Pisosterol Induces G2/M Cell Cycle Arrest and Apoptosis via the ATM/ATR Signaling Pathway in Human Glioma Cells.

BACKGROUND: Pisosterol, a triterpene derived from Pisolithus tinctorius, exhibits potential antitumor activity in various malignancies. However, the molecular mechanisms that mediate the pisosterol-specific effects on glioma cells remain unknown. OBJECTIVE: This study aimed to evaluate the antitumoral effects of pisosterol on glioma cell lines. METHODS: The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue exclusion assays were used to evaluate the effect of pisosterol on cell proliferation and viability in glioma cells. The effect of pisosterol on the distribution of the cells in the cell cycle was performed by flow cytometry. The expression and methylation pattern of the promoter region of MYC, ATM, BCL2, BMI1, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, MDM2, p14ARF and TP53 was analyzed by RT-qPCR, western blotting and bisulfite sequencing PCR (BSP-PCR). RESULTS: Here, it has been reported that pisosterol markedly induced G2/M arrest and apoptosis and decreased the cell viability and proliferation potential of glioma cells in a dose-dependent manner by increasing the expression of ATM, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, p14ARF and TP53 and decreasing the expression of MYC, BCL2, BMI1 and MDM2. Pisosterol also triggered both caspase-independent and caspase-dependent apoptotic pathways by regulating the expression of Bcl-2 and activating caspase-3 and p53. CONCLUSION: It has been, for the first time, confirmed that the ATM/ATR signaling pathway is a critical mechanism for G2/M arrest in pisosterol-induced glioma cell cycle arrest and suggests that this compound might be a promising anticancer candidate for further investigation.

Current Perspectives on Circulating Tumor DNA, Precision Medicine, and Personalized Clinical Management of Cancer.

Circulating tumor DNA (ctDNA) has recently emerged as a minimally invasive "liquid biopsy" tool in precision medicine. ctDNA-genomic DNA fragments that are released into the bloodstream after the active secretion of microvesicles or tumor cell lysis reflects tumor evolution and the genomic alterations present in primary and/or metastatic tumors. Notably, ctDNA analysis might allow the stratification of patients, the monitoring of the therapeutic response, and the establishment of an opportunity for early intervention independent of detection by imaging modalities or clinical symptoms. As oncology moves towards precision medicine, the information in ctDNA provides a means for the individual management of the patient based on their tumor's genetic profile. This review presents current evidence on the potential role for ctDNA in helping to guide individualized clinical treatment decisions for patients with melanoma, castration-resistant prostate cancer, breast cancer, metastatic colorectal cancer, and non-small cell lung cancer.

Biflorin inhibits the proliferation of gastric cancer cells by decreasing MYC expression.

Gastric cancer is the third leading cause of cancer-related death worldwide. To evaluate the anticancer potential and molecular mechanism of biflorin, a prenyl-ortho-naphthoquinone obtained from Capraria biflora L. roots, we used ACP02, a gastric cancer cell line established from a primary diffuse gastric adenocarcinoma. In this study, biflorin was shown to be a potent cytotoxic agent against ACP02 by Alamar Blue and Trypan Blue assays. Morphological analysis indicated cell death with features of necrosis. Furthermore, a decrease in colony formation, migration and invasion of ACP02 cells was observed after treatment with biflorin (1.0, 2.5 and 5.0 µM). Regarding the underlying molecular mechanism of biflorin in ACP02 cells, we observed a decrease in MYC expression and telomere length using FISH. Our findings suggest a novel molecular target of biflorin in ACP02 cells, which may be a significant therapeutic approach for gastric cancer management.

New prognostic markers revealed by RNA-Seq transcriptome analysis after MYC silencing in a metastatic gastric cancer cell line.

MYC overexpression is considered a driver event in gastric cancer (GC), and is frequently correlated with poor prognosis and metastasis. In this study, we evaluated the prognostic value of genes upregulated by MYC in patients with GC. Metastatic GC cells (AGP01) characterized by MYC amplification, were transfected with siRNAs targeting MYC. RNA-seq was performed in silenced and non-silenced AGP01 cells. Among the differentially expressed genes, CIAPIN1, MTA2, and UXT were validated using qRT-PCR, western blot, and immunohistochemistry in gastric tissues of 213 patients with GC; and their expressions were correlated with clinicopathological and survival data. High mRNA and protein levels of CIAPIN1, MTA2, and UXT were strongly associated with advanced GC stages (P < 0.0001). However, only CIAPIN1 and UXT gene expressions were able to predict distant metastases in patients with early-stage GC (P < 0.0001), with high sensitivity (> 92%) and specificity (> 90%). Overall survival rate of patients with overexpressed CIAPIN1 or UXT was significantly lower (P < 0.0001). In conclusion, CIAPIN1 and UXT may serve as potential molecular markers for GC prognosis.

Analysis of 8q24.21 miRNA cluster expression and copy number variation in gastric cancer.

Aim: To analyze gene expression and copy number of five miRNAs (miR-1204, miR-1205, miR-1206, miR-1207 and miR-1208) localized in this chromosome region in gastric cancer (GC). Materials & methods: 65 paired neoplastic and non-neoplastic specimens collected from GC patients and 20 non-neoplastic gastric tissues from cancer-free individuals were included in this study. The expression levels of the five miRNAs were accessed by real time qPCR and were correlated. Results: MiR-1207-3p, miR-1205, miR-1207-5p and miR-1208 were upregulated in approximately 50% of GC tumors in relation to those of adjacent non-neoplastic tissues. MiR-1205 expression was associated with gain of gene copies and was upregulated in adjacent non-neoplastic samples relative to external controls. Conclusion: The coexpression of the 8q24 miRNAs indicated the role of miR-1205 in the initiation of gastric cancer development.

Role of histone acetylation in gastric cancer: implications of dietetic compounds and clinical perspectives.

Histone modifications regulate the structural status of chromatin and thereby influence the transcriptional status of genes. These processes are controlled by the recruitment of different enzymes to a specific genomic site. Furthermore, obtaining an understanding of these mechanisms could help delineate alternative treatment and preventive strategies for cancer. For example, in gastric cancer, cholecalciferol, curcumin, resveratrol, quercetin, garcinol and sodium butyrate are natural regulators of acetylation and deacetylation enzyme activity that exert chemopreventive and anticancer effects. Here, we review the recent findings on histone acetylation in gastric cancer and discuss the effects of nutrients and bioactive compounds on histone acetylation and their potential role in the prevention and treatment of this type of cancer.

Evaluation of in vivo and in vitro toxicological and genotoxic potential of aluminum chloride.

Aluminum and its compounds are common contaminants of water and food, as well as medications and cosmetics. The wide distribution of the element facilitates the demand for detailed studies of its biological and toxicological effects. This work aimed to evaluate the possible genotoxic and toxic activity resulting from in vivo and in vitro exposure to Al. For in vivo analysis, 40 Swiss mice were used, various concentrations of hydrated aluminum chloride were administered orally. They were analyzed for possible genic activity and metal cytotoxicity using a micronucleus test (MN), and for toxicity through histopathological evaluation of the extracted organs. For in vitro analysis, lymphocytes from the peripheral blood of 3 healthy donors were used. These cells were exposed to the same chemical agent in various concentrations. In vivo study revealed a significant increase in the number of MN in all Al concentrations. Furthermore, significant alterations in all the organs evaluated were verified by the presence of irreversible lesions (such as necrosis). Corroborating these findings, a significant increase in the quantity of MN in all concentrations with lymphocytes in vitro. In light of this, we suggest that this metal presents genotoxic potential and is potentially a cause of pathological disorders.

Lack of detection of human papillomavirus DNA in prostate carcinomas in patients from northeastern Brazil.

Prostate cancer is the second most common cancer among men in western populations, and despite its high mortality, its etiology remains unknown. Inflammatory processes are related to the etiology of various types of tumors, and prostate inflammation, in particular, has been associated with prostate cancer carcinogenesis and progression. Human papillomavirus (HPV) is associated with benign and malignant lesions in the anogenital tract of both females and males. The possible role of HPV in prostate carcinogenesis is a subject of great controversy. In this study, we aimed to examine the prevalence of HPV infections in prostate carcinomas of patients from northeastern Brazil. This study included 104 tissue samples from primary prostate carcinoma cases. HPV DNA was purified and then amplified using MY09/11 and GP5+/GP6+ degenerate primer sets that detect a wide range of HPV types, and with specific PCR primers sets for E6 and E7 HPV regions to detect HPV 16. None of the samples showed amplification products of HPV DNA for primer sets MY09/11 and GP5+/GP6+, or the specific primer set for the E6 and E7 HPV regions. HPV infection, thus, does not seem to be one of the causes of prostate cancer in the population studied.

Whole exome sequencing in a case of sporadic multiple meningioma reveals shared NF2, FAM109B, and TPRXL mutations, together with unique SMARCB1 alterations in a subset of tumor nodules.

Meningiomas are common intracranial tumors derived from arachnoid cells. Multiple meningiomas are occasionally present even in patients with no history of neurofibromatosis type 2, a condition that can cause the formation of this neoplasm. Previous studies have shown that most multiple meningiomas are monoclonal in origin. In this study, exome sequencing was performed on four meningiomas and the corresponding peripheral blood DNA from a 61-year-old woman with sporadic multiple meningioma. At least three common mutational events (at the NF2, FAM109B, and TPRXL genes) were detected in the tumors' DNA when they were compared with the lymphocyte DNA from the patient as control. Additionally, an array of unique mutations was detected in each tumor, including in SMARCB1 in two of the samples, a gene whose alteration leads to the development of meningioma. Mutations in other genes, such as IRS4, GULP1, NHSL1, and C10orf53, accounted for one alteration in each meningioma nodule. Our data suggest a monoclonal origin of the meningiomas in this patient, although the numerous alterations contained in each sample indicated multiple secondary variable changes in each tumor nodule. Whether the alterations described in this work are drivers of tumorigenesis or are simply passengers requires further study.

Genome-wide methylation analysis in vestibular schwannomas shows putative mechanisms of gene expression modulation and global hypomethylation at the HOX gene cluster.

Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein-coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA-21, Met Proto-Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets.

Global expression profile in low grade meningiomas and schwannomas shows upregulation of PDGFD, CDH1 and SLIT2 compared to their healthy tissue.

Schwannomas and grade I meningiomas are non­metastatic neoplasms that share the common mutation of gene NF2. They usually appear in neurofibromatosis type 2 patients. Currently, there is no drug treatment available for both tumors, thus the use of wide expression technologies is crucial to identify therapeutic targets. Affymetrix Human Gene 1.0 ST was used to test global gene expression in 22 meningiomas, 31 schwannomas and, as non-tumoral controls, 3 healthy meningeal tissues, 8 non-tumoral nerves and 1 primary Schwann cell culture. A non-stringent P-value cut-off and fold change were used to establish deregulated genes. We identified a subset of genes that were upregulated in meningiomas and schwannomas when compared to their respectively healthy tissues, including PDGFD, CDH1 and SLIT2. Thus, these genes should be thoroughly studied as targets in a possible combined treatment.

Homozygous deletion of TNFRSF4, TP73, PPAP2B and DPYD at 1p and PDCD5 at 19q identified by multiplex ligation-dependent probe amplification (MLPA) analysis in pediatric anaplastic glioma with questionable oligodendroglial component.

BACKGROUND: Pediatric oligodendrogliomas are rare and appear to show a different molecular profile from adult tumors. Some gliomas display allelic losses at 1p/19q in pediatric patients, although less frequently than in adult patients, but this is rare in tumors with an oligodendroglial component. The molecular basis of this genomic abnormality is unknown in pediatric gliomas, but it represents a relatively common finding in pediatric oligodendroglioma-like neoplasms with leptomeningeal dissemination. RESULTS: Multiplex ligation-dependent probe amplification (MLPA) analysis using SALSA P088-B1 for the analysis of the 1p/19q allelic constitution in a pediatric anaplastic (oligodendro)-glioma showed homozygous co-deletion for markers: TNFRSF4 (located at 1p36.33), TP73 (1p36.32), PPAP2B (1pter-p22.1), DPYD (1p21.3), and PDCD5 (19q13.12), and hemizygous deletion of BAX (19q13.3-q13.4). No sequence changes for R132 and R172 of the IDH1/2 genes were identified. CONCLUSIONS: The molecular findings in this pediatric anaplastic glioma do not allow for a clearly definitive pathological diagnosis. However, the findings provide data on a number of 1p/19q genomic regions that, because of homozygotic deletion, might be the location of genes that are important for the development and clinical evolution of some malignant gliomas in children.

Gene expression analysis of aberrant signaling pathways in meningiomas.

Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, NF2, which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningioma.

Global profiling in vestibular schwannomas shows critical deregulation of microRNAs and upregulation in those included in chromosomal region 14q32.

BACKGROUND: Vestibular schwannomas are benign tumors that arise from Schwann cells in the VIII cranial pair and usually present NF2 gene mutations and/or loss of heterozygosity on chromosome 22q. Deregulation has also been found in several genes, such as ERBB2 and NRG1. MicroRNAs are non-coding RNAs approximately 21 to 23 nucleotides in length that regulate mRNAs, usually by degradation at the post-transcriptional level. METHODS: We used microarray technology to test the deregulation of miRNAs and other non-coding RNAs present in GeneChip miRNA 1.0 (Affymetrix) over 16 vestibular schwannomas and 3 control-nerves, validating 10 of them by qRT-PCR. FINDINGS: Our results showed the deregulation of 174 miRNAs, including miR-10b, miR-206, miR-183 and miR-204, and the upregulation of miR-431, miR-221, miR-21 and miR-720, among others. The results also showed an aberrant expression of other non-coding RNAs. We also found a general upregulation of the miRNA cluster located at chromosome 14q32. CONCLUSION: Our results suggest that several miRNAs are involved in tumor formation and/or maintenance and that global upregulation of the 14q32 chromosomal site contains miRNAs that may represent a therapeutic target for this neoplasm.

Microarray analysis of gene expression in vestibular schwannomas reveals SPP1/MET signaling pathway and androgen receptor deregulation.

Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (Nrg1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan low density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.

Inhibition of DNA topoisomerase I activity and induction of apoptosis by thiazacridine derivatives.

Thiazacridine derivatives (ATZD) are a novel class of cytotoxic agents that combine an acridine and thiazolidine nucleus. In this study, the cytotoxic action of four ATZD were tested in human colon carcinoma HCT-8 cells: (5Z)-5-acridin-9-ylmethylene-3-(4-methylbenzyl)-thiazolidine-2,4-dione - AC-4; (5ZE)-5-acridin-9-ylmethylene-3-(4-bromo-benzyl)-thiazolidine-2,4-dione - AC-7; (5Z)-5-(acridin-9-ylmethylene)-3-(4-chloro-benzyl)-1,3-thiazolidine-2,4-dione - AC-10; and (5ZE)-5-(acridin-9-ylmethylene)-3-(4-fluoro-benzyl)-1,3-thiazolidine-2,4-dione - AC-23. All of the ATZD tested reduced the proliferation of HCT-8 cells in a concentration- and time-dependent manner. There were significant increases in internucleosomal DNA fragmentation without affecting membrane integrity. For morphological analyses, hematoxylin-eosin and acridine orange/ethidium bromide were used to stain HCT-8 cells treated with ATZD, which presented the typical hallmarks of apoptosis. ATZD also induced mitochondrial depolarisation and phosphatidylserine exposure and increased the activation of caspases 3/7 in HCT-8 cells, suggesting that this apoptotic cell death was caspase-dependent. In an assay using Saccharomyces cerevisiae mutants with defects in DNA topoisomerases 1 and 3, the ATZD showed enhanced activity, suggesting an interaction between ATZD and DNA topoisomerase enzyme activity. In addition, ATZD inhibited DNA topoisomerase I action in a cell-free system. Interestingly, these ATZD did not cause genotoxicity or inhibit the telomerase activity in human lymphocyte cultures at the experimental levels tested. In conclusion, the ATZD inhibited the DNA topoisomerase I activity and induced tumour cell death through apoptotic pathways.

Expression analysis of tumor-related genes involved in critical regulatory pathways in schwannomas.

PURPOSE: Gene expression array analysis is providing key data on the potential candidate genes and biological pathways involved in schwannoma origin and development. In this way we performed expression array studies on tumor-related genes in schwannomas. METHODS: The GE Array Q Series HS-006 (SuperArray, Bethesda, MD, USA) was used to determine the expression levels of 96 genes corresponding to 6 primary biological regulatory pathways in a series of 23 schwannomas. RESULTS: We identified 15 genes down-regulated, primarily corresponding to signal transduction functions, and 26 genes up-regulated, most frequently involving cell adhesion functions. CONCLUSIONS: In addition to the NF2 inactivation (considered as an early step), variations of other biological regulatory pathways might play a key role in schwannoma.

Prognostic value of the TP53 Arg72Pro single-nucleotide polymorphism and susceptibility to medulloblastoma in a cohort of Brazilian patients.

Medulloblastoma is a highly cellular malignant embryonal neoplasm, being the most common malignant pediatric brain tumor, accounting for 20-25 % of pediatric central nervous system tumors. To investigate the effect of the TP53 Arg72Pro single-nucleotide polymorphism (SNP) on clinicopathological and phenotypic parameters, we performed a case-controlled study of 122 patients and 122 healthy controls from Brazil. No significant associations were found between the TP53 Arg72Pro genotypes and the clinicopathological parameters studied. Compared with Arg/Arg, which is the most common genotype in the study population, both the Arg/Pro and Pro/Pro genotypes did not influence the medulloblastoma development risk [odds ratio (OR) = 1.36 and P = 0.339 for the Arg/Pro genotype; OR = 1.50 and P = 0.389 for the Pro/Pro genotype]. With regard to prognosis, disease-free survival was not significantly different among the TP53 Arg72Pro SNP genotypes (P > 0.05), but the less frequent genotype (Pro/Pro) was associated with shorter overall survival of medulloblastoma patients (P = 0.021). These data suggest that, although there is no association between the TP53 Arg72Pro SNP and medulloblastoma risk, the Pro/Pro genotype is associated with shorter overall survival of patients submitted to adjuvant therapy. Nevertheless, due to the interethnic composition of the Brazilian population, future studies on larger populations from other parts of the world are essential for a definitive conclusion on the function of the TP53 Arg72Pro SNP.

Inhibitory effect of pisosterol on human glioblastoma cell lines with C-MYC amplification.

Despite the remarkable progress in the characterization of the molecular pathogenesis of glioblastoma multiforme (GBM), these tumors remain incurable and, in most cases, refractory to aggressive cytotoxic treatments. We conducted a morphological and cytogenetic study in two GBM cell lines (U343 and AHOL1), before and after treatment with pisosterol (at 0.5, 1.0 and 1.8 µg ml⁻¹), a triterpene isolated from the fungus Pisolithus tinctorius. No significant alteration was observed in the morphology and frequency of chromosomal abnormalities in the cell lines analyzed after treatment with pisosterol. Using fluorescence in situ hybridization analysis with a locus-specific probe for C-MYC showed that 72% of U343 and 65% of AHOL1 cells contained more than two alleles of C-MYC before treatment. After treatment, no effects were detected at lower concentrations of pisosterol (0.5 and 1.0 µg ml⁻¹). However, at 1.8 µg ml⁻¹ of pisosterol, only 33% of U343 cells and 15% of AHOL1 cells presented more than two fluorescent signals, suggesting that pisosterol blocks the cells with gene amplification. Cells that do not show a high degree of C-MYC gene amplification have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.