Nu-Man Tsai1,2, Kai-Fu Chang1,2, Jun-Cheng Wang3. 1. Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan. 2. Institute of Medicine of Chung Shun Medical University, Taichung, Taiwan. 3. Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan.
Abstract
BACKGROUND/AIMS: Herbal materials derived from Juniperus communis (JCo) possess anticancer activity. In this study, we evaluated the efficacy of a JCo berry extract in suppressing glioblastoma growth. METHODS: The effects of JCo extract on the viability of normal and glioma cell lines was analyzed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The synergistic therapeutic effect of JCo extract and temozolomide (TMZ) on glioma cells was examined by MTT analysis. Flow cytometry analysis, the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) test, and western blotting were performed to identify the apoptotic pathway. To determine the in vivo efficacy of the JCo extract, rats were injected with 5 × 104 rat glioma RG2 cells in the back skin and brain hemisphere and then received a subcutaneous injection in the back skin that contained either JCo extract or vehicle. Finally, blood and histologic examinations were performed to evaluate JCo toxicity. RESULTS: The IC50 values of JCo extract were 57-69 µg/mL and 49-67 µg/mL in the glioblastoma cell lines after 24 and 48 h, respectively. However, in non-tumor cell lines, the respective IC50 values of JCo extract were 76-105 µg/mL and 77-108 µg/mL. The JCo extract had a stronger cytotoxicity and a larger range of IC50 values in glioma than in normal cells as compared to those effects caused by temozolomide (TMZ). In addition, the results of flow cytometry analysis, TUNEL test, and western blotting revealed that the JCo extract induced glioma cell cycle arrest through intrinsic and extrinsic apoptotic pathways. In the in vivo studies, a significant reduction of tumor size in JCo-treated rats, as measured by animal MRI, demonstrated that the JCo extract effectively inhibited glioma cell growth and successfully penetrated the blood-brain barrier. The immunohistochemical (IHC) staining detected positive signals of PCNA, VEGFR-1, and VEGFR -2 in 44.49%, 5.88%, and 5.85% of JCo-treated glioma cells, respectively. However, positive signals of PCNA, VEGFR-1, and VEGFR-2 were detected in 73.08%, 9.67%, and 11.70% of vehicle-treated glioma cells, respectively. The IHC examination of PCNA and VEGFR-1 and -2 indicated that JCo extract significantly decreased the degree of neovascularization. However, no significant differences in serum levels of blood cell count and hepatic enzymes, renal function index, and the histologic appearance of vital organs were detected between the JCo and vehicle-treated rats. CONCLUSION: The JCo extract penetrated the blood-brain barrier and significantly induced glioma cell apoptosis by reducing neovascularization via suppression of the PI3K/AKT/mTOR pathway. Furthermore, JCo extract was less cytotoxic to non-neoplastic vital organs than TMZ.
BACKGROUND/AIMS: Herbal materials derived from Juniperus communis (JCo) possess anticancer activity. In this study, we evaluated the efficacy of a JCo berry extract in suppressing glioblastoma growth. METHODS: The effects of JCo extract on the viability of normal and glioma cell lines was analyzed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The synergistic therapeutic effect of JCo extract and temozolomide (TMZ) on glioma cells was examined by MTT analysis. Flow cytometry analysis, the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) test, and western blotting were performed to identify the apoptotic pathway. To determine the in vivo efficacy of the JCo extract, rats were injected with 5 × 104 ratglioma RG2 cells in the back skin and brain hemisphere and then received a subcutaneous injection in the back skin that contained either JCo extract or vehicle. Finally, blood and histologic examinations were performed to evaluate JCotoxicity. RESULTS: The IC50 values of JCo extract were 57-69 µg/mL and 49-67 µg/mL in the glioblastoma cell lines after 24 and 48 h, respectively. However, in non-tumor cell lines, the respective IC50 values of JCo extract were 76-105 µg/mL and 77-108 µg/mL. The JCo extract had a stronger cytotoxicity and a larger range of IC50 values in glioma than in normal cells as compared to those effects caused by temozolomide (TMZ). In addition, the results of flow cytometry analysis, TUNEL test, and western blotting revealed that the JCo extract induced glioma cell cycle arrest through intrinsic and extrinsic apoptotic pathways. In the in vivo studies, a significant reduction of tumor size in JCo-treated rats, as measured by animal MRI, demonstrated that the JCo extract effectively inhibited glioma cell growth and successfully penetrated the blood-brain barrier. The immunohistochemical (IHC) staining detected positive signals of PCNA, VEGFR-1, and VEGFR -2 in 44.49%, 5.88%, and 5.85% of JCo-treated glioma cells, respectively. However, positive signals of PCNA, VEGFR-1, and VEGFR-2 were detected in 73.08%, 9.67%, and 11.70% of vehicle-treated glioma cells, respectively. The IHC examination of PCNA and VEGFR-1 and -2 indicated that JCo extract significantly decreased the degree of neovascularization. However, no significant differences in serum levels of blood cell count and hepatic enzymes, renal function index, and the histologic appearance of vital organs were detected between the JCo and vehicle-treated rats. CONCLUSION: The JCo extract penetrated the blood-brain barrier and significantly induced glioma cell apoptosis by reducing neovascularization via suppression of the PI3K/AKT/mTOR pathway. Furthermore, JCo extract was less cytotoxic to non-neoplastic vital organs than TMZ.
Authors: Ana C Gonçalves; José David Flores-Félix; Paula Coutinho; Gilberto Alves; Luís R Silva Journal: Int J Mol Sci Date: 2022-03-16 Impact factor: 5.923