Search Results (778)

The core clock gene Period2 (PER2) is associated with mammary gland development and lipid synthesis in rodents and has recently been found to have a diurnal variation in the process of lactation, but has not yet been demonstrated in bovine mammary epithelial cells (BMECs). To explore the regulatory function of PER2 on milk fat synthesis in bovine mammary epithelial cells, we initially assessed the expression of clock genes and milk fat metabolism genes for 24 h using real-time quantitative PCR and fitted the data to a cosine function curve. Subsequently, we overexpressed the PER2 in BMECs using plasmid vector (pcDNA3.1-PER2), with empty vector pcDNA3.1-myc as the control. After transfecting BMECs for 48 h, we assessed the protein abundance related to milk fat synthesis by Western blot, the expression of genes coding for these proteins using real time-quantitative PCR, the production of triacylglycerol, and the fatty acid profile. The findings indicated that a total of nine clock genes (PER1/2, CRY1/2, REV-ERBα, BMAL1, NCOR1, NR2F2, FBXW11), seven fatty acid metabolism genes (CD36, ACSS2, ACACA, SCD, FADS1, DGAT1, ADFP), and six nuclear receptor-related genes (INSIG1, SCAP, SREBF1, C/EBP, PPARG, LXR) exhibited oscillation with a period close to 24 h in non-transfected BMECs (R² ≥ 0.7). Compared to the control group (transfected with empty pcDNA3.1-myc), the triglyceride content significantly increased in the PER2 overexpression group (p < 0.05). The lipogenic genes for fatty acid transport and triglyceride synthesis (ACACA, SCD, LPIN1, DGAT1, and SREBF1) were upregulated after PER2 overexpression, along with the upregulation of related protein abundance (p < 0.05). The contents and ratios of palmitic acid (C16:0), oleic acid (C18:1n9c), and trans-oleic acid (C18:1n9t) were significantly increased in the overexpression group (p < 0.05). Overall, the data supported that PER2 participated in the process of milk fat metabolism and is potentially involved in the de novo synthesis and desaturation of fatty acid in bovine mammary epithelial cells. Full article

Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wild-type cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here, we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming, highlighting the critical roles of these frequently mutated genes in driving liver neoplasia. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models of hepatocarcinoma, providing insights into early tumorigenesis mechanisms. Full article

Yak is an excellent germplasm resource on the Tibetan Plateau and is able to live in high-altitude areas with hypoxic, cold, and harsh environments. Studies on induced pluripotent stem cells (iPSCs) in large ruminants commonly involve a combination strategy involving six transcription factors, Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 (OSKMNL). This strategy tends to utilize genes from the same species to optimize pluripotency maintenance. In this study, we cloned the six pluripotency genes (OSKMNL) from yak and constructed a multi-cistronic lentiviral vector carrying these genes. This vector efficiently delivered the genes into yak fibroblasts, aiming to promote the reprogramming process. We verified that the treated cells had several pluripotency characteristics, marking the first successful construction of a lentiviral system carrying yak pluripotency genes. This achievement lays the foundation for subsequent establishment of yak iPSCs and holds significant implications for yak-breed improvement and germplasm-resource conservation. Full article

Though Ginsenoside F2 (GF2), a protopanaxadiol saponin from Panax ginseng, is known to have an anticancer effect, its underlying mechanism still remains unclear. In our model, the anti-glycolytic mechanism of GF2 was investigated in human cervical cancer cells in association with miR193a-5p and the β-catenin/c-Myc/Hexokinase 2 (HK2) signaling axis. Here, GF2 exerted significant cytotoxicity and antiproliferation activity, increased sub-G1, and attenuated the expression of pro-Poly (ADPribose) polymerase (pro-PARP) and pro-cysteine aspartyl-specific protease (procaspase3) in HeLa and SiHa cells. Consistently, GF2 attenuated the expression of Wnt, β-catenin, and c-Myc and their downstream target genes such as HK2, pyruvate kinase isozymes M2 (PKM2), and lactate dehydrogenase A (LDHA), along with a decreased production of glucose and lactate in HeLa and SiHa cells. Moreover, GF2 suppressed β-catenin and c-Myc stability in the presence and absence of cycloheximide in HeLa cells, respectively. Additionally, the depletion of β-catenin reduced the expression of c-Myc and HK2 in HeLa cells, while pyruvate treatment reversed the ability of GF2 to inhibit β-catenin, c-Myc, and PKM2 in GF2-treated HeLa cells. Notably, GF2 upregulated the expression of microRNA139a-5p (miR139a-5p) in HeLa cells. Consistently, the miR139a-5p mimic enhanced the suppression of β-catenin, c-Myc, and HK2, while the miR193a-5p inhibitor reversed the ability of GF2 to attenuate the expression of β-catenin, c-Myc, and HK2 in HeLa cells. Overall, these findings suggest that GF2 induces apoptosis via the activation of miR193a-5p and the inhibition of β-catenin/c-Myc/HK signaling in cervical cancer cells. Full article

In 2022, 2.5 million cases of lung cancer were diagnosed, resulting in 1.8 million deaths. These statistics have motivated us to introduce a new natural product which is feasible in lung cancer therapies. This comprehensive study was performed to study the effects of chia seed extracts (70% ethanol and petroleum ether) on lung cancer in vitro and in vivo models. The invitro cytotoxicity activity of the chia extracts was studied in lung cancer cell lines (A549 cells). After 48 h, chia alcohol and ether extracts showed more inhibitory influence (IC₅₀, 16.08, and 14.8 µg/mL, respectively) on A549 cells compared to Dox (IC₅₀, 13.6 µg/mL). In vivo, administration of chia alcohol and ether extracts (500 mg/kg/day, orally for 20 weeks) recovered 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer, as a significant reduction in the lung cancer biomarkers, including the relative weight of the lung (20.0 and 13.33%), ICAM(31.73 and 15.66%), and c-MYC (80 and 96%) and MMP9(60 and 69%) expression genes, and improvement in these changes were observed by histopathological examinations of the lung tissues compared to the lung control. Chia seeds fought lung cancer via suppression of proliferation, angiogenesis, inflammation, and activation apoptosis. These activities may be attributed to the chemical composition of chia, which is identified by LC-Mass, such as caffeic acid, vanillic acid, kaempferol-3-O-glucuronide, and taxifolin. Finally, we can conclude that chia seeds have an anti-lung cancer effect with a good safety margin. Full article

Breast and ovarian cancers pose significant therapeutic challenges. We explored the synergistic cytotoxicity of histone deacetylase inhibitors (HDACis), poly(ADP-ribose) polymerase inhibitors (PARPis), and decitabine in breast (MDA-MB-231 and MCF-7) and ovarian (HEY-T30 and SKOV-3) cancer cell lines that were exposed to HDACi (panobinostat or vorinostat), PARPi (talazoparib or olaparib), decitabine, or their combinations. HDACi, PARPi, and decitabine combinations had synergistic cytotoxicity (assessed by MTT and clonogenic assays) in all cell lines (combination index < 1). Clonogenic assays confirmed the sensitivity of breast and ovarian cancer cell lines to the three-drug combinations (panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine; vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine). Cell proliferation was inhibited by 48–70%, and Annexin V positivity was 42–59% in all cell lines exposed to the three-drug combinations. Western blot analysis showed protein PARylation inhibition, caspase 3 and PARP1 cleavage, and c-MYC down-regulation. The three-drug combinations induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs, impaired the DNA repair pathways, and altered the epigenetic regulation of gene expression. These results indicate that HDACi, PARPi, and decitabine combinations should be further explored in these tumor types. Further clinical validation is warranted to assess their safety and efficacy. Full article

Parallel-stranded G-quadruplex structures are found to be common in the human promoter sequences. We tested highly fluorescent 9-methoxyluminarine ligand (9-MeLM) binding interactions with different parallel G-quadruplexes DNA by spectroscopic methods such as fluorescence and circular dichroism (CD) titration as well as UV melting profiles. The results showed that the studied 9-MeLM ligand interacted with the intramolecular parallel G-quadruplexes (G4s) with similar affinity. The binding constants of 9-methoxyluminarine with different parallel G4s were determined. The studies upon oligonucleotides with different flanking sequences on c-MYC G-quadruplex suggest that 9-methoxyluminarine may preferentially interact with 3′end of the c-MYC promoter. The high decrease in 9-MeLM ligand fluorescence upon binding to all tested G4s indicates that 9-methoxyluminarine molecule can be used as a selective fluorescence turn-off probe for parallel G-quadruplexes. Full article

Nonsense-mediated mRNA decay (NMD) is a highly conserved post-transcriptional gene expression regulatory mechanism in eukaryotic cells. NMD eliminates aberrant mRNAs with premature termination codons to surveil transcriptome integrity. Furthermore, NMD fine-tunes gene expression by destabilizing RNAs with specific NMD features. Thus, by controlling the quality and quantity of the transcriptome, NMD plays a vital role in mammalian development, stress response, and tumorigenesis. Deficiencies of NMD factors result in early embryonic lethality, while the underlying mechanisms are poorly understood. SMG5 is a key NMD factor. In this study, we generated an Smg5 conditional knockout mouse model and found that Smg5-null results in early embryonic lethality before E13.5. Furthermore, we produced multiple lines of Smg5 knockout mouse embryonic stem cells (mESCs) and found that the deletion of Smg5 in mESCs does not compromise cell viability. Smg5-null delays differentiation of mESCs. Mechanistically, our study reveals that the c-MYC protein, but not c-Myc mRNA, is upregulated in SMG5-deficient mESCs. The overproduction of c-MYC protein could be caused by enhanced protein synthesis upon SMG5 loss. Furthermore, SMG5-null results in dysregulation of alternative splicing on multiple stem cell differentiation regulators. Overall, our findings underscore the importance of SMG5-NMD in regulating mESC cell-state transition. Full article

In the tumor microenvironment (TME), ROS production affects survival, progression, and therapy resistance in colorectal cancer (CRC). H₂O₂-mediated oxidative stress can modulate Wnt/β-catenin signaling and metabolic reprogramming of the TME. Currently, it is unclear how mild/moderate oxidative stress (eustress) modulates Wnt/β-catenin/APC and JNK signaling relationships in primary and metastatic CRC cells. In this study, we determined the effects of the H₂O₂ concentration inducing eustress on isogenic SW480 and SW620 cells, also in combination with JNK inhibition. We assessed cell viability, mitochondrial respiration, glycolysis, and Wnt/β-catenin/APC/JNK gene and protein expression. Primary CRC cells were more sensitive to H₂O₂ eustress combined with JNK inhibition, showing a reduction in viability compared to metastatic cells. JNK inhibition under eustress reduced both glycolytic and respiratory capacity in SW620 cells, indicating a greater capacity to adapt to TME. In primary CRC cells, H₂O₂ alone significantly increased APC, LEF1, LRP6, cMYC and IL8 gene expression, whereas in metastatic CRC cells, this effect occurred after JNK inhibition. In metastatic but not in primary tumor cells, eustress and inhibition of JNK reduced APC, β-catenin, and pJNK protein. The results showed differential cross-regulation of Wnt/JNK in primary and metastatic tumor cells under environmental eustress conditions. Further studies would be useful to validate these findings and explore their therapeutic potential. Full article

In the face of rising global demand and unsustainable production methods, cultivated crustacean meat (CCM) is proposed as an alternative means to produce delicious lobster, shrimp, and crab products. Cultivated meat requires starting stem cells that may vary in terms of potency and the propensity to proliferate or differentiate into myogenic (muscle-related) tissues. Recognizing that regenerating limbs are a non-lethal source of tissue and may harbor relevant stem cells, we selected those of the crayfish Cherax quadricarinatus as our model. To investigate stem cell activity, we conducted RNA-Seq analysis across six stages of claw regeneration (four pre-molt and two post-molt stages), along with histology and real-time quantitative PCR (qPCR). Our results showed that while genes related to energy production, muscle hypertrophy, and exoskeletal cuticle synthesis dominated the post-molt stages, growth factor receptors (FGFR, EGFR, TGFR, and BMPR) and those related to stem cell proliferation and potency (Cyclins, CDKs, Wnts, C-Myc, Klf4, Sox2, PCNA, and p53) were upregulated before the molt. Pre-molt upregulation in several genes occurred in two growth peaks; Stages 2 and 4. We therefore propose that pre-molt limb regeneration tissues, particularly those in the larger Stage 4, present a prolific and non-lethal source of stem cells for CCM development. Full article

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1. Full article

Anti-estrogenic therapy is established in the management of estrogen receptor (ER)-positive breast cancer. However, to overcome resistance and improve therapeutic outcome, novel strategies are needed such as targeting widely recognized aberrant epigenetics. The study aims to investigate the combination of the aromatase inhibitor exemestane and the histone deacetylase (HDAC) inhibitor and antioxidant α-lipoic acid in ER-positive breast cancer cells. First, the enantiomers and the racemic mixture of α-lipoic acid, and rac-dihydro-lipoic acid were investigated for HDAC inhibition. We found HDAC inhibitory activity in the 1–3-digit micromolar range with a preference for HDAC6. Rac-dihydro-lipoic acid is slightly more potent than rac-α-lipoic acid. The antiproliferative IC₅₀ value of α-lipoic acid is in the 3-digit micromolar range. Notably, the combination of exemestane and α-lipoic acid resulted in synergistic behavior under various incubation times (24 h to 10 d) and readouts (MTT, live-cell fluorescence microscopy, caspase activation) analyzed by the Chou–Talalay method. α-lipoic acid increases mitochondrial fusion and the expression of apoptosis-related proteins p21, APAF-1, BIM, FOXO1, and decreases expression of anti-apoptotic proteins survivin, BCL-2, and c-myc. In conclusion, combining exemestane with α-lipoic acid is a promising novel treatment option for ER-positive breast cancer. Full article

c-MYC is overexpressed in 70% of human cancers, including triple-negative breast cancer (TNBC), yet there is no clinically approved drug that directly targets it. Here, we engineered the mRNA-stabilizing poly U sequences within the 3′UTR of c-MYC to specifically destabilize and promote the degradation of c-MYC transcripts. Interestingly, the engineered derivative outcompetes the endogenous overexpressed c-MYC mRNA, leading to reduced c-MYC mRNA and protein levels. The iron oxide nanocages (IO-nanocages) complexed with MYC-destabilizing constructs inhibited primary and metastatic tumors in mice bearing TNBC and significantly prolonged survival by degrading the c-MYC-STAT5A/B-PD-L1 complexes that drive c-MYC-positive TNBC. Taken together, we have described a novel therapy for c-MYC-driven TNBC and uncovered c-MYC-STAT5A/B-PD-L1 interaction as the target. Full article

Classical Hodgkin lymphoma (cHL) is a common B-cell cancer and a significant health concern, especially in Western and Asian countries. Despite the effectiveness of chemotherapy, many relapse cases are being reported, highlighting the need for improved treatments. This study aimed to address this issue by discovering biomarkers through the analysis of gene expression data specific to cHL. Additionally, potential anticancer inhibitors were explored to target the discovered biomarkers. This study proceeded by retrieving microarray gene expression data from cHL patients, which was then analyzed to identify significant differentially expressed genes (DEGs). Functional and network annotation of the upregulated genes revealed the active involvement of matrix metallopeptidase 12 (MMP12) and C-C motif metallopeptidase ligand 22 (CCL22) genes in the progression of cHL. Additionally, the mentioned genes were found to be actively involved in cancer-related pathways, i.e., oxidative phosphorylation, complement pathway, myc_targets_v1 pathway, TNFA signaling via NFKB, etc., and showed strong associations with other genes known to promote cancer progression. MMP12, topping the list with a logFC value of +6.6378, was selected for inhibition using docking and simulation strategies. The known anticancer compounds were docked into the active site of the MMP12 molecular structure, revealing significant binding scores of −7.7 kcal/mol and −7.6 kcal/mol for BDC_24037121 and BDC_27854277, respectively. Simulation studies of the docked complexes further supported the effective binding of the ligands, yielding MMGBSA and MMPBSA scores of −78.08 kcal/mol and −82.05 kcal/mol for MMP12-BDC_24037121 and −48.79 kcal/mol and −49.67 kcal/mol for MMP12-BDC_27854277, respectively. Our findings highlight the active role of MMP12 in the progression of cHL, with known compounds effectively inhibiting its function and potentially halting the advancement of cHL. Further exploration of downregulated genes is warranted, as associated genes may play a role in cHL. Additionally, CCL22 should be considered for further investigation due to its significant role in the progression of cHL. Full article