Juan Molina-Guarneros

Decrease of cyclin D1 in the human lung adenocarcinoma cell line A-427 by 7-hydroxycoumarin

Coumarin in vivo has antitumor activity in various types of cancer. In vitro, coumarin and 7-hydroxycoumarin, its major biotransformation product in humans, inhibit the proliferation of several human tumor cell lines. The molecular mechanisms of these effects are unknown. To gain information about these mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin in the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) cell proliferation; (ii) cell cycle progression; and (iii) expression of cyclins D1, E and A. The inhibitory concentrations 50 (IC(50)) of both compounds were estimated by cytostatic assays of tetrazolium (MTT) reduction. The effects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins D1, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal antibodies for each cyclin. The IC(50) (+/-S.E.M. n=3) of 7-hydroxycoumarin and coumarin at 72 h exposure, were 100+/-4.8 and 257+/-8.8 microg/ml, respectively. 7-Hydroxycoumarin at the concentration of 160 microg/ml (1 mM), inhibited the G(1)/S transition of the cell cycle, an action consistent with the cytostatic effect. No significant decreases of cyclins E and A were observed. In contrast, cyclin D1 significantly decreased, which appears to indicate an action of 7-hydroxycoumarin in early events of phase G(1). However, messenger RNA of cyclin D1, assayed by RT-PCR, did not change. This suggests a posttranscriptional effect. The effects of coumarin were not significant. Cyclin D1 is overexpressed in many types of cancer, and its inhibition has been proposed as a pharmacological and therapeutic target for novel antitumor agents. Knowledge of the decrease of cyclin D1 by 7-hydroxycoumarin may lead to its use in cancer therapy, as well as to the development of more active compounds.

Activation of Protein Kinase A Stimulates the Progesterone-Induced Calcium Influx in Human Sperm Exposed to the Phosphodiesterase Inhibitor Papaverine

Progesterone induces a fast transient calcium influx in human sperm though the activation of nongenomic receptors. During sperm capacitation, a complex process required for sperm to be able to fertilize the egg, the calcium influx induced by progesterone is enhanced. Sperm capacitation is mediated by an increase in cAMP content and subsequent protein kinase A (PKA) activation. In this work, we examined the effect of increasing intracellular cAMP on the calcium influx induced by progesterone in noncapacitated human sperm. To do this, sperm were exposed to the phosphodiesterase inhibitor papaverine for 5 minutes, a treatment that increased both the cAMP content and the PKA activity several-fold. The calcium influx induced by progesterone was increased by papaverine to levels close to those found in capacitated sperm. This effect was partially inhibited by H89 (48%) and by genistein (41%), and the sum of both inhibitors reduced the stimulating effect of papaverine by 89%. The inhibitory effect of genistein on the progesterone-induced calcium influx could be related to its capability to inhibit the papaverine-stimulated increase in cAMP content and PKA activity. The results presented here suggest that the calcium influx induced by progesterone is up-regulated by the PKA activity.

Recovery after a traumatic brain injury depends on diurnal variations effect of cystatin C.

Many studies indicate that the hour of the day at which the onset of stroke occurs is very important in patient recovery. Furthermore, multiple studies have been conducted which show that ischemia in rats produces different magnitudes of injury depending on the hour of the day at which it was induced. Using a traumatic brain injury (TBI) model, we analyzed the effect of the time of day on the recovery of rats and obtained a higher survival rate if TBI was induced at 01:00 h as compared with TBI induced at 13:00 h. We also analyzed the effect of the protease inhibitor cystatin C (CC) on the recovery of rats from TBI and found that it increased mortality and bleeding, and that these effects were more pronounced at 13:00 h.

Cystatin C has a dual role in post-traumatic brain injury recovery.

Cathepsin B is one of the major lysosomal cysteine proteases involved in neuronal protein catabolism. This cathepsin is released after traumatic injury and increases neuronal death; however, release of cystatin C, a cathepsin inhibitor, appears to be a self-protective brain response. Here we describe the effect of cystatin C intracerebroventricular administration in rats prior to inducing a traumatic brain injury. We observed that cystatin C injection caused a dual response in post-traumatic brain injury recovery: higher doses (350 fmoles) increased bleeding and mortality, whereas lower doses (3.5 to 35 fmoles) decreased bleeding, neuronal damage and mortality. We also analyzed the expression of cathepsin B and cystatin C in the brains of control rats and of rats after a traumatic brain injury. Cathepsin B was detected in the brain stem, cerebellum, hippocampus and cerebral cortex of control rats. Cystatin C was localized to the choroid plexus, brain stem and cerebellum of control rats. Twenty-four hours after traumatic brain injury, we observed changes in both the expression and localization of both proteins in the cerebral cortex, hippocampus and brain stem. An early increase and intralysosomal expression of cystatin C after brain injury was associated with reduced neuronal damage.

Present status and perspective of pharmacogenetics in Mexico.

Abstract Drug costs account for up to 24% of the country’s health expenditure and there are 13,000 registered drugs being prescribed. Diabetes is the main cause of death in the country, with over 85% of diabetic patients currently under drug treatment. The importance of knowing interindividual variability in drug metabolism on Mexican populations is thus evident. The purpose of this article is to provide an overlook of the current situation of pharmacogenetic research in Mexico, focusing on drug-metabolizing enzymes, and the possibility of developing a phenotyping cocktail for Mexican populations. So far, 21 pharmacogenetic studies on Mexican population samples (Mestizos and Amerindian) have been published. These have reported interindividual variability through phenotyping and/or genotyping cytochromes: CYP2D6, 2C19, 2C9, 2E1, and phase II enzymes UGT and NAT2. Some cytochromes with important clinical implications have not yet been phenotyped in Mexican populations. The development of a cocktail adapted to them could be a significant contribution to a larger knowledge on drug response variability at a lower price and shorter time. There are validated phenotyping cocktails that present several practical advantages, being valuable, safe, and inexpensive tools in drug metabolism characterization, which require only a single experiment to provide information on several cytochrome activities.

Prescription patterns of antihypertensives in a community health centre in Mexico City: a drug utilization study.

Hypertension is highly prevalent; in Mexico, the 2012 National Health and Nutrition Survey reported a prevalence of hypertension of 31.5% in the adult population. Pharmacological treatment is the commonest intervention and has been shown to reduce cardiovascular mortality and morbidity, and total mortality. Accordingly, the type and number of antihypertensives used and the outcome — in terms of blood pressure (BP) control — are important. Therefore, our purpose is to learn the pattern of antihypertensive drug prescription and explore the determinants of BP control in an urban population in Mexico. A retrospective cross‐sectional drug utilization study was conducted. Medical records from a community health centre were searched to identify those corresponding to patients diagnosed with hypertension; information upon antihypertensives used and control of the disease was carefully retrieved. A logistic regression model was built to know the main determinants of BP control. A sample of 345 clinical records of interest was identified. Most patients received antihypertensives (86.4%); the leading medications used were angiotensin‐converting enzyme inhibitors, 63.8%; beta‐blockers (26.5%), diuretics (19.8%), angiotensin‐receptor blockers (15.8%) and calcium‐channel blockers (6.4%). Only the age (≥55 years) and BMI (>30) of the patients, and the age of the doctors (≥55 years), had an important influence on BP control. Obesity is a particular and important determinant of uncontrolled hypertension; it is worth to act on body weight, on an individual basis. As lack of control has been also tied to elderly doctors, an education programme could be envisaged.

Identification of genetic variants in pharmacogenetic genes associated with type 2 diabetes in a Mexican-Mestizo population

Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic pathologies in the world. In developing countries, such as Mexico, its prevalence represents an important public health and research issue. Determining factors triggering T2DM are environmental and genetic. While diet, exercise and proper weight control are the first measures recommended to improve the quality of life and life expectancy of patients, pharmacological treatment is usually the next step. Within every population there are variations in interindividual drug response, which may be due to genetic background. Some of the most frequent first line T2DM treatments in developing countries are sulfonylureas (SU), whose targets are ATP-sensitive potassium channels (KATP). Single nucleotide polymorphisms (SNPs) of the KATP coding genes, potassium voltage-gated channel subfamily J member 11 (KCNJ11) and ATP binding cassette subfamily C member 8 (ABCC8) have been associated with SU response variability. To date, there is little information regarding the mechanism by which these SNPs work within Mexican populations. The present study describes the distribution of three SNPs [KCNJ11 rs5219 (E23K), ABCC8 rs757110 (S1369A) and rs1799854 (−3C/T)] among Mestizo Mexican (MM) T2DM patients, and compares it with published data on various healthy subjects and T2DM populations. Through this comparison, no difference in the KCNJ11 rs5219 and ABCC8 rs757110 allelic and genotypic frequencies in MM were observed compared with the majority of the reported populations of healthy and diabetic individuals among other ethnic groups; except for African and Colombian individuals. By contrast, ABCC8 rs1799854 genomic and allelic frequencies among MM were observed to be significantly different from those reported by the 1000 Genomes Project, and from diabetic patients within other populations reported in the literature, such as the European, Asian and Latin-American individuals [T=0.704, G=0.296; CC=0.506, CT=0.397, TT=0.097; 95% confidence interval (CI); P≤0.05]; except for South Asian and Iberian populations, which may reflect the admixture origins of the present Mexican population. This genetic similarity has not been observed in the other Latin-American groups. To the best of our knowledge, this is the first study of ABCC8 rs757110 and rs1799854 SNP frequencies in any Mexican population and, specifically with diabetic Mexicans. Knowledge of the genetic structure of different populations is key to understanding the interindividual responses to drugs, such as SU and whether genotypic differences affect clinical outcome.

Pulsatile diastolic increase and systolic decrease in arterial blood pressure: Their mechanism of production and physiological role

The diastolic pulsatile increase in arterial blood pressure is shown to occur earlier in the aorta than in other arteries. It is thus not a reflection of the systolic pressure wave, as has been generally assumed, but an independent pressure wave produced by the sequential contraction of the arterial tree. Conversely, a systolic pulsatile decrease in the rate of blood pressure rise is also produced by an active relaxation of the arterial tree. Simultaneously with the pulsatile changes in arterial blood pressure, there are corresponding changes in arterial blood flow. All these cyclic changes are reflex responses to decreasing diastolic and increasing systolic baroreceptor firing rates, respectively. The two reflexes contribute, together with the known compliance of the large arteries and the great arteriolar blood flow resistance, to the steadiness of capillary blood flow throughout the systolic and the much longer-lasting diastolic phases of the cardiac cycle.

Single-cell microinjection assay indicates that 7-hydroxycoumarin induces rapid activation of caspase-3 in A549 cancer cells.

Coumarins have attracted intense interest in recent years due to their apoptogenic effects. The aim of the present study was to determine whether 7-hydroxycoumarin (7-HC) induces changes in caspase-3 (C-3) activity in A549 human lung carcinoma cells. A range of analytical techniques, including colorimetric and fluorometric assays, western blotting, single-cell microinjection, fluorescence microscopy and image analysis were conducted to elucidate the effects of 7-HC. A 24-h exposure to 1.85 mM 7-HC induced a 65% increase in C-3 activity, and a notable conversion of procaspase-3 to C-3, in addition to poly(ADP-ribose)polymerase cleavage. Furthermore, morphological changes associated with apoptosis were observed. Exposure of the cells to 7-HC for 3 or 6 h increased calcium conductance by 27%. By performing the single-cell microinjection of a specific fluorescent substrate of C-3 into previously 7-HC-exposed cells, a typical enzymatic kinetic profile of C-3 activation was identified a number of hours prior to the morphological and biochemical changes associated with apoptosis being observed. These results suggest that the rapid in vivo activation of C-3 is induced by 7-HC, the most relevant biotransformation product of coumarin in humans.