Epigenetic Evaluation of N-(2-hydroxyphenyl)-2-Propylpentanamide, a Valproic Acid Aryl Derivative with Activity Against HeLa Cells.

BACKGROUND: Valproic acid (VPA) is an HDAC inhibitor (HDACI) with an anticancer activity, but is hepatotoxic. N-(2-hydroxyphenyl)-2-propylpentanamide (o-OH-VPA) is a VPA aryl derivative designed in silico as a selective inhibitor of HDAC8 with biological properties against HeLa, rhabdomyosarcoma and breast cancer cell cultures. OBJECTIVE: We studied the epigenetic mechanism of o-OH-VPA as an HDACI and evaluated whether it was toxic to normal cells. METHODS: HeLa cells and primary human fibroblasts were used for this study as carcinogenic and normal cells, respectively. Cell survival was evaluated by MTT assay, whereas viability and doubling time were determined by the Trypan-blue method. HDAC activity was tested using the colorimetric HDAC activity assay. The expression of p21 was analyzed by PCR and HDAC8 expression was also evaluated by real-time PCR. Cell cycle and caspase-3 activity were analyzed by flow cytometry and caspase-3 colorimetric assay, respectively. RESULTS: o-OH-VPA (IC50 = 0.1 mM) was fifty-eight times more effective than VPA (IC50 = 5.8 mM) to reduce HeLa cell survival. Furthermore, o-OH-VPA increased the doubling time of HeLa cells by 33% with respect to the control. o-OH-VPA acted as HDACI in HeLa cells without affecting the HDAC8 expression, arresting the cell cycle of HeLa cells in the G0/G1 phase due to the increase in p21 expression with the inhibition of caspase-3 activity without exhibiting toxicity toward normal cells. CONCLUSION: Our results revealed that o-OH-VPA is an HDACI with a selective effect against HeLa cells but without the known toxicity exerted by most pan-HDACIs on normal cells.

Molecular modeling and LC-MS-based metabolomics of a glutamine-valproic acid (Gln-VPA) derivative on HeLa cells.

Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.

Docking Studies of Glutamine Valproic Acid Derivative (S)-5- amino-2-(heptan-4-ylamino)-5-oxopentanoic Acid (Gln-VPA) on HDAC8 with Biological Evaluation in HeLa Cells.

In this contribution, we focused on evaluating a novel compound developed by our group. This molecule, derived from glutamine (Gln) and valproic acid (VPA), denominated (S)- 5-amino-2-(heptan-4-ylamino)-5-oxopentanoic acid (Gln-VPA), was submitted to docking studies on histone deacetylase 8 (HDAC8) to explore its non-bonded interactions. The theoretical results were validated in HeLa cells as a cancer cell model and in human dermal fibroblasts as a normal cell model. The effects of Gln-VPA on HeLa and normal fibroblasts in terms of cell survival and the ability to inhibit HDAC activity in nude nuclear proteins and in nuclear proteins of whole cells treated for 24 h were analyzed. The HeLa cell cycle was analyzed after 24 and 48 h of treatment with Gln-VPA. The docking studies show that Gln-VPA can reach the catalytic site of HDAC8. Gln-VPA was organically synthesized with a purity greater than 97%, and its structure was validated using mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. Gln-VPA showed a similar effect to VPA as an HDAC inhibitor but with less toxicity to fibroblasts. Although Gln-VPA was less efficient than VPA in reducing the survival of HeLa cells, it could be studied for use as a cancer cell sensitizer.

ATP-binding cassette transporter ABCB5 gene is expressed with variability in malignant melanoma.

BACKGROUND: Melanoma is a malignant neoplasm with high metastatic disease risk and elevated mortality. Incidence of melanoma varies according to geographic region and genetic BACKGROUND: Epidemiological studies indicate that acral melanoma (AM) is among the most common melanomas in the Mexican population. While extensive studies have identified genes associated with melanoma, little is known about the genes involved in the pathogenesis of AM. OBJECTIVE: To compare the gene expression patterns between primary melanoma and normal skin. METHODS: We used 10 samples of fresh acral melanomas and normal skin for the study of differential gene expression and 22 samples of melanoma for in situ hybridization. RESULTS: We first identified a gene that was present in a sample of AM and absent in normal skin. DNA sequencing of this differentially expressed gene revealed that it corresponded to ABCB5, a gene recently implicated in the regulation of progenitor cell fusion. Furthermore, we detected ABCB5 expression in other melanoma specimens by RT-PCR. We showed that nine out of ten melanomas were positive for ABCB5 while only one melanoma and normal skin samples were negative. All ABCB5 expressing melanomas had variable gene expression according to in situ hybridization studies, suggesting that the ABCB5 gene may be differentially regulated by individual melanomas. CONCLUSIONS: The ABCB5 gene may be related to the properties of chemoresistance and aggressiveness of melanoma. The high expression found in samples of acral melanoma may provide more insight on the pathogenesis of this common type of melanoma in the Mexican population, frequently associated with poor prognosis.

Expresión variable del gen ABCB5 de las transportadoras de casetes de unión a ATP en el melanoma maligno / ATP-binding Cassette Transporter ABCB5 Gene is Expressed with Variability in Malignant Melanoma

Background: Melanoma is a malignant neoplasm with high metastatic disease risk and elevated mortality. Incidence of melanoma varies according to geographic region and genetic background. Epidemiological studies indicate that acral melanoma (AM) is among the most common melanomas in the Mexican population. While extensive studies have identified genes associated with melanoma, little is known about the genes involved in the pathogenesis of AM. Objective: To compare the gene expression patterns between primary melanoma and normal skin. Methods: We used 10 samples of fresh acral melanomas and normal skin for the study of differential gene expression and 22 samples of melanoma for in situ hybridization. Results: We first identified a gene that was present in a sample of AM and absent in normal skin. DNA sequencing of this differentially expressed gene revealed that it corresponded to ABCB5, a gene recently implicated in the regulation of progenitor cell fusion. Furthermore, we detected ABCB5 expression in other melanoma specimens by RT-PCR. We showed that nine out of ten melanomas were positive for ABCB5 while only one melanoma and normal skin samples were negative. All ABCB5 expressing melanomas had variable gene expression according to in situ hybridization studies, suggesting that the ABCB5 gene may be differentially regulated by individual melanomas. Conclusions: The ABCB5 gene may be related to the properties of chemoresistance and aggressiveness of melanoma. The high expression found in samples of acral melanoma may provide more insight on the pathogenesis of this common type of melanoma in the Mexican population, frequently associated with poor prognosis (AU)

Introducción: El melanoma es una neoplasia maligna que presenta una elevada mortalidad y un alto riesgo de desarrollar metástasis. La incidencia de esta enfermedad varía en función de la región geográfica y el trasfondo genético. Estudios epidemiológicos indican que el melanoma acral es uno de los más comunes en la población mexicana. Hay una gran cantidad de estudios sobre genes asociados al melanoma, sin embargo, se sabe muy poco de los genes que se relacionan con el melanoma acral. Objetivo: Comparar el patrón de expresión génica entre melanomas acrales primarios y piel sana. Métodos: Se utilizaron muestras en fresco de 10 lesiones de melanoma acral y piel sana para el estudio de expresión diferencial de genes y 22 muestras de melanoma para la hibridación in situ. Resultados: Identificamos un gen que estaba presente en una muestra de melanoma acral y ausente en la piel normal. La secuenciación de este gen reveló que correspondía al gen ABCB5, recientemente implicado en la regulación de la fusión de células progenitoras. Al realizar RT-PCR de otros melanomas se detectó la expresión de este gen: 9 de 10 melanomas fueron positivos para ABCB5. Todos los melanomas tuvieron una expresión variable de ABCB5 detectado por hibridación in situ, lo cual sugiere que el gen puede ser regulado diferencialmente en melanomas individuales. Conclusiones: El gen ABCB5 podría estar relacionado con las propiedades de resistencia a la quimioterapia y la agresividad del melanoma. La elevada expresión encontrada en las muestras de melanoma acral podría ayudar a la mejor comprensión de la patogenia de esta forma frecuente de melanoma en la población mexicana, que se asocia generalmente con un peor pronóstico (AU)