1、Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex PLoS ONE 2011, 6(3): 1-12美国纽约州立大学；纽约州生物信息学与生命科学卓越中心；瑞典兰德大学；新加坡A*STARApoptosis induced by a human milk protein人乳蛋白诱导细胞凋亡Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8064-8068, August 1995ABSTRACT To the breast-fed infan
2、t, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. W
3、hile investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state-multimeric a-lactalbumin- is a potent Ca2+-eleva
4、ting and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric a-lactalbumin killed all transformed, embryonic, and lymnphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by fu
5、rnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric a-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.对母乳喂养婴儿来说，母乳不仅是营养来源，而且也提供着丰富的分子用以抵御微生物
6、体，如抗体和细菌素，并限制细菌粘附。然而，人们很少想到母乳中也会含有针对宿主细胞的生物活性物质。在调查母乳对细菌粘附到人肺癌细胞株的影响时，我们惊奇地发现母乳杀死了癌细胞。对此结果做进一步分析后得出是母乳中的一种成分，以-乳白蛋白多聚体形式存在，能够提高钙离子浓度，作为细胞凋亡的诱导剂；并有选择性地发挥对细胞的毒性。-乳白蛋白多聚体能杀灭所有转化、胚胎和淋巴样细胞，但对成熟的上皮细胞无作用。这些发现提醒我们：母乳有助于粘膜免疫系统不仅是因为产生了抗菌分子，而且也在于policing the function of lymphocytes and epithelium。最后，分析-乳白蛋白多聚体
7、能在分化的上皮细胞中诱导肿瘤细胞凋亡的机理能为我们设计抗肿瘤药提供思路。HAMLET Kills Tumor Cells by Apoptosis: Structure, Cellular Mechanisms, and Therapy HAMLET通过诱发凋亡杀灭肿瘤细胞：结构，细胞机制和疗效J. Nutr. 135: 12991303, 2005.ABSTRACT: New cancer treatments should aim to destroy tumor cells without disturbing normal tissue. HAMLET (human -lactalbu
8、min made lethal to tumor cells) offers a new molecular approach to solving this problem, because it induces apoptosis in tumor cells but leaves normal differentiated cells unaffected. After partial unfolding and binding to oleic acid, -lactalbumin forms the HAMLET complex, which enters tumor cells a
9、nd freezes their metabolic machinery. The cells proceed to fragment their DNA, and they disintegrate with apoptosis-like characteristics. HAMLET kills a wide range of malignant cells in vitro and maintains this activity in vivo in patients with skin papillomas. In addition, HAMLET has striking effec
10、ts on human glioblastomas in a rat xenograft model. After convection- enhanced delivery, HAMLET diffuses throughout the brain, selectively killing tumor cells and controlling tumor progression without apparent tissue toxicity. HAMLET thus shows great promise as a new therapeutic with the advantage o
11、f selectivity for tumor cells and lack of toxicity. 癌症的新疗法应该着眼于摧毁肿瘤细胞但不干扰正常机体组织。HAMLET（能致肿瘤细胞死亡的人-乳白蛋白）为解决此问题提供了一种新的分子途径，因为它诱导肿瘤细胞凋亡但却不影响正常分化细胞。-乳白蛋白分子部分展开与油酸结合后形成了HAMLET复合物，进入到肿瘤细胞内部并“冻结”其新陈代谢活动。细胞DNA不断分解，出现凋亡状特征。HAMLET 在体外能够杀灭各类恶性细胞，在皮肤乳头状瘤患者体内则继续着发挥这种能力。此外，HAMLET对进行异种组织移植的大鼠体内的人胶质母细胞瘤也有着惊人的影响力。在进
12、入大鼠脑组织后，HAMLET扩散至整个脑部，选择性地杀死肿瘤细胞并控制其发展，而对组织无明显毒性。因其具有对肿瘤细胞的选择性且无毒性，HAMLET具有巨大的应用前景。I. HAMLET: structural aspects（ 略，详见原文 ）II. Cellular targets of HAMLET in tumor cells（ 略，详见原文 ）III. In vivo effects of HAMLET in tumor models HAMLET 在体内对肿瘤的作用（ 自此以下全文翻译 ）HAMLET induces in vitro apoptosis in cells from
13、carcinomas of lung, throat, kidney, colon, bladder, prostate and ovaries; in melanomas; in glioblastomas of the brain; and in leukemias. The broad activity against such different tumors is quite remarkable, but the in vivo effects may be quite different and must be rigorously tested. HAMLET在体外能诱导各类癌
14、细胞凋亡，包括肺癌、咽喉癌、肾癌、结肠癌、膀胱癌、前列腺癌和卵巢癌，以及黑色素瘤、脑胶质母细胞瘤和白血病。HAMLET对上述各类肿瘤细胞的抵抗作用非常明显，但在体内，其效果则大不相同，必须进行严格的测试。Effects of HAMLET on human skin papillomas. HAMLET对人皮肤乳头状瘤的作用。Papillomas are premalignant lesions of the skin and mucosal surfaces 【18-21】. The human papilloma virus (HPV) can cause condyloma acumin
15、atum, and laryngeal and genital papillomas, and skin lesions, which are extremely common. Therapeutic options are limited and often are ineffective or destructive 【23,24】. They include cryotherapy, curettage, cautery, salicylic acid, CO2 laser, photodynamic therapy, antimitotic agents, or immune mod
16、ulators. Currently, HPV vaccines are being developed to prevent HPV infection, but they are not available for use. 乳头状瘤是皮肤和粘膜表层癌变前的病变体。人乳头状瘤病毒（HPV）能引发尖锐湿疣，喉和外阴乳头状瘤，以及皮肤损害，这些都是极其常见的病症。对其采用的治疗手段经常无效或对机体产生破坏性。包括：冷冻，刮除，烧灼，水杨酸，CO 2激光，光动力疗法，抗有丝分裂剂或免疫调节剂。当前，正在开发HPV疫苗，但尚不能使用。Skin papillomas were selected as
17、 a first model to examine the effect of HAMLET in humans (0.9% NaCl) 【25】. Treatment was performed according to a double-blind, placebocontrolled protocol. HAMLET (0.7 mM in 0.9% NaCl) or placebo was applied topically, once a day for 3 wk. The lesions were measured and photographed once a week durin
18、g the treatment period and at follow-up visits, 1 and 2 mo after completed treatment. The treatment was deemed successful if the patient showed a reduction in lesion volume by 75% within 1 mo after completed treatment. 选择皮肤乳头状瘤作为研究人体环境（0.9%NaCl）中HAMLET 功效的第一个模型。按照双盲、安慰剂对照的实验流程进行实验。以0.9% NaCl 为溶剂，将 H
19、AMLET配置成0.7mM的溶液，与安慰剂按照每天1次的频率分别注入病人的病灶部位，持续治疗3周。在此期间，每周观测一次病灶并拍照，在治疗完成后1个月和2个月时对病人跟踪访问。如果在治疗后1个月时病灶体积减少75%，则视治疗成功。HAMLET treatment reduced the papilloma volume in 100% (20/20) of the patients (88/92 papillomas) compared with 15% in the placebo group (3/20 patients, 15/74 papillomas) (P0.001). This
20、effect was reproduced in the open study (10/12 patients, 36/41 lesions). No adverse reactions were reported, and there was no difference in efficacy between immunocompetent and immunosuppressed patients (Fig. 3). 结果显示：HAMLET对皮肤乳头状瘤的治愈成功率达到100%（20/20 人，88/92乳头状瘤），安慰剂组的治愈率达15% （3/20 人，15/74 乳头状瘤）。此疗效在
21、后来的公开研究中被重复（12/20人，36/41病灶）。无不良反应的报道，在免疫正常和免疫力受抑制病人身上的功效也无差异（图3）。FIGURE 3 Effect of HAMLET on human skin papillomas. A. Study design. A double-blind, placebo-controlled study was performed with HAMLET on human skin papillomas. The substance was applied once a day for 3 wk, and the lesions were measu
22、red and photographed once a week during the treatment period and at follow-up visits 1 and 2 mo after completed treatment. B. Morphology of papilloma before and after HAMLET treatment. C. Treatment effect of HAMLET contra placebo. A 75% reduction in lesion volume was observed in 20/20 patients recei
23、ving HAMLET and in 3/20 patients receiving placebo (P0.001).Topical HAMLET application significantly reduced the volume of skin papillomas. We propose that HAMLET should be further explored as a novel therapeutic agent in patients with HPV-induced diseases.经典的HAMLET临床应用显著降低了皮肤乳头状瘤的体积。我们建议应该对 HAMLET进
24、行更深入的研究，以期能成为一种治疗HPV诱发疾病的新型疗法。In vivo effects of HAMLET on glioblastoma xenografts.HAMLET 在体内对胶质母瘤细胞的影响。The majority of intracranial neoplasms originate from neuroglial cells and form a heterogeneous group known as gliomas【26】. They account for 60% of all primary brain tumors and have the most unfav
25、orable prognosis. Patients with glioblastomas of WHO grade IV show a mean survival time of less than 1y【27】, and glioblastomas constitute approximately one-fourth of all intracranial tumors in neurosurgical and neuropathological series. In recent years, surgical treatment of glioblastomas has made s
26、ignificant technical advances. Microsurgery and neuronavigation, as well as new diagnostic high resolution imaging techniques have reduced surgical mortality and morbidity, but there has been no significant improvement in survival. The tumors are inaccessible to complete surgical removal due to thei
27、r invasive nature and diffuse infiltrating growth, and the current treatment of patients with malignant gliomas is palliative, involving surgery, radiotherapy, and chemotherapy【22】. 大多数颅内肿瘤来源于神经胶质细胞，并形成了常称为“胶质瘤”的异质性基团。所有原发性脑肿瘤中约有 60%为胶质瘤，此种瘤会产生最不利的预后。按照 WHO 的分级标准，患有 4 级胶质瘤的患者平均存活时间不超过 1 年，胶质瘤囊括了神经外科
28、和神经病理科领域内全部颅内肿瘤的 1/4。近年来，手术治疗胶质瘤在技术上取得了重要进展。显微外科和神经导航系统，以及新的高分辨率诊断成像技术，已经降低了手术死亡率和发病率，但一直没有明显提高术后的存活率。由于具有侵入性和弥漫性、浸润性生长的特点，想通过手术方式完全移除肿瘤很难办到。目前对恶性胶质瘤患者的治疗采用姑息疗法，包括手术，放疗和化疗。During our survey of tumor-cell lines, we observed that glioblastoma cells undergo apoptosis in response to HAMLET. Native -lact
29、albumin, which was used as a control throughout these studies, did not influence cell viability or cause DNA fragmentation. HAMLET did not induce apoptosis in differentiated brain cells. The normal cells maintained their viability and retained intact DNA after 24 h of exposure to HAMLET. 在对肿瘤细胞株研究期间
30、，我们观察到胶质瘤细胞在HAMLET作用下出现凋亡。天然的- 乳白蛋白作为对照，没有影响细胞活性或引发DNA分解。HAMLET没有诱导已分化的脑细胞凋亡。正常细胞在接触HAMLET 24小时后仍保持着活性和结构完整的 DNA。To investigate the effect of HAMLET on tumor tissue instead of cell lines, human glioblastoma biopsy spheroids were exposed to HAMLET or-lactalbumin in vitro, and apoptotic cells were det
31、ected by the TUNEL assay 【29】. HAMLET was shown to induce apoptosis throughout the tumor spheroids, but-lactalbumin had no effect (Fig. 4A). The effect of HAMLET was investigated in a rat model of human glioblastoma 【28,29 】 . Xenotransplantation of human glioblastoma biopsies into the nude rat brai
32、n offers a unique model to study the human disease under experimental conditions, because the xenografts show the infiltrative growth characteristic of human tumors. In this model, human tumor biopsies are allowed to form spheroids in vitro as an intermediate step to obtain standardized inocula of t
33、umor cells. After xenotransplantation, the rats develop pressure symptoms after 8 wk with little variation, and large tumor masses can be detected by MRI scans. 为了考察HAMLET对肿瘤组织而非细胞的作用，将人胶质瘤活体球在体外暴露于HAMLET或-乳白蛋白中，通过 TUNEL分析法检测凋亡细胞。 HAMLET诱导整个肿瘤体产生凋亡，但-乳白蛋白却无影响（图4A ）。采用移植人胶质瘤的大鼠模型继续研究HAMLET对肿瘤组织的作用。将人
34、脑胶质瘤活体异种移植到大鼠脑中为在实验条件下研究人类疾病提供了一个独特模型，这是因为异种移植体能表现出人类肿瘤浸润式生长的特点。在这种模式下，人肿瘤活体在体外形成球体，可作为一个中间步骤，以获得肿瘤细胞的标准化接种。异种组织移植后，大鼠的压力症状缓慢发展，经过8周后变化不大，可以通过核磁共振成像扫描仪发现大的肿瘤块。The therapeutic potential of HAMLET was investigated in this model. The tumor cells were allowed 1 wk to integrate in the brain 【28,29】. HAML
35、ET was administered by infusion into the brain for 24 h with -lactalbumin as a control, and the rats were observed for another 7 wk (Fig. 4B). HAMLET inhibited tumor development (Fig. 4C), as demonstrated by a delayed onset of pressure symptoms. Rats receiving-lactalbumin developed symptoms signific
36、antly earlier than the HAMLET-treated animals (P0.01). HAMLET的治疗潜力在该模型中被研究。肿瘤细胞在1周内侵入脑部。 HAMLET随后扩散至大脑中24小时，以-乳白蛋白为对照，大鼠继续被观察7周（图4B ）。HAMLET 移植了肿瘤发展（图4C），延迟了压力症状的出现。而接受 -乳白蛋白治疗的大鼠其症状发展明显早于（P0.01）接受HAMLET的大鼠。FIGURE 4 Effect of HAMLET on human glioblastomas. A. Apoptosis induction in vitro (TUNEL stai
37、ning). B. Xenotransplantation model of human glioblastomas. Biopsy spheroids were injected into the brains of immunodeficient nu/nu rats. Convection enhanced delivery (CED) was used to infuse HAMLET or-lactalbumin (0.7 mmol/L). Seven weeks later, the tumor volume was determined by MRI. C. Tumor volu
38、me by MRI. HAMLET showed effect on tumor growth compared with the -lactalbumintreated rats (P 0.01).Apoptosis induction in vivo was examined by subjecting sections from the treated rats to the TUNEL assay. There was extensive apoptosis in the tumor, but the tissue surrounding the tumor did not show
39、TUNEL staining. Furthermore, the infusion of HAMLET did not harm the normal brain and did not produce any neurological symptoms. HAMLET has the potential to act as a selective inducer of apoptosis in patients with malignant glioblastomas.对实验大鼠进行TUNEL分析后显示：大鼠脑内肿瘤出现大面积的凋亡，但周边组织未显示出TUNEK 染色。此外， HAMLET没
40、有侵害正常的脑组织，也没有产生任何的神经症状。HAMLET 具有选择性地诱发恶性肿瘤凋亡的能力。CONCLUSIONSHAMLET illustrates the value of human milk as a rich source of molecules with a beneficial effect on a variety of human-disease conditions. The conditions required to form HAMLET are present in the stomach of the breast-fed child, where the
41、low pH may unfold the protein by the release of calcium, and where acid-sensitive lipases hydrolyze milk triglycerides to release oleic acid. We speculate that HAMLET may kill virus-transformed or premalignant cells from the gastrointestinal tract of the breast-fed child. This effect might include l
42、ymphoid cells in the gut-associated lymphoid tissue, because breast-fed children have a much reduced frequency of lymphomas compared with age-matched but bottle-fed children【30】.HAMLET 显示出人乳因含有丰富的对各类人体疾病均有抑制和抵抗作用的活性分子而具有的价值。形成 HAMLET 需要以下条件：在母乳喂养儿童的胃内，在低 pH 值环境中打开蛋白质分子结构释放出钙，以及对酸敏感的脂肪酶水解母乳中的甘油三酸酯释放出
43、油酸。我们推测，HAMLET 可以杀死母乳喂养儿童胃肠道内由 病毒转化或癌变前的细胞。这种影响可能涉及到肠道相关淋巴组织中的淋巴细胞，因为与年龄相仿的配方奶粉喂养儿童相比，母乳喂养儿童淋巴瘤的发生率大大降低。LITERATURE CITED1. Hakansson, A., Zhivotovsky, B., Orrenius, S., Sabharwal, H. & Svanborg, C. (1995) Apoptosis induced by a human milk protein. Proc. Natl. Acad. Sci. U.S.A. 92: 80648068.2. Svenss
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49、not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells). Protein Sci. 12: 27942804.9. Duringer, C., Hamiche, A., Gustafsson, L., Kimura, H. & Svanborg, C. (2003) HAMLET interacts with histones and chromatin in tumor cell nuclei. J. Biol. Chem. 278: 4213142135.10. Ye, X., Franco, A. A., Santos, H., Nelson, D. M., Kaufman, P. D. & Adams, P. D. (2003) Defective S phase chromatin assembly causes DNA damage, activation of the S phase checkpoint, and S phase arrest. Mol. Cell
