Hedgehog信号通路对胰腺癌细胞株氟尿嘧啶化疗敏感性的影响研究
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摘要
研究背景及目的
胰腺癌属高度恶性肿瘤,其发病率在全球呈上升趋势,确诊后平均生存时间不超过6个月,其诊断和治疗仍存在许多问题亟待解决。虽然手术切除是目前最有效的治疗方法,但85%的患者就诊时已属晚期,只有20%左右可以行手术治疗,大样本临床资料表明胰腺癌的5年生存率低于5%。虽然国内外有关胰腺癌早期诊断及综合治疗的研究报道很多,但至今尚无突破性进展。除手术外,化疗仍是目前治疗胰腺癌、预防术后复发、延长病人生存时间和提高生活质量的重要手段,但胰腺癌化疗过程中出现的复杂的耐药现象极大地影响了其治疗效果。化疗耐药仍是目前胰腺癌化疗中面临的最大困境,而如何逆转已知的化疗耐药成了近年来胰腺癌研究的热点。胰腺癌细胞经过化疗药物处理后,大部分的细胞可以被杀灭,但总会有少量肿瘤细胞残存下来,这部分细胞数量不多,经过一段时间的相对静止和休眠后,细胞又再次进入快速生长期,而且这部分经过药物筛选的细胞的耐药性明显升高。我们在研究中还同时发现,经过化疗药物处理后处于相对休眠状态的这一少部分细胞的某些特性与上述的肿瘤干细胞具有极大的相似性。
Hedgehog(HH)信号通路是肿瘤干细胞的自我更新机制之一,主要由四部分所组成,包括：HH配体、2个跨膜蛋白质受体(PTCH)、(Smo)组成的受体复合物以及下游的转录因子G1i蛋白,通过激活此通路使肿瘤细胞的自我更新能力增强；Cyclopamine：环杷明)是Hedgehog信号通路的抑制剂,通过作用于Smo蛋白阻止该信号往下传递,从而抑制肿瘤细胞的自我更新繁殖。目前国内外的研究已证实Hedgehog通路在多种肿瘤中有表达,Hedgehog通路的研究可以为日后针对肿瘤干细胞治疗提供更进一步的资料。通过适当浓度的化疗药物联合针对干细胞HH信号传导通路的抑制剂可以在体外试验中抑制胰腺癌细胞的生长,提高胰腺癌的化疗效果,为胰腺癌的治疗带来新希望。
研究方法：
(1) SW1990, T3M4、PANC-1三株胰腺癌细胞株的体外培养、传代
(2) SW1990、T3M4、PANC-1三株胰腺癌细胞株的生长曲线测定(MTT法)。
(3)化疗药物5-FU以不同浓度梯度对三株细胞分别进行药物杀伤实验以筛选适宜的药物浓度,MTT法测定三株胰腺癌细胞株对5-FU化疗敏感性的IC50值。
(4)运用针对HH信号传导通路的环杷明,以不同的浓度干预胰腺癌细胞株(实验共分为四组：5-FU化疗药物对照组、1uM和10uM环杷明处理组、番茄碱处理组),并进行以下实验：A. MTT实验；B.提取蛋白,Western Blot方法进行HH信号传导通路的检测；C.多种肿瘤干细胞标记物CD 133、CD44、CD24、EPCAM的联合检测。
结果：
1、SW1990、PANC-1、T3M4三株胰腺癌细胞株的5-FU化疗的IC50分别是31.97±5.21um、1.88±0.42mM、5.59±0.93uM。T3M4对5-FU化疗最为敏感,PANC-1对5-FU化疗不敏感,而SW1990对5-FU的敏感性中等。细胞增殖在48小时至72小时内较为缓慢,但是48小时至72小时后,细胞株增殖明显加快,进入对数生长期,至第5天到达顶峰,并进入平台期,其后细胞增殖缓慢。
2、在T3M4和SW1990细胞株中,进行cyclopamine干预后,其对于5-FU化疗的IC50明显下降(P<0.01),1uM和10uM之间的差异有显著统计学意义(P<0.05)。而PANC-1对于cyclopamine干预的影响不大,不同浓度之间的差异也不具有显著性(P>0.05)。cyclopamine干预后,HH信号通路下游的Gli-1的表达明显下降。
3、SW1990胰腺癌细胞株经过5-FU化疗处理后,肿瘤干细胞标记物(CD44、CD24)的比率明显增加,联合cyclopamine和5-FU干预后,肿瘤干细胞标记物(CD44、CD24)的比率显著下降(P<0.05)。而CD133、EPCAM的表达在各组之间的差别没有显著性(P>0.05)。
结论：
1、SW1990、PANC-1、T3M4三株胰腺癌细胞株中,T3M4对5-FU化疗最为敏感,PANC-1对5-FU化疗不敏感,而SW1990对5-FU的敏感性中等。
2、Cyclopamine干预可以明显提高T3M4和SW1990细胞株对5-FU的化疗敏感性,且其效果与浓度成正比。但是cyclopamine对于PANC-1细胞株5-FU的化疗敏感性的提高相对较迟钝。
3、经5-FU化疗后SW1990细胞株的肿瘤干细胞标记物(CD44、CD24)的表达比率明显增加,而CD133、EPCAM的变化不明显；联合环杷明和5-FU干预后,CD44、CD24表达比率明显下降,而CD133、EPCAM的变化不明显,因此,CD24、CD44可以作为胰腺癌干细胞的表面标志物,而EPCAM的表达率过高,不适宜作为胰腺癌干细胞的表面标志物。CD133的表达经过干预前后变化不明显,有待于进一步深入研究。
4、本实验结果显示5-FU化疗耐药的细胞,具有部分肿瘤干细胞的特性,能被HH通路阻滞剂所抑制。
Background and Objective:
Pancreatic cancer is highly malignant tumor, the incidence rate in the world is rising, the average survival time after diagnosis is not more than 6 months, its diagnosis and treatment there are still many issues that must be resolved. And there is a very low radical surgical resection rate because most cases are already in the advanced stages when diagnosed. The role of chemotherapy has become more important due to its effect towards tumor lesion and metastasis, as well as remission of symptoms. However, Traditional chemotherapy is ineffective. Five-year survival rate is very low for those treated with chemotherapy. Chemo-resistance is currently a major obstacle in tumor chemotherapy. Most of patients present with unresectable disease, resistant to chemotherapy, and high relapse, making prognosis of Pancreatic carcinoma very disappointing.
Hedgehog signaling pathway is one of the self renewal mechanism of cancer stem cell. It is composed of four parts, including HH ligand, two transmembrane protein-acceptor(PTCH)、(Smo) compounds and transcriptive protein Gli in downstream. The self renewal ability of cancer cell is become stronger When the Hedgehog signaling pathway is in activation. Cyclopamine is the specificity inhibitor of Hedgehog signaling pathway and it could bound with protein Smo, so the pathway turn into inactivation and reduce the capability of self renewal. Nowadays, researches identified Hedgehog pathway expressed in many kinds of cancers. Hedgehog researches could bring more useful information for killing cancer cell in the further.
Methods:
1. SW1990, T3M4 and PANC-1 pancreatic cancer cell lines are cultured in vitro, breeding the next generation.
2. Three pancreatic carcinoma cell lines growth curve are analysis (MTT methed).
3. Three cell lines were treated with 5-FU of different concentrations to screen suitable anti-drug concentration. And also MTT method 3 pancreatic cancer cells to determine 5-FU chemotherapy sensitivity of IC50 values.
4. Different concentration of cyclopamine, which is HH signaling pathway inhibitor, are applied to treated the three pancreatic cancer cell lines. Experiment is divided into four groups:5-FU chemotherapy group, luM and 10uM cyclopamine combine with 5-FU group, tomatidine combine with 5-FU group. The following experiments are carryout:A. MTT test; B. Extracted proteins for Western Blot test; C. Multiple cancer stem cell marker CD133, CD44, CD24, EPCAM joint detection.
RESULT:1、SW1990, PANC-1, T3M43 pancreatic cancer cell lines IC50 value of 5-FU chemotherapy were 31.97±5.21um,1.88±0.42mM,5.59±0.93uM. Of the three cell lines, T3M4 of the most sensitive to 5-FU chemotherapy, PANC-1 is not sensitive, and SW1990 is medium.
2、Cell proliferation in 48 hours to 72 hours is slow, but after 48-72 hours, cell proliferation significantly accelerated into the logarithmic phase, and reach its peak after 5 days, and enter the plateau phase of cell proliferation. To T3M4 and SW1990 cell lines, after treated with cyclopamine, IC50 value of 5-FU chemotherapy is decreased dramaticaly (P<0.01), luM and 10uM cyclopamine significant difference between the statistical significance (P<0.05). But to PANC-1 cell line, the change is not significant. After the intervention with cyclopamine, Gli-1 which is HH pathway downstream protein decrease.
3、after 5-FU chemotherapy treatment, cancer stem cell marker (CD44, CD24) increase significantly in SW1990, and decrease significantly after treated with cyclopamine and 5-FU (P<0.05). And CD133, EPCAM expression of differences among the groups is not significant (P> 0.05).
Conclusion:
1. In the three pancreatic cancer cell lines, T3M4 is the most sensitive to 5-FU chemotherapy, PANC-1 is not sensitive, and SW1990 is on the sensitivity of 5-FU medium.
2. Cyclopamine intervention can significantly improve the T3M4 and SW1990 cells to 5-FU chemotherapy sensitivity, and its effect is proportional to the concentration. But to PANC-1 cells, the chemotherapy sensitivity increase is relatively slow.
3. The 5-FU chemotherapy in cancer stem cell marker (CD44. CD24) expression ratio significantly increased, while CD 133 and EPCAM not obvious. the combine intervention of cyclopamine and 5-FU, CD44 and CD24 expression ratio significantly, while the CD133, EPCAM not obvious. Thus, CD24 and CD44 can serve as a pancreatic stem cell surface markers, However, EPCAM and CD 133 are not suitable.
4.5-FU chemotherapy resistant cells, with some of the characteristics of cancer stem cells, can be inhibited by HH signaling pathway blockers.
胰腺癌属高度恶性肿瘤,其发病率在全球呈上升趋势,确诊后平均生存时间不超过6个月,其诊断和治疗仍存在许多问题亟待解决。虽然手术切除是目前最有效的治疗方法,但85%的患者就诊时已属晚期,只有20%左右可以行手术治疗,大样本临床资料表明胰腺癌的5年生存率低于5%。虽然国内外有关胰腺癌早期诊断及综合治疗的研究报道很多,但至今尚无突破性进展。除手术外,化疗仍是目前治疗胰腺癌、预防术后复发、延长病人生存时间和提高生活质量的重要手段,但胰腺癌化疗过程中出现的复杂的耐药现象极大地影响了其治疗效果。化疗耐药仍是目前胰腺癌化疗中面临的最大困境,而如何逆转已知的化疗耐药成了近年来胰腺癌研究的热点。胰腺癌细胞经过化疗药物处理后,大部分的细胞可以被杀灭,但总会有少量肿瘤细胞残存下来,这部分细胞数量不多,经过一段时间的相对静止和休眠后,细胞又再次进入快速生长期,而且这部分经过药物筛选的细胞的耐药性明显升高。我们在研究中还同时发现,经过化疗药物处理后处于相对休眠状态的这一少部分细胞的某些特性与上述的肿瘤干细胞具有极大的相似性。
Hedgehog(HH)信号通路是肿瘤干细胞的自我更新机制之一,主要由四部分所组成,包括：HH配体、2个跨膜蛋白质受体(PTCH)、(Smo)组成的受体复合物以及下游的转录因子G1i蛋白,通过激活此通路使肿瘤细胞的自我更新能力增强；Cyclopamine：环杷明)是Hedgehog信号通路的抑制剂,通过作用于Smo蛋白阻止该信号往下传递,从而抑制肿瘤细胞的自我更新繁殖。目前国内外的研究已证实Hedgehog通路在多种肿瘤中有表达,Hedgehog通路的研究可以为日后针对肿瘤干细胞治疗提供更进一步的资料。通过适当浓度的化疗药物联合针对干细胞HH信号传导通路的抑制剂可以在体外试验中抑制胰腺癌细胞的生长,提高胰腺癌的化疗效果,为胰腺癌的治疗带来新希望。
研究方法：
(1) SW1990, T3M4、PANC-1三株胰腺癌细胞株的体外培养、传代
(2) SW1990、T3M4、PANC-1三株胰腺癌细胞株的生长曲线测定(MTT法)。
(3)化疗药物5-FU以不同浓度梯度对三株细胞分别进行药物杀伤实验以筛选适宜的药物浓度,MTT法测定三株胰腺癌细胞株对5-FU化疗敏感性的IC50值。
(4)运用针对HH信号传导通路的环杷明,以不同的浓度干预胰腺癌细胞株(实验共分为四组：5-FU化疗药物对照组、1uM和10uM环杷明处理组、番茄碱处理组),并进行以下实验：A. MTT实验；B.提取蛋白,Western Blot方法进行HH信号传导通路的检测；C.多种肿瘤干细胞标记物CD 133、CD44、CD24、EPCAM的联合检测。
结果：
1、SW1990、PANC-1、T3M4三株胰腺癌细胞株的5-FU化疗的IC50分别是31.97±5.21um、1.88±0.42mM、5.59±0.93uM。T3M4对5-FU化疗最为敏感,PANC-1对5-FU化疗不敏感,而SW1990对5-FU的敏感性中等。细胞增殖在48小时至72小时内较为缓慢,但是48小时至72小时后,细胞株增殖明显加快,进入对数生长期,至第5天到达顶峰,并进入平台期,其后细胞增殖缓慢。
2、在T3M4和SW1990细胞株中,进行cyclopamine干预后,其对于5-FU化疗的IC50明显下降(P<0.01),1uM和10uM之间的差异有显著统计学意义(P<0.05)。而PANC-1对于cyclopamine干预的影响不大,不同浓度之间的差异也不具有显著性(P>0.05)。cyclopamine干预后,HH信号通路下游的Gli-1的表达明显下降。
3、SW1990胰腺癌细胞株经过5-FU化疗处理后,肿瘤干细胞标记物(CD44、CD24)的比率明显增加,联合cyclopamine和5-FU干预后,肿瘤干细胞标记物(CD44、CD24)的比率显著下降(P<0.05)。而CD133、EPCAM的表达在各组之间的差别没有显著性(P>0.05)。
结论：
1、SW1990、PANC-1、T3M4三株胰腺癌细胞株中,T3M4对5-FU化疗最为敏感,PANC-1对5-FU化疗不敏感,而SW1990对5-FU的敏感性中等。
2、Cyclopamine干预可以明显提高T3M4和SW1990细胞株对5-FU的化疗敏感性,且其效果与浓度成正比。但是cyclopamine对于PANC-1细胞株5-FU的化疗敏感性的提高相对较迟钝。
3、经5-FU化疗后SW1990细胞株的肿瘤干细胞标记物(CD44、CD24)的表达比率明显增加,而CD133、EPCAM的变化不明显；联合环杷明和5-FU干预后,CD44、CD24表达比率明显下降,而CD133、EPCAM的变化不明显,因此,CD24、CD44可以作为胰腺癌干细胞的表面标志物,而EPCAM的表达率过高,不适宜作为胰腺癌干细胞的表面标志物。CD133的表达经过干预前后变化不明显,有待于进一步深入研究。
4、本实验结果显示5-FU化疗耐药的细胞,具有部分肿瘤干细胞的特性,能被HH通路阻滞剂所抑制。
Background and Objective:
Pancreatic cancer is highly malignant tumor, the incidence rate in the world is rising, the average survival time after diagnosis is not more than 6 months, its diagnosis and treatment there are still many issues that must be resolved. And there is a very low radical surgical resection rate because most cases are already in the advanced stages when diagnosed. The role of chemotherapy has become more important due to its effect towards tumor lesion and metastasis, as well as remission of symptoms. However, Traditional chemotherapy is ineffective. Five-year survival rate is very low for those treated with chemotherapy. Chemo-resistance is currently a major obstacle in tumor chemotherapy. Most of patients present with unresectable disease, resistant to chemotherapy, and high relapse, making prognosis of Pancreatic carcinoma very disappointing.
Hedgehog signaling pathway is one of the self renewal mechanism of cancer stem cell. It is composed of four parts, including HH ligand, two transmembrane protein-acceptor(PTCH)、(Smo) compounds and transcriptive protein Gli in downstream. The self renewal ability of cancer cell is become stronger When the Hedgehog signaling pathway is in activation. Cyclopamine is the specificity inhibitor of Hedgehog signaling pathway and it could bound with protein Smo, so the pathway turn into inactivation and reduce the capability of self renewal. Nowadays, researches identified Hedgehog pathway expressed in many kinds of cancers. Hedgehog researches could bring more useful information for killing cancer cell in the further.
Methods:
1. SW1990, T3M4 and PANC-1 pancreatic cancer cell lines are cultured in vitro, breeding the next generation.
2. Three pancreatic carcinoma cell lines growth curve are analysis (MTT methed).
3. Three cell lines were treated with 5-FU of different concentrations to screen suitable anti-drug concentration. And also MTT method 3 pancreatic cancer cells to determine 5-FU chemotherapy sensitivity of IC50 values.
4. Different concentration of cyclopamine, which is HH signaling pathway inhibitor, are applied to treated the three pancreatic cancer cell lines. Experiment is divided into four groups:5-FU chemotherapy group, luM and 10uM cyclopamine combine with 5-FU group, tomatidine combine with 5-FU group. The following experiments are carryout:A. MTT test; B. Extracted proteins for Western Blot test; C. Multiple cancer stem cell marker CD133, CD44, CD24, EPCAM joint detection.
RESULT:1、SW1990, PANC-1, T3M43 pancreatic cancer cell lines IC50 value of 5-FU chemotherapy were 31.97±5.21um,1.88±0.42mM,5.59±0.93uM. Of the three cell lines, T3M4 of the most sensitive to 5-FU chemotherapy, PANC-1 is not sensitive, and SW1990 is medium.
2、Cell proliferation in 48 hours to 72 hours is slow, but after 48-72 hours, cell proliferation significantly accelerated into the logarithmic phase, and reach its peak after 5 days, and enter the plateau phase of cell proliferation. To T3M4 and SW1990 cell lines, after treated with cyclopamine, IC50 value of 5-FU chemotherapy is decreased dramaticaly (P<0.01), luM and 10uM cyclopamine significant difference between the statistical significance (P<0.05). But to PANC-1 cell line, the change is not significant. After the intervention with cyclopamine, Gli-1 which is HH pathway downstream protein decrease.
3、after 5-FU chemotherapy treatment, cancer stem cell marker (CD44, CD24) increase significantly in SW1990, and decrease significantly after treated with cyclopamine and 5-FU (P<0.05). And CD133, EPCAM expression of differences among the groups is not significant (P> 0.05).
Conclusion:
1. In the three pancreatic cancer cell lines, T3M4 is the most sensitive to 5-FU chemotherapy, PANC-1 is not sensitive, and SW1990 is on the sensitivity of 5-FU medium.
2. Cyclopamine intervention can significantly improve the T3M4 and SW1990 cells to 5-FU chemotherapy sensitivity, and its effect is proportional to the concentration. But to PANC-1 cells, the chemotherapy sensitivity increase is relatively slow.
3. The 5-FU chemotherapy in cancer stem cell marker (CD44. CD24) expression ratio significantly increased, while CD 133 and EPCAM not obvious. the combine intervention of cyclopamine and 5-FU, CD44 and CD24 expression ratio significantly, while the CD133, EPCAM not obvious. Thus, CD24 and CD44 can serve as a pancreatic stem cell surface markers, However, EPCAM and CD 133 are not suitable.
4.5-FU chemotherapy resistant cells, with some of the characteristics of cancer stem cells, can be inhibited by HH signaling pathway blockers.
引文
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