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胰腺癌是一种病死率极高的消化道恶性肿瘤 [1]。超过80%以上的患者一旦确诊即是晚期,手术难以根治,需在术后进行辅助化疗、放疗、对症支持治疗等 [2]。盐酸吉西他滨(hcGEM)是治疗胰腺癌的一线化疗药。由于存在半衰期短、产生耐药性及不可避免的毒副作用等问题,其疗效不尽如人意[3]。因此,hcGEM的临床应用需要联合化疗来提高疗效[4]。
铁死亡是一种铁依赖的非凋亡性细胞死亡形式,针对铁死亡的治疗策略可能为传统疗法难以攻克的癌症提供新的治疗思路 [5]。埃拉斯汀 (Era)作为一种高效持久的铁死亡诱导剂,它可以激活多种信号通路来触发癌细胞的铁死亡 [6]。柳氮磺胺吡啶(SASP)是一种能抑制铁死亡相关的胱氨酸-谷氨酸逆向转运蛋白的抗炎药,可通过降低癌细胞对胱氨酸的摄取以及胞内谷胱甘肽水平来抑制胰腺癌细胞的生长[7]。青蒿琥酯(Art)是一种青蒿素的衍生物,除用作抗疟治疗外,可通过促进铁蛋白吞噬来增加细胞内游离铁水平,进而引发癌细胞的铁死亡[8]。铁死亡诱导剂与盐酸吉西他滨联合应用可能是胰腺癌治疗的潜在策略[9]。
本研究分别考察Era、SASP和Art这三种铁死亡诱导剂单独或联合hcGEM使用,对人胰腺癌PANC-1细胞的增殖抑制作用,以期发现具有潜在协同抑制的联合方案,为今后开发胰腺癌新疗法奠定基础。
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如图1所示,hcGEM、Era、SASP、Art单独作用于PANC-1细胞时可明显抑制细胞的增殖,并且这种抑制作用呈现剂量依赖性。hcGEM、Era、SASP、Art作用于PANC-1细胞的IC50值分别为0.175 7、1.884、195.1、23.13 μmol/L。hcGEM单独用药时,小于0.015 625 μmol/L的剂量对PANC-1细胞的生长几乎无抑制作用,存活率>95%(P>0.05);剂量大于2 μmol/L时,细胞的活性受到显著抑制,存活率<15%(P<0.05)。Era单独用药时,大于16 μmol/L 的剂量能显著抑制PANC-1细胞的生长,存活率<10%(P<0.05),而小于0.25 μmol/L的剂量会使80%以上的细胞存活。SASP单独用药时,大于800 μmol/L的剂量能抑制PANC-1细胞的生长,存活率<15%(P<0.05),而小于12.5 μmol/L的剂量仅轻微抑制PANC-1细胞存活,存活率>85%(P>0.05)。Art单独用药时,大于128 μmol/L的剂量能显著抑制PANC-1细胞的生长,存活率<40%(P<0.05),而小于0.5 μmol/L时,仅能轻微抑制细胞增殖,存活率>85%(P>0.05)。
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在研究单药对PANC-1细胞抑制效果的基础上,我们进一步探究了hcGEM-Era 4∶1、1∶1、1∶4和1∶16 联合用药,hcGEM-SASP 4∶1、1∶1、1∶20和1∶400联合用药,以及hcGEM-Art 2∶1、1∶1、1∶4和1∶16联合用药,对PANC-1细胞的抑制效果。如图2所示, hcGEM-Era、hcGEM-SASP以及hcGEM-Art联合用药组的抑制效果随着浓度的增加而提高。其中,hcGEM-Era 4∶1、1∶4、1∶16联合组,以及hcGEM-SASP 1∶400联合组对PANC-1细胞抑制效果在所有浓度梯度范围均优于hcGEM组(P<0.05)。此外,hcGEM-Era 4∶1、1∶4、1∶16联合用药组,hcGEM-SASP 1∶20、1∶400联合组,和hcGEM-Art 1∶16联合组的IC50值均小于0.1757 μmol/L(hcGEM单药组的IC50),说明上述联合用药能在一定程度上提高对PANC-1细胞的抑制效果。不同联合用药组hcGEM的IC50值见表1。
表 1 hcGEM联合三种铁死亡诱导剂对胰腺癌PANC-1细胞作用时的IC50值
组别 摩尔浓度比 IC50(μmol/L,hcGEM) hcGEM-Era联合组 1∶0.25 0.140 3±0.009 1 1∶1 0.340 2±0.018 3 1∶4 0.091 3±0.005 1 1∶16 0.083 3±0.002 5 hcGEM-SASP联合组 1∶0.25 0.297 5±0.016 1 1∶1 0.240 2±0.021 2 1∶20 0.120 8±0.008 9 1∶400 0.092 6±0.006 7 hcGEM-Art联合组 1∶0.5 0.366 4±0.018 7 1∶1 0.344 4±0.026 3 1∶4 0.249 3±0.015 7 1∶16 0.154 6±0.013 5 -
为了研究hcGEM分别与Era、SASP、Art联合用药,对PANC-1细胞是否具有协同抑制作用,我们分别设计了hcGEM与三种铁死亡诱导剂的4种不同比例的联用方案,来探讨不同联合用药组对PANC-1细胞的协同抑制效果。CompuSyn软件分析结果显示,hcGEM-Era 4∶1或1∶4联用组在所有药物浓度下均能对PANC-1细胞产生良好的协同抑制效果(CI<1),且4∶1联用组的协同抑制效果略优于1∶4联用组。对于hcGEM-Era 1∶16联用组,CI值随着联合药物浓度的增加而减小,说明该比例下两药协同抑制效果随着浓度增大有所增强,而hcGEM-Era 1∶1联用组的CI值,仅除1 μmol/LhcGEM+1 μmol/L Era、0.031 25 μmol/L hcGEM+0.031 25 μmol/L Era联用组的CI<1外,其余浓度组CI>1,说明hcGEM-Era 1∶1联用组对PANC-1细胞几乎无协同抑制效果(图3A)。
对于hcGEM与SASP联合用药组对PANC-1细胞的协同抑制效果,CompuSyn分析结果提示,hcGEM-SASP 4∶1联用对PANC-1细胞没有协同作用(CI>1),而hcGEM-SASP 1∶1联用组除了0.25 μmol/L hcGEM+0.25 μmol/L SASP、0.125 μmol/L hcGEM+0.125 μmol/LSASP联用组CI<1外,其余联用浓度组的CI>1,表明hcGEM-SASP 1∶1联合用药对PANC-1细胞几乎无协同抑制作用。hcGEM-SASP 1∶20联合用药对PANC-1细胞,除了1 μmol/LhcGEM+20 μmol/L SASP、2 μmol/L hcGEM+40 μmol/LSASP联用组CI>1外,其余浓度组CI<1,提示hcGEM-SASP 1∶20联合用药对PANC-1细胞主要是协同抑制效果,且协同效果随着浓度的增加而减弱。hcGEM-SASP 1∶400联合用药对PANC-1细胞有良好的协同抑制效果(CI<1),且协同效果随着浓度的增加而增强(图3B)。
对于hcGEM与Art联合用药组对PANC-1细胞的协同抑制效果,CompuSyn结果提示,hcGEM-Art 2∶1联用组在0.031 25 ~0.25 μmol/L hcGEM联合浓度范围内CI <1,且接近1,其余联合浓度组的CI>1,表明hcGEM-Art 2∶1联合用药对PANC-1细胞几乎无协同抑制作用。hcGEM-Art 1∶1联用组CI>1,表明hcGEM-Art 1∶1联合用药对PANC-1细胞无协同抑制作用。hcGEM-Art 1∶4联用组仅在0.031 25~0.125 μmol/L hcGEM联合浓度范围内CI<1,其余联合浓度范围内CI>1,表明hcGEM-Art 1∶4联合用药对PANC-1细胞仅在0.031 25~0.125 μmol/L hcGEM联合浓度范围内存在协同抑制作用。 hcGEM-Art 1∶16联用组在0.015 625~0.25 μmol/L hcGEM联合浓度范围内CI <1,其余联合浓度范围内CI>1,表明hcGEM-Art 1∶16联用组在0.015 625~0.25 μmol/L hcGEM联合浓度范围内对PANC-1细胞有协同抑制作用 (图3C)。
Inhibitory effects of gemcitabine hydrochloride combined with ferroptosis inducers on the proliferation of PANC-1 cells
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摘要:
目的 研究三种铁死亡诱导剂埃拉斯汀(Era)、柳氮磺胺吡啶(SASP)和青蒿琥酯(Art)与盐酸吉西他滨(hcGEM)单独或联合应用对人胰腺癌PANC-1细胞的抑制作用。 方法 采用CCK-8法检测不同浓度的Era、SASP、Art单独或联合hcGEM对PANC-1细胞的增殖抑制作用,并根据联合指数(CI) 判断三种铁死亡诱导剂联合hcGEM对 PANC-1细胞是否具有协同抑制作用。 结果 hcGEM与三种铁死亡诱导剂单独或联合应用均能显著抑制PANC-1细胞活性,并且这种抑制作用随着浓度增大而增强。hcGEM-Era 4∶1或1∶4联用组、hcGEM-SASP 1∶400联用组 CI 值小于1。hcGEM-Art 1∶4或1∶16联用组仅在一定浓度范围内CI值小于1。 结论 hcGEM与三种铁死亡诱导剂单独或联合应用对PANC-1细胞的增殖抑制作用呈现剂量依赖性。hcGEM与三种铁死亡诱导剂联用能协同抑制PANC-1细胞的增殖。 Abstract:Objective To study the effects of three ferroptosis inducers Erastin (Era), sulfasalazine (SASP) and artesunate (Art) alone or combined with gemcitabine hydrochloride (hcGEM) on the proliferation inhibition of Human pancreatic cell line PANC -1. Methods The CCK-8 method was used to detect the inhibitory effects of different concentrations of Era, SASP and Art alone or combined with hcGEM on the proliferation of PANC-1, and the combination index (CI) was used to judge whether three ferroptosis inducers combined with hcGEM had synergistic inhibitory effect on PANC-1. Results The three ferroptosis inducers and hcGEM alone or in combination could significantly inhibit the activity of PANC-1. The inhibitory effects were enhanced with the concentration increasing. The CI values of hcGEM-Era 4∶1 or 1∶4 combination group and hcGEM-SASP 1∶400 combination group were less than 1.The CI values of hcGEM-Art 1∶4 or 1∶16 combination group were less than 1 only within a certain concentration range. Conclusion The inhibitory effects of the three ferroptosis inducers and hcGEM alone or in combination were dose-dependent. The combination of hcGEM and three ferroptosis inducers could synergistically inhibit the proliferation of PANC-1. -
表 1 hcGEM联合三种铁死亡诱导剂对胰腺癌PANC-1细胞作用时的IC50值
组别 摩尔浓度比 IC50(μmol/L,hcGEM) hcGEM-Era联合组 1∶0.25 0.140 3±0.009 1 1∶1 0.340 2±0.018 3 1∶4 0.091 3±0.005 1 1∶16 0.083 3±0.002 5 hcGEM-SASP联合组 1∶0.25 0.297 5±0.016 1 1∶1 0.240 2±0.021 2 1∶20 0.120 8±0.008 9 1∶400 0.092 6±0.006 7 hcGEM-Art联合组 1∶0.5 0.366 4±0.018 7 1∶1 0.344 4±0.026 3 1∶4 0.249 3±0.015 7 1∶16 0.154 6±0.013 5 -
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