known. Study designWe employed a pair of cell lines, CL1-0 and CL1-5, which have the same genetic background but different levels of EGFR expression, to examine the effects of high EGFR level in the sensitivity to SFN. MethodsThe effect of SFN on cell viability and tumorigenicity was examined by trypan blue dye-exclusion assay, clonogenic assays, flow cytometry, and immunoblotting in vitro as well as tumorigenicity study in vivo. ROS levels in cells were assessed by flow cytometry using the ROS-reactive fluorescent indicator CM-H2DCFDA. Knockdown of EGFR in CL1-5 cells was infected with an EGFR-targeting small hairpin (interfering) RNA (shRNA)–containing lentivirus. ResultsWe present evidence that cells with high-level EGFR expression (CL1-5) are more resistant to SFN treatment than those with low-level expression (CL1-0). SFN treatment produced a similar increase in ROS and caused arrest of a cell population at S-phase accompanied by the induction of γH2AX, a DNA damage-response marker, in both cell sublines. However, SFN induced apoptosis only in the high-EGFR-expressing CL1-0 subline. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented SFN-induced apoptosis in CL1-0 cells and production of γH2AX in both CL1-0 and CL1-5 cells. shRNA-mediated knockdown of EGFR in CL1-5 cells rendered the cells susceptible to SFN-induced apoptosis. ConclusionThe cellular effects produced by SFN in NSCLC cells are largely mediated by SFN-induced production of ROS. Cells with higher levels of EGFR were more resistant to SFN treatment and showed resistance to SFN-induced apoptosis, suggesting that high EGFR levels protect cells from SFN-induced apoptosis. Despite this, we found that SFN retained the ability to inhibit the growth of NSCLC tumors with high-level EGFR expression in vivo.