Intrinsic Pathway Research Articles

Discovery of flavonoid-containing compound Lupalbigenin as anti-NSCLC cancer agents via suppression of EGFR and ERK1/2 pathway

Epidermal growth factor receptor exon 20 insertions (EGFR Ex20ins) driver mutations in non–small cell lung cancer (NSCLC) is insensitive to EGFR tyrosine kinase inhibitors (TKIs). Therefore, it is necessary to develop more novel strategy to address the limitations of existing therapies targeting EGFR-mutated NSCLC. Lupalbigenin (LB), a flavonoid compound extracted from Derris scandens, has shown preclinical activity in lung cancer. However, the activity of LB in Ex20ins-driven tumors has not yet been elucidated. In this study, a series of stable BaF/3 cell-line that contains a high proportion (>90 %) of EGFR-eGFP Ex20ins were generated using an IL3-deprivation method. Ba/F3 cell models harboring dissimilar Ex20ins were used to characterize the antineoplastic mechanism of LB. Molecular docking confirmed that the LB could effectively bind to key target EGFR. The in vitro anticancer activity of LB was investigated in engineered Ba/F3 cells bearing diverse uncommon EGFR mutations. LB was shown to be more potent in inhibiting the viability of various uncommon EGFR-mutated cell lines. Mechanistic studies disclosed that LB repressed EGFR phosphorylation and downstream survival pathways in Ba/F3 cells expressing EGFR Ex20ins, resulting in caspase activation by activating the intrinsic apoptotic pathway. Further analyses showed that LB significantly induced G0/G1 cell cycle arrest and apoptosis in cells. LB also reduced the protein expression levels of CDK4, CDK6, CDK8, cyclin D1, cyclin A2, and Bcl2 and promoted the expression of cytochrome C, p27, and p53. In summary, we explored the possible potential targets of LB through network pharmacology and verified the target using in vitro experiments. Furthermore, our results demonstrated that LB showed potential anti-Ex20ins cancer activity through suppression of the EGFR and ERK1/2 signaling pathway in Ba/F3 cells bearing two to three amino acid insertion mutations. These findings suggested that LB might be valuable for further investigation as a potential candidate in the treatment of associated diseases.

Role of autophagy and ferroptosis in the development of endometriotic cysts (Review).

It is considered that the etiology of endometriosis is retrograde menstruation of endometrial tissue. Although shed endometrial cells are constantly exposed to a challenging environment with iron overload, oxidative stress and hypoxia, a few cells are able to survive and continue to proliferate and invade. Ferroptosis, an iron‑dependent form of non‑apoptotic cell death, is known to play a major role in the development and course of endometriosis. However, few papers have concentrated on the dynamic interaction between autophagy and ferroptosis throughout the progression of diseases. The present review summarized the current understanding of the mechanisms underlying autophagy and ferroptosis in endometriosis and discuss their role in disease development and progression. For the present narrative review electronic databases including PubMed and Google Scholar were searched for literature published up to the October 31, 2023. Autophagy and ferroptosis may be activated at early stages in endometriosis development. On the other hand, excessive activation of intrinsic pathways (e.g., estrogen and mechanistic target of rapamycin) may promote disease progression through autophagy inhibition. Furthermore, suppression of ferroptosis may cause further progression of endometriotic lesions. In conclusion, the autophagy and ferroptosis pathways may play a dual role in disease initiation and progression. The present review discussed the temporal transition of non‑apoptotic cell death regulation during disease progression from retrograde endometrium to early lesions to established lesions.

Therapeutic Potential of FXI Inhibitors: Hype or Hope?

Significant advancements have shaped the landscape of anticoagulant therapy in the past two decades, including the introduction of direct oral anticoagulants (DOACs), characterized by favorable safety and efficacy profiles and reduced drug-to-drug or food interaction resulting in excellent patient compliance. However, residual concerns still exist with standard-of-care anticoagulant therapy, including the inability to use DOACs in several clinical settings and the need to further reduce the risk of bleeding. Recent improvements in the understanding of the mechanisms behind thrombus formation have led to the awareness that the intrinsic pathway of the coagulation cascade may play an important role in pathological thrombosis, but not in hemostasis. This has represented the rationale for targeting this pathway with factor XI (FXI) inhibitors, with the aim of uncoupling hemostasis and thrombosis. Clinical evidence from patients with FXI deficiency further supports this concept. A number of compounds with different mechanisms of action have been developed to target FXI (i.e., asundexian, abelacimab, Ionis-FXIRx, milvexian, osocimab, and Xisomab 3G). To date, the majority of available trials have not gone beyond completion of phase 2 and results are conflictive making it difficult to appraise the clinical benefit of these compounds in the different clinical settings where they have been tested (i.e., atrial fibrillation, acute ischemic stroke, acute myocardial infarction, end-stage renal disease, total knee arthroplasty). Moreover, the largest phase 3 randomized trial designed to test the efficacy of asundexian over apixaban in patients with atrial fibrillation, the OCEANIC-AF, has been prematurely stopped as a result of the inferior efficacy of asundexian. In this review we discuss the pharmacological properties and available evidence generated thus far for factor XI inhibitors, providing a perspective on the current state of these drugs.

Effect of Agkistrodon acutus venom (AAVC-I) on apoptosis through modulation of the Keap1/Nrf2 pathway in HSC-3 oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial regions. Patients with OSCC exhibit a poor response to conventional chemoradiotherapies, which are associated with severe side effects. Therefore, it is essential to identify an effective therapeutic method to treat patients with OSCC. An anti-tumor compound, Agkistrodon acutus venom component I (AAVC-I), purified from Agkistrodon acutus venom, has demonstrated anticancer activity both in vitro and in vivo. However, the mechanism of AAVC-I's anticancer activity in cancer cells has yet to be established. This study aimed to investigate the mechanism of AAVC-I-induced apoptosis in HSC-3 OSCC cells and explore its regulatory effect on oxidative stress. Survival rates of human OSCC cell HSC-3 were detected by Cell Counting Kit-8 (CCK-8). The reactive oxygen species (ROS) level was analyzed by flow cytometry and fluorescence microscopy. The mitochondrial membrane potential was analyzed by cytometry and fluorescent microplate reader. Apoptosis of HSC-3 cells was analyzed using flow cytometry. The oxidative stress level was evaluated using glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits. In addition, the target proteins were analyzed by western blot. AAVC-I reduced HSC-3 cells' survival rates in a dose-dependent manner with a 50% inhibiting concentration (IC50) of 8.86 µg/mL. It induced apoptosis of HSC-3 cells and the expression of cleaved caspase-3, cleaved caspase-9, and Cyt-c increased significantly, whereas the expression level of Bcl-2 decreased in AAVC-I-treated HSC-3 cells. Thus, AAVC-I caused apoptosis of HSC-3 via the activation of the intrinsic apoptotic pathway. In addition, AAVC-I reduced the mitochondrial membrane potential in HSC-3, enhanced intracellular ROS, and increased intracellular oxidative stress levels in comparison to that of untreated control cells. Furthermore, AAVC-I increased the expression of Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) levels. These findings demonstrate the inhibitory effects and associated mechanisms of AAVC-I on the HSC-3 OSCC cell line. This insight could be valuable for investigating AAVC-I as a potential therapeutic option for patients with OSCC.

Calcium ion delivery by microbubble-assisted sonoporation stimulates cell death in human gastrointestinal cancer cells

Ultrasound-mediated cell membrane permeabilization – sonoporation, enhances drug delivery directly to tumor sites while reducing systemic side effects. The potential of ultrasound to augment intracellular calcium uptake – a critical regulator of cell death and proliferation – offers innovative alternative to conventional chemotherapy. However, calcium therapeutic applications remain underexplored in sonoporation studies. This research provides a comprehensive analysis of calcium sonoporation (CaSP), which combines ultrasound treatment with calcium ions and SonoVue microbubbles, on gastrointestinal cancer cells LoVo and HPAF-II. Initially, optimal sonoporation parameters were determined: an acoustic wave of 1 MHz frequency with a 50 % duty cycle at intensity of 2 W/cm2. Subsequently, various cellular bioeffects, such as viability, oxidative stress, metabolism, mitochondrial function, proliferation, and cell death, were assessed following CaSP treatment. CaSP significantly impaired cancer cell function by inducing oxidative and metabolic stress, evidenced by increased mitochondrial depolarization, decreased ATP levels, and elevated glucose uptake in a Ca2+ dose-dependent manner, leading to activation of the intrinsic apoptotic pathway. Cellular response to CaSP depended on the TP53 gene's mutational status: colon cancer cells were more susceptible to CaSP-induced apoptosis and G1 phase cell cycle arrest, whereas pancreatic cancer cells showed a higher necrotic response and G2 cell cycle arrest. These promising results encourage future research to optimize sonoporation parameters for clinical use, investigate synergistic effects with existing treatments, and assess long-term safety and efficacy in vivo. Our study highlights CaSP's clinical potential for improved safety and efficacy in cancer therapy, offering significant implications for the pharmaceutical and biomedical fields.

Picrasidine I Regulates Apoptosis in Melanoma Cell Lines by Activating ERK and JNK Pathways and Suppressing AKT Signaling.

World Health Organization data indicate a continuous increase in melanoma incidence, with metastatic melanoma characterized by poor prognosis and drug resistance. The exploration of therapeutics derived from natural products remains an active area of in vitro research. The aim of this study was to determine the antitumor effects of picrasidine I, a natural compound extracted from Picrasma quassioides, against two melanoma cell lines. We selected two metastatic melanoma cell lines, HMY-1 and A2058, for molecular studies, including Western blotting, 4',6-diamidino-2-phenylindole staining, and flow cytometry. Picrasidine I demonstrated cytotoxic effects against the HMY-1 and A2058 melanoma cell lines. It induced cell cycle arrest in the sub-G1 phase and downregulated cell cycle-related proteins (e.g., cyclin A2, D1, cyclin-dependent kinases 4, and 6). In the intrinsic apoptosis pathway, picrasidine I activated proapoptotic proteins (e.g., Bax, Bak, t-Bid, BimL/S) and suppressed the expression of antiapoptotic proteins (e.g., Bcl-2, Bcl-xL), with an observed increase in the quantity of depolarized cells. In addition, the apoptotic effects of picrasidine I were linked to the activation of the c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways and the inhibition of the protein kinase B signaling pathway. A human apoptosis array indicated claspin inhibition upon picrasidine I treatment, suggesting the potential involvement of picrasidine I in apoptosis and cell cycle regulation. Our findings suggest that picrasidine I has potential as a candidate for treating advanced melanoma, and thus these findings warrant further investigation. The modulation of claspin expression by picrasidine I could be investigated further as a potential biomarker to predict its efficacy in related to advanced stages of melanoma.

Anti-Cancer Potential of Isoflavone-Enriched Fraction from Traditional Thai Fermented Soybean against Hela Cervical Cancer Cells.

Cervical cancer is a leading cause of gynecological malignancies and cancer-related deaths among women worldwide. This study investigates the anti-cancer activity of Thua Nao, a Thai fermented soybean, against HeLa cervical carcinoma cells, and explores its underlying mechanisms. Our findings reveal that the ethyl acetate fraction of Thua Nao (TN-EA) exhibits strong anti-cancer potential against HeLa cells. High-performance liquid chromatography (HPLC) analysis identified genistein and daidzein as the major isoflavones in TN-EA responsible for its anti-cancer activity. TN-EA and genistein reduced cell proliferation and induced G2/M phase arrest, while daidzein induced G1 arrest. These responses were associated with the downregulation of cell cycle regulators, including Cyclin B1, cycle 25C (Cdc25C), and phosphorylated cyclin-dependent kinase 1 (CDK-1), and the upregulation of the cell cycle inhibitor p21. Moreover, TN-EA and its active isoflavones promoted apoptosis in HeLa cells through the intrinsic pathway, evidenced by increased levels of cleaved Poly (ADP-ribose) polymerase (PARP) and caspase-3, loss of mitochondrial membrane potential, and the downregulation of anti-apoptotic proteins B-cell leukemia/lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL), cellular inhibitor of apoptosis proteins 1 (cIAP), and survivin. Additionally, TN-EA and its active isoflavones effectively reduced cell invasion and migration by downregulating extracellular matrix degradation enzymes, including Membrane type 1-matrix metalloproteinase (MT1-MMP), urokinase-type plasminogen activator (uPA), and urokinase-type plasminogen activator receptor (uPAR), and reduced the levels of the mesenchymal marker N-cadherin. At the molecular level, TN-EA suppressed STAT3 activation via the regulation of JNK and Erk1/2 signaling pathways, leading to reduced proliferation and invasion of HeLa cells.

Pancreatic β‑cell apoptosis in type 2 diabetes is related to post‑translational modifications of p53 (Review).

Pancreatic β‑cells are the only cells that synthesize insulin to regulate blood glucose levels. Various conditions can affect the mass of pancreatic β‑cells and decrease insulin levels. Diabetes mellitus is a disease characterized by insulin resistance and chronic hyperglycemia, mainly due to the loss of pancreatic β‑cells caused by an increase in the rate of apoptosis. Additionally, hyperglycemia has a toxic effect on β‑cells. Although the precise mechanism of glucotoxicity is not fully understood, several mechanisms have been proposed. The most prominent changes are increases in reactive oxygen species, the loss of mitochondrial membrane potential and the activation of the intrinsic pathway of apoptosis due to p53. The present review analyzed the location of p53 in the cytoplasm, mitochondria and nucleus in terms of post‑translational modifications, including phosphorylation, O‑GlcNAcylation and poly‑ADP‑ribosylation, under hyperglycemic conditions. These modifications protect p53 from degradation by the proteasome and, in turn, enable it to regulate the intrinsic pathway of apoptosis through the regulation of anti‑apoptotic and pro‑apoptotic elements. Degradation of p53 occurs in the proteasome and depends on its ubiquitination by Mdm2. Understanding the mechanisms that activate the death of pancreatic β‑cells will allow the proposal of treatment alternatives to prevent the decrease in pancreatic β‑cells.

KGF-2 ameliorates UVB-triggered skin photodamage in mice by attenuating DNA damage and inflammatory response and mitochondrial dysfunction.

Long-term exposure to UVB induces DNA damage, inflammatory response, mitochondrial dysfunction, and apoptosis in skin cells, thus causing skin photodamage. Research has demonstrated the noteworthy antioxidant, anti-inflammatory, DNA repair, and mitochondrial protective properties of keratinocyte growth factor-2 (KGF-2). To examine the impact of KGF-2 on UVB-triggered skin photodamage in mice, hair-removed mice were initially exposed under UVB radiation and subsequently treated with KGF-2 hydrogel and repeated for 6 days. On day 7, the assessment of histopathological alterations, inflammation, DNA damage, mitochondrial function, and apoptosis in mouse skin was assessed. It was found that KGF-2 could effectively relieve cutaneous photodamage symptoms and inhibit epidermal proliferation in mice. Meanwhile, KGF-2 was found to significantly reduce DNA damage, attenuate the inflammatory response, and inhibit the mitochondria-mediated intrinsic apoptotic pathway in the UVB-exposed mouse skin photodamage model. To summarize, our results indicated that KGF-2 reduces the severity of mouse skin photodamage caused by UVB rays by attenuating DNA damage and the inflammatory response, besides inhibiting the mitochondria-mediated intrinsic apoptosis pathway.

Insights into Molecular Mechanisms of Anticancer Activity of Juniperus communis Essential Oil in HeLa and HCT 116 Cells.

As cancer remains a significant global health challenge, there is an increasing need for novel therapeutic approaches. We investigated the antitumor potential of Juniperus communis berry essential oil on cervical cancer HeLa and colorectal HCT 116 cells. Cytotoxicity was evaluated through the MTT assay, revealing concentration-dependent reductions in cell viability. A clonogenic assay demonstrated long-term cytotoxic effects. Apoptosis markers were assessed via flow cytometric analysis and showed an induction of the intrinsic pathway in both cell lines, demonstrated by the elevated levels of cleaved caspase-3, Bax/Bcl-2 ratio, JC-10 monomer formation, and cytochrome C migration to the cytosol. The treatment inhibited cell-survival pathways in HCT 116 cells and arrested HeLa cells in the S phase. An extensive molecular docking screening provided insight into the binding affinity and interaction patterns of the essential oil components with NADH ubiquinone oxidoreductase and superoxide dismutase enzymes, further confirming the induction of the intrinsic pathway of apoptosis. The obtained in silico and in vitro results indicated the anticancer potential of J. communis berry essential oil as it interferes with cancer cell molecular mechanisms. Our findings highlight J. communis berry essential oil as a promising natural agent with anticancer potential.

The Proapoptotic Action of Pyrrolidinedione-Thiazolidinone Hybrids towards Human Breast Carcinoma Cells Does Not Depend on Their Genotype.

The development of new, effective agents for the treatment of breast cancer remains a high-priority task in oncology. A strategy of treatment for this pathology depends significantly on the genotype and phenotype of human breast cancer cells. We aimed to investigate the antitumor activity of new pyrrolidinedione-thiazolidinone hybrid molecules Les-6287, Les-6294, and Les-6328 towards different types of human breast cancer cells of MDA-MB-231, MCF-7, T-47D, and HCC1954 lines and murine breast cancer 4T1 cells by using the MTT, clonogenic and [3H]-Thymidine incorporation assays, flow cytometry, ELISA, and qPCR. The studied hybrids possessed toxicity towards the mentioned tumor cells, with the IC50 ranging from 1.37 to 21.85 µM. Simultaneously, these derivatives showed low toxicity towards the pseudonormal human breast epithelial cells of the MCF-10A line (IC50 > 93.01 µM). Les-6287 at 1 µM fully inhibited the formation of colonies of the MCF-7, MDA-MB-231, and HCC1954 cells, while Les-6294 and Les-6328 did that at 2.5 and 5 µM, respectively. Les-6287 suppressed DNA biosynthesis in the MCF-7, MDA-MB-231, and HCC1954 cells. At the same time, such an effect on the MCF-10A cells was significantly lower. Les-6287 induces apoptosis using extrinsic and intrinsic pathways via a decrease in the mitochondrial membrane potential, increasing the activity of caspases 3/7, 8, 9, and 10 in all immunohistochemically different human breast cancer cells. Les-6287 decreased the concentration of the metastasis- and invasion-related proteins MMP-2, MMP-9, and ICAM-1. It did not induce autophagy in treated cells. In conclusion, the results of our study suggest that the synthesized hybrid pyrrolidinedione-thiazolidinones might be promising agents for treating breast tumors of different types.

Hormonal orchestra: mastering mitochondria's role in health and disease.

Mitochondria is a subcellular organelle involved in the pathogenesis of cellular stress, immune responses, differentiation, metabolic disorders, aging, and death by regulating process of fission, fusion, mitophagy, and transport. However, an increased interest in mitochondria as powerhouse for ATP production, the mechanisms of mitochondria-mediated cellular dysfunction in response to hormonal interaction remains unknown. Mitochondrial matrix contains chaperones and proteases that regulate intrinsic apoptosis pathway through pro-apoptotic Bcl-2 family's proteins Bax/Bak, and Cyt C release, and induces caspase-dependent and independent cells death. Energy and growth regulators such as thyroid hormones have profound effect on mitochondrial inner membrane protein and lipid compositions, ATP production by regulating oxidative phosphorylation system. Mitochondria contain cholesterol side-chain cleavage enzyme, P450scc, ferredoxin, and ferredoxin reductase providing an essential site for steroid hormones biosynthesis. In line with this, neurohormones such as oxytocin, vasopressin, and melatonin are correlated with mitochondrial integrity, displaying therapeutic implications for inflammatory and immune responses. Melatonin's also displayed protective role against oxidative stress and mitochondrial synthesis of ROS, suggesting a defense mechanism against aging-related diseases. An imbalance in mitochondrial bioenergetics can cause neurodegenerative disorders, cardiovascular diseases, and cancers. Hormone-induced PGC-1α stimulates mitochondrial biogenesis via activation of NRF1 and NRF2, which in turn triggers mtTFA in brown adipose and cardiac myocytes. Mitochondria can be transferred through cells merging, exosome-mediated transfer, and tunneling through nanotubes. By delineating the underlying molecular mechanism of hormonal mitochondrial interaction, this study reviews the dynamics mechanisms of mitochondria and its effects on cellular level, health, diseases, and therapeutic strategies targeting mitochondrial diseases.

Green tea extract reduces viral proliferation and ROS production during Feline Herpesvirus type-1 (FHV-1) infection

BackgroundFeline Herpesvirus type-1 (FHV-1) is a worldwide spread pathogen responsible for viral rhinotracheitis and conjunctivitis in cats that, in the most severe cases, can lead to death. Despite the availability of a variety of antiviral medications to treat this illness, mainly characterized by virostatic drugs that alter DNA replication, their use is often debated. Phytotherapeutic treatments are a little-explored field for FHV-1 infections and reactivations. In this scenario, natural compounds could provide several advantages, such as reduced side effects, less resistance and low toxicity. The purpose of this study was to explore the potential inhibitory effects of the green tea extract (GTE), consisting of 50% of polyphenols, on FHV-1 infection and reactive oxygen species (ROS) production.ResultsCrandell-Reese feline kidney (CRFK) cells were treated with different doses of GTE (10–400 µg/mL) during the viral adsorption and throughout the following 24 h. The MTT and TCID50 assays were performed to determine the cytotoxicity and the EC50 of the extract, determining the amounts of GTE used for the subsequent investigations. The western blot assay showed a drastic reduction in the expression of viral glycoproteins (i.e., gB and gI) after GTE treatment. GTE induced not only a suppression in viral proliferation but also in the phosphorylation of Akt protein, generally involved in viral entry. Moreover, the increase in cell proliferation observed in infected cells upon GTE addition was supported by enhanced expression of Bcl-2 and Bcl-xL anti-apoptotic proteins. Finally, GTE antioxidant activity was evaluated by dichloro-dihydro-fluorescein diacetate (DCFH-DA) and total antioxidant capacity (TAC) assays. The ROS burst observed during FHV-1 infection was mitigated after GTE treatment, leading to a reduction in the oxidative imbalance.ConclusionsAlthough further clinical trials are necessary, this study demonstrated that the GTE could potentially serve as natural inhibitor of FHV-1 proliferation, by reducing viral entry. Moreover, it is plausible that the extract could inhibit apoptosis by modulating the intrinsic pathway, thus affecting ROS production.

Terphenyllin induces CASP3-dependent apoptosis and pyroptosis in A375 cells through upregulation of p53

BackgroundMelanoma, one of the most lethal forms of skin cancer, has the potential to develop in any area where melanocytes are present. Currently, postoperative recurrence due to the emergence of systemic drug resistance represents a significant challenge in the treatment of melanoma. In this study, terphenyllin (TER), a distinctive inhibitory impact on melanoma cells was identified from the natural p-terphenyl metabolite. This study aimed to elucidate the intrinsic mechanism of this inhibitory effect, which may facilitate the discovery of novel chemotherapeutic agents.MethodsA transcriptome sequencing and metabolomic analysis of TER-treated A375 cells was conducted to identify potential pathways of action. The key proteins were knocked out and backfilled using CRISPR-Cas9 technology and molecular cloning. Subsequently, the results of cytosolic viability, LDH release, immunofluorescence and flow cytometry were employed to demonstrate the cell death status of the drug-treated cells.ResultsThe p53 signalling pathway was markedly upregulated following TER treatment, leading to the activation of CASP3 via the intrinsic apoptotic pathway. The activated CASP3 initiated apoptosis, while simultaneously continuing to cleave the GSDME, thereby triggering pyroptosis. The knockout of p53, a key protein situated upstream of this pathway, resulted in a significant rescue of TER-induced cell death, as well as an alleviation of the decrease in cell viability. However, the knockout of key proteins situated downstream of the pathway (CASP3 and GSDME) did not result in a rescue of TER-induced cell death, but rather a transformation of the cells from apoptosis and pyroptosis.ConclusionsThe induction of apoptosis and pyroptosis in A375 cells by TER is mediated via the p53-BAX/FAS-CASP3-GSDME signalling pathway. This lays the foundation for TER as a potential anti-melanoma drug in the future. It should be noted that CASP3 and GSDME in this pathway solely regulate the mode of cell death, rather than determine whether cell death occurs. This distinction may prove valuable in future studies of apoptosis and pyroptosis.

The Feline calicivirus Leader of the Capsid (LC) Protein Contains a Putative Transmembrane Domain, Binds to the Cytoplasmic Membrane, and Exogenously Permeates Cells.

Feline calicivirus (FCV), an important model for studying the biology of the Caliciviridae family, encodes the leader of the capsid (LC) protein, a viral factor known to induce apoptosis when expressed in a virus-free system. Our research has shown that the FCV LC protein forms disulfide bond-dependent homo-oligomers and exhibits intrinsic toxicity; however, it lacked a polybasic region and a transmembrane domain (TMD); thus, it was initially classified as a non-classical viroporin. The unique nature of the FCV LC protein, with no similarity to other proteins beyond the Vesivirus genus, has posed challenges for bioinformatic analysis reliant on sequence similarity. In this study, we continued characterizing the LC protein using the AlphaFold 2 and the recently released AlphaFold 3 artificial intelligence tools to predict the LC protein tertiary structure. We compared it to other molecular modeling algorithms, such as I-Tasser's QUARK, offering new insights into its putative TMD. Through exogenous interaction, we found that the recombinant LC protein associates with the CrFK plasmatic membrane and can permeate cell membranes in a disulfide bond-independent manner, suggesting that this interaction might occur through a TMD. Additionally, we examined its potential to activate the intrinsic apoptosis pathway in murine and human ovarian cancer cell lines, overexpressing survivin, an anti-apoptotic protein. All these results enhance our understanding of the LC protein's mechanism of action and suggest its role as a class-I viroporin.

Perinatal Exposure to Glyphosate or a Commercial Formulation Alters Uterine Mechanistic Pathways Associated with Implantation Failure in Rats.

Perinatal exposure to a glyphosate-based herbicide (GBH) or its active ingredient, glyphosate (Gly), has been demonstrated to increase implantation failure in rats. This study investigates potential mechanisms of action, analyzing uterine preparation towards the receptive state. Pregnant Wistar rats (F0) were treated orally with GBH or Gly (3.8 and 3.9 mg Gly/kg/day, respectively) from gestational day (GD) 9 until weaning. Adult F1 females became pregnant and uterine samples were collected on GD5 (preimplantation period). Histomorphological uterine parameters were assessed. Immunohistochemistry was applied to evaluate cell proliferation and protein expression of estrogen receptors (ERα and ERβ), cell cycle regulators (PTEN, cyclin G1, p27, and IGF1R-α), and the Wnt5a/β-catenin/FOXA2/Lif pathway. Both GBH and Gly females showed increased stromal proliferation, associated with a high expression of ERs. Dysregulation of PTEN and cyclin G1 was also observed in the Gly group. Reduced gland number was observed in both groups, along with decreased expression of Wnt5a/β-catenin/FOXA2/Lif pathway in the glandular epithelium. Overall, GBH and Gly perinatal exposure disrupted intrinsic uterine pathways involved in endometrial proliferation and glandular function, providing a plausible mechanism for glyphosate-induced implantation failure by compromising uterine receptivity. Similar effects between GBH and Gly suggest the active principle mainly drives the adverse outcomes.

Synthesis of gold nanoparticles using Ginkgo biloba leaf extract and its in vitro anticancer effects on nasopharyngeal carcinoma cells

Green synthesis of gold nanoparticles (AuNPs) using Ginkgo biloba leaf aqueous extract was optimized by response surface methodology to yield monodispersed, crystalline nanoparticles of 18 ± 4 nm diameter. Fourier transform infrared spectroscopy confirmed capping by flavonoids, terpenoids and polyphenols providing stability for over 6 months. The biosynthesized AuNPs exhibited promising in vitro dose and time-dependent antiproliferative effects against human nasopharyngeal carcinoma CNE1 cells with IC50 reducing from 65.7 to 39.2 μg/mL over 24–72 hrs. Distinct cytoplasmic damage was observed under microscope. Treatment with 50 μg/mL AuNPs for 48 hrs dramatically upregulated pro-apoptotic Bax, Caspase-3 and p53 by 8.7, 6.5 and 4.3 fold respectively while downregulating anti-apoptotic Bcl-2 by 11.2 fold, indicating apoptosis induction via mitochondrial intrinsic pathway. The green fabricated G. biloba AuNPs displayed preferential toxicity towards carcinoma cells compared to normal fibroblasts, indicating potential for targeted nasopharyngeal cancer therapy.

Mycoplasma hyopneumoniae inhibits the unfolded protein response to prevent host macrophage apoptosis and M2 polarization.

Enzootic pneumonia caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) has inflicted substantial economic losses on the global pig industry. The progression of M. hyopneumoniae induced-pneumonia is associated with lung immune cell infiltration and extensive proinflammatory cytokine secretion. Our previous study established that M. hyopneumoniae disrupts the host unfolded protein response (UPR), a process vital for the survival and immune function of macrophages. In this study, we demonstrated that M. hyopneumoniae targets the UPR- and caspase-12-mediated endoplasmic reticulum (ER)-associated classical intrinsic apoptotic pathway to interfere with host cell apoptosis signaling, thereby preserving the survival of host tracheal epithelial cells (PTECs) and alveolar macrophages (PAMs) during the early stages of infection. Even in the presence of apoptosis inducers, host cells infected with M. hyopneumoniae exhibited an anti-apoptotic potential. Further analyses revealed that M. hyopneumoniae suppresses the three UPR branches and their induced apoptosis. Interestingly, while UPR activation typically drives host macrophages toward an M2 polarization phenotype, M. hyopneumoniae specifically obstructs this process to maintain a proinflammatory phenotype in the host macrophages. Overall, our findings propose that M. hyopneumoniae inhibits the host UPR to sustain macrophage survival and a proinflammatory phenotype, which may be implicated in its pathogenesis in inducing host pneumonia.

Tributyltin induces apoptosis in mammalian cells invivo: a scoping review.

The present review aimed to evaluate the apoptotic effect of tributyltin (TBT) exposure on mammalian tissues and cells invivo. A search was conducted in specialized literature databases including Embase, Medline, Pubmed, Scholar Google, and Scopus for all manuscripts using the following keywords: "tributyltin", "apoptosis", "mammals", "mammalian cells', "eukaryotic cells", 'rodents', "rats", "mice" and "invivo" for all data published until September 2023. A total of 16 studies were included. The studies have demonstrated that TBT exposure induces apoptosis in cells from various mammalian organs or tissues invivo. TBT is capable to increase apoptotic cells, to activate proapoptotic proteins such as calpain, caspases, bax andbeclin-1 and to inhibit antiapoptotic protein bcl-2. Additionally, TBT alters the ratio of bcl-2/bax which favor apoptosis. Therefore, the activation of enzymes such as calpain induces apoptosis mediated by ERS and caspases through the intrinsic apoptosis pathway. This review has demonstrated that TBT exposure induces apoptosis in mammalian tissues and cells invivo.

Oral Administration of Carotenoid-Rich Dunaliella salina Powder Inhibits Colon Carcinogenesis via Modulation of Wnt/β-catenin Signaling Cascades in a Rat Model.

The present study aims to investigate the oral therapeutic and molecular role of carotenoid-rich Dunaliella salina powder (DSP) against 1,2-dimethylhydrazine (DMH)-triggered colon carcinogenesis. In this study, thirty six male Wistar rats were categorized into six distinct groups (G1-G6): G1 group with no intervention, G2 group received only DSP (1000mg/kg), G3 group received only DMH carcinogen (20mg/kg), and G4-G6 group received both DMH and DSP at various phases (pre-initiation, post-initiation and entire phases) for 32weeks. Body weight, tumor incidence, tumor volume, histopathological examination, antioxidants, and detoxification enzymes activities were analyzed in the experimental rats. In addition, the protein expression profile of components involved in the Wnt/β-catenin signaling pathway was determined by western blot analysis. Matrix metalloproteinases (MMP-7 and MMP-9), proliferation marker (PCNA), and pro-apoptotic (Bcl-2 and Bax) proteins were analyzed using immunohistochemistry. Colorimetric assay was used to determine the levels of anti-inflammatory (iNOS and COX-2) and apoptotic proteins (Caspase-3 and Caspase-9). Results showed that concomitant administration of DSP with DMH significantly reduced tumor progression and prevented colon carcinogenesis in rats. However, treatment with DSP before or after DMH exposure did not significantly prevent colon carcinogenesis. DMH and DSP treatment group showed increased activities of antioxidant enzymes with significant reduction in the oxidative stress. Additionally, the detoxification enzymes and colonic histopathology of those rats were restored to that of control rats. The administration of DSP to rats exposed to DMH exhibited antitumor effects via inhibition of the Wnt/β-catenin signaling pathway with induced apoptosis through the Bcl-2/Bax/caspases signaling cascades. Moreover, the same group also showed significant anti-inflammatory activity via regulating iNOS and COX-2 biomarkers. Our findings revealed molecular chemopreventive activity of carotenoid-rich DSP through regulating Wnt/beta-catenin and intrinsic apoptotic pathways. Thus, DSP is propound to function as a potent antioxidant, anti-proliferative, and anti-inflammatory therapeutic agent against colon carcinogenesis.