Advances in the Properties of Incomptine A: Cytotoxic Activity and Downregulation of Hexokinase II in Breast Cancer Cell Lines.

Breast cancer treatments are limited by the cancer subtype and its selectivity towards tumor cells, hence the importance of finding compounds that increase the survival of healthy cells and target any subtype. Incomptine A (IA) is a sesquiterpene lactone with demonstrated cytotoxic activity. In this study, through in vitro assays, it was observed that IA has similar cytotoxic activity between the subtypes triple negative, HER2+, and luminal A of the breast cancer cell lines. IA cytotoxic activity is higher in cancer than in nontumorigenic cells, and its selectivity index for cancer cells is more than that of the drug doxorubicin. Molecular docking and its in silico comparison with the 2-Deoxyglucose inhibitor suggest that IA could bind to Hexokinase II (HKII), decreasing its expression. Since we did not find changes in the expression of the glycolytic pathway, we suppose that IA could affect the antiapoptotic function of HKII in cancer cells. The IA-HKII union would activate the voltage-gated anion channel 1 (VDAC1), resuming apoptosis. Therefore, we suggest that IA could be used against almost any subtype and that its cytotoxic effect could be due to the reactivation of apoptosis in breast cancer cells.

The Transcriptomic Landscape of Pediatric Astrocytoma.

Central nervous system tumors are the most common solid neoplasia during childhood and represent one of the leading causes of cancer-related mortality. Tumors arising from astrocytic cells (astrocytomas) are the most frequently diagnosed, and according to their histological and pathological characteristics, they are classified into four categories. However, an additional layer of molecular classification considering the DNA sequence of the tumorigenesis-associated genes IDH1/2 and H3F3A has recently been incorporated into the classification guidelines. Although mutations in H3F3A are found exclusively in a subtype of grade IV pediatric astrocytoma, mutations in IDH1/2 genes are very rare in children under 14 years of age. The transcriptomic profiles of astrocytoma in adults and children have been extensively studied. However, there is scarce information on these profiles in pediatric populations considering the status of tumorigenesis-associated genes. Therefore, here we report the transcriptomic landscape of the four grades of pediatric astrocytoma by RNA sequencing. We found several well-documented biological functions associated with the misregulated genes in the four grades of astrocytoma, as well as additional biological pathways. Among the four grades of astrocytoma, we found shared misregulated genes that could have implications in tumorigenesis. Finally, we identified a transcriptional signature for almost all grades of astrocytoma that could be used as a transcription-based identification method.

Expanding the Study of the Cytotoxicity of Incomptines A and B against Leukemia Cells.

Heliangolide-type sesquiterpene lactones (HTSLs) are phytocompounds with several pharmacological activities including cytotoxic and antitumor activity. Both bioactivities are related to an α-methylene-γ-lactone moiety and an ester group on carbon C-8 in the sesquiterpene lactone (SL) structure. Two HTSLs, incomptines A (AI) and B (IB) isolated from Decachaeta incompta, were evaluated for their cytotoxic activity on three leukemia cell lines: HL-60, K-562, and REH cells. Both compounds were subjected to a molecular docking study using target proteins associated with cancer such as topoisomerase IIα, topoisomerase IIß, dihydrofolate reductase, methylenetetrahydrofolate dehydrogenase, and Bcl-2-related protein A1. Results show that IA and IB exhibit cytotoxic activity against all cell lines used. The CC50 value of IA was 2-4-fold less than etoposide and methotrexate, two anticancer drugs used as positive controls. The cytotoxic activity of IB was close to that of etoposide and methotrexate. The molecular docking analysis showed that IA and IB have important interaction on all targets used. These findings suggest that IA and IB may serve as scaffolds for the development of new treatments for different types of leukemia.

A novel method of male sex identification of human ancient skeletal remains.

Sex identification of ancient individuals is important to understand aspects of the culture, demographic structure, religious practices, disease association, and the history of the ancient civilizations. Sex identification is performed using anthropometric measurements and molecular genetics techniques, including quantification of the X and Y chromosomes. These approaches are not always reliable in subadult, or fragmented, incomplete skeletons or when the DNA is highly degraded. Most of the methods include the identification of the male and female sexes, but the absence of a specific marker for the males does not mean that the sample obtained was from a female. This study aims (1) to identify new male-specific regions that allow male identification; (2) to contrast the effectiveness of these markers against AMELX/AMELY and anthropometric measurement procedures; and (3) to test the efficacy of these markers in archaeological samples. For the first two aims, we used known sex samples, and for the third aim, we used samples from different archaeological sites. A novel molecular technique to identify male-specific regions by amplification of TTTY7, TSPY3, TTTY2, and TTTY22 genes of the human Y chromosome was developed. The results showed amplification of the specific DNA regions of Y chromosome in male individuals, with no amplification being observed in any of the female samples, confirming their specificity for male individuals. This approach complements the current procedures, such as the AMELX/AMELY test and anthropometric principle.

Incomptine A Induces Apoptosis, ROS Production and a Differential Protein Expression on Non-Hodgkin's Lymphoma Cells.

Sesquiterpene lactones are of pharmaceutical interest due their cytotoxic and antitumor properties, which are commonly found within plants of several genera from the Asteraceae family such as the Decachaeta genus. From Decachaeta incompta four heliangolide, namely incomptines A-D have been isolated. In this study, cytotoxic properties of incomptine A (IA) were evaluated on four lymphoma cancer cell lines: U-937, Farage, SU-DHL-2, and REC-1. The type of cell death induced by IA and its effects on U-937 cells were analyzed based on its capability to induce apoptosis and produce reactive oxygen species (ROS) through flow cytometry with 4',6-diamidino-2-phenylindole staining, dual annexin V/DAPI staining, and dichlorofluorescein 2',7'-diacetate, respectively. A differential protein expression analysis study was carried out by isobaric tags for relative and absolute quantitation (iTRAQ) through UPLC-MS/MS. Results reveal that IA exhibited cytotoxic activity against the cell line U-937 (CC50 of 0.12 ± 0.02 µM) and the incubation of these cells in presence of IA significantly increased apoptotic population and intracellular ROS levels. In the proteomic approach 1548 proteins were differentially expressed, out of which 587 exhibited a fold-change ≥ 1.5 and 961 a fold-change ≤ 0.67. Most of these differentially regulated proteins are involved in apoptosis, oxidative stress, glycolytic metabolism, or cytoskeleton structuration.

Antilymphoma Effect of Incomptine A: In Vivo, In Silico, and Toxicological Studies.

Incomptine A (IA) is a sesquiterpene lactone isolated from Decachaeta incompta that induces apoptosis, reactive oxygen species production, and a differential protein expression on the U-937 (diffuse histiocytic lymphoma) cell line. In this work, the antitumor potential of IA was investigated on Balb/c mice inoculated with U-937 cells and through the brine shrimp lethality (BSL) test. Furthermore, IA was subjected to molecular docking study using as targets proteins associated with processes of cancer as apoptosis, oxidative stress, and glycolytic metabolism. In addition to determining the potential toxicity of IA in human, its acute toxicity was performed in mice. Results reveals that IA showed high antilymphoma activity and BSL with an EC50 of 2.4 mg/kg and LC50 16.7 µg/mL, respectively. The molecular docking study revealed that IA has strong interaction on all targets used. In the acute oral toxicity, IA had a LD50 of 149 mg/kg. The results showed that the activities of IA including antilymphoma activity, BSL, acute toxicity, and in silico interactions were close to the methotrexate, an anticancer drug used as positive control. These findings suggest that IA may serve as a candidate for the development of a new drug to combat lymphoma.

Antitumor Potential of Annona muricata Linn. An Edible and Medicinal Plant in Mexico: In Vitro, In Vivo, and Toxicological Studies.

Annona muricata (Am) is a plant used in traditional Mexican medicine to treat cancer. In this study, ethanol extracts of Am collected in Acapulco and Tecpan from Guerrero state were evaluated orally on Balb/c mice inoculated with 4T1 cells, for cytotoxic activity (CA) on 4T1 cells, in brine shrimp lethality assay (BSLA), and for acute oral toxicity in mice. In addition, ethanol extracts were subjected to high-performance liquid chromatography (HPLC) with diode array detection. Results showed that the extracts collected in December in Acapulco (AcDe) and Tecpan (TeDe) exhibited the most significant antitumor and cytotoxic activity. In the BSLA, the most important effect was observed in the extracts from Acapulco and Tecpan collected in June (AcJu) and August (TeAg), respectively. The samples from Acapulco (AcJu, and AcAg) and Tecpan (TeJu and TeAg) showed the highest toxicity. The analysis of the extracts, AcDe and TeDe, by HPLC revealed that flavonoids, rutin, narcissin, and nicotinflorin were the major components. These findings suggest that extracts from Am collected in Acapulco and Tecpan in the month of December may be an important source to obtain flavonoid glycosides with anticancer potential specifically against breast cancer. This also supports the use of Am to treat cancer in Mexican traditional medicine.

Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

BACKGROUND: Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown. OBJECTIVE: To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound. METHODS: We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog. RESULTS: KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog. CONCLUSIONS: KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.

Correlation of the protein expression of GRP78 and BIK/NBK with prognostic markers in patients with breast cancer and neoadjuvant chemotherapy.

The objective of this study was to determine the frequency of expression of GRP78 and BIK/NBK proteins, as well as to evaluate their correlation with prognostic factors for clinical use in patients with locally advanced breast cancer (LABC) or preoperative chemotherapy (PC). An observational, analytical and retrospective study that evaluated the expression of BIK/NBK and GRP78 by means of immunohistochemistry in paraffin-embedded tumour tissue samples obtained before the start of PC was executed. GRP78 was positive in 93.3% and BIK/NBK was positive in 40% of the samples analysed. Correlation of GRP78 expression with Ki67 expression (Rho = 0.732, p = .039), and of BIK/NBK expression with Ki67 expression (Rho = 0.777, p = .023) and number of residual lymph nodes (Rho = 0.619, p = .014) was observed. Given the correlation observed, association studies of the expression of GRP78 and BIK/NBK with clinical and pathological response, recurrence and survival should be carried out in a greater number of patients with LABC or PC.Impact StatementWhat is already known on this subject? There are reports of the overexpression of GRP78 in various types of cancer cell lines and tumour biopsies, including breast cancer, with participation in resistance to adjuvant chemotherapeutic agents (topoisomerase inhibitors including Adriamycin). On the other hand, BIK/NBK has been observed expressed associated with poor outcomes in breast cancer. There are no studies of the expression of both biomarkers in patients with preoperative chemotherapy, including locally advanced breast cancer (LABC) or their correlation with prognosis markers of clinical use.What the results of this study add? We observed a correlation of higher expression percentage of GRP78 and BIK/NBK with higher expression of Ki67, a marker of poor prognosis in breast cancer, in addition to a significant correlation of higher percentage of BIK/NBK expression with a greater number of residual nodes in patients with LABC or PC.What the implications are of these findings for clinical practice and/or further research? There is a potential prognostic value of GRP78 and BIK/NBK in the pathological response that merits further study addressing their interaction with other proteins, their cellular location in relation to the clinical stages of breast cancer, and their association with prognosis (clinical and pathological response, recurrence, disease free period and survival) in a greater number of patients with LABC or PC.

Association between promoter hypermethylation of the DACT2 gene and tumor stages in breast cancer.

PURPOSE: Aberrant methylation of CpG islands in the promoter is a hallmark of cancer, leading to transcriptional silencing of tumor suppressor genes. The aim of this work was to evaluate the promoter methylation status of the DACT2 gene in breast cancer (BC) tissue and to analyze its possible effect on tumor type or grade. METHODS: CpG island from the DACT2 promoter in region -240 to -14 from transcriptional start site (TSS) were obtained. Through the use of sodium bisulfite DNA conversion analysis, followed by detection with MSP (methylation specific PCR), we analyzed 79 BC and 15 adjacent healthy samples. RESULTS: T he c ases a nalyzed w ere i n s tage I ( 2.5%), I I (38%), or III (59.5%). The most frequent tumor type was invasive ductal carcinoma (71.4%). Methylation analysis comparing tumor tissues with adjacent non-cancerous tissues showed statistical significance. Methylation was observed in 32.9% (26/79) of the samples; no methylation was found in adjacent healthy tissue. DACT2 methylation was associated with tumor stage I-II (p=0.03) and stage III (p=0.004). CONCLUSION: An association was found of DACT2 promoter methylation with advanced tumor stages. This gene has been suggested as a potential biomarker, however, more investigation is required to validate this function.

Antidiarrheal Thymol Derivatives from Ageratina glabrata. Structure and Absolute Configuration of 10-Benzoyloxy-8,9-epoxy-6-hydroxythymol Isobutyrate.

Chemical investigation of the leaves from Ageratina glabrata yielded four new thymol derivatives, namely: 10-benzoyloxy-8,9-dehydro-6-hydroxythymol isobutyrate (4), 10-benzoyloxy-8,9-dehydrothymol (5), 10-benzoyloxythymol (6) and 10-benzoyloxy-6,8-dihydroxy-9-isobutyryl-oxythymol (7). In addition, (8S)-10-benzoyloxy-8,9-epoxy-6-hydroxythymol isobutyrate (1), together with other two already known thymol derivatives identified as 10-benzoyloxy-8,9-epoxy-6-methoxythymol isobutyrate (2) and 10-benzoyloxy-8,9-epoxythymol isobutyrate (3) were also obtained. In this paper, we report the structures and complete assignments of the ¹H and (13)C-NMR data of compounds 1-7, and the absolute configuration for compound 1, unambiguously established by single crystal X-ray diffraction, and evaluation of the Flack parameter. The in vitro antiprotozoal assay showed that compound 1 and its derivative 1a were the most potent antiamoebic and antigiardial compounds. Both compounds showed selectivity and good antiamoebic activity comparable to emetine and metronidazole, respectively, two antiprotozoal drugs used as positive controls. In relation to anti-propulsive effect, compound 1 and 1a showed inhibitory activity, with activities comparable to quercetin and compound 9, two natural antipropulsive compounds used as positive controls. These data suggest that compound 1 may play an important role in antidiarrheal properties of Ageratina glabrata.

Antiamoebic and Antigiardial Activity of Clerodane Diterpenes from Mexican Salvia Species Used for the Treatment of Diarrhea.

Terpenoids from Salvia species have been identified to possess biological properties as antiprotozoal agents. Here, we evaluated the antiamoebic and antigiardial activities of 14 known clerodane and modified clerodane-type diterpenes isolated from five Mexican Salvia species against Entamoeba histolytica and Giardia lamblia, and analyzed the effects of the functionalities in decalin ring or in the whole clerodane framework to visualize the structural requirements necessary to produce an antiprotozoal activity. Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 µM for E. histolytica and of 28.2 µM in the case of G. lamblia. In this context it may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea.

Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women.

BACKGROUND: Breast cancer is a complex multifactorial genetic disease. Among other factors, race and, to an even greater extent, viruses are known to influence the development of this heterogeneous disease. It has been reported that MMTV-like (HMTV) gene sequences with a 90 to 98% homology to mouse mammary tumor virus are found in several populations with a prevalence range of 0 to 74%. In the Mexican population, 4.2% of patients with breast cancer exhibit the presence of HMTV (MMTV-like) sequences. The aim of this study was to evaluate the presence and current prevalence of retroviral HMTV (MMTV-like) sequences in breast cancer in Mexican women. METHODS: We used nested PCR and real-time PCR with a TaqMan probe. As a positive control, we used the C3H MMTV strain inserted into pBR322 plasmid. To confirm that we had identified the HMTV sequences, we sequenced the amplicons and compared these sequences with those of MMTV and HMTV (GenBank AF033807 and AF346816). RESULTS: A total of 12.4% of breast tumors were HMTV-positive, and 15.7% of the unaffected tissue samples from 458 patients were HMTV-positive. A total of 8.3% of the patients had both HMTV-positive tumor and adjacent tissues. The HMTV-positive samples presented 98% similarity to the reported HMTV sequence. CONCLUSIONS: These results confirm that the HMTV sequence is present in breast tumors and non-affected tissues in the Mexican population. HMTV should be considered a prominent causative agent of breast cancer.

Prevalence of anemia and its impact on the state of frailty in elderly people living in the community: SADEM study.

Anemia represents a global health problem that negatively impacts quality of life in elderly population; however, its impact on the geriatric syndrome of frailty is unclear. We examined the prevalence of anemia among elderly and sought a relationship between hemoglobin and the phenotype of frailty. Baseline hemoglobin quintiles and anemia were assessed in relation to frailty status in a prospective study with 1,933 older community-dwelling adults enrolled in the Study on Aging and Dementia in Mexico (SADEM). Logistic regression was used to model the relationship between frailty and Hb, adjusting for risk factors of frailty, sociodemographic data, cognitive decline, chronic diseases, and some risky habits. Prevalence of frailty was 8.3 %. Frailty risk was highest at the lowest hemoglobin quintile (<14.3 g/dL for men; <13.3 g/dL for women), and 160 (8.3 %) were anemic (<13 g/dL for men; <12 g/dL for women). The relationship between frailty and Hb levels, adjusted for age and sex, observed in the first and fifth quintiles, compared with the fourth quintile, were 1.53 (95 % confidence interval (CI), 1.46-1.60) and 1.05 (95 % CI, 1.01-1.15). After multivariate adjustment, the odds ratios (ORs) were 1.23 (95 % CI, 1.17-1.13) and 1.06 (95 % CI, 1.01-1.11). The association was not diminished by risk factors for frailty (body mass index (BMI), comorbidity, cognitive decline, smoking, alcohol consumption, etc.). In community-dwelling older adults, low hemoglobin concentrations and anemia were independently associated with increased frailty risk. This suggests that mild anemia and low Hb levels are independent, modifiable risk factors for frailty.

Polymorphism 677C → T MTHFR gene in Mexican mothers of children with complex congenital heart disease.

Congenital heart defects (CHD) are the third leading cause of death in children <1 year of age in Mexico where there is a high prevalence of the 677C → T polymorphism of the MTHFR gene. This is important because the homozygous 677T/T MTHFR gene and deficiency of folic acid (FA) intake have been associated with CHD. Our objective was to analyze the possible association between the genotype 677T/T of the MTHFR gene and supplementation of FA in Mexican women with the presence of complex CHD in their children. We analyzed genotypes of 31 mothers of children with complex CHD (group I) and 62 mothers of healthy children (group II) and investigated FA supplementation during pregnancy in both study groups. Allele frequencies in group I were 41.9 % for C and 58.1 % for T and 22.6 % for genotype frequencies CC, 38.7 % for CT, and 38.7 % for TT. Allele frequencies in group II were 63.7 % for C and 36.3 % for T and 38.7 % for genotype frequencies CC, 50 % for CT and 11.3 % for TT. Both populations are in Hardy-Weinberg equilibrium. Odds ratio for having a child with a complex CHD was 5.9, p = 0.008 (95 % CI 1.67; 20.63) for the TT genotype. FA supplementation at any time during pregnancy was 90.3 and 87.9 % in groups II and I respectively (p > 0.05). Association was found between the maternal genotype (677/TT MTHFR) with the presence of complex CHD in their offspring. No differences in FA supplementation during any stage were found between groups.

Variants in the Control Region of Mitochondrial Genome Associated with type 2 Diabetes in a Cohort of Mexican Mestizos.

BACKGROUND: According to the International Diabetes Federation, Mexico is seventh place in the prevalence of type 2 diabetes (T2D) worldwide. Mitochondrial DNA variant association studies in multifactorial diseases like T2D are scarce in Mexican populations. AIM OF THE STUDY: The objective of this study was to analyze the association between 18 variants in the mtDNA control region and T2D and related metabolic traits in a Mexican mestizo population from Mexico City. METHODS: This study included 1001 participants divided into 477 cases with T2D and 524 healthy controls aged between 42 and 62 years and 18 mtDNA variants with frequencies >15%. RESULTS: Association analyses matched by age and sex showed differences in the distribution between cases and controls for variants m.315_316insC (p = 1.18 × 10-6), m.489T>C (p = 0.009), m.16362T>C (p = 0.001), and m.16519T>C (p = 0.004). The associations between T2D and variants m.315_316ins (OR = 6.13, CI = 3.42-10.97, p = 1.97 × 10-6), m.489T>C (OR = 1.45, CI = 1.00-2.11, p = 0.006), m.16362T>C (OR = 2.17, CI = 1.57-3.00, p = 0.001), and m.16519T>C (OR = 1.69, CI = 1.23-2.33, p = 0.006) were significant after performing logistic regression models adjusted for age, sex, and diastolic blood pressure. Metabolic traits in the control group through linear regressions, adjusted for age, sex and BMI, and corrected for multiple comparisons showed nominal association between glucose and variants m.263A>G (p <0.050), m.16183A>C (p <0.010), m.16189T>C (p <0.020), and m.16223C>T (p <0.024); triglycerides, and cholesterol and variant m.309_310insC (p <0.010 and p <0.050 respectively); urea, and creatinine, and variant m.315_316insC (p <0.007, and p <0.004 respectively); diastolic blood pressure and variants m.235A>G (p <0.016), m.263A>G (p <0.013), m.315_316insC (p <0.043), and m.16111C>T (p <0.022). CONCLUSION: These results demonstrate a strong association between variant m.315_316insC and T2D and a nominal association with T2D traits.

Understanding the Anti-Diarrhoeal Properties of Incomptines A and B: Antibacterial Activity against Vibrio cholerae and Its Enterotoxin Inhibition.

Incomptines A (IA) and B (IB) are two sesquiterpene lactones with antiprotozoal, antibacterial, cytotoxic, antitumor, spermicidal, and phytotoxic properties. The antibacterial activity of IA and IB against bacteria causing diarrhoea have been reported; however, no information is available regarding their antibacterial activity on Vibrio cholerae. In this work, both compounds were evaluated for their anti-diarrhoeal potential using the bacterium V. cholerae, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on cholera toxin, and a cholera toxin-induced diarrhoea model in male Balb/c mice. In addition, a molecular docking study was carried out to understand the interaction of IA and IB with cholera toxin. In terms of antibacterial activity, IB was three times more active than IA on V. cholerae. In the case of SDS-PAGE analysis and the in silico study, IA was most effective, revealing its potential binding mode at a molecular level. In terms of anti-diarrhoeal activity, IA was 10 times more active than IB and racecadotril, an antisecretory drug used as positive control; the anti-diarrheal activity of IB was also closer than racecadotril. The results obtained from in vitro, in vivo, and computational studies on V. cholerae and cholera toxin support the potential of IA and IB as new anti-diarrhoeal compounds.

A New Approach to Identify the Methylation Sites in the Control Region of Mitochondrial DNA.

Mitochondrial DNA (mtDNA) methylation has the potential to be used as a biomarker of human development or disease. However, mtDNA methylation procedures are costly and time-consuming. Therefore, we developed a new approach based on an RT-PCR assay for the base site identification of methylated cytosine in the control region of mtDNA through a simple, fast, specific, and low-cost strategy. Total DNA was purified, and methylation was determined by RT-PCR bisulfite sequencing. This procedure included the DNA purification, bisulfite treatment and RT-PCR amplification of the control region divided into three subregions with specific primers. Sequences obtained with and without the bisulfite treatment were compared to identify the methylated cytosine dinucleotides. Furthermore, the efficiency of C to U conversion of cytosines was assessed by including a negative control. Interestingly, mtDNA methylation was observed mainly within non-Cphosphate- G (non-CpG) dinucleotides and mostly in the regions containing regulatory elements, such as OH or CSBI, CSBII, and CSBIII. This new approach will promote the generation of new information regarding mtDNA methylation patterns in samples from patients with different pathologies or that are exposed to a toxic environment in diverse human populations.

Reduced mitochondrial DNA copy number is associated with the haplogroup, and some clinical features of breast cancer in Mexican patients.

Mitochondrial DNA (mtDNA) copy number and mitochondrial DNA haplogroups have been associated with different types of cancer, including breast cancer, because they alter cellular energy metabolism. However, whether mtDNA copy number or haplogroups are predictors of oxidative stress-related risks in human breast cancer tissue in Mexican patients remains to be determined. Using quantitative real-time PCR assays and sequencing of the mtDNA hypervariable region, analysis of mtDNA copy numbers in 82 breast cancer tissues (BCT) and matched normal adjacent tissues (NAT) was performed to determine if copy number correlated with clinical features and Amerindian haplogroups (A2, B2, B4, C1 and D1) . The results showed that the mtDNA copy number was significantly decreased in BCT compared with NAT (p = 0.010); it was significantly decreased in BCT and NAT in women > 50 years of age, compared with NAT in women < 50 years of age (p = 0.032 and p = 0.037, respectively); it was significantly decreased in NAT and BCT in the postmenopausal group and in BCT in the premenopausal group compared with NAT in the premenopausal group (p = 0.011, p = 0.010 and, p = 0.018; respectively); and it was also significantly decrease in members of the BCT group classified as having invasive ductal carcinoma I-III (IDC-I, IDC-II and IDC-III) and IDC-II for NAT compared to IDC-I of NAT (p = 0.025, p = 0.022 and p = 0.031 and p = 0.020; respectively). The mtDNA copy number for BCT from patients with haplogroup B2 was decreased compared to patients with haplogroup D1 (p = 0.01); for BCT from patients with haplogroup C1 was also decreased compare with their NAT counterpart (p = 0.006) and with BCT patients belonging to haplogroups A2 and D1 (p = 0.01 and p = 0.03; respectively). In addition, the mtDNA copy number was decrease in the sequences with three deletions relative to the rCRS at nucleotide positions A249del, A290del and A291del, or C16327T polymorphism with the same p = 0.019 for all four variants. Contrary, the copy number increased in sequences containing C16111T, G16319A or T16362C polymorphisms (p = 0.021, =0.048, and = 0.001; respectively). In conclusion, a decrease in the copy number of mtDNA in BCT compared with NAT was shown by the results, which suggests an imbalance in oxidative phosphorylation (OXPHOS) that can affect the apoptosis pathway and cancer progression. It was also observed an increase of the copy number in samples with specific polymorphisms, which may be a good sign of favourable prognosis.

Pediatric ependymoma: GNAO1, ASAH1, IMMT and IPO7 protein expression and 5-year prognosis correlation.

OBJECTIVE: The aim of this work was to evaluate a pediatric ependymoma protein expression that may be useful as a molecular biomarker candidate for prognosis, correlated with clinical features such as age, gender, histopathological grade, ependymal tumor recurrence and patient survival. PATIENTS AND METHODS: Immunohistochemistry assays were performed for GNAO1, ASAH1, IMMT, IPO7, Cyclin D1, P53 and Ki-67 proteins. Kaplan-Meier and Cox analysis were performed for age, gender, histopathological grade, relapse and survival correlation. RESULTS: We found that three proteins correlate with histopathological grade and relapse; two proteins correlate with survival; one protein does not correlate with any clinical feature. CONCLUSION: Our results suggest that, out of the proteins analyzed, five may be considered suitable prognostic biomarkers and one may be considered a predictive biomarker for response to treatment of pediatric ependymoma.