Expression Levels Of Cyclin B1 Research Articles

EIF6 Promotes Gastric Cancer Proliferation and Invasion by Regulating Cell Cycle.

To investigate the role and function of eIF6 in gastric cancer (GC). The expression level of eIF6 in GC tissues and normal tissues was detected in different high-throughput sequencing cohorts. Survival analysis, gene differential analysis, and enrichment analysis were performed in the TCGA cohort. Biological networks centered on eIF6 were constructed through two different databases. Immunohistochemistry (IHC) and Western blot were used to detect protein expression of eIF6, and qRT-PCR was used to detect eIF6 mRNA expression. The correlation between the expression of eIF6 in GC tissues and clinicopathological parameters of GC was analyzed. siRNA knockout of eIF6 was used to study the proliferation, migration, and invasion. The effects of eIF6 on cell cycle and Cyclin B1 were detected by flow cytometry and Western blot. eIF6 was significantly overexpressed in GC tissues and predicted poor prognosis. In addition, 113 differentially expressed genes were detected in cancer-related biological pathways and functions by differential analysis. Biological networks revealed interactions of genes and proteins with eIF6. The expression intensity of eIF6 in cancer tissues was higher than that in adjacent tissues (P = 0.0001), confirming the up-regulation of eIF6 expression in GC tissues. The expression level of eIF6 was statistically significant with pTNM stage (P = 0.006). siRNA knockout of eIF6 significantly reduced the proliferation, colony formation, migration, and invasion ability of GC cells. Silencing of eIF6 also inhibited the cell cycle of GC cells in G2/M phase and decreased the expression level of CyclinB1. Our study suggests that eIF6 is up-regulated in GC and may promote the proliferation, migration, and invasion of GC by regulating cell cycle.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Ahnak depletion accelerates liver regeneration by modulating the TGF-β/Smad signaling pathway

Ahnak, a large protein first identified as an inhibitor of TGF-β signaling in human neuroblastoma, was recently shown to promote TGF-β in some cancers. The TGF-β signaling pathway regulates cell growth, various biological functions, and cancer growth and metastasis. In this study, we used Ahnak knockout (KO) mice that underwent a 70% partial hepatectomy (PH) to investigate the function of Ahnak in TGF-β signaling during liver regeneration. At the indicated time points after PH, we analyzed the mRNA and protein expression of the TGF -β/Smad signaling pathway and cell cycle-related factors, evaluated the cell cycle through proliferating cell nuclear antigen (PCNA) immunostai-ning, analyzed the mitotic index by hematoxylin and eosin staining. We also measured the ratio of liver tissue weight to body weight. Activation of TGF-β signaling was confirmed by analyzing the levels of phospho-Smad 2 and 3 in the liver at the indicated time points after PH and was lower in Ahnak KO mice than in WT mice. The expression levels of cyclin B1, D1, and E1; proteins in the Rb/E2F transcriptional pathway, which regulates the cell cycle; and the numbers of PCNA-positive cells were increased in Ahnak KO mice and showed tendencies opposite that of TGF-β expression. During postoperative regeneration, the liver weight to body weight ratio tended to increase faster in Ahnak KO mice. However, 7 days after PH, both groups of mice showed similar rates of regeneration, following which their active regeneration stopped. Analysis of hepatocytes undergoing mitosis showed that there were more mitotic cells in Ahnak KO mice, consistent with the weight ratio. Our findings suggest that Ahnak enhances TGF-β signaling during postoperative liver regeneration, resulting in cell cycle disruption; this highlights a novel role of Ahnak in liver regeneration. These results provide new insight into liver regeneration and potential treatment targets for liver diseases that require surgical treatment.

Dysregulation of Pseudogenes/lncRNA-Hsa-miR-1-3p-PAICS Pathway Promotes the Development of NSCLC.

Objective Non-small cell lung cancer (NSCLC) explains about 80 percent of whole lung cancers, and its 5-year survival rate is impoverished, as when people are first diagnosed, 68% of whom are identified at a dangerous stage. The molecular mechanisms of NSCLC are still being explored. Methods GSE18842 and GSE19804 were exerted to scan for diversely expressed genes (DEGs) in NSCLC, and then we used GEPIA for the validation of DEGs expression. The prognostic values were determined through Kaplan–Meier analysis. Three target prediction databases indicated potential microRNAs (miRNAs), while miRNet predicted hsa-miR-1-3p′s upstream long non-coding RNAs (lncRNAs) and pseudogenes. UALCAN was utilized to identify the co-expressed genes of PAICS, while enrichment analysis on them was managed with Enrichr. Results We initially found that the gene expression level of cyclin B1 (CCNB1), cyclin-dependent kinases1 (CDK1), and phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) had a notable increase in NSCLC. We predicted 6, 10, and 7 microRNAs to target CCNB1, CDK1, and PAICS, respectively. Among miRNA-mRNA (microRNA-messenger RNA) pairs, we deduced that the hsa-miR-1-PAICS axis was the most potential one to inhibit the occurrence of NSCLC. We also noted that the hsa-miR-1-3p-PAICS axis participated in regulating the process of mitosis with mechanical functions. Moreover, we identified 5 pseudogenes and 33 long non-coding RNAs (lncRNAs) that might inhibit the hsa-miR-1-3p-PAICS axis in NSCLC. Conclusions The pseudogene/lncRNA-hsa-miR-1-3p-PAICS is very important in NSCLC on the basis of this study, thus providing us with effective treatments and promising biomarkers for the diagnosis of NSCLC.

BRAF Inhibitor Resistance Confers Increased Sensitivity to Mitotic Inhibitors.

Single agent and combination therapy with BRAFV600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases BRAF inhibitor (BRAFi) resistance eventually develops. One resistance mechanism is reactivation of the ERK pathway. However, only about half of BRAFi resistance is due to ERK reactivation. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy, used to track mitosis in real time, revealed that parental but not BRAFi-resistant melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint in arrested cells, we found lower Cyclin B1 levels in parental compared with BRAFi-resistant melanoma cells, suggesting that inability to down-regulate Cyclin B1 expression levels may explain the increased vulnerability of resistant cells to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, which was associated with an accumulation of DNA damage, resulting in mitotic failure. This study demonstrates that BRAFi-resistance, in at least a subset of melanoma cells, confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach for overcoming resistance to BRAF/MEK-directed therapies.

The kinesin Eg5 inhibitor K858 exerts antiproliferative and proapoptotic effects and attenuates the invasive potential of head and neck squamous carcinoma cells

Our group recently demonstrated that K858, an inhibitor of motor kinesin Eg5, has important antiproliferative and apoptotic effects on breast cancer, prostatic cancer, melanoma and glioblastoma cells. Since high levels of kinesin Eg5 expression have been correlated with a poor prognosis in laryngeal carcinoma, we decided to test the anticancer activity of K858 toward this tumor, which belongs to the group of head and neck squamous cell carcinomas (HNSCCs). These cancers are characterized by low responsiveness to therapy. The effects of K858 on the proliferation and assembly of mitotic spindles of three human HNSCC cell lines were studied using cytotoxicity assays and immunofluorescence for tubulin. The effect of K858 on the cell cycle was analyzed by FACS. The expression levels of cyclin B1 and several markers of apoptosis and invasion were studied by Western blot. Finally, the negative regulation of the malignant phenotype by K858 was evaluated by an invasion assay. K858 inhibited cell replication by rendering cells incapable of developing normal bipolar mitotic spindles. At the same time, K858 blocked the cell cycle in the G2 phase and induced the accumulation of cytoplasmic cyclin B and, eventually, apoptosis. Additionally, K858 inhibited cell migration and attenuated the malignant phenotype. The data described confirm that kinesin Eg5 is an interesting target for new anticancer strategies and suggest that this compound may be a powerful tool for an alternative therapeutic approach to HNSCCs.

Evaluation of anti-cancer potential of Excoecaria\xa0agallocha (L.) leaf extract on human cervical cancer (SiHa) cell line and assessing the underlying mechanism of action

BackgroundThe incidence of cervical cancer is increasing at an alarming rate in many countries and presently, it is the most common form of malignant cancer being reported among women in India. Development of novel approach for cervical cancer therapy, sparing healthy normal cells overcoming the limitations of prevailing therapies is of prime importance. Mangroves constitute a significant repository of medicinally important plants. Thus, in this study, we aimed to determine the anticancer activity of the mangrove Excoecaria agallocha L. leaf extracts on human cervical cancer (SiHa HPV 16+) cell line with subsequent characterization of the bioactive compounds conferring the anticancer activity and studying the probable underlying mechanism of action of the purified plant extract.ResultsThe plant extract was subjected to silica gel column chromatography and the fractions obtained were analyzed for cytotoxic activity against SiHa cells by MTT assay. One out of the three eluted fractions exhibited selective toxicity against SiHa cells with an IC50 value of 15.538 ± 0.577 µg/mL, while it had no cytotoxic effect on normal healthy human peripheral blood mononuclear cells. High-resolution liquid chromatography mass spectroscopy, coupled to electron spray ionization and diode array detection analysis, led to the structure elucidation and identification of a few pharmacologically important compounds, with Bergenin being present in the highest abundance. Fluorescence microscopy results revealed that the plant extract fraction induced LC3 puncta formation, in EGFP- SiHa cells indicating the onset of autophagy, with simultaneous stimulation of mitophagy. The plant extract also inhibited proliferation of the SiHa-smac-mCherry cells by second mitochondria-derived activator of caspase (SMAC)—induced cytochrome c dependent apoptosis, that was further confirmed with Caspase-3 activation by colorimetric assay. The GFP-dgn in SiHa cells was remarkably protected from proteasomal degradation that might upregulate the survivability of the cells significantly. Flow cytometry followed by Western blot analysis further asserted the ability of the plant extract fraction to cause cell cycle arrest of SiHa cells in the G2/M phase by significantly reducing protein expression levels of cyclin B1 and D1, decreasing Cdc2 level and simultaneously increasing p21 and p53 levels.ConclusionIt could be inferred that the aqueous extract of E. agallocha successfully decreased the proliferation of SiHa cervical cancer cells through induction of autophagy and apoptosis in a concerted manner, with simultaneous stimulation of mitophagy and G2/M phase cell cycle arrest, hinting at Bergenin being the major compound conferring the anti-cancer activity of the plant extract. Thus, isolation of the identified bioactive compounds from E. agallocha and their subsequent purification for drug development might serve as a novel medicinal approach for the treatment of cervical cancer in conjugation with existing therapeutic methods.

Delphinidin induces cell cycle arrest and apoptosis in HER-2 positive breast cancer cell lines by regulating the NF-κB and MAPK signaling pathways.

Delphinidin is an anthocyanidin monomer, commonly found in vegetables and fruits, and has demonstrated antitumor effects in the HER-2-positive MDA-MB-453 breast cancer cell line, with low cytotoxicity on normal breast cells. However, the direct functional mechanisms underlying the effect of delphinidin on HER-2-positive breast cancer cells has not been fully characterized. In the present study, it was found that delphinidin could induce G2/M phase cell cycle arrest by inhibiting the protein expression level of cyclin B1 and Cdk1 in HER-2-positive breast cancer cell lines. In addition, delphinidin promoted the mitochondrial apoptosis pathway by inhibiting the ERK and NF-κB signaling pathway and activating the JNK signaling pathway. Therefore, delphinidin markedly suppressed the viability of the HER-2-positive breast cancer cell lines by modulating the cell cycle and inducing apoptosis. Overall, the findings from the present study demonstrated that delphinidin treatment could induce the mitochondrial apoptosis pathway in human HER-2-positive breast cancer cell lines, providing an experimental basis for the prevention and treatment of HER-2-positive breast cancer by flavonoids.

MiR-3074-5p/CLN8 pathway regulates decidualization in recurrent miscarriage.

Decidualization is essential for the successful establishment of pregnancy, and the dysregulated decidualization may lead to early pregnancy loss. It was previously reported by us that miR-3074-5p could promote apoptosis but inhibit invasion of human extravillous trophoblast (EVT) cells in vitro, and the expression level of miR-3074-5p in villus tissues of recurrent miscarriage (RM) patients was significantly increased. The aim of this study was to preliminarily explore the role of miR-3074-5p played in the decidualization of human endometrial stromal cells (ESCs). It was found that the decidual expression level of miR-3074-5p in RM patients was remarkably higher than that in the control group. The overexpression of miR-3074-5p in the immortalized human ESC line, T-HESCs, showed suppressive effects not only on the cell proliferation, as well as the intracellular expression levels of cyclin B1 (CCNB1), CCND1 and CCNE1 but also on the in vitro-induced decidualization. CLN8 mRNA, encoding an endoplasmic reticulum (ER)-associated membrane protein, was validated to be directly targeted by miR-3074-5p. And, the expression level of CLN8 was continuously increased along with the decidualization process, whereas down-regulated CLN8 expression could inhibit the decidualization of T-HESCs in vitro. Furthermore, contrary to the increased expression level of miR-3074-5p, a significantly decreased CLN8 expression was observed in decidual tissues of RM patients. Collectively, these data suggested that an increased miR-3074-5p expression in ESCs might cause early pregnancy failure by disturbing decidualization of ESCs via the miR-3074-5p/CLN8 pathway, providing a potential diagnostic and therapeutic target for RM.

Anthraquinone derivative C10 inhibits proliferation and cell cycle progression in colon cancer cells via the Jak2/Stat3 signaling pathway

Since its discovery, anthraquinone has become very valuable as a lead compound in the development of anti-cancer drugs. Previously, we designed and synthesized a new type of amide anthraquinone derivative (1-nitro-2-acylanthraquinone glycine, C10) with good activity against colon cancer. However, its effect and the underlying mechanism are unclear. In this study, C10 significantly inhibited the proliferation of HCT116 and HT29 colon cancer cells by blocking the cell cycle at the G2/M phase. C10 also plays a role in cell cycle arrest by reducing the protein and gene expression levels of cyclin B1 and its downstream signaling molecule cyclin-dependent kinase (CDK1). In addition, molecular docking studies showed that C10 has high affinity for Jak2, the first target in the cell cycle-related Jak2/Stat3 signaling pathway. Furthermore, C10 downregulated the expression of Jak2/Stat3 signaling pathway-related signaling molecules proteins and genes, and up-regulated the expression of PIAS-3, the upstream signaling molecule of Stat3, thereby down-regulating Stat3 phosphorylation. C10 reversed the expression of Jak2/Stat3 signaling pathway-related molecules activated by IL-6. Overall, our results indicate for the first time that C10 induces cell cycle arrest and inhibits cell proliferation by inhibiting the Jak2/Stat3 signaling pathway. This study provides new insights into the potential role of Jak2/Stat3 in the regulating cell cycle-related signaling pathways that mediate the inhibitory effects of C10 on colon cancer cell proliferation.

Cucurbitacin B exhibits antitumor effects on CD133+ HepG2 liver cancer stem cells by inhibiting JAK2/STAT3 signaling pathway.

Cancer stem cells (CSCs), a crucial cancer cell subpopulation, possess stemness phenotypic characteristics. Cucurbitacin B (CuB), a tetracyclic triterpenoid isolated from Cucurbitaceae, exerts widely pharmacological activities in many diseases. The aim of this study was to enrich, identify liver CSCs and investigate antitumor effects of CuB as well as explore the underlying molecular mechanisms in these liver CSCs. HepG2 cell lines were used for the enrichment of liver CSCs by serum-free medium culture and magnetic-activated cell sorting. The CSC characteristics were analyzed by immunofluorescent staining, sphere-forming, western blot and xenograft tumorigenicity assay. CuB' antitumor effects and underlying molecular mechanism were measured by cell counting kit-8, colony formation, sphere-forming, cell cycle, xenograft and western blot assay. Our results showed that we could enrich 97.29% CD133+ HepG2 cells, which possessed CSC characteristics including re-renewal capacity, proliferative ability, sorafenib resistance, overexpressed stemness-related molecules and enhanced tumorigenic potential. Furthermore, we also found that CuB inhibited cell viability, sphere formation, colony formation and arrested cell cycle at G2/M phase as well as sensitized CD133+ HepG2 cells to sorafenib in vitro and in vivo. Western blot assay indicated that CuB inhibited expression levels of cyclin B1, CDK1, CD133, p-JAK2 and p-STAT3. In conclusion, our findings indicated that CuB could exhibit antitumor effects on CD133+ HepG2 CSCs by inhibiting the Janus kinase 2/signal transducers and activators of transcription-3 signaling pathway, expanding basic and preclinical investigations on liver CSCs.

Isoorientin induces the apoptosis and cell cycle arrest of A549 human lung cancer cells via the ROS‑regulated MAPK, STAT3 and NF‑κB signaling pathways.

Isoorientin (ISO) is a naturally occurring C‑glycosyl flavone that has various pharmacological properties, such as anti‑bacterial and anti‑inflammatory effects. However, its underlying molecular mechanisms in human lung cancer cells remain unknown. In the present study, the effects of ISO on the induction of apoptosis and relative molecular mechanisms in A549 human lung cancer cells were investigated. The results of Cell Counting Kit‑8 assay (CCK‑8) indicated that ISO exerted significant cytotoxic effects on 3lung cancer cell lines, but had no obvious side‑effects on normal cells. Moreover, flow cytometry and western blot analysis revealed that ISO induced mitochondrial‑dependent apoptosis by reducing mitochondrial membrane potential. ISO also increased the expression levels of Bax, cleaved‑caspase‑3 (cle‑cas‑3) and poly(ADP‑ribose) polymerase (PARP; cle‑PARP), and decreased the expression levels of Bcl‑2 in A549 cells. Furthermore, ISO induced G2/M cell cycle arrest by decreasing the expression levels of cyclin B1 and CDK1/2, and increasing the expression levels of p21 and p27 in A549 cells. As the duration of ISO treatment increased, intracellular reactive oxygen species (ROS) levels in A549 cells also increased. However, pre‑treatment of the cells with the ROS scavenger, N‑acetylcysteine (NAC), inhibited ISO‑induced apoptosis. In addition, ISO increased the expression levels of p‑p38, p‑JNK and IκB‑α; and decreased the expression levels of p‑extracellular signal‑regulated kinase (ERK), p‑signal transducer and activator of transcription (STAT)3, p‑nuclear factor (NF)‑κB, NF‑κB and p‑IκB; these effects were induced by mitogen‑activated protein kinase (MAPK) inhibitors and blocked by NAC. Taken together, the results of the present study indicate that ISO induces the apoptosis of A549 lung cancer cells via the ROS‑mediated MAPK/STAT3/NF‑κB signaling pathway, and thus may be a potential drug for use in the treatment of lung cancer.

Clinicopathologic and protein markers distinguishing POLE and copy-number low group in endometrial cancer.

e18110 Background: Requirement of DNA sequencing to diagnose POLE group is a barrier for adoption of molecular subtyping in endometrial cancer. We aimed to identify clinicopathologic and protein markers distinguishing POLE and copy-number (CN) low group in endometrial cancer. Methods: Ninety-one samples (POLE: 15, CN low 76) classified as POLE or CN low groups by integrative clustering were selected from The Cancer Genome Atlas endometrial cancer dataset. Clinicopathologic variables and normalized reverse phase protein array expression data were extracted. We first selected clinicopathologic variables associated with group (POLE vs CN low) via univariate analysis. Then, we identified protein makers using the logistic regression model with significant clinicopathologic variables as adjusting covariates. The differentially expressed proteins were selected based on q-value of the false discovery rate for multiple comparison. With various q-value cut-off ( < 5%, 10%, 20%), several logistic models including differentially expressed markers were constructed by stepwise selection method using the area under curve (AUC) from 5-fold cross-validation (CV). Results: Among clinicopathologic variables, body mass index (BMI) and tumor grade were associated with group (p = 0.02 and p < 0.01, respectively). Being adjusted for BMI and tumor grade, 5 proteins were associated with group in q-value cut-off of < 5%. The model including clinicopathologic variables and 5 proteins identified BMI, Cyclin B1, Caspase 8, and XBP1 as markers distinguishing POLE and CN low groups. The mean of CV AUC, sensitivity and specificity of the selected model were 0.97, 0.97, and 0.60, respectively. Conclusions: BMI and expression levels of Cyclin B1, Caspase 8, XBP1 are candidate markers distinguishing POLE and CN low group. A further validation study using immunohistochemical staining is necessary to facilitate the adoption of molecular subtyping as daily practice.

Design, synthesis and biological evaluation of 3-nitro-1,8-naphthalimides as potential antitumor agents

A series of 3-nitro-naphthalimides 1(1a–1h) were designed and synthesized as antitumor agents. MTT assay results showed that all these compounds exhibited obvious antiproliferative activity against SKOV3, HepG2, A549, T-24 and SMMC-7721 cancer cell lines, while compound 1a displayed the best antiproliferative activity against HepG2 and T-24 cell lines in comparison with mitonafide, with IC50 of 9.2 ± 1.8 and 4.133 ± 0.9 μM, respectively. In vivo antiproliferative activity assay results showed that compound 1a exhibited good antiproliferative activity in the HepG2 and T-24 models, compared with mitonafide. Action mechanism results showed that compound 1a could induced the damage of DNA and the inhibition topo I, accompanying by inducing the G2-stage arresting and the apoptosis of T-24 cancer cells through up-regulating expression levels of cyclin B1, cdc 2-pTy, Wee1, γH2AX, p21, Bax and cytochrome c and down-regulating expression of Bcl-2.

Single high dose irradiation induces cell cycle arrest and apoptosis in human hepatocellular carcinoma cells through the Ras/Raf/MEK/ERK pathways

Purpose: Stereotactic body radiation therapy (SBRT) is emerging as a new noninvasive treatment in patients with primary liver carcinoma or liver-confined metastatic cancer. However, the radiobiological targets remain a subject of debate. Here, we investigated the potential biological effects of the radiation on the human hepatocellular carcinoma HepG2 cells.Materials and methods: Firstly, HepG2 cells were divided into three groups: control group, 3.5 Gy*8f group (L group), and 15 Gy*1f group (H group). After treatment, cell proliferation was examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and plate colony formation assays. Cell cycle and apoptosis were assessed using propidium iodide and Hoechst 33258 staining, respectively. Furthermore, the mechanisms underlying irradiation-induced cell cycle arrest and cell apoptosis were investigated by Western blot assay.Results: Irradiation could effectively inhibit the proliferation and colony formation of HepG2 cells, and the single high dose irradiation showed stronger inhibitory effects. Irradiation-induced cell cycle arrest at G2/M phase in HepG2 cell, during which the expression levels of cyclin B1, CDK1, and p-CDK1 proteins were downregulated, whereas expression of p21 was upregulated in the irradiated HepG2 cells. After irradiation, typical morphological changes of apoptosis in HepG2 cells were observed; the number of cell apoptosis and the expression of apoptosis associated proteins were significantly increased in HepG2 cells by high dose irradiation compared with low dose irradiation. Additionally, compared with low dose irradiation, high dose irradiation significantly downregulated the phosphorylated proteins in the Ras/Raf/MEK/ERK signaling pathway.Conclusions: Our results suggest that irradiation applied in SBRT, particularly single high dose irradiation, mediates its anti-tumor effects by inducing cell cycle arrest and apoptosis via modulation of the Ras/Raf/MEK/ERK signaling pathway.

Germacrone exerts anti-cancer effects on gastric cancer through induction of cell cycle arrest and promotion of apoptosis

BackgroundGermacrone is one of the natural bioactive compounds found in Rhizoma curcuma essential oils. In this study, the potential anti-cancer effect of germacrone in gastric cancer cell line BGC823 was investigated.MethodsThe cell viability and proliferative activity were assessed, and cell cycle analysis was also performed. Hoechst 33258 and Annexin V/PI double staining was used for detection of cell apoptosis. Protein profiles of cell cycle-related and apoptosis-related proteins were assessed.ResultsMTT assay revealed that germacrone had marked cytotoxicity on BGC823 cells. Germacrone induced cell cycle arrest in the G2/M phase via remarkably decreased expression levels of cyclin B1, cdc 2 and cdc 25c. In addition, the treatment with germacrone induced caspase-3 activity and PARP cleavage. These findings demonstrated the effects of germacrone on inhibiting cell proliferation through induction of G2/M phase cell cycle arrest and promotion of cell apoptosis. It also indicated that germacrone functioned through modulations of cell cycle-associated protein expression and mitochondria-mediated apoptosis.ConclusionThese findings will be valuable as the molecular basis for the germacrone-mediated anti-cancer effect against gastric cancer.

Prognostic significance of cyclin B1 expression plus clinicopathologic features in hormonal positive, HER2 negative early breast cancer in King Chulalongkorn Memorial Hospital During 2010-2015.

75 Background: Approximately one third of hormonal receptor (HR) positive, HER2 negative early breast cancer reported disease relapse after adjuvant treatments. Both clinicopathologic features and multigene assays consider to be predicting factor of relapse. Cyclin B1, one of proliferative markers used in OncotypeDx has been explored previously for predicting recurrence in this subgroup of breast cancers. Our study aims to determine the prognostic significance of cyclin B1 in combination with clinicopathologic factors in recurrent HR positive, HER2 negative breast cancer. Methods: Two-hundred and forty-five HR positive, HER2 negative early breast cancers who were diagnosed during 2010 to 2015 in King Chulalongkorn Memorial Hospital were retrospectively reviewed. All clinicopathologic factors and level of cyclin B1 expression were evaluated. Correlation of cyclin B1 expression with clinicopathologic features was also compared in recurrence and non-recurrence group. Results: In 245 patients, 65 patients were recurrence group while 180 patients were non-recurrence group. Mean age at breast cancer diagnosis were 53 years. Recurrence group had high pathological staging, tumor grade, LVI, Ki-67 and lymph node involvement compared to non-recurrence group ( p < 0.05). Mean cyclin B1 expression was 9.61% (19.62 % in recurrence group and 5.88 % in non-recurrence group; p< 0.001). We have used cut-off of cyclin B1 expression at ≥ 10 % (7th decile) to classify high and low of expression. Cyclin B1 high expression were demonstrated in 53.4% of recurrence group compared to 22.4% of non-recurrence group. In multivariate analysis, tumor grade (OR 8.42, 95%CI 1.04-67.98; p = 0.046), receiving neoadjuvant chemotherapy (NAC) (OR 4.27, 95%CI 1.41-12.87; p = 0.010) and % cyclin B1 expression (OR 1.04, 95%CI 1.00-1.07; p = 0.013) were associated with recurrent disease. Five-year relapse free survival for cyclin B1 low and high expression were 84.9% and 60.1%, respectively. Conclusions: Tumor grade, receiving NAC and % cyclin B1 expression were associated with risk of recurrence in HR positive and HER2 negative early breast cancer.

Silencing of long noncoding RNA HOXD-AS1 inhibits proliferation, cell cycle progression, migration and invasion of hepatocellular carcinoma cells through MEK/ERK pathway.

Accumulating findings reveal that long noncoding RNAs (lncRNAs) as crucial regulatory molecules serve vital functions in the progression of hepatocellular carcinoma (HCC). This study aims to investigate the biological roles and mechanisms of lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) in HCC cells based on transcriptome analysis. The Cancer Genome Atlas data analysis and experimental validation showed that HOXD-AS1 was increased in HCC tissues/cell lines and positively relevant to histologic grade. The subcellular localization results indicated HOXD-AS1 was dispersed both in the nucleus as well as the cytoplasm of HCC cells. In vitro loss-of-function experiments revealed that silencing of HOXD-AS1 could dramatically suppress the proliferation, migration, and invasion, and induce S or/and G2/M phase cell cycle arrest as well as apoptosis of Bel-7402 and MHCC97H cells accompanying the changes in expression levels of cyclin B1, cyclin D1, BCL-2, BAX, and MMP2. In vivo assay also showed that HOXD-AS1 silencing could markedly reduce xenograft tumor volume and weight of HCC cells. Transcriptome and bioinformatic analysis indicated that a total of 1103 genes were significantly altered by HOXD-AS1 silencing, of which 132 genes exhibited a significant correlation with HOXD-AS1 expression in HCC tissues. Gene Ontology (GO) enrichment analysis revealed differentially expressed genes were remarkably enriched in several cancer-related biological processes (cell proliferation, cell cycle, apoptosis, migration, angiogenesis, and hypoxic response). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that HOXD-AS1 has the potential to affect p53, tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK) pathway, and Western blot results further validated that HOXD-AS1 silencing could inhibit the MEK/ERK pathway in Bel-7402 cells. Collectively, HOXD-AS1, as an oncogenic lncRNA, might exert crucial functions in HCC progression and serve as a potential diagnostic biomarker and therapeutic target for HCC.

UBA2 promotes proliferation of colorectal cancer.

Small ubiquitin-like modifier proteins are involved in tumorigenesis; however, the potential effects and functions of the family member ubiquitin-like modifier-activating enzyme 2 (UBA2) on colorectal cancer are not clear. The present study aimed to examine the effects of UBA2 on the proliferation of colorectal cancer cells in vitro and in vivo. The mRNA and protein expression levels of UBA2 in patients with colorectal cancer were measured by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. UBA2 expression levels in colorectal cancer tissues were significantly increased compared with the paracancerous normal tissues. The expression of UBA2 was also associated with higher stage colorectal cancer and poor prognosis. MTT and colony formation assays were used to examine proliferation in colorectal cancer cell lines. Flow cytometry was performed to examine the effects of UBA2 on the cell cycle and apoptosis of colorectal cancer cell lines and protein expression levels were examined by western blotting. Athymic nude mice were used to examine the ability of transfected colorectal cancer cells to form tumors in vivo. Downregulation of UBA2 inhibited the proliferation of colorectal cancer cell lines in vitro and in vivo through the regulation of cell cycle associated protein expression and apoptosis. Furthermore, downregulation of UBA2 decreased the expression levels of cyclin B1, B-cell lymphoma-2, phosphorylated protein kinase B and E3 ubiquitin-protein ligase MDM2 in colorectal cancer cells, whereas the expression levels of p21 and p27 were increased. UBA2 was demonstrated to serve an essential role in the proliferation of colorectal cancer and may be used as a potential biomarker to predict prognosis and as a therapeutic target in colorectal cancer.

Gallic acid caused cultured mice TM4 Sertoli cells apoptosis and necrosis.

ObjectiveThe study was designed to determine the cytotoxic effect of gallic acid (GA), obtained by the hydrolysis of tannins, on mice TM4 Sertoli cells apoptosis.MethodsIn the present study, non-tumorigenic mice TM4 Sertoli cells were treated with different concentrations of GA for 24 h. After treatment, cell viability was evaluated using WST-1, mitochondrial dysfunction, cells apoptosis and necrosis was detected using JC-1, Hoechst 33342 and propidium iodide staining. The expression levels of Cyclin B1, proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (BAX), and Caspase-3 were also detected by quantitative real-time polymerase chain reaction and Western-blotting.ResultsThe results showed that 20 to 400 μM GA inhibited viability of TM4 Sertoli cells in a dose-dependent manner. Treatment with 400 μM GA significantly inhibited PCNA and Cyclin B1 expression, however up-regulated BAX and Caspase-3 expression, caused mitochondrial membrane depolarization, activated Caspase-3, and induced DNA damage, thus, markedly increased the numbers of dead cells.ConclusionOur findings showed that GA could disrupt mitochondrial function and caused TM4 cells to undergo apoptosis and necrosis.