Apoptosis-inducing Therapy For Glioblastoma And The Underlying Molecular Mechanism

Glioblastoma accounts for 12-15% of intracranial tumors. Effective treatmentis complicated by multiple factors, including the diffusely infiltrative nature ofthe disease, which limits complete surgical resection. Although conventionaltreatments have improved significantly, the prognosis for glioblastoma patients isgrim. Average life expectancy after initial diagnosis is usually no more than 1year. Glioblastoma carcinogenesis occurs following the functional inactivation ofseveral tumor suppressor genes with parallel activation of key oncogenes bymutations, which affect the apoptosis and cell cycle. Thus, intensive research hasbeen conducted to develop novel anticancer agents that induce cancer cellapoptosis. Saponins extracted from natural plants are among the most promising.Ardipusilloside I [3-O-(α-L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→3)-(β-D-glucopyranosyl-(1→2))- -L-arabinopyranosyl)-cyclamiretin A], anaturally occurring saponin with anti-inflammatory properties, is isolated fromArdisia pusilla A. DC, a member of the Myrsinaceae family. Primary studies in vivo and in vitro showed that ardipusilloside I inhibits the growth of, and inducesapoptosis in, human cervical adenocarcinoma cells (HeLa), Lewis pulmonarycarcinoma and hepatocarcinoma, demonstrating the potential anticancerproperties of this compound. The mechanisms by which these effects areachieved are not understood fully, although, in HeLa cells, they do involve anincrease of intracellular free calcium.Apoptosis, or programmed cell death, is a highly conserved, tightly controlledcell suicide process that is regulated by many different intracellular andextracellular events to ablate neoplastic cells in normal physiological functions.Apoptosis is controlled by two potential pathways, the mitochondrial pathwayand the death receptor pathway. The mitochondrial pathway is characterized bythe loss of mitochondrial transmembrane potential and release of cytochrome c.The death receptor pathway is mediated by serial activation of Fas [a cell surfacedeath receptor of the tumor necrosis factor (TNF) family of cytokines] and theresultant effector caspase-3. Fas is a cell-surface receptor that mediates apoptoticcell death when triggered mainly by its lgand FasL. The intracellular domains ofFas recruit adapter molecules such as Fas-associated death domain (FADD), thusaccelerating the formation of a death-inducing signaling complex (DISC). DISCformation, in turn, recruits catalytically inactive procaspase-8 to allowintermolecular autocatalytic cleavage, thus releasing active caspase-8 to activatedownstream procaspases. During apoptosis process, cells are degraded by thecleavage of cellular substrates at specific sites containing aspartic acid and thenresult in the biochemical and morphological changes associated with apoptosisthrough the activation of proteases and endonucleases which catalyze key steps inthe death pathway.Herein, we investigated its effect on glioblastoma cell line U87MG cells and primary cultured human glioblastoma cells, and examined the underlyingmechanism of action. Ardipusilloside I substantially decreased the number ofviable cells of both cell lines in a time- and concentration-dependent manner,with a similar IC₅₀ of 4.05μM. Microscopy revealed apoptotic characteristics,including chromatin condensation and cell nucleus fragmentation, demonstratingthat ardipusilloside I induced apoptosis. Ardipusilloside I exposure also graduallyincreased the sub-G1 fraction (the apoptotic cell population) and an Sphase-arrest of both glioblastoma cells. Furthermore, ardipusilloside I increasedthe expression of Fas and its ligand (FasL), and enhanced the activation ofcaspase-8 and caspase-3. Additionally, we observed a significant decreasedapoptosis after the trigger effection of FasL was abolished by the neutralizationantibody anti-FasL antibody and an unchanged apoptosis level when theactivation of caspase-8 was interrupted by specific inhibitor z-lETD-fmk, whichsuggested that a casepase-8 independent FasL/Fas signaling mediated deathreceptor pathway is involved. These data suggested that ardipusilloside I could bedeveloped as a chemotherapeutic agent for the management of glioblatomas.The current study not only confirms the induction of apoptosis byardipusilloside I in both glioblastoma U87MG cells and primary culturedglioblastoma cells, but also clearly demonstrates the minor effect ardipusilloside Iwith a concentration within the therapeutical dose-window has on the viability ofthe nonneoplastic astrocyte line SVGp12 which presented the supporting cellsthat are generously existed in brain parenchyma. The effect of ardipusilloside I onthe viability of U87MG cells, primary cultured glioblastoma cells and SVGp12astrocytes was assessed by the MTT assay. The number of viable U87MG cellsand primary cultured glioblastoma cells decreased significantly in aconcentration- and time-dependent manner compared to vehicle control cells. The OD value decreased compared to vehicle control cell.(p＜0.05). ButArdipusilloside I did not affect the viability of SVGp12 astrocytes whichpresented the supporting cells that are generously existed in brain parenchyma.The OD value did not alter compared with vehicle control cell(p＞0.05).The biologic security of ardipusilloside I which is promising to be applied inthe clinical management for glioblastoma as a novel chemotherapeutic agent has beenapproved. Moreover, we investigated the role of FasL/Fas signaling mediateddeath receptor pathway in the chemotherapeutic effect of ardipusilloside I in vitro,which suggested an experimental base for developing targeting gene therapeuticagent that aims at specific effector factors. It is warranted that developingassistant agents for ardipusilloside I will consummate its clinical application.