（-）棉酚的抗肿瘤作用及其与Imatinib协同诱导人慢性髓性白血病细胞凋亡
摘要
棉酚是从棉籽中提取的一种多酚类化合物。研究表明,它对多种肿瘤都有杀伤作用。其中(-)棉酚成份被证明抗肿瘤效果最强。目前认为(-)棉酚的主要作用机制是抑制抗凋亡蛋白Bcl-2和Bcl-X_L。
慢性髓性白血病(CML)是一种血液系统恶性增生疾病。以细胞内出现嵌合蛋白Bcr-Ab1为主要特征。Bcr-Ab1是一种被持续激活的酪氨酸激酶,并激活多条信号传导通路,引起发病。
Imatinib(又称Gleevec,STI-571)是Bcr-Ab1的选择性抑制剂。在体内、体外实验中,Imatinib引起细胞凋亡,并且对临床上Bcr-Ab1阳性的患者有非常好的疗效。但是Imatinib对CML急变期的患者治疗效果却并不理想,而且很容易产生耐药。这促使人们不断寻找新的方案治疗CML。
本实验探讨了(-)棉酚与Imatinib联合应用对人CML细胞系K562的影响,以及(-)棉酚对K562细胞和K562耐Imatinib细胞系K562/R的抑制作用。
一、(-)棉酚及其与Imatinib合用对K562细胞的抑制作用
采用MTT的方法观察了(-)棉酚以及(-)棉酚与Imatinib联合应用对K562细胞的生长抑制作用。(-)棉酚抑制K562细胞生长,IC_(50)为5.22μmol/L。在6.25μmol/L浓度下,(-)棉酚使Imatinib对K562细胞的IC_(50)由1.56μmol/L降低到0.013μmol/L。说明(-)棉酚增强Imatinib对K562细胞的抑制作用。裸鼠中空纤维模型实验进一步证实,(-)棉酚可以抑制K562细胞在裸鼠体内的生长,并且(-)棉酚15mg/kg与Imatinib 30gmol/kg合用对裸鼠体内K562细胞抑制率达到93.7%。在10:1的比例下,用MTT方法得出(-)棉酚与Imatinib的联合指数＜1,说明二者具有协同作用。
在体外(-)棉酚5μmol/L与Imatinib 0.5μmol/L联合作用使大量K562细胞出现核碎裂这种典型的凋亡形态学改变,而(-)棉酚5μmol/L,Imatinib 0.5μmol/L分别作用引起的改变则不明显。采用流式细胞术技术,Annexin V/PI双染及PI单染两种方法都得出(-)棉酚与Imatinib联合应用使凋亡细胞比例明显增加。另外,(-)棉酚单独作用也可以使K562细胞凋亡增加,并使细胞周期阻滞于G0/G1期,并且呈剂量依赖性。用DiOC_6染色方法观察了线粒体膜电位(△Ψm)的变化,这也是线粒体功能的一种反映。(-)棉酚和Imatinib分别单独作用时均可引起△Ψm降低,(-)棉酚与Imatinib联合作用则使△Ψm降低更为明显。
为了阐明(-)棉酚与Imatinib引起K562细胞凋亡的作用机制,本研究中采用western blot方法检测了caspase3和细胞质内细胞色素C含量的变化。(-)棉酚使K562细胞caspase3前体减少,释放入细胞质的细胞色素C含量增加;(-)棉酚与Imatinib联合应用时细胞色素C释放增多,caspase3前体进一步减少,并出现被水解的小片段,这意味着caspase3被激活。由此可以推断(-)棉酚和Imatinib作用后的K562细胞,细胞色素C释放入细胞质并随后激活caspase3,引起细胞凋亡。
为了进一步研究(-)棉酚与Imatinib引起K562细胞生长抑制并诱导凋亡的机制,采用western blot和RT-PCR方法分析了Bcl-2家族成员蛋白和mRNA水平的变化,以及P21的变化。(-)棉酚使Bcl-X_L、Mcl-1蛋白水平降低,并使Bcl-X_LmRNA表达也降低。与Imatinib合用时Bcl-X_L、Mcl-1进一步降低,并且使Bcl-2,Bcl-X_L,Mcl-1,Bak mRNA表达降低,Bad mRNA表达增加。(-)棉酚可以诱导K562细胞P21表达,但Imatinib却具有相反的作用。在实验中还发现(-)棉酚和Imatinib单独应用并未能使Bcr-Ab1和JNK蛋白含量改变,但二者合用却明显降低Bcr-Ab1和JNK蛋白水平。
由此可见,(-)棉酚通过诱导K562细胞凋亡和周期阻滞于G0/G1期,从而抑制K562细胞的生长。(-)棉酚增强Imatinib引起的K562细胞凋亡,并且二者具有协同作用。(-)棉酚所引起的凋亡至少部分通过线粒体途径,其中抗凋亡蛋白Bcl-X_L、Mcl-1的减少是其机制之一。尽管(-)棉酚对Bcl-2表达水平影响甚微,但是,目前普遍认为(-)棉酚通过结合于Bcl-X_L和Bcl-2而影响其功能,并不能改变其蛋白水平,因此可以推断在Bcl-X_L高表达的K562细胞内,(-)棉酚也降低了Bcl-X_L和Bcl-2的抗凋亡作用。(-)棉酚与Imatinib联合应用时引起了较为广泛的Bcr-Ab1传导通路和凋亡传导通路上的改变,其中包括Bcl-X_L、Mcl-1、Bcl-2、Bcr-Ab1、JNK不同程度的下调。(-)棉酚与Imatinib联合应用对细胞周期影响并不显著,而(-)棉酚引起的G0/G1期阻滞应与P21的诱导表达有关。
二、K562/R细胞株的建立以及(-)棉酚对K562/R细胞的抑制作用
通过逐渐增加Imatinib浓度施加于K562细胞的方法建立了K562耐Imatinib细胞系K562/R。与K562细胞相比,K562/R细胞对Imatinib敏感性降低约15倍,而两个细胞系对(-)棉酚敏感程度基本一致。K562/R细胞Bcr-Ab1以及线粒体Bcl-2表达增高,Mcl-1则无变化。令人费解的是,K562/R细胞Bcl-X_L表达却显著降低,因此初步认为,所建立的细胞系为Bcr-Ab1、Bcl-2高表达细胞系。进一步的鉴定工作有待深入。
MTT方法和裸鼠中空纤维模型实验都证实,(-)棉酚可以抑制K562/R细胞的生长。体外实验IC_(50)为7.97μmol/L,裸鼠体内实验(-)棉酚15mg/kg、30mg/kg剂量的抑制作用与对照组相比有显著性差异。在体外(-)棉酚10μmol/L使K562/R细胞出现典型的凋亡形态学改变。采用流式细胞术技术,(-)棉酚使K562/R细胞凋亡增加,并使细胞周期阻滞于G0/G1期,且呈剂量依赖性。(-)棉酚5μmol/L、10μmol/L引起K562/R细胞△Ψm降低。10μmol/L(-)棉酚引起K562/R细胞色素C释放。为了进一步研究(-)棉酚与Imatinib引起K562/R细胞生长抑制并诱导凋亡的机制,采用western blot和RT-PCR方法分析了一些Bcl-2家族成员蛋白和mRNA水平的变化。(-)棉酚使Bcl-X_L、Mcl-1蛋白水平降低,Bax、Bad、BakmRNA表达增加。与K562细胞不同,(-)棉酚并未诱导K562/R细胞P21表达(结果未给出)。
由此可见,(-)棉酚通过诱导K562/R细胞凋亡和周期阻滞于G0/G1期,抑制K562/R细胞生长;(-)棉酚通过上调促凋亡基因表达,下调抗凋亡蛋白水平从而引起K562/R细胞凋亡;(-)棉酚所引起G0/G1期阻滞可能与P21无关。
综上所述,(-)棉酚可能对CML治疗有效,尤其与Imatinib合用将会更有前景。当然,在应用临床之前,对于(-)棉酚的研究还有待于进一步深入。
棉酚作为一种避孕药,已经证实其还对多种肿瘤细胞系有生长抑制作用。我们通过实验验证了棉酚及其衍生物棉醌对肿瘤细胞的生长抑制作用。其作用强度从高到低依次为(-)棉酚、棉醌、(±)棉酚、(+)棉酚。(-)棉酚可以诱导人黑色素瘤细胞A375产生凋亡,但对A375细胞周期无明显影响。(-)棉酚增强A375细胞P53蛋白表达,可能与其诱导凋亡有关,但对Bcl-2 mRNA表达无影响。研究还发现(-)棉酚可降低B16BL6细胞的侵袭能力和恶性程度。
Gossypol, a natural polyphenol extracted from cottonseed, has been shown to have antiproliferative activity in a variety of cancer cell lines. (-)gossypol was proved the potent component and regarded as a small-molecule inhibitor of antiapoptosis protein Bcl-2 and Bcl-X_L proteins.
Chronic myeloid leukemia (CML) is a hematopoietic neoplasm with the hallmark of chimeric oncoprotein, Bcr-Abl, which displays constitutive tyrosine kinase activity and activates several signaling transduction pathways. Imatinib (Gleevec, STI-571; Novartis, Inc.) is a tyrosine kinase inhibitor that competitively inhibits Bcr-Abl kinases. It specifically induces apoptosis both in vitro and in vivo, Imatinib has shown a substantial clinical effect in BCR/ABL-positive leukemia patients. Although demonstrating impressive clinical activity against chronic-phase CML, in the accelerated and blastic phases of CML (CML-BC) the outcome after imatinib therapy is unacceptably poor and imatinib resistance occur shortly after the treatment. These findings emphasize the need to identify novel therapies for CML.
In the present studies, we discussed the combination effect of (-)gossypol and imatinib on CML cell line K562, along with the effects of (-)gossypol on K562 and K562/R.
1. Inhibition effects of (-)gossypol and its combination with imatinib on K562 cells.
Effects of (-)gossypol and the combination of (-)gossypol with imatinib on the growth of K562 cells were analyzed by MTT assay. The growth of K562 cells was inhibited by (-)gossypol with the IC_(50) value of 5.22μmol/L. At the concentration of 6.25μmol/L (-)gossypol reduced the IC_(50) value of imatinib from 1.56μmol/L to 0.013μmol/L. In vivo result in nude mice bearing hollow fibers showed that (-)gossypol inhibited growth of K562 cells in nude mice, and co-treatment with (-)gossypol 15mg/kg and Imatinib 30μmol/kg inhibited K562 cell growth by 93.7%. At a fixed ratio of 10:1, combination index value of (-)gossypol and imatinib < 1.0, corresponding to synergistic interactions, was obtained in MTT assay.
Morphologically, combination of (-)gossypol 5μmol/L with imatinib 0.5μmol/L dramatically induced typical apoptotic change -- karyorrhexis in K562 cells, whereas the effect of either (-)gossypol 5μmol/L or imatinib 0.5μmol/L was barely seen. Flow cytometric assay was performed to evaluate the cell cycle and apoptosis, by staining with PI as well as Annexin V/PI, similar results came out that percentage of apoptotic K562 cells increased markedly after combination treatment with (-)gossypol and imatinib. PI staining showed that (-)gossypol increased apoptosis percentage and caused G0/G1 arrest of cell cycle in K562 cells in a dose-dependent manner. By using DiOC_6, loss ofΔΨm was also detected in (-)gossypol and imatinib treated K562 cells, which represent mitochondria function. Combination of (-)gossypol 5μmol/L and imatinib 0.5μmol/L leaded toΔΨm loss to a deeper extent in K562 cells .
In order to understand the pathways involved in (-)gossypol and imatinib induced apoptosis, the level of caspase3 and cytoplasmic cytochrome C were tested using western blot. Procaspase3 decreased and cytoplasmic cytochrome C increased after treatment with (-)gossypol or imatinib. Combination with (-)gossypol and imatinib increased cytoplasmic cytochrome C more, and decreased procaspase3 with cleaved fragments of caspase3 detected, which it predicted the activation of caspase3. It can be inferred that treatment with (-)gossypol and imatinib arose cytochrome C release in K562 cells and consequently activated caspase3. To highlight the mechanism of growth inhibition and apoptosis induced by (-)gossypol and imatinib, protein and mRNA expression of several Bcl-2 family members and P21 were analyzed using western blot and RT-PCR assays, (-)gossypol decreased Bcl-X_L, Mcl-1 protein level moderately and Bcl-2 protein level neglectablely. Combination with (-)gossypol and imatinib caused Bcl-X_L, Mcl-1 protein level to decrease markedly and had no obvious effect on Bcl-2 protein level. As far as the mRNA level was concerned, combination with (-)gossypol and imatinib decreased level of Bcl-2, Bcl-X_L, Mcl-1, Bak, and increased level of Bad, whereas (-)gossypol simply decreased Bcl-X_L level. P21 induction was observed in (-)gossypol treated K562 cells, however, imatinib seemed to have reverse effect on P21 induction. Combination with (-)gossypol and imatinib reduced expression of Bcr-Abl and JNK, while neither (-)gossypol nor imatinib caused detectable change of these two proteins at tested concentrations.
Conclusively, (-)gossypol inhibited the growth of CML cell line K562 by inducing apoptosis and G0/G1 arrest in cell cycle, (-)gossypol synergistically potentiated imatinib induced apoptosis in K562 cells. Mictochondria pathway contributed at least partly to (-)gossypol induced apoptosis with the decreased Bcl-X_L, Mcl-1 expression. Although (-)gossypol did not effect Bcl-2 expression significantly, in the well accepted view of that (-)gossypol interrupted function of Bcl-X_L and Bcl-2 by interacting with them, we confer that in K562 cells (-)gossypol also lower the function of Bcl-2 as well as Bcl-X_L which is overexpressed in Bcr-Abl positive cells. Combination with (-)gossypol and imatinib leaded to more extensive changes in Bcr-Abl signaling and apoptosis signaling in K562 cells, including downregulation of Bcl-X_l, Mcl-1, Bcl-2, Bcr-Abl, JNK by a varied extent. Combination treatment seemed to have less effect on cell cycle, although (-)gossypol caused G0/G1 arrest, which may partly owe to the induction of P21.
2. The establishment of K562/R cell line and inhibition of (-)gossypol in K562/R cells.
By increasing imatinib concentration in K562 cell culture step by step, an imatinib resistant K562 cell line was established, referred as K562/R. In contrast with K562 cell line, the sensitivity of K562/R to imatinib decreased 15-fold, while both cell lines had similar responses to (-)gossypol. By comparison, expression of Bcr-Abl and mitochondrial Bcl-2 dramatically increased, Mcl-1 was unchanged. However, Bcl-X_L decreased to an almost undetectable extent perplexingly. Anyway, because of the complex mechanisms in imatinib resistance further identifications between the two cell lines need to be done.
In vivo result from nude mice bearing hollow fibers showed that (-)gossypol inhibited growth of K562/R cells in nude mice. Compared with control group the dose of 15mg/kg and 30mg/kg had P values less than 0.05. Morphologically, (-)gossypol induced typical apoptotic change -- karyorrhexis in K562/R cells at concentration of 10μmol/L. Flow cytometric assay was performed to evaluate the cell cycle and apoptosis. Results showed that being treated with (-)gossypol, percentage of apoptotic K562/R cells increased, and G0/G1 arrest of cell cycle in K562/R cells was observed with a dose-dependent manner. Loss ofΔΨm was also detected in (-)gossypol treated K562/R cells, (-)gossypol caused cytochrome C release was proved by using western blot. To investigate the mechanism of growth inhibition and apoptosis of (-)gossypol in K562/R cells, protein and mRNA expression of several Bcl-2 family members were analyzed using western blot and RT-PCR assays, (-)gossypol decreased Bcl-X_L and Mcl-1 protein level, and increased mRNA level of Bax, Bad and Bak. Unlike in K562 cells (-)gossypol seemed to have no effect on P21 induction in K562/R cells(data not shown).
Conclusively, (-)gossypol inhibited K562/R by inducing apoptosis and G0/G1 arrest of cell cycle, (-)gossypol induced apoptosis by decreasing antiapoptosis proteins and increasing proapoptosis gene transcription. (-)gossypol caused G0/G1 arrest may have no relationship with P21.
In summary, (-)gossypol may be effective in CML treatment and combining it with imatinib may be more promising, although there is still more to be explored before (-)gossypol can be used in clinic.
Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative effects on many kinds of cancer cells in vitro. Here, we tested the antiproliferative effect of gossypol and its derivative gossypolone. The results showed that the rank of intensity was (-)gossypol, gossypolone, (±)gossypol, (+)gossypol. (-)gossypol induced apoptosis in human melanoma cell line A375, but did not cause cell cycle change. (-)gossypol increased P53 protein level in A375, which may contribute partly to the apoptosis, but it had no obvious effect on Bcl-2 mRNA expression. We also found that (-)gossypol lowered antimetastatic ability of B16BL6 cells.
慢性髓性白血病(CML)是一种血液系统恶性增生疾病。以细胞内出现嵌合蛋白Bcr-Ab1为主要特征。Bcr-Ab1是一种被持续激活的酪氨酸激酶,并激活多条信号传导通路,引起发病。
Imatinib(又称Gleevec,STI-571)是Bcr-Ab1的选择性抑制剂。在体内、体外实验中,Imatinib引起细胞凋亡,并且对临床上Bcr-Ab1阳性的患者有非常好的疗效。但是Imatinib对CML急变期的患者治疗效果却并不理想,而且很容易产生耐药。这促使人们不断寻找新的方案治疗CML。
本实验探讨了(-)棉酚与Imatinib联合应用对人CML细胞系K562的影响,以及(-)棉酚对K562细胞和K562耐Imatinib细胞系K562/R的抑制作用。
一、(-)棉酚及其与Imatinib合用对K562细胞的抑制作用
采用MTT的方法观察了(-)棉酚以及(-)棉酚与Imatinib联合应用对K562细胞的生长抑制作用。(-)棉酚抑制K562细胞生长,IC_(50)为5.22μmol/L。在6.25μmol/L浓度下,(-)棉酚使Imatinib对K562细胞的IC_(50)由1.56μmol/L降低到0.013μmol/L。说明(-)棉酚增强Imatinib对K562细胞的抑制作用。裸鼠中空纤维模型实验进一步证实,(-)棉酚可以抑制K562细胞在裸鼠体内的生长,并且(-)棉酚15mg/kg与Imatinib 30gmol/kg合用对裸鼠体内K562细胞抑制率达到93.7%。在10:1的比例下,用MTT方法得出(-)棉酚与Imatinib的联合指数＜1,说明二者具有协同作用。
在体外(-)棉酚5μmol/L与Imatinib 0.5μmol/L联合作用使大量K562细胞出现核碎裂这种典型的凋亡形态学改变,而(-)棉酚5μmol/L,Imatinib 0.5μmol/L分别作用引起的改变则不明显。采用流式细胞术技术,Annexin V/PI双染及PI单染两种方法都得出(-)棉酚与Imatinib联合应用使凋亡细胞比例明显增加。另外,(-)棉酚单独作用也可以使K562细胞凋亡增加,并使细胞周期阻滞于G0/G1期,并且呈剂量依赖性。用DiOC_6染色方法观察了线粒体膜电位(△Ψm)的变化,这也是线粒体功能的一种反映。(-)棉酚和Imatinib分别单独作用时均可引起△Ψm降低,(-)棉酚与Imatinib联合作用则使△Ψm降低更为明显。
为了阐明(-)棉酚与Imatinib引起K562细胞凋亡的作用机制,本研究中采用western blot方法检测了caspase3和细胞质内细胞色素C含量的变化。(-)棉酚使K562细胞caspase3前体减少,释放入细胞质的细胞色素C含量增加;(-)棉酚与Imatinib联合应用时细胞色素C释放增多,caspase3前体进一步减少,并出现被水解的小片段,这意味着caspase3被激活。由此可以推断(-)棉酚和Imatinib作用后的K562细胞,细胞色素C释放入细胞质并随后激活caspase3,引起细胞凋亡。
为了进一步研究(-)棉酚与Imatinib引起K562细胞生长抑制并诱导凋亡的机制,采用western blot和RT-PCR方法分析了Bcl-2家族成员蛋白和mRNA水平的变化,以及P21的变化。(-)棉酚使Bcl-X_L、Mcl-1蛋白水平降低,并使Bcl-X_LmRNA表达也降低。与Imatinib合用时Bcl-X_L、Mcl-1进一步降低,并且使Bcl-2,Bcl-X_L,Mcl-1,Bak mRNA表达降低,Bad mRNA表达增加。(-)棉酚可以诱导K562细胞P21表达,但Imatinib却具有相反的作用。在实验中还发现(-)棉酚和Imatinib单独应用并未能使Bcr-Ab1和JNK蛋白含量改变,但二者合用却明显降低Bcr-Ab1和JNK蛋白水平。
由此可见,(-)棉酚通过诱导K562细胞凋亡和周期阻滞于G0/G1期,从而抑制K562细胞的生长。(-)棉酚增强Imatinib引起的K562细胞凋亡,并且二者具有协同作用。(-)棉酚所引起的凋亡至少部分通过线粒体途径,其中抗凋亡蛋白Bcl-X_L、Mcl-1的减少是其机制之一。尽管(-)棉酚对Bcl-2表达水平影响甚微,但是,目前普遍认为(-)棉酚通过结合于Bcl-X_L和Bcl-2而影响其功能,并不能改变其蛋白水平,因此可以推断在Bcl-X_L高表达的K562细胞内,(-)棉酚也降低了Bcl-X_L和Bcl-2的抗凋亡作用。(-)棉酚与Imatinib联合应用时引起了较为广泛的Bcr-Ab1传导通路和凋亡传导通路上的改变,其中包括Bcl-X_L、Mcl-1、Bcl-2、Bcr-Ab1、JNK不同程度的下调。(-)棉酚与Imatinib联合应用对细胞周期影响并不显著,而(-)棉酚引起的G0/G1期阻滞应与P21的诱导表达有关。
二、K562/R细胞株的建立以及(-)棉酚对K562/R细胞的抑制作用
通过逐渐增加Imatinib浓度施加于K562细胞的方法建立了K562耐Imatinib细胞系K562/R。与K562细胞相比,K562/R细胞对Imatinib敏感性降低约15倍,而两个细胞系对(-)棉酚敏感程度基本一致。K562/R细胞Bcr-Ab1以及线粒体Bcl-2表达增高,Mcl-1则无变化。令人费解的是,K562/R细胞Bcl-X_L表达却显著降低,因此初步认为,所建立的细胞系为Bcr-Ab1、Bcl-2高表达细胞系。进一步的鉴定工作有待深入。
MTT方法和裸鼠中空纤维模型实验都证实,(-)棉酚可以抑制K562/R细胞的生长。体外实验IC_(50)为7.97μmol/L,裸鼠体内实验(-)棉酚15mg/kg、30mg/kg剂量的抑制作用与对照组相比有显著性差异。在体外(-)棉酚10μmol/L使K562/R细胞出现典型的凋亡形态学改变。采用流式细胞术技术,(-)棉酚使K562/R细胞凋亡增加,并使细胞周期阻滞于G0/G1期,且呈剂量依赖性。(-)棉酚5μmol/L、10μmol/L引起K562/R细胞△Ψm降低。10μmol/L(-)棉酚引起K562/R细胞色素C释放。为了进一步研究(-)棉酚与Imatinib引起K562/R细胞生长抑制并诱导凋亡的机制,采用western blot和RT-PCR方法分析了一些Bcl-2家族成员蛋白和mRNA水平的变化。(-)棉酚使Bcl-X_L、Mcl-1蛋白水平降低,Bax、Bad、BakmRNA表达增加。与K562细胞不同,(-)棉酚并未诱导K562/R细胞P21表达(结果未给出)。
由此可见,(-)棉酚通过诱导K562/R细胞凋亡和周期阻滞于G0/G1期,抑制K562/R细胞生长;(-)棉酚通过上调促凋亡基因表达,下调抗凋亡蛋白水平从而引起K562/R细胞凋亡;(-)棉酚所引起G0/G1期阻滞可能与P21无关。
综上所述,(-)棉酚可能对CML治疗有效,尤其与Imatinib合用将会更有前景。当然,在应用临床之前,对于(-)棉酚的研究还有待于进一步深入。
棉酚作为一种避孕药,已经证实其还对多种肿瘤细胞系有生长抑制作用。我们通过实验验证了棉酚及其衍生物棉醌对肿瘤细胞的生长抑制作用。其作用强度从高到低依次为(-)棉酚、棉醌、(±)棉酚、(+)棉酚。(-)棉酚可以诱导人黑色素瘤细胞A375产生凋亡,但对A375细胞周期无明显影响。(-)棉酚增强A375细胞P53蛋白表达,可能与其诱导凋亡有关,但对Bcl-2 mRNA表达无影响。研究还发现(-)棉酚可降低B16BL6细胞的侵袭能力和恶性程度。
Gossypol, a natural polyphenol extracted from cottonseed, has been shown to have antiproliferative activity in a variety of cancer cell lines. (-)gossypol was proved the potent component and regarded as a small-molecule inhibitor of antiapoptosis protein Bcl-2 and Bcl-X_L proteins.
Chronic myeloid leukemia (CML) is a hematopoietic neoplasm with the hallmark of chimeric oncoprotein, Bcr-Abl, which displays constitutive tyrosine kinase activity and activates several signaling transduction pathways. Imatinib (Gleevec, STI-571; Novartis, Inc.) is a tyrosine kinase inhibitor that competitively inhibits Bcr-Abl kinases. It specifically induces apoptosis both in vitro and in vivo, Imatinib has shown a substantial clinical effect in BCR/ABL-positive leukemia patients. Although demonstrating impressive clinical activity against chronic-phase CML, in the accelerated and blastic phases of CML (CML-BC) the outcome after imatinib therapy is unacceptably poor and imatinib resistance occur shortly after the treatment. These findings emphasize the need to identify novel therapies for CML.
In the present studies, we discussed the combination effect of (-)gossypol and imatinib on CML cell line K562, along with the effects of (-)gossypol on K562 and K562/R.
1. Inhibition effects of (-)gossypol and its combination with imatinib on K562 cells.
Effects of (-)gossypol and the combination of (-)gossypol with imatinib on the growth of K562 cells were analyzed by MTT assay. The growth of K562 cells was inhibited by (-)gossypol with the IC_(50) value of 5.22μmol/L. At the concentration of 6.25μmol/L (-)gossypol reduced the IC_(50) value of imatinib from 1.56μmol/L to 0.013μmol/L. In vivo result in nude mice bearing hollow fibers showed that (-)gossypol inhibited growth of K562 cells in nude mice, and co-treatment with (-)gossypol 15mg/kg and Imatinib 30μmol/kg inhibited K562 cell growth by 93.7%. At a fixed ratio of 10:1, combination index value of (-)gossypol and imatinib < 1.0, corresponding to synergistic interactions, was obtained in MTT assay.
Morphologically, combination of (-)gossypol 5μmol/L with imatinib 0.5μmol/L dramatically induced typical apoptotic change -- karyorrhexis in K562 cells, whereas the effect of either (-)gossypol 5μmol/L or imatinib 0.5μmol/L was barely seen. Flow cytometric assay was performed to evaluate the cell cycle and apoptosis, by staining with PI as well as Annexin V/PI, similar results came out that percentage of apoptotic K562 cells increased markedly after combination treatment with (-)gossypol and imatinib. PI staining showed that (-)gossypol increased apoptosis percentage and caused G0/G1 arrest of cell cycle in K562 cells in a dose-dependent manner. By using DiOC_6, loss ofΔΨm was also detected in (-)gossypol and imatinib treated K562 cells, which represent mitochondria function. Combination of (-)gossypol 5μmol/L and imatinib 0.5μmol/L leaded toΔΨm loss to a deeper extent in K562 cells .
In order to understand the pathways involved in (-)gossypol and imatinib induced apoptosis, the level of caspase3 and cytoplasmic cytochrome C were tested using western blot. Procaspase3 decreased and cytoplasmic cytochrome C increased after treatment with (-)gossypol or imatinib. Combination with (-)gossypol and imatinib increased cytoplasmic cytochrome C more, and decreased procaspase3 with cleaved fragments of caspase3 detected, which it predicted the activation of caspase3. It can be inferred that treatment with (-)gossypol and imatinib arose cytochrome C release in K562 cells and consequently activated caspase3. To highlight the mechanism of growth inhibition and apoptosis induced by (-)gossypol and imatinib, protein and mRNA expression of several Bcl-2 family members and P21 were analyzed using western blot and RT-PCR assays, (-)gossypol decreased Bcl-X_L, Mcl-1 protein level moderately and Bcl-2 protein level neglectablely. Combination with (-)gossypol and imatinib caused Bcl-X_L, Mcl-1 protein level to decrease markedly and had no obvious effect on Bcl-2 protein level. As far as the mRNA level was concerned, combination with (-)gossypol and imatinib decreased level of Bcl-2, Bcl-X_L, Mcl-1, Bak, and increased level of Bad, whereas (-)gossypol simply decreased Bcl-X_L level. P21 induction was observed in (-)gossypol treated K562 cells, however, imatinib seemed to have reverse effect on P21 induction. Combination with (-)gossypol and imatinib reduced expression of Bcr-Abl and JNK, while neither (-)gossypol nor imatinib caused detectable change of these two proteins at tested concentrations.
Conclusively, (-)gossypol inhibited the growth of CML cell line K562 by inducing apoptosis and G0/G1 arrest in cell cycle, (-)gossypol synergistically potentiated imatinib induced apoptosis in K562 cells. Mictochondria pathway contributed at least partly to (-)gossypol induced apoptosis with the decreased Bcl-X_L, Mcl-1 expression. Although (-)gossypol did not effect Bcl-2 expression significantly, in the well accepted view of that (-)gossypol interrupted function of Bcl-X_L and Bcl-2 by interacting with them, we confer that in K562 cells (-)gossypol also lower the function of Bcl-2 as well as Bcl-X_L which is overexpressed in Bcr-Abl positive cells. Combination with (-)gossypol and imatinib leaded to more extensive changes in Bcr-Abl signaling and apoptosis signaling in K562 cells, including downregulation of Bcl-X_l, Mcl-1, Bcl-2, Bcr-Abl, JNK by a varied extent. Combination treatment seemed to have less effect on cell cycle, although (-)gossypol caused G0/G1 arrest, which may partly owe to the induction of P21.
2. The establishment of K562/R cell line and inhibition of (-)gossypol in K562/R cells.
By increasing imatinib concentration in K562 cell culture step by step, an imatinib resistant K562 cell line was established, referred as K562/R. In contrast with K562 cell line, the sensitivity of K562/R to imatinib decreased 15-fold, while both cell lines had similar responses to (-)gossypol. By comparison, expression of Bcr-Abl and mitochondrial Bcl-2 dramatically increased, Mcl-1 was unchanged. However, Bcl-X_L decreased to an almost undetectable extent perplexingly. Anyway, because of the complex mechanisms in imatinib resistance further identifications between the two cell lines need to be done.
In vivo result from nude mice bearing hollow fibers showed that (-)gossypol inhibited growth of K562/R cells in nude mice. Compared with control group the dose of 15mg/kg and 30mg/kg had P values less than 0.05. Morphologically, (-)gossypol induced typical apoptotic change -- karyorrhexis in K562/R cells at concentration of 10μmol/L. Flow cytometric assay was performed to evaluate the cell cycle and apoptosis. Results showed that being treated with (-)gossypol, percentage of apoptotic K562/R cells increased, and G0/G1 arrest of cell cycle in K562/R cells was observed with a dose-dependent manner. Loss ofΔΨm was also detected in (-)gossypol treated K562/R cells, (-)gossypol caused cytochrome C release was proved by using western blot. To investigate the mechanism of growth inhibition and apoptosis of (-)gossypol in K562/R cells, protein and mRNA expression of several Bcl-2 family members were analyzed using western blot and RT-PCR assays, (-)gossypol decreased Bcl-X_L and Mcl-1 protein level, and increased mRNA level of Bax, Bad and Bak. Unlike in K562 cells (-)gossypol seemed to have no effect on P21 induction in K562/R cells(data not shown).
Conclusively, (-)gossypol inhibited K562/R by inducing apoptosis and G0/G1 arrest of cell cycle, (-)gossypol induced apoptosis by decreasing antiapoptosis proteins and increasing proapoptosis gene transcription. (-)gossypol caused G0/G1 arrest may have no relationship with P21.
In summary, (-)gossypol may be effective in CML treatment and combining it with imatinib may be more promising, although there is still more to be explored before (-)gossypol can be used in clinic.
Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative effects on many kinds of cancer cells in vitro. Here, we tested the antiproliferative effect of gossypol and its derivative gossypolone. The results showed that the rank of intensity was (-)gossypol, gossypolone, (±)gossypol, (+)gossypol. (-)gossypol induced apoptosis in human melanoma cell line A375, but did not cause cell cycle change. (-)gossypol increased P53 protein level in A375, which may contribute partly to the apoptosis, but it had no obvious effect on Bcl-2 mRNA expression. We also found that (-)gossypol lowered antimetastatic ability of B16BL6 cells.
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