Study On Combined Effect Of BER Inhibitor Methoxyamine And -Elemene On Treatment Of Malignant Glioma

Glioma is the most frequently diagnosed primary tumor in adult central nervous system and, despite advances in surgical techniques, it still remains to be impossible for the thorough surgical removal of this tumor and it seems the relapse of this malignance is almost inevitable.β-elemene is a noval non-cytotoxic anti-tumor drugs which belongs to the national second-class medicine. Pre-clinical experiment have revealed thatβ-elemene is useful for the treatment of many types of tumors and has the advantages such as improving body immune function and working synergistically with radio-and chemo-therapies. However, further in vivo and clinical trials failed to found such distinctive anti-tumor effects as seen in the in vitro experiments. Tumor cells are endowed with strong DNA damage repairing machinery and, therefore, are found resistant to multiple chemo-therapy drugs. Therefore, inhibiting the innate DNA damage repairing process might be helpful to improving the therapeutic efficiency of present chemo-therapy drugs. Nano-liposomes drug delivery system can selectively increase the drug concentration at the desired tissue location, and offer an continuous release of the target drug. Thus, the application ofβ-elemene-nano-liposomes system might be a novel approach for targeted therapy of glioma. Our present study includes two parts:PartⅠ. The tumor-suppression effect of methoxyamine plusβ-elemene on glioma cellsAIM To improve the in vitro and in vivo tumor-suppression effect ofβ-elemene and reduce its side-effects by changing its pharmaceutical delivery form. To clarify whether a synergetic effect exists betweenβ-elemene and methoxyamine on the inhibition of glioma cell growth and provide theoretical basis for the further usage of β-elemene in the treatment of glioma.METHODS(1) in vitro study:p-elemene is combined with methoxyamine, a base-excison repair inhibitor, and tested for their in vitro tumor-suppression effect by MTT, comet assay, quantitative analysis ofγ-H2AX focus and Western-blot.(2) in vivo study:animal xeno-graft tumor model were established with C6 glioma cells in nude mice. Five groups were randomly divided and received the following drugs:control group,β-elemene (ELE) group, methoxyamine (MX) group, ELE+MX group, and Cisplatin (DDP) group (positive control group). The growth of the xeno-graft tumors were regularly observed and measured for their gross volume. Then the tumor growth curve was draw and tumor inhibition rate was calculated.RESULTS(1) in vitro study:β-elemene liposomes can significantly inhibit the growth of C6 and SHG44 glioma cells and its inhibitory effect increased as the drug concentration and treatment time increased.. Comet assay also showed that after treatment with methoxyamine plusβ-elemene, the DNAH%,DNAT%,TL,TM and OTM of C6 glioma cells were 58.26±3.46%,41.74±3.46%,32.77±9.12,13.87±4.77 and 14.07±3.84 respectively (compared with control group, P＜0.05; P＜0.01).γ-H2AX focus formation results revealed that, after treated with methoxyamine plus P-elemene, 14% of the tumor cells tested had 20γ-H2AX focus,in 8 h and 29% of them had more than 20γ-H2AX focus (compared with control group, P＜0.05; P＜0.01).(2) in vivo study:before receiving the drugs, there was no statistically significant differences in the tumor volume between the five experiment groups(P＜0.05). By day 16, all groups treated with drugs had a significant decrease in the tumor size as compared with the control group (P＜0.05; or P＜0.01). The RTV of the ELE+MX group is significantly smaller than the control (P＜0.05; or P＜0.01). Moreover, the T/C of the ELE+MX group is 51.6%, which is smaller than either the ELE group or the MX group. PartⅡ. Preparation of elemene liposomes drug delivery system and its in vitro tumor suppression effect on C6 glioma cellsAIM To study the apoptotic inducing effect of elemene nano-liposome on C6 glioma cells and explore its effect on the expression of capasese-3 gene.METHODSA elemene liposomes drug delivery system was firstly prepared and tested for its quality (entrapment rate, stability, body distribution). C6 glioma cells were cultured with the same amount of elemene nano-liposomes, routine elemene or placebo respectively and their apoptotic states were determined by MTT and flow cytometry. The expression level of Caspase-3 was measured by immunohistochemistry assay.RESULTS(1) MTT assay showed that elemene nano-liposomes can significantly inhibit the growth of C6 glioma cells and its inhibitory effect increased as the drug concentration and treatment time increased.(2) Cells in the elemene nano-liposomes and routine elemene groups underwent significant apoptosis at 48 h and 72 h after drug incubation, as compared with the control group.(3) Cells in the elemene nano-liposomes and routine elemene groups showed significant Caspase-3 protein expression at 48 h and 72 h after drug incubation, as compared with the control group.CONCLUSION(1) The co-administration of methoxyamine withβ-elemene has an significant tumor suppression effect on C6 and SHG44 glioma cells, and this tumor inhibitory effect is dose-and time-dependant.(2) The co-administration of methoxyamine can aggravate the DNA damages caused byβ-elemene in C6 glioma cells, suggesting this might be one of the mechanism by which methoxyamine can enhance the tumor suppression effect ofβ-elemene.(3)γ-H2AX focus formation can be found after treating C6 glioma cells with methoxyamine plusβ-elemene, suggesting that activation of PI3K signal pathway, which subsequently phosphorylate H2AX, might be one of the mechanism mediating the tumor suppression effect of the two drugs.(4)β-elemene has significant apoptosis and capasese-3 inducing effect in C6 glioma cells and its delivery by nano-liposome could facilitate its pro-apoptotic effects.