Myrtenal-induced V-ATPase inhibition - A toxicity mechanism behind tumor cell death and suppressed migration and invasion in melanoma.

BACKGROUND: Metastatic tumor cells have acidic extracellular pH and differential electrochemical H+ gradients generated across their cell membranes by V-type H+-ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice. METHODS: The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach. RESULTS: The inhibition of V-ATPases by 100 µM Myrtenal disrupted the electrochemical H+ gradient across the cell membranes, strongly induced cell death (4-5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15 mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers. CONCLUSIONS: These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H+ fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition. GENERAL SIGNIFICANCE: The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential.

Spectral imaging microscopy demonstrates cytoplasmic pH oscillations in glial cells.

Glial cells exhibit distinct cellular domains, somata, and filopodia. Thus the cytoplasmic pH (pH(cyt)) and/or the behavior of the fluorescent ion indicator might be different in these cellular domains because of distinct microenvironments. To address these issues, we loaded C6 glial cells with carboxyseminaphthorhodafluor (SNARF)-1 and evaluated pH(cyt) using spectral imaging microscopy. This approach allowed us to study pH(cyt) in discrete cellular domains with high temporal, spatial, and spectral resolution. Because there are differences in the cell microenvironment that may affect the behavior of SNARF-1, we performed in situ titrations in discrete cellular regions of single cells encompassing the somata and filopodia. The in situ titration parameters apparent acid-base dissociation constant (pK'(a)), maximum ratio (R(max)), and minimum ratio (R(min)) had a mean coefficient of variation approximately six times greater than those measured in vitro. Therefore, the individual in situ titration parameters obtained from specific cellular domains were used to estimate the pH(cyt) of each region. These studies indicated that glial cells exhibit pH(cyt) heterogeneities and pH(cyt) oscillations in both the absence and presence of physiological HCO(3)(-). The amplitude and frequency of the pH(cyt) oscillations were affected by alkalosis, by acidosis, and by inhibitors of the ubiquitous Na(+)/H(+) exchanger- and HCO(3)(-)-based H(+)-transporting mechanisms. Optical imaging approaches used in conjunction with BCECF as a pH probe corroborated the existence of pH(cyt) oscillations in glial cells.

Analisis ultraestructural del espermatozoide in individuos euspérmicos fértiles y oligozoospérmicos infértiles / An ultrastructural analysis of the spermatozoa in fertile euspermic and infertile oligospermic individuals

Sólo los espermatozoides estructurales y funcionalmente integros son capaces de transportarse desde el fondo de saco vaginal hasta la región ampular de de trompa de Falopio y fertilizar al ovocito homólogo. Por lo tanto, analizamos comparativamente mediante microscopia electrónica de transmisión, la integridad morfológica de los espermatozoides de nueve individuos oligozoospérmicos infértiles y de cinco individuos euspérmicos. Según nuestros resultados, en los espermatozoides de individuos oligozoospérmicos la proporción de acrosomas morfológicamente normales es menor que la observada en individuos fértiles (11.29 ñ 5.11 y 18.69 ñ 1.95%, rspectivamente). Asimismo, en los espermatozoides de indivíduos infértiles se encontraron hasta 4.2 veces menos núcleos ultraestructuralmente normales, en comparación con lo determinado en individuos fértiles. En contraste, a nivel de la pieza media no se observaron diferencias en la estructura fina entre ambos grupos. El análisis ultraestructural integral del espermatozoide (acrosoma, núcleo y pieza media) indicó que en individuos oligozoospérmicos infértiles 4.79 ñ 2.28% de los espermatozoides son ultraestructuralmente normales, en comparación con 17.35 ñ 3.39% en individuos euspérmicos fértiles. Po lo tanto, proponemos que en el análisis de semen de individuos oligozoospérmicos infértiles de etiología idiopática,e s necesario se incluya el análisis ultraestructural del sepermatozoide con la finalidad de obtener un diagnóstico y pronóstico más preciso para esta entidad nosológica