IMP-1 inhibits renal cell carcinoma 786-O cell growth by targeting EphrinB2 signaling pathway

Abstract EphrinB2 plays a critical role in tumor growth. In this study, we studied the antitumor activity of imperatorin derivative IMP-1 in renal cell carcinoma (RCC) by regulating EphrinB2 pathway.. Results showed that IMP-1 inhibited the proliferation of 786-O cells in a dose- and time-dependent manner. More importantly, knockdown and transfection of EphrinB2 altered the inhibitory effect of IMP-1 on the activity of 786-O cells. IMP-1 arrested 786-O cell cycle at G0/G1 phase by decreasing the expression of cyclin D1 and cyclin E. Moreover, IMP-1 regulated Bcl-2 family proteins' expression, thus inducing apoptosis of 786-O cells. IMP-1 down-regulated the expression of EphrinB2, Syntenin1 and PICK1. Then, IMP-1 decreased the phosphorylation of Erk1/2 and AKT. In all, IMP-1 could regulate the EphrinB2 pathway in order to inhibit 786-O cell growth by arresting the cell cycle at G0/G1 phase and inducing cell apoptosis. Thus, IMP-1 may present as a potential strategy for RCC treatment.

Regulation of sphingolipid synthesis by the G1/S transcription factor Swi4.

SBF (Swi4/Swi6 Binding Factor) complex is a crucial regulator of G1/S transition in Saccharomyces cerevisiae. Here, we show that SBF complex is required for myriocin resistance, an inhibitor of sphingolipid synthesis. This phenotype was not shared with MBF complex mutants nor with deletion of the Swi4p downstream targets, CLN1/CLN2. Based on data mining results, we selected putative Swi4p targets related to sphingolipid metabolism and studied their gene transcription as well as metabolite levels during progression of the cell cycle. Genes which encode key enzymes for the synthesis of long chain bases (LCBs) and ceramides were periodically transcribed during the mitotic cell cycle, having a peak at G1/S, and required SWI4 for full transcription at this stage. In addition, HPLC-MS/MS data indicated that swi4Δ cells have decreased levels of sphingolipids during progression of the cell cycle, particularly, dihydrosphingosine (DHS), C24-phytoceramides and C24-inositolphosphoryl ceramide (IPC) while it had increased levels of mannosylinositol phosphorylceramide (MIPC). Furthermore, we demonstrated that both inhibition of de novo sphingolipid synthesis by myriocin or SWI4 deletion caused partial arrest at the G2/M phase. Importantly, our lipidomic data demonstrated that the sphingolipid profile of WT cells treated with myriocin resembled that of swi4Δ cells, with lower levels of DHS, IPC and higher levels of MIPC. Taken together, these results show that SBF complex plays an essential role in the regulation of sphingolipid homeostasis, which reflects in the correct progression through the G2/M phase of the cell cycle.

MicroRNA-23b and microRNA-27b plus flutamide treatment enhances apoptosis rate and decreases CCNG1 expression in a castration-resistant prostate cancer cell line.

The acquisition of a castration-resistant prostate cancer phenotype by prostate cancer cells is the alteration that has the worst prognosis for patients. The aim of this study was to evaluate the role of the microRNAs-23b/-27b as well as the possible CCNG1 target gene in tissue samples from patients with localized prostate cancer that progressed to castration-resistant prostate cancer and in a castration-resistant prostate cancer cell line (PC-3). The microRNAs and target gene expression levels of the surgical specimens were analyzed by quantitative real-time polymerase chain reaction. The prostate cancer cell line, PC-3, was transfected with pre-miR-23b, pre-miR-27b, and their respective controls using Lipofectamine RNAiMAX and exposed or not to flutamide. After transfections, expression levels of both the microRNAs and the gene, CCNG1, were analyzed by quantitative real-time polymerase chain reaction. The apoptosis and cell cycle assays were performed on the mini MUSE cytometer. MicroRNAs-23b/-27b were underexpressed in surgical specimens of prostate cancer; however, their target gene, CCNG1, was overexpressed in 69% of the cases. After transfection with the microRNAs-23b/-27b and flutamide, we observed a reduction in gene expression compared with cells that were treated only with microRNAs or only with flutamide. In the apoptosis assay, we demonstrated cell sensitization following transfection with microRNAs-23b/-27b and potentiation when co-administered with flutamide. The number of cells in apoptosis was almost three times higher with the simultaneous treatments (miR + flutamide) compared with the control (p < 0.05). In the cell cycle assay, only flutamide treatment showed better results; a higher number of cells were found in the G0-G1 phase, and a lower percentage of cells completed the final phase of the cycle (p < 0.05). We conclude that microRNAs-23b/-27b are downexpressed in prostate cancer, and their target gene, CCNG1, is overexpressed. We postulated that microRNAs-23b/-27b sensitize the PC-3 cell line and that after the addition of flutamide in the apoptosis assay, we would observe synergism in the treatments between miR and flutamide. In the cell cycle assay, the use of flutamide was sufficient to decrease the number of cells in mitosis. Therefore, we postulate that microRNAs, along with other drugs, may become very useful therapeutic tools in the treatment of castration-resistant prostate cancer.

Synthetic phosphoethanolamine induces cell cycle arrest and apoptosis in human breast cancer MCF-7 cells through the mitochondrial pathway.

Phosphoethanolamine (Pho-s) is a compound involved in phospholipid turnover, acting as a substrate for many phospholipids of the cell membranes. In a recent study, we showed that Pho-s has antitumor effect in the several tumor cells. In this study we evaluated the antitumor activity of synthetic Pho-s on MCF-7 breast cancer cells. Here we demonstrate that Pho-s is cytotoxic to MCF-7 cells in a dose-dependent manner, while it is cytotoxic to MCF10 only at higher concentrations. In addition, Pho-s induces a disruption in mitochondrial membrane potential (Δψm). Furthermore, Pho-s induces mitochondria aggregates in the cytoplasm and DNA fragmentation of MCF-7 cells visualized by confocal microscopy. In agreement with the reduction on Δψm, we showed that Pho-s induces apoptosis followed by an increase in cytochrome c expression and capase-3-like activity in MCF-7 cells. Our results demonstrate that Pho-s induces a cell cycle arrest in the G1 phase through an inhibition of cyclin D1 and stimulates p53. An additional highlight of this study is the finding that Pho-s inhibits Bcl-2, inducing apoptosis through the mitochondrial pathway. Taken together, these results show that Pho-s is a promising compound in the fight against cancer.

Signaling pathways of cell proliferation are involved in the differential effect of erythropoietin and its carbamylated derivative.

It is now recognized that in addition to its activity upon erythroid progenitor cells, erythropoietin (Epo) is capable of stimulating survival of different non-erythroid cells. Since stimulation of erythropoiesis is unwanted for neuroprotection, Epo-like compounds with a more selective action are under investigation. Although the carbamylated derivative of erythropoietin (cEpo) has demonstrated non-hematopoietic tissue protection without erythropoietic effect, little is known about differential mechanisms between Epo and cEpo. Therefore, we investigated signaling pathways which play a key role in Epo-induced proliferation. Here we show that cEpo blocked FOXO3a phosphorylation, allowing expression of downstream target p27(kip1) in UT-7 and TF-1 cells capable of erythroid differentiation. This is consistent with the involvement of cEpo in slowing down G1-to-S-phase progression compared with the effect of Epo upon cell cycle. In contrast, similar antiapoptotic actions of cEpo and Epo were observed in neuronal SH-SY5Y cells. Inhibition and competition assays suggest that Epo may act through both, the homodimeric (EpoR/EpoR) and the heterodimeric (EpoR/ßcR) receptors in neuronal SH-SY5Y cells and probably in the TF-1 cell type as well. Results also indicate that cEpo needs both the EpoR and ßcR subunits to prevent apoptosis of neuronal cells. Based on evidence suggesting that cell proliferation pathways were involved in the differential effect of Epo and cEpo, we went forward to studying downstream signals. Here we provide the first evidence that unlike Epo, cEpo failed to induce FOXO3a inactivation and subsequent p27(kip1) downregulation, which is clearly shown in the incapacity of cEpo to induce erythroid cell growth.

The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.

DNA-PKcs-dependent NHEJ pathway supports the progression of topoisomerase II poison-induced chromosome aberrant cells.

The role of DNA double strand break (DSB) repair pathways, non-homologous end joining (NHEJ), and homologous recombination (HR) was evaluated to prevent the chromosome instability induced by the topoisomerase II (Top2) poisons, idarubicin, and etoposide in Chinese hamster cell lines. XR-C1 (DNA-PKcs deficient) and V-C8 (BRCA2 deficient) showed higher sensitivity to increased concentrations of Top2 poisons compared with their normal counterparts, CHO9 and V79. Both proficient and deficient cells exhibited a marked DSB induction in all phases of the cell cycle. Additionally, deficient cells showed persistent DNA damage 24 hr post-treatment. Chromosomal aberrations increased in the first mitosis following Top2 poison-treatments in G1 or G2 in proficient and deficient cells. CHO9 and V79 demonstrated chromosome and chromatid exchanges following treatments in G1 and G2 phases, respectively. Deficient cells showed high frequencies of chromatid exchanges following treatments in G1 and G2. Simultaneously, we analyzed the micronuclei (MN) induction in interphase cells after treatments in G1, S, or G2 of the previous cell cycle. Both Top2 poisons induced an important increase in MN in CHO9, V79, and V-C8 cells. XR-C1 exhibited an increased MN frequency when cells were treated in G1 phase but not in S or G2. This MN reduction was due to a cell accumulation at G2/M and death in G2-treated cells. Our data suggest that NHEJ and HR operate differentially throughout the cell cycle to protect from Top2 poison-induced chromosome instability, and that DNA-PKcs-dependent NHEJ pathway allows the survival of chromosome damaged cells during S/G2 to the next interphase.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae.

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.

Butylated hydroxytoluene does not protect Chinese hamster ovary cells from chromosomal damage induced by high-dose rate 192Ir irradiation.

Previous reports showed the protective effect of the synthetic antioxidant butylated hydroxytoluene (BHT) against the chromosomal damage induced by bleomycin (BLM), cadmium chloride and potassium dichromate. To test the hypothesis that this effect was exerted by inhibition and/or scavenging of reactive oxygen species (ROS), the effect of BHT on the chromosomal damage induced by a high dose-rate gamma rays (HDR (192)Ir). Experiments were carried out by irradiating G(1) CHO cells with nominal doses of 1, 2 or 3 Gy. BHT (doses of 1.0, 2.5 or 5.0 microg/ml) was added to the culture immediately before or immediately after irradiation. Cells were then incubated in the presence of BHT for 13 h until harvesting and fixation. Results obtained showed that BHT did not decrease the chromosomal damage induced by radiation in any consistent fashion. On the contrary, in cells post-treated with 5.0 microg/ml of BHT the yield of chromosomal aberrations increased in several experimental points. These results with ionizing radiation suggest that the previous observed protective effects of BHT on the chromosomal damage induced by chemical genotoxicants may not be mediated solely through the scavenging or inactivating reactive oxidative species. The decrease of the yield of chromosomal damage induced by BLM could be due to the union of BHT with a metallic ion, in this case Fe (II), required for the activation of BLM. In the same way, the protective effect of BHT on the chromosomal damage induced by cadmium chloride and potassium dichromate could be due to the decrease of the effective dose of both salts in the cell through the chelation of the cations by BHT.

Evaluation of the mutagenic activity of hydroxyurea on the G1-S-G2 phases of the cell cycle: an in vitro study

Hydroxyurea is considered an antineoplastic drug, which also plays an important role in the treatment of sickle cell anemia patients. We evaluated and compared the clastogenic and cytotoxic effects of hydroxyurea, using chromosomal aberrations and mitotic index, respectively, as endpoints. In vitro short-term cultures of lymphocytes were exposed to several concentrations of this drug, at various cell cycle phases. There was a significant increase in the cytotoxicity of hydroxyurea at G1 and G1/S as well in the G2 phase of the cell cycle. Hydroxyurea did not significantly increase chromosome aberrations. There was an S-dependent cytotoxic effect of hydroxyurea, which is expected based on the known activity of hydroxyurea as an inhibitor of ribonucleotide reductase

Evaluation of the mutagenic activity of hydroxyurea on the G1-S-G2 phases of the cell cycle: an in vitro study.

Hydroxyurea is considered an antineoplastic drug, which also plays an important role in the treatment of sickle cell anemia patients. We evaluated and compared the clastogenic and cytotoxic effects of hydroxyurea, using chromosomal aberrations and mitotic index, respectively, as endpoints. In vitro short-term cultures of lymphocytes were exposed to several concentrations of this drug, at various cell cycle phases. There was a significant increase in the cytotoxicity of hydroxyurea at G1 and G1/S as well in the G2 phase of the cell cycle. Hydroxyurea did not significantly increase chromosome aberrations. There was an S-dependent cytotoxic effect of hydroxyurea, which is expected based on the known activity of hydroxyurea as an inhibitor of ribonucleotide reductase.

The effect of butylated hydroxytoluene on the chromosomal damage induced by bleomycin in Chinese hamster ovary cells.

The effect of butylated hydroxytoluene (BHT) on chromosomal damage induced by bleomycin (BLM) in CHO cells was studied. Treatments were performed in cells at quiescent state (90/95% at G0-G1), 2 h after subculture (G1) or 3 h before fixation (G2). Cells were treated for 20 min with BLM plus BHT and subsequently incubated in the presence of BHT until fixation. Results were compared with those obtained from untreated and DMSO-treated controls, from treatments with BLM or BHT alone, from treatments with BLM followed by treatment with DMSO until fixation, and from treatments with BLM plus BHT for 20 min without post-treatment with BHT. BLM induced chromatid- and chromosome-type aberrations in cells treated at G0/G1 or G1 and chromatid-type aberrations in cells treated at the G2 stage. Post-treatment with BHT strongly decreased the frequency of chromosome- but not of chromatid-type aberrations in G0/G1 and G1 and of chromatid-type aberrations in G2. These results are explained assuming that chromosome-type aberrations induced at G0/G1 and G1, and chromatid-type aberrations induced at G2 are originated by the induction of double-strand breaks by BLM through the formation of free radicals. Thus, the observed effect of BHT post-treatment could be considered as evidence that chromosome aberrations are induced by BLM following a two-step mechanism. On the other hand, it is necessary to differentiate between chromatid-type aberrations induced by BLM at G0/G1 and those produced by G2 treatment on the basis of replication errors for the former and DNA repair errors for the latter. In addition, the induction of chromatid-type aberrations by BHT itself at G0/G1 must be taken into account. As BHT acts as an S-dependent agent, chromatid-type aberrations observed after treatment with BLM and BHT in G0/G1 could arise from single-strand breaks induced by BLM and DNA primary lesions induced by BHT.

Regulation of G0 G1 S transition: a genetic approach

A genetic approach was adopted to analyze the cell cycle G(O)(G (1) (S transition in mouse Balb/ 3T3 fibroblasts (clone A3l). We designed selection procedures to isolate revertant from the EJ-ras transformed Balb/3T3 ribroblasts that had recovered strict -control of the G(O) ( G(1), transition by serum growth factors. The aim was to uncover phenotypic traits associated with malignancy (high growth rate G(1) phase shortening and high tumorigenicity) that segregate independently.

Downregulation of JE and KC genes by glucocorticoids does not prevent the G0----G1 transition in BALB/3T3 cells.

The effects of glucocorticoid hormones on the expression of the growth factor-inducible genes JE, KC, and c-myc were analyzed in parental BALB/3T3 and polyomavirus middle-T antigen-transfected cell lines. Northern (RNA) blot hybridization and run-on transcription analysis showed that (i) glucocorticoid hormones selectively inhibit JE and KC expression at the transcriptional level and (ii) the downregulatory effect of glucocorticoids on JE and KC expression is partial for serum-stimulated and middle T antigen-transformed cells and total for quiescent and exponentially growing cells. Gel mobility assays using AP-1 oligonucleotides showed a positive correlation between glucocorticoid downregulating effect and presence of the AP-1 complex. JE and KC downregulation by means of the AP-1 complex may play a role in the actions of glucocorticoids as anti-inflammatory and antitumor agents. The ability of glucocorticoids to downregulate JE and KC was used to investigate the relevance of these genes to the mitogenic response to serum growth factors. Hydrocortisone did not alter the basal DNA synthesis level displayed by quiescent 3T3 cells, but it potentiated both the mitogenic effect of platelet-derived growth factor and c-myc induction by serum growth factors. Upon serum restimulation, untreated and dexamethasone-treated quiescent 3T3 cultures entered the S phase after an identical time lag (G1). These results suggest that (i) JE and KC are not necessary for the G0----G1----S transition and (ii) c-myc overexpression is likely to be the basis for the potentiating effect of glucocorticoids on serum growth factors.