Identification of the molecular targets and mechanisms of compound mylabris capsules for hepatocellular carcinoma treatment through network pharmacology and bioinformatics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal formulas have been proven to exert an inhibitory effect on tumor. Compound mylabris capsules (CMC) has been used for treating cancer, especially hepatocellular carcinoma (HCC), for years in China. However, its therapeutic mechanisms on HCC remain unclear. AIM OF THE STUDY: This research aimed to elucidate the molecular targets and mechanisms of CMC for treating HCC. MATERIALS AND METHODS: First, the bioactive ingredients and potential targets of CMC, as well as HCC-related targets were retrieved from publicly available databases. Next, the overlapped genes between potential targets of CMC and HCC-related targets were determined using bioinformatics analysis. Then, networks of ingredient-target and gene-pathway were constructed. Finally, cell experiments were carried out to examine the effects of CMC-medicated serum on HCC and validate the core molecular targets. RESULTS: In total, 151 bioactive ingredients and 255 potential targets of CMC were selected, 982 differentially expressed genes of HCC were identified. Among them, 34 overlapped genes were finally selected. In addition, 20 pathways and 429 GO terms were significantly enriched. Protein-protein interaction and gene-pathway networks indicated that Cyclin B1(CCNB1) and Cyclin Dependent Kinase 1(CDK1) were the core gene targets for the treatment of CMC on HCC. Moreover, in vitro studies showed that CMC-medicated serum significantly inhibited the viability of HepG2 cells. Furthermore, CMC downregulated CCNB1 and CDK1 expressions and induced G2/M phase cell cycle arrest. CONCLUSIONS: CMC plays a therapeutic role in HCC via multi-component, -target and -pathway mechanisms, in which CCNB1 and CDK1 may be the core molecular targets. This study indicates that the integration of network pharmacology and bioinformatics analysis, followed by experimental validation, can serves as an effective tool for studying the therapeutic mechanisms of traditional Chinese medicine.

Integrated Analyses Identify Immune-Related Signature Associated with Qingyihuaji Formula for Treatment of Pancreatic Ductal Adenocarcinoma Using Network Pharmacology and Weighted Gene Co-Expression Network.

The study aimed to clarify the potential immune-related targets and mechanisms of Qingyihuaji Formula (QYHJ) against pancreatic cancer (PC) through network pharmacology and weighted gene co-expression network analysis (WGCNA). Active ingredients of herbs in QYHJ were identified by the TCMSP database. Then, the putative targets of active ingredients were predicted with SwissTargetPrediction and the STITCH databases. The expression profiles of GSE32676 were downloaded from the GEO database. WGCNA was used to identify the co-expression modules. Besides, the putative targets, immune-related targets, and the critical module genes were mapped with the specific disease to select the overlapped genes (OGEs). Functional enrichment analysis of putative targets and OGEs was conducted. The overall survival (OS) analysis of OGEs was investigated using the Kaplan-Meier plotter. The relative expression and methylation levels of OGEs were detected in UALCAN, human protein atlas (HPA), Oncomine, DiseaseMeth version 2.0 and, MEXPRESS database, respectively. Gene set enrichment analysis (GSEA) was conducted to elucidate the key pathways of highly-expressed OGEs further. OS analyses found that 12 up-regulated OGEs, including CDK1, PLD1, MET, F2RL1, XDH, NEK2, TOP2A, NQO1, CCND1, PTK6, CTSE, and ERBB2 that could be utilized as potential diagnostic indicators for PC. Further, methylation analyses suggested that the abnormal up-regulation of these OGEs probably resulted from hypomethylation, and GSEA revealed the genes markedly related to cell cycle and proliferation of PC. This study identified CDK1, PLD1, MET, F2RL1, XDH, NEK2, TOP2A, NQO1, CCND1, PTK6, CTSE, and ERBB2 might be used as reliable immune-related biomarkers for prognosis of PC, which may be essential immunotherapies targets of QYHJ.

Unique microRNA alterations in hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice.

Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3'-untranslated regions (3'-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.

Cytogenotoxic potential of a hazardous material, polystyrene microparticles on Allium cepa L.

Plastic pollution represents a global concern for the biodiversity conservation, ecosystem and public health. The polystyrene is one of the dominant pollutants in both terrestrial and aquatic ecosystem. This work measured the hazardous nature of 100 nm micropolystyrene (MPS) using 25, 50, 100, 200, and 400 mg/L concentrations in terms of oxidative stress, morphotoxicity and cytogenotoxicity in Allium cepa. The results were compared with the positive control (PC) (400 mg/L chlorpyrifos). MPS significantly (p < 0.05) reduced the root length while induced the production of hydroxyl, superoxide radicals with a concomitant increase in DPPH scavenging activity and lipid peroxidation as compared to the negative control. The significant decrease in mitotic index with respect to the negative control (MI: 23.855 ±â€¯5.336 %; lowest MI: 3.88 ±â€¯1.042 %) showed the cytotoxic nature of MPS. Genotoxicity was assessed by various chromosomal and nuclear aberrations. The highest 3.029 ±â€¯0.403 % (PC: 3.09 ±â€¯0.535 %) chromosomal abnormality index and 2.31 ±â€¯0.338 % (PC: 1.178 ±â€¯0.095 %) nuclear abnormality index were observed. MPS down-regulated the expression of plant CDKA encoding gene: cdc2, an important cell cycle regulator. The overall results indicated that MPS could induce cytogenotoxicity through the exacerbation of ROS production and inhibition of cdc2.

Inhibition of Cytomegalovirus Replication with Extended-Half-Life Synthetic Ozonides.

Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.

YLT-11, a novel PLK4 inhibitor, inhibits human breast cancer growth via inducing maladjusted centriole duplication and mitotic defect.

Polo-like kinase 4 (PLK4) is indispensable for precise control of centriole duplication. Abnormal expression of PLK4 has been reported in many human cancers, and inhibition of PLK4 activity results in their mitotic arrest and apoptosis. Therefore, PLK4 may be a valid therapeutic target for antitumor therapy. However, clinically available small-molecule inhibitors targeting PLK4 are deficient and their underlying mechanisms still remain not fully clear. Herein, the effects of YLT-11 on breast cancer cells and the associated mechanism were investigated. In vitro, YLT-11 exhibited significant antiproliferation activities against breast cancer cells. Meanwhile, cells treated with YLT-11 exhibited effects consistent with PLK4 kinase inhibition, including dysregulated centriole duplication and mitotic defects, sequentially making tumor cells more vulnerable to chemotherapy. Furthermore, YLT-11 could strongly regulate downstream factors of PLK4, which was involved in cell cycle regulation, ultimately inducing apoptosis of breast cancer cell. In vivo, oral administration of YLT-11 significantly suppressed the tumor growth in human breast cancer xenograft models at doses that are well tolerated. In summary, the preclinical data show that YLT-11 could be a promising candidate drug for breast tumor therapy.

The ethanol extraction of prepared Psoralea corylifolia induces apoptosis and autophagy and alteres genes expression assayed by cDNA microarray in human prostate cancer PC-3 cells.

Prostate cancer is the most common male reproductive system cancer. The prevalence of prostate cancer in Europe and the United States is higher than that in the Asian region. However, the treatment of prostate cancer remains unsatisfactory. Psoralea corylifolia has been used to cure this disease as Chinese medicine in the Asian region. In this study, we analyzed the components of ethanol extraction of unprepared and prepared P. corylifolia by HPLC. Psoralen and isopsoralen content from the prepared P. corylifolia is twofold higher than that from unprepared, so we use the prepared extraction in this study. However, the effects of the ethanol extraction of P. corylifolia (PCE) on PC-3 human prostate cancer cells remain unclear. PC-3 cells were treated with PCE for different time periods and cells were examined for cell morphological change and total viable cells by using contrast phase microscopy and flow cytometer, respectively. Results indicated that PCE induced cell morphological changes and cytotoxic effect in PC-3 cells in dose-dependent manners. PCE induced chromatin condensation of PC-3 cells dose-dependently. PCE also induced apoptosis and autophagy in PC-3 by western blotting and acridine orange (AO) staining, respectively. Furthermore, a complementary DNA microarray analysis demonstrated that PCE treatment led to 944 genes upregulation and 872 genes downregulation. For example, the DNA damage-associated gene DNA-damage-inducible transcript 3 (DDIT 3) had a 62.1-fold upregulation and CDK1 2.68-fold downregulation. The differential genes were classified according to the Gene Ontology. Furthermore, GeneGo software was used for the key genes involved and their possible interaction pathways. Those genes were affected by P. corylifolia, which provided information for the understanding of the antiprostate cancer mechanism at the genetic level and provide additional targets for the treatments of human prostate cancer.

Cytotoxic and chemosensitization effects of Scutellarin from traditional Chinese herb Scutellaria altissima L. in human prostate cancer cells.

Scutellaria altissima L. is a common traditional Chinese medicine used to treat inflammation in some countries. Scutellarin, an active major flavone glycoside isolated from the traditional Chinese medicine Scutellaria altissima L., has been shown to offer various beneficial biochemical effects on cerebrovascular diseases and inflammation. However, the antiproliferative effects of Scutellarin in prostate cancer and the underlying mechanism are not fully elucidated. In the present study, we aimed to ascertain whether Scutellarin inhibits cancer cell growth and to further explore the molecular mechanism. Scutellarin enhanced the sensitivity of prostate cancer cells to cisplatin. MTT assays revealed that cell viability was significantly decreased in the prostate cancer cells treated with Scutellarin. Flow cytometric analysis indicated that Scutellarin suppressed cell proliferation by promoting G2/M arrest and inducing apoptosis. We employed western blotting to delineate the underlying mechanisms involved in the G2/M arrest and apoptosis. Comet assay and γH2AX immunocytochemistry were used to detect levels of DNA damage in PC3 cells exposed to Scutellarin and/or cisplatin. Our data revealed that Scutellarin significantly induced prostate cancer cell apoptosis by activating the caspase cascade. An increase in the Bax/Bcl-2 ratio, depolarization of mitochondrial membrane potential and cell cycle arrest at G2/M phase were accompanied by the apoptosis induction. Additionally, Scutellarin altered the protein expression of cell cycle and apoptosis regulatory genes by downregulating Cdc2, cyclin B1 and Bcl-2 and upregulating caspase-3, caspase-9 and Bax in prostate cancer cells. Furthermore, Scutellarin sensitized PC3 cells to cisplastin treatment in a dose-dependent manner. Taken together, our data confirmed the cytotoxicity of Scutellarin against prostate cancer PC3 cells and provide new findings in regards to Scutellarin sensitizing prostate cancer cells to chemotherapy. Our findings suggest that Scutellarin has potential to be used as a novel antineoplastic therapeutic candidate for prostate cancer patients.

Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.

The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 µmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 µmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 µmol/L group. Appropriate Se level (2.0 µmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3ß-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3ß-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 µmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the ERK signaling pathway and the expression of its downstream genes (StAR and 3ß-HSD), which could be closely related to the regulating roles of Se in male fertility and spermatogenesis.

Sulforaphane inhibits proliferation and invasive activity of everolimus-resistant kidney cancer cells in vitro.

Although the mechanistic target of rapamycin (mTOR) inhibitor, everolimus, has improved the outcome of patients with renal cell carcinoma (RCC), improvement is temporary due to the development of drug resistance. Since many patients encountering resistance turn to alternative/complementary treatment options, an investigation was initiated to evaluate whether the natural compound, sulforaphane (SFN), influences growth and invasive activity of everolimus-resistant (RCCres) compared to everolimus-sensitive (RCCpar) RCC cell lines in vitro. RCC cells were exposed to different concentrations of SFN and cell growth, cell proliferation, apoptosis, cell cycle, cell cycle regulating proteins, the mTOR-akt signaling axis, adhesion to human vascular endothelium and immobilized collagen, chemotactic activity, and influence on surface integrin receptor expression were investigated. SFN caused a significant reduction in both RCCres and RCCpar cell growth and proliferation, which correlated with an elevation in G2/M- and S-phase cells. SFN induced a marked decrease in the cell cycle activating proteins cdk1 and cyclin B and siRNA knock-down of cdk1 and cyclin B resulted in significantly diminished RCC cell growth. SFN also modulated adhesion and chemotaxis, which was associated with reduced expression of the integrin subtypes α5, α6, and ß4. Distinct differences were seen in RCCres adhesion and chemotaxis (diminished by SFN) and RCCpar adhesion (enhanced by SFN) and chemotaxis (not influenced by SFN). Functional blocking of integrin subtypes demonstrated divergent action on RCC binding and invasion, depending on RCC cell sensitivity to everolimus. Therefore, SFN administration could hold potential for treating RCC patients with established resistance towards everolimus.

Interaction effects between genes involved in the AKT signaling pathway and phytoestrogens in gastric carcinogenesis: a nested case-control study from the Korean Multi-Center Cancer Cohort.

SCOPE: To investigate whether genes involved in AKT/nuclear factor kappa B signaling and/or gene-environment interactions between the genes and phytoestrogens may be susceptible factors for gastric cancer. METHODS AND RESULTS: The representative single nucleotide polymorphisms (SNPs) identified during the primary analysis (screening a total of 622 SNPs within ± 5 kbp of the 51 target gene locations) were further investigated in 317 matched case-control sets. The summary odds ratios (ORs) and 95% confidence intervals (CIs) for gastric cancer were calculated. Interaction effects between the SNPs and phytoestrogen biomarkers (genistein, daidzein, equol, and enterolactone) were computed. CDK1 rs4145643, FAS rs6586161, and FAS rs1468063 in the AKT signaling pathway presented significant genetic effects on gastric cancer (OR = 0.81 (95% CI: 0.66-0.99) for CDK1 rs4145643; OR = 1.27 (95% CI: 1.03-1.58) for FAS rs6586161; OR = 1.29 (95% CI: 1.03-1.56) for FAS rs1468063; Cochran Q statistics > 0.10). Risk alleles of FAS rs6586161, FAS rs1468063, MAP3K1 rs16886448, and MAP3K1 rs252902 showed significant interaction effects with enterolactone (p(interaction) < 0.05). CONCLUSION: CDK1 and FAS genes involved in AKT signaling and influenced by anti-carcinogenic property of phytoestrogens can play a role as susceptible genetic factors in gastric carcinogenesis. FAS and MAP3K1 genes significantly interact with enterolactone, thereby modifying the individual's risk for gastric cancer.

Effects of maternal and dietary selenium (Se-enriched yeast) on the expression of p34(cdc2) and CyclinB1 of germ cells of their offspring in goats.

The aim of the study was to evaluate the effect of selenium on the expression of p34(cdc2) and CyclinB1 (two components of MPF regulating cell cycle) of germ cells of their offspring in goats. A herd of 119 Taihang Black Goats, which was randomly divided into 4 treatments, received experimental diet with different Se levels (from Se-enriched yeast) for 174d. The four treatments, fed with a basal diet, were supplemented with 0 (control), 0.5, 2 and 4 mgkg⁻¹ DM Se. Testis samples were collected from the young male goats of each treatment group at the end of the study (30d after weaning) for mRNA expression using real-time PCR and for protein expression by immunohistochemistry assay. Results show that a significant decrease was observed in mRNA expression of p34(cdc2) and CyclinB1 in the testis of Se-deficient (Group 1) and Se-excess (Group 4) animals compared with that in Groups 2 and 3. However, no significant changes were found in mRNA expression of p34(cdc2) between Se-deficient (Group 1) and Se-excess (Group 4). Also the immunohistochemistry assay detected similar results of protein expression of these two genes. These results suggest, that maternal and dietary Se-induced oxidative stress can modulate the mRNA and protein expression of the cell cycle related genes (p34(cdc2) and CyclinB1) in the testis of their offspring. In addition, Se deficiency and Se excess could prevent the completion of the cell cycle.

Aqueous extract of Curcuma aromatica induces apoptosis and G2/M arrest in human colon carcinoma LS-174-T cells independent of p53.

Curcuma aromatica is a common Chinese herb for treating diseases with blood stasis and has been regarded as an anticancer herb in modern clinical practice. However, the anticancer effects and related molecular mechanisms of Curcuma aromatica remain unclear. In the present study, human colon carcinoma LS-174-T cell line with wild-type p53 was used as a model cell to evaluate the anticancer effects of aqueous extract of Curcuma aromatica (AECA). AECA inhibits LS-174-T cell proliferation in a dose- and time-dependent manner and colony formation in a dose-dependent manner. AECA treatment induces apoptosis accompanied by caspase-8, -9, and -3 activation in LS-174-T cells. Moreover, blocking the activities of these caspases with a specific inhibitor significantly protected LS-174-T cells from AECA-induced apoptosis. AECA treatment also induces G2/M phase arrest in LS-174-T cells. Expression of p53 was unchanged after AECA treatment; specific silence of p53 did not influence AECA-induced apoptosis and G2/M phase arrest. Further, the expression of cyclin B1 and CDK1 was reduced by AECA. This study suggests that AECA might be effective as an antiproliferative herb for colon carcinoma, the antitumor activity of AECA may involve both extrinsic and intrinsic apoptosis, and AECA induces G2/M phase arrest via downregulation of cyclin B1 and CDK1 and without the participation of p53.

Regulation of p21, MMP-1, and MDR-1 expression in human colon carcinoma HT29 cells by Tian Xian liquid, a chinese medicinal formula, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tian-Xian liquid (TXL), a commercially available Chinese medicine decoction, has been used as an anticancer dietary agent for more than 10 years without reported side effects. AIM OF THE STUDY: The safety and quality consistency of TXL and its mechanisms of action on antiproliferation, antimetastasis, and reversion of multidrug resistance (MDR) regimens were explored. MATERIALS AND METHODS: In this study, an atomic absorption spectrophotometer and reversed phase high performance liquid chromatography with photodiode array detection (HPLC-DAD) were used to evaluate the main toxic elements and the quality consistency among different batches of TXL extracts, respectively. HT29 human colon cancer cell line and tumor-bearing nude mice were used. TXL was provided by China-Japan Feida Union Company Limited. The effect of TXL on in vitro proliferation of HT29 human colon cancer cell line was examined. The percentages of treated cells distributed in different phases of the cell cycles were analyzed by flow cytometry. Antiproliferative effect after treatment with TXL was assessed by determination of the protein levels of p21, cyclinD1, PCNA, and cdk-2, which are the key regulators for cell cycle progression. Meanwhile, the protein levels of MMP-1 and MDR-1 (multidrug resistance protein-1) were also determined to assess the effect of TXL on antimetastasis and reversion of MDR regimen, respectively. RESULTS: The contents of main toxic elements were lower in TXL extract compared with the standard set by the Department of Health of the Government of Hong Kong Special Administrative Region (SAR). Our HPLC results showed that the relative standard deviations of the amount of the 5 standards were less than 5% in different batches of TXL. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of cyclinD1, PCNA, and cdk-2 in the TXL-treated in vitro models, thereby, impeding cell progression from G1/S phase. Results obtained from the in vivo study also demonstrated that TXL upregulated the protein level of p21 and downregulated the protein levels of MMP-1 and MDR-1. CONCLUSIONS: Results obtained from the present investigation not only demonstrate the safety and quality of TXL extract but also demonstrate that TXL possesses antiproliferative and antimetastatic activities and brings about reversion of MDR on HT29 cell and on xenografted tissue in tumor-implanted nude mice.

Effects of phenylethyl isothiocyanate and its metabolite on cell-cycle arrest and apoptosis in LNCaP human prostate cancer cells.

Cruciferous vegetable consumption is associated with decreased risk of several cancers, including prostate cancer. Gluconasturtiin, one of the predominant glucosinolates in cruciferous vegetables, is hydrolyzed to yield phenylethyl isothiocyanate (PEITC). PEITC absorption and metabolism in humans involves glutathione conjugation followed by conversion via the mercapturic acid pathway to an N-acetylcysteine (NAC) conjugate that is excreted in the urine. We observed an inhibitory effect of PEITC and its metabolite, NAC-PEITC, on cancer cell proliferation, cell-cycle progression, and apoptosis in LNCaP human prostate cancer cells. PEITC and NAC-PEITC suppressed LNCaP cell proliferation in a dose-dependent manner, and exposure to 5 microM PEITC or NAC-PEITC reduced cell proliferation by 25% and 30%, respectively. Cell-cycle analysis revealed that cells treated with 5 microM PEITC or NAC-PEITC arrested at the G(2)/M phase. In addition, the percentage of cells in the S phase decreased from 46% to 25% following 48 h of incubation with PEITC or NAC-PEITC. The G(2)/M-phase cell-cycle arrest of LNCaP cells grown in the presence of PEITC or NAC-PEITC is correlated with the downregulation of Cdk1 and cyclin B(1) protein expression. Apoptosis was observed at the later stages of 24-h and 48-h treatments with 5 microM PEITC and NAC-PEITC. In conclusion, PEITC and NAC-PEITC are potential chemopreventive/chemotherapeutic agents against LNCaP human prostate cancer cells.

Dietary selenium variation-induced oxidative stress modulates CDC2/cyclin B1 expression and apoptosis of germ cells in mice testis.

Oxidative stress has been linked with apoptosis in germ cells and with male infertility. However, the molecular mechanism of oxidative-stress-mediated apoptosis in germ cells has not been clearly defined so far. Because of the involvement of CDC2 and cyclin B1 in cell cycle regulation and their plausible role in apoptosis, the present study aimed to investigate the possibility that selenium (Se)-induced oxidative-stress-mediated modulations of these cell cycle regulators cause DNA damage and apoptosis in germ cells. To create different Se status (deficient, adequate and excess), male Balb/c mice were fed yeast-based Se-deficient diet (Group I) and a deficient diet supplemented with Se as sodium selenite (0.2 and 1 ppm Se in Groups II and III, respectively) for a period of 8 weeks. After the completion of the diet feeding schedule, a significant decrease in Se levels and glutathione peroxidase activity was observed in the Se-deficient group (Group I), whereas the Se-excess group (Group III) demonstrated an increase in Se levels. Increased levels of lipid peroxidation were seen in both Groups I and III when compared to Group II, indicating oxidative stress. The mRNA and protein expressions of both CDC2 and cyclin B1 were found to be significantly decreased in Groups I and III. A decrease in the immunohistochemical localization of these proteins was also observed in spermatogenic cells. The mRNA expressions of apoptotic factors such as Bcl-2, Bax, caspase-3 and caspase-9 were found to be increased in Groups I and III. A decrease in CDC2 kinase activity was also seen in these groups. Increased apoptosis was observed in Group I and Group III animals by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay indicating oxidative-stress-mediated DNA damage. These findings suggest the effect of Se-induced oxidative stress on the cell cycle regulators and apoptotic activity of germ cells, thus providing new dimensions to molecular mechanisms underlying male infertility.

Inhibition of CDC2/Cyclin B1 in response to selenium-induced oxidative stress during spermatogenesis: potential role of Cdc25c and p21.

Various cell cycle regulators control and coordinate the process of cell cycle. Because of the crucial involvement of CDC2, Cyclin B1, Cdc25c, and p21 in cell cycle regulation, the present study was aimed to investigate the possibility that selenium (Se)-induced oxidative stress mediated alterations in Cdc25c and p21 may cause modulations in the CDC2/Cyclin B1 complex responsible for G2/M phase checkpoint during meiosis I of spermatogenesis. To create different Se status-deficient, adequate and excess Se, male Balb/c mice were fed yeast based Se deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet feeding schedule, a significant decrease in the Se and glutathione peroxidase levels were observed in the Se deficient group (I), whereas Se excess group (III) demonstrated an increase in Se levels. Increased levels of lipid peroxidation (LPO) were seen in both group I and group III when compared to group II, thus indicating oxidative stressed conditions. The mRNA and protein expression of CDC2, Cyclin B1, and Cdc25c were found to be significantly decreased in groups I and III. However, the expression of p21, a kinase inhibitor, was found to be elevated in Se deficient and Se excess fed groups. A statistically significant decrease in the CDC2 kinase activity was also seen in the Se deficient and excess groups. These findings suggest that under the influence of Se-induced oxidative stress, the down regulation of CDC2/Cyclin B1 complex is mediated through changes in Cdc25c and p21 leading to the cell cycle arrest and thus providing new dimensions to the molecular mechanisms underlying male infertility.

Indirubin-3'-monoxime, a CDK inhibitor induces growth inhibition and apoptosis-independent up-regulation of survivin in transitional cell cancer.

In traditional Chinese Medicine, the preparation Danggui Longhui Wan has been used for years in the treatment of chronic myelocytic leukemia. The compound indirubin has been shown to be the active constituent. A cell permeable derivative, indirubin-3'-monoxime, is a selective and potent inhibitor of cyclin-dependent kinases (cdk). The ability of indirubin-3'-monoxime to induce apoptosis and tumor cell death in transitional cell cancer cell lines was investigated here. The growth-inhibitory properties were evaluated by EZ4U, a cytotoxic assay; apoptosis induction was determined by immunoblotting of cleaved PARP and flow cytometry of Annexin-V/PI staining during treatment. To evaluate further the underlying molecular action of indirubin-3'-monoxime on the cell cycle, the levels of cdk-1 and survivin, a mitotic spindle checkpoint and apoptosis-regulating protein, respectively, were additionally determined by flow cytometry and immunoblotting. The results indicated that indirubin-3'-monoxime induced reversible growth arrest in all four cell lines and an increase of apoptosis in two of them. The treatment with indirubin-3'-monoxime increased the expression of survivin almost four times in the RT4 cells and more than doubled it in the RT112 and T24 cells. In the SUP cells, the expression of survivin increased more than seven-fold after 72-h incubation. No clear correlation between the low apoptosis induction rate and extent of survivin expression was found. Cdk expression was not significantly altered by indirubin-3'-monoxime. In summary, indirubin-3'-monoxime might be a promising candidate for targeted cancer therapy, however, its molecular action remains to be further evaluated.

CDK1-cyclin B1 mediates the inhibition of proliferation induced by omega-3 fatty acids in MDA-MB-231 breast cancer cells.

Long-chain omega-3 polyunsaturated fatty acids are thought to inhibit the development of breast cancer. We investigated the effects of docosahexaenoic and eicosapentaenoic acids on the proliferation of MDA-MB-231 human mammary epithelial cells. Both docosahexaenoic and eicosapentaenoic acids decreased cell growth with a higher efficiency for docosahexaenoic acid (87% at 100 microM versus 74% for eicosapentaenoic acid). The effect on specific cell cycle phases was studied. G2/M duration was markedly increased by docosahexaenoic and by eicosapentaenoic acids (respectively by more than seven- and six-fold at 50 microM) when cells were synchronized at the G1/S boundary and released in the cell cycle. In contrast, there was no alteration of G1 or S phases. The expression of cyclin A, cyclin B1 and cyclin-dependent kinase 1, the regulators required for the progression from G2 to mitosis, were all decreased by these fatty acids (western blot). Since omega-3 fatty acids had no effect on the S phase, thus ruling out an involvement of cyclin A in their anti-proliferative effect, we examined whether the regulation of the cyclin-dependent kinase 1-cyclin B1 complex was altered. Upon omega-3 fatty acids treatment, cyclin B1 phosphorylation was inhibited and the expression of the cell division cycle 25C phosphatase, which dephosphorylates cyclin-dependent kinase 1, was decreased. We conclude that the anti-proliferative effect of omega-3 fatty acids occurs via the regulation of the cyclin-dependent kinase 1-cyclin B1 complex.

A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis.

Double fertilization of the egg cell and the central cell by one sperm cell each produces the diploid embryo and the typically triploid endosperm and is one of the defining characteristics of flowering plants (angiosperms). Endosperm and embryo develop in parallel to form the mature seed, but little is known about the coordination between these two organisms. We characterized a mutation of the Arabidopsis thaliana Cdc2 homolog CDC2A (also called CDKA;1), which has a paternal effect. In cdc2a mutant pollen, only one sperm cell, instead of two, is produced. Mutant pollen is viable but can fertilize only one cell in the embryo sac, allowing for a genetic dissection of the double fertilization process. We observed exclusive fertilization of the egg cell by cdc2a sperm cells. Moreover, we found that unfertilized endosperm developed, suggesting that a previously unrecognized positive signal from the fertilization of the egg cell initiates proliferation of the central cell.