Cell Cycle Progression Research Articles

Y9, a Gboxin analog, displays anti-tumor effect in non-small cell lung cancer by inducing lysosomal dysfunction and apoptosis

Non-small cell lung cancer (NSCLC) ranks among the most prevalent malignancies globally. Gboxin, a novel inhibitor of mitochondrial complex V that exerts unique anti-tumor effects via oxidative phosphorylation inhibition, but shows no efficacy against NSCLC in vivo. Through chemical structure optimization, we designed and synthesized Gboxin analog Y9, which demonstrates significantly enhanced potency over its predecessor. Specifically, Y9 inhibited NSCLC significantly more strongly than Gboxin and possessed the ability to inhibit cell cycle progression and induce oxidative stress similar to Gboxin. Further investigation revealed that unlike Gboxin, Y9 selectively acidifies lysosomes and induces lysosomal dysfunction. This leads to hyperactive autophagy with impaired substrate clearance, and ultimately resulting in apoptosis. Animal studies confirmed the efficacy of Y9 in suppressing tumor growth in a xenograft mouse model. Collectively, Y9 is a distinctive Gboxin analog that outperforms its prototype by inducing lysosomal dysfunction and apoptosis, and has the potential to be developed as a novel anti-NSCLC lead compound.

Combination of cisplatin and ethyl acetate extract of Vernonia amygdalina Delile induces cell cycle arrest and apoptosis on PANC-1 cells via PI3K/mTOR

Context: Vernonia amygdalina Delile (VAD) is known as a potential plant with a wide variety of medicinal properties, including anticancer. Aims: To evaluate the combination effect of VAD extract with cisplatin (CIS) against PANC-1 cells, focusing on cell cycle arrest and apoptosis activities. Methods: This study is an experimental study using PANC-1 cells as objects. The phytochemical compounds were analyzed with LC-MS/MS. The cytotoxic activity of extracts and their combinations was determined using the MTT assay method in the PANC-1 cell line. Apoptosis, cell cycle arrest, and PI3K/mTOR profiles were analyzed with flow cytometry. Immunocytochemistry was used to determine Bcl-2, Cyclin D1, and p53 expression. Results: The phytochemicals found were five compounds with retention times of 8.96, 9.85, 10.52, 12.59, and 10.36. The results of the MTT assay showed that the IC50 values of extract and CIS were 21.83 ± 0.46 µg/mL and 3.02 ± 0.44 µg/mL, respectively. The combination index (CI) value of extract and CIS had a synergistic effect. Combination extract and CIS induced early and late apoptosis, inhibited cell cycle progression on the G1 phase, inhibited Bcl-2 and cyclin D1 expression, induced p53 expression, and inhibited PI3K and mTOR expression. Conclusions: The combination of extract and CIS showed anticancer activity against PANC-1 cells through induction of apoptosis and cell cycle arrest via inhibition of PI3K and mTOR expressions.

Abstract P197: Identification and Characterization of Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Proteins in Placenta

Preeclampsia is life threatening condition affecting about 10% of all pregnancies. A potential mechanism is via malfunctioning syncytiotrophoblasts in the placenta. RhoBTB1 is an adapter protein for the CUL3 E3 ubiquitin ligase which delivers protein substrates for proteasomal degradation. An analysis of gene expression databases revealed that the highest level of RhoBTB1 expression is in the placenta. RNAscope in situ hybridization data reveals that RhoBTB1 expression is specifically localized to syncytiotrophoblasts. In blood vessels, RhoBTB1 controls the state of nitric oxide mediated vasodilation because it acts as a substrate adaptor for Phosphodiesterase 5. Thus, we wanted to identify potential protein targets of RhoBTB1 in the placenta to assess if RhoBTB1 plays a role in placental function. To simplify the first identification of RhoBTB1 binding proteins, we employed HTR-8 /SVneo (HTR-8) cells as a model of placental trophoblast cells. We biotinylated the HTR-8 proteome utilizing ascorbate peroxidase (APEX2) proximity labelling system followed by mass spectrometry to identify RhoBTB1 targets. We employed a fusion protein consisting of the C-terminal half of RhoBTB1 (B1B2C), the region required for the binding of other protein substrates, and a control lacking the most C-terminal region (B1B2). We next chose proteins with binding to B1B2C > B1B2 of 2-fold or greater that were expressed in the placenta based on the Human Protein Atlas. Rho GTPase Activating Protein 29 (ARHGAP29), which controls the regulation of small GTP binding proteins, and S-phase Kinase associated Protein 2 (SKP2), a regulator of cell cycle progression was studied further. SKP2 is interesting because it exhibits a tissue specific tropism for placental expression with its highest expression in extravillous trophoblasts, cytotrophoblasts, and syncytiotrophoblasts. Expression of ARHGAP29 and SKP2 in HTR-8 cells were markedly upregulated by MLN4924 suggesting they are regulated by the CUL pathway. They were also modestly upregulated after partial inhibition of RhoBTB1 by siRNA suggesting they may be RhoBTB1-CUL3 targets. Studies are ongoing to validate the physical interaction of ARHGAP29 and SKP2 with RhoBTB1 by co-immunoprecipitation. We are also using RNAscope to assess the co-expression of RhoBTB1, ARHGAP29 and SKP2 in the human placenta from normal and preeclamptic pregnancies.

Piperine Enhances the Anticancer Effects of Cisplatin on Tongue Squamous Cell Carcinoma Cell Line by Inducing Cell Apoptosis.

Oral squamous cell carcinoma is ranked as the predominant type of head and neck squamous cell carcinoma, comprising roughly 90% of all oral cancer cases. Natural products have proven to be highly valuable as complementary, or adjunctive in the treatment of cancer. Piperine, a natural compound derived from Piper nigrum, demonstrates anti-proliferative and anti-neoplastic effects across various types of cancer. This study focused on assessing the cytotoxic effect of piperine in conjunction with cisplatin within the OSCC cell line. In this in-vitro study, cultured OSCC cells were divided into four groups: a control group (untreated), a group exposed solely to piperine, a group exposed solely to cisplatin, and a group receiving both piperine and cisplatin. Cell viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay technique. Additionally, flow cytometric analysis was employed to examine cell cycle progression and apoptosis. Assessment of reactive oxygen species activity, morphological changes, and nuclear area factor measurements were carried out. Expression of the apoptotic regulator Bax was assessed through western blotting analysis. Piperine has cytotoxic and apoptotic effects in a concentration-dependent manner. Piperine in combination with cisplatin exhibited a synergistic effect, resulting in more pronounced inhibition of cell viability in OSCC cells compared to using piperine and cisplatin alone. Piperine and cisplatin for 24 h induced apoptosis strongly by increasing Bax protein and ROS activity. Combining piperine with cisplatin demonstrated a greater effectiveness in triggering apoptosis in OSCC cells compared to using cisplatin alone, allowing for a reduction.

CD147 mediates S protein pseudovirus of SARS-CoV-2 infection and its induction of spermatogonia apoptosis.

Male cases diagnosed COVID-19 with more complications and higher mortality compared with females, and the overall consequences of male sex hormones and semen parameters deterioration were observed in COVID-19 patients, whereas the involvement and mechanism for spermatogenic cell remains unclear. The study was aimed to investigate the infection mode of S protein (D614G) pseudovirus (pseu-S-D614G) to spermatogenic cells, as well as the influence on cell growth. Both mouse spermatogonia (GC-1 cell, immortalized spermatogonia) and spermatocyte (GC-2 cell, immortalized spermatocytes) were used to detect the infection of pseu-S-D614G of SARS-CoV-2, and further explored the effect of SARS-CoV-2-spike protein (S-protein) and SARS-CoV-2-spike protein (omicron) (O-protein) on GC-1 cell apoptosis and proliferation. The data showed that the pseu-S-D614G invaded into GC-1 cells through either human ACE2 (hACE2) or human CD147 (hCD147), whereas GC-2 cells were insensitive to viral infection. In addition, the apoptosis and proliferation suppression inflicted by S-protein and O-protein on GC-1 cells was through Bax-Caspase3 signaling rather than arresting cell cycle progression. These findings suggest that CD147, apart from ACE2, may be a potential receptor for SARS-CoV-2 infection in testicular tissues, and that the apoptotic effect was induced in spermatogonia cells by S-protein or O-protein, eventually resulted in the damage to male fertility.

MAFB-mediated CEBPA regulated human urothelium growth through Wnt/β-catenin signaling pathway

MAFB is essential for regulating male-type urethral differentiation, and especially, its variation can contribute to hypospadias in mice. However, the potential mechanism is still unclear. Here we observed that the basic leucine zipper (bZIP) transcription factor MAFB and CCAAT/enhancer-binding protein alpha (CEBPA) could promote human urothelium SV-HUC-1 growth. Moreover, MAFB and CEBPA expression were reduced in the prepuce tissues of hypospadias patients. Based on transcriptome sequencing analysis and Western blot, MAFB knockdown was found to suppress CEBPA protein expression and repress Wnt/β-catenin signaling in urothelium cells. Meanwhile, we observed blocked cell-cycle progression from the G1 to the S phase, inhibited cell proliferation, and activated apoptosis. Furthermore, MAFB could facilitate CEBPA transcription and regulate the proliferation of urothelium. The above results indicated that MAFB-mediated inhibition of urothelial SV-HUC-1 growth resulted from inhibiting the Wnt/β-catenin signaling pathway by down-regulating CEBPA. Our findings provide new insight into the understanding of genes associated with hypospadias and the pathogenic mechanism of this disorder.

Exploring the therapeutic potential of simvastatin in pancreatic neuroendocrine neoplasms: insights into cell cycle regulation and apoptosis.

Pancreatic neuroendocrine neoplasm (pNEN) poses significant challenges in clinical management due to their heterogeneity and limited treatment options. In this study, we investigated the potential of simvastatin (SIM) as an anti-tumor agent in pNEN. We conducted cell culture experiments using QGP-1 and BON-1 cell lines and assessed cell viability, proliferation, migration, and invasion following SIM treatment. To further validate our findings, we performed in vivo experiments using a mouse xenograft model. Additionally, we investigated the underlying molecular mechanisms by analyzing changes in cell cycle progression, apoptosis, and signaling pathways. SIM treatment suppresses pNEN growth both in vitro and in vivo, and led to G1 phase arrest in QGP-1 cells. In contrast, SIM affected both the G1-S and G2-M phase transitions in the BON-1 cell line and induced apoptosis, indicating diverse mechanisms of action. Furthermore, SIM treatment resulted in decreased expression of mutant p53 (mutp53) in BON-1 cells, suggesting a potential therapeutic strategy targeting mutp53. Modulation of the MAPK pathway was also implicated in QGP-1 cells. Our study highlights SIM as a promising candidate for pNEN treatment by inducing cell cycle arrest or apoptosis, potentially through the p53 and MAPK pathways. Further research is warranted to fully elucidate SIM's mechanisms of action and evaluate its therapeutic potential in clinical settings.

AC099850.3 promotes HBV-HCC cell proliferation and invasion through regulating CD276: a novel strategy for sorafenib and immune checkpoint combination therapy

BackgroundThis study investigates the molecular mechanisms of CC@AC&SF@PP NPs loaded with AC099850.3 siRNA and sorafenib (SF) for improving hepatitis B virus-related hepatocellular carcinoma (HBV-HCC).MethodsA dataset of 44 HBV-HCC patients and their survival information was selected from the TCGA database. Immune genes related to survival status were identified using the ImmPort database and WGCNA analysis. A prognostic risk model was constructed and analyzed using Lasso regression. Differential analysis was performed to screen key genes, and their significance and predictive accuracy for HBV-HCC were validated using Kaplan–Meier survival curves, ROC analysis, CIBERSORT analysis, and correlation analysis. The correlation between AC099850.3 and the gene expression matrix was calculated, followed by GO and KEGG enrichment analysis using AC099850.3 and its co-expressed genes. HepG2.2.15 cells were selected for in vitro validation, and lentivirus interference, cell cycle determination, CCK-8 experiments, colony formation assays, Transwell experiments, scratch experiments, and flow cytometry were performed to investigate the effects of key genes on HepG2.2.15 cells. A subcutaneous transplanted tumor model in mice was constructed to verify the inhibitory effect of key genes on HBV-HCC tumors. Subsequently, pH-triggered drug release NPs (CC@AC&SF@PP) were prepared, and their therapeutic effects on HBV-HCC in situ tumor mice were studied.ResultsA prognostic risk model (AC012313.9, MIR210HG, AC099850.3, AL645933.2, C6orf223, GDF10) was constructed through bioinformatics analysis, showing good sensitivity and specificity in diagnostic prediction. AC099850.3 was identified as a key gene, and enrichment analysis revealed its impact on cell cycle pathways. In vitro cell experiments demonstrated that AC099850.3 promotes HepG2.2.15 cell proliferation and invasion by regulating immune checkpoint CD276 expression and cell cycle progression. In vivo, subcutaneously transplanted tumor experiments showed that AC099850.3 promotes the growth of HBV-HCC tumors in nude mice. Furthermore, pH-triggered drug release NPs (CC@AC&SF@PP) loaded with AC099850.3 siRNA and SF were successfully prepared and delivered to the in situ HBV-HCC, enhancing the effectiveness of combined therapy for HBV-HCC.ConclusionsAC099850.3 accelerates the cell cycle progression and promotes the occurrence and development of HBV-HCC by upregulating immune checkpoint CD276 expression. CC@AC&SF@PP NPs loaded with AC099850.3 siRNA and SF improve the effectiveness of combined therapy for HBV-HCC.

Inhibition of PRMT5 moderately suppresses prostate cancer growth in vivo but enhances its response to immunotherapy

Protein arginine methylation is a common post-translational modification (PTM) catalyzed by nine protein arginine methyltransferases (PRMTs). As the major symmetric arginine methyltransferase that methylates both histone and non-histone substrates, PRMT5 plays key roles in a number of biological processes critical for development and tumorigenesis. PRMT5 overexpression has been reported in multiple cancer types including prostate cancer (PCa), but the exact biological and mechanistic understanding of PRMT5 in aggressive PCa remains ill-defined. Here, we show that PRMT5 is upregulated in PCa, correlates with worse patient survival, promotes corrupted RNA splicing, and functionally cooperates with an array of pro-tumorigenic pathways to enhance oncogenesis. PRMT5 inhibition via either genetic knockdown or pharmacological inhibition reduces stemness with paralleled differentiation and arrests cell cycle progression without causing appreciable apoptosis. Strikingly, the severity of antitumor effect of PRMT5 inhibition correlates with disease aggressiveness, with AR+ PCa being less affected. Molecular characterization pinpoints MYC, but not (or at least to a lesser degree) AR, as the main partner of PRMT5 to form a positive feedback loop to exacerbate malignancy in both AR+ and AR— PCa cells. Inspired by the surprising finding that PRMT5 negatively correlates with tumor immune infiltration and transcriptionally suppresses an immune-gene program, we further show that although PRMT5 inhibitor (PRMT5i) EPZ015666 or anti-PD-1 immunotherapy alone exhibits limited antitumor effects, combination of PRMT5i with anti-PD-1 displays superior efficacy in inhibiting castration-resistant PCa (CRPC) in vivo. Finally, to expand the potential use of PRMT5i through a synthetic lethality concept, we also perform a global CRISPR/Cas9 knockout screen to unravel that many clinical-grade drugs of known oncogenic pathways can be repurposed to target CRPC when used in combination with PRMT5i at low doses. Collectively, our findings establish a rationale to exploit PRMT5i in combination with immunotherapy or other targeted therapies to treat aggressive PCa.

Novel strategies in HPV‑16‑related cervical cancer treatment: An in vitro study of combined siRNA-E5 with oxaliplatin and ifosfamide chemotherapy

BackgroundCervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. MethodsThe CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. ResultsE5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10−8 M (45.36 μg/ml) to 6.71 × 10−8 M (26.66 μg/ml) and Ifosfamide from 12.52 × 10−5 M (32.7 μg/ml) to 8.206 × 10−5 M (21.43 μg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. ConclusionThe study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.

Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells.

DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18-24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes.

The Antitumour Mechanisms of Carotenoids: A Comprehensive Review.

Carotenoids, known for their antioxidant properties, have garnered significant attention for their potential antitumour activities. This comprehensive review aims to elucidate the diverse mechanisms by which carotenoids exert antitumour effects, focusing on both well-established and novel findings. We explore their role in inducing apoptosis, inhibiting cell cycle progression and preventing metastasis by affecting oncogenic and tumour suppressor proteins. The review also explores the pro-oxidant function of carotenoids within cancer cells. In fact, although their overall contribution to cellular antioxidant defences is well known and significant, some carotenoids can exhibit pro-oxidant effects under certain conditions and are able to elevate reactive oxygen species (ROS) levels in tumoural cells, triggering mitochondrial pathways that would lead to cell death. The final balance between their antioxidant and pro-oxidant activities depends on several factors, including the specific carotenoid, its concentration and the redox environment of the cell. Clinical trials are discussed, highlighting the conflicting results of carotenoids in cancer treatment and the importance of personalized approaches. Emerging research on rare carotenoids like bacterioruberin showcases their superior antioxidant capacity and selective cytotoxicity against aggressive cancer subtypes, such as triple-negative breast cancer. Future directions include innovative delivery systems, novel combinations and personalized treatments, aiming to enhance the therapeutic potential of carotenoids. This review highlights the promising yet complex landscape of carotenoid-based cancer therapies, calling for continued research and clinical exploration.

Targeted colorectal cancer treatment: In vitro anti-cancer effects of carnosine nanoparticles supported by agar and magnetic iron oxide

The usage of peptides in the colorectal cancer (CRC) treatment promises to be a new anti-cancer therapy with improved treatment efficacy. Carnosine, a natural dipeptide molecule, has been demonstrated to be a potential anti-cancer drug. Nonetheless, it shows an exhibition of high-water solubility and is quickly degraded by carnosinase. Meanwhile, agar and magnetic iron oxide are the most used materials for drug delivery due to some of their advantages such as the low cost and the larger biocompatibility feature. The purpose of this study was to investigate the anti-cancer ability of agar-encapsulated carnosine nanoparticles (AgCa-NPs) and agar-encapsulated carnosine nanoparticles-coated magnetic iron oxide nanoparticles (AgCaN-MNPs) in human CRC cells, HCT-116. We evaluated the effects of AgCa-NPs and AgCaN-MNPs with a variety of concentrations (0, 5, 10, 15, 30, 40, or 50 mM) on HCT-116 cells after 72 h and 96 h by using MTT assay and observation cell morphology. We then analyzed the cell cycle progression and assessed the expression changes of genes related to apoptosis, autophagy, necroptosis, and angiogenesis after treatment for 96 h. The results showed that AgCa-NPs and AgCaN-MNPs in vitro study decreased HCT-116 cells viability. This effect was attributed to arrest of cell cycle, induction of programmed cell death, and suppression of angiogenesis by AgCa-NPs and AgCaN-MNPs. These findings revealed the antitumor efficacy of AgCa-NPs or AgCaN-MNPs for CRC treatment.

Hepatocyte-derived tissue extracellular vesicles safeguard liver regeneration and support regenerative therapy

Tissue-derived extracellular vesicles (EVs) are emerging as pivotal players to maintain organ homeostasis, which show promise as a next-generation candidate for medical use with extensive source. However, the detailed function and therapeutic potential of tissue EVs remain insufficiently studied. Here, through bulk and single-cell RNA sequencing analyses combined with ultrastructural tissue examinations, we first reveal that in situ liver tissue EVs (LT-EVs) contribute to the intricate liver regenerative process after partial hepatectomy (PHx), and that hepatocytes are the primary source of tissue EVs in the regenerating liver. Nanoscale and proteomic profiling further identify that the hepatocyte-specific tissue EVs (Hep-EVs) are strengthened to release with carrying proliferative messages after PHx. Moreover, targeted inhibition of Hep-EV release via AAV-shRab27a in vivo confirms that Hep-EVs are required to orchestrate liver regeneration. Mechanistically, Hep-EVs from the regenerating liver reciprocally stimulate hepatocyte proliferation by promoting cell cycle progression through Cyclin-dependent kinase 1 (Cdk1) activity. Notably, supplementing with Hep-EVs from the regenerating liver demonstrates translational potential and ameliorates insufficient liver regeneration. This study provides a functional and mechanistic framework showing that the release of regenerative Hep-EVs governs rapid liver regeneration, thereby enriching our understanding of physiological and endogenous tissue EVs in organ regeneration and therapy.

PELO regulates erythroid differentiation through interaction with MYC to upregulate KLF10.

Erythropoiesis is a multistep process of erythroid cell production that is controlled by multiple regulatory factors. Ribosome rescue factor PELO plays a crucial role in cell meiotic division and mice embryonic development. However, the function of PELO in erythroid differentiation remains unclear. Here, we showed that knockdown of PELO increased hemin-induced erythroid differentiation of K562 and HEL cells, exhibiting a higher number of benzidine-positive cells and increased mRNA levels of erythroid genes. PELO knockdown inhibited the proliferation and cell cycle progression and promoted apoptosis of K562 cells. Mechanistically, PELO could regulate the expression of KLF10 through interaction with MYC. Moreover, KLF10 knockdown also enhanced erythroid differentiation of K562 and HEL cells induced by hemin. Collectively, our results demonstrated that PELO regulates erythroid differentiation and increases KLF10 expression levels by interacting with MYC.

GRK2-mediated AKT activation controls cell cycle progression and G2 checkpoint in a p53-dependent manner

Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss and the weakening of the G2 checkpoint, rendering cell viability dependent on the strength of the latter through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53’s scaffolding function, triggering FOXO3a phosphorylation, impaired Cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.

The Regulation of SPRY4 Intronic Transcript 1 (SPRY4-IT1) on KIT Signaling and Imatinib Resistance of Gastrointestinal Stromal Tumor (GIST) Cells

BACKGROUND: SPRY4 intronic transcript 1 (SPRY4-IT1), is a long non-coding RNA coded by the intron of SPRY4. SPRY4 is highly expressed in gastrointestinal stromal tumor (GIST) and inhibits the tumorigenesis of GIST, but whether SPRY4-IT1 regulates the tumorigenesis of GIST or not remains unclear. Therefore, in this study, the regulation of SPRY4-IT1 expression and its role in GIST will be investigated.METHODS: GIST-T1 cells, and Ba/F3 cells which express KIT proto-oncogene (KIT) and SPRY4-IT1 were used as cell models. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to examine mRNA expression, while the protein expression and signal transduction were examined by western blot. The association between SPRY4-IT1 and KIT was examined by pull down of KIT and PCR. Cell proliferation, survival, and cell cycle progression were examined by cell counting kit-8 (CCK8) and flow cytometry.RESULTS: KIT mutants increased the expression of SPRY4-IT1 in GIST. SPRY4-IT1 bound to KIT, also enhanced the activation and expression of both wild-type KIT and primary KIT mutants, therefore increasing the activation of downstream signaling proteins AKT and ERK of KIT, GIST cell survival, and proliferation. In addition, SPRY4-IT1 reduced the sensitivity of wild-type KIT, or primary KIT mutants to the first-line targeted therapeutic drug of GIST, imatinib, which can inhibit KIT activation. Gaining drug-resistant secondary KIT mutants might be one of the main reasons of GIST recurrence after targeted therapy. Similar to wild-type KIT and primary KIT mutants, the activation and expression of secondary KIT mutants and their resistance to imatinib were also increased by SPRY4-IT1.CONCLUSION: The results indicated positive feedback between SPRY4-IT1 and wild-type KIT, primary KIT mutants or secondary KIT mutants, and the upregulation of AKT and ERK activation by SPRY4-IT1 in GIST cells, providing a new insight in the KIT signaling regulation in GIST, and the resistance of GIST to targeted therapy.KEYWORDS: SPRY4-IT1, KIT, GIST, SPRY4, signaling

Targeting NUF2 suppresses gastric cancer progression through G2/M phase arrest and apoptosis induction.

Gastric cancer (GC), a malignant tumor with poor prognosis, is one of the leading causes of cancer-related deaths worldwide; consequently, identifying novel therapeutic targets is crucial for its corresponding treatment. NUF2 , a component of the NDC80 kinetochore complex, promotes cancer progression in multiple malignancies. Therefore, this study aimed to explore the potential of NUF2 as a therapeutic target to inhibit GC progression. Clinical samples were obtained from patients who underwent radical resection of GC at Lanzhou University Second Hospital from 2016 to 2021. Cell count assays, colony formation assays, and cell-derived xenotransplantation (CDX) models were used to determine the effects of NUF2 on GC progression. Flow cytometry was used to detect the effect of NUF2 or quercetin on cell cycle progression and apoptosis. A live-cell time-lapse imaging assay was performed to determine the effect of NUF2 on the regulation of mitotic progression. Transcriptomics was used to investigate the NUF2 -associated molecular mechanisms. Virtual docking and microscale thermophoresis were used to identify NUF2 inhibitors. Finally, CDX, organoid, and patient-derived xenograft (PDX) models were used to examine the efficacy of the NUF2 inhibitor in GC. NUF2 expression was significantly increased in GC and was negatively correlated with prognosis. The deletion of NUF2 suppressed GC progression both in vivo and in vitro . NUF2 significantly regulated the mitogen-activated protein kinase (MAPK) pathway, promoted G2/M phase transition, and inhibited apoptosis in GC cells. Additionally, quercetin was identified as a selective NUF2 inhibitor with low toxicity that significantly suppressed tumor growth in GC cells, organoids, CDX, and PDX models. Collectively, NUF2 -mediated G2/M phase transition and apoptosis inhibition promoted GC progression; additionally, NUF2 inhibitors exhibited potent anti-GC activity. This study provides a new strategy for targeting NUF2 to suppress GC progression in clinical settings.

Interaction of high lipogenic states with titanium on osteogenesis

As obesity rates continue to rise, the prevalence of metabolic dysfunction and alcohol-associated steatotic liver disease (MetALD), a new term for Nonalcoholic Fatty Liver Disease (NAFLD), also increases. In an aging population, it is crucial to understand the interplay between metabolic disorders, such as MetALD, and bone health. This understanding becomes particularly significant in the context of implant osseointegration. This study introduces an in vitro model simulating high lipogenesis through the use of human Mesenchymal Stroma Cells-derived adipocytes, 3D intrahepatic cholangiocyte organoids (ICO), and Huh7 hepatocytes, to evaluate the endocrine influence on osteoblasts interacting with titanium. We observed a significant increase in intracellular fat accumulation in all three cell types, along with a corresponding elevation in metabolic gene expression compared to the control groups. Notably, osteoblasts undergoing mineralization in this high-lipogenesis environment also displayed lipid vesicle accumulation. The study further revealed that titanium surfaces modulate osteogenic gene expression and impact cell cycle progression, cell survival, and extracellular matrix remodeling under lipogenic conditions. These findings provide new insights into the challenges of implant integration in patients with obesity and MetALD, offering a deeper understanding of the metabolic influences on bone regeneration and implant success.

Oncogenic tRNA-derived fragment tRF-Leu-CAG promotes tumorigenesis of lung cancer via targeting TCEA3 and increasing autophagy.

Lung cancer is a prevalent and severe form of malignant tumors worldwide. tRF-Leu-CAG, a recently discovered non-coding single-stranded small RNA derived from transfer RNA, has sparked interest in exploring its biological functions and potential molecular mechanisms in lung cancer. The abundance of tRF-Leu-CAG was measured via quantitative real-time polymerase chain reaction (qRT-PCR) in 96 sets of lung cancer tissue samples obtained from clinical patients. Subsequently, both in vivo and in vitro experiments were conducted to validate the biological functions of tRF-Leu-CAG in lung cancer. Furthermore, an exploration of the potential target genes of tRF-Leu-CAG and its association with autophagy and drug resistance in lung cancer was undertaken. Our analysis revealed a significant upregulation of tRF-Leu-CAG in non-small cell lung cancer (NSCLC) tissues. Additionally, we observed that heightened expression of tRF-Leu-CAG significantly augmented the proliferation and migration of NSCLC cells, facilitated cell cycle progression, and suppressed apoptosis. Furthermore, we identified transcription elongation factor A3 (TCEA3) as a direct target gene of tRF-Leu-CAG. TCEA3 inhibited the proliferation and migration of NSCLC, and tRF-Leu-CAG promoted the proliferation and migration of NSCLC by mediating the silencing of TCEA3. Moreover, we demonstrated that the augmentation of paclitaxel resistance by tRF-Leu-CAG was contingent on autophagy. Finally, tRF-Leu-CAG notably accelerated tumor growth and promoted the process of epithelial-mesenchymal transition (EMT) in vivo. tRF-Leu-CAG promotes NSCLC tumor growth and metastasis by targeting TCEA3 and promotes paclitaxel resistance by enhancing cellular autophagy. These results provide potentially effective targets and therapeutic options for the clinical treatment of NSCLC.