Western Blot Assay Research Articles

A lentivirus-vectored feline erythropoietin gene therapy strategy in tissue culture and rodent models for the potential treatment of chronic renal disease-associated anemia

Abstract OBJECTIVE The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) “suicide gene.” ANIMALS CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. METHODS Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. RESULTS We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. CLINICAL RELEVANCE These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats.

USP7 promotes cervical cancer progression by stabilizing MTDH expression through deubiquitination.

Metadherin (MTDH) and ubiquitin specific protease 7 (USP7) have been identified to involve in the tumorigenesis of cervical cancer (CC). USP7 is one of the deubiquitinating enzymes. Here, this study aimed to explore whether USP7 affected CC progression via interacting with MTDH and regulating its stability via deubiquitination. qRT-PCR and western blotting assays detected the levels of genes and proteins. Functional analysis was conducted using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays, respectively. Proteins between USP7 and MTDH were identified by co-immunoprecipitation assay. A mouse xenograft model was established for in vivo analysis. MTDH was highly expressed in CC tissues and cells, silencing of MTDH suppressed CC cell proliferation, migration, invasion, angiogenesis, and macrophage M2 polarization. Mechanistically, USP7 directly bound to MTDH, and maintained its stability by removing ubiquitination on MTDH. CC tissues and cells showed high USP7 expression, and USP7 knockdown also inhibited CC cell proliferation, migration, invasion, angiogenesis and macrophage M2 polarization, and these effects mediated by USP7 knockdown were reversed by MTDH overexpression. Moreover, USP7 knockdown impeded CC growth in vivo by regulating MTDH. Collectively, USP7 promoted CC cell proliferation, migration, invasion, angiogenesis, and macrophage M2 polarization in vitro, as well as tumor growth in vivo by regulating MTDH.

Moringa isothiocyanate-1 mitigates the damage of oxidative stress and apoptosis in diabetic nephropathy mice.

Diabetic nephropathy (DN) is a prevalent cause of end-stage kidney disease worldwide. Moringa isothiocyanate-1 (MIC-1) has shown potential for DN management, however, the exact mechanisms remain unclear. This research intended to evaluate the impact and mechanism of MIC-1 on DN. Six C57BLKS/J-db/m mice served as controls. Eighteen C57BLKS/J-db/db mice were randomly separated into three groups: db/db, db/db + irbesartan (IBS), and db/db + MIC-1. Three weeks post-drug administration, the body weight and kidney weight of mice in each group were measured. Concurrently, serum creatinine (Scr), urine albumin, insulin, glycosylated hemoglobin (GHb), oxidative stress-, and inflammatory-related factors were determined. Additionally, the pathological injury, apoptosis, apoptosis-related markers, NLRP3, and ASC levels in the kidney tissues were examined utilizing H&E, Masson, PAS, TUNEL staining, and Western blot. MIC-1 decreased the body weight, kidney weight, the levels of Glu, Scr, and urine albumin in db/db mice. Moreover, MIC-1 significantly suppressed the levels of MDA, insulin, GHb, TNF-α, IL-1β, and IL-6, while increased the activities of SOD, CAT, and GPX in the serum of db/db mice. MIC-1 also mitigated the kidney tissue injury in db/db mice. Western blot assay showed that MIC-1 enhanced the Bcl-2 level and suppressed the Bax, cleaved caspase-3, cleaved caspase-9, NLRP3, ASC, and caspase-1 levels of the kidney tissues in db/db mice. MIC-1 ameliorated the kidney injury in DN mice, and its mechanism may be associated with the suppression of renal cell apoptosis, oxidative stress, and inflammatory responses.

Knockdown of circXPO1 inhibits the development of oral squamous cell carcinoma cells.

Circular RNAs (circRNAs) have emerged as pivotal regulators of cellular processes in human malignancies, including oral squamous cell carcinoma (OSCC). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect RNA expression levels of circXPO1, miR-524-5p and cyclin D1 (CCND1). Colony formation assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay were performed to analyze cell proliferation, while transwell assay was carried out to investigate the cell migration and invasion. Cell apoptosis was assessed by flow cytometry. Protein expression analysis was implemented by Western blot assay. Additionally, lactate production and glucose consumption were investigated using a lactate assay kit and glucose assay kit, respectively. The invivo tumorigenic potential of circXPO1 was evaluated using a xenograft mouse model assay. Elevated levels of circXPO1 and CCND1, alongside reduced miR-524-5p expression were decreased in OSCC tissues and cells. Knockdown of circXPO1 in OSCC cells inhibited their proliferative, migratory and invasive capacities, as well as glycolysis, prompting apoptosis. Moreover, circXPO1 silencing hindered tumor growth invivo. MiR-524-5p could be sequestered by circXPO1, and its inhibition could counteract the beneficial effects of circXPO1 knockdown on OSCC progression. Knockdown of circXPO1 inhibited OSCC progression by up-regulating miR-524-5p and down-regulating CCND1 expression, which might provide potential targets for OSCC treatment.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/β-Catenin Signaling Pathway.

Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/β-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on β-catenin and p-glycogen synthase kinase-3β (p-GSK-3β). Similarly, the deliberate activation of RBM5 reduced the increase in β-catenin and p-GSK-3β caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/β-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

Abstract P48: Advancing Personalized Anti-Cancer Pharmacotherapy: Magnetic Bioprinting of a Ductal Drug Screening Platform for Salivary Gland Mucoepidermoid Carcinoma

Abstract Mucoepidermoid carcinoma (MEC) is the most common malignant neoplasm of the salivary glands (SG), and it arises from “reserve” stem cells within the ductal network. Recently, our research team has consistently generated acinar-like epithelial SG organoids using a magnetic three-dimensional bioprinting (M3DB) platform. However, 3D ductal constructs with lumenized and functional features have not been established by biofabrication strategies due to challenges related to the lack of epithelial polarity and reproducibility. Consequently, our research unit is developing an in vitro high throughput drug screening platform with bio-printed ductal constructs as avatars of MEC ductal units towards a more effective and personalized anti-cancer pharmacotherapy. M3DB, multiplex immunofluorescence, western blot, and functional assays were performed. M3DB induced the formation of ring-like organoid 3D constructs after the magnetic bioassembly of MEC HTB41 cells overnight. These bio-printed constructs had a lumen formation efficiency of ~70% and contained functional E-cadherin+ cell clusters expressing ZO-1 with mainly K14+ and K5+ ductal progenitor populations with high pro-mitotic activity (Ki-67). These bio-printed ductal-like constructs were then proteomic and functionally evaluated in the presence or absence of 5-fluorouracil (5-FU), a chemotherapy drug. Proteomic assessments revealed changes in cellular antioxidant response (Nrf2) as well as DNA damage repair (γ-H2AX) after 5-FU exposure, potentially safeguarding cells from damage and halting cancer cell cycle progression. Tumor cell invasion capabilities within co-culture in mili-fluidic platforms, and functional evaluation with cholinergic and adrenergic agonists and swelling assays confirmed the 5-FU-induced cytotoxicity, decreased invasion potential, and ductal hypofunction. In summary, our team successfully established novel bio-printed MEC ductal-like avatar platforms that will pave the way for personalized pharmacotherapy and precision oncology in SG malignant tumors. Citation Format: Yamin Oo, Tien T.T. Truong, Toan V. Phan, Khurshid Ahmed, Teerapat Rodboon, Risa Chaisuparat, Glauco R. Souza, Joao N. Ferreira. Advancing Personalized Anti-Cancer Pharmacotherapy: Magnetic Bioprinting of a Ductal Drug Screening Platform for Salivary Gland Mucoepidermoid Carcinoma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P48.

Unveiling the potential of SLURP1 protein as a biomarker for prostate cancer screening

BackgroundProstate cancer (PCa) develops slowly and lacks obvious symptoms in the early stage, which makes early screening and diagnosis difficult. Urine collection is simple and is an ideal source of biomarkers. In this study, we performed urinary proteomic studies in PCa patients to screen proteins and apply them to the non-invasive early diagnosis of PCa.MethodUrine samples from PCa patients, benign prostatic hyperplasia (BPH) patients and normal control group were collected. Mass spectrometry was used for proteomic analysis and screening target proteins. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the results. Correlations with clinical indicators were explored, and receiver operating characteristic (ROC) curves were drawn to evaluate the value of target proteins in PCa.ResultA total of 1065 proteins were identified. Urinary SLURP1 protein was significantly elevated in patients with PCa compared with normal controls and patients with BPH patients. Western blot and ELISA further verified the expression changes of SLURP1. The immunohistochemical staining results revealed a substantial increase in positive SLURP1 expression within PCa tumor tissue. Correlation analysis showed a positive correlation between the expression level of urine SLURP1 protein and serum PSA. ROC curve analysis of the SLURP1 protein in the urine of both normal individuals and PCa patients is determined to be 0.853 (95% CI=0.754 to 0.954).ConclusionThe concentration of SLURP1 protein in urine of PCa patients is increased, which can serve as a biomarker for screening PCa.

MiR-185-5p May Modulate the Chemosensitivity of LUSC to Cisplatin via Targeting PCDHA11: Multi-omics Analysis and Experimental Validation.

Drug resistance is the major difficulty in treatment of lung squamous cell carcinoma (LUSC). This study aims to explore drug response-related miRNAs (DRmiRNAs) based on multi-omics research. We identified DRmiRNAs of LUSC with a multi-omics integrated system that combines expression data of microRNA, lncRNA, mRNA, methylation levels, somatic mutations. After identifying DRmiRNAs, we screened and validated of the target mRNAs of DRmiRNAs through Targetscan and the miRDB database. Then, Real-time PCR and Western blot assays were used to estimate the expression of DRmiRNAs and target protein, and the dual-luciferase assays were used to confirm the interaction of DRmiRNAs and target mRNA. Furthermore, CCK-8 (Cell Counting Kit-8) assays were used to evaluate cell proliferation and drug sensitivity. After integrated analysis, hsa-miR-185-5p was identified as DRmiRNA based on multi-omics data. Through Targetscan and miRDB database, the possible target mRNAs were obtained and PCDHA11 was validated as a target mRNA of miR-185-5p by real-time PCR, Western blot assays and dual-luciferase assays. CCK-8 assays and clone formation assays showed that the proliferation of miR-185-5p mimics was significantly slower than that of miR-185-5p inhibitors, which means overexpression of miR-185-5p enhanced the anticancer effects of cisplatin, whereas the downregulation of miR-185-5p reduced the effects. Furthermore, the proliferation of silencing PCDHA11 was significantly slower than that of overexpression of PCDHA11, which means PCDHA11 overexpression weakened the anticancer effects of cisplatin, and silencing PCDHA11 enhanced the effects. This study demonstrated that miR-185-5p was involved in chemoresistance of LUSC cells to cisplatin partly via down-regulating PCDHA11, which may promote understanding the underlying molecular mechanisms of drug response.

Garlic oil supplementation blocks inflammatory pyroptosis-related acute lung injury by suppressing the NF-κB/NLRP3 signaling pathway via H2S generation.

Acute lung injury (ALI) is a major cause of acute respiratory failure with a high morbidity and mortality rate, and effective therapeutic strategies for ALI remain limited. Inflammatory response is considered crucial for the pathogenesis of ALI. Garlic, a globally used cooking spice, reportedly exhibits excellent anti-inflammatory bioactivity. However, protective effects of garlic against ALI have never been reported. This study aimed to investigate the protective effects of garlic oil (GO) supplementation on lipopolysaccharide (LPS)-induced ALI models. Hematoxylin and eosin staining, pathology scores, lung myeloperoxidase (MPO) activity measurement, lung wet/dry (W/D) ratio detection, and bronchoalveolar lavage fluid (BALF) analysis were performed to investigate ALI histopathology. Real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were conducted to evaluate the expression levels of inflammatory factors, nuclear factor-κB (NF-κB), NLRP3, pyroptosis-related proteins, and H2S-producing enzymes. GO attenuated LPS-induced pulmonary pathological changes, lung W/D ratio, MPO activity, and inflammatory cytokines in the lungs and BALF. Additionally, GO suppressed LPS-induced NF-κB activation, NLRP3 inflammasome expression, and inflammatory-related pyroptosis. Mechanistically, GO promoted increased H2S production in lung tissues by enhancing the conversion of GO-rich polysulfide compounds or by increasing the expression of H2S-producing enzymes in vivo. Inhibition of endogenous or exogenous H2S production reversed the protective effects of GO on ALI and eliminated the inhibitory effects of GO on NF-κB, NLRP3, and pyroptotic signaling pathways. Overall, these findings indicate that GO has a critical anti-inflammatory effect and protects against LPS-induced ALI by suppressing the NF-κB/NLRP3 signaling pathway via H2S generation.

Dual pH/redox-responsive hyperbranched polymeric nanocarriers with TME-trigger size shrinkage and charge reversible ability for amplified chemotherapy of breast cancer

A novel pH/redox-responsive hyperbranched MeO-PEG-b-(NIPAAm-co-PBAE) nanoparticles (NPs) were designed with size shrinkage and charge-reversible potential for targeted delivery of docetaxel (DTX) to MDA-MB-231 cell lines. In the tumor microenvironment (TME), amine protonation induces charge reversal and disulfide bond cleavage under high TME GSH concentration causing size shrinkage, improved deep tumor penetration, and active targeting of the therapeutic agents. These nano drug delivery systems (NDDSs) significantly promoted cancer cell uptake (~ 100% at 0.5 h), facilitating site-specific delivery and deep tumor penetration. The MTT assay revealed significantly higher cytotoxicity (P value < 0.0001) for DTX-loaded NPs compared to free DTX. Cell cycle analysis revealed G2/M (58.3 ± 2.1%) and S (21.5 ± 1.3%) arrest for DTX-loaded NPs, while free DTX caused G2/M (67.9 ± 1.1%) and sub-G1 (10.3 ± 0.8%) arrest. DTX-loaded NPs induced higher apoptosis (P value < 0.001) in MDA-MB-231 cells (71.5 ± 2.8%) compared to free DTX (42.3 ± 3.1%). Western blotting and RT-PCR assays confirmed significant up-regulation of protein levels and apoptotic genes by DTX-loaded NPs compared to free DTX. In conclusion, TME-responsive charge reversal and size-shrinkable smart NDDSs designed based on low pH, and high glutathione (GSH), offer more effective site-specific delivery of therapeutic agents to tumors.

The effects of hyaluronan and proteoglycan link protein 1 (HAPLN1) in ameliorating spinal cord injury mediated by Nrf2.

Excessive inflammatory response and oxidative stress (OS) play an important role in the pathogenesis of spinal cord injury (SCI). Balance of inflammation and prevention of OS have been considered an effective strategy for the treatment of SCI. Hyaluronan and proteoglycan link protein 1 (HAPLN1), also known as cartilage link protein, has displayed a wide range of biological and physiological functions in different types of tissues and cells. However, whether HAPLN1 regulates inflammation and OS during SCI is unknown. Therefore, we aimed to examine whether HAPLN1 can have a protective effect on SCI. In this study, both in vitro and in vivo SCI models were established. Nissl staining and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays were used. Western blotting and enzyme-linked immunosorbent assay were employed to assess the expression of proteins. Our results demonstrate that the administration of HAPLN1 promoted the recovery of motor neurons after SCI by increasing the Basso mouse scale score, increasing the numbers of motor neurons, and preventing apoptosis of spinal cord cells. Additionally, HAPLN1 mitigated OS in spinal cord tissue after SCI by increasing the content of superoxide dismutase SOD and the activity of glutathione peroxidase but reducing the levels of malondialdehyde. Importantly, we found that HAPLN1 stimulated the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and stimulated the expression of heme oxygenase-1 and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1, which mediated the attenuation of HAPLN1 in activation of the NOD-like receptor protein 3 (NLRP3) inflammasome by reducing the levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin-1β. Correspondingly, in vitro experiments show that the presence of HAPLN1 suppressed the NLRP3 inflammasome and prevented cell injury against H2O2 in PC12 cells. These effects were mediated by the Nrf2/ARE pathway, and inhibition of Nrf2 with ML385 abolished the beneficial effects of HAPLN1. Based on these findings, we conclude that HAPLN1 inhibits the NLRP3 inflammasome through the stimulation of the Nrf2/ARE pathway, thereby suppressing neuroinflammation, enhancing motor neuronal survival, and improving the recovery of nerve function after SCI.

Preparation of an inclusion complex of nickel-based β-cyclodextrin: Characterization and accelerating the osteoarthritis articular cartilage repair

Abstract Osteoarthritis is caused by the cartilage destruction of the bones of the joint surfaces and structures that produce synovium fluid. Osteoarthritis treatment includes the use of surgical methods and non-surgical or maintenance treatments including knee orthoses, medical insoles with external edges, use of physiotherapy techniques, exercise, weight loss in obese people, and teaching the principles of joint care. The main goal of treatment in osteoarthritis of the knee is to ameliorate physical function, decrease pain, and reduce the progression of the disease, through correcting the knee alignment and reducing the varus torque. Previous studies have indicated that medicinal plants and herbal nanoparticles (NPs) have the best anti-inflammatory effects. β-Cyclodextrin is a cyclic carbohydrate molecule that is used as a host to prepare inclusion complexes. In this study, the synthesis of nickel NPs is based on β-cyclodextrin (NiBCD NPs) for accelerating the osteoarthritis articular cartilage repair. The FT-IR and XRD techniques confirmed the formula of NiO for the NiBCD NPs. The FE-SEM imaging shows a non-spherical structure for NiBCD NPs with a size of less than 100 nm. In EDX, the signals at the energy levels of 8.3, 7.5, and 0.87 keV are assigned for the electron migration of Ni Kβ, Ni Kα, and Ni Lα. Furthermore, the signals for the elements of oxygen and carbon of BCD appeared at 0.52 and 0.28 keV. The effectiveness of NiBCD NPs in promoting chondrogenesis was examined in orthopedic experiments using primary cultured chondrocytes. Subsequently, we determined the functional restoration following NiBCD NPs’ transplantation in a knee osteoarthritis articular cartilage injury model. We conducted histological, PCR, and Western blot assays. In the immunological analysis, the levels of MMPs, IL-1β, TNF-α, and p-p65 expression were found to be reduced by NiBCD NPs. This reduction may be attributed to the regulation of cellular redox homeostasis through Nrf2. Furthermore, our findings demonstrated the positive impact of NiBCD NPs on stimulating chondrogenesis in vitro. Notably, the NiBCD NPs’ application accelerated the recovery of injury-induced dysfunction. Additionally, the presence of NiBCD NPs at the injury site suppressed abnormal fibrosis and angiogenesis. The histological assay revealed the chondrocytes’ proliferation and increased cartilage matrix synthesis in the NiBCD NPs’ presence.

Tumor-associated macrophage-derived exosomal miR21-5p promotes tumor angiogenesis by regulating YAP1/HIF-1α axis in head and neck squamous cell carcinoma

Extracellular vesicles (EVs) have recently received increasing attention as essential mediators of communication between tumor cells and their microenvironments. Tumor-associated macrophages (TAMs) play a proangiogenic role in various tumors, especially head and neck squamous cell carcinoma (HNSCC), and angiogenesis is closely related to tumor growth and metastasis. This research focused on exploring the mechanisms by which EVs derived from TAMs modulate tumor angiogenesis in HNSCC. Our results indicated that TAMs infiltration correlated positively with microvascular density in HNSCC. Then we collected and identified EVs from TAMs. In the microfluidic chip, TAMs derived EVs significantly enhanced the angiogenic potential of pHUVECs and successfully induced the formation of perfusable blood vessels. qPCR and immunofluorescence analyses revealed that EVs from TAMs transferred miR-21-5p to endothelial cells (ECs). And targeting miR-21-5p of TAMs could effectively inhibit TAM-EVs induced angiogenesis. Western blot and tube formation assays showed that miR-21-5p from TAM-EVs downregulated LATS1 and VHL levels but upregulated YAP1 and HIF-1α levels, and the inhibitors of YAP1 and HIF-1α could both reduce the miR-21-5p enhanced angiogenesis in HUVECs. The in vivo experiments further proved that miR-21-5p carried by TAM-EVs promoted the process of tumor angiogenesis via YAP1/HIF-1α axis in HNSCC. Conclusively, TAM-derived EVs transferred miR-21-5p to ECs to target the mRNA of LATS1 and VHL, which inhibited YAP1 phosphorylation and subsequently enhanced YAP1-mediated HIF-1α transcription and reduced VHL-mediated HIF-1α ubiquitination, contributing to angiogenesis in HNSCC. These findings present a novel regulatory mechanism of tumor angiogenesis, and miR-21-5p/YAP1/HIF-1α might be a potential therapeutic target for HNSCC.

SPOP attenuates the proliferation, invasion, and migration of choriocarcinoma JAR cells by promoting KIF23 degradation

Abstract Objectives Choriocarcinoma is a highly malignant and aggressive trophoblastic tumor. In our previous study, we discovered that the speckle-type POZ protein (SPOP), which recognizes substrates of E3 ubiquitin ligase, plays a crucial role in trophoblast-derived choriocarcinoma cell lines. Therefore, we investigated the correlation between SPOP and the substrate kinesin-like protein KIF23, as well as the role of KIF23 in choriocarcinoma cells. Methods We constructed JAR cells overexpressing SPOP using lentiviral vectors and subsequently screened the related proteins through ubiquitination-modified quantitative proteomic analysis. The relationship between KIF23 and SPOP was determined using western blotting, and CCK-8, plate cloning, flow cytometry, and Transwell assays were used to investigate the effects of KIF23 and SPOP/KIF23. Results We identified the KIF23 protein and observed that SPOP promoted its degradation. The abundance of KIF23 increased after the addition of the protease inhibitor MG132. KIF23 was highly expressed in choriocarcinoma cells. Compared with JAR cells transfected with NC–small-interfering RNA (siRNA), the proliferation, invasion, migration, and percentage of G0/G1 cells in the KIF23-siRNA group were significantly lower, and the activation of the Akt/GSK3β signaling pathway was markedly attenuated. Additionally, the sh-SPOP+KIF23-siRNA group exhibited significantly inhibited JAR cell proliferation, invasion, and migration, along with clearly attenuated activation of the Akt/GSK3β signaling pathway. Conclusions SPOP attenuates the proliferation, invasion, and migration of choriocarcinoma JAR cells by promoting KIF23 degradation.

Cannabidiol Inhibits Epithelial Ovarian Cancer: Role of Gut Microbiome.

Epithelial ovarian cancer is among the deadliest gynecological tumors worldwide. Clinical treatment usually consists of surgery and adjuvant chemo- and radiotherapies. Due to the high rate of recurrence and rapid development of drug resistance, the current focus of research is on finding effective natural products with minimal toxic side effects for treating epithelial ovarian tumors. Cannabidiol is among the most abundant cannabinoids and has a non-psychoactive effect compared to tetrahydrocannabinol, which is a key advantage for clinical application. Studies have shown that cannabidiol has antiproliferative, pro-apoptotic, cytotoxic, antiangiogenic, anti-inflammatory, and immunomodulatory properties. However, its therapeutic value for epithelial ovarian tumors remains unclear. This study aims to investigate the effects of cannabidiol on epithelial ovarian tumors and to elucidate the underlying mechanisms. The results showed that cannabidiol has a significant inhibitory effect on epithelial ovarian tumors. In vivo experiments demonstrated that cannabidiol could inhibit tumor growth by modulating the intestinal microbiome and increasing the abundance of beneficial bacteria. Western blot assays showed that cannabidiol bound to EGFR/AKT/MMPs proteins and suppressed EGFR/AKT/MMPs expression in a dose-dependent manner. Network pharmacology and molecular docking results suggested that cannabidiol could affect the EGFR/AKT/MMPs signaling pathway.

Celastrol enhances the viability of random-pattern skin flaps by regulating autophagy through the AMPK-mTOR-TFEB axis.

In reconstructive and plastic surgery, random-pattern skin flaps (RPSF) are often used to correct defects. However, their clinical usefulness is limited due to their susceptibility to necrosis, especially on the distal side of the RPSF. This study validates the protective effect of celastrol (CEL) on flap viability and explores in terms of underlying mechanisms of action. The viability of different groups of RPSF was evaluated by survival zone analysis, laser doppler blood flow, and histological analysis. The effects of CEL on flap angiogenesis, apoptosis, oxidative stress, and autophagy were evaluated by Western blot, immunohistochemistry, and immunofluorescence assays. Finally, its mechanistic aspects were explored by autophagy inhibitor and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. On the seventh day after surgery, the survival area size, blood supply, and microvessel count of RPSF were augmented following the administration of CEL. Additionally, CEL stimulated angiogenesis, suppressed apoptosis, and lowered oxidative stress levels immediately after elevated autophagy in ischemic regions; These effects can be reversed using the autophagy inhibitor chloroquine (CQ). Specifically, CQ has been observed to counteract the protective impact of CEL on the RPSF. Moreover, it has also been discovered that CEL triggers the AMPK-mTOR-TFEB axis activation in the area affected by ischemia. In CEL-treated skin flaps, AMPK inhibitors were demonstrated to suppress the AMPK-mTOR-TFEB axis and reduce autophagy levels. This investigation suggests that CEL benefits the survival of RPSF by augmenting angiogenesis and impeding oxidative stress and apoptosis. The results are credited to increased autophagy, made possible by the AMPK-mTOR-TFEB axis activation.

VDR promotes pancreatic cancer progression in vivo by activating CCL20-mediated M2 polarization of tumor associated macrophage.

Activation of VDR pathway was a promising anti-tumor therapy strategy. However, numerous clinical studies have demonstrated the effect of activating VDR is limited, which indicates that VDR plays a complex role in vivos. We analyzed the TCGA database to examine the association between VDR expression and immune cell infiltration in pancreatic adenocarcinoma (PAAD). Western blot, ELISA, ChIP, and dual-luciferase reporter assays were performed to determine the mechanism of VDR regulating CCL20. Migration assay and immunofluorescence were used to investigate the role of CCL20 in M2 macrophage polarization and recruitment. We employed multiplexed immunohistochemical staining and mouse models to validate the correlation of VDR on macrophages infiltration in PAAD. Flow cytometry analysis of M2/M1 ratio in subcutaneous graft tumors. VDR is extensively expressed in PAAD, and patients with elevated VDR levels exhibited a significantly reduced overall survival. VDR expression in PAAD tissues was associated with increased M2 macrophages infiltration. PAAD cells overexpressing VDR promote macrophages polarization towards M2 phenotype and recruitment in vitro and vivo. Mechanistically, VDR binds to the CCL20 promoter and up-regulates its transcription. The effects of polarization and recruitment on macrophages can be rescued by blocking CCL20. Finally, the relationship between VDR and M2 macrophages infiltration was evaluated using clinical cohort and subcutaneous graft tumors. A positive correlation was demonstrated between VDR/CCL20/CD163 in PAAD tissues and mouse models. High expression of VDR in PAAD promotes M2 macrophage polarization and recruitment through the secretion of CCL20, which activates tumor progression. This finding suggests that the combination of anti-macrophage therapy may improve the efficacy of VDR activation therapy in PAAD.

Plumbagin inhibits fungal growth, HMGB1/LOX-1 pathway and inflammatory factors in A. fumigatus keratitis

To investigate the anti-inflammatory and antifungal effects of plumbagin (PL) in Aspergillus fumigatus (A. fumigatus) keratitis, the minimum inhibitory concentration (MIC), time-killing curve, spore adhesion, crystal violet staining, calcium fluoride white staining, and Propidium Iodide (PI) staining were employed to assess the antifungal activity of PL in vitro against A. fumigatus. The cytotoxicity of PL was assessed using the Cell Counting Kit-8 (CCK8). The impact of PL on the expression of HMGB1, LOX-1, TNF-α, IL-1β, IL-6, IL-10 and ROS in A. fumigatus keratitis was investigated using RT-PCR, ELISA, Western blot, and Reactive oxygen species (ROS) assay. The therapeutic efficacy of PL against A. fumigatus keratitis was assessed through clinical scoring, plate counting, Immunofluorescence and Hematoxylin-Eosin (HE) staining. Finally, we found that PL inhibited the growth, spore adhesion, and biofilm formation of A. fumigatus and disrupted the integrity of its cell membrane and cell wall. PL decreased IL-6, TNF-α, and IL-1β levels while increasing IL-10 expression in fungi-infected mice corneas and peritoneal macrophages. Additionally, PL significantly attenuated the HMGB1/LOX-1 pathway while reversing the promoting effect of Boxb (an HMGB1 agonist) on HMGB1/LOX-1. Moreover, PL decreased the level of ROS. In vivo, clinical scores, neutrophil recruitment, and fungal burden were all significantly reduced in infected corneas treated with PL. In summary, the inflammatory process can be inhibited by PL through the regulation of the HMGB-1/LOX-1 pathway. Simultaneously, PL can exert antifungal effects by limiting fungal spore adhesion and biofilm formation, as well as causing destruction of cell membranes and walls.

Indisulam synergizes with melphalan to inhibit Multiple Myeloma malignancy via targeting TOP2A.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy which remains uncurable. Numerous drugs have been discovered to inhibit MM cells. Indisulam, an aryl sulfonamide, has a potent anti-myeloma activity in vitro and in vivo. This study aims to explore the new mechanism of indisulam and investigate its potential use in combination with melphalan. We examined DNA damage in MM cells through various methods such as western blotting (WB), immunofluorescence, and comet assay. We also identified the role of topoisomerase IIα (TOP2A) using bioinformatic analyses. The impact of indisulam on the RNA and protein levels of TOP2A was investigated through qPCR and WB. Cell proliferation and apoptosis were assessed using CCK-8 assays, Annexin V/PI assays and WB. We predicted the synergistic effect of the combination treatment based on calculations performed on a website, and further explored the effect of indisulam in combination with melphalan on MM cell lines and xenografts. RNA sequencing data and basic experiments indicated that indisulam caused DNA damage and inhibited TOP2A expression by decreasing transcription and promoting degradation via the proteasome pathway. Functional experiments revealed that silencing TOP2A inhibited cell proliferation and induced apoptosis and DNA damage. Finally, Indisulam/melphalan combination treatment demonstrated a strong synergistic anti-tumor effect compared to single-agent treatments in vitro and in vivo. These findings suggest that combination therapies incorporating indisulam and melphalan have the potential to enhance treatment outcomes for MM.

TDF and TAF inhibit liver cancer cell migration, invasion via p7TP3

Tenofovir disoproxil fumarate (TDF) seems to prevent hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV). However, the mechanism is still little known. This study aimed to investigate the the roles and mechanisms of TDF, tenofovir alafenamide fumarate (TAF), and entecavir (ETV) on the malignant characteristics of liver cancer cells. Using the wound-healing assays, transwell assays, matrigel transwell assays, and cell counting kit-8 (CCK-8) assays, it was possible to identify that TDF/TAF, inhibited migration, invasion, and proliferation of HepG2 cells and Huh7 cells. To investigate the mechanisms, we performed TOP/FOP-Flash system, Western blot, and RT-qPCR assays of liver cancer cells cultured with TDF/TAF and found a lower activity of Wnt/β-catenin signaling pathway compared with control cells. Finally, Hepatitis C virus p7 trans-regulated protein 3 (p7TP3), a tumor suppressor in liver cancers, was significantly increased in HepG2 cells and Huh7 cells that treated with TDF/TAF. However, entecavir (ETV)-treated liver cancer cells showed no significant difference in the malignant characteristics of liver cancer cells, activity of Wnt/β-catenin signaling pathway, and expression of p7TP3, compared with the control groups. To conclude, TDF/TAF maybe novel promising therapeutic strategy for liver cancers, including HCC and hepatoblastoma, via Wnt/β-catenin signaling pathway, by up-regulating expression of the tumor suppressor, p7TP3.