Cdc25C Protein Research Articles

Icaritin increases radiosensitivity of nasopharyngeal carcinoma cells by regulating iron death

To explore the radiosensitizing effect of icaritin on nasopharyngeal carcinoma (NPC) cells and the underlying mechanism. MTT assay and clonal formation assay were used to evaluate the effect of icaritin on proliferation of human NPC HONE1 and HNE1 cells. The effects of icaritin treatment, γ-ray radiation, or both on production of reactive oxygen species (ROS), cell cycle distribution and apoptosis of the NPC cells were assessed using flow cytometry. The expressions of DNA damage markers γ-H2AX, cycle-related proteins CDC25C, p-CDC25C and cyclin B1, and ferroptosis markers ACSL4 and GXP4 were detected using Western blotting. A nude mouse model bearing subcutaneous HONE1 cell xenograft was used to observe the effect of icaritin and radiation on tumor growth. Icaritin dose-dependently inhibited the viability of the NPC cells and enhanced the inhibitory effect of radiation on cell proliferation. Flow cytometry and Western blotting showed that icaritin treatment prior to radiation significantly promoted ROS production and γ-H2AX expression in the NPC cells (P<0.001). Compared with radiation exposure alone, the combined treatment caused cell cycle arrest in G2 phase, down-regulated CDC25C and cyclin B1 expression, and up-regulated p-CDC25C expression in the cells (P<0.01), resulting also in increased cell apoptosis, enhanced expression of ferroptosis protein ACSL4 and lowered expression of GXP4 (P<0.001). In the tumor-bearing mice, icaritin treatment, compared with radiation alone, significantly reduced the tumor growth rate and decreased tumor weight (P<0.001). Icaritin can enhance radiosensitivity of NPC cells both in vitro and in nude mice possibly by enhancing ROS production to promote iron death of the cells.

Effectiveness of Nonfunctionalized Graphene Oxide Nanolayers as Nanomedicine against Colon, Cervical, and Breast Cancer Cells.

Recent studies in nanomedicine have intensively explored the prospective applications of surface-tailored graphene oxide (GO) as anticancer entity. However, the efficacy of nonfunctionalized graphene oxide nanolayers (GRO-NLs) as an anticancer agent is less explored. In this study, we report the synthesis of GRO-NLs and their in vitro anticancer potential in breast (MCF-7), colon (HT-29), and cervical (HeLa) cancer cells. GRO-NLs-treated HT-29, HeLa, and MCF-7 cells showed cytotoxicity in the MTT and NRU assays via defects in mitochondrial functions and lysosomal activity. HT-29, HeLa, and MCF-7 cells treated with GRO-NLs exhibited substantial elevations in ROS, disturbances of the mitochondrial membrane potential, an influx of Ca2+, and apoptosis. The qPCR quantification showed the upregulation of caspase 3, caspase 9, bax, and SOD1 genes in GRO-NLs-treated cells. Western blotting showed the depletion of P21, P53, and CDC25C proteins in the above cancer cell lines after GRO-NLs treatment, indicating its function as a mutagen to induce mutation in the P53 gene, thereby affecting P53 protein and downstream effectors P21 and CDC25C. In addition, there may be a mechanism other than P53 mutation that controls P53 dysfunction. We conclude that nonfunctionalized GRO-NLs exhibit prospective biomedical application as a putative anticancer entity against colon, cervical, and breast cancers.

The effect of paclitaxel on apoptosis, autophagy and mitotic catastrophe in AGS cells

Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

Hispolon Induces Apoptosis, Suppresses Migration and Invasion of Glioblastoma Cells and Inhibits GBM Xenograft Tumor Growth In Vivo.

Hispolon, a polyphenol compound isolated from Phellinus linteus, has been reported to exhibit antioxidant, antiproliferative, and antitumor activities. This study aimed to explore the antitumor effects of hispolon on glioblastoma multiforme (GBM) cells in vitro and in vivo. The results revealed that hispolon significantly inhibited GBM cell proliferation and induced apoptosis through caspase-9 and caspase-3 activation and PARP cleavage. Hispolon also induced cell cycle G2/M phase arrest in GBM cells, as supported by flow cytometry analysis and confirmed by a decrease in cyclin B1, cdc2, and cdc25c protein expressions in a dose- and time-dependent manner. Furthermore, hispolon suppressed the migration and invasion of GBM cells by modulating epithelial–mesenchymal transition (EMT) markers via wound healing, transwell assays, and real-time PCR. Moreover, hispolon significantly reduced tumor growth in DBTRG xenograft mice and activated caspase-3 in hispolon-treated tumors. Thus, our findings revealed that hispolon is a potential candidate for the treatment of GBM.

Iridin Induces G2/M Phase Cell Cycle Arrest and Extrinsic Apoptotic Cell Death through PI3K/AKT Signaling Pathway in AGS Gastric Cancer Cells.

Iridin is a natural flavonoid found in Belamcanda chinensis documented for its broad spectrum of biological activities like antioxidant, antitumor, and antiproliferative effects. In the present study, we have investigated the antitumor potential of iridin in AGS gastric cancer cells. Iridin treatment decreases AGS cell growth and promotes G2/M phase cell cycle arrest by attenuating the expression of Cdc25C, CDK1, and Cyclin B1 proteins. Iridin-treatment also triggered apoptotic cell death in AGS cells, which was verified by cleaved Caspase-3 (Cl- Caspase-3) and poly ADP-ribose polymerase (PARP) protein expression. Further apoptotic cell death was confirmed by increased apoptotic cell death fraction shown in allophycocyanin (APC)/Annexin V and propidium iodide staining. Iridin also increased the expression of extrinsic apoptotic pathway proteins like Fas, FasL, and cleaved Caspase-8 in AGS cells. On the contrary, iridin-treated AGS cells did not show variations in proteins related to an intrinsic apoptotic pathway such as Bax and Bcl-xL. Besides, Iridin showed inhibition of PI3K/AKT signaling pathways by downregulation of (p-PI3K, p-AKT) proteins in AGS cells. In conclusion, these data suggest that iridin has anticancer potential by inhibiting PI3K/AKT pathway. It could be a basis for further drug design in gastric cancer treatment.

Vanadium oxides modify the expression levels of the p21, p53, and Cdc25C proteins in human lymphocytes treated in vitro.

In vitro assays have demonstrated that vanadium compounds interact with biological molecules similar to protein kinases and phosphatases and have also shown that vanadium oxides decrease the proliferation of cells, including human lymphocytes; however, the mechanism, the phase in which the cell cycle is delayed and the proteins involved in this process are unknown. Therefore, we evaluated the effects of vanadium oxides (V2 O3 , V2 O4 and V2 O5 ) in human lymphocyte cultures (concentrations of 2, 4, 8, or 16 μg/ml) on cellular proliferation and the levels of the p53, p21 and Cdc25C proteins. After 24 h of treatment with the different concentrations of vanadium oxides, the cell cycle phases were determined by evaluating the DNA content using flow cytometry, and the levels of the p21, p53 and Cdc25C proteins were assessed by Western blot analysis. The results revealed that the DNA content remained unchanged in every phase of the cell cycle; however, only at high concentrations did protein levels increase. Although, according to previous reports, vanadium oxides induce a delay in proliferation, DNA analysis did not show this occurring in a specific cell cycle phase. Nevertheless, the increases in p53 protein levels may cause this delay.

Resveratrol synergizes with cisplatin in antineoplastic effects against AGS gastric cancer cells by inducing endoplasmic reticulum stress‑mediated apoptosis and G2/M phase arrest.

Gastric cancer (GC) is a common gastrointestinal malignancy, and cisplatin (DDP) is an important component of chemotherapeutic regimens for GC. However, the application of DDP is limited by its dose-dependent systemic toxicity. Resveratrol (RES) is a natural polyphenol compound that has chemopreventive and therapeutic effects against various cancers, including GC. However, whether RES can sensitize GC cells to DDP remains unknown. Following RES/DDP combination treatment, cell viability was determined by Cell Counting Kit-8 and colony-forming assays, and cell apoptosis and the cell cycle were detected by FITC-Annexin V/PI staining assay and PI staining assay, respectively, followed by flow cytometry. Moreover, western blotting was performed to evaluate the protein expression levels, and the intracellular free Ca2+ concentration was determined by a Fluo-4 AM probe after cell cotreatment with RES and DDP. The present results demonstrated that RES/DDP combination treatment significantly inhibited cell viability, promoted cell apoptosis and induced G2/M phase arrest in AGS cells. In addition, it was determined that RES combined with DDP significantly increased the levels of Bax, cleaved poly-ADP-ribose polymerase (PARP), glucose-regulated protein 78 (GRP78), PRKR-like ER kinase (PERK), p-eukaryotic translation initiation factor 2α (p-eIF2α), CCAAT/enhancer binding protein homologous protein (CHOP) and cleaved caspase-12, whereas Bcl-2 expression was downregulated following RES/DDP cotreatment. Moreover, RES/DDP cotreatment significantly upregulated phosphorylated cyclin-dependent kinase 1 (p-CDK1, Tyr15), p21Waf1/Cip1 and p27Kip1 protein levels and downregulated Cdc25C protein levels. In conclusion, RES and DDP synergistically inhibited the growth of the gastric adenocarcinoma cell line AGS by inducing endoplasmic reticulum stress-mediated apoptosis and G2/M phase arrest via activation of the PERK/eIF2α/activating transcription factor 4 (ATF4)/CHOP signaling pathway and caspase-12 and by inactivating the CDK1-cyclin B1 complex. These results indicated that RES is a promising adjuvant for DDP during GC chemotherapy.

Effect of silencing hepatocyte growth factor receptor c-Met expression on biological characteristics of colon cancer cells

Objective: To investigate the effect of silencing hepatocyte growth factor receptor (c-Met) expression on the biological characteristics of HCT116 colon cancer cells. Methods: Cellular model of c-Met transient transfection was established by using small interfering RNA (siRNA), the expression of c-Met in colon cancer cells was detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot. The apoptosis assay, cell invasion assay, cell migration and other experiments were conducted to observe the effects of silencing c-Met on the biological characteristics of colon cancer cells. Results: RT-qPCR results showed that the relative expression levels of c-Met mRNA in siRNA-Met group, blank control group and siRNA negative control (siRNA-NC) group were 0.32±0.26, 1.01±0.03 and 1.05±0.23, respectively, and the difference was statistically significant (P<0.05). Western blot analysis showed that the expression level of c-Met protein in the siRNA-Met group was 0.24±0.03, significantly lower than 1.23±0.06 in the blank control group and 1.18±0.11 in the siRNA-NC group (P<0.05). The cell counting kit-8 (CCK8) results showed that the 72-hour absorbance (A) values of the siRNA-Met group, blank control group and the siRNA-NC group were 1.13±0.05, 1.48±0.08 and 1.53±0.07, respectively, and the difference was statistically significant (P<0.01). Cell cycle results showed that the proportion of cells in G(2)/M phase was (14.65±1.41)% in siRNA-Met group , (5.07±0.70)% in blank control group and (5.63±0.71)% in siRNA-NC group, and the difference was statistically significant (P<0.05). The expression levels of cell cycle regulatory proteins Cdc25c and cyclin B1 in siRNA-Met group were significantly decreased. The apoptotic rate in siRNA-Met group was (5.85±0.35)%, significantly higher than (1.00±0.17)% in blank control group and (0.91±1.14)% in siRNA-NC group (P<0.05). The expression level of apoptosis-related protein Bcl-2 in the siRNA-Met group was significantly decreased while Bcl-2 associated X protein (BAX) expression level was significantly increased. The cell scratching result showed that the cell migration abilities of the siRNA-Met group, blank control group and the siRNA-NC group were (51.33±8.62)%, (100.00±3.72)% and (102.33±6.43)%, respectively, and the difference was statistically significant (P<0.05). The number of cell penetrating into the basement membrane of the siRNA-Met group, blank control group and the siRNA-NC group were 47.50±10.60, 100.00±5.33 and 102.50±10.61, respectively, and the difference was statistically significant (P<0.05). The expressions of invasion related proteins including MMP-2 and MMP-9 in siRNA-Met group were decreased significantly. Conclusions: c-Met plays an important role in maintaining the biological characteristics of colon cancer cells. Inhibition of c-Met may have important values in the treatment of colon cancer.

Apigetrin induces extrinsic apoptosis, autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell

Apigetrin is a flavonoid glycoside phytonutrient derived from fruits and vegetables that is well known for a variety of biological activities such as antioxidant and anti-inflammatory activities. In the current study, we determined the effect of apigetrin on AGS gastric cancer cell. Apigetrin reduced cancer cell proliferation and induced G2/M phase cell cycle arrest by regulating cyclin B1, cdc25c and cdk1 protein expression in AGS cell. Apigetrin treatment caused apoptotic cell death in AGS cells, characterized by the accumulation of apoptosis portion, cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Apigetrin-treated cells increased the expression of extrinsic apoptosis pathway proteins and mRNA. However, intrinsic apoptosis pathway related proteins were not altered. In addition, AGS cells treated with apigetrin increased autophagic cell death, featured by the formation of autophagic vacuole and acidic vesicular organelles. Autophagy marker proteins, such as LC3B-II and beclin-1, were increased, and p62, an autophagy flux marker protein, was also increased by endoplasmic reticulum stress. Also, the phosphorylation of PI3K/AKT/mTOR pathway proteins and its downstream targets in apigetrin-treated AGS cells was identified to be decreased. Taken together, these data suggest that apigetrin-treated AGS cells induced G2/M phase cell cycle arrest, extrinsic apoptosis and autophagic cell death through PI3K/AKT/mTOR pathway, which can lead to the inhibition of gastric cancer development. Thus, our findings strongly indicate that apigetrin is a basic natural derived compound that could be used as a nutrient source with potential anticancer activities against gastric cancer.

Activation of PERK Contributes to Apoptosis and G2/M Arrest by Microtubule Disruptors in Human Colorectal Carcinoma Cells

Microtubule-targeting agents (MTAs) are widely used in cancer chemotherapy, but the therapeutic responses significantly vary among different tumor types. Protein kinase RNA-like endoplasmic reticular (ER) kinase (PERK) is an ER stress kinase, and the role of PERK in the anticancer effects of MTAs is still undefined. In the present study, taxol (TAX) and nocodazole (NOC) significantly induced apoptosis with increased expression of phosphorylated PERK (pPERK; Tyr980) in four human colon cancer cell lines, including HCT-15, COLO205, HT-20, and LOVO cells. Induction of G2/M arrest by TAX and NOC with increases in phosphorylated Cdc25C and cyclin B1 protein were observed in human colon cancer cells. Application of the c-Jun N-terminal kinase (JNK) inhibitors SP600125 (SP) and JNK inhibitor V (JNKI) significantly reduced TAX- and NOC-induced apoptosis and G2/M arrest of human colon cancer cells. Interestingly, TAX- and NOC-induced pPERK (Tyr980) protein expression was inhibited by adding the JNK inhibitors, SP and JNKI, and application of the PERK inhibitor GSK2606414 (GSK) significantly reduced apoptosis and G2/M arrest by TAX and NOC, with decreased pPERK (Tyr980) and pJNK, phosphorylated Cdc25C, and Cyc B1 protein expressions in human colon cancer cells. Decreased viability by TAX and NOC was inhibited by knockdown of PERK using PERK siRNA in COLO205 and HCT-15 cells. Disruption of the mitochondrial membrane potential and an increase in B-cell lymphoma-2 (Bcl-2) protein phosphorylation (pBcl-2; Ser70) by TAX and NOC were prevented by adding the PERK inhibitor GSK and JNK inhibitor SP and JNKI. A cross-activation of JNK and PERK by TAX and NOC leading to anti-CRC actions including apoptosis and G2/M arrest was first demonstrated herein.

Palmitic Acid Methyl Ester Induces G2/M Arrest in Human Bone Marrow-Derived Mesenchymal Stem Cells via the p53/p21 Pathway.

Bone marrow-derived mesenchymal cells (BM-MSCs) are able to differentiate into adipocytes, which can secrete adipokines to affect BM-MSC proliferation and differentiation. Recent evidences indicated that adipocytes can secrete fatty acid metabolites, such as palmitic acid methyl ester (PAME), which is able to cause vasorelaxation and exerts anti-inflammatory effects. However, effects of PAME on BM-MSC proliferation remain unclear. The aim of this study was to investigate the effect of PAME on human BM-MSC (hBM-MSC) proliferation and its underlying molecular mechanisms. hBM-MSCs were treated with PAME for 48 h and then subjected to various analyses. The results from the present study show that PAME significantly reduced the levels of G2/M phase regulatory proteins, cyclin-dependent kinase 1 (Cdk1), and cyclin B1 and inhibited proliferation in hBM-MSCs. Moreover, the level of Mdm2 protein decreased, while the levels of p21 and p53 protein increased in the PAME-treated hBM-MSCs. However, PAME treatment did not significantly affect apoptosis/necrosis, ROS generation, and the level of Cdc25C protein. PAME also induced intracellular acidosis and increased intracellular Ca2+ levels. Cotreatment with PAME and Na+/H+ exchanger inhibitors together further reduced the intracellular pH but did not affect the PAME-induced decreases of cell proliferation and increases of the cell population at the G2/M phase. Cotreatment with PAME and a calcium chelator together inhibited the PAME-increased intracellular Ca2+ levels but did not affect the PAME-induced cell proliferation inhibition and G2/M cell cycle arrest. Moreover, the half-life of p53 protein was prolonged in the PAME-treated hBM-MSCs. Taken together, these results suggest that PAME induced p53 stabilization, which in turn increased the levels of p53/p21 proteins and decreased the levels of Cdk1/cyclin B1 proteins, thereby preventing the activation of Cdk1, and eventually caused cell cycle arrest at the G2/M phase. The findings from the present study might help get insight into the physiological roles of PAME in regulating hBM-MSC proliferation.

Effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia K562 cells and its mechanism

Objective To explore the effect of dehydrocostus lactone on the proliferation of chronic myeloid leukemia cell line K562 and its mechanism. Methods K562 cells in logarithmic growth phase were treated with different concentrations of dehydrocostus lactone. Cell morphology was observed by Wright-Giemsa staining. Cell proliferation was detected by CCK-8 method. Cell cycle, apoptosis and the expressions of cell surface differentiation antigen CD14 and CD11b were detected by flow cytometry. JAK-STAT pathway and the expressions of apoptosis and cell cycle related proteins were detected by Western blot. Results Compared with the control group, the proliferation of K562 cells could be inhibited after treatment with different concentrations (4.0, 6.0, 8.0, 10.0 and 12.0 μmol/L) of dehydrocostus lactone for 24 h, and the difference was statistically significant (F = 109.510, P < 0.05). After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h, the apoptosis rates of K562 cells were (16.1±3.8)% and (29.6±4.3)%, which were higher than that of the control group [(3.1±0.5)%] (F = 83.255, P < 0.05). After treatment with 5.0 and 10.0 μmol/L dehydrocostus lactone for 24 h, the proportion of G2/M phase cells in K562 cells were (17.0±3.2)% and (28.8±3.9)%, which were higher than that of the control group [(9.1±2.3)%] (F = 161.598, P < 0.05); the proportion of S phase cells in K562 cells were (48.1±3.9)% and (61.0±5.4)%, which were higher than that of the control group [(39.6±3.6)%] (F = 192.356, P < 0.05). After treatment with 2.5 and 5.0 μmol/L dehydrocostus lactone for 72 h, the expression rates of CD14 in K562 cells were (28.6±3.9)% and (41.1±4.4)%, which were higher than that in the control group [(3.1±0.5)%] (F = 132.811, P < 0.05); the expression rates of CD11b in K562 cells were (42.4±5.0)% and (61.2±5.7)%, which were higher than that in the control group [(4.2±1.1)%] (F = 179.553, P < 0.05). Dehydrocostus lactone could decrease the expressions of JAK2, STAT5, cyclin E, CDK2, cyclin A, CDC25C, cyclin B1, CDK1 and bcl-2 proteins, and up-regulate the expressions of p21 and bax proteins. Conclusion Dehydrocostus lactone can inhibit the proliferation of chronic myeloid leukemia K562 cells, which may be achieved by cell cycle arrest, induction of apoptosis and differentiation. Key words: Dehydrocostus lactone; Cell cycle; Apoptosis; Differentiation; Leukemia

Bufotalin induces cell cycle arrest and cell apoptosis in human malignant melanoma A375 cells.

Venenum bufonis has been used as an antitumor drug in China for many years. Bufotalin, as an active component of Venenum bufonis, has been proven to exhibit antitumor effects in cancer types. In the present study, the effect of bufotalin on the human melanoma skin cancer cell line A375 was analyzed using MTT and colony formation assays. Bufotalin significantly inhibited the proliferation and colony formation of A375 cells. Further studies demonstrated that bufotalin significantly upregulated the protein levels of ATM serine/threonine kinase and Chk2, downregulated CDC25C protein expression, and subsequently inhibited CDK1 expression, leading to cell cycle arrest at the G2/M phase of the A375 cells. Furthermore, bufotalin significantly increased BAX expression levels, decreased BCL‑2 expression, and then upregulated apoptosis‑related proteins, caspase‑3/-9, followed by A375 cell apoptosis. Taken together, these results show that bufotalin induces cell cycle arrest at the G2/M phase and cell apoptosis, resulting in the inhibition of A375 cell proliferation, thereby suggesting that bufotalin may be utilized in melanoma treatment.

Scutellarein Induces Fas-Mediated Extrinsic Apoptosis and G2/M Cell Cycle Arrest in Hep3B Hepatocellular Carcinoma Cells

Scutellarein (SCU), a flavone found in the perennial herb Scutellaria baicalensis, is known for a wide range of biological activities. In the present study, we investigated the effects of treatment with SCU flavonoids on inducing apoptosis via the extrinsic pathway in Hep3B cells. SCU treatment significantly inhibited Hep3B cell proliferation and induced G2/M phase cell cycle arrest by inhibiting the expression level of the proteins Cdc25C, cdk1 and Cyclin B1. Allophycocyanin (APC)/Annexin V and propidium iodide (PI) double-staining showed upregulation of apoptotic cell death fraction. We further confirmed apoptosis by 4′-6-diamidino-2-phenylindole (DAPI) fluorescent staining and observed DNA fragmentation with agarose gel electrophoresis. Further, immunoblotting results showed that treatment with SCU showed no changes in Bax and Bcl-xL protein levels. In addition, SCU treatment did not affect the mitochondrial membrane potential (MMP) in Hep3B cells. On the contrary, treatment with SCU increased the expression of Fas and Fas ligand (FasL), which activated cleaved caspase-8, caspase-3, and polymeric adenosine diphosphate ribose (PARP), whereas the expression level of death receptor 4 (DR4) decreased. We confirmed that the proteins expressed upon treatment with SCU were involved in the Fas-mediated pathway of apoptosis in Hep3B cells. Thus, our findings in the current study strongly imply that SCU can be a basic natural source for developing potent anti-cancer agents for hepatocellular carcinoma (HCC) treatment.

Evodiamine Inhibits the Proliferation of BGC-823 and SGC-7901 Cells by Inducing Cell Cycle Arrest and Apoptosis in Gastric Cancer

Gastric cancer represents a major cause of cancer-related death worldwide. Although various tactics and anti-tumor drugs have been used to improve curative effects, five-year survival rate of lung cancer patients remains poor. Evodiamine, a sophora alkaloid, has been demonstrated to exert antitumor effects on many types of cancer. However, the molecular mechanism of evodiamine against gastric cancer has not been clearly elucidated. In this study, we investigated the anti-tumor activity and the underlying mechanisms of EVO on gastric cancer cells, and found that it significantly inhibited the proliferation of BGC-823 and SGC-7901 cells by inducing cell cycle arrest at G2/M phase and cell apoptosis in a dose- and time-dependent manner. Its molecular mechanism may be that it reduces the expression of cell cycle- promoting protein Cdc25C and promotes the expression of cell cycle inhibitor p53, as well as prompts the activity of caspases pathways, such as the expression level of cleaved caspase-3 and cleaved caspase-8; cleaved caspase-9 and cleaved PARP-1 are up-regulated, treated with EVO (10 μM) at different points in time (0, 3, 6, 9, 12, 24 h). Collectively, our data demonstrated that EVO was a potential anti-tumor agent against gastric cancer.

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).

Abstract 4287: Whole-genome CRISPR/Cas9 screen of the CHK1 inhibitor prexasertib implicates FAM122A loss as a potential resistance mechanism

Abstract Introduction: Phase I clinical trials of the CHK1 inhibitor prexasertib have been completed in non-small cell lung cancer (NSCLC) and other cancers and several Phase I-II trials are currently recruiting. By inhibiting the G2/M and S-phase checkpoints, prexasertib has been shown to cause increased replication stress, DNA damage and premature entry into mitosis, resulting in mitotic catastrophe and cell death. While the inhibitor is showing therapeutic promise, acquired resistance is of concern. Little is known about how cancer cells may become resistant to CHK1 inhibitors. Understanding potential resistance mechanisms may help predict patient responses, design CHK1 therapy combinations and implement means of re-sensitization. Materials and Methods: NSCLC cell lines A549 and NCI-H460 stably expressing the Cas9 endonuclease were obtained from the Genetic Perturbation Platform at the Broad Institute. Cells were infected with the Brunello v2 lentiviral sgRNA library and grown for 15 days in prexasertib-treated media. Cells were collected pre- and post-drug selection, genomic DNA was extracted, and sgRNA sequences were PCR amplified and Illumina sequenced. Top hits among knockouts that conferred drug resistance were identified by the STARS gene-ranking algorithm. Hit validation was performed with sgRNAs and pooled siRNAs (Qiagen). Prexasertib IC50s were assessed by colony formation and CellTiter-Glo cell viability assays. Cell cycle analysis was performed by propidium iodide flow cytometry. PP2A activity was measured using Ser/Thr phosphatase assay kits (Upstate Biotechnology). Dinaciclib and LB-100 were obtained from Selleck and Apexbio, respectively. Results: CRISPR knockouts of several individual genes were found to promote resistance of both cell lines to prexasertib with false discovery rates &amp;lt; 0.01%. One top hit codes for a little-known but highly conserved protein FAM122A, which has been shown to inhibit PP2A, which dephosphorylates the Chk1 target protein CDC25C. SiRNA knockdown or sgRNA knockout of FAM122A conferred prexasertib resistance. Similarly, prexasertib-mediated G2/M accumulation was inhibited by FAM122A knockdown. FAM122A silencing increased PP2A activity. FAM122A knockdown mediated resistance was reversed with PP2A B subunit α siRNA knockdown or the PP2A inhibitor LB-100. Dinaciclib, an inhibitor of the CDC25C kinase CDK2, increased prexasertib resistance. Conclusion: Although CHK1 inhibitors like prexasertib show promise, one mechanism of resistance may be through other cell-cycle regulating compensatory pathways. Identification of FAM122A loss as a potential mechanism for resistance suggests a predictive biomarker and means for re-sensitization of cancer cells to CHK1 inhibitors. Citation Format: Peter Deraska, Hunter Reavis, Shelby Labe, Alan D'Andrea, David Kozono. Whole-genome CRISPR/Cas9 screen of the CHK1 inhibitor prexasertib implicates FAM122A loss as a potential resistance mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4287.

The Molecular Tweezer CLR01 Stabilizes a Disordered Protein-Protein Interface.

Protein regions that are involved in protein–protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein–protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular “Janus” ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein–protein interface, “freezes” one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.

Gene expression profiling of idiopathic interstitial pneumonias (IIPs): identification of potential diagnostic markers and therapeutic targets

BackgroundChronic fibrosing idiopathic interstitial pneumonia (IIP) is characterized by alveolar epithelial damage, activation of fibroblast proliferation, and loss of normal pulmonary architecture and function. This study aims to investigate the genetic backgrounds of IIP through gene expression profiling and pathway analysis, and to identify potential biomarkers that can aid in diagnosis and serve as novel therapeutic targets.MethodsRNA extracted from lung specimens of 12 patients with chronic fibrosing IIP was profiled using Illumina Human WG-6 v3 BeadChips, and Ingenuity Pathway Analysis was performed to identify altered functional and canonical signaling pathways. For validating the results from gene expression analysis, immunohistochemical staining of 10 patients with chronic fibrosing IIP was performed.ResultsNinety-eight genes were upregulated in IIP patients relative to control subjects. Some of the upregulated genes, namely desmoglein 3 (DSG3), protocadherin gamma-A9 (PCDHGA9) and discoidin domain-containing receptor 1 (DDR1) are implicated in cell-cell interaction and/or adhesion; some, namely collagen type VII, alpha 1 (COL7A1), contactin-associated protein-like 3B (CNTNAP3B) and mucin-1 (MUC1) are encoding the extracellular matrix molecule or the molecules involved in cell-matrix interactions; and the others, namely CDC25C and growth factor independent protein 1B (GFI1B) are known to affect cell proliferation by affecting the progression of cell cycle or regulating transcription. According to pathway analysis, alternated pathways in IIP were related to cell death and survival and cellular growth and proliferation, which are more similar to cancer than to inflammatory response and immunological diseases. Using immunohistochemistry, we further validate that DSG3, the most highly upregulated gene, shows higher expression in chronic fibrosing IIP lung as compared to control lung.ConclusionWe identified several genes upregulated in chronic fibrosing IIP patients as compared to control, and found genes and pathways implicated in cancer, rather than in inflammatory or immunological disease to play important roles in the pathogenesis of IIPs. Moreover, DSG3 is a novel potential biomarker for chronic fibrosing IIP with its significantly high expression in IIP lung.

Mdm2 promotes Cdc25C protein degradation and delays cell cycle progression through the G2/M phase.

Upon different types of stress, the gene encoding the mitosis-promoting phosphatase Cdc25C is transcriptionally repressed by p53, contributing to p53's enforcement of a G2 cell cycle arrest. In addition, Cdc25C protein stability is also decreased following DNA damage. Mdm2, another p53 target gene, encodes a ubiquitin ligase that negatively regulates p53 levels by ubiquitination. Ablation of Mdm2 by siRNA led to an increase in p53 protein and repression of Cdc25C gene expression. However, Cdc25C protein levels were actually increased following Mdm2 depletion. Mdm2 is shown to negatively regulate Cdc25C protein levels by reducing its half-life independently of the presence of p53. Further, Mdm2 physically interacts with Cdc25C and promotes its degradation through the proteasome in a ubiquitin-independent manner. Either Mdm2 overexpression or Cdc25C downregulation delays cell cycle progression through the G2/M phase. Thus, the repression of the Cdc25C promoter by p53, together with p53-dependent induction of Mdm2 and subsequent degradation of Cdc25C, could provide a dual mechanism by which p53 can enforce and maintain a G2/M cell cycle arrest.