Abstract:

Objective To discuss the effect of D-limonene on the proliferation and apoptosis of the glioblastoma （GBM） cells，and to clarify its possible mechanism. Methods The GBM cells were divided into control group （0 mmol·L^-1 D-limonene） and 0.2， 0.4， 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups. CCK-8 method was used to detect the inhibitory rates of proliferation of the cells in various groups； clone formation assay was used to detect the clone formation rates of the cells in various groups； Annexin Ⅴ-FITC/PI method was used to detect the apoptotic rates of the cells in various groups； Western blotting method was used to detect the expression levels of protein kinase B （AKT）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and poly adenosine diphosphate（ADP）-ribose polymerase （PARP） proteins in the cells in various groups； imunofluorescence method was used to detect the expression levels of cleaved Caspase-3 protein in the cells in various groups. Fifteen model mice with subcutaneous tumor xenografts were randomly divided into blank group （0 mg·kg^-1·d^-1 D-limonene）， low dose of D-limonene group （200 mg·kg^-1·d^-1 D-limonene）， and high dose of D-limonene group （400 mg·kg^-1·d^-1 D-limonene）， and there were 5 mice in each group.The inhibitory rates of the tumor in vitro in various groups were calculated； HE staining and immunohistochemical staining were used to observe the morphology of subcutaneous tumor tissue of the mice in various groups and the growth curves of the tumor were drawn； immunohistochemical assay was used to detect the positive expression rates of Ki67 protein in subcutaneous tumor tissue of the mice in various groups； TUNEL staining was used to detect the apoptosis of the tumor cells in various groups. Results In control group， the cells were spindle-shaped， in good condition， growing closely and adherently， with normal organelles and cytoplasm. After treated for 48 h， the cells in 0.6 mmol·L^-1 D-limonene group showed reduced volume， intact but more permeable cell membranes， shrunken cytoplasm， internal vacuole structures， and some fragments floating in the solution. The cells in 0.8 and 1.0 mmol·L^-1 D-limonene groups exhibited significant apoptotic bodies and were in an apoptotic state. The CCK-8 results showed that compared with control group， the inhibitory rates of proliferation of the U87， LN229， and GL261 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）， the inhibitory rates of proliferation of the U87 and GL261 cells were significantly increased （P<0.01）. The clone formation assay results showed that compared with control group， the clone formation rates of the U87， LN229， and GL261 cells in 0.4， 0.6， and 0.8 mmol·L^-1 D-limonene groups were significantly decreased （P<0.05 or P<0.01）. The Annexin Ⅴ-FITC/PI results showed that compared with control group， after treated with D-limonene for 48 h， the apoptotic rates of the LN229 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）. The Western blotting results showed that compared with control group， the expression levels of Bax proteins in the LN229 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）， while the expression levels of AKT and Bcl-2 proteins were significantly decreased （P<0.01）， the expression level of PARP protein in the LN229 cells in 0.8 and 1.0 mmol·L^-1 D-limonene group was significanthy increased（P<0.01）.The immunofluorescence results showed that compared with control group， the expression levels of cleaved Caspase-3 protein in the LN229 cells in 0.6， 0.8， and 1.0 mmol·L^-1 D-limonene groups were significantly increased （P<0.01）. Compared with blank group， the tumor volumes of the mice in low and high doses of D-limonene groups were significantly decreased （P<0.01）. Compared with blank group， the tumor weights of the mice in low and high doses of D-limonene groups were significantly decreased （P<0.05）， and the inhitory rates of tumor were significantly increased （P<0.05）. The tumor cells in blank group were diffusely distributed， with deepened nuclear staining and increased nucleocytoplasmic ratio； a large number of degenerated and necrotic tumor cells were observed in tumor tissue of the mice in low and high doses of D-limonene groups. Compared with blank group， the positive expression rates of Ki67 protein in tumor tissue of the mice in low and high doses of D-limonene groups were significantly decreased （P<0.01）. Compared with blank group， the apoptotic rates of tumor cells of the mice in low and high doses of D-limonene groups were significantly increased （P<0.01）. Conclusion D-limonene has the inhibitory effect on the proliferation of the GBM cells； its mechanism may be related to the regulation of AKT protein expression and the activation of the Caspase-3 pathway to induce the apoptosis.

Key words: D-limonene, Glioblastoma, Apoptosis, Transplantation tumor, Protein kinase B