Mitochondrial-derived peptide humanin as therapeutic target in cancer and degenerative diseases

Introduction: Mitochondrial-derived peptides (MDPs) are encoded within the mitochondrial genome. They signal within the cell or are released to act as autocrine/paracrine/endocrine cytoprotective factors playing a key role in the cellular stress response. The first reported and better characterized MDP is humanin (HN), which exerts robust protective effects against a myriad of cytotoxic stimuli in many cell types. These effects have led to the evaluation of HN and its analogs as therapeutic targets for several chronic diseases.

Areas covered: We describe the latest findings on the mechanism of action of HN and discuss the role of HN as therapeutic target for neurodegenerative and cardiovascular diseases, diabetes, male infertility, and cancer. Since HN can be detected in circulation, we also depict its value as a biomarker for these diseases.

Expert opinion: HN analogs and peptide mimetics have been developed over the last decade and show promising results in preclinical models of degenerative diseases. Local administration of gene therapy vectors that overexpress or silence endogenous HN could also hold therapeutic potential. Controversy on the role of HN in cancer progression and chemoresistance should be addressed before the translation of these therapeutic approaches.     

Genome expression analysis by suppression subtractive hybridization identified overexpression of Humanin, a target gene in gastric cancer chemoresistance

Background

In cancer cells, apoptosis is an important mechanism that influences the outcome of chemotherapy and the development of chemoresistance. To find the genes involved in chemoresistance and the development of gastric cancer, we used the suppression subtractive hybridization method to identify the genes that are overexpressed in gastric cancer tissues compared to normal gastric tissues.

Results

In the suppression subtractive hybridization library we constructed, the most highly overexpressed genes were humanin isoforms. Humanin is a recently identified endogenous peptide that has anti-apoptotic activity and has been selected for further study due to its potential role in the chemoresistance of gastric cancer. Upregulation of humanin isoforms was also observed in clinical samples by using quantitative real-time PCR. Among the studied isoforms, humanin isoform 3, with an expression level of 4.166 ± 1.44 fold, was the most overexpressed isoform in GC.

Conclusions

The overexpression of humanin in gastric cancer suggests a role for chemoresistance and provides new insight into the biology of gastric cancer. We propose that humanin isoforms are novel targets for combating chemoresistance in gastric cancer.     

Humanin detected in skeletal muscles of MELAS patients: a possible new therapeutic agent

Humanin (HN) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimers disease-related insults. We have previously indicated that HN increases cellular ATP levels and speculated that this peptide may rescue energy-deficient cells in mitochondrial disorders. Here, we report, for the first time, increased HN expression in skeletal muscles from patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). HN was strongly positive in all ragged-red fibers (RRFs) and some non-RRFs, and most of them were type 1 fibers generally requiring higher energy than type 2 fibers. HN in these fibers was localized in mitochondria. HN expression was also increased in small arteries that strongly reacted for succinate dehydrogenase. Our experiments on muscular TE671 cells indicated the possibility that synthesized HN increases cellular ATP levels by directly acting on mitochondria. From these in vivo and in vitro findings, we propose that HN expression might be induced in response to the energy crisis within affected fibers and vessels in MELAS muscles and further be a possible therapeutic candidate for MELAS.     

Humanin attenuates apoptosis induced by DRPLA proteins with expanded polyglutamine stretches

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal-dominant neurodegenerative disorder caused by expansion of CAG repeats in the DRPLA gene, which codes for a polyglutamine (polyQ) stretch. The expanded polyQs are known to form intracellular aggregates and to confer neurotoxic activity. Recent studies have indicated that activation of apoptosis signal-regulating kinase 1 (ASK1) is involved in polyQ-induced apoptosis. Humanin (HN) is an endogenous peptide that inhibits neuronal cell death caused by mutant Alzheimer’s disease genes, and this neuroprotective factor has recently been reported to suppress apoptosis by inhibiting activation of ASK1. To test the anti-ASK1 effect of HN on polyQ neurotoxicity, we constructed neuronal PC12 cells expressing expanded polyQs under the control of the Tet-Off™ system. Using this cell line, we showed that HN suppresses apoptotic cell death induced by expanded polyQs. However, the suppression was incomplete, suggesting that polyQs also stimulate other pathogenic cascades unrelated to ASK1. We further showed that HN suppresses polyQ aggregate formation. This result implied the possibility that aggregation is also related to the polyQ-mediated cascade involving ASK1 activation. Although the details remain uncertain, our results suggest that ASK1 is potentially involved in pathogenesis of DRPLA and that HN might contribute partially to the suppression of neurodegeneration in polyQ diseases.     

Amino- and carboxyl-terminal mutants of presenilin 1 cause neuronal cell death through distinct toxic mechanisms: Study of 27 different presenilin 1 mutants

Presenilin (PS)1 and its mutants, which consist of the N-terminal and C-terminal fragments, cause certain familial forms of Alzheimer's disease (FAD). Our earlier studies found that FAD-linked M146L-PS1 causes neuronal cell death through nitrogen oxide synthase (NOS) and that FAD-linked N141I-PS2, another member of the PS family, causes neuronal cell death through NADPH oxidase. In this study, we examined 27 different FAD-linked mutants of PS1, and found that PS1 mutants with mutations in the N-terminal fragment caused NOS inhibitor (NOSI)-sensitive neuronal cell death; in contrast, the PS1 mutants with mutations in the C-terminal fragment caused NOSI-resistant neuronal cell death. The former toxicity was resistant to the specific NADPH oxidase inhibitor apocynin and was inhibited by Humanin (HN), a newly identified neuroprotective factor against Alzheimer's disease (AD)-relevant insults, but not by insulin-like growth factor-I (IGF-I). In contrast, the latter toxicity was sensitive to apocynin and inhibited by both IGF-I and HN. This study indicates for the first time that N- and C-terminal fragment PS1 mutants can generate distinct neurotoxic signals, which will provide an important clue to the understanding of the entire array of neurotoxic signals generated by FAD-causative mutations of PS1.     

Humanin对缺氧诱导的大鼠皮层神经元损伤的保护作用

目的观察Humanin（HN）对缺氧诱导皮层神经细胞损伤的保护作用。方法培养10d的原代皮层神经元在缺氧环境下（氧含量〈2％）放置4h诱导神经细胞损伤。实验组在缺氧之前24h分别加入终浓度为10μmol／L和20μmol／L的HN。通过测定MTT和Calcein—AM染色检测细胞活力。通过测定丙二醛（MDA）、超氧化物歧化酶（SOD）、乳酸脱氢酶（LDH）的变化检测细胞损伤的程度。结果HN可以提高缺氧4h后细胞的存活率,同时可以降低由细胞损伤所引起的MDA、SOD、LDH的升高。20μmol／L的HN具有比10μmol／L更显著的保护作用。结论HN对缺氧诱导的神经元细胞损伤具有保护作用。     

Humanin拮抗Aβ_(31-35)诱导的神经元凋亡

为了研究Humanin(HN)对Aβ31-35诱导的大鼠皮层神经元凋亡的影响,本研究采用原代培养的大鼠皮层神经元,应用流式细胞术、TUNEL法、HO33342染色法检测不同时间(0、8、16h)加入不同浓度的HN对Aβ31-35致神经元凋亡的影响。结果显示:Aβ31-35(25μmol/L)引起培养皮层神经元的凋亡率明显增高,神经元凋亡率由7.43%上升到32.69%;凋亡指数由6.87%上升到28.36%(P<0.05)。与Aβ31-35同时或提前8h给予不同浓度(5,10,20μmol/L)的HN对Aβ31-35(25μmol/L)诱导的神经元凋亡均未产生影响;但20μmol/L的HN提前16h孵育可明显抑制Aβ31-35所致的神经元凋亡,其凋亡率由32.69%下降到20.36%,凋亡指数由28.36%下降到17.57%。本研究结果提示,HN拮抗Aβ31-35致神经元凋亡作用具有剂量和时间依赖性。     

Humanin通过保护线粒体功能对缺氧诱导的神经元损伤发挥保护作用

BACKGROUND： Humanin is a 24-amino acid peptide isolated from the brain of an Alzheimer’s disease patient. Several studies have indicated that Humanin can protect cells against cytotoxicity induced by various insults.
OBJECTIVE： To investigate the protective role of Humanin on hypoxia-induced neuronal death, and to determine the most appropriate therapeutic concentration of Humanin.
DESIGN, TIME AND SETTING： Neuropathophysiological, randomized, controlled experiment, conducted at the Department of Physiology and Neurobiology, Shanxi Medical University, between March 2007 and October 2007.
MATERIALS： Newborn Wistar rats, 5,5＇,6,6＇ tetrachloro-1,1＇,3,3＇-tetraethyl- benzimidazolylcarbo- cyanine iodide （JC-1, USA）, calcein-acetoxymethylester （calcein-AM, USA）, and Humanin （Shanghai, China） were used in this study. METHODS： Primary cortical neurons were cultured with dulbecco＇s modified eagle＇s medium containing 15% fetal bovine serum. Cultures were divided into three groups： control, hypoxia, and hypoxia ＋ Humanin. Various concentrations of Humanin （1, 10, and 20 μmol/L） were added to the cultures 16 hours prior to hypoxia induction. For hypoxic conditions, cells were maintained at 37 ℃ within an incubator chamber filled with 95% N2 and 5% CO2 for 24 hours. Cells in the control group were cultured in normal oxygen.
MAIN OUTCOME MEASURES： Cell viability was determined through the use of the vital dye calcein-AM, and the number of live cells was determined. Mitochondrial membrane potential （△Ψm） was assessed using the fluorescent probe JC-1. Mitochondrial permeability transition pore （mPTP） opening was determined with calcein-AM in the presence of cobalt chloride.
RESULTS： （1） Cell viability： Hypoxia for 24 hours induced death in a large number of neurons. Pre- treatment with 10 μmol/L and 20 μmol/L Humanin, 16 hours prior to hypoxia, protected cells against hypoxia. However, 1 μmol/L Humanin provided little protection. （2） △Ψm： △Ψm was re-duced after 24-hour hypoxia, as assessed by JC-1 and a confocal microscope. Pretreatment with 20 μmol/L Humanin preserved the loss of △Ψm. （3） mPTP： Hypoxia induced the opening of mPTP. Pretreatment with 20 μmol/L Humanin repressed the opening of mPTP, as most of the calcein fluorescence remained in the mitochondria.
CONCLUSION： Humanin （20 μmol/L） protects neuronal cells from hypoxia-induced insults by in- hibiting the opening of mPTP and preserving △Ψm.     

Humanin通过JNK途径减轻Aβ31-35诱导的皮层神经元的凋亡

目的研究JNK信号通路在AAβ31-35诱导神经元凋亡中的作用以及Humanin（HN）的神经保护机制。方法原代培养大鼠皮层神经元随机分为对照组、Aβ31-35组（1h,4h,8h,16h,24h）、抑制剂SP600125组、HN预处理组。应用流式细胞术观察各组神经元凋亡率,应用免疫细胞化学法观察各组磷酸化c—Jun（p-c—Jun）和FasL蛋白表达情况。结果对照组仅有少数神经元发生凋亡;Aβ31-35诱导培养皮层神经元异常凋亡;JNK抑制剂SP600125抑制了Aβ31-35诱导的神经元凋亡;HN（20μmol／L）16h预孵育部分抑制了Aβ31-35的致凋亡作用,各组神经元凋亡率分别为7．43％,32．6％（24h）,8．8％,20．36％。对照组仅有少量p-c—Jun及FasL蛋白的表达;Aβ31-35时间依赖性诱导了p-c—Jun、FasL蛋白的表达;SP600125抑制了Aβ31-35诱导的p-c—Jun、FasL蛋白的表达;20μmol／LHN16h预孵育部分抑制了p-c-Jun及FasL蛋白的表达。结论JNK通路可能参与了Aβ31-35诱导的神经元凋亡过程;HN可在一定程度上阻抑JNK信号通路,减轻Aβ31-35诱导的神经元凋亡。