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.

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation.

BACKGROUND: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. METHODS: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. RESULTS: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states. CONCLUSIONS: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.

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.

Influence of interleukin 1 beta and tumour necrosis factor alpha on the in vitro growth, maturation and mitochondrial distribution of bovine oocytes from small antral follicles.

SummaryThis study aimed to investigate the effects of IL1ß and TNFα on growth and maturation of oocytes from small follicles (1-3 mm) during in vitro culture. To this end, cumulus-oocyte complexes (COCs) with diameters of ~110 µm were cultured in TCM-199 medium alone or supplemented with IL1ß (10 ng/ml), TNFα (10 ng/ml) or both for 48 h. The oocytes were measured at the beginning and at the end of the culture period. COCs were cultured for 20 h in pre-maturation medium and then half of the COCs of each group was destined for in vitro maturation and the remaining COCs were used to evaluate meiotic progression, mitochondrial distribution and the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. The results showed that COCs cultured with TNFα alone or together with IL1ß had higher diameters than those cultured in control medium alone or supplemented with IL1ß. Control oocytes isolated from large antral follicles (>5 mm) had heterogeneous distribution of mitochondria. Oocytes isolated from small antral follicles, that had been grown in vitro in TCM-199 alone or supplemented with TNFα had similar heterogeneous mitochondrial distribution before in vitro maturation (IVM). After IVM, mitochondria were heterogeneously distribution when cultured in TCM-199. However, when cultured with TNFα and/or IL1ß, mitochondria were homogeneously distributed. Presence of TNFα and/or IL1ß in TCM-199 culture medium did not influence the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. In conclusion, TNFα and a mixture of TNFα and IL1ß both stimulated the growth of bovine oocytes during their in vitro culture, but do not influence gene expression in grown oocytes.

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.

Synthetic resveratrol-curcumin hybrid derivative inhibits mitosis progression in estrogen positive MCF-7 breast cancer cells.

Curcumin (1) and resveratrol (2) are bioactive natural compounds that display wide pharmacological properties, including antitumor activity. However, their clinical application has been limited due to their low solubility and bioavailability. Nevertheless, independent studies have considered these compounds as interesting prototypes for developing new chemical structures useful for anticancer therapy. Here in, we report the synthesis of novel curcumin-like hydrazide analogues (3a and 3b), and a series of curcumin-resveratrol hybrid compounds (4a-f), and the evaluation of their cytotoxic potential on three tumor cell lines MCF-7 (breast), A549 (lung), and HepG2 (liver). Cell viability was significantly reduced in all tested cell lines when compounds 4c-4e were used. The IC50 values for these compounds on MCF-7 cells were lower than those for curcumin, resveratrol, or curcumin combined with resveratrol. We evidenced that 4c promoted a drastic increase of G2/M population. The accumulation of cells in mitosis onset in treated cultures was due to, at least in part, the ability of 4c to modulate nuclear kinase proteins, which orchestrate important events in mitosis progression. We have also observed significant reduction of the relative RNAm abundance of CCNB1, PLK1, AURKA, AURKB in samples treated with 4c, with concomitant increase of CDKN1A (p21). Thus, compound 4c is a promising multi-target antitumor agent that should be considered for further in vivo studies.

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.

Comparison of gene expression patterns across 12 tumor types identifies a cancer supercluster characterized by TP53 mutations and cell cycle defects.

Transcriptional profile-based subtypes of cancer are often viewed as identifying different diseases from the same tissue origin. Understanding the mechanisms driving the subtypes may be key in development of novel therapeutics but is challenged by lineage-specific expression signals. Using a t-test statistics approach, we compared gene expression subtypes across 12 tumor types, which identified eight transcriptional superclusters characterized by commonly activated disease pathways and similarities in gene expression. One of the largest superclusters was determined by the upregulation of a proliferation signature, significant enrichment in TP53 mutations, genomic loss of CDKN2A (p16(ARF)), evidence of increased numbers of DNA double strand breaks and high expression of cyclin B1 protein. These correlations suggested that abrogation of the P53-mediated apoptosis response to DNA damage results in activation of cell cycle pathways and represents a common theme in cancer. A second consistent pattern, observed in 9 of 11 solid tumor types, was a subtype related to an activated tumor-associated stroma. The similarity in transcriptional footprints across cancers suggested that tumor subtypes are commonly unified by a limited number of molecular themes.

PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells.

BACKGROUND: Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol's hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). RESULTS: Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. CONCLUSIONS: This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells.

Evaluation of cell cycle protein expression in gastric cancer: cyclin B1 expression and its prognostic implication.

The cell cycle progression is regulated by interactions of specific cyclin-dependent kinases at the G1-S and G2-M checkpoints. In addition, the cell cycle dysregulation plays a major role in carcinogenesis of human cancers. To investigate the role of cell cycle regulators in the pathogenesis and progression of human gastric cancer, the expression of cyclin D1, A, B1, p16(INK4a), p21(CPI1), p27(KIP1), p53, and pRb was investigated in 482 gastric carcinomas using immunohistochemistry in terms of histologic type, tumor invasion, size, location, and metastatic behavior. The cyclin D1, A, and B1 expression (>10%) was observed in 49%, 69%, and 49% of the cases, respectively. Negative cases for p16(INK4a), p21(CPI1), and p27(KIP1) were detected in 90%, 86%, and 50.5%. There were 30% and 68% of the gastric tumors positive for p53 and pRb, respectively. Diffuse carcinomas frequently were positive for cyclin B1 and pRb, and negative for p21. A relationship between p53 expression and intestinal type carcinomas was found. In addition, the expression of cyclin B1 was associated with regional lymph node metastasis and poor prognosis. No relationship was noticed between any other cell cycle proteins expression and age, sex, tumor size, tumor location, and lymph node involvement. These findings have shown alterations in several cell cycle regulators, and it was suggested that cyclin B1 expression is closely associated with poor behavior in gastric cancer.

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.