Cytojournal最新文献

Objective: Lung cancer represents a major global health issue and serves as a leading cause of cancer-related deaths, with non-small cell lung cancer (NSCLC) accounting for a considerable proportion of these cases. This study aimed to investigate the expressions and clinical importance of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in patients with rare mutations of the epidermal growth factor receptor (EGFR) gene in NSCLC.

Material and methods: A retrospective analysis including 121 NSCLC patients with rare EGFR mutations was performed. Immunohistochemistry was conducted to assess PD-L1 expression, and patients were categorized into PD-L1-negative (PLN, n = 95) and PD-L1-positive (PLP, n = 26) groups. PD-1 expression was also evaluated, with patients divided into PD-1-negative (PN, n = 93) and PD-1-positive (PP, n = 25) groups. The associations among PD-L1/PD-1 expression and demographic characteristics, progression-free survival (PFS), overall survival (OS), and a 5-year survival period were analyzed.

Results: Significant negative correlations were observed between PD-L1 expression and PFS (r = -0.202, R² = 0.041, P = 0.026) and OS (r = -0.204, R² = 0.042, P = 0.024). The PLN group exhibited a significantly longer PFS (13.47 ± 3.58 months) than the PLP group (11.67 ± 3.67 months; t = 2.222, P = 0.032) and longer OS (21.39 ± 5.69 months) compared with the PLP group (18.65 ± 4.32 months; t = 2.664, P = 0.010). For PD-1 expression, a negative correlation with PFS was noted (r = -0.325, R² = 0.106, P < 0.001). The PN group displayed longer PFS (14.36 ± 3.18 months) and OS (21.71 ± 5.82 months) compared with the PP group (PFS: 11.98 ± 3.72 months, OS: 20.01 ± 5.18 months).

Conclusion: This study underscored the importance of PD-1 and PD-L1 expression as prognostic and predictive markers in NSCLC patients with uncommon EGFR mutations. These biomarkers are crucial for achieving informed treatment choices and enhancement of prognostic evaluations in this specific group.

Objective: Glioblastoma hinders therapeutic interventions and prognostic outlooks. At the same time, a disintegrin-like and metalloproteinase 15 (ADAM15) influences cellular processes, such as adhesion and migration. Furthermore, protease-activated receptor 1 (PAR1), a vital receptor, impacts tumorigenesis and disease progression. This study aimed to investigate ADAM15 and PAR1 interaction in epithelial-mesenchymal transition (EMT) modulation in glioblastoma behavior and provide insights into therapeutic targets.

Material and methods: The impacts of ADAM15 overexpression and PAR-1/2 inhibition on the proliferation, invasion, and migration of glioblastoma cells U251 and U87 were evaluated using transwell assays, EdU incorporation, clonogenic assay, Ki67 immunohistochemistry, and immunofluorescence staining. Real-time quantitative polymerase chain reaction and Western blot analysis were employed to investigate the impact of ADAM15 on PAR1 expression.

Results: After analyzing the impacts of ADAM15 overexpression on the migration, invasion, and proliferation of human glioblastoma cell lines U251 and U87, the results showed that ADAM15 overexpression significantly enhanced migration (P < 0.001) and invasion rates (P < 0.001), as confirmed by scratch and transwell assays, thus indicating its tumor-promoting effects. This study revealed a significant increase in colony formation (P < 0.001), EdU incorporation (P < 0.001), and Ki67-positive cells (P < 0.001) in the ADAM15 overexpressed group. PAR1 and EMT markers were significantly increased in the ADAM15 overexpressed group (P < 0.001). Treatment with the PAR-1 antagonist SCH79797 inhibited EMT (P < 0.01) and suppressed cell proliferation (P < 0.001), migration (P < 0.001), and invasion (P < 0.001) in U251 and U87 cells overexpressing ADAM15, indicating the involvement of PAR-1 signaling in the effects of ADAM15 on cell behaviors. In comparison, the PAR-2 antagonist FSLLRY-NH2 did not show significant effects on EMT or these cell behaviors.

Conclusion: ADAM15 drives glioblastoma cell lines U251 and U87 progression through PAR1.

Objective: Previous studies reported that esculin could protect against renal ischemia-reperfusion injury and liver injury, but its mechanism of action in skin wound healing is unclear. The Wnt/β-catenin signaling pathway plays a positive role in the wound healing process. This study aimed to investigate the effects of esculin on the rate and quality of skin wound healing in mice and explore its regulatory role in the Wnt/b-catenin signaling pathway.

Material and methods: Circular full-thickness skin wounds with a diameter of 8 mm were created on the backs of C57BL/6 mice, which were administered with 20 and 40 mg•kg^-1 esculin through gastric lavage. Wound healing was monitored, and samples collected on day 14 were analyzed through hematoxylin-eosin and Masson staining to assess granulation tissue formation and collagen deposition. Immunohistochemistry, immunofluorescence, and Western blot evaluated markers of collagen synthesis, proliferation, angiogenesis, and proteins in the Wnt/b-catenin signaling pathway. National institutes of health/3T3 cells treated with esculin (50 and 200 μM) were analyzed for proliferating cell nuclear antigen (PCNA) expression to assess proliferative activity.

Results: Compared with the model group, the esculin-treated groups exhibited significantly enhanced wound healing (P < 0.05), increased skin epithelial thickness (P < 0.01), and promoted extracellular matrix formation in mice. In addition, esculin significantly raised type I collagen alpha-1 chain and type III collagen alpha-1 chain protein levels (P < 0.05), boosted the expression of the cell proliferation marker PCNA and the vascular marker cluster of differentiation 31 in the dermis (P < 0.05), and upregulated proteins related to the Wnt/b-catenin signaling pathway and increased glycogen synthase kinase 3 beta phosphorylation in skin wound and NIH/3T3 cells (P < 0.05).

Conclusion: Esculin could upregulate and activate the Wnt/b-catenin signaling pathway to promote wound healing.

Objective: Endometrial cancer (EC) is one of the most common gynecological malignancies, and it poses a considerable threat to women's lives. Therefore, searching for EC inhibitors and exploring the potential mechanism of action is particularly important. This article aims to investigate the potential effect of collagen type XII α1 chain (COL12A1) on macrophage polarization and its subsequent influence on the biological behavior of EC cells to further elucidate the underlying mechanisms of EC development.

Material and methods: Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of COL12A1 messenger RNA and protein in EC cells. A subcutaneous tumor formation assay was performed in nude mice to evaluate the effect of COL12A1 on EC cell growth in vivo. Flow cytometry was utilized to assess the expression levels of macrophage surface markers under different treatments. Cell counting kit-8, Transwell assay, and Western blot experiments were conducted to investigate the effects of COL12A1 knockdown and various macrophage treatments on the biological behavior of EC cells.

Results: The expression of COL12A1 was upregulated in EC cells. Knockdown of COL12A1 significantly inhibited the viability, invasion, migration, and extracellular matrix abilities of EC cells and tumor growth in vivo. Overexpression of COL12A1 significantly promoted M2-type macrophage polarization, which enhanced the invasion, migration, and epithelial-mesenchymal transition abilities of EC cells.

Conclusion: The expression of COL12A1 is upregulated in EC, and COL12A1 promotes EC cell invasion and migration by activating macrophage M2 polarization.

Objective: Colorectal cancer (CRC) presents significant treatment challenges, including immune evasion and tumor microenvironment (TME) suppression. Chimeric antigen receptor (CAR) T-cell therapy has shown promise in hematologic malignancies, but its effectiveness against solid tumors is hampered by the detrimental effects of the TME. This article aims to explore the potential of bispecific CAR T cells targeting programmed death-ligand 1 (PD-L1) and cancer-associated fibroblasts (CAFs) in CRC treatment.

Material and methods: Dual-targeted CAR-T cells against PD-L1 and CAF were engineered using the GV400 lentiviral vector. Programmed death-1 (PD-1)/nanobody (Nb) and fibroblast activation protein (FAP)/Nb-encoding lentiviral vectors were generated, and CAR T cells were produced through a three-plasmid system in 293T cells. Human peripheral blood mononuclear cells (PBMCs) were separated, transduced with these vectors, and then expanded. Functional characterization of CAR-T cells was performed through enzyme-linked immunosorbent assay (ELISA), Western blot analysis, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and cell counting kit-8 (CCK-8) assay. Migration and invasion assays were conducted using Transwell chambers to assess the ability of FAP-PD-1/Nb CAR-T cells to migrate toward tumor cells and invade the extracellular matrix.

Results: We developed dual-targeted CAR-T cells incorporating PD-L1 and CAF Nbs, which continuously secreted PD-1/Nb. Western blot confirmed PD-1/Nb expression in PD-1/Nb and FAP-PD-1/Nb CAR-T cells, with no expression in the untreated (UTD) group (P < 0.01). Flow cytometry showed a significantly higher cluster of differentiation (CD)25 and CD69 expression in FAP-PD-1/Nb CAR-T cells upon stimulation with FAP-positive target cells compared with the other groups (P < 0.01). TUNEL, flow cytometry, and CCK-8 assays revealed that FAP-PD-1/Nb CAR-T cells exhibited superior cytotoxicity and proliferation inhibition against FAP-positive HCT116 cells (P < 0.01). ELISA demonstrated increased interferon-gamma and tumor necrosis factor-alpha levels and reduced interleukin-10 (P < 0.01), suggesting enhanced cytokine modulation and antitumor immunity. Compared with single-target CAR-T cells and UTD, FAP-PD-1/Nb CAR-T cells showed notably enhanced Matrigel penetration and invasion (P < 0.01). Safety tests confirmed minimal cytotoxicity to normal PBMCs, indicating favorable safety.

Conclusion: This study successfully developed dual-targeted CAR-T cells against PD-L1 and CAF and demonstrated their superior antitumor activity and immunomodulatory effects on CRC treatment. This novel therapeutic strategy was established using CAR T-cell technology for the treatment of CRC.

Objective: Histone deacetylase 6 (HDAC6) has been confirmed to participate in the regulation of liver fibrosis (LF) progression. This study aims to explore the role and mechanism of HDAC6 in the LF process.

Material and methods: Serum samples were collected from liver cirrhosis (LC) patients and normal healthy individuals. Human hepatic stellate cells (HSC; LX-2) were stimulated with transforming growth factor β1 (TGF-β1) to mimic LF cell models. The levels of HDAC6, ribosomal protein S5 (RPS5), embryonic lethal abnormal vision like 1 (ELAVL1), and fibrosis-related markers were determined by quantitative real-time polymerase chain reaction or western blot. Cell proliferation and invasion were detected using cell counting kit 8 assay, 5-ethynyl-2'-deoxyuridine assay, and Transwell assay. The contents of inflammatory factors were examined using enzyme-linked immunosorbent assay. Furthermore, co-immunoprecipitation and RNA immunoprecipitation assays were performed to assess the interaction between HDAC6 and RPS5 or ELAVL1. The effect of ELAVL1 knockdown on HDAC6 mRNA stability was evaluated using Actinomycin D treatment assay.

Results: HDAC6 showed increased expression in LC patients. The knockdown of HDAC6 reduced TGF-β1-induced LX-2 cell proliferation, invasion, fibrosis, and inflammation. Moreover, HDAC6 reduced the acetylation of RPS5, and RPS5 knockdown reversed the inhibition effect of si-HDAC6 on TGF-β1-induced LX-2 cell proliferation, invasion, fibrosis, and inflammation. Meanwhile, ELAVL1 interacted with HDAC6 to stabilize its mRNA, thus inhibiting RPS5 expression.

Conclusion: Our data revealed that ELAVL1-stabilized HDAC6 promoted TGF-β1-induced HSC activation by repressing RPS5 acetylation, thus providing a novel target for alleviating LF progression.

Objective: Epithelial ovarian cancer (EOC) is the most common and lethal type of ovarian cancer, and the cross-talk between tumor cell ferroptosis and macrophages is essential to cancer progression. This study aims to investigate the roles of ubiquitin-specific protease 7 (USP7) and acyl-CoA synthetase long-chain family member 4 (ACSL4) in the pathogenesis of EOC.

Material and methods: The expression patterns of USP7 and ACSL4 in EOC cell lines were first determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. ACSL4 recombinant protein was applied alone or in conjunction with a USP7 overexpression plasmid in EOC cells, and the effects of USP7 and ACSL4 on EOC cell proliferation and apoptosis were assessed using colony formation assays and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling staining. The effects of USP7 and ACSL4 on ferroptosis in EOC cells were evaluated by measuring reactive oxygen species (ROS) fluorescence intensity, malondialdehyde (MDA), glutathione (GSH) levels, and glutathione peroxidase 4 (GPX4) messenger RNA (mRNA) levels. Co-culture of EOC cell-conditioned medium treated with ACSL4 recombinant protein or USP7 overexpression plasmid was performed with Human Acute Monocytic Leukemia Cell Line (THP-1) macrophages, and the expression levels of cluster of differentiation 86 and cluster of differentiation 206 were analyzed by flow cytometry. The expression levels of M1 polarization markers and M2 markers in macrophages were measured by qRT-PCR.

Results: ACSL4 was expressed at low levels in the EOC cell lines, whereas USP7 was expressed at high levels. Treatment with ACSL4 recombinant protein reduced colony formation and increased apoptotic cell levels in the EOC cells (P < 0.001). In addition, ACSL4 treatment increased ROS fluorescence intensity and MDA levels while decreasing GSH levels and GPX4 expression (P < 0.001). Furthermore, ACSL4 treatment promoted the polarization of THP-1 macrophages toward M1, increasing the expression of M1 markers (P < 0.001). USP7 overexpression exerted the opposite effect (P < 0.001).

Conclusion: This study reveals the critical role of USP7 in the progression of EOC. ACSL4 inhibits EOC growth and anti-apoptosis by inhibiting USP7-induced antiferroptosis and anti-M1 macrophage polarization, highlighting this mechanism as a potential therapeutic target in EOC.

Objective: Small-cell lung cancer (SCLC) remains challenging to treat due to its high invasiveness and propensity for drug resistance. Evidence suggests that the regulatory relationship between metallothionein 2A (MT2A) and the yes-associated protein 1 (YAP1) signaling pathway may influence the development of SCLC. Therefore, this study aims to explore the potential mechanisms affecting SCLC progression based on the regulatory interaction between YAP1 and MT2A.

Material and methods: This study utilized reverse transcription quantitative polymerase chain reaction and Western blot analysis to analyze MT2A expression in cells. SCLC cell models with MT2A silencing and overexpression, as well as cotransfected cell models with YAP1 silencing and MT2A overexpression, were constructed. The effect of MT2A/YAP1 on cell growth, migration, and invasion was evaluated through a series of experiments, including cell viability assessment using cell counting kit-8 assay, colony formation examination, 5-ethynyl-2'-deoxyuridine staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, and Transwell analysis. In addition, Western blot analysis was conducted to investigate alterations in crucial proteins associated with the YAP1 pathway and the epithelial-mesenchymal transition ( EMT) markers influenced by MT2A/YAP1. Lung metastasis and Ki67 expression were analyzed through hematoxylin and eosin staining and immunofluorescence analysis in vivo.

Results: In the SCLC cell line ( NCI-H69 cells), MT2A exhibits increased expression, facilitating cell growth, migration, and invasion. YAP1 expression decreases when MT2A is depleted. In addition, our findings validate that MT2A facilitates EMT progression and SCLC invasion and metastasis by upregulating YAP1 expression. In vitro, silencing MT2A inhibits lung metastasis and Ki67 expression.

Conclusion: MT2A facilitates the migration and invasion of SCLC cells by influencing the YAP1 signaling cascade. This investigation offers a fresh avenue for delving deeply into the potential mechanisms involved in the progression of SCLC.

Objective: Uremic encephalopathy presents as central nervous system symptoms in acute and chronic renal failure. Nobiletin (NOB), an extract from chenpi, has demonstrated anti-inflammatory bioactivity and potential neuroprotective effects without remarkable toxicity. This study aims to evaluate the pharmacological effects of NOB on treating uremic brain injury and elucidate its underlying mechanisms.

Material and methods: A uremic encephalopathy mouse model was established by inducing renal failure with cisplatin (DDP). The therapeutic effects of NOB were investigated by assessing its effect on brain damage and neuronal viability. HT22 murine hippocampal neurons were also treated with DDP to induce neurotoxicity, and the effects of NOB on cell viability, apoptosis, and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were examined. The PI3K inhibitor LY294002 was used to further investigate the involvement of the PI3K/Akt pathway in the neuroprotective effects of NOB.

Results: NOB alleviated uremia-induced brain damage in mice, and this function was associated with the activation of the PI3K/Akt signaling pathway. In vitro, NOB improved the DPP-suppressed cell viability in HT22 neurons and restored apoptosis. NOB treatment also restored the phosphorylation levels of PI3K, Akt, and Pyruvate dehydrogenase kinase 1. These effects were partially blocked by the PI3K inhibitor LY294002.

Conclusion: NOB exerts potent neuroprotective effects by activating the PI3K/Akt pathway, mitigating uremia-induced brain injury and preventing DDP-induced neurotoxicity. These findings support the potential therapeutic application of NOB for uremic encephalopathy and provide insights into its underlying mechanisms.