Selective inhibition of HDAC6 by N-acylhydrazone derivative reduces the proliferation and induces senescence in carcinoma hepatocellular cells.

Hepatocellular carcinoma (HCC) is a significant contributor to cancer-related deaths globally. Systemic therapy is the only treatment option for HCC at an advanced stage, with limited therapeutic response. In this study, we evaluated the antitumor potential of four N-acylhydrazone (NAH) derivatives, namely LASSBio-1909, 1911, 1935, and 1936, on HCC cell lines. We have previously demonstrated that the aforementioned NAH derivatives selectively inhibit histone deacetylase 6 (HDAC6) in lung cancer cells, but their effects on HCC cells have not been explored. Thus, the present study aimed to evaluate the effects of NAH derivatives on the proliferative behavior of HCC cells. LASSBio-1911 was the most cytotoxic compound against HCC cells, however its effects were minimal on normal cells. Our results showed that LASSBio-1911 inhibited HDAC6 in HCC cells leading to cell cycle arrest and decreased cell proliferation. There was also an increase in the frequency of cells in mitosis onset, which was associated with disturbing mitotic spindle formation. These events were accompanied by elevated levels of CDKN1A mRNA, accumulation of CCNB1 protein, and sustained ERK1 phosphorylation. Furthermore, LASSBio-1911 induced DNA damage, resulting in senescence and/or apoptosis. Our findings indicate that selective inhibition of HDAC6 may provide an effective therapeutic strategy for the treatment of advanced HCC, including tumor subtypes with integrated viral genome. Further, in vivo studies are required to validate the antitumor effect of LASSBio-1911 on liver cancer.

Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and the expression of maturation-related transcripts during prematuration of oocytes from small and medium-sized bovine antral follicles.

This study evaluated the effects of epidermal growth factor (EGF) and progesterone (P4) on growth, the resumption of meiosis and expression of eukaryotic translation initiation factor 4E(eIF4E), poly(A)-specific ribonuclease (PARN), oocyte-specific histone H1 (H1FOO), oocyte maturation factor Mos (cMOS), growth differentiation factor-9 (GDF9) and cyclin B1 (CCNB1) mRNA in oocytes from small and medium-sized antral follicles after prematuration and maturation invitro. Oocytes from small (<2.0mm) and medium (3.0-6.0mm) antral follicles were cultured in medium containing EGF (10ng mL-1), P4 (100 µM) or both. After culture, growth rate, resumption of meiosis and eIF4E, PARN, H1FOO, cMOS, GDF9 and CCNB1 mRNA levels were evaluated. P4 increased cMOS, H1FOO and CCNB1 mRNA levels after the culture of oocytes from small antral follicles, and EGF increased CCNB1 mRNA levels in these oocytes. In the medium-sized antral follicles, P4 alone or in combination with EGF increased oocyte diameter after prematuration invitro. In these oocytes, the presence of either EGF or P4 in the culture medium increased cMOS mRNA levels. In conclusion, P4 increases cMOS, H1FOO and CCNB1 mRNA levels after the culture of oocytes from small antral follicles. P4 and the combination of EGF and P4 promote the growth of oocytes from medium-sized antral follicles, and both EGF and P4 increase cMOS mRNA levels.

Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4).

Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

Mitochondrial ncRNA targeting induces cell cycle arrest and tumor growth inhibition of MDA-MB-231 breast cancer cells through reduction of key cell cycle progression factors.

The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.

Effect of EGF on expression and localization of maturation-promoting factor, mitogen-activated protein kinase, p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes.

This study aimed to investigate the localization of MPF, MAPK, p34cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus-oocyte complexes. Cumulus-oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.

Toxico-pharmacological evaluations of the small-molecule LQFM166: Inducer of apoptosis and MDM2 antagonist.

Inhibition of p53-MDM2 complex has been emerging as a strategy for antitumoral drug development considering the pro-apoptotic role of functional p53 in tumor cells. In our study, the prototype LQFM166 (2), designed through molecular simplification strategy inspired in the Nutlins compounds, was synthetized, characterized and the mechanisms of cell death were investigated. In addition, we estimated the starting doses for acute oral systemic toxicity tests according to the OECD Guidance Document No.129 - 3T3 NRU. The cytotoxic profile of LQFM166 (2) was determined in K-562 cells, a p53-null cell line, since previous studies also showed activity of LQFM166 (2) on this cells. After 24, 48 or 72 h of compound treatment, using MTT reduction assay, the IC50 values found were 100.1 µM, 56.76 µM and 45.11 µM, respectively. LQFM166 (2) was cytotoxic for leukemia cells in a concentration-time-dependent manner. Cell death mechanisms studies of LQFM166 on K-562 cells, revealed that the compound induced cell cycle arrest, increased the expression of caspase 3/7, 8 and 9, cytochrome c, Bax, p21 and p27. Additionally, a decrease in the expression of the Bcl-2 and cyclin-B1 was observed. The apoptotic inducer profile of the compound was confirmed by phosphatidylserine externalization. Investigation of complexation of p53/MDM2 was carried out by ELISA assay using 3T3 cell, showing a decrease in the p53-MDM2 complex induced by the compound. Furthermore, the cytotoxicity in basal fibroblasts 3T3 was determined to estimate LD50. LQFM166 (2) reduced 3T3 cells viability with the IC50 of 185.3 µM and estimated LD50 of 706.7 mg/kg (category 4 of GHS). The rationally designed of the prototype LQFM166 (2) induced cell death by apoptotic mechanisms in leukemic cells and showed MDM2 complexation antagonism in 3T3 cells.

Effects of tumour necrosis factor-alpha and interleukin-1 beta on in vitro development of bovine secondary follicles.

The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.

Cytotoxic effects of natural and semisynthetic cucurbitacins on lung cancer cell line A549.

Cucurbitacins and their derivatives are triterpenoids that are found in various plant families, and are known for their pharmacological and biological activities, including anti-cancer effects. Lung cancer represents a major public health problem, with non-small-cell lung cancer (NSCLC) being the most frequent and aggressive type of lung cancer. The objective of this work was to evaluate four cucurbitacins (CUCs) for their cytotoxic activity, effects on apoptosis induction, cell cycle progression, anti-migratory, and anti-invasive effects on the human NSCLC cell line (A549 cells). Our findings showed that these CUCs could suppress human NSCLC cell growth in vitro through their effects on the PI3Kinase and MAPK pathways, which lead to programmed cell death induction, as well as inhibition of cell migration and cell invasion. Additionally, these effects culminate in apoptosis induction and G2/M cell cycle arrest by modulating cyclin B1 expression, and in the mitigation of strategic steps of lung cancer metastasis, including migration and invasion of A549 cells. These results suggest that two natural (DDCB and CB) and two novel semisynthetic derivatives of cucurbitacin B (ACB and DBCB) could be considered as promising compounds with antitumor potential.

Dehydroleucodine inhibits tumor growth in a preclinical melanoma model by inducing cell cycle arrest, senescence and apoptosis.

Malignant melanoma represents the fastest growing public health risk of all cancer types worldwide. Several strategies and anti-cancer drugs have been used in an effort to improve treatments, but the development of resistance to anti-neoplastic drugs remains the major cause of chemotherapy failure in melanomas. Previously, we showed that the sesquiterpene lactone, dehydroleucodine (DhL), promotes the accumulation of DNA damage markers, such as H2AX and 53BP1, in human tumor cells. Also DhL was shown to trigger either cell senescence or apoptosis in a concentration-dependent manner in HeLa and MCF7 cells. Here, we evaluated the effects of DhL on B16F0 mouse melanoma cells in vitro and in a pre-clinical melanoma model. DhL inhibited the proliferation of B16F0 cells by inducing senescence or apoptosis in a concentration-dependent manner. Also, DhL reduced the expression of the cell cycle proteins cyclin D1 and B1 and the inhibitor of apoptosis protein, survivin. In melanomas generated by subcutaneous injection of B16F0 cells into C57/BL6 mice, the treatment with 20 mg DhL /Kg/day in preventive, simultaneous and therapeutic protocols reduced tumor volumes by 70%, 60% and 50%, respectively. DhL treatments reduced the number of proliferating, while increasing the number of senescent and apoptotic tumor cells. To estimate the long-term effects of DhL, a mathematical model was applied to fit experimental data. Extrapolation beyond experimental time points revealed that DhL administration following preventive and therapeutic protocols is predicted to be more effective than simultaneous treatments with DhL in restricting tumor growth.

Copper Uptake in Mammary Epithelial Cells Activates Cyclins and Triggers Antioxidant Response.

The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion.

G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound.

Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4'-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies.

The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.

Inhibition of PI3K/Akt pathway impairs G2/M transition of cell cycle in late developing progenitors of the avian embryo retina.

PI3K/Akt is an important pathway implicated in the proliferation and survival of cells in the CNS. Here we investigated the participation of the PI3K/Akt signal pathway in cell cycle of developing retinal progenitors. Immunofluorescence assays performed in cultures of chick embryo retinal cells and intact tissues revealed the presence of phosphorylated Akt and 4E-BP1 in cells with typical mitotic profiles. Blockade of PI3K activity with the chemical inhibitor LY 294002 (LY) in retinal explants blocked the progression of proliferating cells through G2/M transition, indicated by an expressive increase in the number of cells labeled for phosphorylated histone H3 in the ventricular margin of the retina. No significant level of cell death could be detected at this region. Retinal explants treated with LY for 24 h also showed a significant decrease in the expression of phospho-Akt, phospho-GSK-3 and the hyperphosphorylated form of 4E-BP1. Although no change in the expression of cyclin B1 was detected, a significant decrease in CDK1 expression was noticed after 24 h of LY treatment both in retinal explants and monolayer cultures. Our results suggest that PI3K/Akt is an active pathway during proliferation of retinal progenitors and its activity appears to be required for proper CDK1 expression levels and mitosis progression of these cells.

Oral squamous cell carcinoma versus oral verrucous carcinoma: an approach to cellular proliferation and negative relation to human papillomavirus (HPV).

Human papillomavirus (HPV) has been cited as a possible initiating agent in the pathogenesis of oral cancer. However, the literature tends to be both controversial and inconclusive about the prevalence of HPV and its potential for proliferation in oral squamous cell carcinoma (SCC). The aim of this study was to investigate the cellular proliferation and the presence of HPV in SCC and verrucous carcinoma (VC). Forty-seven samples of SCC were selected and divided into three groups: 39 SCC, 8 VC, and 9 of normal mucosa (control-CT). Quantitative analyses of all groups showed a greater expression of PCNA, followed by Ki-67 and cyclin B1. A significant difference was observed in cyclin B1 expression in the SCC group compared with VC. PCNA, Ki-67, and cyclin B1 were statistically significant when comparing the SCC and CT groups. However, when SCC and VC were compared, there was no difference in Ki-67 expression. Our results showed that only cyclin B1 had an association with histological grade, and that poorly differentiated tumors presented a higher expression of cyclin B1. Therefore, considerable differences in the cellular proliferation between SCC and VC were observed, and no correlation with HPV was established, since all samples were negative for HPV.

Effect of cyclin-dependent kinase (CDK) inhibition on expression, localization and activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) in bovine oocytes.

This study aimed to assess the effects of cyclin-dependent kinase (CDK) inhibition on factors involved in the control of meiosis in bovine oocytes: maturation promoting factor (MPF) (p34(cdc2) and cyclin B1) and mitogen activated protein kinase (MAPK). Oocytes were maintained at germinal vesicle (GV) stage in vitro with 10 µM of the CDK inhibitor butyrolactone I (BLI) for 24 h (inhibited). After this period, some of the oocytes were transferred to in vitro maturation (IVM) culture for 24 h (inhibited and matured). Control oocytes were assessed immediately after follicle aspiration (immature) or after in vitro maturation for 24 h (matured). Real-time PCR analyses showed that transcripts for p34(cdc2) and MAPK were detected in immature and inhibited oocytes and decreased after maturation, irrespective of CDK inhibition with BLI. Cyclin B1 was detected at similar levels in all oocyte groups. The p34(cdc2) and MAPK proteins were detected by Western blotting at similar levels in all oocyte groups, and cyclin B1 protein was detected only after maturation. Immunofluorescence detection showed that p34(cdc2) was localized in the cytoplasm and GV of immature oocytes, and then throughout the cytoplasm after maturation. Cyclin B1 and MAPK were detected in the cytoplasm in all oocyte groups. Maturation promoting factor and MAPK activities were similar throughout most of maturation for oocytes treated with or without BLI. In conclusion, CDK inhibition did not affect the expression (mRNA and protein levels) and localization of MPF and MAPK, and had nearly no effect on kinase activities during maturation.