Cyclin B-Cdc2 Research Articles

Chemical Modification of Curcumin Increases Its Potency against Hypopharyngeal Carcinoma

Hypopharyngeal carcinoma is notorious for its poor prognosis among all head and neck cancers, posing a persistent challenge in clinical settings. The continuous hyperactivation of the NFκB signaling pathway has been noted in various cancer types, including hypopharyngeal carcinoma. In our quest to develop a novel drug that targets hypopharyngeal cancer via the NFκB pathway, we employed curcumin, a well-known lead compound, and performed chemical modifications to create a mono-carbonyl analog called L42H17. This compound exhibited exceptional stability and displayed an enhanced binding affinity to myeloid differentiation protein 2 (MD2). Consistent with expectations, L42H17 demonstrated the ability to inhibit TNF-α-induced phosphorylation of IKK, prevent IκB degradation, and subsequently impede NFκB-p65 nuclear translocation in hypopharyngeal cancer cells. Additionally, L42H17 exhibited a remarkable capacity to induce cell cycle arrest at the G2-M phase by inactivating the cdc2-cyclin B1 complex. Moreover, it facilitated cell apoptosis by reducing Bcl-2 levels and augmenting the expression of cle-PARP and cle-caspase3. Importantly, we observed a significant enhancement in the anti-cancer efficacy of L42H17 in a patient-derived tumor xenograft (PDTX) model of hypopharyngeal carcinoma. In conclusion, our findings strongly suggest that L42H17 holds promise as a potential candidate drug for the treatment of hypopharyngeal carcinoma in the future.

Pro-Apoptotic and Immunotherapeutic Effects of Carbon Nanotubes Functionalized with Recombinant Human Surfactant Protein D on Leukemic Cells.

Nanoparticles are efficient drug delivery vehicles for targeting specific organs as well as systemic therapy for a range of diseases, including cancer. However, their interaction with the immune system offers an intriguing challenge. Due to the unique physico-chemical properties, carbon nanotubes (CNTs) are considered as nanocarriers of considerable interest in cancer diagnosis and therapy. CNTs, as a promising nanomaterial, are capable of both detecting as well as delivering drugs or small therapeutic molecules to tumour cells. In this study, we coupled a recombinant fragment of human surfactant protein D (rfhSP-D) with carboxymethyl-cellulose (CMC) CNTs (CMC-CNT, 10–20 nm diameter) for augmenting their apoptotic and immunotherapeutic properties using two leukemic cell lines. The cell viability of AML14.3D10 or K562 cancer cell lines was reduced when cultured with CMC-mwCNT-coupled-rfhSP-D (CNT + rfhSP-D) at 24 h. Increased levels of caspase 3, 7 and cleaved caspase 9 in CNT + rfhSP-D treated AML14.3D10 and K562 cells suggested an involvement of an intrinsic pathway of apoptosis. CNT + rfhSP-D treated leukemic cells also showed higher mRNA expression of p53 and cell cycle inhibitors (p21 and p27). This suggested a likely reduction in cdc2-cyclin B1, causing G2/M cell cycle arrest and p53-dependent apoptosis in AML14.3D10 cells, while p53-independent mechanisms appeared to be in operation in K562 cells. We suggest that CNT + rfhSP-D has therapeutic potential in targeting leukemic cells, irrespective of their p53 status, and thus, it is worth setting up pre-clinical trials in animal models.

PKCβ1 regulates meiotic cell cycle in mouse oocyte

ABSTRACT PKCβI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCβI in mouse oocyte meiotic maturation. PKCβI and p-PKCβI (phosphor-PKCβI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCβI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCβI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCβI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCβI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCβI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCβI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint

Proteasome mediated degradation of CDC25C and Cyclin B1 in Demethoxycurcumin treated human glioma U87 MG cells to trigger G2/M cell cycle arrest

Recently, we have reported that Demethoxycurcumin induced Reactive oxygen species via inhibition of Mitochondrial Superoxide Dismutase is an initial event to trigger apoptosis through caspase-8 and 9 activation and to inhibit Akt/NF-κB survival signaling in human glioma U87 MG cells (Kumar et al., 2018). Although cell-cycle disruption had been suggested to be the possible mechanism for DMC inhibitory effect on human glioma U87 MG cells, comprehensive mechanisms of cell-cycle arrest caused by DMC are not fully understood. The present study was designed to elucidate the DMC induced mechanism of cell cycle arrest in human glioma U87 MG cells. In this study, the results illustrated that DMC induced Reactive oxygen species (ROS) leads to reduced expression of CDC25C, Cyclin B1 and CDK1 (Thr161) triggers G2/M cell cycle arrest in U87 MG glioma cells. Moreover, the DMC induced ROS generation activates ubiquitination and proteasome degradation of CDC25C and Cyclin B1 in U87MG glioma cells. In addition, the immunoprecipitation results showed that significant dissociation of CDK1or CDC2-Cyclin B1 complex leads to G2/M cell cycle arrest. To explore the possibility of direct involvement of DMC in the dissociation of CDK1/Cyclin B1 complex, the molecular docking and MD simulation studies were carried. The results showed that DMC nicely fitted into the binding site of CDK1 and Cyclin B1 with minimum binding energy (ΔG) of -9.46 kcal/mol (Ki = 0.11 μM) and − 9.90 kcal/mol (Ki = 0.05 μM) respectively. Therefore, this is the first study demonstrating CDC25C and Cyclin B1 proteins could be used as potential target for anticancer therapy and DMC may be explored as new therapeutic agent in the cure of Glioblastoma (GBM).

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and in vivo through induction of cell cycle arrest at the G2-M phase.

e16024 Background: Protein Kinase D (PKD) is implicated in tumor growth, death, invasion, and progression. CRT0066101 is an inhibitor of PKD and has antitumor activity in several types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer remain unknown. Methods: The MTS assay was used to evaluate the ability of CRT0066101 to inhibit cellular proliferation in bladder cancer cells. Cell cycle was analyzed by flow cytometry. Protein expression and phosphorylation were assessed by western blotting. Results: We showed that CRT0066101 suppressed the proliferation and migration of 4 bladder cancer cell lines in vitro. We also demonstrated that CRT0066101 inhibited tumor growth in an in vivo mouse model of bladder cancer. To verify the role of PKD in bladder tumor, we found that PKD2 was highly expressed in 8 bladder cancer lines and that RNA interference-mediated silencing of the PKD2 gene dramatically reduced bladder cancer growth in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was confirmed by demonstrating that the levels of PKD2 and phospho-PKD2 (Ser-876) were markedly decreased in CRT0066101-treated bladder cancer. In addition, our cell cycle analysis by flow cytometry revealed that CRT0066101 arrested bladder cancer cells at the G2-M phase. We further validated these data by immunoblotting showing that treatment of bladder carcinoma cells with CRT0066101 downregulated the expression of cyclin B1, cdc2 and cdc25C, but elevated the levels of p27kip1, gadd45a, chk1/2, and wee1. Finally, CRT0066101 was found to increase the phosphorylation of cdc2 and cdc25C, which lead to reduction in cdc2-cyclin B1 activity. Conclusions: These novel findings suggest that CRT0066101 inhibits bladder cancer growth through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression. QQL and IH contributed equally to this work.

Effects of etoposide alone and in combination with piroxicam on canine osteosarcoma cell lines

Osteosarcoma (OSA) is the most common primary bone tumour in dogs. The poor survival rate in dogs with OSA highlights the need for new therapeutic approaches. This study evaluated the cytotoxic effects of etoposide, alone and in combination with piroxicam, on canine OSA cell cultures. Etoposide alone significantly suppressed cell growth and viability, whereas etoposide in combination with piroxicam exhibited concentration dependent cytotoxicity. The anti-proliferative effect was a result of inactivity of the Cdc2-cyclin B1 complex, which correlated with an increase in the G2/M fraction. This subsequently activated the apoptosis cascade, as indicated by elevated apoptosis levels and up-regulation of poly (ADP-ribose) polymerase proteolytic cleavage. Down-regulation of survivin expression induced by the combination treatment may have contributed to the enhanced cytotoxicity. The results of this study suggest that further investigation of etoposide and piroxicam as a therapeutic combination for canine OSA is warranted.

AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest.

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.MethodsWe isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.ResultsCombination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.ConclusionsThese findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.

The mitotic regulator Hec1 is a critical modulator of prostate cancer through the long non-coding RNA BX647187 invitro.

Hec1 (highly expressed in cancer) is a member of a conserved Ndc80 (nuclear division cycle 80) complex that regulates mitotic processes. Its overexpression is seen in various tumours and is associated with cancer progression. However, its expression pattern and role inhuman prostate cancer (PCa) still not clear. The aim of our study is to investigate the expression and functional role of Hec1in human PCa. Hec1 expression was measured in 10 pairs of PCa cancerous and non-cancerous tissue samples by quantitative real-time (qRT)-PCR. The effects of Hec1 on PCa cells were studied by RNAi approach. Apoptosis and cell cycle were analysed by flow cytometry. Cells viability was evaluated using cell counting Kit-8. Cyclin B1-Cdc2 (cell division cycle 2) activity was measured by ELISA assay. Long non-coding (Lnc)RNAs regulated by Hec1 were gained from bioinformatics analysis. The role of LncRNA BX647187, regulated by Hec1, was finally characterized in PCa cells by siRNA. Our results showed that Hec1 mRNA and protein were significantly overexpressed in Human PCa tissues and several PCa cell lines. Silencing Hec1 markedly suppressed proliferation, promoted apoptosis and induced cell-cycle arrest in G2/M-phase in PCa cells. Through bioinformatics analysis and knockdown Hec1in PCa cells, we found LncRNA BX647187 was positively regulated by Hec1. We further demonstrated that suppression of BX647187in PCa cells significantly reduced cell proliferation and promoted apoptosis. Thus, we conclude that Hec1 is consistently overexpressed in human PCa and Hec1 is closely linked with human PCa progression through the meditator LncRNA BX647187. Our studies may contribute to understand the molecular mechanism of PCa pathogenesis and clinical therapy.

P53 Configures the G2/M Arrest Response of Nucleostemin-Deficient Cells.

Nucleostemin (NS) protects the genome from replication-induced DNA damage and has an indispensable role in maintaining the continuous proliferation of both p53-wild-type and mutant cells. Yet, some outcomes of NS-deficient cells appear to be shaped by their p53 status, which stimulates conflicting claims on the role of p53 in executing the NS function. This disparity was conveniently attributed to the usual suspect of cell-type variations. To provide a definitive resolution, we investigated the interplay between NS and p53 in two pairs of isogenic cells, that is, genetically modified mouse embryonic fibroblast (MEF) cells and HCT116 human colon cancer cells. In MEF cells, p53 deletion further compromises rather than rescues the proliferative potential of NS-depleted cells without changing their G2/M arrest fate before prophase entry. The detrimental effect of p53 loss in NS-depleted MEF cells correlates with a dramatic increase of polyploid giant cells (PGCs) (up to 24%), which indicates aberrant mitosis. To determine how p53 shapes the response of cells to NS depletion at the molecular level, we showed that p53 turns on the expression of reprimo and MDM2 in NS-deficient MEF cells. In absence of p53, NS-deficient MEF cells exhibit increased levels of phosphorylated cdc2 (Y15) protein and cyclin B1. In cancer (HCT116) cells, NS loss leads to G2/M arrest under both p53wt and p53ko conditions and increases phosphorylated cdc2 more in p53ko than in p53wt cells, as it does in MEF cells. Unlike its effect in MEF cells, NS depletion decreases tumor growth and increases the expression of reprimo and cyclin B1 in a p53-independent manner in HCT116 cells. Our data indicate that the p53 status of NS-deficient cells orchestrates how they respond to G2/M arrest in a normal versus cancer cell distinct fashion.

Silibinin Combination with Arsenic Strongly Inhibits Survival and Invasiveness of Human Prostate Carcinoma Cells

Effects of silibinin, a naturally occurring flavanone, on prostate carcinoma (PCa) cells in presence of arsenic are not known. Arsenic is clinically approved for leukemia treatment; however, studies are not enough to support its role in the management of solid tumors. In the present study, we observed that silibinin (100 µM) modulated the oxidative status of human PCa DU145 cells exposed to arsenic (0.5 or 5 µM) and inhibited cell growth and survival by primarily inducing autophagy and apoptosis. The silibinin–arsenic combination also inhibited the growth, survival, and clonogenic potential of 22Rv1 PCa cells. Silibinin with 0.5 or 5 µM arsenic induced G1 or G2/M phase arrest, respectively, and decreased the protein levels of CDK2, -4, and -6 and cyclin D1, D3, and E and increased CDK inhibitors p21 and p27. Arsenic alone increased cyclin B1 level and Cdc2 kinase activity which were reduced in silibinin combination. Cell motility and invasiveness along with expression of MMP-2 and vimentin were suppressed. Together, these in vitro findings suggest that in presence of arsenic, silibinin strongly inhibits tumorigenic and metastatic potential of PCa cells.

The Role of CHMP4C on Proliferation in the Human Lung Cancer A549 Cells

The human lung cancer has high incidence rate and mortality among the carcinoma. The research on enhancing the efficacy of therapy for lung cancer is significant. A resent research found that as a subunit of ESCRT-III, CHMP4C functioned to retard abscission timing to coordinate midbody resolution and prevent accumulation of DNA damage in the abscission checkpoint through phosphorylated by AuroraB. In the current study, we evaluated the possible mechanism of the effects of CHMP4C inhibition on cell cycle and cell survival in A549 cells. We found that CHMP4C knockdown caused lagging S phase in cell cycle through enhancing the phosphorylation of Rb, raising the expression of cyclin B1-cdc2 and suppressing the activation of cyclin A. Meanwhile, CHMP4C deletion depressed cell survival via decreasing cell viability and increasing caspase 3/7 activity. This study may promote new significant reference and advance for the mechanism underlying specific function of CHMP4C as well as further research on enhancing therapy effect on non-small lung cancer.

Role of Greatwall kinase in release of mouse oocytes from diplotene arrest.

In eukaryotes, mitosis entry is induced by activation of maturation-promoting factor (MPF), which is regulated by a network of kinases and phosphatases. It has been suggested that Greatwall (GWL) kinase was crucial for the M-phase entry and could maintain cyclin B-Cdc2 activity through regulation of protein phosphatase 2A (PP2A), a counteracting phosphatase of MPF. Here, the role of GWL was assessed during release of mouse oocytes from prophase I arrest. GWL was crucial for meiotic maturation in mouse oocytes. As a positive regulator for meiosis resumption, GWL was continually expressed in germinal vesicle (GV) and MII stage oocytes and two-cell stage embryos. Additionally, GWL localized to the nucleus and dispersed into cytoplasm during GV breakdown (GVBD). Furthermore, downregulation of GWL or overexpression of catalytically-inactive GWL inhibited partial meiotic maturation. This prophase I arrest induced by GWL depletion could be rescued by the PP2A inhibition. However, both GWL-depleted and rescued oocytes had severe spindle defects that hardly reached MII. In contrast, oocytes overexpressing wild-type GWL resumed meiosis and progressed to MII stage. Thus, our data demonstrate that GWL acts in a pathway with PP2A which is essential for prophase I exit and metaphase I microtubule assembly in mouse oocytes.

The tumor suppressive role of NUMB isoform 1 in esophageal squamous cell carcinoma

Esophageal quamous cell carcinoma (ESCC) is the predominant histological type of esophageal carcinoma in Asian populations. To date, few biomarkers have been identified for ESCC. In present study, we found a tumor suppressor, NUMB isoform 1 (NUMB-1), as a promising prognostic biomarker for patients with ESCC. NUMB-1 mRNA was downregulated in 66.7% of primary ESCC tissues when compared with matched adjacent non-tumor tissues. The low expression of NUMB-1 was significantly associated with high tumor recurrence (p=0.029) and poor post-operative overall survival (p=0.016). To further explore the underlying mechanisms by which NUMB-1 regulates ESCC, we demonstrated that ectopic expression of NUMB-1 inhibited cell proliferation through inducing G2/M phase arrest, which was accompanied by an increase in p21 and cyclin B1-cdc2 levels. However, it had no impact on apoptosis of ESCC cells. In addition, overexpression of NUMB-1 prevented epithelial-mesenchymal transition, inhibited invasion of ESCC cells and NOTCH pathway, suppressed Aurora-A activity by preventing phosphorylation of Aurora-A at T288 which resulted in cell cycle arrest. Taken together, our findings suggested NUMB-1 functions as a tumor-suppressor and serves as a prognositc biomarker for ESCC patients; thus, NUMB-1 may be a potential novel therapeutic target for treatment of ESCC.

Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells.

BackgroundChk1 forms a core component of the DNA damage response and small molecule inhibitors are currently being investigated in the clinic as cytotoxic chemotherapy potentiators. Recent evidence suggests that Chk1 inhibitors may demonstrate significant single agent activity in tumors with specific DNA repair defects, a constitutively activated DNA damage response or oncogene induced replicative stress.MethodsGrowth inhibition induced by the small molecule Chk1 inhibitor V158411 was assessed in a panel of human leukemia and lymphoma cell lines and compared to cancer cell lines derived from solid tumors. The effects on cell cycle and DNA damage response markers were further evaluated.ResultsLeukemia and lymphoma cell lines were identified as particularly sensitive to the Chk1 inhibitor V158411 (mean GI50 0.17 μM) compared to colon (2.8 μM) or lung (6.9 μM) cancer cell lines. Chk1 inhibition by V158411 in the leukemia and lymphoma cell lines induced DNA fragmentation and cell death that was both caspase dependent and independent, and prevented cells undergoing mitosis. An analysis of in vitro pharmacodynamic markers identified a dose dependent decrease in Chk1 and cyclin B1 protein levels and Cdc2 Thr15 phosphorylation along with a concomitant increase in H2AX phosphorylation at Ser139 following V158411 treatment.ConclusionsThese data support the further evaluation of Chk1 inhibitors in hematopoietic cancers as single agents as well as in combination with standard of care cytotoxic drugs.

RGC-32 is essential for TGF-β mediated Th17 cell differentiation (LYM3P.728)

Abstract Our group has cloned and characterized the Response Gene to Complement (RGC)-32 which is induced by C5b-9 and exerts an important role in cell cycle activation via cyclin B1-CDC2 activation and Akt phosphorylation. RGC-32 is also a downstream target of TGF-β and plays a role in fibrogenesis. As Th17 and induced T regulatory cells (iTreg) differentiate under the control of TGF-β, we determined whether RGC-32 plays a role in this process using RGC-32 KO mice generated in our lab. RGC-32 was not required for peripheral T cell homeostasis as we observed normal cellularity, T cell subset composition and phenotypic markers. However, RGC-32 KO CD4+ T cells failed to differentiate normally to Th17 lineage as they exhibited a significant reduction in IL-17 and RORγt mRNA and protein expression in vitro and a decreased proportion of IL-17+ cells in lamina propria lymphocytes in vivo. The defect in IL-17 expression appeared to be mediated by decreased expression of IL-6R and TGF-βRI at baseline and under Th17 conditions and was not restored by IL-2 inhibition. A trend for increased iTreg differentiation was noted in RGC-32 KO CD4+ T cells under Treg skewing conditions but the difference was not statistically significant. Differentiation of CD4+ T cells into Th1 cells was decreased in RGC-32 KO mice while Th2 cells developed normally. These results suggest that RGC-32 is a novel mediator of Th17 differentiation in vitro and in vivo through modulation of IL-6R and TGF-βRI expression.

Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

Hip2 ubiquitin-conjugating enzyme overcomes radiation-induced G2/M arrest

Radiation induces cell cycle arrest and/or cell death in mammalian cells. In the present study, we show that Hip2, a ubiquitin-conjugating enzyme, can overcome radiation-induced G2/M cell cycle arrest and trigger the entry into mitosis. Ionizing radiation increased the levels of Hip2 by preventing its degradation but not its gene transcription. The stability of Hip2 in irradiated cells was further confirmed using live cell fluorescence imaging. Flow cytometric and molecular analyses revealed that Hip2 abrogated radiation-induced G2/M arrest, promoting entry into mitosis. Bimolecular fluorescence complementation assays and co-immunoprecipitation experiments showed that Hip2 interacted with and targeted p53 for degradation via the ubiquitin proteasome system, resulting in the activation of cdc2-cyclin B1 kinase to promote mitotic entry. These results contribute to our understanding of the mechanisms that regulate cell cycle progression and DNA damage-induced G2/M checkpoint cellular responses.

DAT-230, a Novel Microtubule Inhibitor, Induced Aberrant Mitosis and Apoptosis in SGC-7901 Cells

2-Methoxy-5-(2-(3,4,5-trimethoxyphenyl)thiophen-3-yl) aniline (DAT-230) is a novel synthesized compound of combretastatin-A-4 derivative with more stability. The present study is to investigate its anti-tumor activity and molecular mechanisms in human gastric adenocarcinoma SGC-7901 cells. DAT-230 inhibited SGC-7901 cells growth. The treatment of DAT-230 resulted in microtubule de-polymerization and G2/M phase arrest. Besides the accumulation and translocation of Cyclin B1, reduction of p-14/15-cdc2 and mitosis delay denoted the Cyclin B1-cdc2 complex active and M phase arrest in SGC-7901 cells treated with DAT-230. Mitochondria pathway participated in apoptosis after G2/M arrest in SGC-7901 cells treated with DAT-230, which was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP), activation of caspase-3 and caspase-9, changes of Bcl-2 and Bax expression, decrease of mitochondrial membrane potential and release of cytochrome c from mitochondria. In vivo, DAT-230 delayed tumor growth in BALB/c nude mice with human gastric adenocarcinoma xenografts. Besides apoptosis was detected with terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay in tumor tissue. In conclusion, DAT-230 is a promising microtubule inhibitor with great anti-tumor activity to SGC-7901, in vitro and in vivo. Its potential to be a candidate of anti-cancer agent is worth of being further investigated.

Jaceosidin, isolated from dietary mugwort (Artemisia princeps), induces G2/M cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation

Jaceosidin, a flavonoid derived from Artemisia princeps (Japanese mugwort), has been shown to inhibit the growth of several human cancer cells, However, the exact mechanism for the cytotoxic effect of jaceosidin is not completely understood. In this study, we investigated the molecular mechanism involved in the antiproliferative effect of jaceosidin in human endometrial cancer cells. We demonstrated that jaceosidin is a more potent inhibitor of cell growth than cisplatin in human endometrial cancer cells. In contrast, jaceosidin-induced cytotoxicity in normal endometrial cells was lower than that observed for cisplatin. Jaceosidin induced G2/M phase cell cycle arrest and modulated the levels of cyclin B and p-Cdc2 in Hec1A cells. Knockdown of p21 using specific siRNAs partially abrogated jaceosidin-induced cell growth inhibition. Additional mechanistic studies revealed that jaceosidin treatment resulted in an increase in phosphorylation of Cdc25C and ATM-Chk1/2. Ku55933, an ATM inhibitor, reversed jaceosidin-induced cell growth inhibition, in part. Moreover, jaceosidin treatment resulted in phosphorylation of ERK, and pretreatment with the ERK inhibitor, PD98059, attenuated cell growth inhibition by jaceosidin. These data suggest that jaceosidin, isolated from Japanese mugwort, modulates the ERK/ATM/Chk1/2 pathway, leading to inactivation of the Cdc2-cyclin B1 complex, followed by G2/M cell cycle arrest in endometrial cancer cells.

Effects of SASH1 on melanoma cell proliferation and apoptosis in vitro

The SAM and SH3 domain containing1 (SASH1) gene was originally identified as a potential tumor suppressor gene in breast cancer, mapped on chromosome 6q24.3. The expression of SASH1 plays a prognostic role in human colon cancer. Its expression is frequently downregulated in several human malignancies. However, the biological function of SASH1 in melanoma cells is yet to be determined. In this study, in order to investigate the tumor suppressive effects of the SASH1 gene, an A-375 stable melanoma cell line was established, overexpressing the SASH1 gene. The stable cell line was examined using proliferation assay, apoptosis assay, cell cycle analysis and real-time PCR. The results indicated that the tumor suppressive activity of SASH1 derived from G2/M arrest in A-375 cells, and that the phosphorylation of Cdc2 or the disruption of cyclinB-Cdc2 binding may be responsible for the G2/M arrest.