Western Blot Studies Research Articles

Transferrin Immobilized Graphene Oxide Nanocomposite for Targeted Cancer Chemodynamic Therapy via Increasing Intracellular Labile Fe2+ Concentration.

Recently, different alternative regulated cell death (RCD) pathways, viz., necroptosis, pyroptosis, ferroptosis, cuproptosis etc., have been explored as important targets for the development of cancer medications in recent years, as these can change the immunogenicity of the tumor microenvironment (TME) and will finally lead to the inhibition of cancer progression and metastasis. Here, we report the development of transferrin immobilized graphene oxide (Tfn@GOAPTES) nanocomposite as a therapeutic strategy toward cancer cell killing. The electrostatic immobilization of Tfn on the GOAPTES surface was confirmed by different spectroscopy and microscopy techniques. The Tfn immobilization was found to be ∼74 ± 4%, whereas the stability of the protein on the GO surface suggested a robust nature of the nanocomposite. The MTT assay suggested that Tfn@GOAPTES exhibited cytotoxicity toward HeLa cells via increased lipid peroxidation and DNA damage. Western blot studies resulted in decreased expression of acetylation on lysine 40 of α-tubulin and increased expression of LC3a/b for Tfn@GOAPTES treated HeLa cells, suggesting autophagy to be the main cause of the cell death mechanism. Overall, we predict that the present approach can be used as a therapeutic strategy for cancer cell killing via selective induction of a high concentration of intracellular iron.

Synthesis of novel triazole-urea hybrids and their antiproliferative activity against pancreatic cancer through suppression of eEF2K and induction of apoptosis

Pancreatic cancer is one of the deadliest cancers with its highly aggressive and metastatic character and there is a huge unmet need for new drugs treating pancreatic cancer. In the present study, a series of 1,2,4-triazole-urea conjugates have been designed and synthesized as novel candidates of antiproliferative agents against pancreatic cancer cells. Among them, compounds 33, 34, 35 and 38 possesing IC50 values between 0.231 and 0.488 μM against PANC-1 cells demonstrated the highest anti-proliferative activity. These compounds presenting the highest antiproliferative activity were evaluated for further biological studies. The same four compounds inhibited colony formation in pancreatic cancer cells dose dependently. Western blot study on the selected compounds showed that compounds 33 and 38 significantly reduced eEF2K protein levels in cancer cells. These compounds displayed an effective eEF2K activity suppression by down-regulated levels of unphosphorylated eEF2 in PANC-1 cells. Compounds 33, 34, 35 and 38 were also demonstrated to induce apoptosis and activate caspase 3/7. In silico studies were performed to predict the druggability and ADMET/ properties of the active molecules. In summary, 1,2,4-triazole-urea conjugates developed in this study represent a novel and promising lead structure with anticancer activity against pancreatic cancer achieved through eEF2K activity suppression. Compounds being referred to are the first "triazole-urea hybrid" molecules found to be effective against pancreatic cancer.

PARP1 promotes EGFR-TKI drug-resistance via PI3K/AKT pathway in non-small-cell lung cancer.

Tyrosine kinase inhibitor (TKI) resistance is the main type of drug resistance in lung cancer patients with epidermal growth factor receptor (EGFR) mutations, but its underlying mechanism remains unclear. The purpose of this work was to investigate the mechanism by which PARP1 regulates EGFR-TKI resistance to identify potential targets for combating drug resistance. The GEO databases, TCGA databases, western blot and qPCR studies were used to investigate the expression of PARP1 in lung cancer cells and tissues and its correlation with the prognosis of lung cancer. The expression of PARP1 in lung cancer TKI resistant cell PC9-ER and TKI sensitive cell PC9 was analyzed by qPCR and western blot. After knocking down of PARP1, CCK-8 assays, colony formation, flow cytometry were used to investigate its impact on erlotinib sensitivity, cell survival, cell cycle, and apoptosis. RNA-seq was used to investigate the mechanism by which PARP1 participates in EGFR-TKI resistance, and the results were validated in vitro and in vivo studies. PARP1 was highly expressed in both lung cancer tissues and cells. Subsequently, increased PARP1 expression was observed in PC9-ER compared with its parental cell line. Knockdown of PARP1 increased erlotinib sensitivity, promoted cell apoptosis, and suppressed cell growth. RNA-seq and previous studies have shown that the PI3K/AKT/mTOR/P70S6K pathway is involved in PARP1-mediated TKI resistance, and these results were confirmed by Western blot in vitro and in vivo. PARP1 may serve as a potential therapeutic target for reversing EGFR-TKI resistance in NSCLC via the PI3K/AKT/mTOR/P70S6K pathway.

Abstract LB075: Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models

Abstract Chimeric antigen receptor (CAR)-T cell therapy has been shown to effectively treat solid tumors in syngeneic murine models, but antigen loss and T cell trafficking into the tumors pose major obstacles to its long-term efficacy. Here we explored a combination therapy against advanced stages of colorectal and ovarian syngeneic murine tumor models using a tumor-specific small molecule (PAC-1) that triggers the engagement of the host immune system and synergizes with a Tn-specific adoptively transferred 237 CAR-T immunotherapy. PAC-1 has been shown to induce apoptosis in tumor cells with high expression of procaspase 3, via chelation of inhibitory zinc and release of active executioner caspase 3. PAC-1 also recently completed a phase I trial (NCT02355535) and was shown to be well-tolerated in late-stage cancer patients. RNA sequencing of CT26 colorectal cells stimulated with PAC-1 for 24 hours revealed that PAC-1 induced global changes at the transcriptome level, up-regulating genes involved in immune activation and apoptosis, and down-regulating genes involved in DNA repair, cell cycle regulation, and myc-driven proliferation. Validation of RNAseq data was performed using a combination of RT-qPCR, Western blot, flow cytometry, and co-culture studies. A combination of transcriptome and exome sequencing analyses in murine colorectal cell line CT26 revealed that the PAC-1-mediated down-regulation of DNA repair machinery led to somatic alterations in the genome and a higher mutational load in tumor cells, subsequently presented as neo-antigens by antigen-presenting cells. As a result, host CD8+ cytotoxic T cells were able to infiltrate tumors and reduce the tumor burden. Tn-dependent CARs have been used recently to demonstrate the proof of concept in the targeting of cancer-specific Tn-antigens such as the Tn-MUC1 antigen in humans, and an analogous Tn-OTS8 target in mice. In our murine ID8 ovarian tumor model, untreated mice with disseminated tumors have a median survival of 71 days. As a single agent administered intraperitoneally, PAC-1 extended the median survival to 90 days (p<0.0001), while 237 CAR-T treated mice have a median survival of 103 days (p<0.0001). A combination of the two treatment modalities (PAC-1 administered before 237 CAR-T) further extended the median survival to 145 days (p=0.048). Administering PAC-1 after 237 CAR-T also modestly extended the median survival to 132 days (p=0.06). Taken altogether, our results demonstrate that PAC-1 engages the immune system to treat cancer by inducing expression of pro-inflammatory cytokines in tumor cells allowing the recruitment of CD8+ effector T cells to the tumor microenvironment. Combination strategies that boost T cell trafficking shows great promise to enhance the anti-tumor efficacy and feasibility of adoptive CAR-T therapy against solid tumors. Citation Format: Diana R. Ranoa, Yifei Kang, Jenny Drnevich, Gloria Rendon, Christopher J. Fields, Keith Bailey, Edward J. Roy, Timothy M. Fan, David M. Kranz, Paul J. Hergenrother. Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB075.

CCDC25 suppresses clear cell renal cell carcinoma progression by LATS1/YAP-mediated regulation of the hippo pathway

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most prevalent renal cancers, and the molecular mechanisms underlying its progression are still not fully understood. The expression of CCDC25, a notably underexpressed gene in many tumors, has been understudied in ccRCC. This research aims to explore the role of CCDC25 in ccRCC's clinical outcomes and uncover the molecular pathways influenced by it.MethodsA multi-tiered approach was adopted involving bioinformatic analysis, tissue sample evaluation, in vitro and in vivo experiments. CCDC25 expression levels in tumor vs. normal tissues were quantified using Western blot and immunofluorescence studies. Cell proliferation and migration were analyzed using CCK8, EDU, Transwell assays, and wound healing assays. RNA sequencing was performed to elucidate the molecular pathways affected, followed by detailed protein–protein interaction studies and mouse xenograft models.ResultsCCDC25 was predominantly underexpressed in ccRCC tumors and associated with advanced clinical stages and poor prognosis. Overexpression of CCDC25 in renal cancer cell lines resulted in reduced proliferation and migration. RNA sequencing revealed significant alterations in the Hippo pathway. Overexpression of CCDC25 inhibited the expression of downstream Hippo pathway proteins ITGA3 and CCND1 and promoted YAP phosphorylation. Mechanistic studies showed that CCDC25 interacts with YAP and influences YAP phosphorylation through LATS1. In vivo, CCDC25 overexpression inhibited tumor growth and promoted apoptosis.ConclusionCCDC25 acts as a potential tumor suppressor in ccRCC by inhibiting cell proliferation and migration, potentially through regulating the Hippo signaling pathway. These findings highlight the potential of CCDC25 as a therapeutic target in ccRCC treatment.Graphical

Glucosamine conjugated gold nanoparticles modulate protein aggregation induced autophagic neuronal cell death via regulation of intracellular Parkin homeostasis

Recent studies have suggested that ∼ 25% of the brain glycogen is composed of glucosamine (Gln), though its role in maintaining the balance in nervous system is yet to be fully understood. Here, we have reported the synthesis and protective effect of glucosamine conjugated gold nanoparticles (Gln@CA-AuNP) on the oligomeric and fibrillar fraction of hen egg white lysozyme (HEWL). The synthesized Gln@CA-AuNP (∼ 30.1±3.7 nm) was characterized by different spectroscopy and microscopy techniques. The corresponding biophysical studies suggested that Gln@CA-AuNP could effectively inhibit the HEWLO formation and simultaneously, could restrict the nucleation step of protein aggregation. Studies carried out with human neuroblastoma cells (SH-SY5Y) suggested that Gln@CA-AuNP alleviated protein aggregation induced cell death via inhibition of oligomer formation (∼ 2.4 times), reduction of intracellular oxidative stress (∼ 3.6 times) by uplifting the mitochondrial health. The intracellular delivery of Gln was found to modulate cytosolic Parkin oxidation and restrict autophagic neuronal cell death as confirmed by western blot studies. Further, Gln@CA-AuNP has been found to enhance sulfated glycosaminoglycans (sGAGs) production which suggested its role in protecting the extracellular matrix (ECM) during protein aggregation induced toxicity. Also, neuronal synapse of the in vivo Caenorhabditis elegans model could be preserved with the pre-treatment of Gln@CA-AuNP. Overall, we predict that this approach of neuroprotection via simultaneous targeting of early and late aggregates may serve as a better therapeutic strategy for the treatment of neurodegenerative diseases.

Abstract 2105: Targeting the “Achilles Heel” of androgen receptor activity in castration resistant prostate cancer

Abstract Background: Each year, millions of men are diagnosed with prostate cancer (CaP). The unchecked activation of the Androgen Receptor (AR) spurs CaP development and progression. Mutations or the presence of AR splice variants can add complexity to tumor ecology, leading to chemoresistance. This study aims to develop novel inhibitors targeting the “Achilles Heel” of AR activity: the N-terminal domain (NTD). This would circumvent the limitations of LBD (Ligand Binding Domain) therapies and offer a superior treatment option for patients. Though the AR-NTD is an intrinsically disordered protein, which complicates structure-based drug design, we have developed a unique small molecule inhibitor, ASR600, which specifically targets AR-NTD and promotes AR and AR- variants degradation by ubiquitination at previously unknown sites. Methods: Mass-spec analysis was performed to identify distinct ubiquitination sites in AR's NTD. To perform spatial transcriptome analysis, CytAssist Visium (10X genomics) was used. Furthermore, we used western blotting, immunofluorescence, immunoprecipitation, and PDX mice studies in CRPC mouse models to examine the impact of ASR600 on CRPC. Results: Four AR ubiquitination sites: K845, K847, and K913 (LBD) and K311 (NTD) are already identified. Interestingly, ASR600 continued to inhibit AR expression even with mutated ubiquitination sites. Mass spectrometry analysis of ASR-mediated mono-ubiquitinated N-terminal AR identified K16 as a unique ubiquitin acceptor. Altering this ubiquitination site rescues AR expression from ASR600-mediated degradation, emphasizing AR N-terminus as an ASR600 target. To discern the role of the K16 ubiquitination site in AR protein turnover, we evaluated AR protein stability in LNCaP cells that stably expressed ARWT or AR-K16R constructs. Our data indicates the pivotal importance of K16 for AR transcriptional activity. Invivo, ASR600, when used as a sole agent, exhibited inhibitory responses in PDX of clinically aggressive, AR-expressing tumors. Spatial gene expression analysis revealed marked expression differences in AR signature genes between control and ASR600-treated PDX tissues. Crucially, AR signaling, and related markers mainly aligned with epithelial markers rather than stromal ones. In contrast, the ASR600-treated PDX tumor showcased reduced AR signaling within epithelial cells. Conclusion: Targeting AR-NTD has received a lot of attention, but its innately disordered structure has hindered progress. ASR600 targets AR-NTD specifically by ubiquitinating the protein at a novel location, K16. Our in vivo, PDX studies, combined with the spatial analysis, lay a strong foundation for initiating IND-enabling TOX studies, followed by a phase-I clinical trial for ASR-600 in CRPC patients. Citation Format: Ashish Tyagi, Balaji Chandrasekaran, Arun K. Sharma, Chendil Damoadaran. Targeting the “Achilles Heel” of androgen receptor activity in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2105.

Abstract 1346: Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations

Abstract Extracellular vesicles (EVs) derived from immune cells (e.g., NK cells, dendritic cells) may be able to treat malignancies that are resistant to other treatments (e.g., chemotherapy) relying on the hypothesis that EVs carry almost the same cargo of the parent cells. In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib resistant lung cancer (H1975R) with EGFR mutations(L858R). NK-EVs were isolated by ultracentrifugation and nanoparticle tracking analysis revealed a size distribution of 89.5 ± 3.4 nm, zeta potential of -31.38 ± 0.25 mV. Atomic force microscopy imaging revealed vesicles with a spherical form and comparable sizes, meeting the criteria of exosomal EVs. Further, western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by using proteomic analysis which demonstrated that EVs had proteins for natural killer cell mediated cytotoxicity (Granzyme B) and T cell activation (Perforin and Plastin-2). Gene oncology (GO) analysis also showed that these are differentially expressed proteins (DEPs) that are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs (1*1011 particles/mL) were cytotoxic to H1975R cells in vitro with 55% and 68% cell viability in 2D and 3D cell cultures respectively against control. Carboplatin’s IC50 was reduced by approximately 1.9 and 1.7-fold (p less than 0.001) in 2D and 3D cell culture respectively when combined with NK-EVs. The EVs were then combined with carboplatin (25 mg/kg) and administered by i.p. route to H1975R tumor xenografts and significant reduction in tumor volume in vivo (≈1000 mm3 compared to ≈2000 mm3 of control group, p less than 0.001) was observed after 10 days. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint and induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB and TGF-ß. MicroRNAs regulating cytotoxic proteins like perforin, granzyme and plastin were also identified using sequencing and the miRNAs: hsa-miR-5193/149-5P/3133/193-5p are picked for further studies. These miRNAs have been encapsulated in NK-EVs by electroporation and will be evaluated against Lung PDX xenografts along with carboplatin loaded NK-EVs. These miRNAs are directly involved in T cell or NK cell mediated cytotoxicity and are being currently investigated. The ability to isolate functional NK-EVs on a large scale and using them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and carboplatin as a viable immunochemotherapeutic strategy for resistant cancers. Citation Format: Aakash Nathani, Li Sun, Islauddin Khan, Mounika Aare, Arvind Bagde, Yan Li, Mandip Sachdeva. Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1346.

Liraglutide Attenuates Diabetic Cardiomyopathy via the ILK/PI3K/AKT/PTEN Signaling Pathway in Rats with Streptozotocin-Induced Type 2 Diabetes Mellitus.

One of the possible candidates for the treatment of diabetic cardiomyopathy is liraglutide, a glucagon-like peptide-1 receptor (GLP1R) agonist. In this study, the impacts of liraglutide on the integrin-linked kinase (ILK)-related PI3K/AKT axis in rats with type 2 diabetes induced via streptozotocin were examined. Twenty-four Wistar albino rats were distributed in four different groups, and a high-fat diet and streptozotocin were used to induce type 2 in two groups. Rats in the untreated control groups were administered 0.9% NaCl solution over a 6-week period, and those in the treatment groups were administered 0.9% NaCl for 3 weeks, followed by subcutaneous injection of liraglutide (150 μg/kg) for an additional 3 weeks. In the liraglutide-treated diabetic group, the heart-to-body weight ratio was significantly reduced, levels of cardiac biomarkers, troponin I and creatine-kinase-MB, were improved; activities of antioxidant enzymes, glutathione peroxidase and superoxide dismutase, were increased; and levels of malondialdehyde were decreased. Western blotting and immunohistochemical studies revealed increased levels of ILK, P-PI3K, P-AKT, and BCL2, as well as those of caspase 3, BAX, and P-PTEN, indicating mitigation of cardiomyocyte apoptosis. Our results show that liraglutide, by targeting GLP1Rs, enhances the expression of proteins in the ILK/PI3K/AKT/PTEN pathway and thereby exerts its cardioprotective effects in rats with DCM.

Influenza A Virus PB1-F2 Induces Affective Disorder by Interfering Synaptic Plasticity in Hippocampal Dentate Gyrus.

Influenza A virus (IAV) infection, which leads to millions of new cases annually, affects many tissues and organs of the human body, including the central nervous system (CNS). The incidence of affective disorders has increased after the flu pandemic; however, the potential mechanism has not been elucidated. PB1-F2, a key virulence molecule of various influenza virus strains, has been shown to inhibit cell proliferation and induce host inflammation; however, its role in the CNS has not been studied. In this study, we constructed and injected PB1-F2 into the hippocampal dentate gyrus (DG), a region closely associated with newborn neurons and neural development, to evaluate its influence on negative affective behaviors and learning performance in mice. We observed anxiety- and depression-like behaviors, but not learning impairment, in mice injected with PB1-F2. Furthermore, pull-down and mass spectrometry analyses identified several potential PB1-F2 binding proteins, and enrichment analysis suggested that the most affected function was neural development. Morphological and western blot studies revealed that PB1-F2 inhibited cell proliferation and oligodendrocyte development, impaired myelin formation, and interfered with synaptic plasticity in DG. Taken together, our results demonstrated that PB1-F2 induces affective disorders by inhibiting oligodendrocyte development and regulating synaptic plasticity in the DG after IAV infection, which lays the foundation for developing future cures of affective disorders after IAV infection.

Synthesis, characterization, and anti-inflammatory activity of tetrahydropiperine, piperic acid, and tetrahydropiperic acid via down regulation of NF-κB pathway

The present study describes the synthesis, characterization, and evaluation of tetrahydropiperine (THP), piperic acid (PA), and tetrahydropiperic acid (THPA) as anti-inflammatory agents. THPA demonstrated potent anti-inflammatory activity among all the compounds. The anti-inflammatory potential was investigated in both in-vitro and in-vivo experimental models. Our findings demonstrated that THPA effectively suppressed the production of pro-inflammatory mediators, including nitric oxide and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in both in vitro and in vivo. Additionally, THPA attenuated the expression of i-NOS and COX-2 in RAW 264.7 macrophages. The oral administration of THPA significantly reduced carrageenan induced paw edema thickness and alleviated liver, lung, and kidney injury induced by LPS. THPA also reduced the infiltration of inflammatory cells, prevented the occurrence of significant lesions, and mitigated tissue damage. Moreover, THPA significantly improved the survival rate of mice challenged with LPS. Our western blot studies also found that LPS induced NF-κB activation was downregulated by treatment with THPA in an in vivo system. These results collectively illustrated the potential of THPA as a therapeutic agent for treating inflammatory diseases.

Polarization to M1-type microglia in the hippocampus is involved in depression-like behavior in a mouse model of olfactory dysfunction

Impaired olfactory function may be associated with the development of psychiatric disorders such as depression and anxiety; however, knowledge on the mechanisms underlying psychiatric disorders is incomplete. A reversible model of olfactory dysfunction, zinc sulfate (ZnSO4) nasal-treated mice, exhibit depression-like behavior accompanying olfactory dysfunction. Therefore, we investigated olfactory function and depression-like behaviors in ZnSO4-treated mice using the buried food finding test and tail suspension test, respectively; investigated the changes in the hippocampal microglial activity and neurogenesis in the dentate gyrus by immunohistochemistry; and evaluated the inflammation and microglial polarity related-proteins in the hippocampus using western blot study. On day 14 after treatment, ZnSO4-treated mice showed depression-like behavior in the tail suspension test and recovery of the olfactory function in the buried food finding test. In the hippocampus of ZnSO4-treated mice, expression levels of ionized calcium-binding adapter molecule 1 (Iba1), cluster of differentiation 40, inducible nitric oxide synthase, interleukin (IL)-1β, IL-6, tumor necrosis factor-α, cleaved caspase-3, as well as the number of Iba1-positive cells and cell body size increased, and arginase-1 expression and neurogenesis decreased. Except for the increased IL-6, these changes were prevented by a microglia activation inhibitor, minocycline. The findings suggest that neuroinflammation due to polarization of M1-type hippocampal microglia is involved in depression accompanied with olfactory dysfunction.

Targeting antibody-mediated complement-independent mechanism in bullous pemphigoid with diacerein

BackgroundBullous pemphigoid (BP) is an antibody-mediated blistering disease predominantly affecting the elderly. The pathogenesis involves both complement-dependent and complement-independent mechanisms. The therapeutic potential of targeting complement-independent mechanism has not yet been determined. The mainstay of treatment, corticosteroid, has many side effects, indicating the needs of better treatments. ObjectiveWe tempted to establish an in vitro model of BP which resembles complement-independent mechanism and to examine the therapeutic potential of a novel anti-inflammatory agent, diacerein. MethodsCultured HaCaT cells were treated with purified antibodies from BP patients, with or without diacerein to measure the cell interface presence of BP180, protein kinase C, and the production of proinflammatory cytokines. An open-label, randomized, phase 2 trial was conducted to compare topical diacerein and clobetasol ointments in patients with mild-to-moderate BP (NCT03286582). ResultsThe reduced presentation of BP180 at cell interface after treating with BP autoantibodies was noticed in immunofluorescence and western blotting studies. The phenomenon was restored by diacerein. Diacerein also reduced the autoantibody-induced increase of pro-inflammatory cytokines. Reciprocal changes of BP180 and protein kinase C at the cell interface were found after treating with BP autoantibodies. This phenomenon was also reversed by diacerein in a dose-dependent manner. The phase 2 trial showed that topical diacerein reduced the clinical symptoms which were comparable to those of topical clobetasol. ConclusionDiacerein inhibited BP autoantibody-induced reduction of BP180 and production of proinflammatory cytokines in vitro and showed therapeutic potential in patients with BP. It is a novel drug worthy of further investigations.

The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1

Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1.Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated.Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively.In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.

Nebulization of low-dose aspirin ameliorates Huntington's pathology in N171-82Q transgenic mice.

Huntington Disease (HD), a devastating hereditary neurodegenerative disorder, is caused by expanded CAG trinucleotide repeats in the huntingtin gene (Htt) on chromosome 4. Currently, there is no effective therapy for HD. Although aspirin, acetylsalicylic acid, is one of the most widely-used analgesics throughout the world, it has some side effects. Even at low doses, oral aspirin can cause gastrointestinal symptoms, such as heartburn, upset stomach, or pain. Therefore, to bypass the direct exposure of aspirin to stomach, here, we described a new mode of use of aspirin and demonstrated that nebulization of low-dose of aspirin (10 μg/mouse/d=0.4 mg/kg body wt/d roughly equivalent to 28 mg/adult human/d) alleviated HD pathology in N171-82Q transgenic mice. Our immunohistochemical and western blot studies showed that daily aspirin nebulization significantly reduced glial activation, inflammation and huntingtin pathology in striatum and cortex of N171-82Q mice. Aspirin nebulization also protected transgenic mice from brain volume shrinkage and improved general motor behaviors. Collectively, these results highlight that nebulization of low-dose aspirin may have therapeutic potential in the treatment of HD.

Salmonella-mediated methionine deprivation drives immune activation and enhances immune checkpoint blockade therapy in melanoma

BackgroundAlthough immune checkpoint inhibitor (ICI)-based therapy is advantageous for patients with advanced melanoma, resistance and relapse are frequent. Thus, it is crucial to identify effective drug combinations and develop new...

Abstract TP285: Chronic Remote Ischemic Conditioning (C-RIC) and Physical Exercise Attenuate NLRP3 Inflammasome Activation Induced by Chronic Cerebral Hypoperfusion in the BCAS Mouse Model of VCID

Background and Purpose: Inflammation plays a significant role in acute and chronic cerebral ischemia. The bilateral carotid artery stenosis (BCAS)-induced chronic cerebral hypoperfusion activates the NLRP inflammasome in brain tissue. We aimed to determine if C-RIC and physical exercise (EXR) attenuated the increase in the NLRP3 inflammasome and related cytokines in the BCAS model. Methods: Microcoil-induced BCAS was used for chronic hypoperfusion, a model for VCID. Middle-young (5 months old) and old-aged (14 months old) C57Bl/6 mice were randomly assigned to Sham, BCAS, and BCAS+EXR (four weeks treadmill exercise) for middle-young mice and to Sham, BCAS, and BCAS+RIC (four months C-RIC treatment) for old-aged mice of both sexes. qPCR was run for NLRP3, TNFa, IL-1B, IL-6, IL-10, ICAM1, VCAM1 in brain tissue.Biochemical assays and histopathological staining of NLRP3 were also assessed on the brain tissues. Results: Four weeks of exercise in middle-aged mice and 4 months of C-RIC in aged RIC groups attenuated the increased NLRP3, cytokines (IL-1B, IL-6, IL-10, TNFα), and inflammatory markers (ICAM-1 & VCAM-1) induced by BCAS in middle-aged (exercise) and aged mice (CRIC). Western blot and immunohistopathological studies also attenuated the increased NLRP3 expression after physical exercise and C-RIC treatment. Conclusions: C-RIC and Physical exercise attenuated the increase of NLRP3 inflammasome and cytokines (IL-1B, IL-6, IL-10, TNFα) induced by chronic cerebral hypoperfusion in the BCAS mouse model of VCID. This may be an important mechanism of action of physical exercise and C-RIC in VCID.

Proteomic analysis identifies HSP90AA1, PTK2B, and ANXA2 in the human entorhinal cortex in Alzheimer's disease: Potential role in synaptic homeostasis and Aβ pathology through microglial and astroglial cells.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder worldwide, is clinically characterized by cognitive deficits. Neuropathologically, AD brains accumulate deposits of amyloid-β (Aβ) and tau proteins. Furthermore, these misfolded proteins can propagate from cell to cell in a prion-like manner and induce native proteins to become pathological. The entorhinal cortex (EC) is among the earliest areas affected by tau accumulation along with volume reduction and neurodegeneration. Neuron-glia interactions have recently come into focus; however, the role of microglia and astroglia in the pathogenesis of AD remains unclear. Proteomic approaches allow the determination of changes in the proteome to better understand the pathology underlying AD. Bioinformatic analysis of proteomic data was performed to compare ECs from AD and non-AD human brain tissue. To validate the proteomic results, western blot, immunofluorescence, and confocal studies were carried out. The findings revealed that the most disturbed signaling pathway was synaptogenesis. Because of their involvement in synapse function, relationship with Aβ and tau proteins and interactions in the pathway analysis, three proteins were selected for in-depth study: HSP90AA1, PTK2B, and ANXA2. All these proteins showed colocalization with neurons and/or astroglia and microglia and with pathological Aβ and tau proteins. In particular, ANXA2, which is overexpressed in AD, colocalized with amoeboid microglial cells and Aβ plaques surrounded by astrocytes. Taken together, the evidence suggests that unbalanced expression of HSP90AA1, PTK2B, and ANXA2 may play a significant role in synaptic homeostasis and Aβ pathology through microglial and astroglial cells in the human EC in AD.

Design and Synthesis of Cytotoxic Water-Soluble Rocaglaol Derivatives against HEL Cells by Inhibiting Fli-1.

Rocaglaol, embedding a cyclopenta[b]benzofuran scaffold, was isolated mainly from the plants of Aglaia and exhibited nanomolar level antitumor activity. However, the drug-like properties of these compounds are poor. To improve the physicochemical properties of rocaglaol, 36 nitrogen-containing phenyl-substituted rocaglaol derivatives were designed and synthesized. These derivatives were tested for the inhibitory effects on three tumor cell lines, HEL, MDA-231, and SW480, using the MTT assay. Among them, 22 derivatives exhibited good cytotoxic activities with IC50 values between 0.11 ± 0.07 and 0.88 ± 0.02 μM. Fourteen derivatives exhibited stronger cytotoxicity than the positive control, adriamycin. In particular, a water-soluble derivative revealed selective cytotoxic effects on HEL cells (IC50 = 0.19 ± 0.01 μM). This compound could induce G1 cell cycle arrest and apoptosis in HEL cells. Western blot assays suggested that the water-soluble derivative could downregulate the expression of the marker proteins of apoptosis, PARP, caspase-3, and caspase-9, thus inducing apoptosis. Further CETSA and Western blot studies implied that this water-soluble derivative might be an inhibitor of friend leukemia integration 1 (Fli-1). This water-soluble derivative may serve as a potential antileukemia agent by suppressing the expression of Fli-1.

Fangji Huangqi decoction ameliorates membranous nephropathy through the upregulation of BNIP3-mediated mitophagy

Ethnopharmacological relevanceFangji Huangqi Decoction (FJHQ), a traditional Chinese medicinal formula outlined in Zhang Zhongjing's “Jin Gui Yao Lue” during the Han Dynasty, is often used to treat conditions characterized by symptoms like edema and dysuria, including membranous nephropathy (MN). Despite its proven clinical effectiveness, the exact mechanisms through which FJHQ acts on MN remain elusive. Aim of the studyThis study aimed to investigate whether FJHQ enhances BNIP3-mediated mitophagy in podocytes by promoting BNIP3 expression and whether this improvement leads to the amelioration of MN. Materials and methodsIn this study, by establishing passive Heymann nephritis (PHN) rats, an experimental rat model of MN induced by sheep anti-rat Fx1A serum, we evaluated the effects of FJHQ in vivo. In vitro experiments were carried out by treating primary podocytes with experimental rat serum. Furthermore, the potential mechanism by which FJHQ acts through BNIP3 was further examined by transfecting primary podocytes with the siRNA of BNIP3 or the corresponding control vector. ResultsAfter 4 weeks, significant kidney damage was observed in the rats in the model group, comparatively, FJHQ markedly decreased urine volume, 24-h urinary protein, blood urea nitrogen (BUN), creatinine (Scr), and increased serum total albumin (ALB). Histology showed that FJHQ caused significant improvements in glomerular hyperplasia, and IgG immune complex deposition in MN rats. JC-1 fluorescence labelling and flow cytometry analysis showed that FJHQ could significantly increase mitochondrial membrane potential in vivo. In the mitochondria of MN model rats, FJHQ was able to down-regulate the expression of P62 and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I, according to Western blot and immunofluorescence studies. Furthermore, FJHQ has been shown to significantly up-regulate mitochondrial membrane potential, down-regulate P62 expression in mitochondria, and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I in mitochondria at the cellular level. After the administration of the autophagy inhibitor chloroquine, the serum of rats treated with FJHQ further increased the expression of LC3 II/LC3 I in primary podocytes, showing higher autophagy flow. After the interference of BNIP3 in podocytes, the effect of FJHQ on mitochondrial membrane potential and autophagy-related proteins almost disappeared. ConclusionFJHQ enhanced mitophagy in podocytes by promoting the expression of BNIP3, thereby contributing to the amelioration of MN. This work reveals the possible underlying mechanism by which FJHQ improves MN and provides a new avenue for MN treatment.