Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Prognostic value of mitotic checkpoint protein BUB3, cyclin B1, and pituitary tumor-transforming 1 expression in prostate cancer.

The mitotic checkpoint protein BUB3, cyclin B1 (CCNB1) and pituitary tumor-transforming 1 (PTTG1) regulates cell division, and are sparsely studied in prostate cancer. Deregulation of these genes can lead to genomic instability, a characteristic of more aggressive tumors. We aimed to determine the expression levels of BUB3, CCNB1, and PTTG1 as potential prognostic markers of recurrence after radical prostatectomy. Protein levels were determined by immunohistochemistry on three formalin-fixed paraffin-embedded tissue sections from each of the 253 patients treated with radical prostatectomy. Immunohistochemistry scores were obtained by automated image analysis for CCNB1 and PTTG1. Recurrence, defined as locoregional recurrence, distant metastasis or death from prostate cancer, was used as endpoint for survival analysis. Tumors having both positive and negative tumor areas for cytoplasmic BUB3 (30%), CCNB1 (28%), or PTTG1 (35%) were considered heterogeneous. Patients with ≥1 positive tumor area had significantly increased risk of disease recurrence in univariable analysis compared with patients where all tumor areas were negative for cytoplasmic BUB3 (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.41-3.36), CCNB1 (HR = 2.98, 95% CI 1.93-4.61) and PTTG1 (HR = 1.91, 95% CI 1.23-2.97). Combining the scores of cytoplasmic BUB3 and CCNB1 improved risk stratification when integrated with the Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score (difference in concordance index = 0.024, 95% CI 0.001-0.05). In analysis of multiple tumor areas, prognostic value was observed for cytoplasmic BUB3, CCNB1, and PTTG1.

Purpurogallin is a novel mitogen-activated protein kinase kinase 1/2 inhibitor that suppresses esophageal squamous cell carcinoma growth in vitro and in vivo.

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.

Crocodile choline from Crocodylus siamensis induces apoptosis of human gastric cancer.

Crocodile choline, an active compound isolated from Crocodylus siamensis, was found to exert potent anti-cancer activities against human gastric cancer cells in vitro and in vivo. Our study revealed that crocodile choline led to cell cycle arrest at the G2/M phase through attenuating the expressions of cyclins, Cyclin B1, and CDK-1. Furthermore, crocodile choline accelerated apoptosis through the mitochondrial apoptotic pathway with the decrease in mitochondrial membrane potential, the increase in reactive oxygen species production and Bax/Bcl-2 ratio, and the activation of caspase-3 along with the release of cytochrome c. In addition, this study, for the first time, shows that Notch pathway is remarkably deregulated by crocodile choline. The combination of crocodile choline and Notch1 short interfering RNA led to dramatically increased cytotoxicity than observed with either agent alone. Notch1 short interfering RNA sensitized and potentiated the capability of crocodile choline to suppress the cell progression and invasion of gastric cancer. Taken together, these data suggested that crocodile choline was a potent progression inhibitor of gastric cancer cells, which was correlated with mitochondrial apoptotic pathway and Notch pathway. Combining Notch1 inhibitors with crocodile choline might represent a novel approach for gastric cancer.

Platelet rich plasma releasate promotes proliferation of skeletal muscle cells in association with upregulation of PCNA, cyclins and cyclin dependent kinases.

Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The purpose of this study is to investigate the effect and molecular mechanism of PRP releasate on proliferation of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP releasate. Cell proliferation was evaluated by 3-[4,5-Dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and immunocytochemistry with Ki-67 stain. Flow cytometric analysis was used to evaluate the cell cycle progression. Western blot analysis was used to evaluate the protein expressions of PCNA, cyclin E1, cyclin A2, cyclin B1, cyclin dependent kinase (cdk)1 and cdk2. The results revealed that PRP releasate enhanced proliferation of skeletal muscle cells by shifting cells from G1 phase to S phase and G2/M phases. Ki-67 stain revealed the increase of proliferative capability after PRP releasate treatment. Protein expressions including cyclin A2, cyclin B1, cdk1, cdk2 and PCNA were up-regulated by PRP releasate in a dose-dependent manner. It was concluded that PRP releasate promoted proliferation of skeletal muscle cells in association with the up-regulated protein expressions of PCNA, cyclin A2, cyclin B1, cdk1 and cdk2.

Docosahexaenoic acid-acylated phloridzin, a novel polyphenol fatty acid ester derivative, is cytotoxic to breast cancer cells.

Docosahexaenoic acid-acylated phloridzin (PZ-DHA), a novel polyphenol fatty acid ester derivative, was synthesized through a regioselective acylation reaction with the aim of increasing the bioactivity of phloridzin (PZ) and docosahexaenoic acid (DHA). In this study, PZ-DHA's cytotoxic activity was explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA was selectively cytotoxic for mammary carcinoma (MDA-MB-231, MDA-MB-468, 4T1, MCF-7 and T-47D) cells compared to non-malignant human mammary epithelial cells (HMEC and MCF-10A) and fibroblasts by MTS assay and Annexin-V-FLUOS/propidium iodide staining. Flow cytometric analysis of Oregon Green 488- and Ki-67-stained MDA-MB-231 cells showed antiproliferative activity of PZ-DHA at a subcytotoxic concentration. PZ-DHA also arrested MDA-MB-231 cell division at the G2/M phase and down-regulated expression of cyclin B1 and cyclin-dependent kinase 1 (CDK1). PZ-DHA-induced apoptosis in MDA-MB-231 cells was confirmed by caspase 3/7 activation in a luminescence assay and DNA fragmentation by TUNEL staining. Moreover, MDA-MB-231 xenograft growth in non-obese diabetic severe combined immunodeficient mice was suppressed by intra-tumoral administration of PZ-DHA. This study shows that PZ-DHA is selectively cytotoxic to breast cancer cells in vitro and in vivo, suggesting that further investigations of PZ-DHA are warranted as a potential treatment for breast cancer.

A monomer purified from Paris polyphylla (PP-22) triggers S and G2/M phase arrest and apoptosis in human tongue squamous cell carcinoma SCC-15 by activating the p38/cdc25/cdc2 and caspase 8/caspase 3 pathways.

Recent studies have shown that the aqueous, ethanolic extracts and a monomer compound of Paris polyphylla exhibit anticancer activity toward several types of cancer cell lines, but the anticancer activity of (3ß,17α,25R)-spirost-5-ene-3,17-diol 3-O-α-L-rhamnopyranosyl-(1 â†’ 2)-ß-D-glucopyranoside, a monomer isolated from P. polyphylla (PP), named PP-22, has not been reported previously. In this study, we investigated the effect of PP-22 on human tongue squamous cell carcinoma SCC-15 cells in vitro. MTT assays showed that PP-22 inhibited the growth of SCC-15 cells and had no obvious inhibitory effects on human liver L02 cells. Flow cytometry assays showed that the percentages of apoptotic cells were increased. In addition, cleaved caspase-8, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) could be detected by Western blotting. Flow cytometry also showed that PP-22 triggered S and G2/M phases arrest in SCC-15 cells, and on the other hand, the expression of cyclin A, cyclin E2, cyclin B1, phospho-cell division cycle2 (p-cdc2)(Tyr15), p-Wee1, Myt1, and p53 was upregulated. Moreover, p-p38 levels increased, p-extracellular signal-regulated kinase (ERK) levels decreased, and cdc25B expression was inhibited. Furthermore, the p38/mitogen-activated protein kinase (MAPK) inhibitor SB203580 reversed the increase of the expression level of p38, p-cdc2 (Tyr15), cleaved caspase 3, cleaved PARP, p-p53, and p53 and reversed the decrease in cdc25B expression. In conclusion, these results demonstrated that PP-22 activated p38, inhibited cdc25B, increased p-cdc2 (Tyr15), and triggered S and G2/M phase arrest, as well as activated p53 through the p38-p53 pathway, inhibited the MAPK/ERK pathway, activated the caspase 8/caspase 3 pathway, and triggered the extrinsic apoptotic pathway in SCC-15 cells.

The milk-derived fusion peptide, ACFP, suppresses the growth of primary human ovarian cancer cells by regulating apoptotic gene expression and signaling pathways.

BACKGROUND: ACFP is an anti-cancer fusion peptide derived from bovine milk protein. This study was to investigate the anti-cancer function and underlying mechanisms of ACFP in ovarian cancer. METHODS: Fresh ovarian tumor tissues were collected from 53 patients who underwent initial debulking surgery, and primary cancer cells were cultured. Normal ovarian surface epithelium cells (NOSECs), isolated from 7 patients who underwent surgery for uterine fibromas, were used as normal control tissue. Anti-viabilities of ACFP were assessed by WST-1 (water-soluble tetrazolium 1), and apoptosis was measured using a flow cytometry-based assay. Gene expression profiles of ovarian cancer cells treated with ACFP were generated by cDNA microarray, and the expression of apoptotic-specific genes, such as bcl-xl, bax, akt, caspase-3, CDC25C and cyclinB1, was assessed by real time PCR and western blot analysis. RESULTS: Treatment with ACFP inhibited the viability and promoted apoptosis of primary ovarian cancer cells but exhibited little or no cytotoxicity toward normal primary ovarian cells. Mechanistically, the anti-cancer effects of ACFP in ovarian cells were shown to occur partially via changes in gene expression and related signal pathways. Gene expression profiling highlighted that ACFP treatment in ovarian cancer cells repressed the expression of bcl-xl, akt, CDC25C and cyclinB1 and promoted the expression of bax and caspase-3 in a time- and dose-dependent manner. CONCLUSIONS: Our results suggest that ACFP may represent a potential therapeutic agent for ovarian cancer that functions by altering the expression and signaling of cancer-related pathways in ovarian cancer cells.

Obestatin directly controls chicken ovarian cell functions.

The aim of the present in-vitro study was to examine the role of obestatin in the direct control of basic avian ovarian granulosa cell functions – proliferation, apoptosis and secretory activity. In addition, the effects of obestatin on hormone release by cultured ovarian granulosa cells and follicular fragments (containing both granulosa and theca cells) were examined. We identified the effect of obestatin addition (0.1, 10 or 100 ng/ml medium) on the accumulation of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2) and nuclear (TdT) and cytoplasmic (bax, caspase 3) apoptosis, as well as the release of progesterone (P), testosterone (T) and estradiol (E) by cultured chicken granulosa cells. Furthermore, the action of obestatin addition (0.1, 10 or 100 ng/ml medium) on the release of P, T, E and argininevasotocin (AVT) by cultured fragments of chicken ovarian follicles was examined. The accumulation of proliferation and apoptosis markers was assessed by immunocytochemistry and SDS PAGE-Western immunoblotting. The release of hormones was determined by an EIA. It was observed that obestatin addition could inhibit the accumulation of proliferation markers (PCNA and cyclin B1, but not of MAPK/ERK1,2), promote the expression of nuclear (TdT) and cytoplasmic (bax, caspase 3) apoptosis markers and suppress P, T, and E release by cultured granulosa cells. In cultured ovarian follicular fragments, obestatin promoted P, T, and E, but not AVT, release. These observations represent the first demonstration that (i) obestatin can directly control avian ovarian cell proliferation, apoptosis and hormone release and (ii) the interrelationship between theca and granulosa cells can determine the characteristics of obestatin action on ovarian secretory activity.

Hematopoietic pre-B cell leukemia transcription factor interacting protein is overexpressed in gastric cancer and promotes gastric cancer cell proliferation, migration, and invasion.

Hematopoietic pre-B cell leukemia transcription factor interacting protein (HPIP) has been shown to play an important role in the development and progression of some cancers. However, the role of HPIP in gastric cancer (GC) is unclear. Here, we show that HPIP is upregulated in most GC patients and promotes GC cell proliferation, migration, and invasion. In GC patients, HPIP positively associates with tumor size and nodal metastasis, and negatively associates with tumor differentiation. Hematopoietic pre-B cell leukemia transcription factor interacting protein increases GC cell proliferation through activation of G1 /S and G2 /M cell cycle transitions, accompanied by a marked increase of the positive cell cycle regulators, including cyclin D1, cyclin A, and cyclin B1. Hematopoietic pre-B cell leukemia transcription factor interacting protein enhances GC cell migration and invasion, and modulates epithelial-mesenchymal transition, which plays a key role in cancer cell migration and invasion. These data underscore the critical role of HPIP in GC cell proliferation and progression and suggest that HPIP inhibition may be a useful therapeutic strategy for GC treatment.

LG308, a Novel Synthetic Compound with Antimicrotubule Activity in Prostate Cancer Cells, Exerts Effective Antitumor Activity.

Microtubule plays many different essential roles in the process of tumorigenesis in many eukaryotes, and targeting mitotic progression by disturbing microtubule dynamics is used as a common strategy for cancer treatment. Microtubule-targeted drugs, including paclitaxel and Vinca alkaloids, were previously considered to work primarily by increasing or decreasing the cellular microtubule mass. The tubulin/microtubule system, which is an integral component of the cytoskeleton, is a therapeutic target for prostate cancer. In this study, we found a novel synthetic compound, 8-fluoro-N-phenylacetyl-1, 3, 4, 9-tetrahydro-ß-carboline (LG308), which disrupted the microtubule organization via inhibiting the polymerization of microtubule in PC-3M and LNCaP prostate cancer cell lines. Further study proved that LG308 induced mitotic phase arrest and inhibited G2/M progression significantly in LNCaP and PC-3M cell lines in a dose-dependent manner, and these were associated with the upregulation of cyclin B1 and mitotic marker MPM-2 and the dephosphorylation of cdc2. Besides, the cell proliferation and colony formation of PC-3M and LNCaP cells were effectively inhibited by LG308. Furthermore, LG308 induced apoptosis and cell death in PC-3M and LNCaP cell lines in vitro. In vivo, LG308 dramatically suppressed the growth and metastasis of prostate cancer in both xenograft and orthotopic models. All these data indicate that LG308 is a promising anticancer candidate with antimitotic activity for the treatment of prostate cancer.

MicroRNA-211, a direct negative regulator of CDC25B expression, inhibits triple-negative breast cancer cells' growth and migration.

The non-coding microRNAs (miRNAs) have tissue- and disease-specific expression patterns. Dysregulation of miRNAs has been associated with initiation and progression of oncogenesis in humans. The abnormal expression of CDC25B phosphatases detected in a number of tumors implies that their dysregulation is involved in malignant transformation. Using miRNA target prediction software, we found that miR-211 could target the 3'UTR sequence of CDC25B. To shed light on their roles of miR-211 in breast cancer, the expression of miR-211 was examined by real-time RT-PCR in breast cancer and normal tissues. MiR-211 is significantly downregulated in breast cancer. MiR-211 re-expression suppressed cell growth, cell cycle, migration, and invasion in triple-negative breast cancer (TNBC) cell line MDA-MB231. Luciferase expression from a reporter vector containing the CDC25B -3'UTR was decreased when this construct was transfected with miR-211. The over-expression of miR-211 suppressed the endogenous CDC25B protein level in TNBC cells. For the first time, we demonstrate that miRNA-211 is a direct negative regulator of CDC25B expression in TNBC cells, alters other related target proteins CCNB1 and FOXM1, and then inhibits breast cancer cells growth, migration, and invasion and lead G2/M arrest. The transcriptional loss of miR-211 and the resultant increase in CDC25B expression facilitate increased genomic instability at an early stage of tumor development.

Lysophosphatidic acid improves porcine oocyte maturation and embryo development in vitro.

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is present in human follicular fluid. The aim of the present study was to compare the developmental competence of porcine embryos created via in vitro fertilization (IVF) and parthenogenetic activation (PA) in culture medium supplemented with LPA, in comparison with a control group. The effects of LPA on porcine oocyte maturation and pre-implantation embryonic development were also examined. Addition of 10 µM LPA to the oocyte maturation medium significantly increased the proportion of oocytes reaching metaphase I (MI) or metaphase II (MII), and enhanced embryonic developmental potential. When present during oocyte maturation, LPA significantly increased the abundance of phosphorylated ERK1/2 in MI and MII oocytes, showing that LPA enhanced nuclear maturation via activation of the mitogen-activated protein kinase (MAPK) pathway. In addition, Cyclin B1 levels were elevated in MI- and MII-stage oocytes, suggesting that LPA plays a role in both nuclear and cytoplasmic maturation of oocytes. After fertilization, the frequency of polyspermy in embryos obtained using LPA-treated oocytes was less than that in the control group. Further, blastocyst formation and blastocyst cell number were enhanced and apoptosis was reduced upon LPA treatment of embryos created either by IVF and PA. LPA treatment of blastocysts derived by IVF or PA resulted in increased expression of the anti-apoptotic BCL2L1 gene while reducing expression of the pro-apoptotic genes BAX and CASP3. Together, our data indicate that LPA supplementation improves porcine oocyte maturation and subsequent in vitro development of pre-implantation embryos.

Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation.

BACKGROUND: Chondrosarcoma is characterized for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and low rates of survival. Research within the field of development and expansion of new treatment options for unresectable or metastatic diseases is of particular priority. Diacerein, a symptomatic slow acting drug in osteoarthritis (SYSADOA), implicates a therapeutic benefit for the treatment of chondrosarcoma by an antitumor activity. METHODS: After treatment with diacerein the growth behaviour of the cells was analyzed with the xCELLigence system and MTS assay. Cell cycle was examined using flow cytometric analysis, RT-PCR, and western blot analysis of specific checkpoint regulators. The status for phosophorylation of mitogen-activated protein kinases (MAPKs) was analyzed with a proteome profiler assay. In addition, the possible impact of diacerein on apoptosis was investigated using cleaved caspase 3 and Annexin V/PI flow cytometric analysis. RESULTS: Diacerein decreased the cell viability and the cell proliferation in two different chondrosarcoma cell lines in a dose dependent manner. Flow cytometric analysis showed a classical G2/M arrest. mRNA and protein analysis revealed that diacerein induced a down-regulation of the cyclin B1-CDK1 complex and a reduction in CDK2 expression. Furthermore, diacerein treatment increased the phosphorylation of p38α and p38ß MAPKs, and Akt1, Akt2, and Akt 3 in SW-1353, whereas in Cal-78 the opposite effect has been demonstrated. These observations accordingly to our cell cycle flow cytometric analysis and protein expression data may explain the G2/M phase arrest. In addition, no apoptotic induction after diacerein treatment, neither in the Cal-78 nor in the SW-1353 cell line was observed. CONCLUSIONS: Our results demonstrate for the first time that the SYSADOA diacerein decreased the viability of human chondrosarcoma cells and induces G2/M cell cycle arrest by CDK1/cyclin B1 down-regulation.

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes.

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3' untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3' UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.

Biological effects of (125)i seeds radiation on A549 lung cancer cells: G2/M arrest and enhanced cell death.

External beam radiation (EBRT) and (125)I seeds continuous low dose rate radiation (CLDR) were used to treat patients with lung cancer. We herein investigated the biological effects of EBRT and CLDR on lung cancer cells. A549 human lung cancer cell line was thus exposed to different doses of EBRT and CLDR. CLDR was more efficient to inhibit cell growth than EBRT. CLDR induced increased DNA damage as evidenced by long-lasting p-H2AX activity. The enhanced inhibitory effects of CLDR on lung cancer cell growth may be, at least in part, due to the increased Bax/Bcl2 ratio and cyclin B1-mediated G2/M arrest.

CDK1 switches mitotic arrest to apoptosis by phosphorylating Bcl-2/Bax family proteins during treatment with microtubule interfering agents.

Microtubule interfering agents (MIAs), that can stabilise or depolymerise microtubules, are an important class of cancer chemotherapeutic drugs. They can lead to mitotic arrest and subsequent apoptosis. We demonstrate that cell cycle-dependent kinase 1 (CDK1) is important in switching cells from mitotic arrest to apoptosis during MIAs treatment. Overexpression of non-degradable cyclin B1 sustained CDK1 activation and mitotic arrest, followed by caspase-3 dependent apoptosis. CDK1 is responsible for the phosphorylation of several pro- and anti-apoptotic Bcl-2 family proteins during MIAs treatment. CDK1-mediated Bcl-2 serine 70 phosphorylation enhances its pro-apoptotic function, whereas CDK1-mediated Bad serine 128 phosphorylation promotes apoptosis. Blockage of CDK1 activity with a specific pharmacological inhibitor suppresses Mcl-1 phosphorylation, degradation and its anti-apoptotic function. Therefore, the death of cancer cells under MIAs treatment was caused by imbalance between CDK1-induced alterations in the pro-apoptotic and anti-apoptotic functions of phosphorylated Bcl-2 family proteins.

MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells.

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.

Upregulation of Cyclin B1 by miRNA and its implications in cancer.

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3'UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth.

Inhibitory effect of oleanolic acid on hepatocellular carcinoma via ERK-p53-mediated cell cycle arrest and mitochondrial-dependent apoptosis.

Incidence of hepatocellular carcinoma (HCC) is dramatically increasing and is the third cause of cancer death worldwide. One key approach to control HCC is chemoprevention by naturally occurring agents. This study aims at investigating the antitumor effect of oleanolic acid (OA) and the molecular mechanisms. BALB/c mice were injected subcutaneously with HepG2 cells to establish transplanted tumors. Apoptosis and cell cycle arrest-related markers and signaling cascades were determined by western blot, immunofluorescence, reverse transcriptase-polymerase chain reaction and flow cytometric analysis. OA exhibited inhibitory effect on HCC through induction of apoptosis and cell cycle arrest both in transplanted tumors and in HepG2 cells. OA induced apoptosis through mitochondrial pathway, evidenced by inhibition of Akt/mammalian target of rapamycin pathway, mitochondrial dysfunction, transient increase of adenosine triphosphate, increase of Bax/Bcl-2 ratio, increased release of cytochrome c and activation of caspase/poly (ADP-ribose) polymerase. Activation of mitochondrial apoptotic pathway may be due to reactive oxygen species generated by mitochondrial fatty acid oxidation, resulted from enhancement of lipolysis regulated by cyclic adenosine 3',5'-monophosphate response element-binding protein-hormone-sensitive lipase/peroxisome proliferator-activated receptor γ signaling. OA induced G2/M cell cycle arrest through p21-mediated downregulation of cyclin B1/cdc2. Cyclooxygenase-2 (COX-2) and p53 were involved in OA-exerted effect, and extracellular signal-regulated kinase-p53 signaling played a central role in OA-activated cascades responsible for apoptosis and cell cycle arrest. OA demonstrated significant antitumor activities in HCC in vivo and in vitro models. These data provide new insights into the mechanisms underlying the antitumor effect of OA.