Increased Protein Expression Research Articles

Exploration of beauvericin's toxic effects and mechanisms in human astrocytes and N-acetylcysteine's protective role

Beauvericin (BEA) is a newly identified mycotoxin produced by various Fusarium species, and its contamination in food and animal feed is widespread globally. This mycotoxin demonstrates cytotoxic effects by inducing oxidative stress in multiple models. Furthermore, evidence indicates that BEA possesses diverse toxic activities, making it a promising candidate for toxicological research. Recent studies have highlighted the ability of BEA to traverse the blood-brain barrier, suggesting its potential neurotoxicity. However, limited information is available regarding the neurotoxic effects of BEA on human astrocytes. Therefore, this study aimed to assess the neurotoxic effects of BEA on the Gibco® Human Astrocyte (GHA) cell line and elucidate the underlying mechanisms. Additionally, the study aimed to investigate the protective effects of the antioxidant N-acetylcysteine (NAC) against BEA-induced toxicity. The data show that exposure to BEA within the 2.5–15 μM concentration range resulted in concentration-dependent cytotoxicity. BEA-treated cells exhibited significantly increased levels of reactive oxygen species (ROS), while intracellular glutathione (GSH) content was significantly reduced. Western blot analysis of cells treated with BEA revealed altered protein levels of Bax, cleaved caspase-9, and caspase-3, along with an increased Bax/Bcl-2 ratio, indicating the induction of apoptosis. Additionally, BEA exposure triggered antioxidant responses, as evidenced by increased protein expression of Nrf2, HO-1, and NQO1. Significantly, pretreatment with NAC partially attenuated the significant toxic effects of BEA. In conclusion, our findings suggest that BEA-induced cytotoxicity in GHA cells involves oxidative stress-associated apoptosis. Furthermore, NAC demonstrates potential as a protective agent against BEA-induced oxidative damage.

Lapatinib combined with doxorubicin causes dose-dependent cardiotoxicity partially through activating the p38MAPK signaling pathway in zebrafish embryos

Because of its enhanced antitumor efficacy, lapatinib (LAP) is commonly used clinically in combination with the anthracycline drug doxorubicin (DOX) to treat metastatic breast cancer. While it is well recognized that this combination chemotherapy can lead to an increased risk of cardiotoxicity in adult women, its potential cardiotoxicity in the fetus during pregnancy remains understudied. Here, we aimed to examine the combination of LAP chemotherapy and DOX-induced cardiotoxicity in the fetus using a zebrafish embryonic system and investigate the underlying pathologic mechanisms. First, we examined the dose-dependent cardiotoxicity of combined LAP and DOX exposure in zebrafish embryos, which mostly manifested as pericardial edema, bradycardia, cardiac function decline and reduced survival. Second, we revealed that a significant increase in oxidative stress concurrent with activated MAPK signaling, as indicated by increased protein expression of phosphorylated p38 and Jnk, was a notable pathophysiological event after combined LAP and DOX exposure. Third, we showed that inhibiting MAPK signaling by pharmacological treatment with the p38MAPK inhibitor SB203580 or genetic ablation of the map2k6 gene could significantly alleviate combined LAP and DOX exposure-induced cardiotoxicity. Thus, we provided both pharmacologic and genetic evidence to suggest that inhibiting MAPK signaling could exert cardioprotective effects. These findings have implications for understanding the potential cardiotoxicity induced by LAP and DOX combinational chemotherapy in the fetus during pregnancy, which could be leveraged for the development of new therapeutic strategies.

Effects of electroacupuncture with "intestinal disease prescription" on NLRP3 inflammasome and intestinal mucosal barrier in rats with acute ulcerative colitis.

To observe the effects of electroacupuncture (EA) with "intestinal disease prescription" on the intestinal mucosal barrier and NLRP3 inflammasome in rats with dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC), and explore the underlying mechanism of EA with "intestinal disease prescription" for the treatment of UC. Thirty-two healthy male SPF-grade SD rats were randomly divided into a blank group, a model group, a medication group, and an EA group, with 8 rats in each group. Except for the blank group, the UC model was established by administering 5% DSS solution for 7 days. After modeling, the rats in the medication group were treated with mesalazine suspension (200 mg/kg) by gavage, while the rats in the EA group were treated with acupuncture at bilateral "Tianshu" (ST 25), "Shangjuxu" (ST 37) and "Zhongwan" (CV 12), with the ipsilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37) connected to the electrodes of the EA instrument, using disperse-dense wave, with a frequency of 10 Hz/50 Hz, and each intervention lasted for 20 minutes. Both interventions were performed once daily for 3 days. The general conditions of rats were observed daily. After intervention, the disease activity index (DAI) score was calculated; colon tissue morphology was observed using HE staining; serum levels of pro-inflammatory cytokines (interleukin [IL]-18, IL-1β) were measured by ELISA; protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in colon tissues was detected by Western blot; positive expression of zonula occludens-1 (ZO-1) and Occludin in colon tissues was examined by immunofluorescence. Compared with the blank group, the rats in the model group exhibited poor general conditions, slow body weight gain, shortened colon length (P<0.01), increased DAI score and spleen index (P<0.01), elevated serum IL-18 and IL-1β levels, and increased protein expression of NLRP3, ASC, and Caspase-1 in colon tissues (P<0.01), along with decreased positive expression of ZO-1 and Occludin in colon tissues (P<0.01). Compared with the model group, the rats in the medication group and the EA group exhibited improved general conditions, accelerated body weight gain, increased colon length (P<0.05), reduced DAI scores and spleen indexes (P<0.05), decreased serum IL-18 and IL-1β levels, and lower protein expression of NLRP3, ASC and Caspase-1 in colon tissues (P<0.05), as well as increased positive expression of ZO-1 and Occludin in colon tissues (P<0.05). There were no significant differences in the above indexes between the medication group and the EA group (P>0.05). Compared with the blank group, the rats in the model group exhibited disrupted colon mucosal morphology, disordered gland arrangement, and atrophy of crypts, along with significant inflammatory cell infiltration. Compared with the model group, the rats in both the medication group and the EA group showed relatively intact colon mucosal morphology, with restored and improved gland and crypt structures, and reduced inflammatory cell infiltration. EA with "intestinal disease prescription" has a significant therapeutic effect on DSS-induced UC, possibly by regulating the expression of NLRP3 inflammasome and proteins related to the intestinal mucosal barrier, thereby alleviating symptoms of ulcerative colitis.

Single cell lineage tracing reveals clonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer

BackgroundMost primary Triple Negative Breast Cancers (TNBCs) show amplification of the Epidermal Growth Factor Receptor (EGFR) gene, leading to increased protein expression. However, unlike other EGFR-driven cancers, targeting this receptor in TNBC yields inconsistent therapeutic responses.MethodsTo elucidate the underlying mechanisms of this variability, we employ cellular barcoding and single-cell transcriptomics to reconstruct the subclonal dynamics of EGFR-amplified TNBC cells in response to afatinib, a tyrosine kinase inhibitor (TKI) that irreversibly inhibits EGFR.ResultsIntegrated lineage tracing analysis revealed a rare pre-existing subpopulation of cells with distinct biological signature, including elevated expression levels of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2). We show that IGFBP2 overexpression is sufficient to render TNBC cells tolerant to afatinib treatment by activating the compensatory insulin-like growth factor I receptor (IGF1-R) signalling pathway. Finally, based on reconstructed mechanisms of resistance, we employ deep learning techniques to predict the afatinib sensitivity of TNBC cells. ConclusionsOur strategy proved effective in reconstructing the complex signalling network driving EGFR-targeted therapy resistance, offering new insights for the development of individualized treatment strategies in TNBC.

Co-exposure of arsenic and polystyrene-nanoplastics induced kidney injury by disrupting mitochondrial homeostasis and mtROS-mediated ferritinophagy and ferroptosis

Arsenic (As) and polystyrene nanoplastics (PSNPs) co-exposure induced biotoxicity and ecological risks have attracted wide attention. However, the combined effects of As and PSNPs on the kidney and their underlying mechanisms of toxicities remain to be explored. Here, we investigated the effects of As and PSNPs co-exposure on structure and function in mice kidney, and further explored the possible mechanisms. In this study, we identified that co-exposure to As and PSNPs exhibited conspicuous renal structural damage and pathological changes, accompanied by renal tissue fibrosis (increased protein expression of Collagen I and α-SMA and deposition of collagen fibers), whereas alone exposure to As or PSNPs does not exhibit nephrotoxicity. Subsequently, our results further showed that combined action of As and PSNPs induced mitochondrial oxidative damage and impaired mitochondrial dynamic balance. Furthermore, co-treatment with As and PSNPs activated NCOA4-mediated ferritinophagy and ferroptosis in mice kidney and TCMK-1 cells, which was confirmed by the changes in the expression of ferritinophagy and ferroptosis related indicators (NCOA4, LC3, ATG5, ATG7, FTH1, FTL, GPX4, SLC7A11, FSP1, ACSL4 and PTGS2). Meaningfully, pretreatment with the mtROS-targeted scavenger Mito-TEMPO significantly attenuated As and PSNPs co-exposure induced mitochondrial damage, ferritinophagy and ferroptosis. In conclusion, these findings demonstrated that mtROS-dependent ferritinophagy and ferroptosis are important factors in As and PSNPs co-exposure induced kidney injury and fibrosis. This study provides a new insight into the study of combined toxicity of nanoplastics and heavy metal pollutants.

Acupressure bladder meridian alleviates anxiety disorder in rats by regulating MAPK and BDNF signal pathway

The aim of this study was to investigate the effects of acupressure bladder meridian (ABM) on anxiety in rats. Chronic stress was induced rats to establish rat anxiety model. Shuttle experiment and open field experiments of were used to measure behaviors. The levels of cytokines in serum and hippocampus of rats were detected. Brain neurotransmitters (dopamine, 5- hydroxy tryptamine, norepinephrine) were detected by Enzyme linked immunosorbent assay (ELISA) kits. Immunohistochemistry and western blotting were used to detect MAPK and BDNF signal pathway in hippocampus of rats. ABM significantly improve behaviors, decreased cytokine levels in serum and hippocampus. ABM restored the changes of neurotransmitters and significantly decreased protein expressions of MAPK signal pathway and increased protein expressions of BDNF signal pathway in hippocampus of rats. The results shown that ABM significantly improved anxiety via inhibition of MAPK signal pathway and increased BDNF signal pathway.

RNF213 variant and autophagic impairment: A pivotal link to endothelial dysfunction in moyamoya disease

Moyamoya disease (MMD) is closely associated with the Ring Finger Protein 213 (RNF213), a susceptibility gene for MMD. However, its biological function remains unclear. We aimed to elucidate the role of RNF213 in the damage incurred by human endothelial cells under oxygen-glucose deprivation (OGD). We analyzed autophagy in peripheral blood mononuclear cells (PBMCs) derived from patients carrying either RNF213 wildtype (WT) or variant (p.R4810K). Subsequently, human umbilical vein endothelial cells (HUVECs) were transfected with RNF213 WT (HUVECWT) or p.R4810K (HUVECR4810K) and exposed to OGD for 2 h. Immunoblotting was used to analyze autophagy marker proteins, and endothelial function was analyzed by tube formation assay. Autophagic vesicles were observed using transmission electron microscopy. Post-OGD exposure, we administered rapamycin and cilostazol as potential autophagy inducers. The RNF213 variant group during post-OGD exposure (vs. pre-OGD) showed autophagy inhibition, increased protein expression of SQSTM1/p62 (p < 0.0001) and LC3-II (p = 0.0039), and impaired endothelial function (p = 0.0252). HUVECR4810K during post-OGD exposure (versus pre-OGD) showed a remarkable increase in autophagic vesicles. Administration of rapamycin and cilostazol notably restored the function of HUVECR4810K and autophagy. Our findings support the pivotal role of autophagy impaired by the RNF213 variant in MMD-induced endothelial cell dysfunction.

Compound 225# inhibits the proliferation of human colorectal cancer cells by promoting cell cycle arrest and apoptosis induction.

Colorectal cancer (CRC) ranks as the second leading cause of cancer‑related death worldwide due to its aggressive nature. After surgical resection, >50% of patients with CRC require adjuvant therapy. As a result, eradicating cancer cells with medications is a promising method to treat patients with CRC. In the present study, a novel compound was synthesized, which was termed compound 225#. The inhibitory activity of compound 225# against CRC was determined by MTT assay, EdU fluorescence labeling and colony formation assay; the effects of compound 225# on the cell cycle progression and apoptosis of CRC cells were detected by flow cytometry and western blotting; and the changes in autophagic flux after the administration of compound 225# were detected using the double fluorescence fusion protein mCherry‑GFP‑LC3B and western blotting. The results demonstrated that compound 225# exhibited antiproliferative properties, inhibiting the proliferation and expansion of CRC cell lines in a time‑ and dose‑dependent manner. Furthermore, compound 225# triggered G2/M cell cycle arrest by influencing the expression of cell cycle regulators, such as CDK1, cyclin A1 and cyclin B1, which is also closely related to the activation of DNA damage pathways. The cleavage of PARP and increased protein expression levels of PUMA suggested that apoptosis was triggered after treatment with compound 225#. Moreover, the increase in LC3‑II expression and stimulation of autophagic flux indicated the activation of an autophagy pathway. Notably, compound 225# induced autophagy, which was associated with endoplasmic reticulum (ER) stress. In accordance with the in vitro findings, the in vivo results demonstrated that compound 225# effectively inhibited the growth of HCT116 tumors in mice without causing any changes in their body weight. Collectively, the present results demonstrated that compound 225# not only inhibited proliferation and promoted G2/M‑phase cell cycle arrest and apoptosis, but also initiated cytoprotective autophagy in CRC cells by activating ER stress pathways. Taken together, these findings provide an experimental basis for the evaluation of compound 225# as a novel potential medication for CRC treatment.

후코이단/이고들빼기 혼합물에 의한 HepG2 간암세포의 apoptosis 유도

In the present study, we investigated whether a mixture of fucoidan and Crepidiastrum denticulatum extract (FCE) had the potential to improve the therapeutic efficacy of cancer treatment. The results demonstrated that FCE significantly reduced cell viability and induced the release of LDH (lactate dehydrogenase) and DNA fragmentation in HepG2 cells in a dose-dependent manner. In addition, FCE treatment also increased the protein expression level of p53, the release of cytochrome c, and the loss of mitochondrial membrane potential. Moreover, FCE dose-dependently increased protein expression levels of Bax, and cleaved caspase-3 and -9. However, FCE decreased the protein expression level of Bcl-2. These results suggest that FCE inhibits cell proliferation and induces apoptosis via the mitochondrial-mediated intrinsic pathway. The present study demonstrates that FCE can be used as an anti-cancer agent for liver cancer based on apoptosis mechanism.

The MET Oncogene: Thirty Years of Insights into Molecular Mechanisms Driving Malignancy.

The discovery and subsequent research on the MET oncogene's role in cancer onset and progression have illuminated crucial insights into the molecular mechanisms driving malignancy. The identification of MET as the hepatocyte growth factor (HGF) receptor has paved the path for characterizing the MET tyrosine kinase activation mechanism and its downstream signaling cascade. Over the past thirty years, research has established the importance of HGF/MET signaling in normal cellular processes, such as cell dissociation, migration, proliferation, and cell survival. Notably, genetic alterations that lead to the continuous activation of MET, known as constitutive activation, have been identified as oncogenic drivers in various cancers. The genetic lesions affecting MET, such as exon skipping, gene amplification, and gene rearrangements, provide valuable targets for therapeutic intervention. Moreover, the implications of MET as a resistance mechanism to targeted therapies emphasize the need for combination treatments that include MET inhibitors. The intriguing "flare effect" phenomenon, wherein MET inhibition can lead to post-treatment increases in cancer cell proliferation, underscores the dynamic nature of cancer therapeutics. In human tumors, increased protein expression often occurs without gene amplification. Various mechanisms may cause an overexpression: transcriptional upregulation induced by other oncogenes; environmental factors (such as hypoxia or radiation); or substances produced by the reactive stroma, such as inflammatory cytokines, pro-angiogenic factors, and even HGF itself. In conclusion, the journey to understanding MET's involvement in cancer onset and progression over the past three decades has not only deepened our knowledge, but has also paved the way for innovative therapeutic strategies. Selective pharmacological inactivation of MET stands as a promising avenue for achieving cancer remission, particularly in cases where MET alterations are the primary drivers of malignancy.

GSDMD induces hepatocyte pyroptosis to trigger alcoholic hepatitis through modulating mitochondrial dysfunction

BackgroundMechanisms and consequences of Gasdermin D (GSDMD) activation in alcoholic hepatitis (AH) are unclear. In the present study, we investigated whether GSDMD induces hepatocyte pyroptosis by regulating mitochondrial dysfunction in AH.ResultsLiver damage in AH mice was assessed by HE staining, serum levels of AST, ALT, TC, and TG. The levels of IL-1β, IL-18, LDH, inflammasome-associated proteins and hepatocyte death were assessed to determine pyroptosis. Mitochondrial dysfunction was assessed through various parameters including mitochondrial DNA (mtDNA) levels, ROS generation, mitochondrial membrane potential, ATP contents, levels of mitochondrial function-related proteins and morphological changes of mitochondria. AH induced gasdermin D (GSDMD) activation, leading to increased protein expression of N-terminal GSDMD (GSDMD-N), NLRP3, and Caspase 11 in liver tissues. Downregulation of GSDMD alleviated alcohol-induced hepatocyte pyroptosis. Alcohol also causes mitochondrial dysfunction in hepatocytes in AH, which was improved by inhibiting GSDMD. Furthermore, enhancing mitochondrial function suppressed alcohol-induced hepatocyte pyroptosis. Further, knockdown of GSDMD or dynamin-related protein 1 (Drp1) improved AH-induced liver injury, accompanied by a decrease in hepatocyte pyroptosis.ConclusionGSDMD induces hepatocyte pyroptosis by modulating mitochondrial dysfunction during AH-induced inflammation and liver injury. These findings may pave the way to develop new therapeutic treatments for AH.

ATP releasing channels and the ameliorative effects of high intensity interval training on diabetic heart: a multifaceted analysis

Type 2 diabetes (T2D) can cause severe cardiac complications at functional, histologic and molecular levels. These pathological complications could be mediated by ATP-releasing channels such as Panx1 and ATP receptors, in particular P2X7. The aim of our study was to investigate the effect of high-intensity interval training (HIIT) on T2D-induced cardiac complications at the functional, histopathological and molecular levels, with a particular focus on ATP-releasing channels. 48 male Wistar rats at the age of 8 weeks were randomly allocated into four groups: control (Con), Diabetes (T2D), Training (TR), and Diabetes + Training (T2D + TR). T2D was induced by a high-fat diet plus a low dose (35 mg/kg) of STZ administration. Rats in the TR and T2D + TR groups underwent an 8-weeks training program involving intervals ranging from 80 to 100% of their maximum running speed (Vmax), with 4–10 intervals per session. Protein expression of Interleukin 1β (IL1β), Interleukin 10 (IL-10), Pannexin 1 (Panx1), P2X7R (purinergic P2X receptor 7), NLRP1 (NLR Family Pyrin Domain Containing 1), BAX, and Bcl2 were measured in the heart tissue. Additionally, we assessed heart function, histopathological changes, as well as insulin resistance using the homeostasis model assessment of insulin resistance (HOMA-IR). In contrast to the T2D group, HIIT led to increased protein expression of Bcl2 and IL-10 in the heart. It also resulted in improvements in systolic and diastolic blood pressures, heart rate, ± dp/dt (maximum and minimum changes in left ventricular pressure), while reducing protein expression of IL-1β, Panx1, P2X7R, NLRP1, and BAX levels in the heart. Furthermore, left ventricular diastolic pressure (LVDP) was reduced (P ≤ 0.05). Moreover, heart lesion scores increased with T2D but decreased with HIIT, along with a reduction in fibrosis percentage (P ≤ 0.05). The results of this study suggest that the cardioprotective effects of HIIT on the diabetic heart may be mediated by the modulation of ATP-releasing channels. This modulation may lead to a reduction in inflammation and apoptosis, improve cardiac function, and attenuate cardiac injury and fibrosis.

Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines.

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 μM and 19.92±1 μM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.

Abstract 5093: Analysis of protein expression in head and neck cancers as therapeutic targets for antibody drug conjugates

Abstract Introduction: Antibody Drug Conjugates (ADCs) represent a new category of therapeutic molecular targeting for solid tumors. To date 6 ADCs have been approved in solid tumors against 5 targets (HER2/ERBB2, Folate alpha, Tissue Factor, Nectin-4, and Trop-2). Thus far, no ADCs have been approved for head and neck cancers. Methods: We queried protein expression data from a publically available consortium of datasets (TNMPlot.com) containing protein expression data from over 56,000 tumor and normal samples from GEO, GTex, TCGA, and TARGET databases. Genes examined for protein targets, included those with ADC targets of both presently approved and investigational drugs from clinical trials.The level of overexpression was determined as the percent of tumors over the 75th percentile of expression in normal tissue. Results: Protein expression data were available for 99 HNSCC samples. Among targets with ADCs available, increased protein expression was seen in HNSCC for 0% of ERBB2 (n=0/99), 38% of FOLR1 (n=38/99), 0% of TACSTD2 (n=0/99), and 42% of F2 (n=42/99) genes.Among gene targets with investigational ADCs, increased protein expression was seen in HNSCC for 35% of of AXL (n=35/99), 13% of B7H3/CD27 (n=12/99), 50% of EGFR (n=50/99), 63% of VTCN1 (n=63/99), 10% of MSLN (n=10/99), 77% of CDH3 (n=76/99), and 39% of PDGFRA (n=39/99). Conclusion: Increased protein expression for approved ADC targets were infrequent in HNSCC. However, increased protein expression is seen for several ADCs under clinical investigation supporting the potential therapeutic approaches for investigational ADC targets. Citation Format: Justin Tyler Moyers, Seth L. Smith, Miranda M. Mora, Roberto Carmagnani Pestana. Analysis of protein expression in head and neck cancers as therapeutic targets for antibody drug conjugates [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 5093.

Abstract 5866: TESC mediates resistance to lenvatinib in hepatocellular carcinoma by regulating cell autophagy

Abstract Objective: To investigate the mechanism by which TESC (Tescalcin) induces lenvatinib resistance in hepatocellular carcinoma (HCC) through autophagy mediation. Methods:1) Utilized established liver cancer organoids from over 70 patients for lenvatinib drug sensitivity screening and transcriptome sequencing to identify lenvatinib-resistant genes. 2) Constructed stable TESC overexpression and downregulation liver cancer cell lines. Employed CCK8 assays to plot IC50 drug sensitivity curves, elucidating changes in lenvatinib sensitivity in liver cancer cells after TESC overexpression or downregulation. 3) Used TESC-stable cells to establish subcutaneous tumor models and intrahepatic tumor models in nude mice. After tumor formation, administered lenvatinib orally to observe whether TESC affects liver cancer sensitivity to lenvatinib in vivo. 4) Conducted Western blot (WB) analysis to assess the expression of autophagy-related molecules (LC3B II/I, P62, and Beclin1 proteins) in TESC downregulated or overexpressed stable cell lines and their control cell lines. Utilized the LC3-GFP-mRFP fluorescence dual-labeling lentivirus system and laser confocal microscopy to examine autophagic flux. 5)Treated TESC downregulated cells with the autophagy inhibitor 3-MA. Conducted WB to assess the expression levels of autophagy-related proteins and plotted drug sensitivity curves using IC50 experiments to evaluate the impact of 3-MA on reversing liver cancer cell sensitivity to lenvatinib. Results: Compared to the control group, HCC cells overexpressing TESC showed an increased IC50 value and reduced sensitivity to lenvatinib, while TESC downregulation resulted in a decreased IC50 value and decreased sensitivity to lenvatinib. Subcutaneous tumor experiments in nude mice indicated that, compared to the control group, tumors with reduced TESC expression were more sensitive to lenvatinib. Additionally, in HCC cells overexpressing TESC, protein expression levels of LC3B II/I and Beclin1 significantly decreased, while P62 expression increased. Conversely, TESC downregulation significantly increased protein expression levels of LC3B II/I and Beclin1, while P62 expression decreased. Furthermore, results from the LC3-GFP-mRFP autophagy dual-labeling lentivirus system showed a significant reduction in fluorescence spots in the TESC overexpression group compared to the control group, indicating weakened autophagic flux. Treatment of TESC downregulated liver cancer cells with 3-MA resulted in an increased IC50 value compared to the control group. Conclusion: TESC expression levels are associated with the sensitivity of liver cancer cells to lenvatinib, possibly by inhibiting autophagy and affecting the sensitivity of liver cancer cells to lenvatinib. Citation Format: Hao Zhuang, Tingting Zhang, Jiaqi Chen. TESC mediates resistance to lenvatinib in hepatocellular carcinoma by regulating cell autophagy [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 5866.

Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors.

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70g/L vs 2.56g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6g/L, accompanied by an improved acidic variants attribute of 20.6%-22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications.

Sex-specific changes in protein expression of membrane transporters in the brain cortex of 5xFAD mouse model of Alzheimer's disease.

Membrane transporters playing an important role in the passage of drugs, metabolites and nutrients across the membranes of the brain cells have been shown to be involved in pathogenesis of Alzheimer's disease (AD). However, little is known about sex-specific changes in transporter protein expression at the brain in AD. Here, we investigated sex-specific alterations in protein expression of three ATP-binding cassette (ABC) and five solute carriers (SLC) transporters in the prefrontal cortex of a commonly used model of familial AD (FAD), 5xFAD mice. Sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic analysis was applied for absolute quantification of transporter protein expression. We compared the changes in transporter protein expressions in 7-month-old male and female 5xFAD mice versus sex-matched wild-type mice. The study revealed a significant sex-specific increase in protein expression of ABCC1 (p = 0.007) only in male 5xFAD mice as compared to sex-matched wild-type animals. In addition, the increased protein expression of glucose transporter 1 (p = 0.01), 4F2 cell-surface antigen heavy chain (p = 0.01) and long-chain fatty acid transport protein 1 (p = 0.02) were found only in female 5xFAD mice as compared to sex-matched wild-type animals. Finally, protein expression of alanine/serine/cysteine/threonine transporter 1 was upregulated in both male (p = 0.02) and female (p = 0.002) 5xFAD mice. The study provides important information about sex-specific changes in brain cortical transporter expression in 5xFAD mice, which will facilitate drug development of therapeutic strategies for AD targeting these transporters and drug delivery research.

Identification of novel homozygous asthenoteratospermia-causing ARMC2 mutations associated with multiple morphological abnormalities of the sperm flagella.

To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.

Silver nanoparticles induced synaptic degeneration via Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22 cells

Silver nanoparticles (AgNPs) have been widely used in biomedicine and cosmetics, increasing their potential risks in neurotoxicity. But the involved molecular mechanism remains unclear. This study aims to explore molecular events related to AgNPs-induced neuronal damage by RNA-seq, and elucidate the role of Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22cells synaptic degeneration induced by AgNPs. This study found that cell viabilities were decreased by AgNPs in a dose/time-dependent manner. AgNPs also increased protein expression of PINK1, Parkin, synaptophysin, and inhibited PGC-1α, MAP2 and APP protein expression, indicating AgNPs-induced synaptic degeneration involved in disturbance of mitophagy and mitochondrial biogenesis in HT22cells. Moreover, inhibition of AgNPs-induced Ca2+/CaMKII activation and Drp1/ROS rescued mitophagy disturbance and synaptic degeneration in HT22cells by reserving aforementioned protein express changes except for PGC-1α and APP protein. Thus, AgNPs-induced synaptic degeneration was mediated by Ca2+/CaMKII signal and Drp1-dependent mitochondrial disorder in HT22cells, and mitophagy is the sensitive to the mechanism. Our study will provide in-depth molecular mechanism data for neurotoxic evaluation and biomedical application of AgNPs.

ATPase Copper Transporting Beta (ATP7B) Is a Novel Target for Improving the Therapeutic Efficacy of Docetaxel by Disulfiram/Copper in Human Prostate Cancer.

Docetaxel has been the standard first-line chemotherapy for lethal metastatic prostate cancer (mPCa) since 2004, but resistance to docetaxel treatment is common. The molecular mechanisms of docetaxel resistance remain largely unknown and could be amenable to interventions that mitigate resistance. We have recently discovered that several docetaxel-resistant mPCa cell lines exhibit lower uptake of cellular copper and uniquely express higher levels of a copper exporter protein ATP7B. Knockdown of ATP7B by silencing RNAs (siRNA) sensitized docetaxel-resistant mPCa cells to the growth-inhibitory and apoptotic effects of docetaxel. Importantly, deletions of ATP7B in human mPCa tissues predict significantly better survival of patients after their first chemotherapy than those with wild-type ATP7B (P = 0.0006). In addition, disulfiram (DSF), an FDA-approved drug for the treatment of alcohol dependence, in combination with copper, significantly enhanced the in vivo antitumor effects of docetaxel in a docetaxel-resistant xenograft tumor model. Our analyses also revealed that DSF and copper engaged with ATP7B to decrease protein levels of COMM domain-containing protein 1 (COMMD1), S-phase kinase-associated protein 2 (Skp2), and clusterin and markedly increase protein expression of cyclin-dependent kinase inhibitor 1 (p21/WAF1). Taken together, our results indicate a copper-dependent nutrient vulnerability through ATP7B exporter in docetaxel-resistant prostate cancer for improving the therapeutic efficacy of docetaxel.