Gap 2/mitosis Research Articles

CCT6A Regulates Cervical Cancer Cell Glycolysis and Proliferation under Hypoxic Conditions via the Telomerase Cajal Body Protein 1/Telomerase Reverse Tranase.

Cervical cancer is one of the most common female malignancies worldwide, a hypoxic microenvironment usually causes enhanced viability and glycolytic capacity of cervical cancer cells. The aim of this study was to investigate the association between chaperonin containing t-complex polypeptide 1 subunit 6A (CCT6A) and cell proliferation as well as hypoxic glycolysis. Bioinformatics analysis was conducted to explore the association between cervical cancer and its partner protein under hypoxic conditions, namely, CCT6A. Subsequently, the expression of CCT6A was silenced, and the effects of CCT6A silencing on cervical cancer cell proliferation, cell cycle, glycolysis-related proteins, and telomerase activity were examined. Bioinformatics analysis was performed to investigate the expression of CCT6A in cervical cancer under hypoxic conditions. The expression of CCT6A was silenced in cervical cancer cells, HeLa and SiHa, to study its effects on cell proliferation and hypoxic glycolysis. The localization of telomerase activity-related proteins, telomerase CTAB1 and TERT, was detected using immunofluorescence, and their interaction was assessed using immunoprecipitation. A cellular hypoxia model was established, and the products of the glycolysis reaction were detected. A nude mouse tumor model was constructed, and the changes in glycolysis-related proteins in tumor tissues were examined using Western blot, while Ki67 expression in tumor tissues was evaluated by immunohistochemistry. Telomerase activity was found to be enhanced in CCT6A-silenced cervical cancer cells, along with an increase in telomerase TCAB1 and TERT protein binding associated with telomerase activity. Additionally, the proportion of cells in the Gap 2/mitosis (G2/M) stage and the 5-ethynyl-2'-deoxyuridine (EdU) positivity rate were decreased in CCT6A-silenced cells, indicating a reduction in cell proliferation. The expression of cell cycle-related proteins, including cyclin E, CCNA2, and cyclin-dependent kinase 2 (CDK2), was suppressed. Furthermore, under a hypoxic environment, silencing CCT6A led to a significant reduction in cell viability and downregulation of glycolysis-related proteins, such as lactate dehydrogenase A (LDHA) and hexokinase 2 (HK2). Mechanistically, silencing CCT6A may reduce telomerase activity by inhibiting the TCAB1/TERT interaction. Additionally, TERT was found to activate the promoter region of the HK2 gene, and inhibition of TERT activity reduced the transcriptional level of HK2. The study primarily explored the involvement of CCT6A in cervical cancer, yet it did not account for the myriad of other elements potentially influencing cell proliferation and glycolysis. It is essential to recognize that cervical cancer's etiology is multifaceted, shaped by an array of genetic variations, environmental influences, and protein interactions. Silencing CCT6A could effectively attenuate the upregulation of cell proliferation and glycolytic function mediated by TCAB1/TERT in cervical cancer cells.

Ethanol extract of Imperata Cylindrica leaves inhibits proliferation and migration of HSC-3 cell lines

Background: Squamous cell carcinoma (SCC) is classified as the most common type of oral cancer up to 90% of all malignant neoplasm in the oral cavity. Currently, the only treatments for SCC are surgery and/or radiation or chemotherapy, which can cause various side effects. Cogon grass leaves (Imperata cylindrica) have been considered an alternative cancer treatment that may reduce side effects. Imperata cylindrica (I. cylindrica) leaf extract can inhibit cancer cell proliferation and migration by withholding the cell cycle in the gap 1/synthesis (G1/S) and gap 2/mitosis (G2/M) phases. Therefore, the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) is decreased and cancer is not progressing. Purpose: The study aims to determine the effect of I. cylindrica leaf extract on the proliferation and migration of human oral squamous carcinoma-3 (HSC-3) cell lines. Methods: This in vitro experimental study was conducted with nine study groups. The treatment group was divided into seven concentrations—640 ppm, 320 ppm, 160 ppm, 80 ppm, 40 ppm, 20 ppm and 10 ppm. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium-bromide (MTT) assay and scratch assay were carried out to assess the effect of I. cylindrica leaf extract on HSC-3 cell proliferation and migration. Results: Ethanol extract of I. cylindrica has a significant effect compared to the negative control towards the proliferation and migration of HSC-3 cells. Conclusion: This study shows that I. cylindrica ethanol leaf extract can inhibit proliferation and migration of HSC-3 cells.

Hemin, a major heme molecule, induced cellular and genetic alterations in normal colonic and colon cancer cells

Heme, a molecule abundant in red meat, is assumed to exert carcinogenic effects on normal colonic cells and tumour suppressive effects on cancer cells, though the hypothesis has not been explicitly proven yet. The present study aims to investigate hemin induced cytotoxic, genetic and biological alterations in both normal and cancerous colonic epithelial cells, which may imply its carcinogenic and anticarcinogenic properties. Normal colonic epithelial cells and colon carcinoma cells were treated with a 0–500 µM concentration of hemin for 1–4 days following which cytotoxicity and wound healing assays, western blot, rt-PCR and cell cycle analysis were performed. Interestingly, hemin was cytotoxic to normal colonic cells, but carcinoma cells were more resistant. Cell migration potential of both normal colonic cells and colon carcinoma cells was impeded by hemin. Hemin caused upregulation of both P53 and β-catenin gene and proteins expression in normal colonic cells with concomitant cell cycle arrest at G1(Gap 1) and G2/M (Gap 2/ Mitosis). G1 and G2 cell cycle arrests were also observed in colon carcinoma cells. In conclusion, the present study confirms that hemin, a main heme molecule present in red meat, facilitates behavioural, genetic and cell cycle kinetic alterations in both normal colonic epithelial and colon carcinoma cells.

Overcoming Radioresistance in Tumor Therapy by Alleviating Hypoxia and Using the HIF-1 Inhibitor.

Radiotherapy has been extensively used to treat cancer patients because it can effectively damage most solid tumors without penetration limits. A hypoxic microenvironment in solid tumors leads to severe radioresistance and expression of hypoxic inducible factor-1 (HIF-1), which results in poor efficacy of radiotherapy alone. Herein, we report the excellent efficacy of radiotherapy achieved using a new type of yolk-shell Cu2-xSe@PtSe (CSP) nanosensitizer functionalized with the HIF-1α inhibitor acriflavine (ACF). We prepare the CSP nanosensitizer through the interfacial redox reactions between chloroplatinic acid and Cu2-xSe nanoparticles (CS) and then functionalize the nanosensitizer with ACF through their electrostatic interactions. We show that the synthesized CSP nanosensitizer can arrest the cell cycle (i.e., at the gap 2/mitosis (G2/M) phases) of tumor cells to enhance their sensitivity to X-rays and decompose endogenous H2O2 into O2 to reduce hypoxia and increase the production of reactive oxygen species, which leads to severe damage to DNA double strands and apoptosis of tumor cells. We also show that the ACF on the surface of CSP nanoparticles can effectively reduce the expression of HIF-1α. All these effects lead to a low vascular endothelial growth factor, low density of microvessels in tumor, decreased cell proliferation, and increased cell apoptosis, which synergistically and drastically enhance the efficacy of radiotherapy. This work provides insights and guidance for developing novel nanosensitizers to enhance the efficacy of radiotherapy.

Enhanced UV Resistance Role of Death Domain–Associated Protein in Human MDA-MB-231 Breast Cancer Cells by Regulation of G2 DNA Damage Checkpoint

Purpose: Death domain–associated protein (DAXX) is a multifunctional nuclear protein involved in apoptosis, transcription, deoxyribonucleic acid damage response, and tumorigenesis. However, the role of DAXX in breast cancer development and progression remains elusive. In this study, we examined the expression patterns and function of DAXX in human breast cancer samples and cell lines. Methods: Immunohistochemistry was used to analyze the expression and localization patterns of DAXX. Additionally, we investigated whether DAXX played an intrinsic role in the cellular response to damage induced by ultraviolet (UV) irradiation in MDA-MB-231 breast cancer cells (isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma). Results: Our results showed that nucleus size, chromatin organization, and DAXX localization were altered in breast cancer tissues compared with those in control tissues. Compared with cytoplasmic and nuclear expression in benign breast tissues, DAXX was colocalized with promyelocytic leukemia in nuclei with a granular distribution. Endogenous DAXX messenger ribonucleic acid levels were upregulated upon UV radiation in MDA-MB-231 cells. DAXX-deficient cells tended to be more sensitive to irradiation than control cells. Conversely, DAXX-overexpressing cells exhibited reduced phosphorylated histone H2AX (γ-H2AX) accumulation, increased cell survival, and resistance to UV-induced damage. The protective effects of DAXX may be related to the activation of the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (ATM-CHK2)-cell division cycle 25c (CDC25c) signaling pathways in Gap2/Mitosis (G2/M) checkpoint and ultimately cell cycle arrest at G2/M phase. Conclusions: Taken together, these results suggested that DAXX may be an essential component in breast cancer initiation, malignant progression, and radioresistance.

Isorhamnetin Induces Cell Cycle Arrest and Apoptosis Via Reactive Oxygen Species-Mediated AMP-Activated Protein Kinase Signaling Pathway Activation in Human Bladder Cancer Cells.

Isorhamnetin is an O-methylated flavonol that is predominantly found in the fruits and leaves of various plants, which have been used for traditional herbal remedies. Although several previous studies have reported that this flavonol has diverse health-promoting effects, evidence is still lacking for the underlying molecular mechanism of its anti-cancer efficacy. In this study, we examined the anti-proliferative effect of isorhamnetin on human bladder cancer cells and found that isorhamnetin triggered the gap 2/ mitosis (G2/M) phase cell arrest and apoptosis. Our data showed that isorhamnetin decreased the expression of Wee1 and cyclin B1, but increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, and increased p21 was bound to Cdk1. In addition, isorhamnetin-induced apoptosis was associated with the increased expression of the Fas/Fas ligand, reduced ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein (Bax) expression, cytosolic release of cytochrome c, and activation of caspases. Moreover, isorhamnetin inactivated the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway by diminishing the adenosine triphosphate (ATP) production due to impaired mitochondrial function. Furthermore, isorhamnetin stimulated production of intracellular reactive oxygen species (ROS); however, the interruption of ROS generation using a ROS scavenger led to an escape from isorhamnetin-mediated G2/M arrest and apoptosis. Collectively, this is the first report to show that isorhamnetin inhibited the proliferation of human bladder cancer cells by ROS-dependent arrest of the cell cycle at the G2/M phase and induction of apoptosis. Therefore, our results provide an important basis for the interpretation of the anti-cancer mechanism of isorhamnetin in bladder cancer cells and support the rationale for the need to evaluate more precise molecular mechanisms and in vivo anti-cancer properties.

Anticancer Activity of Ramalin, a Secondary Metabolite from the Antarctic Lichen Ramalina terebrata, against Colorectal Cancer Cells.

Colorectal cancer is a leading cause of death worldwide and occurs through the highly complex coordination of multiple cellular pathways, resulting in carcinogenesis. Recent studies have increasingly revealed that constituents of lichen extracts exhibit potent pharmaceutical activities, including anticancer activity against various cancer cells, making them promising candidates for new anticancer therapeutic drugs. The main objective of this study was to evaluate the anticancer capacities of ramalin, a secondary metabolite from the Antarctic lichen Ramalina terebrata, in the human colorectal cancer cell line HCT116. In this study, ramalin displayed concentration-dependent anticancer activity against HCT116 cells, significantly suppressing proliferation and inducing apoptosis. Furthermore, ramalin induced cell cycle arrest in the gap 2/mitosis (G2/M) phase through the modulation of hallmark genes involved in the G2/M phase transition, such as tumour protein p53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin-dependent kinase 1 (CDK1) and cyclin B1 (CCNB1). At both the transcriptional and translational level, ramalin caused a gradual increase in the expression of TP53 and its downstream gene CDKN1A, while decreasing the expression of CDK1 and CCNB1 in a concentration-dependent manner. In addition, ramalin significantly inhibited the migration and invasion of colorectal cancer cells in a concentration-dependent manner. Taken together, these data suggest that ramalin may be a therapeutic candidate for the targeted therapy of colorectal cancer.

BRCA1 and HSP90 cooperate in homologous and non-homologous DNA double-strand-break repair and G2/M checkpoint activation

Expression of functional breast cancer susceptibility gene 1 (BRCA1) in human breast and ovarian cancers is associated with resistance to platinum-based chemotherapeutics and poly(ADP ribose) polymerase (PARP) inhibitors. BRCA1 is a nuclear tumor suppressor that is critical for resolving double-strand DNA breaks (DSBs) and interstrand crosslinks (ICLs) by homologous recombination (HR). In vitro, animal and human clinical data have demonstrated that BRCA1-deficient cancers are highly sensitive to ICL-inducing chemotherapeutic agents, are amenable to synthetic lethal approaches that exploit defects in DSB/ICL repair, and may be associated with improved survival. Conversely, high or restored expression of BRCA1 in breast and ovarian cancer is associated with therapeutic resistance and poor prognosis. There has been much interest in identifying agents that interfere with BRCA1-dependent DSB/ICL repair to restore or enhance sensitivity to cancer therapeutics. We demonstrate that the heat-shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin [17-AAG (Tanespimycin)], currently in Phase II/III clinical evaluation for several cancers, induces BRCA1 ubiquitination and proteasomal degradation, resulting in compromised repair of ionizing radiation- and platinum-induced DNA damage. We show that loss of HSP90 function abolishes BRCA1-dependent DSB repair and that BRCA1-deficient cells are hypersensitive to 17-AAG due to impaired Gap 2/Mitosis (G2/M) checkpoint activation and resultant mitotic catastrophe. In summary, we document an upstream HSP90-dependent regulatory point in the Fanconi anemia/BRCA DSB/ICL repair pathway, illuminate the role of BRCA1 in regulating damage-associated checkpoint and repair responses to HSP90 inhibitors, and identify BRCA1 as a clinically relevant target for enhancing sensitivity in refractory and/or resistant malignancies.

Induction of growth arrest by miR-542-3p that targets survivin

Survivin is a protein which functions as a mitotic regulator as well as apoptosis inhibitor. In this study, we show that introduction of synthetic miR-542-3p mimetic reduced both mRNA and protein levels of survivin. In A549 cells, luciferase reporter assay revealed that miR-542-3p targeted predicted binding sites in the 3′-untranslated region (3′-UTR) of survivin. We also demonstrate that ectopic expression of miR-542-3p inhibited cell proliferation by inducing Gap 1 (G1) and Gap 2/Mitosis (G2/M) cell cycle arrest. Collectively, these results suggest that survivin is a direct target of miR-542-3p and growth inhibition by miR-542-3p may have a potential utility as an anti-cancer therapy.

Comparison of Cellular Metabolic Responses of18F-FDG According to the Effect ofβ-Irradiation in p53 Wild and Deleted Cell Lines

The tumor suppressor gene, p53, plays a pivotal role for cell survival and apoptosis, which could cause different responses to therapeutic agents. Rhenium-188 ((188)Re) decays with the emission of a beta-particle with high energy and is expected to be an important candidate of radiotherapy. We investigated the cellular response of (18)F-fluorodeoxyglucose ((18)F-FDG) uptake and the effect of p53 according to beta-irradiation by (188)Re. The HCT116 human colon adenocarcinoma cell lines, containing a wild-type p53 (p53(+/+)) and a p53-deleted derivative (p53(-/-)), were gifts from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). Cells were plated in 24-well plates at 1.0 x 10(5) cells, then (188)Re perrhenate was added and incubated for 24 hours. After irradiation, we performed a cellular uptake assay of (18)F-FDG (370 kBq, 60 minutes). We assayed the hexokinase, cell viability, and cell cycle. p53-deleted HCT116 cells showed a higher (18)F-FDG uptake and increased hexokinase activity after (188)Re treatment. p53-deleted cells showed a higher G2/M (Gap2/Mitosis) arrest in a relatively low dose of beta-irradiation. However, cell viability was not different, according to the p53 status, after (188)Re treatment. Therefore, p53 seemed to have a significant role in cellular glucose metabolism and G2/M checkpoint, according to beta-irradiation, and could cause a different therapeutic response of (18)F-FDG uptake in cancer cells.

Docosahexaenoic Acid (DHA), a Primary Tumor Suppressive Omega-3 Fatty Acid, Inhibits Growth of Colorectal Cancer Independent of p53 Mutational Status

Human colon carcinoma COLO 205, carrying wild type p53, grown subcutaneously in athymic mice was inhibited 80% by a high fat menhaden oil diet containing a mixture of omega-3 fatty acids compared to the low fat corn oil diet containing omega-6 fatty acids. Feeding a high fat diet of golden algae oil containing docosahexaenoic acid (DHA) as the sole long chain omega-3 fatty acid resulted in 93% growth inhibition. Similar findings were previously reported for WiDr colon carcinoma containing mutated p53 (His237). In vitro, 125 μM DHA inhibited COLO 205 growth by 81%, WiDr by 42%, while eicosapentaenoic acid (EPA) marginally inhibited growth of both lines by approximately 30%. DHA inhibited cell proliferation by 41% in WiDr but did not significantly inhibit proliferation in COLO 205. Cell cycle analysis revealed that DHA arrested cell cycle at Resting/Gap 1 (G0/G1 phase) in WiDr and at Gap 2/Mitosis (G2/M) phase in COLO 205. DHA induced apoptosis in COLO 205 but not in WiDr, and EPA did not induce apoptosis in either line. Taken together, these findings suggest DHA is the primary tumor suppressive ω-3 fatty acid in vivo and in vitro and inhibits cancer growth by p53 dependent and independent pathways, while the marginal inhibition by EPA is p53 independent.

Human Herpesvirus 7 Infection Induces Profound Cell Cycle Perturbations Coupled to Disregulation of cdc2 and Cyclin B and Polyploidization of CD4+ T Cells

Human herpesvirus 7 (HHV-7) infection of both primary CD4+ T lymphocytes and SupT1 lymphoblastoid T-cell line induced a progressive accumulation of cells exibiting a gap 2/mitosis (G2/M) and polyploid content coupled to an increased cell size. The expression of both cyclin-dependent kinase cdc2 and cyclin B was increased in HHV-7–infected cells with respect to the uninfected ones. Moreover, the simultaneous flow cytometric analysis of cyclin B and DNA content showed that cyclin B expression was not only increased but also unscheduled with respect to its usual cell cycle pattern. However, the levels of kinase activity associated to cdc2 were decreased in HHV-7–infected cells with respect to uninfected cultures. To elucidate the origin of the enlarged HHV-7–infected cells, extensive electron and confocal microscopy analyses were performed. Membrane fusion events associated to cytoplasmic bridges, which characterize the formation of syncytia, were never observed. On the other hand, analysis of serial sections of the same cells strongly suggested that enlarged HHV-7–infected cells contained a single polylobated nucleus. This was confirmed by flow cytometry analysis performed on nuclei isolated from HHV-7–infected cells, which showed multiple peaks with a DNA content >4n. Taken together, these data indicate that giant cells, which represent the hallmark of in vitro HHV-7 infection, arise from single CD4+ T cells undergoing a process of polyploidization.© 1998 by The American Society of Hematology.

Human Herpesvirus 7 Infection Induces Profound Cell Cycle Perturbations Coupled to Disregulation of cdc2 and Cyclin B and Polyploidization of CD4+ T Cells

Human herpesvirus 7 (HHV-7) infection of both primary CD4+ T lymphocytes and SupT1 lymphoblastoid T-cell line induced a progressive accumulation of cells exibiting a gap 2/mitosis (G2/M) and polyploid content coupled to an increased cell size. The expression of both cyclin-dependent kinase cdc2 and cyclin B was increased in HHV-7–infected cells with respect to the uninfected ones. Moreover, the simultaneous flow cytometric analysis of cyclin B and DNA content showed that cyclin B expression was not only increased but also unscheduled with respect to its usual cell cycle pattern. However, the levels of kinase activity associated to cdc2 were decreased in HHV-7–infected cells with respect to uninfected cultures. To elucidate the origin of the enlarged HHV-7–infected cells, extensive electron and confocal microscopy analyses were performed. Membrane fusion events associated to cytoplasmic bridges, which characterize the formation of syncytia, were never observed. On the other hand, analysis of serial sections of the same cells strongly suggested that enlarged HHV-7–infected cells contained a single polylobated nucleus. This was confirmed by flow cytometry analysis performed on nuclei isolated from HHV-7–infected cells, which showed multiple peaks with a DNA content >4n. Taken together, these data indicate that giant cells, which represent the hallmark of in vitro HHV-7 infection, arise from single CD4+ T cells undergoing a process of polyploidization.© 1998 by The American Society of Hematology.

Mechanisms of cell cycle blocks at the G2/M transition and their role in differentiation and development.

One of the most critical events in the life of a cell is segregation of chromosomes at meiosis and mitosis. Improper segregation of the genetic material during both mitosis and meiosis would be detrimental to the daughter cells. Therefore, cells have evolved many controls and checkpoints to ensure that a critical size is met, DNA is undamaged and correctly replicated, and that the centrosome and spindle apparatus are intact before cell division occurs (Hartwell and Weinert 1989; Murray 1992). A principal point for control of the cell cycle is at the Gap2/mitosis (G2/M) transition. This chapter will concentrate on the mechanisms of negative growth control at the G2/M transition in multicellular eukaryotes. Also, we will review how cells alter these G2/M controls in both cell differentiation and development. Since the genetics of cell-cycle checkpoints in yeast have been recently reviewed, these studies will not be extensively covered; instead, the yeast studies will be referred to where they best illustrate the point (Stewart and Enoch 1996; Elledge 1996).

Fibrillation

Heme, a molecule abundant in red meat, is assumed to exert carcinogenic effects on normal colonic cells and tumour suppressive effects on cancer cells, though the hypothesis has not been explicitly proven yet. The present study aims to investigate hemin induced cytotoxic, genetic and biological alterations in both normal and cancerous colonic epithelial cells, which may imply its carcinogenic and anticarcinogenic properties. Normal colonic epithelial cells and colon carcinoma cells were treated with a 0–500 µM concentration of hemin for 1–4 days following which cytotoxicity and wound healing assays, western blot, rt-PCR and cell cycle analysis were performed. Interestingly, hemin was cytotoxic to normal colonic cells, but carcinoma cells were more resistant. Cell migration potential of both normal colonic cells and colon carcinoma cells was impeded by hemin. Hemin caused upregulation of both P53 and β-catenin gene and proteins expression in normal colonic cells with concomitant cell cycle arrest at G1(Gap 1) and G2/M (Gap 2/ Mitosis). G1 and G2 cell cycle arrests were also observed in colon carcinoma cells. In conclusion, the present study confirms that hemin, a main heme molecule present in red meat, facilitates behavioural, genetic and cell cycle kinetic alterations in both normal colonic epithelial and colon carcinoma cells.