蝎毒素选择性诱导非霍奇金淋巴瘤细胞凋亡及其作用机制的研究
|
摘要
研究背景和研究目的
恶性淋巴瘤是淋巴结或淋巴结外部分淋巴组织的免疫细胞肿瘤,一般分为霍奇金淋巴瘤(Hodgkin's Lymphoma)和非霍奇金淋巴瘤(non-Hodgkin'sLymphoma,NHL)两大类。我国霍奇金淋巴瘤的发病率总体较低,非霍奇金淋巴瘤是最常见的淋巴系统恶性肿瘤。据世界卫生组织统计,在我国近年来非霍奇金淋巴瘤的发病率呈上升趋势,在过去20年,它的发病率增加了75%,在发病率增长最快的肿瘤中位居第3位。因此NHL的防治工作一直是我国医药卫生领域的重点,寻找安全有效的药物是肿瘤治疗中的重要课题。近年来动物来源药物越来越受重视,充分利用我国动物资源开发新的抗肿瘤药物,成为肿瘤研究领域的重要内容。
蝎毒作为中医传统药材已经应用几千年了。在中国第一部药典《本草纲目》中,全蝎被用于治疗多种疾病。蝎毒为传统药材全蝎的主要活性物质,存在于蝎尾部毒囊内,鳌刺时由鳌针排出。现代药理学研究表明,蝎毒主要由蛋白质和非蛋白质两部分组成,蝎毒的主要活性成分是蛋白质。蝎毒具有广泛的药理学性质,近年来国外对蝎毒的研究主要集中在膜通道阻滞的作用和抗毒素方面,罕见关于蝎毒抗肿瘤方面的研究报道,国内的研究侧重其抗肿瘤、抗风湿、抗癫痫、抗炎、纤溶、镇痛以及对心血管病的作用等方面;国内学者对蝎毒的抗肿瘤作用进行了较多研究,从蝎及蝎毒的抗肿瘤作用、蝎毒的成分和主要成分的药理作用、全蝎及蝎毒抗肿瘤的临床应用等方面分析,结果与结论显示,蝎毒可抑制多种人类肿瘤细胞生长,诱导肿瘤细胞凋亡。但是,蝎毒素对肿瘤细胞杀伤或生长抑制具有一定的肿瘤差异性,有一定的肿瘤谱;而且有关蝎毒抗肿瘤作用与靶细胞基因表达的关联问题目前未见文献报道,其诱导细胞凋亡的具体分子机制至今尚未完全阐明,这是一个值得探讨的领域。
肿瘤的发生通常被认为是由于细胞原癌基因的激活和抑癌基因的失活,扰乱了正常的增殖、分化和凋亡的调控,导致细胞增殖过度而凋亡不足。PTEN(第10号染色体缺失性磷酸酶-张力蛋白同源性基因)基因是一种新发现的肿瘤抑制基因。它的蛋白产物具有脂磷酸酶活性,其主要作用底物是磷脂酰肌醇-3,4,5-三磷酸(PIP3)。PIP3是PI3K/Akt信号转导通路的关键性组分,负责刺激正常细胞生长以及抑制肿瘤细胞的生长。PTEN使PIP3形成PIP2,降低PIP3水平,抑制了Akt的活性,因此降低了Bad磷酸化,导致细胞的凋亡;促进了p27的表达,引起细胞周期停滞。已发现这种基因在淋巴瘤细胞中5%可发生突变,导致对肿瘤的生长失去抑制作用。
非霍奇金淋巴瘤(NHL)作为我国最常见淋巴系统的恶性肿瘤,近年来虽然放疗、化疗及造血干细胞移植的有了极大发展,淋巴瘤的死亡率并没有降低。人们致力于寻找新的、疗效肯定、毒副作用小的化疗药物,尤其是作用于肿瘤发生环节的药物。为了探讨蝎毒在NHL淋巴瘤治疗中的应用价值,我们研究了蝎毒对NHL淋巴瘤细胞株Raji和Jurkat细胞及正常人外周血淋巴细胞增殖、细胞周期和凋亡的影响。通过采用分子生物学技术检测凋亡相关蛋白PTEN、Akt、p-Akt、Bad、p-Bad、p27和PTEN mRNA的改变,以进一步阐明蝎毒的抗肿瘤作用的分子机制。
研究内容
1、蝎毒素诱导NHL细胞株Raji和Jurkat细胞凋亡的作用;
2、蝎毒素对Raji和Jurkat细胞周期分布的影响;
3、蝎毒素诱导Raji和Jurkat细胞凋亡的重要分子及信号通路;
4、蝎毒素对Raji和Jurkat胞周期相关蛋白的影响。
研究方法
1.甲基四唑蓝(MTT)法检测不同浓度、不同时间点蝎毒素(BmK)对NHL细胞株及人正常外周血淋巴细胞的抑制作用
选用NHL细胞株Raji和Jurkat及人正常外周血淋巴细胞为研究对象,采用台盼蓝染色,细胞计数仪计数,MTT法检测细胞存活率,由此确定各组的的IC50值。
2.细胞凋亡检测方法如下
2.1倒置相差显微镜观察药物作用后细胞形态学变化;
2.2 Hoechst3342荧光染色并利用荧光显微镜,观察细胞核形态学变化;
2.3 Annexin V-FITC和PI染色,流式细胞仪(FCM)检测细胞凋亡情况,计算细胞凋亡率;
2.4 LY294002联合蝎毒素对细胞凋亡的影响
在蝎毒素作用细胞前20分钟加入终浓度对20μM的LY294002,然后蝎毒素处理细胞48 h,FCM检测细胞凋亡率。
3.PI染色流式细胞仪(FCM)检测细胞周期分布情况。
4.Western blot进一步检测蝎毒素处理前后细胞周期、细胞凋亡及信号转导途径的相关蛋白PTEN、Akt、p-Akt、Bad、p-Bad、p27蛋白水平变化。
5.半定量逆转录聚合酶链反应(PT-PCR)检测PTEN mRNA的表达。
6.统计学处理:本实验所有数据用均数±标准差表达,实验组与对照组比较用t检验,组间均数比较采用单因素方差分析。
研究结果
1.台盼蓝染色,细胞计数仪计数和MTT法均显示:蝎毒素对NHL细胞株Raji和Jurkat细胞具有生长的抑制作用,呈浓度、时间相关性。蝎毒素对NHL细胞株和人外周血淋巴细胞(PBLs)作用48后,IC50分别为275.40μg/ml(Raji)、360.60μg/ml(Jurkat)和1110μg/mL(PBLs),各组间经统计学处理有显著性差异(p＜0.05)。以上结果提示:蝎毒素对NHL细胞株Raji和Jurkat细胞的增殖抑制作用强于人外周血淋巴细胞(PBLs),低浓度(≤500μg/ml)时对PBLs细胞的生长抑制作用不明显。两种NHL细胞株中,Raji细胞对蝎毒素较敏感,强于Jurkat细胞。
2.蝎毒对淋巴瘤细胞凋亡的影响:
2.1倒置相差显微镜观察发现:蝎毒处理Raji和Jurkat细胞48 h后,细胞大小不一,呈现细胞碎片及胞浆内颗粒增多等特征,并最终死亡。
2.2 Hoechst3342染色荧光显微镜检测发现:与对照组相比,蝎毒素处理组中Raji和Jurkat细胞核均呈现碎块状致密浓染,荧光染色增强;随着浓度增高,凋亡小体产生,符合凋亡的形态学改变。
2.3 Annexin V-FITC/PI染色FCM检测显示:蝎毒素能够诱导Raji和Jurkat细胞株凋亡,其凋亡率随用药剂量增加而增加,并且Raji细胞的早期凋亡率(43±7%)明显高于Jurkat细胞(24±5%),有统计学意义(P＜0.05)。蝎毒素作用PBLs细胞48小时后,各浓度组凋亡率较空白对照组的差异未显示统计学意义(P＞0.05)。以上结果提示:蝎毒素可选择性作用于NHL细胞株诱导其凋亡,而对人正常外周血淋巴细胞影响较小。
3.PI染色流式细胞仪(FCM)检测发现:蝎毒素引起NHL细胞株Raji和Jurkat细胞G0/G1期阻滞。
4.Western blot检测发现:蝎毒素促使Rajii细胞PTEN蛋白表达增加,同时p-Akt、p-Bad蛋白表达减少;而对Jurkat细胞以上蛋白表达无影响。提示蝎毒素诱导PTEN蛋白表达上调,且该PTEN蛋白为功能性蛋白,可以下调Akt、Bad磷酸化水平,从而影响PI3K/Akt信号转导通路,这可能是蝎毒素诱导Raji细胞凋亡的重要机制。
5.为了进一步验证PI3K/Akt信号转导通路在蝎毒素诱导Raji细胞凋亡中的作用,LY294002(PI3K特异抑制剂)联合蝎毒素对Raji细胞的影响。发现联合用药呈现叠加效应,不仅使Raji细胞凋亡率明显增加,同时使Akt磷酸化水平进一步降低。由此我们判断,蝎毒素通过上调PTEN表达水平,激活PI3K/Akt信号转导通路是蝎毒素诱导Raji细胞凋亡的重要机制之一。
6.RT-PCR法测PTEN mRNA的表达水平,Raji细胞中发现PTEN mRNA表达呈蝎毒素浓度依赖性增加,说明蝎毒素是在PTEN基因转录水平影响PTEN蛋白表达。
7.Western blot检测发现:蝎毒素作用Raji和Jurkat细胞48 h后,伴随着G1期阻滞,p27蛋白表达呈蝎毒素浓度依赖性增加。提示蝎毒素促使Raji和Jurkat细胞凋亡的机制还与上调p27蛋白进而导致G1期细胞阻滞有关。
结论
蝎毒素能够抑制NHL细胞株Raji和Jurkat细胞的增殖,并且呈时间、浓度依赖性。可以诱导Raji和Jurkat细胞凋亡和细胞周期G1期阻滞的能力。可能是通过以下的机制发挥作用的:1、在PTEN基因转录水平促进Raji细胞PTEN蛋白表达,抑制Akt、Bad的磷酸化,从而激活PI3K/Akt信号转导通路,这可能是蝎毒素诱导Raji细胞凋亡的重要机制之一。2、还可以通过非PTEN依赖途径,上调p27蛋白的表达,引起G1期细胞周期停滞,这是蝎毒诱导Raji和Jurkat细胞凋亡的共同通道。
BACKGROUND AND OBJECTIVE:
Malignant tumors are serious diseases endangering human health.E very year about 1,600,000 patients with cancers were newly increased in our country and about 1,300,000 patients died from tumors.With the growth of population and the acceleration of aging population,the incidence and mortality of tumors are on the rise.Therefore,the prevention and treatment of tumors have long been the focal work in the Chinese medicine.It is then an important subject to find medicines both safe and effective in the treatment of tumors.Recently more attentions have been attached to the drugs from animals,and antineoplastics have been developed in using fully the animal sources of our country,which has then become the important content in the research work of tumors.
The scorpion Buthus martensii Karsch(BmK) had been used as a Chinese traditional medicine for thousands of years.In the first authoritative Chinese pharmacopoeia "Compendium of Material Medica" edited by Li Shizhen in 1578, dried scorpion bodies were used to treat a variety of diseases.Scorpion venoms are complex mixtures of molecules,most of which are peptides displaying various kinds of biological activity.During the past two decades,several of these compounds have been identified.Toxins modulating Na~+,K~+,Ca~(2+) and Ckl~- currents across cell membranes have been described.Some enzymes like proteases and hyaluronidases as well as chlorotoxin-like peptides from this venom may have therapeutic potential,including anticancer activity.Recent studies have demonstrated that BmK venom can induce apoptotic cell death,and possesses anti-tumor effects.However,the molecular mechanisms that underlie the induction of apoptosis by BmK venom are poorly understood,and require further exploration.
PTEN(phosphatase and tensin homolog deleted from chromosome 10) gene is a new tumor suppressor gene proved to be a lipid phosphatase.The target lipid, called phosphatidylinositol-3,4,5-trisphosphate,PIP3,and it is a key component of the major cell growth control pathways,acting both to stimulate cell growth and to inhibit tumor cell proliferation blocking apoptosis.PTEN mutations have been linked to a variety of human cancers.Mutations of PTEN during tumorigenesis allow the mutated cells to grow unchecked when they should die.
Lymphoma is a cancer in the lymphatic system.Despite recent advances in radiotherapy,chemotherapy,and stem cell transplantation,the severe morbidity from lymphoma has not been alleviated.Much effort has been focused on the discovery and development of new chemopreventive agents,especially agents targeted at mechanisms known to be involved in the process of carcinogenesis. Therefore,we have sought to identify novel agents that can effectively prevent lymphoma carcinogenesis but have minimal toxicity to healthy cells.In this study, we examined the effects of BmK venom on two human lymphoma cell lines(Raji and Jurkat) compared with peripheral blood lymphocytes(PBLs),and focused on changes in the PTEN and the PI3K/Akt signal pathway and to further investigate the molecular mechanisms involved in these effects.
STUDY CONTENTS:
1.Identification of apoptosis induction in Raji and Jurkat cells by BmK venom.
2.Analysis of the effects of BmK venom on cell cycle distribution and p27 protein level.
3.Analysis of the apoptosis pathways activated by the small molecules in Raij and Jurkat cells.
METHODS:
1.MTT assay for cell viability,by which small molecules were screened initially.
2.Methods for apoptosis identification:
2.1 Observation of cell morphological changes by Phase Contrast Microscope.
2.2 Observation of nuclear morphological changes by Hoechst 3342 staining combined with fluorescence microscopy.
2.2 Analysis of the changes in apoptosis by flow cytometry.
3.Analysis of the changes in cell cycle distribution by flow cytometry.
4.Analysis of the changes in PTEN、Akt、p-Akt、Bad、p-Bad protein level by Western blot assay.
5.Analysis of the changes in PTEN mRNA level by PT-PCR assay.
RESULTS:
1.BmK venom inhibited the viability of Raji and Jurkat cells in a dose-and time-dependent manner,and the IC50 value calculated to be 275.40μg/ml(Raji) and 360.60μg/ml(Jurkat).
2.BmK venom-induced apoptosis:
When Raji and Jurkat cells were exposed to BmK venom for 24-48 h,cell shrinkage,membrane blebbing and apoptotic bodies occurred.Ultimately,many cells underwent death;
Hoechst 3342 staining combined with Fluorescence Microscopy showed that compared with the nuclei in the control group,the nuclei were stained into brightly blue after treated by BmK venom for 48h.
Flow cytometry showed that in Raji cells,the levels of p-Akt and p-Bad clearly decreased in a dose-dependent manner,while the levels of total Akt and Bad protein were stable.PTEN expression was inversely correlated with phosphorylation of Akt, indicating that the PTEN was functional.
3.Flow cytometry showed that with increasing concentrations of BmK venom,the number of Jurkat and Raji cells in the G_0/G_1 phase increased significantly compared with control cells,while the cells in S and G_2/M phase decreased accordingly.
4.In Raji cells,BmK venom up regulated the expression of PTEN accompanied by decreased levels of Akt and Bad phosphorylation.Treatment with BmK venom and LY294002(an inhibitor of Akt) synergistically enhanced apoptosis. The expression of p27 was increased in both PYEN-positive Raji and PTEN-negative Jurkat cells exposed to BmK venom.
CONCLUSION:
In summary,it was demonstrated that BmK venom could inhibit cell proliferation and induces apoptosis on human NHLs cell lines,Raji and Jurkat cells. The cytostatic effect was dose- and time-dependent.One possible mechanism may be suppression of Akt signal pathway via PTEN and up regulation of p27 with affecting auxiliary pathways.These data suggest that BMK may be effective in the treatment of non-Hodgkin's lymphoma.
恶性淋巴瘤是淋巴结或淋巴结外部分淋巴组织的免疫细胞肿瘤,一般分为霍奇金淋巴瘤(Hodgkin's Lymphoma)和非霍奇金淋巴瘤(non-Hodgkin'sLymphoma,NHL)两大类。我国霍奇金淋巴瘤的发病率总体较低,非霍奇金淋巴瘤是最常见的淋巴系统恶性肿瘤。据世界卫生组织统计,在我国近年来非霍奇金淋巴瘤的发病率呈上升趋势,在过去20年,它的发病率增加了75%,在发病率增长最快的肿瘤中位居第3位。因此NHL的防治工作一直是我国医药卫生领域的重点,寻找安全有效的药物是肿瘤治疗中的重要课题。近年来动物来源药物越来越受重视,充分利用我国动物资源开发新的抗肿瘤药物,成为肿瘤研究领域的重要内容。
蝎毒作为中医传统药材已经应用几千年了。在中国第一部药典《本草纲目》中,全蝎被用于治疗多种疾病。蝎毒为传统药材全蝎的主要活性物质,存在于蝎尾部毒囊内,鳌刺时由鳌针排出。现代药理学研究表明,蝎毒主要由蛋白质和非蛋白质两部分组成,蝎毒的主要活性成分是蛋白质。蝎毒具有广泛的药理学性质,近年来国外对蝎毒的研究主要集中在膜通道阻滞的作用和抗毒素方面,罕见关于蝎毒抗肿瘤方面的研究报道,国内的研究侧重其抗肿瘤、抗风湿、抗癫痫、抗炎、纤溶、镇痛以及对心血管病的作用等方面;国内学者对蝎毒的抗肿瘤作用进行了较多研究,从蝎及蝎毒的抗肿瘤作用、蝎毒的成分和主要成分的药理作用、全蝎及蝎毒抗肿瘤的临床应用等方面分析,结果与结论显示,蝎毒可抑制多种人类肿瘤细胞生长,诱导肿瘤细胞凋亡。但是,蝎毒素对肿瘤细胞杀伤或生长抑制具有一定的肿瘤差异性,有一定的肿瘤谱;而且有关蝎毒抗肿瘤作用与靶细胞基因表达的关联问题目前未见文献报道,其诱导细胞凋亡的具体分子机制至今尚未完全阐明,这是一个值得探讨的领域。
肿瘤的发生通常被认为是由于细胞原癌基因的激活和抑癌基因的失活,扰乱了正常的增殖、分化和凋亡的调控,导致细胞增殖过度而凋亡不足。PTEN(第10号染色体缺失性磷酸酶-张力蛋白同源性基因)基因是一种新发现的肿瘤抑制基因。它的蛋白产物具有脂磷酸酶活性,其主要作用底物是磷脂酰肌醇-3,4,5-三磷酸(PIP3)。PIP3是PI3K/Akt信号转导通路的关键性组分,负责刺激正常细胞生长以及抑制肿瘤细胞的生长。PTEN使PIP3形成PIP2,降低PIP3水平,抑制了Akt的活性,因此降低了Bad磷酸化,导致细胞的凋亡;促进了p27的表达,引起细胞周期停滞。已发现这种基因在淋巴瘤细胞中5%可发生突变,导致对肿瘤的生长失去抑制作用。
非霍奇金淋巴瘤(NHL)作为我国最常见淋巴系统的恶性肿瘤,近年来虽然放疗、化疗及造血干细胞移植的有了极大发展,淋巴瘤的死亡率并没有降低。人们致力于寻找新的、疗效肯定、毒副作用小的化疗药物,尤其是作用于肿瘤发生环节的药物。为了探讨蝎毒在NHL淋巴瘤治疗中的应用价值,我们研究了蝎毒对NHL淋巴瘤细胞株Raji和Jurkat细胞及正常人外周血淋巴细胞增殖、细胞周期和凋亡的影响。通过采用分子生物学技术检测凋亡相关蛋白PTEN、Akt、p-Akt、Bad、p-Bad、p27和PTEN mRNA的改变,以进一步阐明蝎毒的抗肿瘤作用的分子机制。
研究内容
1、蝎毒素诱导NHL细胞株Raji和Jurkat细胞凋亡的作用;
2、蝎毒素对Raji和Jurkat细胞周期分布的影响;
3、蝎毒素诱导Raji和Jurkat细胞凋亡的重要分子及信号通路;
4、蝎毒素对Raji和Jurkat胞周期相关蛋白的影响。
研究方法
1.甲基四唑蓝(MTT)法检测不同浓度、不同时间点蝎毒素(BmK)对NHL细胞株及人正常外周血淋巴细胞的抑制作用
选用NHL细胞株Raji和Jurkat及人正常外周血淋巴细胞为研究对象,采用台盼蓝染色,细胞计数仪计数,MTT法检测细胞存活率,由此确定各组的的IC50值。
2.细胞凋亡检测方法如下
2.1倒置相差显微镜观察药物作用后细胞形态学变化;
2.2 Hoechst3342荧光染色并利用荧光显微镜,观察细胞核形态学变化;
2.3 Annexin V-FITC和PI染色,流式细胞仪(FCM)检测细胞凋亡情况,计算细胞凋亡率;
2.4 LY294002联合蝎毒素对细胞凋亡的影响
在蝎毒素作用细胞前20分钟加入终浓度对20μM的LY294002,然后蝎毒素处理细胞48 h,FCM检测细胞凋亡率。
3.PI染色流式细胞仪(FCM)检测细胞周期分布情况。
4.Western blot进一步检测蝎毒素处理前后细胞周期、细胞凋亡及信号转导途径的相关蛋白PTEN、Akt、p-Akt、Bad、p-Bad、p27蛋白水平变化。
5.半定量逆转录聚合酶链反应(PT-PCR)检测PTEN mRNA的表达。
6.统计学处理:本实验所有数据用均数±标准差表达,实验组与对照组比较用t检验,组间均数比较采用单因素方差分析。
研究结果
1.台盼蓝染色,细胞计数仪计数和MTT法均显示:蝎毒素对NHL细胞株Raji和Jurkat细胞具有生长的抑制作用,呈浓度、时间相关性。蝎毒素对NHL细胞株和人外周血淋巴细胞(PBLs)作用48后,IC50分别为275.40μg/ml(Raji)、360.60μg/ml(Jurkat)和1110μg/mL(PBLs),各组间经统计学处理有显著性差异(p＜0.05)。以上结果提示:蝎毒素对NHL细胞株Raji和Jurkat细胞的增殖抑制作用强于人外周血淋巴细胞(PBLs),低浓度(≤500μg/ml)时对PBLs细胞的生长抑制作用不明显。两种NHL细胞株中,Raji细胞对蝎毒素较敏感,强于Jurkat细胞。
2.蝎毒对淋巴瘤细胞凋亡的影响:
2.1倒置相差显微镜观察发现:蝎毒处理Raji和Jurkat细胞48 h后,细胞大小不一,呈现细胞碎片及胞浆内颗粒增多等特征,并最终死亡。
2.2 Hoechst3342染色荧光显微镜检测发现:与对照组相比,蝎毒素处理组中Raji和Jurkat细胞核均呈现碎块状致密浓染,荧光染色增强;随着浓度增高,凋亡小体产生,符合凋亡的形态学改变。
2.3 Annexin V-FITC/PI染色FCM检测显示:蝎毒素能够诱导Raji和Jurkat细胞株凋亡,其凋亡率随用药剂量增加而增加,并且Raji细胞的早期凋亡率(43±7%)明显高于Jurkat细胞(24±5%),有统计学意义(P＜0.05)。蝎毒素作用PBLs细胞48小时后,各浓度组凋亡率较空白对照组的差异未显示统计学意义(P＞0.05)。以上结果提示:蝎毒素可选择性作用于NHL细胞株诱导其凋亡,而对人正常外周血淋巴细胞影响较小。
3.PI染色流式细胞仪(FCM)检测发现:蝎毒素引起NHL细胞株Raji和Jurkat细胞G0/G1期阻滞。
4.Western blot检测发现:蝎毒素促使Rajii细胞PTEN蛋白表达增加,同时p-Akt、p-Bad蛋白表达减少;而对Jurkat细胞以上蛋白表达无影响。提示蝎毒素诱导PTEN蛋白表达上调,且该PTEN蛋白为功能性蛋白,可以下调Akt、Bad磷酸化水平,从而影响PI3K/Akt信号转导通路,这可能是蝎毒素诱导Raji细胞凋亡的重要机制。
5.为了进一步验证PI3K/Akt信号转导通路在蝎毒素诱导Raji细胞凋亡中的作用,LY294002(PI3K特异抑制剂)联合蝎毒素对Raji细胞的影响。发现联合用药呈现叠加效应,不仅使Raji细胞凋亡率明显增加,同时使Akt磷酸化水平进一步降低。由此我们判断,蝎毒素通过上调PTEN表达水平,激活PI3K/Akt信号转导通路是蝎毒素诱导Raji细胞凋亡的重要机制之一。
6.RT-PCR法测PTEN mRNA的表达水平,Raji细胞中发现PTEN mRNA表达呈蝎毒素浓度依赖性增加,说明蝎毒素是在PTEN基因转录水平影响PTEN蛋白表达。
7.Western blot检测发现:蝎毒素作用Raji和Jurkat细胞48 h后,伴随着G1期阻滞,p27蛋白表达呈蝎毒素浓度依赖性增加。提示蝎毒素促使Raji和Jurkat细胞凋亡的机制还与上调p27蛋白进而导致G1期细胞阻滞有关。
结论
蝎毒素能够抑制NHL细胞株Raji和Jurkat细胞的增殖,并且呈时间、浓度依赖性。可以诱导Raji和Jurkat细胞凋亡和细胞周期G1期阻滞的能力。可能是通过以下的机制发挥作用的:1、在PTEN基因转录水平促进Raji细胞PTEN蛋白表达,抑制Akt、Bad的磷酸化,从而激活PI3K/Akt信号转导通路,这可能是蝎毒素诱导Raji细胞凋亡的重要机制之一。2、还可以通过非PTEN依赖途径,上调p27蛋白的表达,引起G1期细胞周期停滞,这是蝎毒诱导Raji和Jurkat细胞凋亡的共同通道。
BACKGROUND AND OBJECTIVE:
Malignant tumors are serious diseases endangering human health.E very year about 1,600,000 patients with cancers were newly increased in our country and about 1,300,000 patients died from tumors.With the growth of population and the acceleration of aging population,the incidence and mortality of tumors are on the rise.Therefore,the prevention and treatment of tumors have long been the focal work in the Chinese medicine.It is then an important subject to find medicines both safe and effective in the treatment of tumors.Recently more attentions have been attached to the drugs from animals,and antineoplastics have been developed in using fully the animal sources of our country,which has then become the important content in the research work of tumors.
The scorpion Buthus martensii Karsch(BmK) had been used as a Chinese traditional medicine for thousands of years.In the first authoritative Chinese pharmacopoeia "Compendium of Material Medica" edited by Li Shizhen in 1578, dried scorpion bodies were used to treat a variety of diseases.Scorpion venoms are complex mixtures of molecules,most of which are peptides displaying various kinds of biological activity.During the past two decades,several of these compounds have been identified.Toxins modulating Na~+,K~+,Ca~(2+) and Ckl~- currents across cell membranes have been described.Some enzymes like proteases and hyaluronidases as well as chlorotoxin-like peptides from this venom may have therapeutic potential,including anticancer activity.Recent studies have demonstrated that BmK venom can induce apoptotic cell death,and possesses anti-tumor effects.However,the molecular mechanisms that underlie the induction of apoptosis by BmK venom are poorly understood,and require further exploration.
PTEN(phosphatase and tensin homolog deleted from chromosome 10) gene is a new tumor suppressor gene proved to be a lipid phosphatase.The target lipid, called phosphatidylinositol-3,4,5-trisphosphate,PIP3,and it is a key component of the major cell growth control pathways,acting both to stimulate cell growth and to inhibit tumor cell proliferation blocking apoptosis.PTEN mutations have been linked to a variety of human cancers.Mutations of PTEN during tumorigenesis allow the mutated cells to grow unchecked when they should die.
Lymphoma is a cancer in the lymphatic system.Despite recent advances in radiotherapy,chemotherapy,and stem cell transplantation,the severe morbidity from lymphoma has not been alleviated.Much effort has been focused on the discovery and development of new chemopreventive agents,especially agents targeted at mechanisms known to be involved in the process of carcinogenesis. Therefore,we have sought to identify novel agents that can effectively prevent lymphoma carcinogenesis but have minimal toxicity to healthy cells.In this study, we examined the effects of BmK venom on two human lymphoma cell lines(Raji and Jurkat) compared with peripheral blood lymphocytes(PBLs),and focused on changes in the PTEN and the PI3K/Akt signal pathway and to further investigate the molecular mechanisms involved in these effects.
STUDY CONTENTS:
1.Identification of apoptosis induction in Raji and Jurkat cells by BmK venom.
2.Analysis of the effects of BmK venom on cell cycle distribution and p27 protein level.
3.Analysis of the apoptosis pathways activated by the small molecules in Raij and Jurkat cells.
METHODS:
1.MTT assay for cell viability,by which small molecules were screened initially.
2.Methods for apoptosis identification:
2.1 Observation of cell morphological changes by Phase Contrast Microscope.
2.2 Observation of nuclear morphological changes by Hoechst 3342 staining combined with fluorescence microscopy.
2.2 Analysis of the changes in apoptosis by flow cytometry.
3.Analysis of the changes in cell cycle distribution by flow cytometry.
4.Analysis of the changes in PTEN、Akt、p-Akt、Bad、p-Bad protein level by Western blot assay.
5.Analysis of the changes in PTEN mRNA level by PT-PCR assay.
RESULTS:
1.BmK venom inhibited the viability of Raji and Jurkat cells in a dose-and time-dependent manner,and the IC50 value calculated to be 275.40μg/ml(Raji) and 360.60μg/ml(Jurkat).
2.BmK venom-induced apoptosis:
When Raji and Jurkat cells were exposed to BmK venom for 24-48 h,cell shrinkage,membrane blebbing and apoptotic bodies occurred.Ultimately,many cells underwent death;
Hoechst 3342 staining combined with Fluorescence Microscopy showed that compared with the nuclei in the control group,the nuclei were stained into brightly blue after treated by BmK venom for 48h.
Flow cytometry showed that in Raji cells,the levels of p-Akt and p-Bad clearly decreased in a dose-dependent manner,while the levels of total Akt and Bad protein were stable.PTEN expression was inversely correlated with phosphorylation of Akt, indicating that the PTEN was functional.
3.Flow cytometry showed that with increasing concentrations of BmK venom,the number of Jurkat and Raji cells in the G_0/G_1 phase increased significantly compared with control cells,while the cells in S and G_2/M phase decreased accordingly.
4.In Raji cells,BmK venom up regulated the expression of PTEN accompanied by decreased levels of Akt and Bad phosphorylation.Treatment with BmK venom and LY294002(an inhibitor of Akt) synergistically enhanced apoptosis. The expression of p27 was increased in both PYEN-positive Raji and PTEN-negative Jurkat cells exposed to BmK venom.
CONCLUSION:
In summary,it was demonstrated that BmK venom could inhibit cell proliferation and induces apoptosis on human NHLs cell lines,Raji and Jurkat cells. The cytostatic effect was dose- and time-dependent.One possible mechanism may be suppression of Akt signal pathway via PTEN and up regulation of p27 with affecting auxiliary pathways.These data suggest that BMK may be effective in the treatment of non-Hodgkin's lymphoma.
引文
[1]王毓銮,米振国.现代非霍奇金淋巴瘤学.北京:人民军医出版社,2003,9-12.
[2]Weber A,Rahemtullah A,Ferry J.Hodgkin and non-Hodgkin lymphoma of the head and neck:clinical,pathologic,and imaging evaluation[J].Neuroimaging Clin N Am 2003;13(3):371-92.
[3]Jemal A,Siegel R,Ward E,Hao Y,Xu J,Murray T,Thun MJ.Cancer Statistics,2008[J].Cancer J Clin 2008;58(2):71-96.
[4]朱东亚,朱敏生,朱有华,等.蝎提取物对S180肉瘤抑制作用的实验观察[J],江苏医药,1990(9):513-514.
[5]周新华.蝎毒的生化研究及临床应用[J].生物化学与生物物理进展,1984,11(2):20-22.
[6]Oukkache N,Rosso JP,Alami M,Ghalim N,Saile R,Hassar M,Bougis PE,Martin-Eauclaire MF.New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom[J].Toxicon 2008;51:835-852.
[7]Feng L,Gao R,Gopalakrishnakone P.Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensii[J].Comp Biochem Physiol C Toxicol Pharmacol 2008;148:250-257.
[8]Mamelak AN,Jacoby DB.Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin(TM-601)[J].Expert Opin Drug Deliv 2007;4:175-186.
[9]张甫同,许在生,杨建勤,等.中药全蝎抗肿瘤作用初探3.530号粗提物的抗肿瘤作用[J].皖南医学院学报,1990,9(1):1-3.
[10]于丽君,孔力.蝎毒逆转人乳腺癌细胞株耐药性研究[J].吉林大学学报医学版,2003,(30)2:239-242.
[11]郑智敏,王成裕,田中岭,等.蝎毒对人肿瘤细胞株和动物移植性肿瘤的作用[J].河南医科大学学报,1995,30(4):351-354.
[12]董伟华,孔天翰,韩雪飞,等.蝎毒抗癌多肽对4种人肿瘤细胞系的作用[J]. 河南医科大学学报,1999,34(1):52-55.
[13]韩雪飞,董伟华,王永奎,等.蝎毒抗癌多肽对人低分化鼻咽癌上皮细胞的移植作用[J].河南医科大学学报,1999,34(1):58-60.
[14]杨建斌,王庸晋,董伟华,等.东亚钳蝎毒抗癌多肽对ECA109细胞和Hela 细胞的毒性作用[J].中国病理生理杂志,1999,15(12):1116-1119.
[15]汤耀光,陈斌,梁业玲,刘丹霞,李爱华,贺艳杰,杨昌山,何慧华,董伟华.蛇毒多肽和蝎毒多肽对小鼠肝癌移植瘤的抑制作用和Caspase-3表达的影响[J].广州医学院学报,2006,34(5):140-143.
[16]付士波,鞠桂芝,杨英.蝎毒对B16黑色素瘤细胞生长的抑制作用[J].吉林大学学报(医学版),2004,30(2):178-181.
[17]王燕,张维东,贾青,王兆朋,宋守芹,王朝霞,张月.蝎毒多肽提取物对前列腺癌细胞侵袭力影响的体外研究[J].中华肿瘤防治杂志,2008,15(14):1045-1049.
[18]Lippens G;Najib J,Wodak S J,et al.NMR sequential assignments and solution structure of chlorotoxin,a small scorpion toxin that blocks chloride channels[J].Biochemistry 1995;34(1):13-21.
[19]Soroceanu L,Gillespie Y,Khazaeli M B,et al.Use of chlorotoxin for targeting of primary brain tumors[J].Cancer Res 1998,58(21):4871-4879.
[20]Soroceanu L,Manning T J Jr,Sontheimer H.Modulation of glioma cell migration and invasion using Cl~- and K~+ ion channel blockers[J].J Neurosci 1999;19(14):5942-5954.
[21]Lyons S A,O'Neal J,Sontheimer H.Chlorotoxin,a scorpion-derived peptide,specifically binds to gliomas and tumors of neuroectodermal origin[J],Glia 2002;39(2):162-173.
[22]Deshane J,Garner C C,Sontheimer H.Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2[J].J Biol Chem 2003;278(6):4135-4144.
[23]Hockaday D C,Shen S,Fiveash J,et al.Imaging glioma extent with ~(131)I-TM-601[J].J Nucl Med 2005;46(4):580-586.
[24]Shen S,Khazaeli M B,Gillespie G Y,et al.Radiation dosimetry of ~(131)I -chlorotoxin for targeted radiotherapy in glioma-beafing mice[J].J Neurooncol 2005;71(2):113-119.
[25]Veiseh O,Sun C,Gunn J,et al.Optical and MRI multifunctional nanoprobe for targeting gliomas[J].Nano Lett 2005;5(6):1003-1008.
[26]Mathew R,White E.FLIPping the Balance between Apoptosis and Proliferation in Thyroid Cancer[J].Clinical Cancer Research 2006;12:3648-3651.
[27]Evan GI,Vousden KH.Proliferation,cell cycle and apoptosis in cancer[J].Nature 2001;411:342-348.
[28]J(a|¨)(a|¨)ttel(a|¨) M,Tschopp J,Caspase-indepent cell death in lymphocytes[J].Nat immunol 2003;4:416-423.
[29]Kass GE,Eriksson JE,Weis M,Orrenius S,Chow SC.Chromation condensation during apoptosis requires ATP[J].BioChem J 1996;318:749-752.
[30]Zamzami N,Kroemer G.Condensed matter in cell death[J].Nature 1999;401:127-128.
[31]Liu YF,Hu J,Zhang JH,Wang SL,Wu CF.Isolation,purification,and N-terminal partial sequence of an antitumor peptide from the venom of the Chinese scorpion Buthus martensii Karsch[J].Prep Biochem Biotechnol 2002;32:317-327.
[32]Liu YF,Ma RL,Wang SL,Duan ZY,Zhang JH,Wu LJ,Wu CF.Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli[J].Protein Expr Purif 2003;27:253-258.
[33]Das Gupta S,Debnath A,Saha A,Giri B,Tripathi G,Vedasiromoni JR,Gomes A.Indian black scorpion(Heterometrus bengalensis Koch) venom induced antiproliferative and apoptogenic activity against human leukemic cell lines U937 and K562[J].Leuk Res 2007;31:817-825.
[34]Wang WX,Ji YH.Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro[J].J Neurooncol 2005;73:1-7.
[35]朱灵,韦晓谋,杨春旭.蝎毒对人肝癌细胞Bel-7404凋亡及端粒酶逆转录酶基因的作用[J].广西医药,2008,30(2):158-160
[36]Wang WX,Ji YH.Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro[J].J Neurooncol 2005;73:1-7.
[37]Das Gupta S,Debnath A,Saha A,Giri B,Tripathi G,Vedasiromoni JR,Gomes A.Indian black scorpion(Heterometrus bengalensis Koch) venom induced antiproliferative and apoptogenic activity against human leukemic cell lines U937and K562[J].Leuk Res 2007,31:817-25.
[38]Collins JA,Schandi CA,Young KK,Vesely J,Willingham MC.Major DNA fragmentation is a late event in apoptosis [J]. Histochem Cytochem 1997; 45, 923-934.
[39] Elledge SJ, Haper JW. Cdk inhibitors: on the threshold of checkpoints and development [J]. Cur Opin Cell Biol 1994; 6847-6852.
[40] Zi XL, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: Implications for prostate cancer intervention [J]. Proc Natl Acad Sci USA 1999; 96: 7490-7495.
[41] Deshane J, Garner C C, Sontheimer H. Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2 [J]. J Biol Chem 2003; 278(6): 4135-4144.
[42] Li J, Yen C, Lian D,et al. PTEN, a putative protein tyrosine Phosphatase gene mutated in human brain, breast, and prostate cancer[J]. Science 1997; 275: 1943-1947.
[43] Steck PA, PershouseMA, Jasser SA,et al. Identification of a candidate tumor suppressor gene, MMAC1, at chromosome 10q23. 3 that ismutated in multiple advanced cancers[J]. NatGenet 1997; 15: 356-362.
[44] Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphataseregulated by transforming growth factorβ[J]. Cancer Res 1997; 57: 2124-2129.
[45] Tamura M, Gu J, Matsumoto K, et al. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN [J]. Science 1998; 280: 1614-1617.
[46] Bellacosa A, Testa JR, Staal SP, et al. A retroviral oncogene, akt, encoding a serine/threonine kinase containing all SH2-like region [J]. Science 1991; 254(5029): 274-277.
[47] Hamada K, Sasald T, Koni PA, et al. The PTEN/P13K pathway governs normal vascular development and tumor angiogenesis [J]. Genes Dev 2005; 19: 2054-2065.
[48] Maehama T, Dixon JE. The tumor suppressor, PTEN//MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4, 5 -trisphosphate [J]. J Biol Chem 1998, 273: 13375-13378.
[49] Datta S, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival ignals to the cell-intrinsic death machinery [J]. Cell 1997; 91:231-241.
[50] Ramaswamy S, Nakamura N, Vazquez F, et al. Regulation of Gl progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway [J]. Proc Natl Acad Sci USA 1999; 96:211-215.
[51] Myers MP, Stolarov YP, Eng C, et al. PTEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity Phosphatase [J]. Proc N atl Acad Sci USA 1997, 94(17): 9052-9057.
[52]常青,郭丽娜．PTEN／MMAC1／TEP1基因研究进展[J]．国外医学肿瘤学分册,2000．27：193-196．
[53] Shim JH, Kim YS, Bahk YY. Proteome profile change that are differentially regulated by lipid and protein Phosphatase activities of tumor suppressor PTEN-expressing U-87 MG human glioblastoma cells [J]. Proteomices 2006; 6(1):81-93.
[54] Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide -regulated kinases: kinase activation by phosphoinositide -dependent phosphorylation [J]. Annu Rev Biochem 1999; 68:965-1014.
[55] Di Cristofano, A., and Pandolfi, P. P. The multiple roles of PTEN in tumor suppression. Cell 2000; 100: 387-390.
[56] Lee J O, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, Dixon J E, Pandolfi P, and Pavletich N P. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide Phosphatase activity and membrane association. Cell 1999; 99: 323-334.
[57] Cantley, LC, and Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci USA 1999; 96: 4240-4245.
[58] Deichmann M, Thome M, Benner M, et al. PTEN/MMAC1 expression in melanoma resection specimens [J]. Br J Cancer 2002, 87(12): 1431 -1436.
[59] Stambolic V, Suzuki A, de la Pompa J L, Brothers G M, Mirtsos C, Sasaki T, Ruland J, Penninger J M, Siderovski D P, and Mak T W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998; 95: 29-39.
[60] Maehama T, Dixon JE. The Tumor Suppressor, PTEN/MMAC1, Dephosphorylates the Lipid Second Messenger, Phosphatidylinositol 3,4, 5 -Trisphosphate [J]. Journal of Biological Chemistry 1998; 273:13375-13378.
[61] Hamada K, Sasaki T, Koni PA, et al. The PTEN/P13K pathway governs normal vascular development and tumor ansiogenesis [J]. Genes Dev 2005; 19(17): 2054-2065.
[62] Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, and Yamada K M. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science (Wash. DC) 1998; 280:1614-1617.
[63] Huang JH, Kontos CD. PTEN modulates vascular cndothelial growth factor mediated signaling and angiogenic effects [J]. J Biol Chem 2002; 277(13): 10760-10766.
[64] Myers MP, Pass I, Batty IH, et al. The lipid Phosphatase activity of PTEN is critical for its tumor suppressor function [J]. Proc NatlAcad Sci USA 1998; 95: 13513-1351.
[65] Furnari FB, Huang HJ, Cavenee WK.et al. The phosphoinositol Phosphatase activity of PTEN mediates aserum-sensitive G1 growth arrest in glioma cells [J]. CancerRes 1998; 58: 5002-5008.
[66] Vivanco I, Sawyers CL. The phosphatidylinosite 3-kinase AKT pathway in human cancer [J]. Nat Rev Cancer 2002; 2(5): 489-501.
[67] Sulis ML, Parsons R. PTEN: from pathology to biology [J]. Trends Cell Biol 2003; 3(9): 478-483.
[68] Mita MM, Mita A, Rowinsky EK. Mammalian target of rapamycin: a new molecular target for breast cancer [J]. Clin B reast Cancer 2003;4(2):l 26-1 37.
[69] Wu R C, Li X, Schonthal A H. Transcriptionala ctivation of p21 WAF1 by PTEN/MMAC1 tumor suppressor [J]. Mol Cell Biochem 2000; 203:59-71.
[70] Zhu X, Kwon CH, Schlosshauer PW, et al. PTEN induces Gl cell cycle arrest and decreases cyclin D3 levels in endometrial carcinoma cells [J]. Cancer Res 2001; 61:4569-4575.
[71] Moon SK, Kim HM, Kim CH. PTEN induces Gl cell cycle arrest and inhibits MMP-9 expression via the regulation of NF-kappaB and AP-1 in vascular smooth muscle cells [J]. Arch Biochem Biophys 2004; 421: 267-276.
[72] Ginn-Pease ME, Eng C. Increased nuclear Phosphatase and tensin homologue deleted on chromosome I 0 is associated with G0-G1 in MCF-7 cells [J]. Cancer Res 2003; 63(2): 282-286.
[73] Tian XX, Pang JCS, et al. Restoration of wild-type PTEN expression leads to apoptosiss, induces differentiation, and reduces telomerase activity in human glioma cells [J]. J Neuropathol Exp Neurol 1999; 58(5): 472-479.
[74] Waite KA, Eng C. Protean PTEN: form and function [J]. Am J Hum Genet 2002; 70 (4): 829-844.
[75] Mawrin C, Diete S, Treuheit T, et al. Prognostic relevance of MAPK expression in glioblastoma multiforme [J]. Int J Oncol 2003; 23(3): 641-648.
[76] Tanaka Y, Gavrielides MV, Mitsuuchi Y, et al. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway [J]. Biol Chem 2003; 278(36):33753-337562.
[77] Sakai A, Thieblemont C, Wellmann A, et al. PTEN gene alterations in lymphoid neoplasms [J]. Blood 1998; 92:3410-3415.
[78] Nakahara Y, Nagai H, Kinoshita T, et al. Mutational analysis of the PTEN/MMAC1 gene in non-Hodgkin's lymphoma [J]. Leukemia 1998; 12:1277-1280.
[79] Ali IU, Schniml LM, Dean M. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphate activity [J]. J Natl Cancer Inst 1999; 91(22): 1922-1932.
[80] Bellacosa A, Testa JR, Staal SP, et al. A retroviral oncogene akt,encoding a serine/threonine kinase containing a SI-122 like region [J]. Science 1991; 254: 274-277.
[81] Carpenter CL, Cantley LC. Phosphoinositide 3-kinase and the regulation of cell growth [J]. Biochim Biophys Acta. 1996; 1288(1): M11-M16.
[82] Franke TF, Kaplan DR, Cantley LC, et al. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol- 3,4 -bisphosphate [J]. Science 1997; 273:665-668.
[83] Henshall DC, Arakit T, Schindler CK, et al. Activation of Bcl-2-associated death protein and counter-response of Akt within cell populations during seizure-induced neuronal death [J]. J Neurosci 2002; 22(19): 8458-8465.
[84] Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation [J]. J Biol Chem 2005; 280(11): 10781-10789.
[85] Alessi D R, and Cohen P. Mechanism of activation and function of protein kinase B. Curr. Opin Genet Dev, 1998; 8: 55-62.
[86] Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation [J]. Science 1998; 282: 1318-1321.
[87] Datta S R, Brunet A, and Greenberg M E. Cellular survival: a play in three Akts. Genes Dev 1999; 13: 2905-2927.
[88] Bartling B, Tostlebe H, Darmer D, et al. Shear stress-dependent expression of apoptosis- regulating genes in endothelial cells [J]. Biochem Biophys Res Commun 2000; 278(3): 740-746.
[89] Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, and Sellers W R. Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN [J]. Mol. Cell Biol 2000; 20: 8969-8982.
[90] Lin, Banin S, Qu Yang H, et al. ATM is required for Ikappa B kinase (IKKk) activation in response to DNA double strand breaks [J]. J Biol Chem 2001; 276(12): 8898-8903.
[91] Persad S, Troussard A A, McPhee T R, Mulholland D J, and Dedhar S. Tumor suppressor PTEN inhibits nuclear accumulation of β-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation [J]. J Cell Biol 2001; 153: 1161-1174.
[92] Gibson EM, Henson ES, Haney N, et al. Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand- induced apoptosis by inhibiting cytochrome C release [J]. Cancer Res 2002; 62(2): 488-496.
[93] Nam SY, Jung GA, Hut GC, et al. Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosis in human gastric cancers [J]. Cancer Sci 2003; 94(12): 1066-1073.
[94] Sakai A, Thieblemont C, Wellmann A, Jaffe ES, Raffeld M. PTEN Gene Alterations in Lymphoid Neoplasms [J]. Blood 1998; 92:3410-3415.
[95] Shan X, Czar M J, Bunnell S C, Liu P, Liu Y, Schwartzberg, P L, and Wange R L. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol 2000; 20: 6945-6957.
[96] Xu Z, Stokoe D, Kane LP, Weiss A. The Inducible Expression of the Tumor Suppressor Gene PTEN Promotes Apoptosis and Decreases Cell Size by Inhibiting the PI3K/Akt Pathway in Jurkat T Cells [J]. Cell Growth and Differentiation 2002; 13:285-296.
[97] Shan X, Czar MJ, Bunnell SC, Liu P, Liu Y, Schwartzberg PL, Wange RL. Deficiency of PTEN in Jurkat T Cells Causes Constitutive Localization of Itk to the Plasma Membrane and Hyperresponsiveness to CD3 Stimulation [J]. Molecular and Cellular Biology 2000; 20:6945-57.
[98] McGowan CH. Regulation of the eukaryotic cell cycle [J]. Prog Cell Cycle Res 2003; 5:1-4.
[99] Polyak K, Lee MH, Erdjument-Bromage H, et al. Cloning of p27~(Kip1) , a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals [J]. Cell 1994; 78(1): 59-66.
[100] Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression [J]. Genes Dev 1999; 13(12): 1501-1512.
[101] Cohen GM. Caspases: the executioners of apoptosis [J]. Biochem J 1997; 326(Pt 1):1-16.
[102] Li D M, and Sun H. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. Proc Natl Acad Sci USA, 1998; 95: 15406-15411.
[103] Sun H, Lesche R, Li D M, Liliental J, Zhang H, Gao J, Gavrilova N, Mueller B, Liu X, and Wu H. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci USA, 1999; 96: 6199-6204.
[104] Wu RC, Li X, Schonthal AH. Transcriptional activation of p21WAFl by PTEN/MMAC1 tumor suppressor [J]. Mol Cell Biochem 2000; 203:59-71.
[2]Weber A,Rahemtullah A,Ferry J.Hodgkin and non-Hodgkin lymphoma of the head and neck:clinical,pathologic,and imaging evaluation[J].Neuroimaging Clin N Am 2003;13(3):371-92.
[3]Jemal A,Siegel R,Ward E,Hao Y,Xu J,Murray T,Thun MJ.Cancer Statistics,2008[J].Cancer J Clin 2008;58(2):71-96.
[4]朱东亚,朱敏生,朱有华,等.蝎提取物对S180肉瘤抑制作用的实验观察[J],江苏医药,1990(9):513-514.
[5]周新华.蝎毒的生化研究及临床应用[J].生物化学与生物物理进展,1984,11(2):20-22.
[6]Oukkache N,Rosso JP,Alami M,Ghalim N,Saile R,Hassar M,Bougis PE,Martin-Eauclaire MF.New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom[J].Toxicon 2008;51:835-852.
[7]Feng L,Gao R,Gopalakrishnakone P.Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensii[J].Comp Biochem Physiol C Toxicol Pharmacol 2008;148:250-257.
[8]Mamelak AN,Jacoby DB.Targeted delivery of antitumoral therapy to glioma and other malignancies with synthetic chlorotoxin(TM-601)[J].Expert Opin Drug Deliv 2007;4:175-186.
[9]张甫同,许在生,杨建勤,等.中药全蝎抗肿瘤作用初探3.530号粗提物的抗肿瘤作用[J].皖南医学院学报,1990,9(1):1-3.
[10]于丽君,孔力.蝎毒逆转人乳腺癌细胞株耐药性研究[J].吉林大学学报医学版,2003,(30)2:239-242.
[11]郑智敏,王成裕,田中岭,等.蝎毒对人肿瘤细胞株和动物移植性肿瘤的作用[J].河南医科大学学报,1995,30(4):351-354.
[12]董伟华,孔天翰,韩雪飞,等.蝎毒抗癌多肽对4种人肿瘤细胞系的作用[J]. 河南医科大学学报,1999,34(1):52-55.
[13]韩雪飞,董伟华,王永奎,等.蝎毒抗癌多肽对人低分化鼻咽癌上皮细胞的移植作用[J].河南医科大学学报,1999,34(1):58-60.
[14]杨建斌,王庸晋,董伟华,等.东亚钳蝎毒抗癌多肽对ECA109细胞和Hela 细胞的毒性作用[J].中国病理生理杂志,1999,15(12):1116-1119.
[15]汤耀光,陈斌,梁业玲,刘丹霞,李爱华,贺艳杰,杨昌山,何慧华,董伟华.蛇毒多肽和蝎毒多肽对小鼠肝癌移植瘤的抑制作用和Caspase-3表达的影响[J].广州医学院学报,2006,34(5):140-143.
[16]付士波,鞠桂芝,杨英.蝎毒对B16黑色素瘤细胞生长的抑制作用[J].吉林大学学报(医学版),2004,30(2):178-181.
[17]王燕,张维东,贾青,王兆朋,宋守芹,王朝霞,张月.蝎毒多肽提取物对前列腺癌细胞侵袭力影响的体外研究[J].中华肿瘤防治杂志,2008,15(14):1045-1049.
[18]Lippens G;Najib J,Wodak S J,et al.NMR sequential assignments and solution structure of chlorotoxin,a small scorpion toxin that blocks chloride channels[J].Biochemistry 1995;34(1):13-21.
[19]Soroceanu L,Gillespie Y,Khazaeli M B,et al.Use of chlorotoxin for targeting of primary brain tumors[J].Cancer Res 1998,58(21):4871-4879.
[20]Soroceanu L,Manning T J Jr,Sontheimer H.Modulation of glioma cell migration and invasion using Cl~- and K~+ ion channel blockers[J].J Neurosci 1999;19(14):5942-5954.
[21]Lyons S A,O'Neal J,Sontheimer H.Chlorotoxin,a scorpion-derived peptide,specifically binds to gliomas and tumors of neuroectodermal origin[J],Glia 2002;39(2):162-173.
[22]Deshane J,Garner C C,Sontheimer H.Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2[J].J Biol Chem 2003;278(6):4135-4144.
[23]Hockaday D C,Shen S,Fiveash J,et al.Imaging glioma extent with ~(131)I-TM-601[J].J Nucl Med 2005;46(4):580-586.
[24]Shen S,Khazaeli M B,Gillespie G Y,et al.Radiation dosimetry of ~(131)I -chlorotoxin for targeted radiotherapy in glioma-beafing mice[J].J Neurooncol 2005;71(2):113-119.
[25]Veiseh O,Sun C,Gunn J,et al.Optical and MRI multifunctional nanoprobe for targeting gliomas[J].Nano Lett 2005;5(6):1003-1008.
[26]Mathew R,White E.FLIPping the Balance between Apoptosis and Proliferation in Thyroid Cancer[J].Clinical Cancer Research 2006;12:3648-3651.
[27]Evan GI,Vousden KH.Proliferation,cell cycle and apoptosis in cancer[J].Nature 2001;411:342-348.
[28]J(a|¨)(a|¨)ttel(a|¨) M,Tschopp J,Caspase-indepent cell death in lymphocytes[J].Nat immunol 2003;4:416-423.
[29]Kass GE,Eriksson JE,Weis M,Orrenius S,Chow SC.Chromation condensation during apoptosis requires ATP[J].BioChem J 1996;318:749-752.
[30]Zamzami N,Kroemer G.Condensed matter in cell death[J].Nature 1999;401:127-128.
[31]Liu YF,Hu J,Zhang JH,Wang SL,Wu CF.Isolation,purification,and N-terminal partial sequence of an antitumor peptide from the venom of the Chinese scorpion Buthus martensii Karsch[J].Prep Biochem Biotechnol 2002;32:317-327.
[32]Liu YF,Ma RL,Wang SL,Duan ZY,Zhang JH,Wu LJ,Wu CF.Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli[J].Protein Expr Purif 2003;27:253-258.
[33]Das Gupta S,Debnath A,Saha A,Giri B,Tripathi G,Vedasiromoni JR,Gomes A.Indian black scorpion(Heterometrus bengalensis Koch) venom induced antiproliferative and apoptogenic activity against human leukemic cell lines U937 and K562[J].Leuk Res 2007;31:817-825.
[34]Wang WX,Ji YH.Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro[J].J Neurooncol 2005;73:1-7.
[35]朱灵,韦晓谋,杨春旭.蝎毒对人肝癌细胞Bel-7404凋亡及端粒酶逆转录酶基因的作用[J].广西医药,2008,30(2):158-160
[36]Wang WX,Ji YH.Scorpion venom induces glioma cell apoptosis in vivo and inhibits glioma tumor growth in vitro[J].J Neurooncol 2005;73:1-7.
[37]Das Gupta S,Debnath A,Saha A,Giri B,Tripathi G,Vedasiromoni JR,Gomes A.Indian black scorpion(Heterometrus bengalensis Koch) venom induced antiproliferative and apoptogenic activity against human leukemic cell lines U937and K562[J].Leuk Res 2007,31:817-25.
[38]Collins JA,Schandi CA,Young KK,Vesely J,Willingham MC.Major DNA fragmentation is a late event in apoptosis [J]. Histochem Cytochem 1997; 45, 923-934.
[39] Elledge SJ, Haper JW. Cdk inhibitors: on the threshold of checkpoints and development [J]. Cur Opin Cell Biol 1994; 6847-6852.
[40] Zi XL, Agarwal R. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: Implications for prostate cancer intervention [J]. Proc Natl Acad Sci USA 1999; 96: 7490-7495.
[41] Deshane J, Garner C C, Sontheimer H. Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2 [J]. J Biol Chem 2003; 278(6): 4135-4144.
[42] Li J, Yen C, Lian D,et al. PTEN, a putative protein tyrosine Phosphatase gene mutated in human brain, breast, and prostate cancer[J]. Science 1997; 275: 1943-1947.
[43] Steck PA, PershouseMA, Jasser SA,et al. Identification of a candidate tumor suppressor gene, MMAC1, at chromosome 10q23. 3 that ismutated in multiple advanced cancers[J]. NatGenet 1997; 15: 356-362.
[44] Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphataseregulated by transforming growth factorβ[J]. Cancer Res 1997; 57: 2124-2129.
[45] Tamura M, Gu J, Matsumoto K, et al. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN [J]. Science 1998; 280: 1614-1617.
[46] Bellacosa A, Testa JR, Staal SP, et al. A retroviral oncogene, akt, encoding a serine/threonine kinase containing all SH2-like region [J]. Science 1991; 254(5029): 274-277.
[47] Hamada K, Sasald T, Koni PA, et al. The PTEN/P13K pathway governs normal vascular development and tumor angiogenesis [J]. Genes Dev 2005; 19: 2054-2065.
[48] Maehama T, Dixon JE. The tumor suppressor, PTEN//MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4, 5 -trisphosphate [J]. J Biol Chem 1998, 273: 13375-13378.
[49] Datta S, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival ignals to the cell-intrinsic death machinery [J]. Cell 1997; 91:231-241.
[50] Ramaswamy S, Nakamura N, Vazquez F, et al. Regulation of Gl progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway [J]. Proc Natl Acad Sci USA 1999; 96:211-215.
[51] Myers MP, Stolarov YP, Eng C, et al. PTEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity Phosphatase [J]. Proc N atl Acad Sci USA 1997, 94(17): 9052-9057.
[52]常青,郭丽娜．PTEN／MMAC1／TEP1基因研究进展[J]．国外医学肿瘤学分册,2000．27：193-196．
[53] Shim JH, Kim YS, Bahk YY. Proteome profile change that are differentially regulated by lipid and protein Phosphatase activities of tumor suppressor PTEN-expressing U-87 MG human glioblastoma cells [J]. Proteomices 2006; 6(1):81-93.
[54] Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide -regulated kinases: kinase activation by phosphoinositide -dependent phosphorylation [J]. Annu Rev Biochem 1999; 68:965-1014.
[55] Di Cristofano, A., and Pandolfi, P. P. The multiple roles of PTEN in tumor suppression. Cell 2000; 100: 387-390.
[56] Lee J O, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, Dixon J E, Pandolfi P, and Pavletich N P. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide Phosphatase activity and membrane association. Cell 1999; 99: 323-334.
[57] Cantley, LC, and Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci USA 1999; 96: 4240-4245.
[58] Deichmann M, Thome M, Benner M, et al. PTEN/MMAC1 expression in melanoma resection specimens [J]. Br J Cancer 2002, 87(12): 1431 -1436.
[59] Stambolic V, Suzuki A, de la Pompa J L, Brothers G M, Mirtsos C, Sasaki T, Ruland J, Penninger J M, Siderovski D P, and Mak T W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998; 95: 29-39.
[60] Maehama T, Dixon JE. The Tumor Suppressor, PTEN/MMAC1, Dephosphorylates the Lipid Second Messenger, Phosphatidylinositol 3,4, 5 -Trisphosphate [J]. Journal of Biological Chemistry 1998; 273:13375-13378.
[61] Hamada K, Sasaki T, Koni PA, et al. The PTEN/P13K pathway governs normal vascular development and tumor ansiogenesis [J]. Genes Dev 2005; 19(17): 2054-2065.
[62] Tamura M, Gu J, Matsumoto K, Aota S, Parsons R, and Yamada K M. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science (Wash. DC) 1998; 280:1614-1617.
[63] Huang JH, Kontos CD. PTEN modulates vascular cndothelial growth factor mediated signaling and angiogenic effects [J]. J Biol Chem 2002; 277(13): 10760-10766.
[64] Myers MP, Pass I, Batty IH, et al. The lipid Phosphatase activity of PTEN is critical for its tumor suppressor function [J]. Proc NatlAcad Sci USA 1998; 95: 13513-1351.
[65] Furnari FB, Huang HJ, Cavenee WK.et al. The phosphoinositol Phosphatase activity of PTEN mediates aserum-sensitive G1 growth arrest in glioma cells [J]. CancerRes 1998; 58: 5002-5008.
[66] Vivanco I, Sawyers CL. The phosphatidylinosite 3-kinase AKT pathway in human cancer [J]. Nat Rev Cancer 2002; 2(5): 489-501.
[67] Sulis ML, Parsons R. PTEN: from pathology to biology [J]. Trends Cell Biol 2003; 3(9): 478-483.
[68] Mita MM, Mita A, Rowinsky EK. Mammalian target of rapamycin: a new molecular target for breast cancer [J]. Clin B reast Cancer 2003;4(2):l 26-1 37.
[69] Wu R C, Li X, Schonthal A H. Transcriptionala ctivation of p21 WAF1 by PTEN/MMAC1 tumor suppressor [J]. Mol Cell Biochem 2000; 203:59-71.
[70] Zhu X, Kwon CH, Schlosshauer PW, et al. PTEN induces Gl cell cycle arrest and decreases cyclin D3 levels in endometrial carcinoma cells [J]. Cancer Res 2001; 61:4569-4575.
[71] Moon SK, Kim HM, Kim CH. PTEN induces Gl cell cycle arrest and inhibits MMP-9 expression via the regulation of NF-kappaB and AP-1 in vascular smooth muscle cells [J]. Arch Biochem Biophys 2004; 421: 267-276.
[72] Ginn-Pease ME, Eng C. Increased nuclear Phosphatase and tensin homologue deleted on chromosome I 0 is associated with G0-G1 in MCF-7 cells [J]. Cancer Res 2003; 63(2): 282-286.
[73] Tian XX, Pang JCS, et al. Restoration of wild-type PTEN expression leads to apoptosiss, induces differentiation, and reduces telomerase activity in human glioma cells [J]. J Neuropathol Exp Neurol 1999; 58(5): 472-479.
[74] Waite KA, Eng C. Protean PTEN: form and function [J]. Am J Hum Genet 2002; 70 (4): 829-844.
[75] Mawrin C, Diete S, Treuheit T, et al. Prognostic relevance of MAPK expression in glioblastoma multiforme [J]. Int J Oncol 2003; 23(3): 641-648.
[76] Tanaka Y, Gavrielides MV, Mitsuuchi Y, et al. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway [J]. Biol Chem 2003; 278(36):33753-337562.
[77] Sakai A, Thieblemont C, Wellmann A, et al. PTEN gene alterations in lymphoid neoplasms [J]. Blood 1998; 92:3410-3415.
[78] Nakahara Y, Nagai H, Kinoshita T, et al. Mutational analysis of the PTEN/MMAC1 gene in non-Hodgkin's lymphoma [J]. Leukemia 1998; 12:1277-1280.
[79] Ali IU, Schniml LM, Dean M. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphate activity [J]. J Natl Cancer Inst 1999; 91(22): 1922-1932.
[80] Bellacosa A, Testa JR, Staal SP, et al. A retroviral oncogene akt,encoding a serine/threonine kinase containing a SI-122 like region [J]. Science 1991; 254: 274-277.
[81] Carpenter CL, Cantley LC. Phosphoinositide 3-kinase and the regulation of cell growth [J]. Biochim Biophys Acta. 1996; 1288(1): M11-M16.
[82] Franke TF, Kaplan DR, Cantley LC, et al. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol- 3,4 -bisphosphate [J]. Science 1997; 273:665-668.
[83] Henshall DC, Arakit T, Schindler CK, et al. Activation of Bcl-2-associated death protein and counter-response of Akt within cell populations during seizure-induced neuronal death [J]. J Neurosci 2002; 22(19): 8458-8465.
[84] Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation [J]. J Biol Chem 2005; 280(11): 10781-10789.
[85] Alessi D R, and Cohen P. Mechanism of activation and function of protein kinase B. Curr. Opin Genet Dev, 1998; 8: 55-62.
[86] Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation [J]. Science 1998; 282: 1318-1321.
[87] Datta S R, Brunet A, and Greenberg M E. Cellular survival: a play in three Akts. Genes Dev 1999; 13: 2905-2927.
[88] Bartling B, Tostlebe H, Darmer D, et al. Shear stress-dependent expression of apoptosis- regulating genes in endothelial cells [J]. Biochem Biophys Res Commun 2000; 278(3): 740-746.
[89] Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M, and Sellers W R. Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN [J]. Mol. Cell Biol 2000; 20: 8969-8982.
[90] Lin, Banin S, Qu Yang H, et al. ATM is required for Ikappa B kinase (IKKk) activation in response to DNA double strand breaks [J]. J Biol Chem 2001; 276(12): 8898-8903.
[91] Persad S, Troussard A A, McPhee T R, Mulholland D J, and Dedhar S. Tumor suppressor PTEN inhibits nuclear accumulation of β-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation [J]. J Cell Biol 2001; 153: 1161-1174.
[92] Gibson EM, Henson ES, Haney N, et al. Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand- induced apoptosis by inhibiting cytochrome C release [J]. Cancer Res 2002; 62(2): 488-496.
[93] Nam SY, Jung GA, Hut GC, et al. Upregulation of FLIP(S) by Akt, a possible inhibition mechanism of TRAIL-induced apoptosis in human gastric cancers [J]. Cancer Sci 2003; 94(12): 1066-1073.
[94] Sakai A, Thieblemont C, Wellmann A, Jaffe ES, Raffeld M. PTEN Gene Alterations in Lymphoid Neoplasms [J]. Blood 1998; 92:3410-3415.
[95] Shan X, Czar M J, Bunnell S C, Liu P, Liu Y, Schwartzberg, P L, and Wange R L. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol 2000; 20: 6945-6957.
[96] Xu Z, Stokoe D, Kane LP, Weiss A. The Inducible Expression of the Tumor Suppressor Gene PTEN Promotes Apoptosis and Decreases Cell Size by Inhibiting the PI3K/Akt Pathway in Jurkat T Cells [J]. Cell Growth and Differentiation 2002; 13:285-296.
[97] Shan X, Czar MJ, Bunnell SC, Liu P, Liu Y, Schwartzberg PL, Wange RL. Deficiency of PTEN in Jurkat T Cells Causes Constitutive Localization of Itk to the Plasma Membrane and Hyperresponsiveness to CD3 Stimulation [J]. Molecular and Cellular Biology 2000; 20:6945-57.
[98] McGowan CH. Regulation of the eukaryotic cell cycle [J]. Prog Cell Cycle Res 2003; 5:1-4.
[99] Polyak K, Lee MH, Erdjument-Bromage H, et al. Cloning of p27~(Kip1) , a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals [J]. Cell 1994; 78(1): 59-66.
[100] Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression [J]. Genes Dev 1999; 13(12): 1501-1512.
[101] Cohen GM. Caspases: the executioners of apoptosis [J]. Biochem J 1997; 326(Pt 1):1-16.
[102] Li D M, and Sun H. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. Proc Natl Acad Sci USA, 1998; 95: 15406-15411.
[103] Sun H, Lesche R, Li D M, Liliental J, Zhang H, Gao J, Gavrilova N, Mueller B, Liu X, and Wu H. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci USA, 1999; 96: 6199-6204.
[104] Wu RC, Li X, Schonthal AH. Transcriptional activation of p21WAFl by PTEN/MMAC1 tumor suppressor [J]. Mol Cell Biochem 2000; 203:59-71.