Cytojournal最新文献

Objective: Mycobacterium tuberculosis (MTB) manipulates macrophage functions, thus mediating tuberculosis (TB) progression. Whether the switch/sucrose non-fermenting-related matrix-associated actin-dependent regulator of chromatin subfamily a member 5 (SMARCA5) mediates MTB-induced macrophage polarization remains unclear.

Material and methods: Human Promyelocytic Leukemia Cell Line was induced into macrophages and then treated with MTB. Cell viability and apoptosis were tested with cell counting kit 8 assay and flow cytometry. Classically activated macrophages (M1) polarization and inflammation were measured by detecting CD86⁺ cell rate and inflammatory factor levels. The levels of SMARCA5, methyltransferase 3 (METTL3), and insulin-like growth factor 2 binding protein 1 (IGF2BP1) were assessed using quantitative real-time polymerase chain reaction or Western blot. The interaction between SMARCA5 and METTL3 or IGF2BP1 was confirmed by methylated RNA immunoprecipitation (RIP) and RIP assays. The effect of METTL3 knockdown on SMARCA5 messenger RNA (mRNA) stability was evaluated using actinomycin D treatment.

Results: MTB treatment suppressed the viability and promoted the apoptosis and M1 polarization and inflammation of macrophages (P < 0.05), and SMARCA5 knockdown abolished these effects (P < 0.05). METTL3 mediated the m6A methylation of SMARCA5 to enhance the mRNA stability of the latter, and this modification was recognized by IGF2BP1. SMARCA5 upregulation reverted the si-METTL3-mediated inhibition of MTB-induced macrophage M1 polarization and inflammation (P < 0.05).

Conclusion: METTL3-mediated SMARCA5 facilitates macrophage M1 polarization and inflammation, providing a novel target for TB treatment.

Objective: Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, and targeted therapies for CRC are urgently needed. This study aimed to investigate the mechanisms through which rhein induces apoptosis in CRC cells, focusing on its influence on the myeloid differentiation primary response gene 88 (MYD88)/toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway.

Material and methods: Cell Counting Kit-8 assay was conducted, with three non-cytotoxic concentrations of rhein selected for further analysis. Cells were allocated into four groups: control, 10 μM rhein, 20 μM rhein, and 50 μM rhein. Migration ability was evaluated through wound healing assay, and invasive potential was assessed using Transwell invasion assay. Apoptotic rates were determined through terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. The expression levels of apoptosis-related proteins and the key components of the MYD88/TLR4/NF-κB pathway were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blotting after rhein treatment.

Results: The CRC HT-29 and SW480 cells' capacity to migrate and invade was markedly reduced by rhein treatment. (P < 0.05) while markedly enhancing the apoptotic rates (P < 0.05). This finding was marked by a reduction in the expression levels of B-cell lymphoma 2 (BCL-2) protein and messenger RNA (mRNA, P < 0.05), along with a notable increase in the levels of Bcl-2-associated X and cysteinyl aspartate-specific protease 3 proteins and mRNAs (P < 0.05). The expression levels of MYD88, TLR4, and NF-κB proteins and mRNAs were substantially downregulated (P < 0.05). Adding the TLR4 agonist lipopolysaccharide partially reversed the inhibitory effects of rhein on this signaling pathway, thereby restoring some cellular functional behavior.

Conclusion: Rhein appears to promote apoptosis in CRC cells through the MYD88/TLR4/NF-κB signaling pathway, thus inhibiting tumor initiation and progression.

Objective: Liposarcomas are rare tumors, and it is difficult to collect cases in less densely populated areas. Therefore, we aimed to document more cases over a relatively long period to provide more data about the characteristics of liposarcomas. In this study, the clinicopathological features of liposarcomas were investigated in 27 patients.

Material and methods: All cases were confirmed by diagnosis through hematoxylin and eosin staining, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH). Combined IHC analysis was performed for murine double minute 2 (MDM2), cyclin-dependent kinase 4 (CDK4), multiple tumor suppressor 1 (P16), and Cyclin D1. FISH was performed to detect MDM2 amplification in atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS), and DNA damage inducible transcript 3 ( DDIT3) rearrangements in myxoid liposarcoma (MLPS).

Results: Seven cases of liposarcoma were located in the paratesticular region (25.9%, 7/27), 12 in the retroperitoneum (44.4%, 12/27), and eight in the limbs (29.6%, 8/27). Histological analysis showed that there were 13 cases of ALT/WDLPS (48.1%, 13/27), nine cases of DDLPS (33.3%, 9/27), three cases of MLPS (11.1%, 3/27), and two cases of pleomorphic liposarcoma (7.4%, 2/27). IHC analysis revealed that 26 cases were MDM2-positive (96.3%, 26/27), 22 were CDK4-positive (81.5%, 22/27), 26 were P16-positive (96.3%, 26/27), and 27 were cyclin D1-positive (100%, 27/27). FISH analysis revealed 20 cases of MDM2 positivity (90.9%, 20/22) and one case of DDIT3 positivity (50%, 1/2). The clinical outcomes were available for 21 patients. Four patients died (4/21, 19.0%), five experienced recurrence (5/21, 23.8%), and 12 (12/21, 57.1%) survived with no other disease.

Conclusion: A combined IHC examination of the four indicators may be used to diagnose ALT/WDLPS and DDLPS, and FISH is recommended as an important supporting method.

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.