Phagocyte-related S100 proteins and cytokines in acute lymphoblastic leukemia and their prognostic value.

In this retrospective cohort study, we evaluated the level of biomarkers of inflammation like phagocyte-related S100 proteins and a panel of cytokines in 128 children with pre-B ALL and 22 with T-ALL. The biomarkers were evaluated at diagnosis and during antileukemic therapy (day 29 and after six months) and we evaluated their correlation with basic laboratory values. Further, for the children with pre-B ALL, we evaluated whether the biomarkers could predict the outcome of ALL expressed as minimal residual disease (MRD), relapse, and death.The levels of S100A9, S100A12, IL-1beta, IL-12p70, IL-13, IL-17, IL-18, and MPO serum levels increased significantly as chemotherapy was initiated. The difference was most pronounced for S100A9 and S100A12, which had strong positive correlations with the neutrophil counts. In contrast, TNF-alpha, IL-6, IL-10, CCL-2, MMP-3, and CD25 serum levels decreased after chemotherapy. Although none of these biomarkers appear to be an independent predictor of outcomes, in predictive models with MRD as the outcome, AUC increased from 76% (95% CI 68-84%) when using initial risk group stratification alone to 83% (95% CI 73-91%) in a multivariate predictive model including initial risk group stratification and the biomarkers S100A12, TNF-alpha, and IL-10.

S100A1 is Involved in Myocardial Injury Induced by Exhaustive Exercise.

Many studies have confirmed that exhaustive exercise has adverse effects on the heart by generating reactive oxygen species (ROS). S100A1 calcium-binding protein A1 (S100A1) is a regulator of myocardial contractility and a protector against myocardial injury. However, few studies have investigated the role of S100A1 in the regulation of myocardial injury induced by exhaustive exercise. In the present study, we suggested that exhaustive exercise led to increased ROS, downregulation of S100a1, and myocardial injury. Downregulation of S100a1 promoted exhaustive exercise-induced myocardial injury and overexpression of S100A1 reversed oxidative stress-induced cardiomyocyte injury, indicating S100A1 is a protective factor against myocardial injury caused by exhaustive exercise. We also found that downregulation of S100A1 promoted damage to critical proteins of the mitochondria by inhibiting the expression of Ant1, Pgc1a, and Tfam under exhaustive exercise. Our study indicated S100A1 as a potential prognostic biomarker or therapeutic target to improve the myocardial damage induced by exhaustive exercise and provided new insights into the molecular mechanisms underlying the myocardial injury effect of exhaustive exercise.

Rapeseed Protein-Derived Antioxidant Peptide RAP Ameliorates Nonalcoholic Steatohepatitis and Related Metabolic Disorders in Mice.

Rapeseed protein hydrolysates have recently shown in vitro antioxidant and anti-inflammatory activities. However, scant data exist about their in vivo activities. Here, we report that the peptide DHNNPQIR (hereinafter referred to as RAP-8), a bioactive peptide originated from rapeseed protein, exhibits excellent in vivo efficacy in mouse models of nonalcoholic steatohepatitis (NASH) and hepatic fibrosis. We demonstrated that RAP-8 significantly reduced hepatic steatosis and improved insulin resistance and lipid metabolism. Furthermore, RAP-8 showed markedly reduced hepatic inflammation, fibrosis, liver injury, and metabolic deterioration. In particular, RAP-8 directly suppressed fibrosis-associated gene expression, including α-smooth muscle actin (α-Sma) and collagen type I (Col-1α) in the liver of mice in vivo. In addtion, RAP-8 significantly decreased macrophage infiltration and reduced pro-inflammatory cytokines secretion. Finally, we found that RAP-8 administration significantly decreased oxidative stress-induced apoptosis in liver injury induced by CCl4. Therefore, our results suggest that RAP-8 could be available for treatment of NASH and NASH-related metabolic disorders as a potential therapeutic candidate.

S100A11 protects against neuronal cell apoptosis induced by cerebral ischemia via inhibiting the nuclear translocation of annexin A1.

The subcellular location of annexin A1 (ANXA1) determines the ultimate fate of neurons after ischemic stroke. ANXA1 nuclear translocation is involved in neuronal apoptosis after cerebral ischemia, and extracellular ANXA1 is also associated with regulation of inflammatory responses. As the factors and mechanism that influence ANXA1 subcellular translocation remain unclear, studies aiming to determine and clarify the role of ANXA1 as a cell fate 'regulator' within cells are critically needed. In this study, we found that intracerebroventricular injection of the recombinant adenovirus vector Ad-S100A11 (carrying S100A11) strongly improved cognitive function and induced robust neuroprotective effects after ischemic stroke in vivo. Furthermore, upregulation of S100A11 protected against neuronal apoptosis induced by oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. Surprisingly, S100A11 overexpression markedly decreased ANXA1 nuclear translocation and subsequently alleviated OGD/R-induced neuronal apoptosis. Notably, S100A11 exerted its neuroprotective effect by directly binding ANXA1. Importantly, S100A11 directly interacted with ANXA1 through the nuclear translocation signal (NTS) of ANXA1, which is essential for ANXA1 to import into the nucleus. Consistent with our previous studies, ANXA1 nuclear translocation after OGD/R promoted p53 transcriptional activity, induced mRNA expression of the pro-apoptotic Bid gene, and activated the caspase-3 apoptotic pathway, which was almost completely reversed by S100A11 overexpression. Thus, S100A11 protects against cell apoptosis by inhibiting OGD/R-induced ANXA1 nuclear translocation. This study provides a novel mechanism whereby S100A11 protects against neuronal cells apoptosis, suggesting the potential for a previously unidentified treatment strategy in minimizing apoptosis after ischemic stroke.

Valores de referencia de la proteína S100beta en población pediátrica / Serum S100Beta protein reference values in a paediatric population

INTRODUCCIÓN: La proteína S100β se ha propuesto como posible biomarcador en patología neurológica, tanto crónica como aguda. Los valores normales de esta proteína están bien definidos en adultos, no así en niños, en los que los valores séricos parecen variar con la edad. Nuestro objetivo es describir valores de referencia de S100β sérica en niños de 0 a 14 años. MATERIAL Y MÉTODOS: Estudio prospectivo en 257 niños sanos. Se establecieron 3 grupos por edad (menores de 12 meses, de 12 a 24 meses y mayores de 24 meses). RESULTADOS: Se incluyó a 179 niños y 78 niñas. La edad media ± DE fue de 5,5 ± 3,75 años. La concentración sérica media de la proteína S100β en todo el grupo fue 0,156 (0,140-0,172) μg/l. En los menores de 12 meses, la concentración sérica de S100β fue de 0,350 (0,280-0,421) μg/l; 0,165 (0,139-0,190) μg/l en el grupo entre 12 y 24 meses y 0,121 (0,109-0,133) μg/l en el grupo de niños mayores de 24 meses. Se observó una relación inversa entre la edad y la concentración sérica de S100β, que desciende conforme se incrementa la edad. No se observaron diferencias en cuanto al sexo. CONCLUSIONES: La concentración de S100β permanece estable a partir de los 2 años de edad, siendo posible establecer unos valores de referencia de S100β para mayores de 2 años. En los 2 primeros años de vida, la concentración de S100β sérica es más elevada cuanto menor es la edad del niño. No se observan diferencias en el valor de S100β sérica entre ambos sexos

INTRODUCTION: S100β protein has been proposed as a potential biomarker for both chronic and acute neurological disorders. Reference values of this protein are well defined in adults but not in children, in whom serum levels appear to vary with age. Reference values for serum S100β in children from 0 to 14 years are presented. MATERIALS AND METHODS: A prospective study was conducted on 257 healthy children, who were divided into three age groups (under 12 months, 12 to 24 months and over 24 months). RESULTS: The study included179 boys and 78 girls, with a mean age of 5.5 (3.75) years. The mean serum concentration of protein S100β was 0.156 (0.140-0.172) μg/l. In children under 12 months, serum S100β concentration was 0.350 (0.280-0.421) μg/l; 0.165 (0.139-0.190) μg/l in the group between 12 and 24 months and 0.121 (0.109-0.133) μg/l in children older than 24 months. An inverse relationship was observed between age and serum S100β, which declines as age increases. No differences were observed between sexes. CONCLUSIONS: The concentration of S100β remains stable after two years of age, being possible to establish a baseline of S100β for over two years. During the first two years of life, S100β serum concentration is higher, the lower the age of the child. No differences in serum S100β levels between sexes are observed

Respuesta a valores de referencia de la proteína S100β en población pediátrica / Response to serum S100β protein reference values in a paediatric population

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Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide.

The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.

Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model.

Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.

Peptide mimetic of the S100A4 protein modulates peripheral nerve regeneration and attenuates the progression of neuropathy in myelin protein P0 null mice.

We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.

Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model.

As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)-S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca(2+)) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)-S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

A novel model to measure the regenerative potential of the peripheral nervous system after experimental immunological demyelination.

BACKGROUND: Immunological demyelination is a proposed strategy to improve nerve regeneration in the peripheral nervous system. To investigate the remyelinating potential of Schwann cells in vivo in the peripheral nervous system, the authors have reproduced and expanded upon a novel model of immunological demyelination in the adult rat sciatic nerve. The authors demonstrate (1) the peripheral nervous system's quantitative, regenerative response to immunological demyelination and (2) whether Schwann cells within a region of demyelination are induced to divide in the presence of demyelinated axons. METHODS: The sciatic nerves of female Sprague-Dawley rats were exposed and injected with demyelinating agent bilaterally. At 3 days (n = 3), 7 days (n = 3), and 14 days (n = 3), the animals were euthanized for histological evaluation. A second group of animals (n = 3) was similarly injected with demyelinating agent and then exposed to bromodeoxyuridine between 48 and 72 hours after the onset of demyelination. These animals were euthanized soon after the last injection of bromodeoxyuridine. The tissue was analyzed for Schwann cells (labeled with antibodies to S100) and bromodeoxyuridine assay. RESULTS: A single epineural injection of complement proteins plus antibodies to galactocerebroside resulted in demyelination followed by Schwann cell remyelination. At 3 days after injection, peripheral nerve demyelination and Schwann cell proliferation were evident. Maximum demyelination was seen at 7 days; however, Schwann cell proliferation and remyelination peaked at 14 days after injection. CONCLUSIONS: These studies demonstrate an immunological model of demyelination and remyelination in the peripheral nervous system and quantitatively measure regenerative potential. This model will be used to isolate nerve segments and to measure their regenerative potential when given demyelinating agent after acute contusion and transection injuries.

Anti-intercellular adhesion molecule-1 antibody's effect on noise damage.

OBJECTIVES/HYPOTHESIS: The purpose of this study was to investigate possible preventive effects of anti-intercellular adhesion molecule-1 antibody (anti-ICAM-1 Ab) on noise-induced cochlear damage as assessed by changes in auditory thresholds and cochlear blood flow. STUDY DESIGN: A controlled animal study. Pretreated rats with anti-ICAM-1 Ab or saline control, followed with exposure to 72 continuous hours of broad band noise (107 dB SPL), and 24 hours after noise exposure treated again with anti-ICAM-1 Ab or saline. METHODS: Eighteen healthy male Fischer rats (200-250 g) were used. Sixteen were randomly selected to study noise-induced temporary threshold shifts. The remaining two rats were used to study cochlear blood flow (CBF), using laser Doppler flowmetry and blood pressure measurements. RESULTS: Rats treated with anti-ICAM-1 Ab (1.875 mg/kg, intravenously) showed attenuated temporary threshold shifts (TTS) compared to controls. Both groups showed a partial threshold recovery 72 hours following noise exposure, normal for this noise exposure paradigm. Comparisons of baseline and post-treatment measurements of CBF and mean arterial blood pressure revealed no significant changes. Anti-ICAM-1 Ab animals displayed significantly lower mean auditory threshold shifts at all five test frequencies (P < .05) when compared to control. CONCLUSIONS: Blocking the cascade of reactive oxygen species (ROS) generation by using anti-ICAM-Ab protects against noise-induced hearing loss.

S100A1 binds to the calmodulin-binding site of ryanodine receptor and modulates skeletal muscle excitation-contraction coupling.

S100A1, a 21-kDa dimeric Ca2+-binding protein, is an enhancer of cardiac Ca2+ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca2+ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca2+ release in S100A1-/- muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca2+-dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.

Recent findings into the potential of gene therapy to reverse heart failure.

The incidence of heart failure (HF) is ever growing and the mortality of HF patients is similar to patients suffering from cancer disease. The central clinical problem is a lack of therapies to target the underlying molecular defects that lead to chronic ventricular dysfunction. Substantial evidence points to a final common pathway in failing myocardium, including distinct changes in intracellular Ca2+-cycling and beta-adrenergic receptor signaling. An attractive strategy to address these alterations is cardiac gene therapy and several distinct approaches have been undertaken during the last decade with impressing therapeutic benefit, at least in animal HF models. The present focus of research is the clinical translation of cardiac gene therapy including the optimization of vectors, delivery strategies and testing the compatibility with established pharmacologic treatment to improve the prognosis of HF in the near future.

Enhanced hippocampal neurogenesis by intraventricular S100B infusion is associated with improved cognitive recovery after traumatic brain injury.

Evidence of injury-induced neurogenesis in the adult hippocampus suggests that an endogenous repair mechanism exists for cognitive dysfunction following traumatic brain injury (TBI). One factor that may be associated with this restoration is S100B, a neurotrophic/mitogenic protein produced by astrocytes, which has been shown to improve memory function. Therefore, we examined whether an intraventricular S100B infusion enhances neurogenesis within the hippocampus following experimental TBI and whether the biological response can be associated with a measurable cognitive improvement. Following lateral fluid percussion or sham injury in male rats (n = 60), we infused S100B (50 ng/h) or vehicle into the lateral ventricle for 7 days using an osmotic micro-pump. Cell proliferation was assessed by injecting the mitotic marker bromodeoxyuridine (BrdU) on day 2 postinjury. Quantification of BrdU-immunoreactive cells in the dentate gyrus revealed an S100B-enhanced proliferation as assessed on day 5 post-injury (p < 0.05), persisting up to 5 weeks (p < 0.05). Using cell-specific markers, we determined the relative numbers of these progenitor cells that became neurons or glia and found that S100B profoundly increased hippocampal neurogenesis 5 weeks after TBI (p < 0.05). Furthermore, spatial learning ability, as assessed by the Morris water maze on day 30-34 post-injury, revealed an improved cognitive performance after S100B infusion (p < 0.05). Collectively, our findings indicate that an intraventricular S100B infusion induces neurogenesis within the hippocampus, which can be associated with an enhanced cognitive function following experimental TBI. These observations provide compelling evidence for the therapeutic potential of S100B in improving functional recovery following TBI.

S100B and response to treatment in major depression: a pilot study.

S100B is a protein which exerts both detrimental and neurotrophic effects, depending on its concentration in brain tissue. An increase of S100B in micromolar concentrations is observed in traumatic brain conditions and is associated with poor outcome. Micromolar levels of extracellular S100B in vitro may have deleterious effects. However, in nanomolar concentrations S100B has multiple neurotrophic effects in vitro may in vivo be regarded as a hallmark of neuroprotective efforts. This pilot study addresses the hypothesis that S100B serum concentrations may be of predictive validity for the response to antidepressant treatment in patients with major depression. S100B plasma levels were determined in 25 patients with major depression and 25 matched healthy controls using an immunofluorimetric sandwich assay. S100B plasma levels were significantly higher in major depressive patients than in healthy controls and positively correlated with treatment response after 4 weeks of treatment. In a linear regression model, a significant predictive effect was found only for S100B and severity of depressive symptoms upon admission. These results suggest that neuroprotective functions of S100B counterbalance neurodegenerative mechanisms that are involved in the pathophysiology of major depression and in the response to antidepressant treatment.

[Calcium-binding protein S100A2 in the retardation of growth and proliferation of hepatocellular carcinoma cells].

OBJECTIVE: To study the retardation effect of calcium-binding protein S100A2 on the growth and proliferation of hepatocellular carcinoma QGY7701 cells. METHODS: After the plasmid of EGFP-S100A2 had been regrouped and introduced into the hepatocellular carcinoma QGY7701 cells with lipofectin, the expression and location of the products were observed by fluorescent microscopy. The cell growth and proliferation were monitored through cell colony formation in vitro and xenografting subcutaneously in the nude mice in vivo. The effect of S100A2 on the QGY7701 cell cycle was examined with flow cytometry. RESULTS: The chimera protein of S100A2 and green fluorescent protein were detected and appeared to be localized in the cytoplasm and nucleus, while the green fluorescent protein was found to be localized only in the cytoplasm. The cell colony formation of QGY7701/A2 was significantly reduced as compared with the controls. The xenografted tumor of QGY7701/A2 in the nude mice showed a growth at a considerably slower rate than that of QGY7701/pc and QGY7701 groups. The cell cycle review showed retardation of QGY7701/A2 cells in the G1 phase and the DNA content was obviously reduced as compared with the controls. CONCLUSION: The exogenous S100A2 is able to check the QGY7701 cell cycle, stop the cell growth and proliferation either in vitro or in vivo in the QGY7701 cells.