丙酮酸脱氢酶激酶-1在结肠癌中的表达及在细胞增殖和化疗中的作用
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摘要
背景与目的
结直肠癌是临床最常见的恶性肿瘤之一,由于生活习惯和饮食结构的改变,其发病率和死亡率均有逐年升高的趋势,资料显示我国结直肠癌的发病率正以年均4.2%的速度增长。手术和化疗是结直肠癌的常规治疗手段,虽然在一定程度上缓解了患者的痛苦,延长其寿命,但还存在着一定的缺陷。对于中晚期结直肠癌患者而言,手术效果差,化疗却面临越来越频繁的耐药难题,因此,探寻治疗结直肠癌和提高化疗效果的新策略是目前非常迫切的任务。
早在20世纪20年代,Otto Warburg就提出,肿瘤细胞能够通过糖酵解途径在缺乏氧的条件下获得能量,有些肿瘤细胞甚至在获得充足氧气时仍继续依赖糖酵解途径产能,肿瘤细胞这一特殊生化表型,被称为Warburg效应。因此,活跃的糖酵解代谢是恶性肿瘤细胞显著的生化特征,是肿瘤细胞能量ATP的重要来源。临床上已利用肿瘤细胞特异性的Warburg效应,通过正电子发射断层显像(positron emission tomography,PET)技术来诊断恶性肿瘤,而探索通过干预糖酵解代谢途径来治疗恶性肿瘤的策略正越来越受瞩目。
丙酮酸脱氢酶激酶(pyruvate dehydrogenase kinase,PDK)是细胞糖酵解关键限速酶丙酮酸脱氢酶(PDH)的调节酶。在人类和啮齿类动物中目前鉴定出有4种PDK同工酶:PDKI、PDK2、PDK3以及PDK4。其中PDK1在心脏、胰岛和骨骼肌中被检测到微量表达,在PDH磷酸化过程中发挥重要的功能,尤其在PDH长期调节中尤为重要。研究发现PDK1的表达可被缺氧诱导因子HIF-1α调控,可能与肿瘤缺氧的环境有关。近年研究表明,PDK1除了具有糖代谢酶的催化活性,对细胞凋亡拮抗还有一定的影响,药物抑制PDK1可诱导肿瘤细胞凋亡。体外细胞培养显示,HIF-1α和PDK1在头颈部癌、非小细胞肺癌、乳腺癌等细胞中表达增高。但HIF-1α和PDK1在人结肠癌组织中的表达和关系尚不清楚,PDK1在结肠癌中的表达、治疗意义以及其相关作用机制还尚不明确。
化疗作为结肠癌的重要治疗手段,目前效果并不理想,尤其单药化疗已经被联合化疗所取代。PDK1蛋白兼有促进能量ATP合成和影响凋亡的双重功能,因此推测PDK1与结直肠癌化疗有关,抑制PDK1可能增强化疗药物的治疗效果。
线粒体功能的调节在细胞的生长、增殖、凋亡等过程中发挥重要的功能。以往研究表明,恶性肿瘤细胞中线粒体的结构和功能均发生了一定的变化,线粒体膜结构不完整,且糖酵解途径获能导致线粒体膜静息电位高于正常,这些使得恶性肿瘤细胞的增殖被促进而凋亡被抑制。相关研究指出抑制PDK1能改善结构和功能,降低线粒体膜电位。但是这一效是否也是引起结直肠癌细胞增殖和凋亡的机制之一尚需实验证实。
本课题拟检测HIF-1α和PDK1在人结肠癌组织中的表达,观察HIF-1α与PDK1的关系;比较PDK1在正常组织和结肠癌组织中表达的区别与病理联系,以期说明PDK1的表达与结肠癌发生发展的关系。通过抑制PDK1,观察结肠癌细胞增殖、化疗药物协同作用、凋亡相关因子和线粒体结构和功能的变化,以探讨PDK1在结肠癌细胞增殖和化疗中的作用及机制。
方法
1、通过免疫组织化学染色及免疫荧光双标染色技术观察结直肠癌组织标本中HIF-1α与PDK1的表达和分布情况,了解二者在结直肠癌组织中表达的关系及与肿瘤分期、淋巴转移等临床病理参数的关系。
2、采用RT-PCR、real-time PCR和免疫荧光、Western印迹法,检测人结肠癌组织和4种人结肠癌细胞株PDK1基因和蛋白表达。
3、应用MTT、BrdU掺入法、Hoechst33258核染色及Annex-V双染色法,观察PDK1阻断剂(Dichloroacetate,DCA)对结肠癌细胞活性、增殖、凋亡和化疗的影响。
4、通过流式细胞仪检测线粒体膜电位(mitochondria membrane voltage,ΔΨm)、胞内钙离子浓度,探讨抑制PDK1与线粒体功能异常导致的细胞凋亡的关系。
5、在体内实验中,将LoVo细胞接种于裸鼠右侧腋下,用PDK1抑制剂和两种结肠癌常用化疗药物处理4周,测量皮下瘤体积并称重,免疫组织化学染色法检测肿瘤组织Caspase-3和PCNA表达。
结果
1、结直肠癌组织观察到HIF-1α主要表达在肿瘤细胞的胞浆和(或)胞核,肿瘤坏死明显的区域内和肿瘤浸润边缘尤为多见,PDK1表达主要定位于肿瘤细胞的胞浆;HIF-1α的表达阳性率为78.04%,PDK1表达的阳性率为76.20%,与癌旁结肠壁组织相比差异有统计学意义(P<0.01)。二者的表达不受患者性别、年龄、肿瘤位置和肿瘤大小的影响(P>0.05);低分化肿瘤HIF-1α与PDK1的阳性表达率高于高分化肿瘤(P<0.05);肿瘤有淋巴结转移者HIF-1α与PDK1的阳性表达率高于无淋巴结转移者(P<0.05);随着Dukes分期进展,HIF-1α与PDK1阳性表达率逐渐增高,C、D期表达情况较A、B期有显著差异(P<0.05);二者在结直肠癌组织中的表达呈显著正相关(γ=0.727,P<0.01);免疫荧光双标染色结果显示HIF-1α与PDK1在结直肠癌组织中明确存在共表达现象。
2、PDK1基因mRNA和蛋白在人结肠癌LS173T、LoVo、HT-29和SW620细胞中均明显表达,其中LoVo细胞中表达最高。
3、不同浓度(0-90mmol/L)DCA分别作用LoVo、LS174T、HT-29、SW620细胞48h,结肠癌细胞活性和增殖均受到明显抑制。小剂量DCA(2.5mmol/L、5 mmol/L)分别与化疗药物(5-FU和OXA)联合作用LoVo细胞48h。检测显示,与5FU、OXA单独作用LoVo细胞相比,联合小剂量DCA对细胞活性和增殖的抑制作用更加明显,数据分析后显示小剂量DCA与5FU、OXA均有协同作用。
4、流式细胞术检测不同浓度(0-30mmol/L)DCA分别作用LoVo、LS174T、HT-29、SW620细胞48h,结肠癌细胞凋亡明显增加。小剂量DCA联合5FU和OXA后凋亡细胞比例明显高于5FU和OXA单独作用,且具有统计学意义(P<0.05)。同时,在DCA作用下,LoVo细胞线粒体膜电位和胞内钙离子浓度均下降,且联合5FU和OXA后下降效果更加明显。
5、裸鼠体内实验中,三组LoVo移植瘤分别用DCA、OXA及两者联合用药处理后,皮下瘤平均体积和平均重量均明显小于对照组移植瘤(P<0.01)。与对照组比,三组药物处理的LoVo细胞皮下瘤增殖指数显著下降,而凋亡指数则明显升高。
结论
1、HIF-1α与PDK1在结直肠癌组织中存在过表达和共表达现象,其表达不受患者性别、年龄、肿瘤位置、肿瘤大小的影响,但在不同分化程度、不同Dukes分期和是否伴随淋巴结转移等因素中二者表达阳性率呈显著差异,且二者表达呈显著正相关。
2、PDK1阻断剂DCA可通过降低线粒体膜电位,增加线粒体膜通透性等机制来诱导结肠癌细胞凋亡。
3、抑制PDK1可通过增加线粒体膜通透性,降低线粒体膜电位,降低胞内钙离子浓度,增加细胞凋亡等机制来发挥与化疗药物的协同作用。
Background and aims
Colorectal cancer is the one of the most common malignant tumor in the world, and the change of people’s lives and diet lead to the high morbidity and mortality of colorectal cancer. It is reported that the morbidity increases 4.2% every year. Now the effctive treatment includs operation and chemotherapy, which ease the pains of patients partly. However, the current treatment still has deficiency. The efficacy of surgery and chemotherapy is not significant for advanced stage patients.Therefore,other adjuvant therapies are required for colorectal cancer and enhanced effect of chemotherapy.
Early in the 1920s, Otto Warburg proposed that cancer cells obtained energy from glycolysis in hypoxia enviroment. Some cancer cells depended on glycolysis even in the presence of available oxygen, and such an enhancement of glycolysis becomes known as the“Warburg effect”. The most striking biochemical phenotype of malignant tumor cells is their elevated glycolysis,which is one of the main energy sources of ATP. Based on Warburg effect,positron emission tomography(PET)is widely used in the clinical diagnosis of cancers.Importantly,the therapeutic strategies targeting tumor bioenergetics are being explored.
Among the four mammalian pyruvate dehydrogenase kinase 1(PDK1)types(I-IV), which catalyze the pyruvate dehydrogenase (PDH), which is one of the most important steps of glycolysis. PDK1 is predominantly overexpressed in the most poorly differentiated malignant tumors.In such normal tissues as cardiac and skeletal muscles,and pancreatic island, PDK1 often maintains very low levels. And PDK1 is quite important in the process of PDH phosphorylation, and regulates PDH in long term. It is repoted that PDK1 could be regulated by HIF-1α,which is associated with the hypoxia enviroment of tumor.Recent studies have shown that PDK1 plays important roles in both glucose metabolism and cellular apoptosis,and inhibition of PDK1 could induce apoptosis of malignant tumor cells. In vitro study, it has been found that HIF-1αand PDK1 over express in head and neck squamous cancer, Non-Small-Cell Lung Cancer and brest cancer. It has not been reported whether high HIF-1αor PDK1 activity was found in in human colorectal cancer tissues, or high PDK1 in cultured colorectal cancer cells and animal models of experimental colorectal cancer. As well as the expression of PDK1, its mechanisms and their therapeutic significance are not clear in colorectal cancer.
The result of chemotherapy is poor although it is a major treatment. It is known that colon cancer cells are not very sensitive to single dose chemotherapy. PDK1 integrates energy metabolism of tumor cell with control of apoptosis,which may contribute to treatment, and inhibitiong of PDK1 may enhance the effectiveness of current chemotherapy. Regulation of mitochondria influences the growth,proliferation and apoptosis of cells.
It was indeedly demonstrated that the structure and function of mitochondria changes in cancer cells which resulted in incomplete of mitochondria membrane and high resting membrane potential of mitochondria membrane. As a result, the proliferation is inhibited and apoptosis is induced. It is proposed that inhibition of PDK1 regulates the membrane structure and function and decreases the membrane potential. Whether it is related to the proliferation and apoptosis with colorectal cancer remain be studied.
The aim of this study is to exam the expression and distribution of HIF-1αand PDK1 in human colorectal cancer tissue, as well as the expression of PDK1 in four colorectal cancer cell lines. Also, we observed the effect of PDK1 inhibition on cell proliferation, apoptosis and chemotherapeutic treatment in colorectal cancer cells. Furthermore, the effect of mitochondria function was also analyzed .
Methods
1、The expression and distribution of hypoxia-inducible factor-1α(HIF-1α) and PDK1 in human colorectal cancer tissue were detected in human colon carcinoma tissue by immunohistochemistry using streptavidin/peroxidase(SP) and double-label immunofluorescence methods. And the relationship with the certain clinicopathological features, such as tumor location, differentiation degrees, Dukes’stages and lymphatic invasion were analyzed.
2、The mRNA and protein expression of PDK1 in human colorectal cancer tissue and four kinds of human colon cancer cell lines were detected by immunohistochemistry, RT-PCR, real-time PCR , immunofluorescence and Western blot analysis respectively.
2、The effect of PDK1 inhibitor(Dichloroacetate,DCA)on colon cancer cell proliferation,apoptosis and chemotherapeutic sensitivity were analyzed via MTT assay, BrdU uptake test, Hoechst 33258 stain and Annex-V staining respectively.
3、The mechanism that DCA induced cellular apoptosis were assessed by means of exploring mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration.
4、In vivo, LoVo cells were inoculated in the right front armpits of the mice, then the effect of DCA and OXA on the mass of tumor was observed the tumors were measured and weighed after a 4-week follow-up period. Then,the expression of Caspase-3 and PCNA were detected by immunohistochemistry.
Results
1、Positive expression of HIF-1αans PDK1 was found in 78.04% and 76.20% of cases of colorectal cancer respectively,which were much different than that in normal samples(P<0.01).The positive expression of PDK1 correlated with the Duke’s stages of cancer cells and lymph node metastasis(P<0.05),but not related to either patient’s gender,age,tumor location, tumor size or Dukes’stage(P>0.05). Association analysis displays that the expression of HIF-1αprotein is correlated significantly with PDK1.
2、High mRNA and protein expression of PDK1 were observed in colorectal cancer tissue and four kinds of human colon cancer cells(LS174T,LoVo,HT-29 and SW-620) respectively. And LoVo expression is the highest.
2、Treated with DCA at different concentrations(0-90mmol/L)for 48h, there were obvious growth inhibition of LoVo,LS174T,HT-29 and SW620 cells respectively. Combined with low dose of DCA(2.5mmol/l,5 mmol/l)for 48h, the inhibition rate of 5-fluorouracil(5FU,5,10μg/ml) and Oxaliplatin(OXA,0.5,1μg/ml) is higher than 5FU or OXA alone on LoVo cell proliferation. After analysis of data, it is showen that DCA has synergetic effect with 5FU or OXA.
3、When cells are treated with DCA at different concentrations(0-30mmol/L), cellular apoptosis is found via Hoechst 33258 stain and flow cytometry. Combined with low dose of DCA(2.5mmol/l,5 mmol/l)for 48h, the apoptosis rate of 5-fluorouracil(5FU,5,10μg/ml) and Oxaliplatin(OXA,0.5,1μg/ml) is higher than 5FU or OXA alone on LoVo cell apoptosis. And the decreasment of mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration is lower than 5FU and OXA alone.
4、In nude mice experiment, three groups of LoVo transplantable tumor were treated with DCA or OXA or combination. The average tumor volume and weight were significant lower than those of the control respectively(both,P<0.01).As compared with control, proliferation indexes of the LoVo cells treated with three groups of drugs were significant lower, and their apoptosis indexes were significant higher.
Conclusion
1、Overexpression and coexpression of HIF-1αandPDK1 protein do exist in human colorectal cancer tissue.The expression of HIF-1αandPDK1 is not correlated with gender, age,location,and differentiation degree.But the expression of HIF-1αand PDK1 at different Dukes’stages and whether involved in lymphatic invasion shows a significant difference. Association analysis displays that the expression of HIF-1αprotein is correlated significantly with PDK1.
2、PDK1 inhibitor DCA induced colorectal cancer cells apoptosis via decreasing the mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration, and increasing mitochondrial membrane permeability.
3、Inhibition of PDK1 increases the chemosnsitivity of colorectal cancer LoVo cells via decreasing the mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration, and increasing mitochondrial membrane permeability.
结直肠癌是临床最常见的恶性肿瘤之一,由于生活习惯和饮食结构的改变,其发病率和死亡率均有逐年升高的趋势,资料显示我国结直肠癌的发病率正以年均4.2%的速度增长。手术和化疗是结直肠癌的常规治疗手段,虽然在一定程度上缓解了患者的痛苦,延长其寿命,但还存在着一定的缺陷。对于中晚期结直肠癌患者而言,手术效果差,化疗却面临越来越频繁的耐药难题,因此,探寻治疗结直肠癌和提高化疗效果的新策略是目前非常迫切的任务。
早在20世纪20年代,Otto Warburg就提出,肿瘤细胞能够通过糖酵解途径在缺乏氧的条件下获得能量,有些肿瘤细胞甚至在获得充足氧气时仍继续依赖糖酵解途径产能,肿瘤细胞这一特殊生化表型,被称为Warburg效应。因此,活跃的糖酵解代谢是恶性肿瘤细胞显著的生化特征,是肿瘤细胞能量ATP的重要来源。临床上已利用肿瘤细胞特异性的Warburg效应,通过正电子发射断层显像(positron emission tomography,PET)技术来诊断恶性肿瘤,而探索通过干预糖酵解代谢途径来治疗恶性肿瘤的策略正越来越受瞩目。
丙酮酸脱氢酶激酶(pyruvate dehydrogenase kinase,PDK)是细胞糖酵解关键限速酶丙酮酸脱氢酶(PDH)的调节酶。在人类和啮齿类动物中目前鉴定出有4种PDK同工酶:PDKI、PDK2、PDK3以及PDK4。其中PDK1在心脏、胰岛和骨骼肌中被检测到微量表达,在PDH磷酸化过程中发挥重要的功能,尤其在PDH长期调节中尤为重要。研究发现PDK1的表达可被缺氧诱导因子HIF-1α调控,可能与肿瘤缺氧的环境有关。近年研究表明,PDK1除了具有糖代谢酶的催化活性,对细胞凋亡拮抗还有一定的影响,药物抑制PDK1可诱导肿瘤细胞凋亡。体外细胞培养显示,HIF-1α和PDK1在头颈部癌、非小细胞肺癌、乳腺癌等细胞中表达增高。但HIF-1α和PDK1在人结肠癌组织中的表达和关系尚不清楚,PDK1在结肠癌中的表达、治疗意义以及其相关作用机制还尚不明确。
化疗作为结肠癌的重要治疗手段,目前效果并不理想,尤其单药化疗已经被联合化疗所取代。PDK1蛋白兼有促进能量ATP合成和影响凋亡的双重功能,因此推测PDK1与结直肠癌化疗有关,抑制PDK1可能增强化疗药物的治疗效果。
线粒体功能的调节在细胞的生长、增殖、凋亡等过程中发挥重要的功能。以往研究表明,恶性肿瘤细胞中线粒体的结构和功能均发生了一定的变化,线粒体膜结构不完整,且糖酵解途径获能导致线粒体膜静息电位高于正常,这些使得恶性肿瘤细胞的增殖被促进而凋亡被抑制。相关研究指出抑制PDK1能改善结构和功能,降低线粒体膜电位。但是这一效是否也是引起结直肠癌细胞增殖和凋亡的机制之一尚需实验证实。
本课题拟检测HIF-1α和PDK1在人结肠癌组织中的表达,观察HIF-1α与PDK1的关系;比较PDK1在正常组织和结肠癌组织中表达的区别与病理联系,以期说明PDK1的表达与结肠癌发生发展的关系。通过抑制PDK1,观察结肠癌细胞增殖、化疗药物协同作用、凋亡相关因子和线粒体结构和功能的变化,以探讨PDK1在结肠癌细胞增殖和化疗中的作用及机制。
方法
1、通过免疫组织化学染色及免疫荧光双标染色技术观察结直肠癌组织标本中HIF-1α与PDK1的表达和分布情况,了解二者在结直肠癌组织中表达的关系及与肿瘤分期、淋巴转移等临床病理参数的关系。
2、采用RT-PCR、real-time PCR和免疫荧光、Western印迹法,检测人结肠癌组织和4种人结肠癌细胞株PDK1基因和蛋白表达。
3、应用MTT、BrdU掺入法、Hoechst33258核染色及Annex-V双染色法,观察PDK1阻断剂(Dichloroacetate,DCA)对结肠癌细胞活性、增殖、凋亡和化疗的影响。
4、通过流式细胞仪检测线粒体膜电位(mitochondria membrane voltage,ΔΨm)、胞内钙离子浓度,探讨抑制PDK1与线粒体功能异常导致的细胞凋亡的关系。
5、在体内实验中,将LoVo细胞接种于裸鼠右侧腋下,用PDK1抑制剂和两种结肠癌常用化疗药物处理4周,测量皮下瘤体积并称重,免疫组织化学染色法检测肿瘤组织Caspase-3和PCNA表达。
结果
1、结直肠癌组织观察到HIF-1α主要表达在肿瘤细胞的胞浆和(或)胞核,肿瘤坏死明显的区域内和肿瘤浸润边缘尤为多见,PDK1表达主要定位于肿瘤细胞的胞浆;HIF-1α的表达阳性率为78.04%,PDK1表达的阳性率为76.20%,与癌旁结肠壁组织相比差异有统计学意义(P<0.01)。二者的表达不受患者性别、年龄、肿瘤位置和肿瘤大小的影响(P>0.05);低分化肿瘤HIF-1α与PDK1的阳性表达率高于高分化肿瘤(P<0.05);肿瘤有淋巴结转移者HIF-1α与PDK1的阳性表达率高于无淋巴结转移者(P<0.05);随着Dukes分期进展,HIF-1α与PDK1阳性表达率逐渐增高,C、D期表达情况较A、B期有显著差异(P<0.05);二者在结直肠癌组织中的表达呈显著正相关(γ=0.727,P<0.01);免疫荧光双标染色结果显示HIF-1α与PDK1在结直肠癌组织中明确存在共表达现象。
2、PDK1基因mRNA和蛋白在人结肠癌LS173T、LoVo、HT-29和SW620细胞中均明显表达,其中LoVo细胞中表达最高。
3、不同浓度(0-90mmol/L)DCA分别作用LoVo、LS174T、HT-29、SW620细胞48h,结肠癌细胞活性和增殖均受到明显抑制。小剂量DCA(2.5mmol/L、5 mmol/L)分别与化疗药物(5-FU和OXA)联合作用LoVo细胞48h。检测显示,与5FU、OXA单独作用LoVo细胞相比,联合小剂量DCA对细胞活性和增殖的抑制作用更加明显,数据分析后显示小剂量DCA与5FU、OXA均有协同作用。
4、流式细胞术检测不同浓度(0-30mmol/L)DCA分别作用LoVo、LS174T、HT-29、SW620细胞48h,结肠癌细胞凋亡明显增加。小剂量DCA联合5FU和OXA后凋亡细胞比例明显高于5FU和OXA单独作用,且具有统计学意义(P<0.05)。同时,在DCA作用下,LoVo细胞线粒体膜电位和胞内钙离子浓度均下降,且联合5FU和OXA后下降效果更加明显。
5、裸鼠体内实验中,三组LoVo移植瘤分别用DCA、OXA及两者联合用药处理后,皮下瘤平均体积和平均重量均明显小于对照组移植瘤(P<0.01)。与对照组比,三组药物处理的LoVo细胞皮下瘤增殖指数显著下降,而凋亡指数则明显升高。
结论
1、HIF-1α与PDK1在结直肠癌组织中存在过表达和共表达现象,其表达不受患者性别、年龄、肿瘤位置、肿瘤大小的影响,但在不同分化程度、不同Dukes分期和是否伴随淋巴结转移等因素中二者表达阳性率呈显著差异,且二者表达呈显著正相关。
2、PDK1阻断剂DCA可通过降低线粒体膜电位,增加线粒体膜通透性等机制来诱导结肠癌细胞凋亡。
3、抑制PDK1可通过增加线粒体膜通透性,降低线粒体膜电位,降低胞内钙离子浓度,增加细胞凋亡等机制来发挥与化疗药物的协同作用。
Background and aims
Colorectal cancer is the one of the most common malignant tumor in the world, and the change of people’s lives and diet lead to the high morbidity and mortality of colorectal cancer. It is reported that the morbidity increases 4.2% every year. Now the effctive treatment includs operation and chemotherapy, which ease the pains of patients partly. However, the current treatment still has deficiency. The efficacy of surgery and chemotherapy is not significant for advanced stage patients.Therefore,other adjuvant therapies are required for colorectal cancer and enhanced effect of chemotherapy.
Early in the 1920s, Otto Warburg proposed that cancer cells obtained energy from glycolysis in hypoxia enviroment. Some cancer cells depended on glycolysis even in the presence of available oxygen, and such an enhancement of glycolysis becomes known as the“Warburg effect”. The most striking biochemical phenotype of malignant tumor cells is their elevated glycolysis,which is one of the main energy sources of ATP. Based on Warburg effect,positron emission tomography(PET)is widely used in the clinical diagnosis of cancers.Importantly,the therapeutic strategies targeting tumor bioenergetics are being explored.
Among the four mammalian pyruvate dehydrogenase kinase 1(PDK1)types(I-IV), which catalyze the pyruvate dehydrogenase (PDH), which is one of the most important steps of glycolysis. PDK1 is predominantly overexpressed in the most poorly differentiated malignant tumors.In such normal tissues as cardiac and skeletal muscles,and pancreatic island, PDK1 often maintains very low levels. And PDK1 is quite important in the process of PDH phosphorylation, and regulates PDH in long term. It is repoted that PDK1 could be regulated by HIF-1α,which is associated with the hypoxia enviroment of tumor.Recent studies have shown that PDK1 plays important roles in both glucose metabolism and cellular apoptosis,and inhibition of PDK1 could induce apoptosis of malignant tumor cells. In vitro study, it has been found that HIF-1αand PDK1 over express in head and neck squamous cancer, Non-Small-Cell Lung Cancer and brest cancer. It has not been reported whether high HIF-1αor PDK1 activity was found in in human colorectal cancer tissues, or high PDK1 in cultured colorectal cancer cells and animal models of experimental colorectal cancer. As well as the expression of PDK1, its mechanisms and their therapeutic significance are not clear in colorectal cancer.
The result of chemotherapy is poor although it is a major treatment. It is known that colon cancer cells are not very sensitive to single dose chemotherapy. PDK1 integrates energy metabolism of tumor cell with control of apoptosis,which may contribute to treatment, and inhibitiong of PDK1 may enhance the effectiveness of current chemotherapy. Regulation of mitochondria influences the growth,proliferation and apoptosis of cells.
It was indeedly demonstrated that the structure and function of mitochondria changes in cancer cells which resulted in incomplete of mitochondria membrane and high resting membrane potential of mitochondria membrane. As a result, the proliferation is inhibited and apoptosis is induced. It is proposed that inhibition of PDK1 regulates the membrane structure and function and decreases the membrane potential. Whether it is related to the proliferation and apoptosis with colorectal cancer remain be studied.
The aim of this study is to exam the expression and distribution of HIF-1αand PDK1 in human colorectal cancer tissue, as well as the expression of PDK1 in four colorectal cancer cell lines. Also, we observed the effect of PDK1 inhibition on cell proliferation, apoptosis and chemotherapeutic treatment in colorectal cancer cells. Furthermore, the effect of mitochondria function was also analyzed .
Methods
1、The expression and distribution of hypoxia-inducible factor-1α(HIF-1α) and PDK1 in human colorectal cancer tissue were detected in human colon carcinoma tissue by immunohistochemistry using streptavidin/peroxidase(SP) and double-label immunofluorescence methods. And the relationship with the certain clinicopathological features, such as tumor location, differentiation degrees, Dukes’stages and lymphatic invasion were analyzed.
2、The mRNA and protein expression of PDK1 in human colorectal cancer tissue and four kinds of human colon cancer cell lines were detected by immunohistochemistry, RT-PCR, real-time PCR , immunofluorescence and Western blot analysis respectively.
2、The effect of PDK1 inhibitor(Dichloroacetate,DCA)on colon cancer cell proliferation,apoptosis and chemotherapeutic sensitivity were analyzed via MTT assay, BrdU uptake test, Hoechst 33258 stain and Annex-V staining respectively.
3、The mechanism that DCA induced cellular apoptosis were assessed by means of exploring mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration.
4、In vivo, LoVo cells were inoculated in the right front armpits of the mice, then the effect of DCA and OXA on the mass of tumor was observed the tumors were measured and weighed after a 4-week follow-up period. Then,the expression of Caspase-3 and PCNA were detected by immunohistochemistry.
Results
1、Positive expression of HIF-1αans PDK1 was found in 78.04% and 76.20% of cases of colorectal cancer respectively,which were much different than that in normal samples(P<0.01).The positive expression of PDK1 correlated with the Duke’s stages of cancer cells and lymph node metastasis(P<0.05),but not related to either patient’s gender,age,tumor location, tumor size or Dukes’stage(P>0.05). Association analysis displays that the expression of HIF-1αprotein is correlated significantly with PDK1.
2、High mRNA and protein expression of PDK1 were observed in colorectal cancer tissue and four kinds of human colon cancer cells(LS174T,LoVo,HT-29 and SW-620) respectively. And LoVo expression is the highest.
2、Treated with DCA at different concentrations(0-90mmol/L)for 48h, there were obvious growth inhibition of LoVo,LS174T,HT-29 and SW620 cells respectively. Combined with low dose of DCA(2.5mmol/l,5 mmol/l)for 48h, the inhibition rate of 5-fluorouracil(5FU,5,10μg/ml) and Oxaliplatin(OXA,0.5,1μg/ml) is higher than 5FU or OXA alone on LoVo cell proliferation. After analysis of data, it is showen that DCA has synergetic effect with 5FU or OXA.
3、When cells are treated with DCA at different concentrations(0-30mmol/L), cellular apoptosis is found via Hoechst 33258 stain and flow cytometry. Combined with low dose of DCA(2.5mmol/l,5 mmol/l)for 48h, the apoptosis rate of 5-fluorouracil(5FU,5,10μg/ml) and Oxaliplatin(OXA,0.5,1μg/ml) is higher than 5FU or OXA alone on LoVo cell apoptosis. And the decreasment of mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration is lower than 5FU and OXA alone.
4、In nude mice experiment, three groups of LoVo transplantable tumor were treated with DCA or OXA or combination. The average tumor volume and weight were significant lower than those of the control respectively(both,P<0.01).As compared with control, proliferation indexes of the LoVo cells treated with three groups of drugs were significant lower, and their apoptosis indexes were significant higher.
Conclusion
1、Overexpression and coexpression of HIF-1αandPDK1 protein do exist in human colorectal cancer tissue.The expression of HIF-1αandPDK1 is not correlated with gender, age,location,and differentiation degree.But the expression of HIF-1αand PDK1 at different Dukes’stages and whether involved in lymphatic invasion shows a significant difference. Association analysis displays that the expression of HIF-1αprotein is correlated significantly with PDK1.
2、PDK1 inhibitor DCA induced colorectal cancer cells apoptosis via decreasing the mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration, and increasing mitochondrial membrane permeability.
3、Inhibition of PDK1 increases the chemosnsitivity of colorectal cancer LoVo cells via decreasing the mitochondrial membrane voltage(?Ψm)and intracellular Ca serum concentration, and increasing mitochondrial membrane permeability.
引文
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