Study On The Anti-Glioblastoma Effect Of Natural Product Cucurbitin D And The Underline Mechanism

Glioma is the most common primary brain tumor in clinic.The median survival period of patients is only 12 to 14 months,and the 5-year survival rate is less than 5%.A small proportion of glioma stem cells(GSC)are thought to be the main cause of recurrence and drug resistancy after therapy.By now,there are still no effective drugs for glioma treatments.In this study,we constructed a reporting cell line with Nestin-GFP transgene and applied in a high-throughput screening of natural products.We found that Cucurbitacin D(CuD)can inhibit the activity and growth of glioma cells(A172,U87-MG,U251)in a dose-dependent manner and its corresponding IC50 values are 700 nM,150 nM and 200 nM,respectively.Furthermore,CuD reduced the migration and invasion of glioma cells by inhibiting metalloproteinase(MMP).Flow cytometry analysis showed that CuD-treated cells were impeded at G2/M phase due to the increase of P53 expression and the decrease of ERK phosphorylation,thus the expression of cell proliferation relevant protein Ki-67 decreased and DNA synthesis was repressed.Meanwhile,CuD treatment resulted in the damage of respiratory chain and the increase of reactive oxygen species(ROS)level which further induced cellular apoptosis.The increase of Annexin V positive cells by Annexin V/PI staining exhibited the increase of apoptotic cells.In combination with temozolomide(TMZ),the first line anti-glioma drug CuD showed significant synergistic effect.To uncover the underline mechanism,Western blot and qPCR were run and the results indicated that the phosphorylation levels of STAT3 and AKT1 decreased,which then induced the decrease of stemness genes,such as Sox2,Oct3/4,htert and the GSC markers like CD133,Nestin,Olig2.Conversely,the glial differentiation markers such as Gfap and Mbp increased.At last,in following xenograft experiments,CuD effectively inhibited the growth of xenografted tumor without any obvious side effect.In conclusion,CuD could be used as an effective anti-cancer drug or an adjuvant for glioma therapy.