Cell Colony Formation Assays Research Articles

Extracellular vesicle-packaged miR-4253 secreted by cancer-associated fibroblasts facilitates cell proliferation in gastric cancer by inducing macrophage M2 polarization

ABSTRACT Cancer-associated fibroblasts (CAFs) can interact with macrophages in the tumor microenvironment by secreting extracellular vesicles (EVs), thereby affecting tumor progression. However, the mechanisms of CAF-secreted EVs in gastric cancer (GC) remain not well understood. Here, we investigated the effect of CAF-EVs on macrophage polarization in GC and the underlying mechanisms. Macrophage polarization was evaluated using flow cytometry and quantitative real-time polymerase chain reaction. GC cell proliferation was determined using cell counting kit-8, EdU, and colony formation assays. The molecular mechanism was explored using microarray analysis, dual-luciferase reporter assay, and RNA pull-down analysis. The results showed that CAFs secreted EVs that inhibit macrophage M1 polarization and promote M2 polarization. Moreover, miR-4253 expression was increased in CAF-EVs, and inhibition of miR-4253 reversed the macrophage polarization induced by EVs. IL6R was identified as the target of miR-4253. Additionally, macrophages treated with EVs that encapsulated miR-4253 promote GC cell proliferation. In conclusion, CAF-secreted EVs packaging miR-4253 facilitate macrophage polarization from M1 to M2 phenotype by targeting IL6R, thereby accelerating GC cell proliferation. The findings suggest that EV-encapsulated miR-4253 may be a promising therapeutic target of GC.

Tanshinone II A Facilitates Chemosensitivity of Osteosarcoma Cells to Cisplatin via Activation of p38 MAPK Pathway.

To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.

Significance of LIF/LIFR Signaling in the Progression of Obesity-Driven Triple-Negative Breast Cancer.

American women with obesity have an increased incidence of triple-negative breast cancer (TNBC). The impact of obesity conditions on the tumor microenvironment is suspected to accelerate TNBC progression; however, the specific mechanism(s) remains elusive. This study explores the hypothesis that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and assesses the efficacy of LIFR inhibition with EC359 in blocking TNBC progression. TNBC cell lines were co-cultured with human primary adipocytes, or adipocyte-conditioned medium, and treated with EC359. The effects of adiposity were measured using cell viability, colony formation, and invasion assays. Mechanistic studies utilized RNA-Seq, Western blotting, RT-qPCR, and reporter gene assays. The therapeutic potential of EC359 was tested using xenograft and patient-derived organoid (PDO) models. The results showed that adipose conditions increased TNBC cell proliferation and invasion, and these effects correlated with enhanced LIFR signaling. Accordingly, EC359 treatment reduced cell viability, colony formation, and invasion under adipose conditions and blocked adipose-mediated organoid growth and TNBC xenograft tumor growth. RNA-Seq analysis identified critical pathways modulated by LIF/LIFR signaling in diet-induced obesity mouse models. These findings suggest that adiposity contributes to TNBC progression via the activation of the LIF/LIFR pathway, and LIFR inhibition with EC359 represents a promising therapeutic approach for obesity-associated TNBC.

4-Methyl-N-(Piperidin-1-Ylmethylene) Benzenesulfonamide (PMSA) Promotes Ferroptosis of Tumor Cells by Targeting the KEAP1-NRF2-GPX4 Axis.

We aimed to investigate the effect of 4-methyl-N-(piperidin-1-ylmethylene) benzenesulfonamide (PMSA) on tumor cell proliferation, migration, ferroptosis, and the potential molecular mechanism of ferroptosis in tumor cells. PMSA was produced in the marine biomedical research institute of Guangdong Medical University (Zhanjiang, China) and used for tumor cells treatment. MTT and cell colony formation assays were used to measure the inhibition of tumor cell proliferation, the scratch assay was used to identified the suppression of tumor cell migration, the death of tumor cells was measured by Annexin-V-FITC/PI staining, the level of ferroptosis-relative lipid ROS in tumor cells was measured by flow cytometry and MDA detection kit, and the expression of ferroptosis-relative protein was measured by Western blot. The Discovery Studio system was used for molecular docking and the binding ability was measured by cellular thermal shift assay. The PMSA we produced inhibited tumor cell proliferation, colony formation, migration and triggered cell death, and Fer-1 could reverse these effects. The amount of ROS and MDA levels in tumor cells was also markedly raised by PMSA. PMSA treatment significantly reduced the expression of SLC7A11/XCT, NRF2, and GPX4 in tumor cells. The phosphorylation level of NRF2 was also decreased. Through molecular docking, it was discovered that PMSA could bind to NRF2 and thereby block its activity. The KEAP1-NRF2-GPX4 axis was the target of PMSA's anti-tumor action, which results in ferroptosis of tumor cells. This demonstrated that the compound has the potential to be used as a candidate for anti-tumor drugs.

Multi-omics analysis unveils the predictive value of IGF2BP3/SPHK1 signaling in cancer stem cells for prognosis and immunotherapeutic response in muscle-invasive bladder cancer

BackgroundMuscle invasive bladder cancer (MIBC) is a life-threatening malignant tumor characterized by high metastasis rates, poor prognosis, and limited treatment options. Immune checkpoint inhibitors (ICIs) targeting PD-1 and PD-L1 represent an emerging treatment for MIBC immunotherapy. However, the characteristics of patients likely to benefit from immunotherapy remain unclear.MethodsWe performed single-cell mass cytometry (CyTOF) analysis of 179,483 single cells to characterize potential immunotherapy-related cancer stem cells (CSCs)-like populations in the tumor microenvironment of 38 MIBC tissues. The upregulated expression of IGF2BP3 in CD274 + ALDH + CSC-like cells, which was associated with poor clinical prognosis, was analyzed by bulk RNA-sequencing data from an in-house cohort. The functional role of IGF2BP3 was determined through cell proliferation, colony formation, cell apoptosis and sphere formation assays. The regulation of SPHK1 expression by IGF2BP3 was investigated using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and bulk RNA-sequencing (bulk RNA-seq). We further utilized single-nucleus RNA sequencing (snRNA-seq) data from 67,988 cells of 25 MIBC tissues and single-cell RNA sequencing (scRNA-seq) data from MIBC patient-derived organoids to characterize the molecular features of bladder cancer cells co-expressing IGF2BP3 and SPHK1. Spatial transcriptomics (ST) and co-detection by indexing (CODEX) analysis were used to describe the spatial distribution and interactions of IGF2BP3 + SPHK1 + bladder cancer cells and immune cells.ResultsA subset of CD274 + ALDH + CSC-like cells was identified, associating with immunosuppression and low survival rates in MIBC patients. IGF2BP3, an m6A reader gene, was found to be upregulated in the CD274 + ALDH + CSC-like cell population and linked to poor clinical prognosis in MIBC. Knockout of IGF2BP3 dramatically promoted cell apoptosis and reduced cell proliferation in T24 cells. By integrating MeRIP-seq and bulk RNA-seq analyses, we identified SPHK1 served as a substrate for IGF2BP3 in an m6A-dependent manner. Further snRNA-seq, scRNA-seq, ST, and CODEX analysis revealed a closer topographical distance between IGF2BP3 + SPHK1 + bladder cancer cells and exhausted CD8 + T cells, providing one explanation for the superior response to immunotherapy in IGF2BP3 + SPHK1 + bladder cancer cells-enriched patients. Finally, an ICI-associated signature was developed based on the enriched genes of IGF2BP3 + SPHK1 + bladder cancer cells, and its potential ability to predict the response to immunotherapy was validated in two independent immunotherapy cohort.ConclusionsOur study highlighted the critical involvement of the IGF2BP3/SPHK1 signaling in maintaining the stemness of CSCs and promoting MIBC progression. Additionally, these findings suggested that the IGF2BP3/SPHK1 signaling might serve as a biomarker for prognosis and immunotherapy response in MIBC.

Profiling hypoxia signaling reveals a lncRNA signature contributing to immunosuppression in high-grade glioma.

Hypoxic conditions in glioma are linked to tumor aggressiveness, poor prognosis, and treatment resistance. Long non-coding RNAs (lncRNAs) play key roles in the hypoxic and immune microenvironment of cancers, but their link to hypoxia-induced immunosuppression in high-grade glioma (HGG) is not well-studied. Gene expression profiles from TCGA and CGGA, along with clinical and genomic data, were analyzed. Bioinformatics methods including Consensus Clustering, Pearson correlation, and Cox regression analyses were used. Cell proliferation was assessed using cell counting kit-8 and colony formation assays. Glioma-macrophage interactions were evaluated using a co-culture model. Hypoxia subtype clustering showed hypoxic stress correlates with worse HGG prognosis. Eight hypoxia-related lncRNAs (AP000695.4, OSMR-AS1, AC078883.3, RP11-545E17.3, LINC01057, LINC01503, TP73-AS1, and LINC00672) with prognostic value were identified, forming a risk signature that separated patients into distinct prognostic groups. Multivariate Cox regression confirmed the signature as an independent prognostic factor. High-risk patients had greater hypoxia, leading to an immunosuppressive environment and immunotherapy resistance via tumor-associated macrophages (TAMs). TP73-AS1 significantly influenced hypoxia-induced TAM infiltration and M2 polarization. We profiled hypoxic stress in HGG and developed an 8-lncRNA hypoxia-related signature predicting patient survival and immunotherapy response, emphasizing its role in hypoxia-induced immunosuppression.

Ultrasound-mediated PLGA-PEI Nanobubbles Carrying STAT6 SiRNA Enhances NSCLC Treatment via Repolarizing Tumor-associated Macrophages from M2 to M1 Phenotypes.

Tumor-associated macrophages (TAMs) are crucial for non-small cell lung cancer (NSCLC) development. In this study, polylactic acid-co-glycolic acid (PLGA)-polyethylenimine (PEI) nanobubbles (NBs) carrying STAT6 siRNA were prepared and combined with ultrasound-mediated nanobubbles destruction (UMND) to silence the STAT6 gene, ultimately repolarizing TAMs from the M2 to the M1 phenotype, treating NSCLC in vitro. PLGA-PEI NBs-siRNA were prepared and characterised, and their respective ultrasound imaging, biological stabilities and cytotoxicities were detected. Transfection efficiency was evaluated by fluorescence microscopy and flow cytometry. Repolarization of THP-1-derived M2-like macrophages was determined by qPCR and flow cytometry. NSCLC cells (A549) were co-cultured with transfected M2-like macrophages or their associated conditioned medium (CM). Western blotting was used to detect STAT6 gene silencing in M2-like macrophages and markers of epithelial and mesenchymal in A549 cells. The proliferation of A549 cells was detected using CCK-8 and cell colony formation assays. Transwell assays were used to detect the migration and invasion of A549 cells. PLGA-PEI NBs-siRNA had an average size of 223.13 ± 0.92 nm and a zeta potential of about -5.59 ± 0.97 mV. PLGA-PEI NBs showed excellent ultrasonic imaging capability in addition to biological stability to protect siRNA from degradation. UMND enhanced PLGA-PEI NBs-STAT6 siRNA transfection in M2-like macrophages, which made M2-like macrophages repolarize to M1-like macrophages and prevented proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in A549 cells. UMND enhanced PLGA-PEI NBs-STAT6 siRNA to repolarize TAMs from the M2 to the M1 phenotype, thus treating NSCLC. These findings provide a promising therapeutic approach for enhancing NSCLC immunotherapy.

MiR-140-3p Improves Sensitivity to Docetaxel by Suppressing PD-L1/ABCG2/MVP Expression in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC) accounts for the majority of non-small cell lung cancer (NSCLC), and overexpression of programmed death ligand 1 (PD-L1) in these cells is known to induce tumor immune evasion or drug resistance. However, detailed studies are needed to determine whether microRNAs (miRNAs) that reduce PD-L1 expression can suppress drug resistance in NSCLC. Kaplan Meier plotter and Receiver Operating Characteristic plotter were used to determine the effect of specific miRNAs on survival and chemotherapy response in NSCLC patients. Cell viability, colony formation and invasion assays, and qPCR analyses were also performed. The expression of miRNA-140-3p (miR-140-3p) was lower in LUAD patients, compared to the normal group, and low expression of miR-140-3p was associated with poor survival of LUAD patients, but not in LUSC. The miR-140-3p mimic inhibited proliferation, colony formation, and invasion of LUAD cells. Interestingly, the expression of miR-140-3p was significantly lower in the group of LUAD patients who did not respond to docetaxel. In LUAD cells, combined treatment with miR-140-3p and docetaxel significantly reduced cell viability as well as the expression of ABCG2 and MVP, genes associated with drug resistance, compared to either treatment alone. Additionally, combined injection of miR-140-3p mimic and docetaxel significantly inhibited tumor growth compared to treatment with docetaxel alone. These results suggest that the high expression of miR-140-3p in LUAD is correlated with good patient prognosis and may contribute to the treatment of LUAD, especially by increasing responsiveness to docetaxel.

Exploring the anti-cancer and antimetastatic effect of Silymarin against lung cancer

Lung cancer metastasis remains a significant challenge in cancer therapy, necessitating the exploration of novel treatment modalities. Silymarin, a natural compound derived from milk thistle, has demonstrated promising anticancer properties. This work explored the inhibitory effects of silymarin on lung cancer metastasis and revealed the underlying processes, focusing on matrix metalloproteinase (MMP) 2 and MMP-9 activities. Using a combination of in vitro and molecular docking analyses, we found that silymarin effectively reducing the lung cancer cells' motility and invasion by modulation of expression of MMP-2 and MMP-9. Furthermore, MTT assays revealed a dose-dependent inhibition of cell proliferation upon silymarin treatment and found the IC50 value at 58 μM. We observe that apoptotic morphology characteristic in silymarin treated groups. Cell cycle analysis exhibit the cell cycle arrest at G1 phase, 25.8 % increased apoptosis in silymarin treated groups, as evidenced by Annexin V staining. Moreover, silymarin treatment shows the lipid peroxidation in elevated level and reduced in enzymatic antioxidant level, indicating its potential role in mitigating oxidative stress induce cell death. Gelatin zymography assay indicates the silymarin has ability to inhibit the MMP-2 and MMP-9 expression in lung cancer. Additionally, cell migration assays and colony formation assays demonstrated impaired migratory and colony-forming abilities of lung cancer cells when treated with silymarin. Molecular docking studies further supported the binding affinity of silymarin with MMP-2 and MMP-9, demonstrate the −10.26 and −6.69 kcal/mol of binding energy. Collectively, our findings highlight the multifaceted anticancer properties of silymarin against lung cancer metastasis, providing insights into its therapeutic potential as an adjuvant treatment strategy.

5-Azacytidine treatment inhibits the development of lung cancer models via epigenetic reprogramming and activation of cellular pathways with anti-tumor activity.

Non-small cell lung cancer (NSCLC) treatment is challenged by late detection and limited therapeutic options. Aberrant DNA methylation, a common epigenetic alteration in NSCLC, offers new therapeutic avenues. This study aims to evaluate the combined effects of 5-Azacytidine (5-Aza), an epigenetic modifier, and ionizing radiation (IR) on NSCLC, exploring the underlying molecular mechanisms and therapeutic potential. In this study, we examined the effects of 5-Aza combined with IR in both in vitro and in vivo models of NSCLC. Five human NSCLC cell lines were treated with 5-Aza and IR. Cell viability, colony formation, wound healing, and transwell migration assays were performed to assess treatment effects. Microarray and qPCR analyses were conducted to identify gene expression changes. Additionally, subcutaneous and orthotopic xenograft models were used to evaluate the treatment's efficacy in vivo. Treatment with 5-Aza and IR resulted in significant reductions in cell viability, colony formation, and migration in NSCLC cell lines. Microarray analysis revealed significant changes in gene expression, including the upregulation of apoptosis-related genes and the downregulation of cell proliferation-related genes. In vivo studies demonstrated a notable reduction in tumor growth and metastasis in both subcutaneous and orthotopic NSCLC models following 5-Aza and IR treatment. Histological and bioluminescent imaging confirmed the therapeutic effects of the combined treatment. The combination of 5-Aza and IR shows promise as an effective treatment for NSCLC, enhancing apoptosis and reducing tumor growth through epigenetic modulation.

Inhibition of Colorectal Cancer Metastasis by Total Flavones of Abelmoschus Manihot via Lncrna AL137782-mediated STAT3/EMT Pathway Regulation.

Colorectal cancer (CRC) ranks among the most lethal malignancies globally, particularly following metastasis which results in poor prognosis. In recent years, CRC incidence in China has persistently increased. Total flavonoids (TFA) from Abelmoschus manihot, a natural compound, are recognized for their anti-inflammatory, analgesic, and antioxidant properties. However, despite extensive research into the therapeutic potential of TFA, coverage of its role in cancer treatment is notably lacking. To address this research void, our study aims to unveil the role and potential mechanisms of TFA in treating CRC. We conducted a series of experiments to assess the impact of TFA on CRC cells. Two specific CRC cell lines, DLD-1 and HCT116, were employed in cell proliferation, colony formation, flow cytometry, and cell migration assays. Additionally, to test the in vivo effects of TFA, we developed a nude mouse xenograft tumor model to assess TFA's impact on tumor growth and liver metastasis. Furthermore, we meticulously analyzed the gene expression differences between CRC cells pretreated with TGF-β and those treated with TFA using RNA-seq technology. We also examined the molecular mechanisms of TFA and assessed the expression of proteins related to the STAT3/EMT signaling pathway through Western blotting and siRNA technology. Our research findings reveal for the first time the effect of TFA on CRC cells. Result shows that TFA could suppress cell proliferation, migration, and induce apoptosis. In vivo results showed that TFA inhibited tumor growth and liver metastasis. Molecular mechanism studies have shown that TFA exerts these effects by upregulating the expression of non-coding RNA AL137782, inhibiting the EMT/STAT3 signaling pathway. These results suggest that TFA is a potential candidate for mitigating CRC metastasis. However, further research is needed to comprehensively evaluate the efficacy and safety of TFA in animal models and clinical settings. These findings bring great hope for the development of innovative CRC treatment methods.

Wee1 inhibitor PD0166285 sensitized TP53 mutant lung squamous cell carcinoma to cisplatin via STAT1

BackgroundLung squamous cell carcinoma (LUSCs) is associated with high mortality (20–30%) and lacks of effective treatments. Almost all LUSC exhibit somatic mutations in TP53. Wee1, a tyrosine kinase, regulates the cell cycle at the G2/M checkpoint. In TP53-deficient cells, the dependence on G2/M checkpoints increases. PD0166285 is the first reported drug with inhibitory activity against both Wee1 and PKMYT1.MethodsProtein expression was determined by Western blot analysis. Cell proliferation was assessed using cell colony formation and CCK-8 assays. Cell cycle was performed by PI staining with flow cytometry. Apoptosis was evaluated using Annexin V-Phycoerythrin double staining and flow cytometry. DNA damage was detected through comet assay and immunofluorescence assay. In vivo, apoptosis and anti-tumor effects were assessed using the TUNEL assay, a nude mouse model, and immunohistochemistry (IHC). Co-immunoprecipitation assay was used to detect protein–protein interactions. We analyzed Wee1, PKMYT1, and Stat1 expression in pan-cancer studies using the Ualcan public database and assessed their prognostic implications with Kaplan–Meier curves.ResultPD0166285, a Wee1 inhibitor, effectively inhibits Wee1 activity, promoting cell entry into a mitotic crisis. Moreover, PD0166285 sensitizes cells to cisplatin, enhancing clinical outcomes. Our study demonstrated that PD016628 regulates the cell cycle through Rad51 and results in cell cycle arrest at the G2/M phase. We observed increased apoptosis in tumor cells treated with PD0166285, particularly when combined with cisplatin, indicating an enhanced apoptotic response. The upregulation of γ-H2AX serves as an indicator of mitotic catastrophe. Co-immunoprecipitation and data analysis revealed that apoptosis in LUSC is mediated through the Stat1 pathway, accompanied by decreased levels of Socs3. Furthermore, IHC staining confirmed significant differences in the expression of Phospho-CDK1 and γ-H2AX in LUSCs, suggesting involvement in DNA damage.ConclusionsIn summary, our study suggests that PD0166285, an inhibitor of Wee1, sensitizes LUSC cells to cisplatin and modulates DNA damage and apoptosis pathways through Rad51 and Stat1, respectively. These findings highlight the combination of PD0166285 and cisplatin as a promising therapeutic approach for treating LUSC.

SLC25A19 drives colorectal cancer progression by regulating p53.

Investigating the molecular mechanism of colorectal cancer (CRC), a common lethal malignancies worldwide, is of great clinical significance. Solute carrier family 25 member 19 (SLC25A19) is a member of the solute carrier family that contribute to cellular functions, including tumor biology. Recently, many studies have attention on uncovering the relationship of SLC25A19 with malignant cancers, but its precise involvement in the regulation of CRC has not been thoroughly understood. This study sought to uncover the role and mechanism of SLC25A19 in CRC development. The GEPIA database and immunohistochemical staining were utilized to detect the expression of SLC25A19 in CRC tissues. The functional influences of SLC25A19 on CRC cell phenotypes were evaluated through a series of assays including celigo cell count, colony formation, CCK-8, flow cytometry, wound healing, and transwell assays following knocking down SLC25A19. Subsequently, the xenograft tumor model was constructed to evaluate the effect of SLC25A19 on tumor growth invivo. The underlying mechanisms of SLC25A19 silencing were investigated using the human phospho-kinase array. This study demonstrated the upregulation of SLC25A19 in CRC and its significant correlation with unfavorable prognosis in CRC patients. Suppression of SLC25A19 resulted in significant inhibition of cell proliferation, colony formation, and cell migration, alongside a boost in cell apoptosis. Invivo experiments revealed that silenced SLC25A19 displayed reduced growth rates and formed smaller xenografts. Mechanistically, the p53 pathway was found to be upregulated by SLC25A19 knockdown and mediated the function of SLC25A19. Consequently, SLC25A19 was identified as a novel molecule with key regulatory ability in CRC development.

Hsa_circ_0000129 targets miR-383-5p/tropomyosin 3 axis to facilitate ovarian cancer progression.

Ovarian cancer is one of the most prevalent malignancies among women. CircRNAs play key roles in the progression of ovarian cancer. This study aimed to investigate the mechanism of action of hsa_circ_0000129 and its effects on ovarian cancer. Expression of hsa_circ_0000129, tropomyosin 3 (TPM3), and miR-383-5p in ovarian cancer cell lines and tissue specimens was detected using qRT-PCR or western blotting. Cell counting kit-8 (CCK-8), colony formation, and transwell assays were performed to assess viability, proliferation, and migration of ovarian cancer cells. A xenograft model was used to study tumorigenicity of ovarian cancer cells in vivo. Luciferase and RNA immunoprecipitation assays were performed to determine binding between miR-383-5p and hsa_circ_0000129 or TPM3. Upregulation of hsa_circ_0000129 and TPM3 and downregulation of miR-383-5p were observed in ovarian cancer. Low hsa_circ_0000129 and TPM3 expression repressed viability, migration, and proliferation of ovarian cancer cells. Inhibition of miR-383-5p had a contrary effect. Furthermore, knockdown of hsa_circ_0000129 restricted the tumorigenicity of ovarian cancer cells. Mechanistically, hsa_circ_0000129 has a sponging effect on miR-383-5p, which targets TPM3. Hsa_circ_0000129 stimulated development of the malignant ovarian cancer phenotype by sponging miR-383-5p and releasing TPM3.

Oncolytic Virus Senecavirus A Inhibits Hepatocellular Carcinoma Proliferation and Growth by Inducing Cell Cycle Arrest and Apoptosis.

Hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options and high mortality. Senecavirus A (SVA) has shown potential in selectively targeting tumors while sparing healthy tissues. This study aimed to investigate the effects of SVA on HCC cells in vitro and in vivo and to elucidate its mechanisms of action. The cell counting kit-8 assay and colony formation assay were conducted to examine cell proliferation. Flow cytometry and nuclear staining were employed to analyze cell cycle distribution and apoptosis occurrence. A subcutaneous tumor xenograft HCC mouse model was created in vivo using HepG2 cells, and Ki67 expression in the tumor tissues was assessed. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay and hematoxylin and eosin staining were employed to evaluate HCC apoptosis and the toxicity of SVA on mouse organs. In vitro, SVA effectively suppressed the growth of tumor cells by inducing apoptosis and cell cycle arrest. However, it did not have a notable effect on normal hepatocytes (MIHA cells). In an in vivo setting, SVA effectively suppressed the growth of HCC in a mouse model. SVA treatment resulted in a significant decrease in Ki67 expression and an increase in apoptosis of tumor cells. No notable histopathological alterations were observed in the organs of mice during SVA administration. SVA inhibits the growth of HCC cells by inducing cell cycle arrest and apoptosis. It does not cause any noticeable toxicity to vital organs.

ADCY5 act as a putative tumor suppressor in glioblastoma: An integrated analysis

BackgroundAdenylyl cyclase (AC) isoforms played a key role in the multiple cancer pathology, However, the expression, prognostic value and function of ADCY5 in Glioblastoma (GBM) have not been reported yet. This research intends to discover the expression, epigenetic alteration and biological function of ADCY5 in GBM and its value on patients' prognosis. Methods① Transcriptional level, epigenetic alteration, prognostic value and molecular network of ADCY5 were analyzed by using of public online datasets. ② The mRNA expression profile of ADCY5 was explored by using GEPIA database and protein expression levels were detected by HPA Database. ③ The prognostic value of ADCY5 was determined by Kaplan-Meier Plotter, GEPIA and CGGA database. ④ The epigenetic characteristics of ADCY5 were determined by DiseaseMeth database. ⑤ Identification of genes co-expressed with ADCY5 and potential mechanism analyses were performed by using DAVID cBioPorta and STRING. ⑥ Reverse transcription-polymerase chain reaction (RT-PCR), cell counting kit-8 (CCK-8), colony formation, wound-healing scratch and transwell assay were applied to detect relative mRNA expression and biological function of ADCY5 in GMB cells. ResultsADCY5 mRNA and protein were downregulated in GBM compared with normal tissues. Analysis of the genetics and epigenetics of ADCY5 suggested that its expression was negatively correlated with DNA methylation. High expression of ADCY5 was significantly associated with age, grade, IDH mutation, 1p19q_codeletion, radiotherapy and chemotherapy and acted as an independent prognostic factor in GBM. ADCY5 mRNA also down-expressed in GBM cell lines and re-expressed of ADCY5 could inhibit cell proliferation, viability, migration/invasion and epithelial-mesenchymal transition (EMT) in vitro. In the analysis of genes co-expressed with ADCY5, we found that cAMP/AKT pathway, cGMP-PKG pathway, Wnts pathway were dissimilarly enriched. ConclusionOur study indicated that ADCY5 could act as an epigenetic biomarker in GBM, as well as a prognosis target in patients with GBM.

Circular RNA LDLRAD3 promotes gastric cancer progression by upregulating COL4A5 through sponging miR-h37.

Circular RNAs play an important role in the development of gastric cancer (GC). circ-low-density lipoprotein receptor class A domain containing 3 (LDLRAD3) has been confirmed to be related to GC progression. miR-137 is also a suppressor in GC. However, the impact of the interaction between circ-LDLRAD3 and miR-137 on the progression of GC remains unclear at present. The study identified expression level differences of circ-LDLRAD3, miR-137, and COL4A5 in GC pathological specimens compared to normal tissue samples. Furthermore, through in vitro experiments, including flow cytometry, cell counting kit-8 (CCK-8) assays, wound healing, Western blotting, and colony formation assays, we further explored the molecular regulatory mechanisms by which these factors promote the progression of GC. In this study, circ-LDLRAD3 was confirmed to have higher expression, and miR-137 had lower expression in GC tissues and cell lines. circ-LDLRAD3 knockdown and miR-137 overexpression promoted apoptosis and inhibited proliferation, migration, and invasion in GC cell lines. Further experiments validated that COL4A5 had a positive relationship with GC and that circ-LDLRAD3 promoted the expression of COL4A5. circ-LDLRAD3 could be sponged and inhibited by miR-137 in GC cells. As a result, the promotional effect of circ-LDLRAD3 on COL4A5 was counteracted by miR-137. Our study showed that the knockdown of circ-LDLRAD3 suppressed the development of GC by regulating the miR-137/COL4A5 axis.

Lonidamine Increases the Cytotoxic Effect of 1-[(4-Amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea via Energy Inhibition, Disrupting Redox Homeostasis, and Downregulating MGMT Expression in Human Lung Cancer Cell Line.

Lung cancer ranks as the second most diagnosed cancer and the leading cause of cancer-related deaths worldwide. Novel chemotherapeutic strategies are crucial to efficiently target tumor cells while minimizing toxicity to normal cells. In this study, we proposed a combination strategy using energy blocker lonidamine (LND) and cytotoxic drug nimustine (ACNU, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea) to enhance the killing of a human lung cancer cell line and investigated the potential chemo-sensitizing mechanism of LND. LND was found to remarkably increase the cytotoxicity of ACNU to A549 and H1299 cells without significantly affecting normal lung BEAS2B cells. The combination of LND and ACNU also produced significant effects on cell apoptosis, colony formation, cell migration, and invasion assays compared to single drug treatment. Mechanistically, LND decreased intracellular ATP levels by inhibiting glycolysis and inducing mitochondrial dysfunction. Furthermore, the combination of LND and ACNU could intensify cellular oxidative stress, decrease cellular GSH contents, and increase reactive oxygen species (ROS) production. Notably, LND alone dramatically downregulated the expression of DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase), enhancing DNA interstrand cross-link formation induced by ACNU. Overall, LND represents a potential chemo-sensitizer to enhance ACNU therapy through energy inhibition, disrupting redox homeostasis and downregulating MGMT expression in human lung cancer cell line under preclinical and clinical background.

Fatty acid metabolism affects hepatocellular carcinoma progression via the PPAR-γ signaling pathway and fatty acid β-oxidation

PurposeThis study aimed to explore novel targets for hepatocellular carcinoma (HCC) treatment by investigating the role of fatty acid metabolism. MethodsRNA-seq and clinical data of HCC were obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatic analyses were employed to identify differentially expressed genes (DEGs) related to prognosis. A signature was then constructed using the Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression to classify HCC patients from the TCGA database into low-risk and high-risk groups. The predictive performance of the signature was evaluated through principal components analysis (PCA), Kaplan Meier (KM) survival analysis, receiver operating characteristics (ROC) curves, nomogram, genetic mutations, drug sensitivity analysis, immunological correlation analysis, and enrichment analysis. Single-cell maps were constructed to illustrate the distribution of core genes. Immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR), and western blot were employed to verify the expression of core genes. The function of one core gene was validated through a series of in vitro assays, including cell viability, colony formation, wound healing, trans-well migration, and invasion assays. The results were analyzed in the context of relevant signaling pathways. ResultsBioinformatic analyses identified 15 FAMGs that were related to prognosis. A 4-gene signature was constructed, and patients were divided into high- and low-risk groups according to the signature. The high-risk group exhibited a poorer prognosis compared to the low-risk group in both the training (P < 0.001) and validation (P = 0.020) sets. Furthermore, the risk score was identified as an independent predictor of OS (P < 0.001, HR = 8.005). The incorporation of the risk score and clinicopathologic features into a nomogram enabled the effective prediction of patient prognosis. The model was able to effectively predict the immune microenvironment, drug sensitivity to chemotherapy, and gene mutation for each group. Single-cell maps demonstrated that FAMGs in the model were distributed in tumor cells. Enrichment analyses revealed that the cell cycle, fatty acid β oxidation and PPAR signaling pathways were the most significant pathways. Among the four key prognostically related FAMGs, Spermine Synthase (SMS) was selected and validated as a potential oncogene affecting cell cycle, PPAR-γ signaling pathway and fatty acid β oxidation in HCC. ConclusionsThe risk characteristics based on FAMGs could serve as independent prognostic indicators for predicting HCC prognosis and could also serve as evaluation criteria for gene mutations, immunity, and chemotherapy drug therapy in HCC patients. Meanwhile, targeted fatty acid metabolism could be used to treat HCC through related signaling pathways.

Systematic bioinformatics analysis reveals the role of shikonin in blocking colon cancer progression by identifying senescence-induced genes.

Shikonin, a naturally occurring naphthoquinone compound extracted from comfrey plants, has antitumor, anti-inflammatory, and antimicrobial properties. Cell senescence plays a key role in preventing tumor progression. It is unclear whether shikonin has an effect on cell senescence in colon cancer. In the current study, we first determine the IC50 values of shikonin on colon cancer cell lines HT29 and HCT116. Then, we verified the inhibitory effects of shikonin on the proliferation and migration abilities of colon cancer cell lines HT29 and HCT116 using cell counting kit-8, colony formation, and wound healing assays. Next, we identified a series of potential targets using high-throughput mRNA sequencing and identified 210 upregulated and 296 downregulated genes. KEGG profiling revealed eight downregulated genes associated with cell senescence: CCNB3, IL-1α, CXCL8, CDKN2A, MYC, IGFBP3, SQSTM1, and GADD45G. Among them, CXCL8 and CDKN2A were associated with poor prognosis in patients with colon cancer, suggesting that their downregulation by shikonin could improve patient survival. Furthermore, SA-β-galactosidase staining revealed that the percentage of cellular senescence in colon cancer cells was significantly increased after shikonin treatment. Molecular docking revealed that shikonin suppressed colon cancer progression by blocking CXCL8 activity. Based on these findings, we deem that shikonin might induce senescence and exert antitumor activity in colon cancer cells by downregulating CDKN2A and CXCL8. This provides a new molecular mechanism and potential therapeutic target for shikonin to inhibit colon cancer progression.