Molecular Biology Reports最新文献

Background: The hypoxic, nutrient-deprived tumour microenvironment (TME), a hallmark of solid tumours, imposes cellular stress that can also paradoxically promote survival and resistance. While TP53 is the most frequently mutated gene in cancer, approximately 60% of hepatocellular carcinoma (HCC) cases retain wild-type TP53 (WTp53), suggesting its isoforms as potential tumorigenic modulators that override canonical tumour-suppressive functions. Therefore, this study aims to delineate p53 isoform profiles in response to short-term hypoxia and nutrient deprivation, recapitulating key stressors in the tumour biology.

Method: We hereby established a 7-day HepG2 tumoursphere model by seeding 15,000 cells/well, which transitioned from normoxia (4 days) to a hypoxic (1% O₂), low-serum (1% FBS) (HLS) condition (3 days). Expression of p53 isoforms and downstream targets was assessed.

Results: The formation of HepG2 tumoursphere at different seeding densities determined the diameter, viability and proliferation profiles, with 15,000 cells/well producing optimal, viable tumourspheres with the highest yield. HLS conditions significantly reduced tumoursphere size, proliferation capacity and viability. Strikingly, multiplex long-amplicon ddPCR revealed substantial upregulation of FLp53α/Δ40p53α, Δ40p53α and Δ133p53α/Δ160p53α mRNA transcripts. While FLp53α/Δ40p53α remained dominant, Δ40p53α and Δ133p53α/Δ160p53α progressively increased, altering the balance among the isoforms. This shift correlated with enhanced expression of the pro-proliferative and survival markers (PCNA and BCL2) and reduced expression of the pro-apoptotic marker (BAX) and cell cycle inhibitor (CDKN1A), suggesting a potential functional role of these isoforms in promoting tumour cell adaptation under stress.

Conclusion: This study highlights stress-induced p53 isoform modulation as a potential survival mechanism in WTp53 HCC in response to TME stress, which warrants further exploration of isoform-specific p53 functions in understanding heterogeneity, resistance and cancer recurrence.

Background: We previously established HEK293 cells deficient in SEL1L, a key component of endoplasmic reticulum-associated protein degradation (ERAD), and attempted to identify factors affected by ERAD dysfunction through comprehensive MS analysis. Among the factors increased by SEL1L-deficiency, lysyl oxidase (LOX) which plays a role in extracellular matrix cross-linking was involved.

Methods and results: To elucidate the regulatory mechanisms of LOX mRNA and protein, we applied several ERAD-deficient cells and drug treatments. In SEL1L-deficient cells, LOX mRNA expression levels were observed to increase approximately twofold, and the increase in high molecular weight preproLOX protein was more remarkable in each SEL1L deletion. SEL1L-deficiency also slightly increased the amount of secreted LOX protein cleaved by proteases. Increased proproLOX was also observed in cells lacking Hrd1 that associates with SEL1L. Interestingly, LOX protein was hardly increased in cells deficient for EDEM2 and TXNDC11, which are involved in mannose trimming of N-glycosylated proteins. Since ERAD failure may induce sustained ER stress, we examined the effects of three ER stress inducers on LOX expression. Changes in LOX mRNA following 6-h treatment with each reagent was negligible compared to the major ER stress-inducible mRNA. In contrast, long-term treatment with thapsigargin and tunicamycin increased intracellular LOX protein, but significantly decreased secreted LOX protein. Finally, we examined the effects of proteasome and lysosome inhibitors on LOX expression and, unexpectedly, each reagent hardly increased LOX mRNA or protein levels.

Conclusions: These results suggest that expression of intrinsic LOX protein is regulated by SEL1L and Hrd1 in a ubiquitin-proteasome-independent manner.

Background: Breast cancer remains one of the leading causes of cancer-related mortality in women worldwide. Breast cancer stem cells (BCSCs) contribute to tumor initiation, metastasis, recurrence, and resistance to therapy. The Wnt signaling pathway is a major regulator of stemness properties and is associated with poor clinical outcomes. BIBR1532 is a selective telomerase inhibitor widely used in cancer research due to its ability to inhibit telomerase activity in tumor cells with minimal toxicity in normal tissues. This study aimed to investigate the effects of BIBR1532 on the Wnt signaling pathway in breast cancer cells and BCSCs.

Methods: Cytotoxicity of BIBR1532 was evaluated in MCF-7 breast cancer cells, MCF10A normal breast epithelial cells, and BCSCs using a 48-hour treatment. IC₅₀ values were calculated, and apoptosis induction was assessed. Changes in the expression of Wnt pathway-related genes following BIBR1532 treatment were analyzed using RT-qPCR.

Results: The IC₅₀ values of BIBR1532 at 48 h were 35.29 µM for MCF-7, 28.16 µM for MCF10A, and 30.42 µM for the BCSC line. BIBR1532 markedly induced apoptosis in MCF-7 and BCSCs, whereas minimal apoptotic changes were observed in MCF10A cells. RT-qPCR analysis revealed significant modulation of genes in the Wnt signaling pathway: several oncogenic components were downregulated, while multiple tumor-suppressive genes were upregulated. Additionally, expression changes in less-studied Wnt-related genes may provide new insights for future breast cancer research.

Conclusions: BIBR1532 exerts selective cytotoxic and pro-apoptotic effects on breast cancer cells and BCSCs and modulates key components of the Wnt signaling pathway. These findings suggest that telomerase inhibition may influence stemness-related signaling in breast cancer.

Background: ETS2 expression in prostate cancer (PCa) shows variability, with overexpression and loss reported. ETS2 can be dysregulated through proteasomal degradation, but p53 missense mutants may stabilize ETS2 by preventing this process. TP53 mutations are more frequent in metastatic PCa, and their identification in primary tumors could serve as an early marker for patient stratification. In addition, TMPRSS2-ERG fusion often arises from an intrachromosomal deletion including ETS2 locus.

Methods and results: ETS2, p53 and ERG protein expression were assessed by immunohistochemistry in a surgically treated PCa patients' cohort (N = 199). ETS2 and TMPRRS2-ERG mRNA were evaluated by qPCR in an independent series (N = 80). Relationship between molecular markers, and with clinicopathological characteristics was analyzed. High ETS2 protein expression was observed in 34.7% of tumors. Aberrant p53 immunostaining was detected in 5.5% of cases, being associated with high ETS2 expression (p = 0.050). p53 positivity was related to GG5 (p = 0.007) and unifocality (p = 0.011). Shorter time to PSA recurrence was observed in p53-positive patients (p = 0.002). Cases with high ETS2 expression but negative p53 staining had the longest time to recurrence, whereas p53-positive cases, regardless of ETS2, had the shortest (p = 0.002). ERG protein expression -surrogate marker of TMPRSS2-ERG- was strongly associated with high ETS2 (p < 0.001). At mRNA level, low ETS2 expression correlated with TMPRSS2-ERG fusion (p = 0.018), and showed a trend toward association with intrachromosomal deletion (p = 0.113) and high-stage (p = 0.054).

Conclusions: In conclusion, p53 staining was consistently associated with aggressive PCa and may serve as a reliable prognostic marker. High ETS2 expression correlates with delayed PSA recurrence in p53-negative tumors.

Background: Blueberry (Vaccinium spp.) is one of the most important small fruit crops due to its taste and its antioxidant capacity.

Methods and results: To understand the genetic basis of these antioxidant properties, we evaluated the antioxidant activity of 156 diverse blueberry genotypes, including cultivars, hybrids, and wild clones. Phenotypic characterization revealed diverse antioxidant activities ranging from 0.088 to 1.338 mg gallic acid equivalent per gram of fresh leaf weight. On average, 94.51% of the sequences were mapped to the blueberry reference genome. A total of 66,216 genome-wide single-nucleotide polymorphisms (SNPs) were obtained by sequencing and used for genome-wide association studies (GWAS). GWAS with a Fixed and Random Model Circulating Probability Unification model using the Genomic Association and Prediction Integrated Tool in R revealed 9 SNPs significantly associated with the antioxidant activity of blueberry. Functional annotation using BLASTx resulted in three SNP-containing regions producing hits in the NCBI nonredundant protein database. These proteins have been shown to be involved in various developmental processes and responses to biotic and abiotic stress in plants. The remaining six SNP-containing regions resulted in 4,577 RNA hits, consisting of multiple types of RNA molecules. Therefore, complex antioxidant activity is affected by both protein and RNA in blueberry plants.

Conclusion: Overall, our study provides a valuable allele pool to support the improvement of blueberry quality, mainly focused on antioxidant activity.

Background: Dopamine is one of the neurotransmitters that have been implicated in schizophrenia. Researchers suggest that certain D2 receptors (DRD2) gene polymorphisms alter dopaminergic signaling and influence prefrontally-mediated executive functions. The present study aims to test the effects of DRD2 rs6277, rs6275 and rs2242592 polymorphisms on cognitive and motor measures of antisaccade task (AS) in healthy subjects and schizophrenia patients.

Methods and results: In total 162 male subjects participated in the study, 101 of whom completed AS session. Error rate, mean latency and latency variability of correct saccades were analyzed. Dominant model analysis was carried out to compare participants with homozygous major alleles genotypes and minor allele carriers in patient and control groups independently. Genotype x Diagnosis interaction was significant for rs6277 and rs2242592 in relation to error rate: healthy minor alleles (possibly associated with risk of schizophrenia) carriers committed significantly fewer errors compared to dominant homozygotes individuals, minor alleles carriers in schizophrenia group had increased error rate. In both groups the carriers of minor alleles performed AS with shorter latencies and decreased latency variability, the significance of Genotype was revealed in the pooled samples for rs6275 and rs6277.

Conclusions: The role of possible DRD2 genotype-related striatal changes in prefrontal cortex dysfunction in schizophrenia was suggested. A high level of DRD2 expression in striatum corresponding to minor alleles improved motor indices of saccades in both groups. The findings have implications for understanding the mechanisms of individual variability in executive functioning and response to antipsychotic medication in schizophrenia.