Expression Of CDC25B Research Articles

E2F1 Facilitates the Proliferation and Stemness of Gastric Cancer Cells by Activating CDC25B Transcription and Modulating the MAPK Pathway.

Gastric cancer (GC) is a health problem that concerns people around the world. CDC25B is an essential cell cycle regulatory factor that is overexpressed in a variety of tumor cells. CDC25B plays a vital part in the progression and proliferation of malignant tumors. However, it is not yet clear that how CDC25B affects the stemness of GC cells. The study used bioinformatics to detect the expression of E2F1 and CDC25B in GC tissues and their correlation, as well as pathways enriched by CDC25B. We detected the expression of E2F1 and CDC25B in GC cell lines using quantitative reverse transcription polymerase chain reaction and tested the combination relationship between E2F1 and CDC25B using chromatin immunoprecipitation (ChIP) and dual-luciferase assays. We measured cell viability using CCK-8 assay, evaluated sphere-forming efficiency using sphere formation assay, and determined cell proliferation ability using colony formation assay. We also analyzed the expression of stemness markers and MAPK pathway-related proteins using western blot. In GC tissues and cells, CDC25B was upregulated. Silencing CDC25B could affect the MAPK pathway, thereby repressing the proliferation and stemness of GC cells. As predicted by bioinformatics, CDC25B had an upstream transcription factor, E2F1, which also had a high expression level in GC. Dual-luciferase and ChIP assays confirmed the combination relationship between the two. Rescue experiments uncovered that overexpression of CDC25B could reverse the impact induced by E2F1 knockdown on proliferation and stemness of cells. In conclusion, E2F1 could activate CDC25B transcription to regulate the MAPK pathway and enhance the proliferation and stemness of GC cells. We revealed a potential regulatory pathway of stemness of GC cells that was mediated by CDC25B, providing new ideas for improving and innovating GC treatment.

Transcription factor E2F3 activates CDC25B to regulate DNA damage and promote mitoxantrone resistance in stomach adenocarcinoma.

CDC25B, as a member of the cell cycle regulating protein family, is located in the cytoplasm and is involved in the transition of the cell cycle and mitosis. CDC25B is highly expressed in various tumors and is a newly discovered oncogene. This study aimed to investigate the impact of CDC25B on mitoxantrone resistance in stomach adenocarcinoma (STAD) and its possible mechanisms. This study analyzed the expression of CDC25B and its potential transcription factor E2F3 in STAD, as well as the IC50 values of tumor tissues by bioinformatics analysis. Expression levels of CDC25B and E2F3 in STAD cells were measured by qRT-PCR. MTT was utilized to evaluate cell viability and IC50 values of STAD cells, and comet assay was utilized to analyze the level of DNA damage in STAD cells. Western blot was used to analyze the expression of DNA damage-related proteins. The targeting relationship between E2F3 and CDC25B was validated by dual-luciferase and ChIP assays. Bioinformatics analysis and molecular experiments showed that CDC25B and E2F3 were highly expressed in STAD, and CDC25B was enriched in the mismatch repair and nucleotide excision repair pathways. The IC50 values of tumor tissues with high expression of CDC25B were relatively high. Dual-luciferase and ChIP assays confirmed that CDC25B could be transcriptionally activated by E2F3. Cell experiments revealed that CDC25B promoted mitoxantrone resistance in STAD cells by regulating DNA damage. Further research found that low expression of E2F3 inhibited mitoxantrone resistance in STAD cells by DNA damage, but overexpression of CDC25B reversed the impact of E2F3 knockdown on mitoxantrone resistance in STAD cells. This study confirmed a novel mechanism by which E2F3/CDC25B mediated DNA damage to promote mitoxantrone resistance in STAD cells, providing a new therapeutic target for STAD treatment.

FDI-6 inhibits the expression and function of FOXM1 to sensitize BRCA-proficient triple-negative breast cancer cells to Olaparib by regulating cell cycle progression and DNA damage repair

Inducing homologous-recombination (HR) deficiency is an effective strategy to broaden the indications of PARP inhibitors in the treatment of triple-negative breast cancer (TNBC). Herein, we find that repression of the oncogenic transcription factor FOXM1 using FOXM1 shRNA or FOXM1 inhibitor FDI-6 can sensitize BRCA-proficient TNBC to PARP inhibitor Olaparib in vitro and in vivo. Mechanistic studies show that Olaparib causes adaptive resistance by arresting the cell cycle at S and G2/M phases for HR repair, increasing the expression of CDK6, CCND1, CDK1, CCNA1, CCNB1, and CDC25B to promote cell cycle progression, and inducing the overexpression of FOXM1, PARP1/2, BRCA1/2, and Rad51 to activate precise repair of damaged DNA. FDI-6 inhibits the expression of FOXM1, PARP1/2, and genes involved in cell cycle control and DNA damage repair to sensitize TNBC cells to Olaparib by blocking cell cycle progression and DNA damage repair. Simultaneously targeting FOXM1 and PARP1/2 is an innovative therapy for more patients with TNBC.

Expression of CyclinD1, CDC25B and p27 protein in gastric carcinoma And it s clinical significance

Background: Gastric cancer is a common digestive tract tumor in our China, with high morbidity and mortality. Similar with other tumors, the occurrence of gastric cancer was also a complex pathophysiological process, regulated by a variety of oncogenes and tumor suppressor gene. Aims and Objectives: This study aims to investigate the expression of cyclin D1, CDC25B, and p27 in gastric carcinoma and to study their relationship with the occurrence and development of gastric cancer. Materials and Methods: SP immunohistochemical and Western blot analysis were used to detect the expression of cyclin D1, CDC25B, and p27 in 42 cases of gastric carcinoma, 42 cases of paracancer, and 42 cases of normal gastric tissue and then their relationship with clinical and pathological factors was analyzed. Results: (1) The expression of cyclin D1 increased gradually in normal gastric tissue, paracancer, and gastric carcinoma (P>0.05). The expression of CDC25B was higher in gastric carcinoma (P<0.05) while p27 was lower (P<0.05). (2) The result of Western blot shows that the expression of cyclin D1 and CDC25B was higher in gastric carcinoma than that in normal gastric tissue while p27 lower in gastric carcinoma (P<0.05), same with immunohistochemical. (3) The expressions of cyclin D1 and CDC25B had correlation with lymph nodes metastasis, and p27 had correlation with degrees of pathological differentiation, invasion depth, and lymph nodes metastasis. (4) There was a positive relationship between the expression of cyclin D1 and CDC25B in same sample (r=−0.392, P<0.05). Conclusion: Expression of cyclin D1, CDC25B, and p27 protein can be helpful in the prediction of the biological behavior and prognosis of gastric carcinoma.

Abstract 2964: Saffron has enhanced anti-proliferative effect against colorectal cancer cells with deficient DNA mismatch repair

Abstract Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. A subtype of CRC is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy, however, these roles in the context of of dMMR have not been explored. In this study, we aimed to study the effects of saffron in CRC cell lines with dMMR. Methods: Saffron crude extracts and specific compounds (crocin, crocetin and safranal) were used in colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MH3) and HCT116+3+5 (Inserted MLH1 and MSH3). CDC25b, p-H.H2AX, TPDP1, and GAPDH were analyzed by Western blot. Cell cycle was analyzed by FACS. Proliferation and cytotoxicity were analyzed by MTT. Wound and migration assays were also performed. Results: Saffron crudes restricted the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. Wound healing assay indicates that deficient MMR cells are doing better than proficient MMR cells. The expression of CDC25b and TDP1 were upregulated in proficient MMR cell compared to deficient MMR cell, while H2AX was significantly upregulated in both cells type particularly at >10 mg/ml saffron in a concentration-dependent manner (after normalization with GADPH as control).The reduction in cellular proliferation and motility might be due to cell cycle arrest at G2-phase rather than to apoptosis. The major active saffron compounds, safranal and crocin reproduced total saffron crudes effects but with lesser outcome. Conclusion: Saffron's anti-proliferative and healing effects is significant in cells with deficient MMR MLH1 and MSH3 genes. This effect may have a therapeutic implications and benefits for the CRC MSI tumors that are generally not recommended for 5FU-based treatment. Citation Format: Amr Amin, Akbar Soleimani, Chandraprabha Murali, Lucie Beresova, Hamid Shoraka, Hassan Brim, Hassan Ashktorab. Saffron has enhanced anti-proliferative effect against colorectal cancer cells with deficient DNA mismatch repair [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2964.

CircHIPK3 Facilitates the G2/M Transition in Prostate Cancer Cells by Sponging miR-338-3p.

BackgroundCircular RNAs (circRNAs) play a crucial role in gene expression regulation. CircHIPK3 is a circRNA derived from Exon 2 of HIPK3 gene and its role in prostate cancer (PCa) is still unclear.MethodsCCK8 assays, flow cytometry and colony formation assays were performed to assess the effects of circHIPK3 in PCa cells. Bioinformatics analysis, RNA pull-down assay, RNA immunoprecipitation assay (RIP), and luciferase activity assay were performed to dissect the mechanism underlying circHIPK3-mediated G2/M transition in PCa cells.ResultsCircHIPK3 expression was upregulated in PCa cells and prostate cancer tissues. Overexpression of circHIPK3 or circHIPK3 silencing altered PCa viability, proliferation and apoptosis in vitro. CircHIPK3 could sponge miR-338-3p and inhibit its activity, resulting in increased expression of Cdc25B and Cdc2 in vitro.ConclusionCircHIPK3 promotes G2/M transition and induces PCa cell proliferation by sponging miR-338-3p and increasing the expression of Cdc25B and Cdc2. CircHIPK3 may play an oncogenic role in PCa.

Involvement of CaMKII in regulating the release of diplotene-arrested mouse oocytes by pAkt1 (Ser473)

ABSTRACT Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) had been reported to play a role in the process of fertilization. However, the role of CaMKII in the release of diplotene-arrested oocytes is poorly understood. In this study, we explored the potential effect of CaMKII on Akt1 and the relationship among CaMKII, Akt1 and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) during the meiotic resumption of mouse oocytes. We found that inhibition of CaMKII aggravated diplotene arrest. We detected the expression and distribution of pCaMKII (Thr286), pAkt1 (Ser473), Cdc25B and pCdc2 (Tyr15) when oocytes were treated with KN-93, SH-6, LY294002 or PIP3, respectively. Our data showed that down-regulated CaMKII by KN-93 decreased the levels of pAkt1 (Ser473) and rearranged the distribution of pAkt1 (Ser473). Meanwhile, down-regulated pAkt1 (Ser473) by SH-6 also decreased the levels of pCaMKII (Thr286), Cdc25B and pCdc2 (Tyr15) significantly and rearranged the distributions of pCaMKII (Thr286). Furthermore, our data showed that exogenous PIP3 up-regulated GVBD rates significantly and increased the levels of pCaMKII (Thr286) and pAkt1 (Ser473). On the contrary, down-regulation of PIP3 by LY294002 decreased GVBD rates and the levels of pCaMKII (Thr286) and pAkt1 (Ser473), respectively. Our results showed that Akt1 and CaMKII regulated each other, and PIP3 may be involved in these regulations during the release of mouse oocytes from diplotene arrest.

PKCδ promotes fertilization of mouse embryos in early development via the Cdc25B signaling pathway.

Protein kinase C type δ (PKCδ) is involved in B-cell signaling and the regulation of growth, apoptosis and differentiation of a variety of cell types. Cell division cycle 25 (Cdc25) is a key mediator of cell cycle progression that activates cyclin-dependent kinase complexes that drive the cell cycle and participates in the regulation of DNA damage checkpoints. Cdc25B is a member of the Cdc25 family of phosphatases. The present study investigated the role and mechanism of PKCδ in regulating the fertilization of mouse embryos in early development. The expression and subcellular localization of PKCδ and Cdc25B were detected using reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence in one-cell stage mouse embryos. Specific small interfering RNAs targeting PKCδ were used to knockdown the expression of PKCδ. Subsequently, Scansite software was used to predict the target of phosphorylated Cdc25B. Western blotting was used to measure the effects of phosphorylation and dephosphorylation in one-cell stage mouse embryos at different cell cycle phases. PKCδ was expressed during M phase and served a positive role in one-cell stage mouse embryos. Immunofluorescence data revealed that PKCδ and Cdc25B were expressed during G1, S, G2 and M phases of the cell cycle. Furthermore, phosphorylated levels of Cdc25B-Ser96 were observed during G2 and M phases. Microinjection with mimics of phosphorylated Cdc25B-Ser96 mRNA promoted the development of one-cell stage mouse embryos. When PKCδ was suppressed, microinjection with mimics of phosphorylated Cdc25B-Ser96 mRNA reversed the inhibition of PKCδ. To conclude, PKCδ serves a positive role in the first cell cycle of mouse embryos by phosphorylating Cdc25B-Ser96, and provides novel insights for the regulation of early embryonic development.

Cytotoxic Mechanism for Silver Nanoparticles Based High-Content Cellomics and Transcriptome Sequencing.

The aim of this study was to investigate the toxic mechanism for differently sized silver nanoparticles (SNPs) on human dermal fibroblasts (HDFs), by combining high content cellomics and transcriptome sequencing. First, the influences of five SNPs (SNP-5, SNP-20, SNP-50, SNP-100, and SNP-200) on O-₂, focal adhesion, cytoskeleton and ATP content in HDFs were studied with high content screening and colorimetric method, and the role to cytotoxicity was analysed. Transcriptome sequencing technique was then to filter differentially expressed genes induced by SNPs after 4 h treatment. Key pathways in SNP-induced cytotoxicity were also screened via biological pathway analysis. Furthermore, key genes in HDFs after SNP-induced cytotoxicity were determined through matching analysis with previously obtained important microRNAs and their expression levels were verified with qRT-PCR. Cytological experiments showed that the SNP-5 had the strongest effects on O-₂, focal adhesion, cytoskeleton and ATP content, while SNP-20 had the smallest effects. Transcriptome sequencing results showed that 3848, 4213, 2999, 3251 and 5104 genes were found to be differentially expressed in HDFs after treatment with five SNPs. Biological pathway analysis for 1643 uniformly differentially expressed genes revealed that MAPK signaling pathway was the key pathway in SNP-induced cytotoxicity. Two key genes, SOS1 and CDC25B, which are involved in MAPK signaling pathway were finally identified through matching analysis with important microRNAs and verification. In conclusion, the cytotoxic mechanism for SNPs induced cytotoxicity in HDFs involved SNPs down-regulated expression of SOS1 and CDC25B through miR-424-5p in the key MAPK signaling pathway, through blocking of cell cycle, promotion of apoptosis, ultimately leading to cytotoxicity.

CDC25B and CDC25C overexpression in nonmelanoma skin cancer suppresses cell death

Non-melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV-induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin-dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14-3-3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser473 , increased inhibitory phosphorylation of proapoptotic BAD on Ser136 , and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.

The subcellular location of cyclin B1 and CDC25 associated with the formation of polyploid giant cancer cells and their clinicopathological significance

Polyploid giant cancer cells (PGCCs) are key contributors to cancer heterogeneity, and the formation of PGCCs is associated with changes in the expression of cell-cycle-related proteins. This study investigated the intracellular localization and expression level of multiple cell-cycle-related proteins in PGCCs derived from BT-549 and HEY cells. In addition, the formation of PGCCs and the clinicopathological significance of cell-cycle-related proteins in human breast and ovarian cancer were examined. The expression levels of cell-cycle-related proteins, including cyclin B1, CDC25B, CDC25C, and other cell cycle phosphoproteins, including Chk2, and Aurora-A kinase, were determined using immunostaining and western blotting both in vitro and in vivo. Migration, invasion, and proliferation in control cells, cyclin B1 knockdown cells and their PGCCs following CoCl2 treatment were compared. In addition, human breast and ovarian cancer samples were collected to determine the correlation of number of PGCCs, expression of cell-cycle-related proteins, and tumor pathologic grade and metastasis. Our results confirm that cyclin B1 was localized in the cytoplasm of PGCCs and in the nuclei of their budding daughter cells. The phosphorylated proteins Chk2 and Aurora-A kinase regulated the expression and subcellular localization of cyclin B1, CDC25B, and CDC25C. The rate of positive cytoplasmic staining of cyclin B1 and positive nuclear staining of both CDC25B and CDC25C increased with increase in tumor grade and lymph node metastasis. Cell-cycle-related proteins, including cyclin B1, CDC25B, and CDC25C play an important role in regulating the formation of PGCCs. The inhibition of cyclinB1 and CoCl2 treatment significantly promoted cell proliferation, invasion, and migration abilities. The subcellular localization of these cell-cycle-related proteins was regulated by other cell cycle phosphoproteins, and was associated with pathologic grade and metastasis of tumors in cases of human breast and ovarian cancer.

Propensity in low-grade oocytes for delayed germinal vesicle breakdown compromises the developmental ability of sub-optimal grade Bubalus bubalis oocytes.

SummaryMaturing oocytes have diverse developmental potential and good quality oocytes exhibit a better ability to attain physiological milestones in a time-dependent manner. This situation necessitates the confirmation of oocyte developmental status more precisely under an in vitro embryo production (IVEP) regime. The aim of this study was to explain timely events in germinal vesicle breakdown (GVBD), an important milestone of oocyte nuclear maturation, to delineate the developmental capacity of Bubalus bubalis oocytes. In addition, the expression profile of genes responsible for GVBD was assessed in order to understand the molecular context responsible for GVBD. The chronology of GVBD events at different time intervals during in vitro maturation (IVM) suggests that the rate at which oocytes undergo GVBD was strikingly different in the brilliant cresyl blue (BCB)+ and BCB- groups. The expression of AKT and CDC25B genes for BCB+ oocytes was maximum at 8 h of IVM, and CCNB (cyclin B) peaked at around 10 h, which suggested that GVBD was finished after 10 h in BCB+ oocytes, whereas the expression of AKT and CDC25B was found to peak at around 12-14 h of IVM. This difference consequently delays the GVBD event by 2-4 h in BCB- oocytes. Poor abundance of gene transcripts was mainly implicated in delay and lower rate of GVBD in BCB- oocytes which in turn strongly affected the translational ability of oocytes to blastocysts. The findings of this study support the idea that there is a propensity in sub-optimal grade oocytes for delayed GVBD that compromises the developmental ability of low grade buffalo oocytes. The study highlights the very small, but importantly vital and separate, time window of the GVBD event during which the competence levels of buffalo oocytes are altered along with their translational ability to develop into the prospective embryos.

Identification of Biomarkers Correlated with the TNM Staging and Overall Survival of Patients with Bladder Cancer.

Objective: To identify candidate biomarkers correlated with clinical prognosis of patients with bladder cancer (BC).Methods: Weighted gene co-expression network analysis was applied to build a co-expression network to identify hub genes correlated with tumor node metastasis (TNM) staging of BC patients. Functional enrichment analysis was conducted to functionally annotate the hub genes. Protein-protein interaction network analysis of hub genes was performed to identify the interactions among the hub genes. Survival analyses were conducted to characterize the role of hub genes on the survival of BC patients. Gene set enrichment analyses were conducted to find the potential mechanisms involved in the tumor proliferation promoted by hub genes.Results: Based on the results of topological overlap measure based clustering and the inclusion criteria, top 50 hub genes were identified. Hub genes were enriched in cell proliferation associated gene ontology terms (mitotic sister chromatid segregation, mitotic cell cycle and, cell cycle, etc.) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (cell cycle, Oocyte meiosis, etc.). 17 hub genes were found to interact with ≥5 of the hub genes. Survival analysis of hub genes suggested that lower expression of MMP11, COL5A2, CDC25B, TOP2A, CENPF, CDCA3, TK1, TPX2, CDCA8, AEBP1, and FOXM1were associated with better overall survival of BC patients. BC samples with higher expression of hub genes were enriched in gene sets associated with P53 pathway, apical junction, mitotic spindle, G2M checkpoint, and myogenesis, etc.Conclusions: We identified several candidate biomarkers correlated with the TNM staging and overall survival of BC patients. Accordingly, they might be used as potential diagnostic biomarkers and therapeutic targets with clinical utility.

Expression of microRNA-429 in gastric precancerous lesions

Objective To investigate the expression and function of microRNA (miRNA, miR)-429 in human gastric precancerous lesions. Methods The expression of miR-429 in gastric cancer tissues (n=50), gastric tissues with chronic atrophic gastritis (n=60) and normal gastric tissues (n=60) was detected by real-time quantitative polymerase chain reaction (PCR). Human Gastric Epithelial cell strain-1 (GES-1) was transformed into gastric cancer precancerous lesion (PLGC) cell model by tumor necrosis factor-α (TNF-α). PLGC cells were detected by cell counting kit-8 (CCK-8) method using the miR-429 lentivirus and lentivirus negative control. The target gene of miR-429 was predicted by targetscan, and the expression level of target gene was deteceted by Western blotting. Results The expression of miR-429 in gastric tissue of 54 patients (90%, 54/60) with chronic atrophic gastritis was lower than that of normal gastric tissue, of which 37 cases (61.7%, 37/60) decreased more than 2 times. What’s more, the expression of miR-429 in the chronic atrophic gastric tissues with metaplasia and atypical hyperplasia was statistically much more significant (P=0.012). The expression of miR-429 in all gastric cancer tissues was lower than that in normal gastric tissues and chronic atrophic gastric tissues (all P=0.003), and the expression level was negatively correlated with lymph node metastasis, adjacent vascular invasion and TNM staging. The growth of PLGC cells was significantly inhibited at 5th day after transfection with miR-429, and the absorbance (0.852±0.023) was significantly reduced as compared with the control group (1.488±0.031, P=0.000). The expression of CDC25B in the transfection group (the target gene of miR-429 found by targetscan) was significantly lower than that of control group. Conclusion The expression of miR-429 in the gastric tissue of chronic atrophic gastritis and gastric cancer tissues was significantly down-regulated, and it could inhibit the development of gastric precancerous lesions to gastric cancer and the invasion and metastasis of gastric cancer. CDC25B might be the target gene of miR-429. Key words: Gastric precancerous lesions; Gastric cancer; Chronic atrophic gastritis; MicroRNA; Gene CDC25B

Abstract 5120: Cryptolepine a plant alkaloid, inhibits the growth of nonmelanoma skin cancer cells through inhibition of topoisomerase and induction of DNA damage

Abstract Topoisomerases are highly specialized nuclear enzymes that remove superhelical tension on chromosomal DNA that allows replication and transcription of DNA. Many cancer chemotherapeutic drugs used in the clinics inhibit tumor growth by targeting topoisomerase functions resulting in DNA damage and cancer cell death. Cryptolepine, a major alkaloid isolated from Cryptolepis sanguinolenta plant’s roots, has shown anti-malarial, anti-bacterial, anti-fungal, and anti-inflammatory activities. In the present study, we examined the therapeutic effect of cryptolepine on non-melanoma skin cancer cells (NMSCC), SCC-13 and A431 as an in vitro model, and underlying mechanism of action with special emphasis on topoisomerases and DNA damage check points. Western blot analysis and enzyme activity evaluation assays demonstrated that SCC-13 and A431 cells express comparatively higher levels of topoisomerases and higher enzymatic activities compared with normal human epidermal keratinocytes (NHEK). Topoisomerase expression and activity was greatly reduced after 24 h treatment with 2.5, 5.0 and 7.5 µM cryptolepine. Inhibition of topoisomerases expression and function by cryptolepine resulted in significant DNA damage and enhanced expression of DNA-PK expressions. Cryptolepine induced DNA damage activated DNA damage response proteins as demonstrated by increased phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and γH2AX. Consequences of cryptolepine induced DNA damage were also associated with activation of p53 signaling cascade. Cryptolepine induced post-translational modifications such as phosphorylation and acetylation of p53, and inhibition of mdm2 protein expression resulted in activation and accumulation of p53 in SCC-13 and A431 cells. In turn, activated p53 enhanced protein expression of cyclin-dependent kinase inhibitors p16 and p21. Activation of p16 and p21 proteins downregulated expression of cyclin-dependent kinase 2 (CDK2), cyclin A, cyclin D1 and cyclin E and thus induced S-phase cell cycle arrest in NMSC cells with reduced expression of cell division proteins Cdc25a and Cdc25b. Cryptolepine treatment also enhanced release of cytochrome c from mitochondria due to increased disruption of mitochondrial membrane potential. Inhibition of topoisomerase activity and activation of DNA damage response signaling culminated in significantly reduced cell viability and enhanced cell death of SCC-13 and A431 cells. However, NHEK cells were least sensitive to cryptolepine induced cell death. Results of our study strongly suggest for detailed preclinical investigations in animal models to further assess its anti-skin cancer potential and its possible use in human patients. Note: This abstract was not presented at the meeting. Citation Format: Harish C. Pal, Santosh K. Katiyar. Cryptolepine a plant alkaloid, inhibits the growth of nonmelanoma skin cancer cells through inhibition of topoisomerase and induction of DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5120. doi:10.1158/1538-7445.AM2017-5120

Impaired DNA Damage Repair in Severe Congenital Neutropenia Patients

Severe congenital neutropenia (CN) is a pre-malignant bone marrow failure syndrome with maturation arrest of granulopoiesis at the level of promyelocytes in the bone marrow. We hypothesized that increased genetic instability in hematopoietic stem and progenitor cells (HSPC) of CN patients caused by inherited mutations in ELANE (neutrophil elastase) or HAX1(mitochondrial HCLS1-associated protein X-1) may lead to high risk of malignant transformation. Treatment of CN patients with granulocyte-colony stimulating factor (G-CSF) overcomes maturation arrest by forcing unfit HSPC to proliferate and differentiate despite the presence of inherited mutations and thus increasing the risk of leukemogenic transformation.We first investigated differences in DNA damage susceptibility of CD34+ and CD33+ bone marrow cells from CN-ELANE (n = 3) and CN-HAX1 (n = 3) patients, as compared to healthy donors using short-term treatment (5 minutes) with bleomycin to induce DNA double-strand breaks. To detect DNA lesions we used the LORD-Q method, a high-sensitivity long-run real-time PCR-based technique for DNA damage quantification (Lehle S. et al., Nucleic Acids Research, 2014). We found no differences in DNA damage induction between both groups of CN patients and healthy donors. Therefore, we hypothesized that not DNA damage but DNA repair mechanisms may be affected in these patients. Indeed, Gene Set Enrichment Analysis (GSEA) of microarray data revealed a marked inhibition of gene expression in pathways associated with DNA double-strand break (DSB) repair, mismatch repair as well as cell cycle regulation in HSPC from CN patients as compared to cells from healthy individuals. Validation by qRT-PCR confirmed severe downregulation of genes related to DSB repair (BRCA1 and RAD51), mismatch repair (MSH2 and PCNA) as well ascell cycle regulation (CHEK2 and CDKN2C) in CD33+ of both CN groups as compared to healthy individuals. Interestingly, CN-ELANE and CN-HAX1 groups behaved similarly with some exceptions showing decreased expression of CDC25B, RAD50 and ATR expression in the CN-HAX1 group only and of MRE11A in the CN-ELANEgroup only.Taken together, disrupted DNA repair and impaired expression of cell cycle regulating genes resulting from inherited mutations in ELANE and HAX1 indicate that HSPC of CN patients are more susceptible to malignant transformation. DisclosuresNo relevant conflicts of interest to declare.

Safflower polysaccharide induces NSCLC cell apoptosis by inhibition of the Akt pathway.

Lung cancer is the leading cause of cancer death in the world. Safflower polysaccharide (SPS) has been used for the improvement of immunomodulatory activities and treatment of cancers. However, studies on the effect of SPS on the progression of lung cancer have rarely been reported. To study the antitumor effect of SPS on human lung cancer and its potential mechanism, non-small cell lung cancer cell lines (NSCLC), A549 and YTMLC-90 were treated with SPS at various concentrations ranging from 0.04 to 2.56mg/ml and BALB/c nude tumor-bearing mice were injected intraperitoneally with SPS at concentrations ranging from 15 to 135mg/kg. Results showed that SPS suppressed the proliferation of A549 and YTMLC-90 cells and induced apoptosis by increasing mRNA levels of bax and caspase-3, and inhibited tumor growth invivo. SPS induced cell cycle arrest in the G2/M phase by decreasing the expression of cdc25B and cyclin B1. Moreover, SPS decreased the expression of Akt, p-Akt and PI3K. In mice, SPS injection enhanced immunomodulatory activities by increasing levels of TNF-α and IL-6 in tumor-bearing mice. Our findings suggest that SPS suppresses tumor growth by enhancing immunomodulatory activities and blocking the PI3K/Akt pathway. This study provides new insight into the anticancer mechanism of SPS.

CDC25B Involved in Proliferation of Sertoli Cells of New Born Calves Through FSH and Possibly Being Key Regulating Factor

The effects of FSH on the proliferation of sertoli cells of new born calves were studied in order to provide some data for theoretical research and practical use of spermatogenesis in vitro. Different concentrations of FSH (0, 0.01, 0.02, 0.04, and 0.08 IU · mL−1) were taken to treat bovine sertoli cells in vitro culture, the number of sertoli cells and the expression of seven genes were determined at 6, 12 and 24 h after FSH treatments. FSH could significantly promote the proliferation of in vitro cultured sertoli cells. FSH had no significant effects on the expression of CDC25A and could significantly improve the expression of CDC25B. 0.04 IU · mL−1 and 0.08 IU · mL−1 FSH treatments decreased the expression of CDC25C at 12 h. 0.08 IU · mL−1 FSH treatment decreased the expression of CDC25C at 24 h. 0.04 IU · mL−1 FSH could significantly decrease the expression of GSK-3β and improve the expression of β-catenin at 6, 12 and 24 h. 0.02, 0.04 and 0.08 IU · mL−1 FSH treatments enhanced the expressions of CYCLIND1 and C-MYC. In conclusion, FSH promoted the proliferation of sertoli cells and 0.04 IU · mL−1 FSH concentration could significantly promote the proliferation of in vitro cultured sertoli cells. FSH promoted the proliferation of sertoli cells by CDC25B and WNT/β-catenin and CDC25B might be the key regulator to the proliferating rate of sertoli cells of bovine calf.

Abstract 3774: Investigating the role of CDC25B in inhibition of cellular proliferation

Abstract CDC25B is a dual specificity phosphatase that activates cyclin-dependent kinases to trigger entry into mitosis and is required for re-entry into mitosis after DNA damage. Four isoforms of CDC25B are known to be expressed and some differences in their role in dividing cells versus continuation of the cell cycle after DNA damage have been shown. As an oncogene, CDC25B is upregulated by c-Myc and activated by Aurora-A. In humans, CDC25B is overexpressed in multiple tumor types and increased levels correlate with worsening tumor grade. In a transgenic mouse model, overexpression of CDC25B in the mammary glands led to accelerated mammary epithelial proliferation resulting in hyperplasia and the formation of tumors when exposed to the carcinogen DMBA. In vitro, co-transfection of CDC25B with Ras allows for transformation of MEF’s. Surprisingly, given the establishment of CDC25B as an oncogene, colony formation assays using ectopic expression of CDC25B in transformed cell lines reveal an anti-proliferative effect. Although CDC25B has been shown to cause DNA damage and activates p53, our data show that colony formation is decreased independent of p53 status. Preliminary data reveal a role for Ras inhibition as a cause of this anti-proliferative effect. Co-transfection of CDC25B and activated Ras results in decreased expression of the ectopic Ras protein in several cell lines tested. Furthermore overexpression of CDC25B abrogates the G2 arrest and decreases cell viability after pulse treatment with doxorubicin. Taken together, these data suggest a novel mechanism by which CDC25B may inhibit proliferation by decreasing Ras expression. Citation Format: Caleb C. Lee, James Manfredi. Investigating the role of CDC25B in inhibition of cellular proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2015-3774

The BET bromodomain inhibitor JQ1 suppresses growth of pancreatic ductal adenocarcinoma in patient-derived xenograft models.

The primary aim of this study was to evaluate the antitumor efficacy of the bromodomain inhibitor JQ1 in pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (tumorgraft) models. A secondary aim of the study was to evaluate whether JQ1 decreases expression of the oncogene c-Myc in PDAC tumors, as has been reported for other tumor types. We used five PDAC tumorgraft models that retain specific characteristics of tumors of origin to evaluate the antitumor efficacy of JQ1. Tumor-bearing mice were treated with JQ1 (50 mg/kg daily for 21 or 28 days). Expression analyses were performed with tumors harvested from host mice after treatment with JQ1 or vehicle control. An nCounter PanCancer Pathways Panel (NanoString Technologies) of 230 cancer-related genes was used to identify gene products affected by JQ1. Quantitative RT-PCR, immunohistochemistry and immunoblots were carried out to confirm that changes in RNA expression reflected changes in protein expression. JQ1 inhibited the growth of all five tumorgraft models (P<0.05), each of which harbors a KRAS mutation; but induced no consistent change in expression of c-Myc protein. Expression profiling identified CDC25B, a regulator of cell cycle progression, as one of the three RNA species (TIMP3, LMO2 and CDC25B) downregulated by JQ1 (P<0.05). Inhibition of tumor progression was more closely related to decreased expression of nuclear CDC25B than to changes in c-Myc expression. JQ1 and other agents that inhibit the function of proteins with bromodomains merit further investigation for treating PDAC tumors. Work is ongoing in our laboratory to identify effective drug combinations that include JQ1.