Activation Of Caspase Cascade Research Articles

Newcastle disease virus infection induces parthanatos in tumor cells via calcium waves

Parthanatos is distinct from caspase-dependent apoptosis in that it does not necessitate the activation of caspase cascades; Instead, it relies on the translocation of Apoptosis-inducing Factor (AIF) from the mitochondria to the nucleus, resulting in nuclear DNA fragmentation. Newcastle Disease Virus (NDV) is an oncolytic virus that selectively targets and kills tumor cells by inducing cell apoptosis. It has been reported that NDV triggers classic apoptosis through the mitochondrial pathway. In this study, we observed that NDV infection induced endoplasmic reticulum stress (ERS), which caused a rapid release of endogenous calcium ions (Ca2+). This cascade of events resulted in mitochondrial depolarization, loss of mitochondrial membrane potential, and structural remodeling of the mitochondria. The overload of Ca2+ also initiated an increase in mitochondrial membrane permeability, facilitating the transfer of AIF to the nucleus to induce apoptosis. Damaged mitochondria produced excessive reactive oxygen species (ROS), which further exacerbated mitochondrial damage and increased mitochondrial membrane permeability, thus promoting additional intracellular Ca2+ accumulation and ultimately triggering an ROS burst. Collectively, these findings indicate that NDV infection promotes excessive calcium accumulation and ROS generation, leading to mitochondrial damage that releases more calcium and ROS, creating a feedback loop that exacerbates AIF-dependent parthanatos. This study not only provides a novel perspective on the oncolytic mechanism of NDV but also highlights new targets for antiviral research.

PX-478 induces apoptosis in acute myeloid leukemia under hypoxia by inhibiting the PI3K/AKT/mTOR pathway through downregulation of GBE1

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy characterized by limited therapeutic options and a pronounced tendency for relapse. PX-478, a novel inhibitor of hypoxia-inducible factor 1-alpha (HIF-1α), has demonstrated antitumor activity across various cancer models, but its specific role in AML remains unexplored. This study aimed to explore the potential target and mechanism of PX-478-induced AML cell apoptosis. First, PX-478 induced AML cell apoptosis in vitro under hypoxia via modulation of the Bcl-2 family and activation of the mitochondria-mediated caspase cascade, exhibiting a concentration-dependent effect. Additionally, in vivo administration of PX-478 led to notable inhibition of subcutaneous AML xenograft growth in mice, coupled with increased tumor cell apoptosis. RNA sequencing and cellular studies revealed downregulation of the PI3K/AKT/mTOR signaling pathway in PX-478-treated cells. Consistently, cellular studies also implicated PI3K/AKT/mTOR pathway in PX-478-induced AML cell apoptosis. Furthermore, by screening for RNA sequencing differential genes and subsequent experimental verification, Glycogen branching enzyme 1 (GBE1) may be involved in PX-478-induced apoptosis in AML cells. We found that inhibiting GBE1 expression in AML cells (siGBE1) led to downregulation of the PI3K/AKT/mTOR pathway and induced apoptosis. In experiments using AML cells with reduced GBE1 expression (shGBE1), PX-478 treatment did not further downregulate the pathway or enhance apoptosis. Re-expression of GBE1 in shGBE1 cells alleviated apoptosis and reduced PX-478- induced apoptosis and pathway downregulation. In conclusion, our findings provide convincing evidence that PX-478 induces apoptosis by inhibiting the PI3K/AKT/mTOR pathway through downregulation of GBE1 in AML cells

A ketogenic diet alleviates the apoptosis of granulosa cells by inhibiting the activation of cGAS-STING signaling pathway in PCOS mice

BackgroundPolycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility. The ketogenic diet (KD), a diet high in fat and low in carbohydrates, has been applied clinically for the treatment of obese women with PCOS. We have previously demonstrated that KD improved the reproductive phenotype in an androgen-induced PCOS mouse model, yet the underlying molecular mechanisms remain largely unclear. The aim of the present study was to investigate the effect of KD on the reproductive phenotype of a letrozole-induced PCOS mouse model.MethodsFemale C57BL/6N mice were divided into three groups, designated control, letrozole, and letrozole + KD groups. Mice of control and letrozole groups were fed the control diet, whereas letrozole + KD mice were fed a KD with 89.9% (kcal) fat for 3 weeks after the PCOS mouse model was generated. β-hydroxybutyrate (BHB), the most abundant ketone body in the body, was used to treat KGN cells with testosterone (T) to simulate the KD effect on PCOS mouse ovaries in vitro.ResultsOur data showed that KD treatment significantly increased blood ketone levels and reduced body weight. Ovarian functions were improved in some letrozole + KD mice. Results from in vitro experiments indicated mitochondrial damage owing to high T levels, which resulted in the leakage of cytochrome C and mitochondrial DNA into the cytosol and thus induced the activation of the intracellular caspase cascade and the cGAS-STING-NF-κB pathway, leading to granulosa cell inflammation and apoptosis. BHB exhibited certain protective effects on mitochondria of T-treated KGN cells via inhibiting the cGAS-STING pathway. Moreover, the cGAS-STING pathway was activated in ovaries of letrozole mice and was down-regulated in letrozole + KD mice.ConclusionThese findings, for the first time, revealed that hyperandrogenism induced ovarian dysfunction possibly through activation of the cGAS-STING pathway, which could be partially inhibited by ketone bodies produced from KD administration.

Blue Light-Induced Mitochondrial Oxidative Damage Underlay Retinal Pigment Epithelial Cell Apoptosis

Reactive oxygen species (ROS) play a pivotal role in apoptosis. We reported that Blue Light (BL) induced oxidative stress in human retinal pigment epithelial (RPE) cells in vitro and increased drusen deposition and RPE cell apoptosis in human eyes. Here, we investigated the mechanisms underlying BL-induced damage to RPE cells. Cells were exposed to BL with or without the antioxidant N-acetylcysteine. Cells were analyzed for levels of ROS, proliferation, viability, and mitochondria membrane potential (ΔΨM) fluctuation. We performed proteomic analyses to search for differentially expressed proteins. ROS levels increased following RPE cell exposure to BL. While ROS production did not affect RPE cell proliferation, it was accompanied by decreased ΔΨM and increased cell apoptosis due to the caspase cascade activation in a ROS-dependent manner. Proteomic analyses revealed that BL decreased the levels of ROS detoxifying enzymes in exposed cells. We conclude that BL-induced oxidative stress is cytotoxic to RPE cells. These findings bring new insights into the involvement of BL on RPE cell damage and its role in the progression of age-related macular degeneration. The use of antioxidants is an avenue to block or delay BL-mediated RPE cell apoptosis to counteract the disease progression.

Proteome-wide CETSA reveals diverse apoptosis-inducing mechanisms converging on an initial apoptosis effector stage at the nuclear periphery

Cellular phenotypes of apoptosis, as well as the activation of apoptosis caspase cascades, are well described. However, sequences and locations of early biochemical effector events after apoptosis initiation are still only partly understood. Here, we use integrated modulation of protein interaction states-cellular thermal shift assay (IMPRINTS-CETSA) to dissect the cellular biochemistry of early stages of apoptosis at the systems level. Using 5 families of cancer drugs and a new CETSA-based method to monitor the cleavage of caspase targets, we discover the initial biochemistry of the effector stage of apoptosis for all the studied drugs being focused on the peripheral nuclear region rather than the cytosol. Despite very different candidate apoptosis-inducing mechanisms of the drug families, as revealed by the CETSA data, they converge into related biochemical modulations in the peripheral nuclear region. This implies a higher control of the localization of the caspase cascades than previously anticipated and highlights the nuclear periphery as a critical vulnerability for cancer therapies.

Dark and Dronc activation in Drosophila melanogaster

The onset of apoptosis is characterized by a cascade of caspase activation, where initiator caspases are activated by a multimeric adaptor complex known as the apoptosome. In Drosophila melanogaster, the initiator caspase Dronc undergoes autocatalytic activation in the presence of the Dark apoptosome. Despite rigorous investigations, the activation mechanism for Dronc remains elusive. Here, we report the cryo-EM structures of an auto-inhibited Dark monomer and a single-layered, multimeric Dark/Dronc complex. Our biochemical analysis suggests that the auto-inhibited Dark oligomerizes upon binding to Dronc, which is sufficient for the activation of both Dark and Dronc. In contrast, the previously observed double-ring Dark apoptosome may represent a non-functional or "off-pathway" conformation. These findings expand our understanding on the molecular mechanism of apoptosis in Drosophila.

Curcumin Analog L48H37 Induces Apoptosis in Human Oral Cancer Cells by Activating Caspase Cascades and Downregulating the Inhibitor of Apoptosis Proteins through JNK/p38 Signaling.

L48H37 is a synthetic curcumin analog that has anticancer potentials. Here, we further explored the anticancer effect of L48H37 on oral cancer cells and its mechanistic acts. Cell cycle distribution was assessed using flow cytometric analysis. Apoptosis was elucidated by staining with PI/Annexin V and activation of the caspase cascade. Cellular signaling was explored using apoptotic protein profiling, Western blotting, and specific inhibitors. Our findings showed that L48H37 significantly reduced the cell viability of SCC-9 and HSC-3 cells, resulting in sub-G1 phase accumulation and increased apoptotic cells. Apoptotic protein profiling revealed that L48H37 increased cleaved caspase-3, and downregulated cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) in SCC-9 cells, and the downregulated cIAP1 and XIAP in both oral cancer cells were also demonstrated by Western blotting. Meanwhile, L48H37 triggered the activation of caspases and mitogen-activated protein kinases (MAPKs). The involvement of c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) in the L48H37-triggered apoptotic cascade in oral cancer cells was also elucidated by specific inhibitors. Collectively, these findings indicate that L48H37 has potent anticancer activity against oral cancer cells, which may be attributed to JNK/p38-mediated caspase activation and the resulting apoptosis. This suggests a potential benefit for L48H37 for the treatment of oral cancer.

Biogenic Zinc Oxide Nanoparticles synthesized from Tinospora Cordifolia induce oxidative stress, mitochondrial damage and apoptosis in Colorectal Cancer.

Cancer chemotherapy remains a serious challenge, and new approaches to therapy are urgently needed to build novel treatment regimens. The methanol extract of the stem of Tinospora Cordifolia was used to synthesize biogenic zinc oxide nanoparticles (ZnO-NPs) that display anticancer activities against colorectal cancer. Biogenic ZnO-NPs synthesized from methanol extract of Tinospora Cordifolia stem (ZnO-NPs TM) were tested against HCT-116 cell lines to assess anticancer activity. UV-Vis, FTIR, XRD, SEM, and TEM analysis characterized the biogenic ZnO-NPs. To see how well biogenic ZnO-NPs fight cancer, cytotoxicity, AO/EtBr staining, Annexin V/PI staining, mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) analysis, and caspase cascade activity analysis were performed to assess the anticancer efficacy of biogenic ZnO-NPs. The IC50 values of biogenic ZnO-NPs treated cells (HCT-116 and Caco-2) were 31.419 ± 0.682μg/ml and 36.675 ± 0.916μg/ml, respectively. qRT-PCR analysis showed that cells treated with biogenic ZnO-NPs Bax and P53 mRNA levels increased significantly (p ≤ 0.001). It showed to have impaired MMP and increased ROS generation. In a corollary, our in vivo study showed that biogenic ZnO-NPs have an anti-tumour effect. Biogenic ZnO-NPs TM showed both in vitro and in vivo anticancer effects that could be employed as anticancer drugs.

Polyphenol galangin induces the ROS and ER stress-mediated intrinsic and extrinsic apoptotic pathways towards human colon carcinoma HCT-116 cells

A natural anti-oxidant galangin, a flavonoid compound rich in the root of galangal, has been proved to exert anti-proliferation and apoptosis induction towards various cancer cells. However, its activities and molecular mechanism against colorectal cancer cell lines (e.g. HCT-116) is still unclear. The aim of this study was to reveal the inhibition and apoptosis induction of galangin in the HCT-116 cells. The results of CCK-8 assay demonstrated that galangin at 20-160 μmol/L inhibited the HCT-116 cells growth in a dose-time-dependent manner. Galangin changed cell morphology, arrested cell cycle at G0/G1 phase, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species (ROS) and Ca2+ and induced early apoptosis in the HCT-116 cells. Based on western blotting results, on one hand, galangin at 80-160 μmol/L up-regulated pro-apoptotic proteins expression such as AIF, PIG3, and Bax, and induced mitochondrial pathway by up-regulating the levels of cleaved caspase-8, cleaved caspase-7, cleaved caspase-9 and cleaved caspase-3, as well as cleavage of poly (ADP-ribose) polymerase (PARP). On the other hand, galangin also induced endoplasmic reticulum (ER) stress in the cell via up-regulation of CHOP and DR5 and then activation of caspase cascades. The results illustrated that galangin induced apoptosis towards the HCT-116 cells through the ROS- and ER stress-mediated both intrinsic and extrinsic apoptotic pathways. KEYWORDS: galangin; human colon carcinoma cells; apoptotic mechanism; reactive oxygen species; endoplasmic reticulum stress  

A guide to cell death pathways.

Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Apoptosis, necroptosis and pyroptosis are three such programmed cell death modalities. The caspase family of cysteine proteases serve as key regulators of programmed cell death. During apoptosis, a cascade of caspase activation mediates signal transduction and cellular destruction, whereas pyroptosis occurs when activated caspases cleave gasdermins, which can then form pores in the plasma membrane. Necroptosis, a form of caspase-independent programmed necrosis mediated by RIPK3 and MLKL, is inhibited by caspase-8-mediated cleavage of RIPK1. Disruption of cellular homeostatic mechanisms that are essential for cell survival, such as normal ionic and redox balance and lysosomal flux, can also induce cell death without invoking programmed cell death mechanisms. Excitotoxicity, ferroptosis and lysosomal cell death are examples of such cell death modes. In this Review, we provide an overview of the major cell death mechanisms, highlighting the latest insights into their complex regulation and execution, and their relevance to human diseases.

Tailored horseshoe-shaped nicotinonitrile scaffold as dual promising c-Met and Pim-1 inhibitors: Design, synthesis, SAR and in silico study

For the horseshoe tactic to succeed in inhibiting c-Met and Pim-1, the nicotinonitrile derivatives (2a-n) were produced in high quantities by coupling acetyl phenylpyrazole (1) with the proper aldehydes and ethyl cyanoacetate under basic conditions. Consistent basic and spectroscopic data (NMR, IR, Mass, and HPLC) supported the new products' structural findings. With IC50 potency in nanomolar ranges, these compounds had effectively repressed them, particularly compounds 2d and 2 h, with IC50 values below 200 nM. The most potent compounds (2d and 2 h) were tested for their antitumor effects against prostate (PC-3), colon (HCT-116), and breast (MDA-MB-231) and were evaluated in comparison to the anticancer drug tivantinib using the MTT assay. Similar to tivantinib, these compounds showed good antiproliferative properties against the HCT-116 tumor cells while having low cytotoxicity towards healthy fetal colon (FHC) cells. In the HCT-116 cell line, their ability to trigger the apoptotic cascade was also investigated by looking at the level of Bax and Bcl-2 as well as the activation of the proteolytic caspase cascade. When HCT-116 cells were exposed to compounds 2d and 2 h in comparison to the control, active caspase-3 levels increased. The HCT-116 cell line also upregulated Bcl-2 protein levels and downregulated Bax levels. Additionally, when treated with compound 2d, the HCT-116 cell cycle was primarily stopped at the S phase. Compared to the control, compound 2d treatment significantly inhibited the protein expression levels of c-Met and Pim-1 kinases in the treated HCT-116 cells. Thorough molecular modeling analyses, such as molecular docking and dynamic simulation, were performed to ascertain the binding mechanism and stability of the target compounds.

Natural isoflavone glabridin targets PI3Kγ as an adjuvant to increase the sensitivity of MDA-MB-231 to tamoxifen and DU145 to paclitaxel

Glabridin is a natural isoflavone with estrogen receptor agonism and significant anti-tumor activity. Additionally, glabridin has a regulation effect on PI3K/AKT/mTOR pathway, but its exact target remains unclear. In this study, we evaluated the antitumor activity of glabridin against breast cancer and prostate cancer cells, and further clarified its targeting to PI3K. We found that glabridin could significantly inhibit the cell viability of human breast cancer and prostate cancer cell lines. It induced caspase activation cascade and cell apoptosis through decreasing the mitochondrial transmembrane potential and increasing the intracellular reactive oxygen species (ROS). Moreover, glabridin could attenuate epithelial-mesenchymal transition (EMT) progression by inhibiting cell migration. PharmMapper calculation showed that PI3Kγ might be the most potential target protein because of the highest Normal Fit score (0.9735) and z'-score (0.9797). Molecular docking and bio-layer interferometry (BLI) analysis further demonstrated the PI3Kγ targeting of glabridin. In vivo experiments showed that glabridin can effectively inhibit the tumor growth of breast cancer xenograft model, and does not show obvious hepatorenal toxicity. Moreover, glabridin could effectively promote the anti-proliferation and pro-apoptotic effects of tamoxifen on MDA-MB-231 cell and taxol on DU145 cell. Elucidating the targeting of glabridin to PI3K may lay a theoretical foundation for the structural derivatization of glabridin, which is expected to greatly promote the application and development of glabridin in the field of cancer therapy.

HO-3867, a curcumin analog, elicits cell apoptosis and p38-mediated caspase activation in hepatocellular carcinoma.

HO-3867, a synthetic curcumin analog, has displayed various tumor-suppressive characteristics and improved bioabsorption over its parent compound. However, its influences on the development of hepatocellular carcinoma (HCC) are poorly defined. To address this, we tested the anticarcinogenic impact of HO-3867 and investigated the underlying mechanisms in fighting liver cancer. Our result demonstrated that HO-3867 reduced the viability of HCC cells, accompanied by promotion of cell cycle arrest at the sub-G1 stage and apoptotic responses. Furthermore, a distinctive profile of apoptosis associated proteins, encompassing elevated heme oxygenase-1 (HO-1) level and caspase activation, was detected in HO-3867-stimulated HCC cells. In addition, such HO-3867-mediated elevation in caspase activation was dampened by pharmacological suppression of p38 activities. Taken together, our findings unveiled that HO-3867 triggered cell cycle arrest and apoptotic events in liver cancer, involving a p38-mediated activation of caspase cascades. These data highlighted a usefulness of curcumin or its analogs on the management of hepatocarcinogenesis.

Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway.

Natural products are considered effective sources for new therapeutic research and development. The numerous therapeutic properties of natural substances in traditional medicine compel us to investigate the anti-cancer properties of Nimbin (N1) and its semi-natural analog Nimbic acid (N3) from Azadirachta indica against MG-63 Osteosarcoma cells. The therapeutic efficacy of N1 and N3 were screened for their toxicity and cytotoxic activity using L6 myotubes, zebrafish larvae and MG-63 osteosarcoma cells. The mitochondrial membrane potential was evaluated using the Rhodamine 123 stain. Further, the nuclear and cellular damage was distinguished using Hoechst and Acridine orange/EtBr stain. The mechanism of cell cycle progression, cellular proliferation and caspase cascade activation was screened using scratch assay, flow cytometry, and mRNA expression analysis. The Nimbin and analogue N3 were found to be non-toxic to normal L6 cells (Rat skeletal muscles), exhibited cytotoxicity in MG-63 cells, and were exposed to be an active inhibitor of cell proliferation and migration. Analogs N1 and N3 induced negative mitochondrial membrane potential when stained with Rhodamine 123, leading to nuclear damage and apoptosis stimulation using AO/EtBr and Hoechst. Further, N1 and N3 induced cell cycle arrest in G0/G1 phase in flow cytometry using PI staining and induced apoptosis by activating the caspase cascade and upregulated Caspase 3 and caspase 9. The study demonstrated cytotoxic activity against MG-63 osteosarcoma cells while being non-toxic to normal L6 cells. These compounds inhibited cell proliferation and migration, induced mitochondrial dysfunction, nuclear damage, and apoptosis stimulation. Furthermore, N1 and N3 caused cell cycle arrest and activated the caspase cascade, ultimately leading to apoptosis. These findings indicate that N1 and N3 hold promise as potential candidates used alone or combined with existing drugs for further investigation and development as anti-cancer agents.

Recipient TIM4 signaling regulates ischemia reperfusion-induced ER stress and metabolic responses in liver transplantation: from mouse-to-human.

T-cell immunoglobulin and mucin (Tim)4 is expressed on APCs, including macrophages, as one of the main amplifiers in the mechanism of liver ischemia-reperfusion injury (IRI) following orthotopic liver transplantation (OLT). Though donor Tim4 selectively expressed on Kupffer cells serves as a checkpoint regulator of innate immune-driven IRI cascades, its role on cells outside the OLT remains unclear. To dissect the role of donor vs. recipient-specific Tim4 signaling in IR-induced stress and hepatocellular function, we employed a murine OLT model utilizing Tim4-knockout (KO) mice as either donor or recipient (WT → WT, WT → Tim4-KO, Tim4-KO → WT). In the experimental arm, disruption of donor Tim4 attenuated IRI-OLT damage, while recipient Tim4-null mutation aggravated hepatic IRI concomitant with disturbed lipid metabolism, enhanced endoplasmic reticulum stress, and activated pro-apoptotic signaling in the grafts. In the in vitro study, murine hepatocytes co-cultured with Tim4-null adipose tissue showed enhanced C/EBP homologous protein (CHOP) expression pattern and susceptibility to hepatocellular death accompanied by activated caspase cascade in response to TNF-α stimulation. In the clinical arm, liver grafts from forty-one transplant patients with enhanced TIM4 expression showed higher body mass index, augmented hepatic endoplasmic reticulum stress, enhanced pro-apoptotic markers, upregulated innate/adaptive immune responses, exacerbated hepatocellular damage, and inferior graft survival. In conclusion, although TIM4 is considered a principal villain in peri-transplant early tissue injury, recipient TIM4 signaling may serve as a savior of IR-triggered metabolic stress in mouse and human OLT recipients.

Involvement and targeted intervention of benzo(a)pyrene-regulated apoptosis related proteome modification and muti-drug resistance in hepatocellular carcinoma

During the development of hepatocellular carcinoma (HCC), the mutual adaptation and interaction of HCC cells and the microenvironment play an important role. Benzo(a)pyrene (B[a]P) is a common environmental pollutant, which can induce the initiation of various malignant tumors, including HCC. However, the effects of B[a]P exposure on progression of HCC and the potential mechanisms remains largely uninvestigated. Here we found that, after the long-term exposure of HCC cells to low dose of B[a]P, it activated glucose-regulated protein 75 (GRP75), which then induced a modification of apoptosis-related proteome. Among them, we identified the X-linked inhibitor of apoptosis protein (XIAP) as a key downstream factor. XIAP further blocked the caspase cascade activation and promoted the acquisition of the anti-apoptosis abilities, ultimately leading to multi-drug resistance (MDR) in HCC. Furthermore, the abovementioned effects were markedly attenuated when we inhibited GRP75 by using 3,4-dihydroxycinnamic acid (caffeic acid, CaA). Collectively, our present study revealed the effects of B[a]P exposure on the progression of HCC, and identified GRP75 was a meaningful factor involved in.

Ethanolic Extract of Artemisia vulgaris Leaf Promotes Apoptotic Cell Death in Non-Small-Cell Lung Carcinoma A549 Cells through Inhibition of the Wnt Signaling Pathway.

The Wnt signaling pathway is reported to be associated with lung cancer progression, metastasis and drug resistance, and thus it is an important therapeutic target for lung cancer. Plants have been shown as reservoirs of multiple potential anticancer agents. In the present investigation, the ethanolic leaf extract of Artemisia vulgaris (AvL-EtOH) was initially analyzed by means of gas chromatography-mass spectrometry (GC-MS) to identify the important phytochemical constituents. The GC-MS analysis of AvL-EtOH exhibited 48 peaks of various secondary metabolites such as terpenoids, flavonoids, carbohydrates, coumarins, amino acids, steroids, proteins, phytosterols, and diterpenes. It was found that the treatment with increasing doses of AvL-EtOH suppressed the proliferation and migration of lung cancer cells. Furthermore, AvL-EtOH induced prominent nuclear alteration along with a reduction in mitochondrial membrane potential and increased ROS (reactive oxygen species) generation in lung cancer cells. Moreover, AvL-EtOH-treated cells exhibited increased apoptosis, demonstrated by the activation of caspase cascade. AvL-EtOH also induced downregulation of Wnt3 and β-catenin expression along with cell cycle protein cyclin D1. Thus, the results of our study elucidated the potential of bioactive components of Artemisia vulgaris in the therapeutic management of lung cancer cells.

BDE-47 Induces Immunotoxicity in RAW264.7 Macrophages through the Reactive Oxygen Species-Mediated Mitochondrial Apoptotic Pathway

Polybrominated diphenyl ethers (PBDEs) are classic and emerging pollutants that are potentially harmful to the human immune system. Research on their immunotoxicity and mechanisms suggests that they play an important role in the resulting pernicious effects of PBDEs. 2,2',4,4'-Tetrabrominated biphenyl ether (BDE-47) is the most biotoxic PBDE congener, and, in this study, we evaluated its toxicity toward RAW264.7 cells of mouse macrophages. The results show that exposure to BDE-47 led to a significant decrease in cell viability and a prominent increase in apoptosis. A decrease in mitochondrial membrane potential (MMP) and an increase in cytochrome C release and caspase cascade activation thus demonstrate that cell apoptosis induced by BDE-47 occurs via the mitochondrial pathway. In addition, BDE-47 inhibits phagocytosis in RAW264.7 cells, changes the related immune factor index, and causes immune function damage. Furthermore, we discovered a significant increase in the level of cellular reactive oxygen species (ROS), and the regulation of genes linked to oxidative stress was also demonstrated using transcriptome sequencing. The degree of apoptosis and immune function impairment caused by BDE-47 could be reversed after treatment with the antioxidant NAC and, conversely, exacerbated by treatment with the ROS-inducer BSO. These findings indicate that oxidative damage caused by BDE-47 is a critical event that leads to mitochondrial apoptosis in RAW264.7 macrophages, ultimately resulting in the suppression of immune function.

A Synergistic pH-Responsive Serum Albumin-Based Drug Delivery System Loaded with Doxorubicin and Pentacyclic Triterpene Betulinic Acid for Potential Treatment of NSCLC.

Nanosized drug delivery systems (DDS) have been studied as a novel strategy against cancer due to their potential to simultaneously decrease drug inactivation and systemic toxicity and increase passive and/or active drug accumulation within the tumor(s). Triterpenes are plant-derived compounds with interesting therapeutic properties. Betulinic acid (BeA) is a pentacyclic triterpene that has great cytotoxic activity against different cancer types. Herein, we developed a nanosized protein-based DDS of bovine serum albumin (BSA) as the drug carrier combining two compounds, doxorubicin (Dox) and the triterpene BeA, using an oil-water-like micro-emulsion method. We used spectrophotometric assays to determine protein and drug concentrations in the DDS. The biophysical properties of these DDS were characterized using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy, confirming nanoparticle (NP) formation and drug loading into the protein structure, respectively. The encapsulation efficiency was 77% for Dox and 18% for BeA. More than 50% of both drugs were released within 24 h at pH 6.8, while less drug was released at pH 7.4 in this period. Co-incubation viability assays of Dox and BeA alone for 24 h demonstrated synergistic cytotoxic activity in the low μM range against non-small-cell lung carcinoma (NSCLC) A549 cells. Viability assays of the BSA-(Dox+BeA) DDS demonstrated a higher synergistic cytotoxic activity than the two drugs with no carrier. Moreover, confocal microscopy analysis confirmed the cellular internalization of the DDS and the accumulation of the Dox in the nucleus. We determined the mechanism of action of the BSA-(Dox+BeA) DDS, confirming S-phase cell cycle arrest, DNA damage, caspase cascade activation, and downregulation of epidermal growth factor receptor (EGFR) expression. This DDS has the potential to synergistically maximize the therapeutic effect of Dox and diminish chemoresistance induced by EGFR expression using a natural triterpene against NSCLC.

Carvacrol instigates intrinsic and extrinsic apoptosis with abrogation of cell cycle progression in cervical cancer cells: Inhibition of Hedgehog/GLI signaling cascade.

Recent times have seen a strong surge in therapeutically targeting the hedgehog (HH)/GLI signaling pathway in cervical cancer. HH signaling pathway is reported to be a crucial modulator of carcinogenesis in cervical cancer and is also associated with recurrence and development of chemoresistance. Moreover, our previous reports have established that carvacrol (CAR) inhibited the proliferation of prostate cancer cells via inhibiting the Notch signaling pathway and thus, it was rational to explore its antiproliferative effects in cervical cancer cell lines. Herein, the present study aimed to investigate the anticancer and apoptotic potential of CAR on C33A cervical cancer cells and further explore the underlying mechanisms. We found that CAR significantly suppressed the growth of C33Acells, induced cell cycle arrest, and enhanced programmed cell death along with augmentation in the level of ROS, dissipated mitochondrial membrane potential, activation of caspase cascade, and eventually inhibited the HH signaling cascade. In addition, CAR treatment increased the expression of pro-apoptotic proteins (Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c) and concomitantly reduced the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) in C33Acells. CAR mediates the activation of caspase-9 and -3 (intrinsic pathway) and caspase-8 (extrinsic pathway) accompanied by the cleavage of PARP in cervical cancer cells. Thus, CAR induced apoptosis by both the intrinsic and extrinsic apoptotic pathways. CAR efficiently inhibited the growth of cervical cancer cells via arresting the cell cycle at G0/G1 phase and modulated the gene expression of related proteins (p21, p27, cyclin D1 and CDK4). Moreover, CAR inhibited the HH/GLI signaling pathway by down regulating the expression of SMO, PTCH and GLI1 proteins in cervical carcinoma cells. With evidence of the above results, our data revealed that CAR treatment suppressed the growth of HPV-C33A cervical cancer cells and further elucidated the mechanistic insights into the functioning of CAR.