Hypoxia as a modulator of cytochromes P450: Overexpression of the cytochromes CYP2S1 and CYP24A1 in human liver cancer cells in hypoxia.

Low levels of oxygen (hypoxia) have been reported in solid tumours. This hypoxic microenvironment modulates the expression of genes linked to a more aggressive disease. However, it is unclear if the expression of drug-metabolizing enzymes as cytochromes P450 (CYPs) is affected by hypoxia in cancer. We aimed to define which cytochromes are affected by hypoxia using a liver cancer model in vitro. For this purpose, we assessed whole-genome expression microarrays of HepG2 liver cancer cell line from free repository databases, looking for gene expression hypoxia-associated profiles and selected those cytochromes with significant differences. Then, we corroborated their mRNA expression and protein levels by RT-qPCR and western blot, respectively, as well as immunofluorescence. Based on microarray analysis, we found that the expression of CYP2S1 and CYP24A1 were up-regulated with at least twice fold change compared with normoxia. The levels of mRNA and protein of CYP2S1 and CYP24A1 were increased significantly in hypoxic conditions (P < .05), and this tendency was also observed by immunofluorescence assays. Our data show that the expression of cytochromes CYP2S1 and CYP24A1 are induced in hypoxia, being the first time that CYP24A1 expression is associated with tumour hypoxia; which might have consequences in cancer progression and drug resistance. SIGNIFICANCE OF THE STUDY: Hypoxia is among the most important factors for cellular adaptation to stress. Especially in cancer, a major public health issue, hypoxia plays a substantial role in angiogenesis, metastasis and resistance to therapy. Tumoral hypoxia has been described at least in the brain, breast, cervical, liver, renal, lung, pancreatic and renal cancer. However, the understanding of how hypoxia drives cancer progression is still a major challenge. One emerging question is the role of hypoxia over the expression of drug-metabolizing enzymes, with a significant impact on drug treatment. In this context, our paper focus on the effect of hypoxia on CYPs, which is an essential group of drug-metabolizing enzymes. We show that hypoxia induces the expression of two members of the CYPs family: CYP2S1 and CYP24A1. Importantly, CYP2S1 is a major metabolizer of carcinogenic substances being relevant that hypoxia could promote this function. Interestingly, CYP24A1 limits the action of the active form of vitamin D, which is an anti-proliferative factor in cancer. Our evidence shows for the first time that hypoxia can induce CYP24A1 expression, with a potential effect on cancer progression. Our contribution clarifies a particular effect of tumoral hypoxia and the implications will be useful in the understanding of the progression of cancer, the resistance to treatment and the development of alternative therapies.

The rs776746 variant of CYP3A5 is associated with intravenous midazolam plasma levels and higher clearance in critically ill Mexican paediatric patients.

WHAT IS KNOWN AND OBJECTIVE: Midazolam is a drug that is metabolized by cytochrome P450 (CYP450) enzymes, particularly CYP3A4 and CYP3A5. The presence of single-nucleotide polymorphisms (SNPs) in the genes encoding these enzymes, such as CYP3A4*1B which is associated with low enzyme expression and activity and CYP3A5*3, has been associated with decrease in enzymatic activity and reduced drug clearance, with potential effects on drug levels and/or toxicity. The present study was conducted to determine the frequencies of the allelic variants of the CYP3A4 (rs2740574) and CYP3A5 (rs776746) genes and their effects on the plasma levels and clearance of intravenous midazolam in critically ill Mexican paediatric patients. METHODS: Seventy-two DNA samples were genotyped by real-time PCR with TaqMan probes. Plasma midazolam levels were determined at 3 and 24 h post infusion by high-performance liquid chromatography. RESULTS AND DISCUSSION: The allelic variant rs776746 (CYP3A5*3) was associated with high midazolam plasma levels; the median concentration in patients with the normal genotype (CC) <0.01 ng/ml (Q25 0.01-Q75 196.09), whereas patients with the allelic variant (TT+TC) had a median midazolam concentration of 320.3 ng/ml (Q25 37.51-Q75 529.51), p = 0.001. The median pharmacokinetic clearance rates were 0.10 L/kg/h (Q25 0.01-Q75 0.34) in patients with the allelic variant (TT+TC) and 0.03 L/kg/h (Q25 0.002-Q75 0.13) in patients with the normal genotype (CC), p = 0.042. WHAT IS NEW AND CONCLUSION: This is the first study that reports the frequency of the rs776746 polymorphism in critically ill paediatric patients, which is relevant, since carriers of the *1 allele synthesizing a functional enzyme may need higher doses to achieve adequate sedation. Our results show that compared with carriers of the normal allele, patients with the CYP3A5*3 allelic variant (rs776746) had increased plasma midazolam levels at 3 h after infusion discontinuation (320.3 ng/ml) and greater clearance (0.10 L/kg/h) of the drug.

Hypoxia as a Modulator of Inflammation and Immune Response in Cancer.

A clear association between hypoxia and cancer has heretofore been established; however, it has not been completely developed. In this sense, the understanding of the tumoral microenvironment is critical to dissect the complexity of cancer, including the reduction in oxygen distribution inside the tumoral mass, defined as tumoral hypoxia. Moreover, hypoxia not only influences the tumoral cells but also the surrounding cells, including those related to the inflammatory processes. In this review, we analyze the participation of HIF, NF-κB, and STAT signaling pathways as the main components that interconnect hypoxia and immune response and how they modulate tumoral growth. In addition, we closely examine the participation of the immune cells and how they are affected by hypoxia, the effects of the progression of cancer, and some innovative applications that take advantage of this knowledge, to suggest potential therapies. Therefore, we contribute to the understanding of the complexity of cancer to propose innovative therapeutic strategies in the future.

Microenvironment-regulated lncRNA-HAL is able to promote stemness in breast cancer cells.

Multicellular Tumor Spheroids culture (MCTS) is an in vitro model mimicking the characteristics of the tumor microenvironment, such as hypoxia and acidosis, resulting in the presence of both proliferating and quiescent cell populations. lncRNA's is a novel group of regulatory molecules that participates in the acquisition of tumorigenic phenotypes. In the present work we evaluated the oncogenic association of an uncharacterized lncRNA (lncRNA-HAL) in the tumorigenic phenotype induced by the MCTS microenvironment. We measured lncRNA-HAL expression level in MCF-7-MCTS populations and under different hypoxic conditions by RT-qPCR. Afterwards, we silenced lncRNA-HAL expression by shRNAs and evaluated its effect in MCF-7 transcriptome (by RNAseq) and validated the modified cellular processes by proliferation, migration, and stem cells assays. Finally, we analyzed which proteins interacts with lncRNA-HAL by ChIRP assay, to propose a possible molecular mechanism for this lncRNA. We found that lncRNA-HAL is overexpressed in the internal quiescent populations (p27 positive populations) of MCF-7-MCTS, mainly in the quiescent stem cell population, being hypoxia one of the microenvironmental cues responsible of its overexpression. Transcriptome analysis of lncRNA-HAL knockdown MCF7 cells revealed that lncRNA-HAL effect is associated with proliferation, migration and cell survival mechanisms; moreover, lncRNA-HAL silencing increased cell proliferation and impaired cancer stem cell proportion and function, resulting in decreased tumor grafting in vivo. In addition, we found that this lncRNA was overexpressed in triple-negative breast cancer patients. Analysis by ChIRP assay showed that this nuclear lncRNA binds to histones and hnRNPs suggesting a participation at the chromatin level and transcriptional regulation. The results obtained in the present work suggest that the function of lncRNA-HAL is associated with quiescent stem cell populations, which in turn is relevant due to its implications in cancer cell survival and resistance against treatment in vivo. Altogether, our data highlights a new lncRNA whose expression is regulated by the tumor microenvironment and associated to stemness in breast cancer.

Hypoxia increases chemoresistance in human medulloblastoma DAOY cells via hypoxia­inducible factor 1α­mediated downregulation of the CYP2B6, CYP3A4 and CYP3A5 enzymes and inhibition of cell proliferation.

Medulloblastomas are among the most frequently diagnosed pediatric solid tumors, and drug resistance remains as the principal cause of treatment failure. Hypoxia and the subsequent activation of hypoxia­inducible factor 1α (HIF­1α) are considered key factors in modulating drug antitumor effectiveness, but the underlying mechanisms in medulloblastomas have not yet been clearly understood. The aim of the present study was to determine whether hypoxia induces resistance to cyclophosphamide (CPA) and ifosfamide (IFA) in DAOY medulloblastoma cells, whether the mechanism is dependent on HIF­1α, and whether involves the modulation of the expression of cytochromes P450 (CYP)2B6, 3A4 and 3A5 and the control of cell proliferation. Monolayer cultures of DAOY medulloblastoma cells were exposed for 24 h to moderate (1% O2) or severe (0.1% O2) hypoxia, and protein expression was evaluated by immunoblotting. Cytotoxicity was studied with the MTT assay and by Annexin V/PI staining and flow cytometry. Cell proliferation was determined by the trypan­blue exclusion assay and cell cycle by propidium iodide staining and flow cytometry. Hypoxia decreased CPA and IFA cytotoxicity in medulloblastoma cells, which correlated with a reduction in the protein levels of CYP2B6, CYP3A4 and CYP3A5 and inhibition of cell proliferation. These responses were dependent on hypoxia­induced HIF­1α activation, as evidenced by chemical inhibition of its transcriptional activity with 2­methoxyestradiol (2­ME), which enhanced the cytotoxic activity of CPA and IFA and increased apoptosis. Our results indicate that by stimulating HIF­1α activity, hypoxia downregulates the expression of CYP2B6, CYP3A4 and CYP3A5, that in turn leads to decreased conversion of CPA and IFA into their active forms and thus to diminished cytotoxicity. These results support that the combination of HIF­1α inhibitors and canonical antineoplastic agents provides a potential therapeutic alternative against medulloblastoma.

Data mining of pediatric medulloblastoma microarray expression reveals a novel potential subdivision of the Group 4 molecular subgroup.

Medulloblastoma is the most common type of solid brain tumor in children. This type of embryonic tumor is highly heterogeneous and has been classified into 4 molecular subgroups based on their gene expression profiles: WNT, SHH, Group 3 (G3) and Group 4 (G4). WNT and SHH tumors exhibit the specific dysregulation of genes and pathways, whereas G3 and G4 tumors, two of the more frequent subtypes, are the least characterized. Thus, novel markers to aid in the diagnosis, prognosis and management of medulloblastoma are required. In the present study, microarray gene expression data was downloaded from the Gene Expression Omnibus database, including data from the 4 subgroups of medulloblastoma and healthy cerebellum tissue (CT). The data was utilized in an in silico analysis to characterize each subgroup at a transcriptomic level. Using Partek Genomics Suite software, the data were visualized via hierarchical clustering and principal component analysis. The differentially expressed genes were uploaded to the MetaCore portal to perform enrichment analysis using CT gene expression as baseline, with fold change thresholds of <-5 and >5 for differential expression. The data mining analysis of microarray gene expression data enabled the identification of a range of dysregulated molecules associated with each subgroup of medulloblastoma. G4 is the most heterogeneous subgroup, as no definitive pathway defines its pathogenesis; analysis of the gene expression profiles were associated with the G4α and G4ß subcategories. TOX high mobility group box family member 3, synuclein α interacting protein and, potassium voltage-gated channel interacting protein 4 were identified as three novel potential markers for distinguishing the α and ß subcategories of G4. These genes may be associated with medulloblastoma pathogenesis, and thus may provide a basis for researching novel targeted treatment strategies for G4 medulloblastoma.