C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in osteosarcoma cells

Chemotherapy has significantly improved the prognosis of high-grade osteosarcoma (OS), but over 30% of OS patients can still not be cured. Pemetrexed, the newly-developed anti-folate chemotherapy drug, exerted lower efficacy against OS cells. Here, we aimed to increase pemetrexed efficiency, and found that the cell-permeable short-chain ceramide (C6) significantly enhanced pemetrexed-induced viability reduction and death in cultured OS cell lines (U2OS and MG-63). Pemetrexed induced moderate apoptosis in OS cells, which was dramatically augmented by C6 ceramide. The apoptosis inhibitor z-VAD-fmk largely inhibited C6 ceramide plus pemetrexed-induced cytotoxicity and apoptosis in OS cells. By using pharmacological and siRNA-knockdown strategies, we showed that Akt–mammalian TOR (mTOR) over-activation was an important pemetrexed resistance factor in OS cells, and C6 ceramide-mediated pemetrexed sensitization effect was mediated, at least in part, by Akt–mTOR inhibition. Finally, we found that Akt–S6 Kinase 1 (S6K1, an indicator of mTOR activation) was over-activated in human OS tissues. On the other hand, the osteoblastic MC3T3-E1 cells, which expressed lower Akt–S6K1 phosphorylation, were resistant to pemetrexed and/or C6 ceramide. Together, we conclude that C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in OS cells probably through in-activation of Akt–mTOR signaling.     

培美曲塞联合罗格列酮抑制肺癌A549 细胞增殖研究

目的:研究培美曲塞联合过氧化物酶体增殖物激活受体γ(Peroxisome proliferators-activated receptorγ,PPARγ)激动剂罗格列酮(Rosiglitazone,RSG)对人肺癌A549细胞株增殖能力的影响。方法:蛋白质印迹法检测肺癌细胞系中PPARγ的表达水平,筛选高表达PPARγ的肺癌细胞株利用该细胞株并分别给予培美曲塞、培美曲塞联合罗格列酮、培美曲塞联合罗格列酮及PPARγ拮抗剂GW9662处理,之后检测细胞增殖能力及PPARγ、PTEN、pAKT表达水平的变化。结果:PPARγ在A549细胞系中显著高表达;培美曲塞组、培美曲塞联合罗格列酮组、培美曲塞联合罗格列酮及GW9662组,肺癌细胞增殖均受到抑制,吾美曲塞和罗格列酮联合应用对肺癌细胞的增殖抑制具有协同效应,与单用培美曲塞组相比有统计学差异,且该效应可被GW9662有效阻断;给予培美曲塞和罗格列酮联合应用,可明显上调PPARγ、PTEN表达,下调pAKT表达;给予PPARγ拮抗剂GW9662后,PPARγ、PTEN的表达显著下调,pAKT表达上调。结论:PPARγ激动剂RSG对培美曲塞抑制肺癌细胞的增殖具有协同效应且其分子机制可能是通过激活PPARγ/PTEN/pAKT信号通路从而抑制肺癌A549细胞增殖。