Intracellular Reactive Oxygen Species Research Articles

Vismodegib Potentiates Marine Antimicrobial Peptide Tilapia Piscidin 4-Induced Cytotoxicity in Human Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is a common cancer with several accepted treatments, such as chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, and immune checkpoint inhibitors. Nevertheless, NSCLC cells often become insensitive to these treatments, and therapeutic resistance is a major reason NSCLC still has a high mortality rate. The induction of therapeutic resistance in NSCLC often involves hedgehog, and suppression of hedgehog can increase NSCLC cell sensitivity to several conventional therapies. In our previous work, we demonstrated that the marine antimicrobial peptide tilapia piscidin 4 (TP4) exhibits potent anti-NSCLC activity in both EGFR-WT and EGFR-mutant NSCLC cells. Here, we sought to further explore whether hedgehog might influence the sensitivity of NSCLC cells to TP4. Our results showed that hedgehog was activated by TP4 in both WT and EGFR-mutant NSCLC cells and that pharmacological inhibition of hedgehog by vismodegib, a Food and Drug Administration-approved hedgehog inhibitor, potentiated TP4-induced cytotoxicity. Mechanistically, vismodegib acted by enhancing TP4-mediated increases in mitochondrial membrane potential and intracellular reactive oxygen species (ROS). MitoTempo, a specific mitochondrial ROS scavenger, abolished vismodegib/TP4 cytotoxicity. The combination of vismodegib with TP4 also reduced the levels of the antioxidant proteins catalase and superoxide dismutase, and it diminished the levels of chemoresistance-related proteins, Bcl-2 and p21. Thus, we conclude that hedgehog regulates the cytotoxic sensitivity of NSCLC cells to TP4 by protecting against mitochondrial dysfunction and suppressing oxidative stress. These findings suggest that combined treatment of vismodegib and TP4 may be a promising therapeutic strategy for NSCLC.

The male reproductive toxicity after 5-Fluorouracil exposure: DNA damage, oxidative stress, and mitochondrial dysfunction in vitro and in vivo

5-Fluorouracil (5-FU), a chemotherapeutic drug used to treat a variety of cancers, can enter the environment through different routes, causing serious public health and environmental concerns. It has been reported that 5-FU exposure adversely affects male reproductive function, and its effects on this system cannot be avoided. In this study, using western blotting and quantitative polymerase chain reaction studies, we found that 5-FU promoted testicular injury by inducing oxidative stress, which was accompanied by the inhibition of nuclear factor erythroid 2-related factor 2/antioxidant response element signaling. Accumulation of reactive oxygen species (ROS) aggravated 5-FU-mediated mitochondrial dysfunction and apoptosis in murine cell lines and testes, indicating oxidative stress and mitochondrial-dependent apoptotic signaling play crucial roles in the damage of spermatogenic cells caused. N-Acetyl-L-cysteine, an antioxidant that scavenges intracellular ROS, protected spermatogenic cells from 5-FU-induced oxidative damage and mitochondrial dysfunction, revealing the important role of ROS in testicular dysfunction caused by 5-FU. We found that 5-FU exposure induces testicular cell apoptosis through ROS-mediated mitochondria pathway in mice. In summary, our findings revealed the reproductive toxicological effect of 5-FU on mice and its mechanism, provided basic data reference for adverse ecological and human health outcomes associated with 5-FU contamination or poisoning.

Mesoporous MOFs with ROS scavenging capacity for the alleviation of inflammation through inhibiting stimulator of interferon genes to promote diabetic wound healing

Excessive production of reactive oxygen species (ROS) and inflammation are the key problems that impede diabetic wound healing. In particular, dressings with ROS scavenging capacity play a crucial role in the process of chronic wound healing. Herein, Zr-based large-pore mesoporous metal–organic frameworks (mesoMOFs) were successfully developed for the construction of spatially organized cascade bioreactors. Natural superoxide dismutase (SOD) and an artificial enzyme were spatially organized in these hierarchical mesoMOFs, forming a cascade antioxidant defense system, and presenting efficient intracellular and extracellular ROS scavenging performance. In vivo experiments demonstrated that the SOD@HMUiO-MnTCPP nanoparticles (S@M@H NPs) significantly accelerated diabetic wound healing. Transcriptomic and western blot results further indicated that the nanocomposite could inhibit fibroblast senescence and ferroptosis as well as the stimulator of interferon genes (STING) signaling pathway activation in macrophages mediated by mitochondrial oxidative stress through ROS elimination. Thus, the biomimetic multi-enzyme cascade catalytic system with spatial ordering demonstrated a high potential for diabetic wound healing, where senescence, ferroptosis, and STING signaling pathways may be potential targets.Graphical Schematic diagram showing the proposed mechanism that S@M@H NPs promoted diabetic wound healing

Osthole exhibits the remedial potential for polycystic ovary syndrome mice through Nrf2-Foxo1-GSH-NF-κB pathway.

Polycystic ovary syndrome (PCOS) is the primary cause of female infertility with a lack of universal therapeutic regimen. Although osthole exhibits numerous pharmacological activities in treating various diseases, its therapeutic effect on PCOS is undiscovered. The present study found that application of osthole improved the symptoms of PCOS mice through preventing ovarian granulosa cells (GCs) production of more estrogen and alleviating the liberation of pro-inflammatory cytokineinterleukin(IL)-1β, IL-6, andtumor necrosis factor alpha. Meanwhile, osthole enhanced ovarian antioxidant capacity and alleviated intracellular reactive oxygen species (ROS) accumulation with a concurrent attenuation for oxidative stress, while intervention of antioxidant enzymic activity and glutathione (GSH)synthesis neutralized the salvation of osthole on GCs secretory disorder and chronic inflammation. Further analysis revealed that osthole restored the expression of nuclear factor erythroid 2-related factor 2 (Nrf2)and forkhead box O 1 (Foxo1) whose repression antagonized the amelioration of osthole on the insufficiency of antioxidant capacity and accumulation of ROS. Moreover, Nrf2 served as an intermedium to mediate the regulation of osthole on Foxo1. Additionally, osthole restricted the phosphorylation of IκBα and nuclear factor kappa B (NF-κB)subunit p65 by DHEA and weakened the transcriptional activity of NF-κB, but this effectiveness was abrogated by the obstruction of Nrf2 and Foxo1, whereas adjunction of GSH renewed the redemptive effect of osthole on NF-κB whose activation caused an invalidation of osthole in rescuing the aberration of GCs secretory function and inflammation response. Collectively, osthole might relieve the symptoms of PCOS mice via Nrf2-Foxo1-GSH-NF-κB pathway.

AQP8 Modulates Mitochondrial H2O2 Transport to Influence Glioma Proliferation

Background Aquaporin-8 (AQP8) is involved in impacting glioma proliferation and can effect tumour growth by regulating Intracellular reactive oxygen species (ROS) signalling levels. In addition to transporting H2O2, AQP8 has been shown to affect ROS signaling, but evidence is lacking in gliomas. In this study, we aimed to investigate how AQP8 affects ROS signaling in gliomas. Materials and methods We constructed A172 and U251 cell lines with AQP8 knockdown and AQP8 rescue by CRISPR/Cas9 technology and overexpression of lentiviral vectors. We used CCK-8 and flow cytometry to test cell proliferation and cycle, immunofluorescence and Mito-Tracker CMXRos to observe the distribution of AQP8 expression in glioma cells, Amplex and DHE to study mitochondria release of H2O2, mitochondrial membrane potential (MMP) and NAD+/NADH ratio to assess mitochondrial function and protein blotting to detect p53 and p21 expression. Result We found that AQP8 co-localised with mitochondria and that knockdown of AQP8 inhibited the release of H2O2 from mitochondria and led to increased levels of ROS in mitochondria, thereby impairing mitochondrial function. We also discovered that AQP8 knockdown resulted in suppression of cell proliferation and was blocked at the G0/G1 phase with increased expression of mitochondrial ROS signalling-related p53/p21. Conclusions This finding provides further evidence for mechanistic studies of AQP8 as a prospective target for the treatment of gliomas.

Effect of RNF113A deficiency on oxidative stress-induced NRF2 pathway

ABSTRACT The ring finger protein 113A (RNF113A) serves as an E3 ubiquitin ligase and a subunit of the spliceosome. Mutations in the RNF113A gene are associated with X-linked trichothiodystrophy (TTD). However, the cellular roles of RNF113A remain largely unknown. In this study, we performed transcriptome profiling of RNF113A knockout (KO) HeLa cells using RNA sequencing and revealed the upregulation of NRF2 pathway-associated genes. Further analysis confirmed that the KO of RNF113A promotes nuclear localization of the NRF2 protein and elevates the mRNA levels of NRF2 target genes. RNF113A KO cells showed high levels of intracellular reactive oxygen species (ROS) and decreased resistance to cell death following H2O2 treatment. Additionally, RNF113A KO cells more sensitively formed stress granules (SGs) under arsenite-induced oxidative stress. Moreover, RNF113A KO cells exhibited a decrease in glutathione levels, which could be attributed to a reduction in GLUT1 expression levels, leading to decreased glucose uptake reactions and lower intracellular glucose levels. These alterations potentially caused a reduction in ROS scavenging activity. Taken together, our findings suggest that the loss of RNF113A promotes oxidative stress-mediated activation of the NRF2 pathway, providing novel insights into RNF113A-associated human diseases.

Knocking Down PIAS3 Reduces H2O2-induced Oxidative Stress Injury in HT22 Cells.

Oxidative stress is involved in the pathological processes of many neurodegenerative diseases. Protein modification by small ubiquitin-like modifiers (SUMOs) has been implicated in oxidative stress injury. By conjugating SUMOs to their selective protein substrates, SUMO ligases play critical roles in regulating functions of proteins involved in oxidative stress injury. In this study, we screened siRNAs to knockdown the SUMO ligase PIAS3 to assess its role in H2O2-induced injury in HT22 cells. H2O2 stimulation increased total protein SUMOylation, facilitated intracellular reactive oxygen species (ROS) release, increased cleaved caspase-3 levels, promoted p38 and JNK activation (phosphorylation), upregulated apoptosis, and decreased cell viability. The siRNA against PIAS3 329-347 (siPIAS3-329) markedly downregulated the protein expression of PIAS3 and reversed these effects, whereas siNC (negative control) had no effect. Our findings demonstrate that PIAS3-mediated SUMOylation facilitates oxidative stress injury and p38/JNK-mediated cell apoptosis and that PIAS3 is a potential target to protect against oxidative stress injury.

Butyl isothiocyanate exhibits antifungal and anti-biofilm activity against Candida albicans by targeting cell membrane integrity, cell cycle progression and oxidative stress.

The prevalence of Candida albicans infection has increased during the past few years, which contributes to the need for new, effective treatments due to the increasing concerns regarding antifungal drug toxicity and multidrug resistance. Butyl isothiocyanate (butylITC) is a glucosinolate derivative, and has shown a significant antifungal effect contrary to Candida albicans. Additionally, how butylITC affects the virulence traits of C. albicans and molecular mode of actions are not well known. Present study shows that at 17.36 mM concentration butylITC inhibit planktonic growth. butylITC initially slowed the hyphal transition at 0.542 mM concentration. butylITC hampered biofilm development, and inhibits biofilm formation at 17.36 mM concentration which was analysed using metabolic assay (XTT assay) and Scanning Electron Microscopy (SEM). In addition, it was noted that butylITC inhibits ergosterol biosynthesis. The permeability of cell membranes was enhanced by butylITC treatment. Moreover, butylITC arrests cells at S-phase and induces intracellular Reactive Oxygen Species (ROS) accumulation in C. albicans. The results suggest that butylITC may have a dual mode of action, inhibit virulence factors and modulate cellular processes like inhibit ergosterol biosynthesis, cell cycle arrest, induces ROS production which leads to cell death in C. albicans.

Cornus mas L. Extract-Mediated Modulations of the Redox State Induce Cytotoxicity in Schizosaccharomyces pombe

The Cornus mas L. fruit is well known for having a high presence of bioactive substances that include phenolic compounds, anthocyanins, vitamins, flavonoids, carotenoids, and ursolic acid. The health-promoting effects of those substances are mainly associated with their antimicrobial, anti-inflammatory, and antioxidant properties. In this study, we investigated the role of Cornus mas L. fruit ethanolic extract on the cell vitality of a model eukaryotic organism, the yeast Schizosaccharomyces pombe. The effect of Cornus mas L. fruit ethanolic extract on cell viability was determined by analyses of cell growth and cell doubling time during exposure to the extract. To determine the ability of Cornus mas L. to reduce or induce oxidative stress, quantification of intracellular reactive oxygen species and malondialdehyde levels was performed. Additionally, the enzyme activity of superoxide dismutase and catalase was evaluated together with the determination of changes in the expression of genes related to oxidative stress response. The data suggest a dose-dependent cytotoxic effect of the Cornus mas L. fruit ethanol extract, as a higher concentration (2%) led to increased oxidative stress and reduced cell viability of S. pombe cells, while a lower concentration (0.5%) showed only a subtle effect on the analyzed parameters. This study provides a new perspective on the possible antimicrobial or medicinal properties of Cornus mas L. fruit ethanol extract due to its ability to induce oxidative stress in the cell.

Activity of NAD(P)H-Oxidoreductases in Ovarian Cancer

Reactive oxygen species (ROS) play an important and controversial role in carcinogenesis. Microsomal redox chains containing NADH- and NADPH-dependent oxidoreductases are among the main sites of intracellular ROS synthesis, but their role in the oxidative balance has not been fully studied. Here, we studied the activity of cytochrome b5 reductase (CYB5R) and cytochrome P450 reductase (CYPOR) in ovarian cancer tissues and cells isolated from peritoneal fluid, along with the antioxidant capacity of peritoneal fluid. We used the developed a chemiluminescence assay based on stimulation with NADH and NADPH, which reflects the activity of CYB5R and CYPOR, respectively. The activity of CYB5R and CYPOR was significantly higher in moderately and poorly differentiated ovarian adenocarcinomas compared with well-differentiated adenocarcinomas and cystadenomas. For the chemotherapy-resistant tumors, the activity of tissue CYB5R and CYPOR was lower compared to the non-resistant tumors. In the peritoneal fluid, the antioxidant capacity significantly increased in this series, benign tumors < well-differentiated < moderately and poorly differentiated adenocarcinomas, so the antioxidant excess was observed for moderately and poorly differentiated adenocarcinomas. The antioxidant capacity of peritoneal fluid and the activity of CYB5R and CYPOR of cells isolated from peritoneal fluid were characterized by a direct moderate correlation for moderately and poorly differentiated adenocarcinomas. These results indicate the significant role of NAD(P)H oxidoreductases and the antioxidant potential of peritoneal fluid in cancer biochemistry. The parameters studied are useful for diagnostics and prognostics. The developed assay can be used to analyze CYB5R and CYPOR activity in other tissues and cells.

The promotion of non-small cell lung cancer progression by collagen and calcium binding EGF domain 1 is mediated through the regulation of ERK/JNK/P38 phosphorylation by reactive oxygen species.

Reactive oxygen species (ROS) are metabolic by-products of cells, and abnormal changes in their levels are often associated with tumor development. Our aim was to determine the role of collagen and calcium binding EGF domain 1 (CCBE1) in oxidative stress and tumorigenesis in non-small cell lung cancer cells (NSCLC). We investigated the tumorigenic potential of CCBE1 in NSCLC using in vitro and in vivo models of CCBE1 overexpression and knockdown. Immunohistochemical staining results showed that the expression of CCBE1 in cancer tissues was significantly higher than that in adjacent tissues. Cell counting Kit 8, clonal formation, wound healing, and transwell experiments showed that CCBE1 gene knockdown significantly inhibited the migration, invasion, and proliferation of NSCLC cell lines. In terms of mechanism, the silencing of CCBE1 can significantly promote the morphological abnormalities of mitochondria, significantly increase the intracellular ROS level, and promote cell apoptosis. This change of oxidative stress can affect cell proliferation, migration, and invasion by regulating the phosphorylation level of ERK/JNK/P38 MAPK. Specifically, the downregulation of CCBE1 inhibits the phosphorylation of ERK/P38 and promotes the phosphorylation of JNK in NSCLC, and this regulation can be reversed by the antioxidant NAC. In vivo experiments confirmed that downregulating CCBE1 gene could inhibit the growth of NSCLC in BALB/c nude mice. Taken together, our results confirm the tumorigenic role of CCBE1 in promoting tumor invasion and migration in NSCLC, and reveal the molecular mechanism by which CCBE1 regulates oxidative stress and the ERK/JNK/P38 MAPK pathway.

Oxidative stress-related cellular aging causes dysfunction of the Kv3.1/KCNC1 channel reverted by melatonin.

The voltage-gated Kv3.1/KCNC1 channel is abundantly expressed in fast-spiking principal neurons and GABAergic inhibitory interneurons throughout the ascending auditory pathway and in various brain regions. Inactivating mutations in the KCNC1 gene lead to forms of epilepsy and a decline in the expression of the Kv3.1 channel is involved in age-related hearing loss. As oxidative stress plays a fundamental role in the pathogenesis of epilepsy and age-related hearing loss, we hypothesized that an oxidative insult might affect the function of this channel. To verify this hypothesis, the activity and expression of endogenous and ectopic Kv3.1 were measured in models of oxidative stress-related aging represented by cell lines exposed to 100 mM d-galactose. In these models, intracellular reactive oxygen species, thiobarbituric acid reactive substances, sulfhydryl groups of cellular proteins, and the activity of catalase and superoxide dismutase were dysregulated, while the current density of Kv3.1 was significantly reduced. Importantly, the antioxidant melatonin reverted all these effects. The reduction of function of Kv3.1 was not determined by direct oxidation of amino acid side chains of the protein channel or reduction of transcript or total protein levels but was linked to reduced trafficking to the cell surface associated with Src phosphorylation as well as metabolic and endoplasmic reticulum stress. The data presented here specify Kv3.1 as a novel target of oxidative stress and suggest that Kv3.1 dysfunction might contribute to age-related hearing loss and increased prevalence of epilepsy during aging. The pharmacological use of the antioxidant melatonin can be protective in this setting.

Thymoquinone oxime synthesis and its effects on melanoma cells: cytotoxic, genotoxic, and apoptotic evaluation

Strong evidence supports the anticancer properties of natural plant product isolates. The cytotoxic, genotoxic, and apoptotic properties of an oxime derivative of thymoquinone (TQ) in melanoma cancer cells were investigated. The structure of TQ-Oxime was elucidated through nuclear magnetic resonance, and its effect on B16F10 and L929 cell lines was assessed using a luminometric adenosine triphosphate assay. Intracellular reactive oxygen species (iROS) were quantified via fluorometry, mitochondrial membrane potential (MMP) was assessed using flow cytometry, glutathione (GSH) levels were measured using a luminometric GSH/oxidized glutathione assay, DNA damage via comet assay, and apoptosis was detected using acridine orange/ethidium bromide staining. Concentrations (0.5-20 μM) of TQ-Oxime significantly increased cytotoxicity, DNA damage, apoptosis, and iROS, in a concentration-dependent manner compared (p < 0.001). In addition, MMP and GSH levels decreased significantly with increasing concentrations compared with the control (p < 0.001). Overall, these findings contribute to our understanding of the therapeutic potential of TQ and its derivatives in cancer treatment.

Cell-cycle related centromere protein F deficiency suppresses ovarian cancer cells growth by inducing ferroptosis.

This study aimed to investigate the involvement of the cell cycle-related protein centriole protein F (CENPF) in the development of ovarian cancer (OC) and explored its relationship with ferroptosis. The databases were analyzed to identify differential expression of cell cycle-related proteins between individuals with ovarian cancer and normal individuals. Immunohistochemistry and statistical analysis were conducted on ovarian tissues obtained from 40 patients with epithelial ovarian cancer and 20 normal individuals. In vitro experiments were performed using SKOV3 and HEY epithelial ovarian cancer cell lines. The mRNA microarray data set, consisting of GSE14001, GSE54388, GSE40595, and GSE14407, was downloaded from the Gene Expression Omnibus (GEO) database to investigate the genes associated with cell cycle regulation in ovarian cancer cells. CENPF was selected as the subject of study through differential analysis. Assessed the expression of CENPF in both ovarian cancer (OC) patients and normal ovarian tissues using immunohistochemistry. Lentivirus infection was employed to downregulate CENPF expression, and subsequent experiments including Cell Counting Kit-8 (CCK-8) assay, cell cycle analysis, transwell assay, and wound-healing assay were conducted to investigate the effects of CENPF on proliferation, invasion, migration, and cell cycle regulation in OC cells. The Reactive oxygen species (ROS) and the malondialdehyde (MDA) assays were performed to assess the involvement of CENPF in cellular redox reactions. Western blot analysis was conducted to examine the expression levels of ferroptosis-related proteins (GPX4, SLC7A11, DMT1, and P53). By querying and integrating cell cycle-related genes from the GEO database, in silico analyses using The Cancer Genome Atlas (TCGA) database combined with immunohistochemical studies, we discovered that CENPF is upregulated in ovarian cancer tissues and is related to survival. Downregulation of CENPF inhibited biological function of OC cells, increased intracellular ROS and MDA levels, and downregulated the GPX4 protein and the SLC7A11/xCT protein, but upregulated the DMT1 protein and the tumor protein 53(P53) protein expression to induce ferroptosis. This study did not investigate ferroptosis-related studies following CENPF overexpression, and the findings have not been validated in animal studies. Our findings demonstrated that the deficiency of CENPF played a crucial anti-oncogenic role in the progression of OC through the mechanism of ferroptosis.

Cancer cell membrane cloaked fluorescent liposomes loaded with metal-phenolic complex nanoparticles for combinatorial therapy against breast cancer

The development of innovative strategies for effective cancer treatment remains a critical research focus. In this study, we present a novel approach by designing cancer cell membrane-cloaked fluorescent liposomes loaded with metal-phenolic complex nanoparticles for combinatorial therapy against breast cancer. This multifaceted therapeutic system leverages the unique properties of cancer cell membranes, fluorescent liposomes, and metal-phenolic complexes to create a highly targeted and versatile treatment platform. Cancer cell membranes serve as natural camouflage, allowing these nanoparticles to evade the immune system and specifically target breast cancer cells. The inclusion of fluorescent liposomes not only aids in real-time imaging of treatment progress but also enhances the therapeutic effect by providing photodynamic therapy. Metal-phenolic complexes provide a multifunctional payload, offering both thermally assisted cytotoxicity and the ability to modulate cellular pathways critical in cancer progression. The enhanced internalization found in 4T1 cell lines demonstrates the targeted efficiency of our nanosystem. We also observed a significant increase in intracellular reactive oxygen species (ROS), increased cell death, and a significant decrease in colony formation treated with the synthesized nanoparticles. Alteration in mitochondrial membrane potential indicated the onset of cellular apoptosis, which eventually resulted in cell death. The biomimetic CSpFeTA NPs exhibited remarkable deep tumor tissue penetration and apoptotic cell death assisted by NIR irradiation. The development of such a sophisticated nanosystem represents a significant advancement in cancer therapy, opening new avenues for personalized and effective breast cancer treatment.

Polyphyllin VII Promotes Apoptosis in Breast Cancer by Inhibiting MAPK/ERK Signaling Pathway through Downregulation of SOS1.

Polyphyllin VII is a biologically active herbal monomer extracted from the traditional Chinese herbal medicine Chonglou. Many studies have demonstrated the anticancer activity of polyphyllin VII against various types of cancers, such as colon, liver, and lung cancer, but its effect on breast cancer has not been elucidated. In this study, we demonstrate that polyphyllin VII inhibited proliferation, increased production of intracellular reactive oxygen species, and decreased mitochondrial membrane potential in breast cancer cells. Notably, polyphyllin VII also induced apoptosis via the mitochondrial pathway. Transcriptome sequencing was used to analyze the targets of PPVII in regulating breast cancer cells. Mechanistic studies showed that polyphyllin VII downregulated Son of Sevenless1 (SOS1) and inhibited the MAPK/ERK pathway. Furthermore, PPVII exerted strong antitumor effects in vivo in nude mice injected with breast cancer cells. Our results suggest that PPVII may promote apoptosis through regulating the SOS1/MAPK/ERK pathway, making it a possible candidate target for the treatment of breast cancer.

In vitro study on anti-proliferative and anti-cancer activity of picrosides in triple-negative breast cancer.

Picrorhiza kurroa, an "Indian gentian," a known Himalayan medicinal herb with rich source of phytochemicals like picrosides I, II, and other glycosides, has been traditionally used for the treatment of liver and respiratory ailments. Picrosides anti-proliferative, anti-oxidant, anti-inflammatory and other pharmacological properties were evaluated in treating triple-negative breast cancer (TNBC). Picroside I and II were procured from Sigma-Aldrich and were analyzed for anti-cancer activity in triple-negative breast cancer (MDA-MB-231) cells. Cell viability was analyzed using MTT and trypan blue assays. Apoptosis was analyzed through DNA fragmentation and Annexin V/PI flow cytometric analysis. Wound healing and cell survival assays were employed to determine the inhibition of invasion capacity and anti-proliferative activity of picrosides in MDA-MB-231 cells. Measurement of intracellular ROS was studied through mitochondrial membrane potential assessment using DiOC6 staining for anti-oxidant activity of picrosides in MDA-MB-231 cells. Both Picroside I and II have shown decreased cell viability of MDA-MB-231 cells with increasing concentrations. IC50 values of 95.3µM and 130.8µM have been obtained for Picroside I and II in MDA-MB-231 cells. Early apoptotic phase have shown an increase of 20% (p < 0.05) with increasing concentrations (0, 50, 75, and 100µM) of Picroside I and 15% (p < 0.05) increase with Picroside II. Decrease in mitochondrial membrane potential of 2-2.5-fold (p < 0.05) was observed which indicated decreased reactive oxygen species (ROS) generation with increasing concentrations of Picroside I and II. An increasing percentage of 70-80% (p < 0.05) cell population was arrested in G0/G1 phase of cell cycle after Picroside I and II treatment in cancer cells. Our results suggest that Picroside I and II possess significant anti-proliferative and anti-cancer activity which is mediated by inhibition of cell growth, decreased mitochondrial membrane potential, DNA damage, apoptosis, and cell cycle arrest. Therefore, Picroside I and II can be developed as a potential anti-cancer drug of future and further mechanistic studies are underway to identify the mechanism of anti-cancer potential.

The Power of Synergism: A Novel Drug Combination for Improved Therapy of Glioblastoma

AbstractOne of the biggest obstacles in treating glioblastoma multiforme (GBM) is the plasticity and adaptability of GBM cells, which renders monotherapy ineffective. Novel drug combinations that target multiple pathways are required to overcome this challenge. In the current study, the PI3K/AKT/mTOR pathway is identified as a crucial component in GBM cell survival using the KEGG database and literature search. Hence, it is hypothesized that a potential synergistic therapy can be achieved by targeting different stages of this pathway by combining disulfiram (DSF) and 7‐ethyl‐10‐hydroxycamptothecin (SN‐38). The efficacy of this drug combination is evaluated using 2D and 3D in vitro assays, which exhibited a significant synergism with 5 × 104 times lower IC50 than that of temozolomide (TMZ), the gold‐standard drug. The combination treatment increases intracellular ROS (reactive oxygen species) production, and ROS inhibition by using N‐Acetyl Cysteine (NAC) reduced the cytotoxicity substantially, indicating that ROS played a crucial role in the synergistic cytotoxicity. The combination treatment inhibited GBM cell proliferation, migration, and stem cell marker expression. Mechanistically, the DSF+SN‐38 combination is found to increase p53 expression, inhibit PI3K signaling, and activate caspase 9. Altogether, this study demonstrates that the DSF+SN‐38 combination can present a promising therapeutic strategy for treating GBM.

Triiodo-L-thyronine (T3) downregulates Npr1 gene (coding for natriuretic peptide receptor-A) transcription in H9c2 cells: involvement of β-AR-ROS signaling.

Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart. We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of β-adrenergic receptor (β-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied. Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and β-MHC), β-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]. A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of β-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells. Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-β-AR-ROS and NPR-A signaling.

Panax quinquefolius saponins and panax notoginseng saponins attenuate myocardial hypoxia-reoxygenation injury by reducing excessive mitophagy.

Panax quinquefolius saponins (PQS) and Panax notoginseng saponins (PNS) are key bioactive compounds in Panax quinquefolius L. and Panax notoginseng, commonly used in the treatment of clinical ischemic heart disease. However, their potential in mitigating myocardial ischemia-reperfusion injury remains uncertain. This study aims to evaluate the protective effects of combined PQS and PNS administration in myocardial hypoxia/reoxygenation (H/R) injury and explore the underlying mechanisms. To investigate the involvement of HIF-1α/BNIP3 mitophagy pathway in the myocardial protection conferred by PNS and PQS, we employed small interfering BNIP3 (siBNIP3) to silence key proteins of the pathway. H9C2 cells were categorized into four groups: control, H/R, H/R + PQS + PNS, and H/R + PQS + PNS+siBNIP3. Cell viability was assessed by Cell Counting Kit-8, apoptosis rates determined via flow cytometry, mitochondrial membrane potential assessed with the JC-1 fluorescent probes, intracellular reactive oxygen species detected with 2',7'-dichlorodihydrofluorescein diacetate, mitochondrial superoxide production quantified with MitoSOX Red, and autophagic flux monitored with mRFP-GFP-LC3 adenoviral vectors. Autophagosomes and their ultrastructure were visualized through transmission electron microscopy. Moreover, mRNA and protein levels were analyzed via real-time PCR and Western blotting. PQS + PNS administration significantly increased cell viability, reduced apoptosis, lowered reactive oxygen species levels and mitochondrial superoxide production, mitigated mitochondrial dysfunction, and induced autophagic flux. Notably, siBNIP3 intervention did not counteract the cardioprotective effect of PQS + PNS. The PQS + PNS group showed downregulated mRNA expression of HIF-1α and BNIP3, along with reduced HIF-1α protein expression compared to the H/R group. PQS + PNS protects against myocardial H/R injury, potentially by downregulating mitophagy through the HIF-1α/BNIP3 pathway.