Background: Gliomas remain among the most difficult cancers to treat, with a 5-year overall survival no greater than 5%. Many saponins showed a wide spectrum of anti-cancer activities at low concentration.Polyphyllin II is one of the common saponins from Paris polyphylla. However, the effect of Polyphyllin II on glioma cells has not been evaluated. Objective of the present study was to investigate whether Polyphyllin II have inhibition on glioma cells, and the possible mechanisms. Methods: The viability of U87 and U251 cells was detected by cell counting kit-8, cell counting real time cellular analysis and cell clone formation methods. Transwell was used to estimate the aggression of U87 and U251. The cell apoptosis rate was tested by flow cytometry. The morphological change was determined by transmission electron microscopy. The levels of AKT, phosphorylation of AKT, Bax, Bcl-2, cytochrome c, and cleaved caspase 3 proteins were assessed by Western blot. N-acetyl-L-cysteine was used to check the role of ROS in polyphyllin II inhibition to glioma cells. Results: Polyphyllin II showed significant suppress to proliferation and aggression of U87 and U251 in a dose-and time-dependent manner. Result of flow cytometry confirmed that Polyphyllin II induced apoptosis to U87 and U251 cells. Transmission electron microscopy observation revealed majority of the glioma cells treated with Polyphyllin II had turgidity of mitochondrion, disarrangement, diminution and vacuolization, those refer to mitochondrial apoptosis. Western blot indicated that Polyphyllin II promoted cyt-c, Bax, caspase 3 and cleaved-caspase 3, and decreased Bcl-2, AKT and p-AKT. Rescue experiments using N-acetyl-L-cysteine, a reactive oxygen species scavenger, reversed the levels of Bax and cyt-c, and the inhibition in Polyphyllin II-treated U87 and U251 cells. Conclusions:The present findings revealed that polyphyllin II may be a potential drug against glioma.

Many glioma patients develop resistance to temozolomide (TMZ) treatment, resulting in reduced efficacy and survival rates. TMZ‐resistant cell lines SHG44R and U87R, which highly express O6‐methylguanine DNA methyltransferase (MGMT) and P‐gp, were established. CN‐3, a new asterosaponin, showed cytotoxic effects on TMZ‐resistant cells in a dose‐ and time‐dependent manner via reactive oxygen species (ROS)‐mediated apoptosis and autophagy. Transmission electron microscopy and monodansylcadaverine (MDC) staining showed turgidity of the mitochondria and autophagosomes in CN‐3‐treated SHG44R and U87R cells. The autophagy inhibitor 3‐methyladenine was used to confirm the important role of autophagy in CN‐3 cytotoxicity in TMZ‐resistant cells. The ROS scavenger N‐acetyl‐ l‐cysteine (NAC) attenuated the levels of ROS induced by CN‐3 and, therefore, rescued the CN‐3 cytotoxic effect on the viability of SHG44R and U87R cells by Cell Counting Kit‐8 assays and JuLI‐Stage videos. MDC staining also confirmed that NAC rescued an autophagosome increase in CN‐3‐treated SHG44R and U87R cells. Western blotting revealed that CN‐3 increased Bax, cleaved‐caspase 3, cytochrome C, PARP‐1, LC3‐Ⅱ, and Beclin1, and decreased P‐AKT, Bcl‐2, and p62. Further rescue experiments revealed that CN‐3 induced apoptosis and autophagy through ROS‐mediated cytochrome C, cleaved‐caspase 3, Bcl‐2, P‐AKT, PARP‐1, and LC3‐Ⅱ. In addition, CN‐3 promoted SHG44R and U87R cells sensitive to TMZ by reducing the expression of P‐gp, MGMT, and nuclear factor kappa B p65, and it had a synergistic cytotoxic effect with TMZ. Moreover, CN‐3 disrupted the natural cycle arrest and inhibited the migration of SHG44R and U87R cells by promoting cyclin E1 and D1, and by decreasing P21, P27, N‐cadherin, β‐catenin, transforming growth factor beta 1, and Smad2.

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