Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.     

Synthesis and structure-activity relationships of new antiproliferative and proapoptotic retinoid-related biphenyl-4-yl-acrylic acids

Atypical retinoids, or retinoid-related molecules (RRMs), represent a class of proapoptotic agents with a promising potential in the treatment of neoplastic diseases. In the present work, the synthesis and structure-activity relationship of a series of 3'-adamantan-1-yl-biphenyl-4-yl-acrylic acids substituted in ring A were studied. The synthesized compounds were evaluated for their antiproliferative activity in a human promyelocitic leukemia cell line (NB4), and in an ovarian carcinoma cell system including IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of at least one oxygenated substituent in positions 4' or 5' appears determinant for the antiproliferative activity. With two substituents of this kind the activity increases, particularly in the case of alkylenedioxy compounds. The activation of DNA damage response as indicated by phosphorylation of H2AX histone, RPA-2 protein, and p53 at serine 15 by the most apoptotic compounds provides additional support to the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this group.     

Defining targets of modulation of human tumor cell response to cisplatin

We previously observed that in yeast cisplatin activates different pathways accounting for stress response. Here, we investigated whether genes involved in yeast drug response were modulated by cisplatin in human tumor cell lines (A2780, IGROV-1, A431, U2-OS) including cisplatin-resistant sublines (A2780/BBR, IGROV-1/Pt1, A431/Pt and U2-OS/Pt). Factors and pathways involved in stress response (glutathione-S-transferase, proteasome, checkpoint control and recombinational repair) were increased by cisplatin in human tumor sensitive and resistant cells. Moreover, sensitization to cisplatin by pharmacologically targeting glutathione or proteasome was observed in sensitive and resistant cells. Interestingly, only in IGROV-1/Pt1 cells, in which cisplatin up-regulated HSP70 and HSP90, targeting of HSP90 resulted in sensitization of resistant cells, suggesting a protective role of stress response. In conclusion, the present findings support the potential relevance of interfering with heat shock protein response to increase cisplatin cytotoxicity in resistant cells. Overall, pathways activated by cisplatin in human tumor cells appear cell-type specific, at least in part reflecting the stress response observed in yeast.     

Synergistic antitumor effects of novel HDAC inhibitors and paclitaxel in vitro and in vivo

Preclinical studies support the therapeutic potential of histone deacetylases inhibitors (HDACi) in combination with taxanes. The efficacy of combination has been mainly ascribed to a cooperative effect on microtubule stabilization following tubulin acetylation. In the present study we investigated the effect of paclitaxel in combination with two novel HDACi, ST2782 or ST3595, able to induce p53 and tubulin hyperacetylation. A synergistic effect of the paclitaxel/ST2782 (or ST3595) combination was found in wild-type p53 ovarian carcinoma cells, but not in a p53 mutant subline, in spite of a marked tubulin acetylation. Such a synergistic interaction was confirmed in additional human solid tumor cell lines harboring wild-type p53 but not in those expressing mutant or null p53. In addition, a synergistic cytotoxic effect was found when ST2782 was combined with the depolymerising agent vinorelbine. In contrast to SAHA, which was substantially less effective in sensitizing cells to paclitaxel-induced apoptosis, ST2782 prevented up-regulation of p21(WAF1/Cip1) by paclitaxel, which has a protective role in response to taxanes, and caused p53 down-regulation, acetylation and mitochondrial localization of acetylated p53. The synergistic antitumor effects of the paclitaxel/ST3595 combination were confirmed in two tumor xenograft models. Our results support the relevance of p53 modulation as a major determinant of the synergistic interaction observed between paclitaxel and novel HDACi and emphasize the therapeutic interest of this combination.     

Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection.

We have previously shown that p53 mutations are associated with cisplatin resistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely elucidated; in particular, the biological mechanisms behind the acquired drug-resistant p53-mutant phenotype were not clearly explained. Thus, in this study, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (270 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexistent in the cisplatin-sensitive parental cell line (IGROV-1) or were induced by drug (genotoxic) treatment. For this purpose, an allele-specific PCR approach was used. Primers carrying the desired mutations (T-->A codon 270, C-->T codon 282) in the 3' terminus, and the corresponding wild-type primers were used to amplify genomic DNA from the original IGROV-1 cell line used to select the mutant IGROV-1/Pt 1. To increase sensitivity, we hybridized blots of the PCRs with the radiolabeled PCR fragment from IGROV-1/Pt 1. Amplification was obtained for IGROV-1 DNA with the mutated allele-specific primers, indicating the preexistence of a mutated population in the IGROV-1 cell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%. Single-strand conformation polymorphism and allele-specific PCR analysis of the IGROV-1/Pt 0.1 cells, which are less resistant to cisplatin than IGROV-1/Pt 1 cells and which carry both mutant and wild-type p53 alleles with a wild-type predominance, suggested a progressive selection of the mutant population by cisplatin treatment. This is the first observation that indicates that a subpopulation of p53 mutant cells can occasionally be selected by cisplatin treatment. Thus, considering the susceptibility to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, the selection process resulting in emergence of p53 mutant tumors is a possible origin of resistance of ovarian carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.     

Increased Sensitivity to Chemotherapy Induced by CpG-ODN Treatment Is Mediated by microRNA Modulation

We recently reported that peritumoral CpG-ODN treatment, activating TLR-9 expressing cells in tumor microenvironment, induces modulation of genes involved in DNA repair and sensitizes cancer cells to DNA-damaging cisplatin treatment. Here, we investigated whether this treatment induces modulation of miRNAs in tumor cells and their relevance to chemotherapy response. Array analysis identified 20 differentially expressed miRNAs in human IGROV-1 ovarian tumor cells from CpG-ODN-treated mice versus controls (16 down- and 4 up-regulated). Evaluation of the role of the 3 most differentially expressed miRNAs on sensitivity to cisplatin of IGROV-1 cells revealed significantly increased cisplatin cytotoxicity upon ectopic expression of hsa-miR-302b (up-modulated in our array), but no increased effect upon reduced expression of hsa-miR-424 or hsa-miR-340 (down-modulated in our array). Accordingly, hsa-miR-302b expression was significantly associated with time to relapse or overall survival in two data sets of platinum-treated ovarian cancer patients. Use of bio-informatics tools identified 19 mRNAs potentially targeted by hsa-miR-302b, including HDAC4 gene, which has been reported to mediate cisplatin sensitivity in ovarian cancer. Both HDAC4 mRNA and protein levels were significantly reduced in IGROV-1 cells overexpressing hsa-miR-302b. Altogether, these findings indicate that hsa-miR-302b acts as a “chemosensitizer” in human ovarian carcinoma cells and may represent a biomarker able to predict response to cisplatin treatment. Moreover, the identification of miRNAs that improve sensitivity to chemotherapy provides the experimental underpinning for their possible future clinical use.     

Comparative analysis of primary structure of the mutant tubulins with resistance to antimicrotubular drugs for prediction of new mutations with analogous properties

All known for today complete amino acid sequences of alpha- and beta-tubulins were aligned and analyzed to reveal the regularity of location of mutations result in the resistance to antimicrotubular compounds and a prediction of positions of new similar mutations. It was shown, that known positions of amino acid changes lead to decrease a affinity to antimicrotubular agents with depolymerizing mechanism of action are consensus and located in proximity to the residues involved in intradimeric/interdimeric interactions and interactions with nucleotides (within of six residues), but never coincide with them. For changes lead to resistance to stabilizing antimicrotubular compounds, similar dependence is not traced. The identified regularity enables to predict the positions of new mutations lead to resistance to agents with depolymerizing mechanism of action.     

Ursodeoxycholic acid is a GPBAR1 agonist and resets liver/intestinal FXR signaling in a model of diet-induced dysbiosis and NASH

Obeticholic acid (OCA) is a farnesoid-X-receptor (FXR) ligand, shown effective in reducing steatosis and fibrosis in NASH patients. However, OCA causes major side effects including pruritus, while increases the risk for liver decompensation in cirrhotic patients. Ursodeoxycholic acid (UDCA), is a safe and unexpensive bile acid used in the treatment of liver disorders whose mechanism of action is poorly defined. Here we have compared the effects of OCA and UDCA in a mouse model of NASH. In mice exposed to a diet rich in fat/cholesterol and fructose (HFD-F), treatment with OCA or UDCA effectively prevented body weight gain, insulin resistance, as demonstrated by OGTT, and AST plasma levels. After 12 weeks HFD-F mice developed liver microvesicular steatosis, inflammation and mild fibrosis, increased expression of inflammatory (TNFα, IL6, F4/80) and fibrosis (αSma, Col1α1, Tgfβ) markers, reduced liver expression of FXR, dysregulated liver FXR signaling and elevated levels of Tauro-α and β-muricholic acid (T-α and βMCA), two FXR antagonists in mice. Both compounds prevented these changes and improved liver histopathology. OCA reduced primary bile acid synthesis worsening the T-CA/T-βMCA ratio. UDCA effectively transactivated GPBAR1 in vitro. By RNAseq analysis we found that among over 2400 genes modulated by the HFD-F, only 32 and 60 genes were modulated by OCA and UDCA, with only 3 genes (Dbp, Adh7, Osgin1) being modulated by both agents. Both agents partially prevented the intestinal dysbiosis.ConclusionsUDCA is a GPBAR1 ligand and exerts beneficial effects in a rodent model of NASH by activating non-overlapping pathway with OCA.     

Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning,expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.     

Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning,expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.     

Smac mimetics induce inflammation and necrotic tumour cell death by modulating macrophage activity

Smac mimetics (SMs) comprise a class of small molecules that target members of the inhibitor of apoptosis family of pro-survival proteins, whose expression in cancer cells hinders the action of conventional chemotherapeutics. Herein, we describe the activity of SM83, a newly synthesised dimeric SM, in two cancer ascites models: athymic nude mice injected intraperitoneally with IGROV-1 human ovarian carcinoma cells and immunocompetent BALB/c mice injected with murine Meth A sarcoma cells. SM83 rapidly killed ascitic IGROV-1 and Meth A cells in vivo (prolonging mouse survival), but was ineffective against the same cells in vitro. IGROV-1 cells in nude mice were killed within the ascites by a non-apoptotic, tumour necrosis factor (TNF)-dependent mechanism. SM83 administration triggered a rapid inflammatory event characterised by host secretion of TNF, interleukin-1β and interferon-γ. This inflammatory response was associated with the reversion of the phenotype of tumour-associated macrophages from a pro-tumoural M2- to a pro-inflammatory M1-like state. SM83 treatment was also associated with a massive recruitment of neutrophils that, however, was not essential for the antitumoural activity of this compound. In BALB/c mice bearing Meth A ascites, SM83 treatment was in some cases curative, and these mice became resistant to a second injection of cancer cells, suggesting that they had developed an adaptive immune response. Altogether, these results indicate that, in vivo, SM83 modulates the immune system within the tumour microenvironment and, through its pro-inflammatory action, leads cancer cells to die by necrosis with the release of high-mobility group box-1. In conclusion, our work provides evidence that SMs could be more therapeutically active than expected by stimulating the immune system.     

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM‐gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de‐repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein‐1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM‐specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.     

Antitumor quinols: role of glutathione in modulating quinol-induced apoptosis and identification of putative cellular protein targets

Novel heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) are promising novel anticancer agents. They exhibit in vitro antiproliferative activity against colon, renal, and breast carcinoma cell lines as well as in vivo antitumor activity in colon, renal, and breast tumor xenografts. Elucidation of the mechanism of antitumor action of these compounds is of great importance. We show in this study that the compounds induced apoptosis as demonstrated by caspase 3 and PARP cleavage at doses causing G(2)/M cell cycle arrest. Glutathione was found to play an important role in modulating quinol-mediated cytotoxicity. In HCT 116 cells, treatment with 1 and 2 caused a 2- to 3-fold increase in the total glutathione content, suggestive of a glutathione-mediated antioxidant response. Indeed, buthionine sulfoximine (BSO)-induced glutathione depleted cells were 6-10 times more sensitive to 1 and 2, while glutathione monoethyl ester supplementation decreased the antitumor potencies by 2-3 times. In further studies we determined other cellular proteins which bind to an immobilized quinol analog, and identified several proteins including beta-tubulin, heat shock protein 60, and peroxiredoxin 1 as potential molecular targets of quinols that may contribute to their proapoptotic and antiproliferative effects.     

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.