Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

Immunohistochemistry analysis of proteins related with apoptosis as prognostic factor in epidermoid carcinomaof penis / Análisis inmunohistoquímico de proteínas relacionadas con la apoptosis como factor pronóstico en el carcinoma epidermoide de pene

The aim was to analyze the protein expression of apoptotic genes caspase-3, caspase-8 and bcl-2 with the immunohistochemistry technique, correlating with tumor grade (I, II and III) and with the patient survival in order to understand the basic mechanism of tumoral transformation. The immunohistochemistry reactions on 50 samples of squamous cell carcinoma were carried out with the avidin-biotin immunoperoxidase method and antigen recovery. The analyses were made using the graduation method "in crosses" (0 to 4 crosses - no stain to more than 75 percent of positives cells) and in categories (low, intermediate, high) of the cytoplasm immunoreactivity of the epidermoid penile carcinoma cells. It was observed a statistically significant difference when the expression of caspase-3 were compared with the grades I and II of the tumor (p=0.0010) and when comparing the patient survival with the grades I and II of the tumor (p=0.0212). The protein bcl-2 was more expressed than caspase-3 and caspase-8 proteins, suggesting that the apoptotic rate in this carcinoma is low. The higher expression of the anti-apoptotic protein bcl-2 suggests a higher preservation of the tumoral cells...

El objetivo fue analizar la expresión de las proteínas de genes de apoptosis caspasa-3, caspasa-8 y Bcl-2-con la técnica de inmunohistoquímica, en correlación con el grado tumoral (I, II y III) y la supervivencia del paciente con el fin de comprender el mecanismo básico de la transformación tumoral. Se analizaron las reacciones inmunohistoquímicas sobre 50 muestras de carcinoma de células escamosas mediante el método de la inmunoperoxidasa avidina-biotina y la recuperación de antígeno. Los análisis se realizaron utilizando el método de graduación "en cruces" (0 a 4 cruces - no tinción a más del 75 por ciento de las células positivas) y en categorías (baja, media, alta) de la inmunorreactividad citoplasmática de las células de carcinoma epidermoide de pene. Se observó una diferencia estadísticamente significativa cuando la expresión de la caspasa-3 se comparó con los grados I y II del tumor (p = 0,0010) y cuando se comparan la supervivencia de los pacientes con los grados I y II del tumor (p = 0,0212). La proteína bcl-2 se expresa más que la caspasa-3 y caspasa-8, lo que sugiere que la tasa de apoptosis en este carcinoma es baja. La mayor expresión de la proteína anti-apoptótica bcl-2 sugiere una mayor preservación de las células tumorales...