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Summary: Cryptorchidism is a frequent genitourinary malformation considered as an important risk factor for infertility and testicular malignancy. The aetiology of cryptorchidism is multifactorial in which certain SNPs, capable of inhibiting the development of the gubernaculum, are implicated. We analysed 16 SNPs by allelic discrimination and automated sequencing in 85 patients and 99 healthy people, with the objective to identify the association between these variants and isolated cryptorchidism. In two different patients with unilateral cryptorchidism, we found the variants rs121912556 and p.R105R of INSL3 gene in a heterozygous form associated with cryptorchidism, so we could considered them as risk factors for cryptorchidism. On the other hand, SNPs rs10421916 of INSL3 gene, as well as the variants rs1555633 and rs7325513 in the RXFP2 gene, and rs3779456 variant of the HOXA10 gene were statistically significant, when the patients and controls were compared and could be considered as protective factors since are predominantly present in controls. The genotype–phenotype correlation did not show statistical significance. With these results, we could conclude that these polymorphisms can be considered as important variants in our population and would contribute in the future knowledge of the aetiology and physiopathology of cryptorchidism. [ABSTRACT FROM AUTHOR]

The article presents a study which evaluates the safety and efficacy of epigenetic therapy and cisplatin chemoradiation in International Federation of Gynecology and Obstetrics (FIGO) Stage IIIB cervical cancer patients. The study uses high-performance liquid chromatographic (HPLC) to determine hydralazine in plasma and fluorescence polarization immunoassay technology to measure valproic acid. It shows that hydralazine and valproic acid are safe when administered with cisplatin chemoradiation.

The article presents a study of the mutation of BRCA1 and BRCA2 genes in Mexican women with breast and ovarian cancer. It stated that the genes were screened in 40 patients through modifying high performance liquid chromatography analyses. It mentions that two frameshift mutations were found in BRCA1 gene and one missense mutation in each gene.

Arylamine N-acetyltransferase 2 has been related to drug side effects and cancer susceptibility; its protein structure and acetylation capacity results from the polymorphism's arrays on the NAT2 gene. Absorption, distribution, metabolism, and excretion, cornerstones of the pharmacological effects, have shown diversity patterns across populations, ethnic groups, and even interethnic variation. Although the 1000 Genomes Project database has portrayed the global diversity of the NAT2 polymorphisms, several populations and ethnicities remain underrepresented, limiting the comprehensive picture of its variation. The NAT2 clinical entails require a detailed landscape of its striking diversity. This systematic review spans the genetic and acetylation patterns from 164 articles from October 1992 to October 2020. Descriptive studies and controls from observational studies expanded the NAT2 diversity landscape. Our study included 243 different populations and 101 ethnic minorities, and, for the first time, we presented the global patterns in the Middle Eastern populations. Europeans, including its derived populations, and East Asians have been the most studied genetic backgrounds. Contrary to the popular perception, Africans, Latinos and Native Americans have been significantly represented in recent years. NAT2*4, *5B, and *6A were the most frequent haplotypes globally. Nonetheless, the distribution of *5B and *7B were less and more frequent in Asians, respectively. Regarding the acetylator status, East Asians and Native Americans harboured the highest frequencies of the fast phenotype, followed by South Europeans. Central Asia, the Middle East, and West European populations were the major carriers of the slow acetylator status. The detailed panorama presented herein, expands the knowledge about the diversity patterns to genetic and acetylation levels. These data could help clarify the controversial findings between acetylator states and the susceptibility to diseases and reinforce the utility of NAT2 in precision medicine., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gutiérrez-Virgen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

(1) Background: The epigenetic regulator EZH2 is a subunit of the polycomb repressive complex 2 (PRC2), and methylates H3K27, resulting in transcriptional silencing. It has a critical role in lymphocyte differentiation within the lymph node. Therefore, mutations at this level are implicated in lymphomagenesis. In fact, the mutation at the Y641 amino acid in the EZH2 gene is mutated in up to 40% of B-cell lymphomas. (2) Methods: We compared the presence of exon 16 EZH2 mutations in tumor samples and ctDNA in a prospective trial. These mutations were determined by Sanger sequencing and ddPCR. (3) Results: One hundred and thirty-eight cases were included. Ninety-eight were germinal center, and twenty had EZH2 mutations. Mean follow-up (IQR 25-75) was 23 (7-42) months. The tumor samples were considered the standard of reference. Considering the results of the mutation in ctDNA by Sanger sequencing, the sensibility (Se) and specificity (Sp) were 52% and 99%, respectively. After adding the droplet digital PCR (ddPCR) analysis, the Se and Sp increased to 95% and 100%, respectively. After bivariate analysis, only the presence of double-hit lymphoma ( p = 0.04) or EZH2 mutations were associated with relapse. The median Progression free survival (PFS) (95% interval confidence) was 27.7 (95% IC: 14-40) vs. 44.1 (95% IC: 40-47.6) months for the mutated vs. wild-type (wt) patients. (4) Conclusions: The ctDNA is useful for analyzing EZH2 mutations, which have an impact on PFS.

Background: Worldwide, gastric cancer is ranked the fifth malignancy in incidence and the third malignancy in mortality. Gastric cancer causes an altered metabolism that can be therapeutically exploited., Objective: The objective of this study is to provide an overview of the significant metabolic alterations caused by gastric cancer and propose a blockade., Methods: A comprehensive and up-to-date review of descriptive and experimental publications on the metabolic alterations caused by gastric cancer and their blockade. This is not a systematic review., Results: Gastric cancer causes high rates of glycolysis and glutaminolysis. There are increased rates of de novo fatty acid synthesis and cholesterol synthesis. Moreover, gastric cancer causes high rates of lipid turnover via fatty acid β-oxidation. Preclinical data indicate that the individual blockade of these pathways via enzyme targeting leads to antitumor effects in vitro and in vivo. Nevertheless, there is no data on the simultaneous blockade of these five pathways, which is critical as tumors show metabolic flexibility in response to the availability of nutrients. This means tumors may activate alternate routes when one or more are inhibited. We hypothesize there is a need to simultaneously block them to avoid or decrease the metabolic flexibility that may lead to treatment resistance., Conclusion: There is a need to explore the preclinical efficacy and feasibility of combined metabolic therapy targeting the pathways of glucose, glutamine, fatty acid synthesis, cholesterol synthesis, and fatty acid oxidation. This may have therapeutical implications because we have clinically available drugs that target these pathways in gastric cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cancer therapy advances have yet to impact global cancer mortality. One of the factors limiting mortality burden reduction is the high cost of cancer drugs. Cancer drug repurposing has already failed to meet expectations in terms of drug affordability. The three FDA-approved cancer drugs developed under repurposing: all-trans-retinoic acid, arsenic trioxide, and thalidomide do not differ in price from other drugs developed under the classical model. Though additional factors affect the whole process from inception to commercialization, the repurposing of widely used, commercially available, and cheap drugs may help. This work reviews the concept of the malignant metabolic phenotype and its exploitation by simultaneously blocking key metabolic processes altered in cancer. We elaborate on a combination called BAPST, which stands for the following drugs and pathways they inhibit: Benserazide (glycolysis), Apomorphine (glutaminolysis), Pantoprazole (Fatty-acid synthesis), Simvastatin (mevalonate pathway), and Trimetazidine (Fatty-acid oxidation). Their respective primary indications are: • Parkinson's disease (benserazide and apomorphine). • Peptic ulcer disease (pantoprazole). • Hypercholesterolemia (simvastatin). • Ischemic heart disease (trimetazidine). When used for their primary indication, the literature review on each of these drugs shows that they have a good safety profile and lack predicted pharmacokinetic interaction among them. Based on that, we propose that the BAPST regimen merits preclinical testing., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Novel prognostic factors in patients with diffuse large B-cell lymphoma (DLBCL) are required in the era of Rituximab., Objective: The objective of the study was to study the prognostic impact of exon-16 enhancer-of-zeste homolog-2 (EZH2) mutations in patients with DLBCL., Methods: In a cohort of patients with DLBCL treated between 2015 and 2017, we analyzed the presence of EZH2 mutations and their association with clinical response (CR), relapse, progression-free survival (PFS), and overall survival (OS)., Results: A total of 198 patients were included; of them, 30 (15.2%) had mutations at codon 641, in exon 16 of EZH2. Response was achieved in 151 patients (76.3%), and 43 (21.7%) relapsed or progressed during follow-up. EZH2 mutations were associated with relapse/progression (risk ratio [RR] 1.18; 95% confidence interval [CI] 0.98-1.42; p = 0.031), while a trend for not achieving a complete response was observed (RR: 0.876; 95%CI 0.74-1.038; p = 0.071). Of note, Tyr641His and Tyr641Ser EZH2 mutations were associated with shorter PFS (hazard ratio 3.234; 95% CI 1.149-9.1; p = 0.026)., Conclusion: The presence of EZH2 mutations was negatively associated with relapse/progression and showed a trend for lack of complete response. Further studies are needed to define better the prognostic significance of these mutations in Mexican-Mestizo DLBCL patients.

Astrocytoma is the most common type of primary brain tumor. The risk factors for astrocytoma are poorly understood; however, germline genetic variants account for 25% of the risk of developing gliomas. In this study, we assessed the risk of astrocytoma associated with variants in AGT, known by its role in angiogenesis, TP53, a well-known tumor suppressor and the DNA repair gene MGMT in a Mexican population. A case-control study was performed in 49 adult Mexican patients with grade II-IV astrocytoma. Sequencing of exons and untranslated regions of AGT, MGMT, and TP53 from was carried in an Ion Torrent platform. Individuals with Mexican Ancestry from the 1000 Genomes Project were used as controls. Variants found in our cohort were then assessed in a The Cancer Genome Atlas astrocytoma pan-ethnic validation cohort. Variants rs1926723 located in AGT (OR 2.74, 1.40-5.36 95% CI), rs7896488 in MGMT (OR 3.43, 1.17-10.10 95% CI), and rs4968187 in TP53 (OR 2.48, 1.26-4.88 95% CI) were significantly associated with the risk of astrocytoma after multiple-testing correction. This is the first study where the AGT rs1926723 variant, TP53 rs4968187, and MGMT rs7896488 were found to be associated with the risk of developing an astrocytoma.

Background: Patients with familial erythrocytosis type 2 have no increased risk of von Hippel-Lindau-associated tumors, although mutations in the VHL gene cause both pathologies., Case Report: We present a case of a compound heterozygote patient with von Hippel-Lindau disease and familial erythrocytosis type 2. One of the mutations found in our patient, c.416C>G (p.Ser139Cys) of the VHL gene, has not been previously reported. This case is the second one reported where von Hippel-Lindau disease and familial erythrocytosis type 2 coexist in the same individual., Conclusions: Despite the low frequency of familial erythrocytosis type 2 in patients with von Hippel-Lindau disease, the possibility of this diagnosis should be considered to avoid unnecessary invasive studies to explain the polyglobulia in these patients and guarantee an adequate follow-up and vigilance of both diseases.

Objectives: Cryptorchidism is the most common genitourinary birth defect in live newborn males and is considered as an important risk factor for testicular germ cell tumors and infertility. The Androgen Receptor gene is important in this pathology due to its participation, mainly, in the inguinoscrotal phase of testicular descent. We determine the length of the CAG tract in the Androgen Receptor ( AR ) gene in Mexican patients with nonsyndromic cryptorchidism., Methods: One hundred and 15 males were included; of these, 62 had nonsyndromic cryptorchidism and 53 were healthy volunteers. DNA was extracted from a peripheral blood samples, subsequently, the CAG tract in exon 1 of AR gene was amplified by PCR and sequenced., Results: Mexican patients with nonsyndromic cryptorchidism presented 25.03 ± 2.58 repeats of CAG tract in the AR gene compared to 22.72 ± 3.17 repeats of CAG tract in Mexican healthy individuals (p≤0.0001; t value of 4.3). Furthermore, the deletion of codon 57 that corresponds to the deletion of a leucine residue at position 57 (Del L57) in the AR gene was found for the first time in a nonsyndromic cryptorchidism patient. This molecular alteration has been related previously to testicular germ cell tumor (TGCT)., Conclusions: The CAG tract in the AR gene is longer in patients with nonsyndromic cryptorchidism than in healthy individuals, supporting the association between this polymorphism of the AR gene and nonsyndromic cryptorchidism in the Mexican population., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Nowadays, extracellular DNA or circulating cell-free DNA is considered to be a molecule with clinical applications (diagnosis, prognosis, monitoring of treatment responses, or patient follow-up) in diverse pathologies, especially in cancer. Nevertheless, because of its molecular characteristics, it can have many other functions. This review focuses on the participation of extracellular DNA (exDNA) in fundamental processes such as cell signaling, coagulation, immunity, evolution through horizontal transfer of genetic information, and adaptive response to inflammatory processes. A deeper understanding of its role in each of these processes will allow development of better tools to monitor and control pathologies, as well as helping to generate new therapeutic options, beyond the applicability of DNA in liquid biopsy.

Lonidamine, 6-Diazo-5-oxo-L-norleucine (DON) and orlistat are well known inhibitors of glycolysis, glutaminolysis and of de novo fatty acid synthesis, respectively. Although their antitumor effects have been explored in detail, the potential inhibition of the malignant metabolic phenotype and its influence on the expression of chemokines and growth factors involved in colon cancer, have not been previously reported to the best of our knowledge. In the present study, dose-response curves with orlistat, lonidamine or DON were generated from cell viability assays conducted in SW480 colon cancer cells. In addition, the synergistic effect of these compounds was evaluated in SW480 human colon cancer cells. The determination of the doses used for maximum synergistic efficacy led to the exploration of the mRNA levels of the target genes hexokinase-2 ( HK2 ), glutaminase-1 ( GLS-1 ) and fatty acid synthase ( FASN ) in human SW480 and murine CT26.WT colon cancer cells. The cell viability was evaluated following transfection with small interfering (si)RNA targeting these genes and was assessed with trypan blue. The expression levels of chemokines and growth factors were quantified in the supernatant of SW480 cells with LEGENDplex™. The combination of lonidamine, DON and orlistat resulted in a synergistic cytotoxic effect and induced the transcription of the corresponding gene targets but their corresponding proteins were actually downregulated. The downregulation of the expression levels of HK2, GLS-1 and FASN following transfection of the cells with the corresponding siRNA sequences decreased their viability. The treatment significantly reduced the expression levels of 9 chemokines [interleukin-9, C-X-C motif chemokine ligand (CXCL) 10, eotaxin, chemokine ligand (CCL) 9, CXCL5, CCL20, CXCL1, CXCL11 and CCCL4] and one growth factor (stem cell factor). These changes were associated with decreased phosphorylated-nuclear factor κB-p65. The data demonstrate that lonidamine, DON and orlistat in combination reduce the expression levels of chemokines and growth factors in colon cancer cells. Additional research is required to investigate the exact way by which both tumor and stromal cells regulate the expression levels of chemokines and growth factors., (Copyright © 2019, Spandidos Publications.)

Ribavirin exhibits inhibitory effects on the epigenetic enzyme enhancer of zeste homolog 2 (EZH2), which participates in lymphomagenesis. Additionally, preclinical and clinical studies have demonstrated the anti‑lymphoma activity of this drug. To further investigate the potential of ribavirin as an anticancer treatment for lymphoma, the tumor‑suppressive effects of ribavirin were analyzed in lymphoma cell lines. The effects of ribavirin on the viability and clonogenicity of the B‑cell lymphoma cell line Pfeiffer (EZH2‑mutant), Toledo (EZH2 wild‑type) and cutaneous T‑cell lymphoma Hut78 cell line were assessed. Expression of EZH2 and trimethylation status of histone 3, lysine 27 trimethylated (H3K27m3) was also determined in response to ribavirin. The transcriptional effects of ribavirin on Hut78 cells were analyzed by microarray expression and the results were validated by reverse transcription‑quantitative polymerase chain reaction, western blotting and knockout of signal transducer and activator of transcription 1 (STAT1). The results of the present study demonstrated that ribavirin suppressed the growth and clonogenicity of cells in a dose‑dependent manner. Ribavirin did not affect the expression of EZH2 nor altered its activity as evaluated by H3K27 trimethylation status. Furthermore, the results of transcriptome analysis indicated that the majority of the canonical pathways affected by ribavirin were associated with the immune system, including 'antigen presentation', 'communication between innate and adaptive immune cells' and 'cross‑talk between dendritic and natural killer cells'. The results of gene expression analysis were confirmed, by demonstrating at the RNA and protein levels, downregulation of stearoyl‑CoA desaturase and upregulation of STAT1. Depletion of STAT1, which was proposed as a key regulator of the aforementioned pathways, exerted growth inhibitory effects almost to the same extent as ribavirin. In conclusion, ribavirin was proposed to exert growth inhibitory effects on lymphoma cell lines, particularly Hut78 cells, a cutaneous T‑cell lymphoma cell line. Of note, these effects may depend on, at least in part, the activation of canonical immune pathways regulated by the key factors STAT1 and interferon‑γ. Our results provide insight into the anti‑lymphoma potential of ribavirin; however, further investigations in preclinical and clinical studies are required to determine the effectiveness of ribavirin as a therapeutic agent for treating lymphoma.

Introduction: Metastasis involves the accumulation of genetic and epigenetic alterations leading to activation of prometastatic genes and inactivation of antimetastatic genes. Among epigenetic alterations, DNA hypermethylation and histone hypoacetylation are the focus of intense translational research because their pharmacological inhibition has been shown to produce antineoplastic activity in a variety of experimental models., Aims: This study aimed to evaluate the antimetastatic effect of the DNA-methylation inhibitor, hydralazine, and the histone deacetylase inhibitor, valproic acid., Methods: NIH 3T3- Ras murine cells were treated with hydralazine and valproic acid to evaluate their effects upon cell proliferation, cell motility, chemotaxis, gelatinase activity, and gene expression. Lung metastases were developed by intravenous injection of NIH 3T3- Ras cells in BALB/c nu/nu mice and then treated with the drug combination., Results: Treatment induced a growth-inhibitory effect on NIH 3T3- Ras cells, showed a trend toward increased gelatinase activity of MMP2 and MMP9, and inhibited chemotaxis and cell motility. The combination led to a strong antimetastatic effect in lungs of nude mice., Conclusion: Hydralazine and valproic acid, two repositioned drugs as epigenetic agents, exhibit antimetastatic effects in vitro and in vivo and hold potential for cancer treatment., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Introduction: Renin-angiotensin system (RAS) in brain cancer represents a scarcely explored field in neuro-oncology. Recently, some pre- and clinical studies have reported that RAS components play a relevant role in the development and behavior of gliomas. The angiotensinogen (AGT) rs5050 genetic variant has been identified as a crucial regulator of the transcription of AGT mRNA, which makes it a logical and promising target of research. The aim of this study was to determine the relationship between the AGT rs5050 genetic variant in blood with prognosis in astrocytoma., Methods: A prospective pilot study was performed on forty-eight astrocytoma patients, who received the standard-of-care treatment. Blood samples were taken prior to surgery and DNA was sequenced using Ion Torrent next-generation sequencing and analyzed by Ion Reporter software. Descriptive, bivariate, multivariate, and survival analyses were performed using SPSS v21, STATA 12 and GraphPad Prism 7., Results: Median follow-up was 41 months (range 1-48). Survival analysis showed a significant difference between the rs5050 genotypes (p = .05). We found lower survival rates in individuals with the GG-genotype of rs5050 AGT compared to patients with the TT- and TG-genotype (2 months vs. 11.5 months, respectively [p = .01]). In bivariate and multivariate analyses, GG-genotype was negatively associated with survival., Conclusions: In patients with astrocytoma, AGT rs5050 GG-genotype was associated with poor prognosis. We propose this germline genetic variant as a complementary biomarker, which can be detected practically and safely in blood samples or saliva., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Cancer cells have increased glycolysis and glutaminolysis. Their third feature is increased de novo lipogenesis. As such, fatty acid (FA) synthesis enzymes are over-expressed in cancer and their depletion causes antitumor effects. As fatty acid synthase (FASN) plays a pivotal role in this process, it is an attractive target for cancer therapy., Areas Covered: This is a review of the lipogenic phenotype of cancer and how this phenomenon can be exploited for cancer therapy using inhibitors of FASN, with particular emphasis on orlistat as a repurposing drug., Expert Opinion: Disease stabilization only has been observed with a highly selective FASN inhibitor used as a single agent in clinical trials. It is too early to say whether the absence of tumor responses other than stabilization results because even full inhibition of FASN is not enough to elicit antitumor responses. The FASN inhibitor orlistat is a 'dirty' drug with target-off actions upon at least seven targets with a proven role in tumor biology. The development of orlistat formulations suited for its intravenous administration is a step ahead to shed light on the concept that drug promiscuity can or not be a virtue.

Introduction: Treatment in metastatic colorectal cancer (mCRC) has expanded with monoclonal antibodies targeting epidermal growth factor receptor, but is restricted to patients with a wild-type (WT) KRAS mutational status. The most sensitive assays for KRAS mutation detection in formalin-fixed paraffin embedded (FFPE) tissues are based on real-time PCR. Among them, high resolution melting analysis (HRMA), is a simple, fast, highly sensitive, specific and cost-effective method, proposed as adjunct for KRAS mutation detection. However the method to categorize WT vs mutant sequences in HRMA is not clearly specified in available studies, besides the impact of FFPE artifacts on HRMA performance hasn't been addressed either., Methods: Avowedly adequate samples from 104 consecutive mCRC patients were tested for KRAS mutations by Therascreen™ (FDA Validated test), HRMA, and HRMA with UDG pre-treatment to reverse FFPE fixation artifacts. Comparisons of KRAS status allocation among the three methods were done. Focusing on HRMA as screening test, ROC curve analyses were performed for HRMA and HMRA-UDG against Therascreen™, in order to evaluate their discriminative power and to determine the threshold of profile concordance between WT control and sample for KRAS status determination., Results: Comparing HRMA and HRMA-UDG against Therascreen™ as surrogate gold standard, sensitivity was 1 for both HRMA and HRMA-UDG; and specificity and positive predictive values were respectively 0.838 and 0.939; and 0.777 and 0.913. As evaluated by the McNemar test, HRMA-UDG allocated samples to a WT/mutated genotype in a significatively different way from HRMA (p > 0.001). On the other hand HRMA-UDG did not differ from Therascreen™ (p = 0.125). ROC-curve analysis showed a significant discriminative power for both HRMA and HRMA-UDG against Therascreen™ (respectively, AUC of 0.978, p > 0.0001, CI 95% 0.957-0.999; and AUC of 0.98, p > 0.0001, CI 95% 0.000-1.0). For HRMA as a screening tool, the best threshold (degree of concordance between sample curves and WT control) was attained at 92.14% for HRMA (specificity of 0.887), and at 92.55% for HRMA-UDG (specificity of 0.952)., Conclusions: HRMA is a highly sensitive method for KRAS mutation detection, with apparently adequate and statistically significant discriminative power. FFPE sample fixation artifacts have an impact on HRMA results, so for HRMA on FFPE samples pre-treatment with UDG should be strongly suggested. The choice of the threshold for melting curve concordance has also great impact on HRMA performance. A threshold of 93% or greater might be adequate if using HRMA as a screening tool. Further validation of this threshold is required., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of the present study was to investigate in vivo the feasibility and efficacy of the combination of lonidamine (LND), 6-diazo-5-oxo-L-norleucine (DON) and orlistat to simultaneously target glycolysis, glutaminolysis and de novo synthesis of fatty acids, respectively. The doses of LND and DON used in humans were translated to mouse doses (77.7 mg/kg and 145.5 mg/kg, respectively) and orlistat was used at 240 mg/kg. Three schedules of LND, DON and orlistat at different doses were administered by intraperitoneal injection to BALB/c mice in a 21-day cycle (schedule 1: LND, 0.5 mg/day; DON, 0.25 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 2: LND, 0.1 mg/day; DON, 0.5 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 3: LND, 0.5 mg/day; DON, 0.08 mg/day 1, 5 and 9; orlistat, 360 mg/kg/day) to assess tolerability. To determine the antitumor efficacy, a syngeneic tumor model in BALB/c mice was created using colon cancer CT26.WT cells, and a xenogeneic tumor model was created in nude mice using the human colon cancer SW480 cell line. Mice were treated with schedule 1. Animals were weighed, clinically inspected during the experiment and the tumor volume was measured at day 21. The 3 schedules assessed in the tolerability experiments were well tolerated, as mice maintained their weight and no evident clinical signs of toxicity were observed. Combination treatment with schedule 1 significantly decreased tumor growth in each mouse model. No evident signs of toxicity were observed and mice maintained their weight during treatment. The triple metabolic blockade of the malignant phenotype appears feasible and promising for cancer therapy.

Background Cell-free DNA circulates in cancer patients and induces in vivo cell transformation and cancer progression in susceptible cells. Based on this, we hypothesized that depletion of circulating DNA with DNAse I and a protease mix could have antitumor effects. Study design The study aimed to demonstrate that DNAse I and a protease mix can degrade in vitro DNA and proteins from the serum of healthy individuals and cancer patients, and in vivo in serum of Wistar rats,. Moreover, the antitumor effect of the systemically administered enzyme mix treatmentwas evaluated in nude mice subcutaneously grafted with the human colon cancer cell line SW480. Results The serum DNA of cancer patients or healthy individuals was almost completely degraded in vitro by the enzymatic treatment, but no degradation was found with the enzymes given separately. The intravenous administration of the enzymes led to significant decreases in DNA and proteins from rat serum. No antitumor effect was observed in immunodeficient mice treated with the enzymes given separately. In contrast, the animals that received both enzymes exhibited a marked growth inhibition of tumors, 40% of them having pathological complete response. Conclusion This study demonstrated that systemic treatment with DNAse I and a protease mix in rats decreases DNA and proteins from serum and that this treatment has antitumor effects. Our results support the hypothesis that circulating DNA could have a role in tumor progression, which can be offset by depleting it. Further studies are needed to prove this concept., (© The Author(s) 2016.)

Introduction: Natural Killer cells (NK) are key for the innate immune response against tumors and viral infections. Several viral proteins evade host immune response and target the NK cell receptor NKG2D and its ligands. Areas covered: This review aimed to describe the viruses and their proteins that interfere with the NKG2D receptor and their ligands, and how these interactions lead to immune evasion, host protection, and tissue damage from acute and chronic viral infections. Expert opinion: The study of viral proteins has already impacted the field of oncology. A prime example is the HBV vaccine and the development of antiviral drugs for HIV, Hepatitis C, and the family of Herpesviridae viruses. The NKG2D system seems to be a rational therapeutic target. Nevertheless, an effective cytotoxic response by NK cells is mediated by a network of activating and inhibitory receptors, the integration of which determines if the NK cell becomes cytotoxic or permissive. Immunotherapeutic agents that increase the antitumor lytic activity of NK cells through modulating activation and inhibitory signaling of NK cells are being developed. Nevertheless, more research is needed to dissect the integrative mechanism of NK cells function to fully exploit their antitumor and antiviral effector mechanisms.

Background: Hyper-CVAD is the treatment for patients with acute lymphoblastic leukemia in our institution., Objective: To evaluate the impact of single nucleotide polymorphisms at genes associated with methotrexate metabolism on survival., Methods: The presence of the single nucleotide polymorphisms G80A at reduced folate carrier-1 gene and C677T in the methylenetetrahydrofolate reductase gene was determined by denaturing high performance liquid chromatography and validated by sequencing. Both single nucleotide polymorphisms were evaluated in 71 healthy donors and in an exploratory pilot trial with acute lymphoblastic leukemia patients to determine the influence of these single nucleotide polymorphisms on clinical outcome. Clinical characteristics, response, and outcome were registered. A Cox regression analysis was done to evaluate factors influencing response and overall survival., Results: There were no differences in the frequency of single nucleotide polymorphisms between volunteers and acute lymphoblastic leukemia patients according to the Hardy-Weinberg test. Sensitivity and specificity were 72 and 91% for the G80A, and 64 and 75% for the C677T, respectively. The multivariate analysis showed that the T-immunophenotype and the presence of single nucleotide polymorphism G80A reduced folate carrier-1 were associated with a shorter relapse-free survival and overall survival., Conclusions: The presence of G80A single nucleotide polymorphism at reduced folate carrier-1 gene in acute lymphoblastic leukemia patients was associated with a poorer prognosis.

The aim of the present study was to demonstrate that ribavirin, a known inhibitor of eIF4E and inosine 5'-phosphate dehydrogenase (IMPDH), also inhibits histone methyltransferase zeste homolog 2 (EZH2). A computational searching revealed that ribavirin has a high structural similarity to 3-deazaneplanocin A (DZNep). The growth inhibitory effects of ribavirin as well as its effects upon epigenetic enzymes were evaluated in various cancer cell lines. siRNA assays were used to downregulate eIF4E, EZH2 and IMPDH to determine the contribution of these targets to the growth inhibitory effects of ribavirin. Ribavirin decreased EZH2 expression, inhibited histone methyltransferase activity and decreased H3K27 trimethylation. Ribavirin induced variable growth inhibition in a number of cell lines and downregulation of the targets, EZH2, eIF4E and IMPDH1 and 2 by siRNA led to comparable growth inhibition while no significant further reduction in viability was observed when siRNA transfected cells were treated with ribavirin. The results showed that ribavirin inhibits these cancer targets and should thus be studied for cancer therapy.

Carcinogenesis is driven by the accumulation of mutations and abnormal DNA methylation patterns, particularly the hypermethylation of tumor‑suppressor genes. Changes in genomic DNA methylation patterns are established by the DNA methyltransferases (DNMTs) family: DNMT1, DNMT3a and DNMT3b. The DNMTs are known to be overexpressed in tumors. However, when the DNMTs expression profile is altered in earlier stages of carcinogenesis remains to be elucidated. The resistant hepatocyte model (RHM) allows the analysis of the hepatocellular carcinoma (HCC) from the formation of altered cell foci to the appearance of tumors in rats. To investigate the DNMTs expression in this model, we first observed that timp3, rassf1a and p16 genes became methylated during cancer development by methylation‑specific PCR (MSP) and the bisulphate sequencing PCR (BSP) of timp3. The differential expression at the RNA and protein level of the three DNMTs was also assessed. dnmt1 expression was higher in tumors than in normal and early cancer stages. However, no evident overexpression of the enzyme was identified by immunohistochemistry. By contrast, DNMT3a and DNMT3b were consistently subexpressed in tumors. In the present study, we report a carcinogenesis model that does not feature the overexpression of DNMT1 but exhibits a transient expression of DNMT3a and DNMT3b.

Introduction: DNA methylation (DNMTi) and histone deacetylase inhibitors (HDACi) are in development for cancer therapy. So far, four epigenetic drugs are approved for myelodysplastic syndrome (MDS) and cutaneous T-cell lymphoma (CTCL). The combination of hydralazine-valproate (TRANSKRIP(™)) is being repositioned as an oral DNMT and HDAC inhibitor., Areas Covered: Brief discussion on the current status of epigenetic drugs and studies published on the preclinical and clinical development of the hydralazine-valproate combination., Expert Opinion: Drug repositioning is a strategy for prompt and cost-efficient drug discovery. There is evidence that combining DNMTi with HDACi would be more efficacious than administering each agent on its own. Hydralazine-valproate is safe when used alone or in combination with chemotherapy or chemoradiation. The fact that both drugs are orally administered is another advantage over current epigenetic drugs. This combination is promising but larger studies are needed. Among these, the randomized Phase III trials in advanced and in locally advanced cervical cancer combined with chemotherapy and cisplatin-radiation respectively, would eventually confirm its efficacy. Studies on MDS and CTCL would also eventually prove the efficacy of hydralazine valproate so that in the coming years hydralazine-valproate could have a role in cancer epigenetic therapy.

Excessive weight gain and obesity are major public health concerns. Childhood obesity is growing at an alarming rate. Polymorphisms in the Calpain-10 gene and the reduced expression of this gene in muscle cells and adipocytes have been associated with an increased risk of type 2 diabetes mellitus in several populations. In the present study, we explored the contribution of Calpain-10 in the development of metabolic impairment in childhood. We evaluated the presence of risk polymorphisms in the CAPN10 gene (SNP-44, SNP-43, InDel-19 and SNP-63) and the associated changes in the Calpain-10 mRNA levels in a pediatric population. A total of 161 Mexican children between 4 and 18 years old were included in this study. This population was classified into three groups according to international growth references: healthy weight (HW), overweight (OW) and obese (OB). Association studies of the anthropometric data, clinical values, genotyping and expression assays showed a decrease in the Calpain-10 mRNA and protein expression in the OW and OB groups with respect to the HW group. This decrease in the Calpain-10 mRNA expression was more evident in individuals homozygous for SNP-44 (T/T) and InDel-19 (3/3), alone (p<0.001 and p=0.015, respectively) or in combination (p=0.017). These polymorphisms were also associated with elevated BMI, weight percentiles, z-scores, waist circumferences, fasting glucose levels and beta cell functions in the OW and OB groups (p<0.05). Moreover, our results indicate a statistically significant decrease in the expression of the 75-kDa Calpain-10 isoform in the OW+OB group. The presence of polymorphisms and alterations in the expression of the CAPN10 gene at early ages might result in metabolic impairment in adulthood and should be further investigated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Unlabelled: The epigenetic drugs hydralazine and valproate were administered in a compassionate manner to 8 patients with chronic myeloid leukemia (CML) refractory to imatinib. Two patients had a complete hematologic response (25%),1 major cytogenetic response, 1 complete cytogenetic response (25% any cytogenetic response), and 3 (37.5%)stable disease. No grade 3 or 4 toxicity was observed. These results show the ability of epigenetic therapy to revert imatinib resistance., Background: Epigenetic alterations participate in the development of acquired resistance to imatinib, hence, the DNA methylation, and histone deacetylase inhibitors hydralazine and valproate, respectively, has the potential to overcome it., Patient and Methods: A series of 8 patients with chronic myeloid leukemia (CML) refractory to imatinib mesylate with no access to second-generation tyrosine kinase inhibitors were treated with hydralazine and valproate in a compassionate manner. Clinical efficacy and safety of these drugs added to imatinib mesylate were evaluated., Results: Two patients were in the blast phase, 5 were in the accelerated phase, and 1 was in the chronic phase. All the patients continued with the same dose of imatinib that they had been receiving at the time of development of resistance, with a median dose of 600 mg daily (range, 400-800 mg). The median time from diagnosis of CML to the start of hydralazine and valproate was 53.6 months (range, 19-84 months). Two (25%) patients had a complete hematologic response, one (12.5%) had an major cytogenetic response, and one (12.5%) had a complete cytogenetic response. Three (37.5%) patients had stable disease, and only one (12.5%) patient failed to respond. At a median follow-up time of 18 months (range, 3-18 months), the median survival had not been reached, and the projected overall survival was 63%. All the patients had mild neurologic toxicity, including distal tremor and somnolence. No grade 3 or 4 toxicity was observed., Conclusions: Our results suggest that the epigenetic drugs hydralazine and valproate revert the resistance to imatinib in patients with CML., (Copyright © 2012 Elsevier Inc. All rights reserved.)

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.

Background: Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells., Methodology/principal Findings: The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity., Conclusions/significance: Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.

N-acetyltransferase 2 (NAT2) catalyzes the bioactivation and/or detoxification of drugs and carcinogens. The aim of this study was to establish the correlation between the NAT2 genotype and the acetylating phenotype in a Mexican population using sulfamethazine as a probe. From a total of 122 individuals, 73 (59.8%) were slow and 49 (40.2%) were fast acetylators. Eleven individuals (9%) had the wild-type genotype (NAT2*4/NAT2*4). The most frequent genotype was NAT2*4/NAT2*5B observed in 20.66% of individuals. In conclusion, our results show that an accurate prediction of the acetylation phenotype by genotyping can be achieved in around half of the population. Further studies with a larger number of individuals are required to establish correlations between phenotype and genotype in half of that patients having a genotype combined with slow/rapid alleles.

The reversing of epigenetic aberrations using the inhibitors of DNA methylation and histone deacetylases may have therapeutic value in cervical cancer. This is a randomized phase III, placebo-controlled study of hydralazine and valproate (HV) added to cisplatin topotecan in advanced cervical cancer. Patients received hydralazine at 182 mg for rapid, or 83 mg for slow acetylators, and valproate at 30 mg/kg, beginning a week before chemotherapy and continued until disease progression. Response, toxicity, and PFS were evaluated, and 36 patients (17 CT + HV and 19 CT + PLA) were included. The median number of cycles was 6. There were four PRs to CT + HV and one in CT + PLA. Stable disease in five (29%) and six (32%) patients, respectively, whereas eight (47%) and 12 (63%) showed progression (P = 0.27). At a median follow-up time of 7 months (1-22), the median PFS is 6 months for CT + PLA and 10 months for CT + HV (P = 0.0384, two tailed). Although preliminary, this study represents the first randomized clinical trial to demonstrate a significant advantage in progression-free survival for epigenetic therapy over one of the current standard combination chemotherapy in cervical cancer. Molecular correlates with response and survival from this trial are pending to analyze.

Cervical cancer (CC) is the second most common cancer in Mexican women. Human papillomavirus (HPV) infection is necessary but not sufficient for CC development. Furthermore, genetic factors as polymorphisms could be important susceptibility factors. Controversial results regarding TP53 polymorphisms specifically in codon 72 of CC have been reported. In the present work, the exon 4 sequence of TP53 gene in CC and healthy Mexican-mestizo women were analyzed. A group of 111 women with CC and 126 healthy women (control) were included. Peripheral blood cells for polymorphism analysis and cervical scrape for HPV detection were used. PCR of exon 4 of TP53 were subjected to denaturing high-performance liquid chromatography (DHPLC) analysis and sequencing. HPV detection was subjected to PCR and sequencing. The statistical analysis was carried out using the Arlequin software. Codon 72 Arg/Arg was the most common SNP detected, and Hardy-Weinberg analysis showed equilibrium in control and CC samples (P>0.05). Wild type sequence of TP53 exon 4 was detected in 66 and 57% in control and CC samples, respectively. For codon 72 Arg/Arg, differences between control and CC women were found (P=0.043). An association between HPV 16/18 infection and 72 Arg/Arg in woman with CC was found (P=0.026). Haplotype GC (codon 36 and 72) was statistically significantly associated with CC (P=0.011). HPV 16 was the most common viral type. Codon 72 Arg/Arg is the most common polymorphism in the Mexican population and could be associated to HPV 16 and/or HPV 18 infection in CC.