scholarly journals New insights into the mechanisms underlying 5-fluorouracil-induced intestinal toxicity based on transcriptomic and metabolomic responses in human intestinal organoids

2021 ◽  
Author(s):  
Daniela Rodrigues ◽  
Terezinha de Souza ◽  
Luke Coyle ◽  
Matteo Di Piazza ◽  
Bram Herpers ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Cell Signalling ◽  
Mrna Translation ◽  
Atp Synthesis ◽  
Metabolome Analysis ◽  
Tissue Specific ◽  
Intestinal Toxicity

Abstract5-Fluorouracil (5-FU) is a widely used chemotherapeutical that induces acute toxicity in the small and large intestine of patients. Symptoms can be severe and lead to the interruption of cancer treatments. However, there is limited understanding of the molecular mechanisms underlying 5-FU-induced intestinal toxicity. In this study, well-established 3D organoid models of human colon and small intestine (SI) were used to characterize 5-FU transcriptomic and metabolomic responses. Clinically relevant 5-FU concentrations for in vitro testing in organoids were established using physiologically based pharmacokinetic simulation of dosing regimens recommended for cancer patients, resulting in exposures to 10, 100 and 1000 µM. After treatment, different measurements were performed: cell viability and apoptosis; image analysis of cell morphological changes; RNA sequencing; and metabolome analysis of supernatant from organoids cultures. Based on analysis of the differentially expressed genes, the most prominent molecular pathways affected by 5-FU included cell cycle, p53 signalling, mitochondrial ATP synthesis and apoptosis. Short time-series expression miner demonstrated tissue-specific mechanisms affected by 5-FU, namely biosynthesis and transport of small molecules, and mRNA translation for colon; cell signalling mediated by Rho GTPases and fork-head box transcription factors for SI. Metabolomic analysis showed that in addition to the effects on TCA cycle and oxidative stress in both organoids, tissue-specific metabolic alterations were also induced by 5-FU. Multi-omics integration identified transcription factor E2F1, a regulator of cell cycle and apoptosis, as the best key node across all samples. These results provide new insights into 5-FU toxicity mechanisms and underline the relevance of human organoid models in the safety assessment in drug development.

scholarly journals The anticancer molecular mechanism of Carnosol in human cervical cancer cells: An in vitro study

2020 ◽  
pp. 88-98
Author(s):  
Rand R. Hafidh ◽  
Ahmed S. Abdulamir
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Cycle Arrest ◽  
Anticancer Agents ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
In Vitro Study ◽  
Future Research ◽  
Cycle Arrest

Carnosol, a phenolic diterpene, is one of the effective anticancer agents naturally occurring in rosemary, sage, parsley, and oregano. The chemoresistance problem increased with the routinely used chemotherapy. Therefore, the efforts to find a substitute with safe and low cost have become crucial worldwide. The current study attempts to inspect the anticancer molecular mechanisms of Carnosol on modulating up- and down- regulation of multiple genetic carcinogenesis pathways. The cytotoxicity of Carnosol on Hela cells was evaluated by MTS assay. Flow cytometry was used to assess apoptosis and cell cycle arrest. The apoptotic morphological changes were obvious by dual apoptosis assay. The differential gene expression after treatment with Carnosol was investigated by qRT-PCR. Up to 80% of the treated cells with Carnosol IC50 underwent apoptosis. Apoptosis together with cell cycle arrest in G0/G1 phase were induced significantly after treatment with Carnosol IC50. Fifteen out of nineteen genes studied were found to be remarkably up- or down- regulated after treatment with Carnosol. Six up-regulated genes (EREG, FOS-2, ID2, CRYAB, DUSP5, and TICAM2) and nine down-regulated genes (FN1, KRAS2, CCNB1-1, FEN1, MCM4, MCM5, GTSE1, CXCL1, and RALA) were recorded. These genes are candidates for future research for elucidating anticancer molecular targeted therapies, cancerous signaling and cancer development pathways in cervical cancer; moreover, elucidating the role of apoptosis, inflammation, cell proliferation, and cell differentiation in the development of cervical cancer.

scholarly journals Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis

Oncogene ◽  
2021 ◽  
Author(s):  
Satoshi Takagi ◽  
Yuki Sasaki ◽  
Sumie Koike ◽  
Ai Takemoto ◽  
Yosuke Seto ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Pulmonary Metastasis ◽  
Lysophosphatidic Acid ◽  
Therapeutic Intervention ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Osteosarcoma Cells ◽  
Invasion And Metastasis ◽  
Platelet Releasate

AbstractOsteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA–LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

scholarly journals LMNB2 promotes the progression of colorectal cancer by silencing p21 expression

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Chen-Hua Dong ◽  
Tao Jiang ◽  
Hang Yin ◽  
Hu Song ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Gene Set Enrichment Analysis ◽  
Molecular Therapy ◽  
Cycle Progression ◽  
Cancer Tissues

AbstractColorectal cancer is the second common cause of death worldwide. Lamin B2 (LMNB2) is involved in chromatin remodeling and the rupture and reorganization of nuclear membrane during mitosis, which is necessary for eukaryotic cell proliferation. However, the role of LMNB2 in colorectal cancer (CRC) is poorly understood. This study explored the biological functions of LMNB2 in the progression of colorectal cancer and explored the possible molecular mechanisms. We found that LMNB2 was significantly upregulated in primary colorectal cancer tissues and cell lines, compared with paired non-cancerous tissues and normal colorectal epithelium. The high expression of LMNB2 in colorectal cancer tissues is significantly related to the clinicopathological characteristics of the patients and the shorter overall and disease-free cumulative survival. Functional analysis, including CCK8 cell proliferation test, EdU proliferation test, colony formation analysis, nude mouse xenograft, cell cycle, and apoptosis analysis showed that LMNB2 significantly promotes cell proliferation by promoting cell cycle progression in vivo and in vitro. In addition, gene set enrichment analysis, luciferase report analysis, and CHIP analysis showed that LMNB2 promotes cell proliferation by regulating the p21 promoter, whereas LMNB2 has no effect on cell apoptosis. In summary, these findings not only indicate that LMNB2 promotes the proliferation of colorectal cancer by regulating p21-mediated cell cycle progression, but also suggest the potential value of LMNB2 as a clinical prognostic marker and molecular therapy target.

scholarly journals A Study of Late Ventral Body Wall Degeneration in the Embryonic Chick, with Special Reference to the Cell Cycle

2021 ◽  
Author(s):  
◽  
Peter Barwell
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Body Wall ◽  
Morphological Changes ◽  
Cell Loss ◽  
Tissue Growth ◽  
Embryonic Chick ◽  
Cell Death Pathway ◽  
Medial Migration

<p>The cell kinetics and morphological changes during late ventral body wall development of the embryonic chick were studied, particularly midline degeneration and the medial migration of lateral tissues. An histological examination of these events was undertaken, along with autoradiography to determine the duration of the cell cycle, followed by teratological studies involving the prevention of differentiative events in the cell death pathway, using BrDU and Janus B Green as agents. The effects of cell cycle blockade on rates of cell death were also examined, as was the tissues ability to express differentiative features in vitro. Ventral body wall (VBW) cell death was classified as apoptosis, and was involved in two distinct events. Medial migration of lateral tissues began at day 5 of development, with widespread VBW apoptosis being seen by day 6, limited to the original mesoderm of the region. A later precise line of apoptosis (the VBL), involving both ectodermal cells of the midline ectodermal ruffle and the underlying mesodermal cells, was observed at day 7, spreading in a rostral to caudal fashion down the embryo, appearing as the migratory lateral tissues fused in the midline body wall. Increases in the amount of cell death are matched by decreases in the MI, such that at its peak (day 7.5 of development) the cell death rate is sufficiently greater than both the cell proliferation and immigration rates that a state of negative tissue growth ensues. The histological half-life of the apoptotic bodies approximates 3.8 hours. The ability to undergo apoptosis at day 7 is dependent upon a differentiative event around day 4 of incubation, and involves signal mechanisms intrinsic to the VBW tissues. BrDU application was found to inhibit apoptotic differentiation, in contrast to Janus B Green, which had a more generalised teratogenic effect on the region as a whole. Tissue culturing experiments revealed that an ectodermal-mesodermal interaction is important in regulating the extent of mesodermal apoptosis, the ectoderm playing a maintenance role for the mesoderm. Dead cells derive from the cycling cell population, as shown by the occurrence of labelled dead cells after autoradiography, and by the prevention of apoptosis by a cell cycle blockade, and by the production of a semi-synchronised wave of apoptoses after release of this blockade. These cell blockading results further suggest that entry into the apoptotic death program requires cells to be in a particular cell cycle stage, and it seems most likely that the decision to die was made in early G1. Tissue and cell growth rates, cell loss and death rates, cell birth rates and cell immigration rates were all determined for the VBW region throughout the time period studied.</p>

scholarly journals Positive feedback between lncRNA FLVCR1-AS1 and KLF10 may inhibit pancreatic cancer progression via the PTEN/AKT pathway

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jiewei Lin ◽  
Shuyu Zhai ◽  
Siyi Zou ◽  
Zhiwei Xu ◽  
Jun Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Positive Feedback ◽  
Molecular Mechanisms ◽  
Tumor Tissues ◽  
And Migration

Abstract Background FLVCR1-AS1 is a key regulator of cancer progression. However, the biological functions and underlying molecular mechanisms of pancreatic cancer (PC) remain unknown. Methods FLVCR1-AS1 expression levels in 77 PC tissues and matched non-tumor tissues were analyzed by qRT-PCR. Moreover, the role of FLVCR1-AS1 in PC cell proliferation, cell cycle, and migration was verified via functional in vitro and in vivo experiments. Further, the potential competitive endogenous RNA (ceRNA) network between FLVCR1-AS1 and KLF10, as well as FLVCR1-AS1 transcription levels, were investigated. Results FLVCR1-AS1 expression was low in both PC tissues and PC cell lines, and FLVCR1-AS1 downregulation was associated with a worse prognosis in patients with PC. Functional experiments demonstrated that FLVCR1-AS1 overexpression significantly suppressed PC cell proliferation, cell cycle, and migration both in vitro and in vivo. Mechanistic investigations revealed that FLVCR1-AS1 acts as a ceRNA to sequester miR-513c-5p or miR-514b-5p from the sponging KLF10 mRNA, thereby relieving their suppressive effects on KLF10 expression. Additionally, FLVCR1-AS1 was shown to be a direct transcriptional target of KLF10. Conclusions Our research suggests that FLVCR1-AS1 plays a tumor-suppressive role in PC by inhibiting proliferation, cell cycle, and migration through a positive feedback loop with KLF10, thereby providing a novel therapeutic strategy for PC treatment.

scholarly journals Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

scholarly journals Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ethan P. Metz ◽  
Erin L. Wuebben ◽  
Phillip J. Wilder ◽  
Jesse L. Cox ◽  
Kaustubh Datta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Cycle Machinery

Abstract Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

2004 ◽  
Vol 286 (3) ◽  
pp. L506-L513 ◽  
Author(s):  
Christopher E. Helt ◽  
Rhonda J. Staversky ◽  
Yi-Jang Lee ◽  
Robert A. Bambara ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Nuclear Antigen ◽  
Cell Cycle Regulators ◽  
Amino Terminal ◽  
Binding Domains ◽  
Green Fluorescent

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1 when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of the other Cip or INK4 G1 kinase inhibitors. To confirm the role of p21 in G1 arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1 arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1 during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1 arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

scholarly journals Differentiation of Mesenchymal Stem Cells to Neuroglia: in the Context of Cell Signalling

2019 ◽  
Vol 15 (6) ◽  
pp. 814-826 ◽  
Author(s):  
Sajan George ◽  
Michael R. Hamblin ◽  
Heidi Abrahamse
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adult Stem Cells ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Cell Signalling ◽  
Specific Cell ◽  
Ongoing Research ◽  
Neural Lineage

Abstract The promise of engineering specific cell types from stem cells and rebuilding damaged or diseased tissues has fascinated stem cell researchers and clinicians over last few decades. Mesenchymal Stem Cells (MSCs) have the potential to differentiate into non-mesodermal cells, particularly neural-lineage, consisting of neurons and glia. These multipotent adult stem cells can be used for implementing clinical trials in neural repair. Ongoing research identifies several molecular mechanisms involved in the speciation of neuroglia, which are tightly regulated and interconnected by various components of cell signalling machinery. Growing MSCs with multiple inducers in culture media will initiate changes on intricately interlinked cell signalling pathways and processes. Net result of these signal flow on cellular architecture is also dependent on the type of ligands and stem cells investigated in vitro. However, our understanding about this dynamic signalling machinery is limited and confounding, especially with spheroid structures, neurospheres and organoids. Therefore, the results for differentiating neurons and glia in vitro have been inconclusive, so far. Added to this complication, we have no convincing evidence about the electrical conductivity and functionality status generated in differentiating neurons and glia. This review has taken a step forward to tailor the information on differentiating neuroglia with the common methodologies, in practice.

scholarly journals Cyclin D3-CDK6 Complex Facilitates Tumorigenesis by Regulating the C-Myc/miR-15a/16 Axis in a Feedback Loop in Gastric Cancer

2020 ◽  
Author(s):  
Yeting Hong ◽  
Wei He ◽  
Jianbin Zhang ◽  
Lu Shen ◽  
Chong Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Feedback Loop ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Cyclin D3 ◽  
Luciferase Reporter

Abstract Background: Cyclin D3-CDK6 complex is a component of the core cell cycle machinery that regulates cell proliferation. By using Human Protein Atlas database, a higher expression level of this complex was found in gastric cancer. However, the function of this complex in gastric cancer remain poorly understood. This study aims to determine the expression pattern of this complex in gastric cancer and to investigate its biological role during tumorigenesis.Methods: To demonstrate that Cyclin D3-CDK6 regulate the c-Myc/miR-15a/16 axis in a feedback loop in gastric cancer, a series of methods were conducted both in vitro and in vivo experiments, including qRT-PCR, western blot analysis, EdU assay, flow cytometry, luciferase reporter assay and immunohistochemical staining. SPSS and Graphpad prism software were used for data analysis.Results: In this study, we found that Cyclin D3 and CDK6 were significantly upregulated in gastric cancer and correlated with poorer overall survival. Further study proved that this complex significantly promoted cell proliferation and cell cycle progression in vitro and accelerated xenografted tumor growth in vivo. Furthermore, we explored the molecular mechanisms through which the complex mediated Rb phosphorylation and then promoted c-Myc expression in vitro, we also found c-Myc could suppress miR-15a/16 expression in gastric cancer cell. Finally, we found that miR-15a/16 can simultaneously regulate Cyclin D3 and CDK6 expression as direct target genes.Conclusions: Our findings uncover the Cyclin D3-CDK6/c-Myc/miR-15a/16 feedback loop axis as a pivotal role in the regulation of gastric cancer tumorigenesis, and this regulating axis may provide a potential therapeutic target for gastric cancer treatment.

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