Human Chromogranin Research Articles

Quantification of Chromogranin A and Its Fragments in Biological Fluids.

Human chromogranin A (CgA), a 439-residue long neurosecretory protein, can serve as a circulating biomarker for a wide range of neuroendocrine tumors. Increased levels of immunoreactive CgA are also present in the blood of patients with cardiovascular, gastrointestinal, or inflammatory diseases with, in certain cases, important diagnostic and prognostic implications. A growing body of evidence suggest that CgA and various CgA-derived fragments have complex roles in the regulation of cardiovascular system, metabolism, innate immunity, angiogenesis, and tissue repair, sometime with opposite biological effects. For example, while full-length CgA (CgA1-439) inhibits angiogenesis, the CgA1-373 fragment, at certain doses, is proangiogenic. Thus, the selective quantification of CgA and its fragments in the blood of patients (and in other biological fluids) is of great experimental and clinical interest. Here, we describe methods to produce CgA1-439 and CgA1-373 and to develop ELISAs capable of detecting these polypeptides in a very selective manner. The same approach can be used, in principle, also for developing assays for other fragments.

Chromogranin B in blood serum of patients with neuroendocrine tumors.

Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms from cells of the diffuse neuroendocrine system. Chromogranin B (CgB) is an acidic protein of the granin family, which can be used to detect the tumours of neuroendocrine nature. Analysis of levels and evaluation of the diagnostic efficiency of CgB in the blood serum of patients with NETs of various localizations. Patients with NETs (n=121) without specific treatment were examined. In the study were presented next localizations: 74 - pancreas, 20 - stomach, 12 - large intestine, 15 - other localizations (lungs, mammary gland, prostate gland, NETs with unidentified primary). 54 practically healthy donors were examined as control group. The determination of CgB in blood serum was performed with ELISA method on BEP 2000 analyzer using a standardized test system Human Chromogranin B (USCN, China). CgB levels in common NET group (median 18.9 ng/mL) were statistically significantly higher than in the control group (8.8 ng/mL). The highest median was obtained in group of intestinal NETs (21.2 ng/ml), which exceeded the median of the control group by more than 2.4 times. According to ROC analysis in the common NET group relative to the control group, the area under the curve AUC was 0.88 (95% CI 0.83-0.929). According to cut-off level of CgB - 15.8 ng/ml, the diagnostic sensitivity was 69.4%, with a specificity of 96.3%. The highest diagnostic sensitivity was in the group of the intestinal NETs (75.0%) and pancreas (71.2%). The study showed the significance of CgB as a potential biochemical marker of NETs with various localizations, alternative to CgA.

Neuropilin-1 and Integrins as Receptors for Chromogranin A-Derived Peptides.

Human chromogranin A (CgA), a 439 residue-long member of the "granin" secretory protein family, is the precursor of several peptides and polypeptides involved in the regulation of the innate immunity, cardiovascular system, metabolism, angiogenesis, tissue repair, and tumor growth. Despite the many biological activities observed in experimental and preclinical models for CgA and its most investigated fragments (vasostatin-I and catestatin), limited information is available on the receptor mechanisms underlying these effects. The interaction of vasostatin-1 with membrane phospholipids and the binding of catestatin to nicotinic and b2-adrenergic receptors have been proposed as important mechanisms for some of their effects on the cardiovascular and sympathoadrenal systems. Recent studies have shown that neuropilin-1 and certain integrins may also work as high-affinity receptors for CgA, vasostatin-1 and other fragments. In this case, we review the results of these studies and discuss the structural requirements for the interactions of CgA-related peptides with neuropilin-1 and integrins, their biological effects, their mechanisms, and the potential exploitation of compounds that target these ligand-receptor systems for cancer diagnosis and therapy. The results obtained so far suggest that integrins (particularly the integrin avb6) and neuropilin-1 are important receptors that mediate relevant pathophysiological functions of CgA and CgA fragments in angiogenesis, wound healing, and tumor growth, and that these interactions may represent important targets for cancer imaging and therapy.

Сравнительное исследование хромогранина А и хромогранина В у больных с нейроэндокринными опухолями желудка и поджелудочной железы

Introduction. Immunoenzyme assay of biochemical markers is one of the most important methods for examination of pa- tients with neuroendocrine tumors (NETs). Along with the generally accepted NET marker chromogranin A (CgA), another member of the granin family, chromogranin B (CgB), can serve as a complementary marker. Objectives. Analysis of CgB as an additional to CgA biochemical marker in the blood serum of patients with gastric and pancreatic neuroendocrine tumors. Materials and methods. We examined 79 patients with gastic ( n = 14) and pancretic ( n = 65) NETS, and 42 particularly healthy people, who were included in the control group. CgB and CgA were determined with ELISA method using the Human Chromogranin B (USCN, China) and Chromogranin A NEOLISA (Eurodiagnostica, Sweden) test systems. , Results. CgB levels in gastric and pancreatic NETs were significantly higher than in control group. CgB concentrations were independent of tumor spread and its biological activity. ROC analysis in common group of NETs relative to control group showed AUC for CgB = 0.869 and for CgA AUC = 0.82. According to results in common group of NET patients when used isolated, CgA and CgB have comparable diagnostic sensitivity, which increases in complex use to 82.5 %. In the group of NET patients with low levels of CgA (<100 ng/ml), an increase in CgB concentration above the cut-off level (>15.7 ng/ml) was observed in 53.6 % of cases. Conclusion. The combination of CgB and CgA in gastric and pancreatic NETs could increase the diagnostic efficacy of bio- chemical diagnostics. The received data confirms the significance of CgB as a complementary biomarker of NETs.

Purified recombinant human Chromogranin A N46 peptide with remarkable anticancer effect on human colon cancer cells

Human Chromogranin A N46 (CGA-N46) is a weak cationic α-helical peptide with wide-spectrum antibacterial, fungal, and anticancer activities. In this study, the recombinant human CGA-N46 peptide was expressed successfully in Escherichia coli. The gene of CGA-N46 was cloned into the expression vector pET-15b without a fusion tag at the N terminus and the peptide was expressed using Isopropyl-β-d-thiogalactoside (IPTG) as an inducer. Using 8 M guanidinium HCl, inclusion bodies containing the peptide were purified and solubilized. The rhCGA-N46 peptide was purified using Q-FF anion exchange column. The cytotoxicity of the purified rhCGA-N46 peptide was investigated on WI-38 human lung normal cell line. The anticancer activity was studied on human colon cancer cell line; HCT-116 cell line. Besides, the possible involvement of rhCGA-N46 peptide in regulating apoptotic signal pathways was analyzed by detecting the expression levels of BCL2, BID, and CAS-8 in the treated cells. The results concluded that the active peptide recovery was up to 41.98%. The purified rhCGA-N46 was safe on normal cells with IC50 = 227.74 µg/ml (40.67 µM) and had an obvious anticancer effect on colon cancer cells with IC50 = 1.997 µg/ml (356.6 nM). The expression level of BCL2 was down-regulated and BID and CAS-8 were up-regulated significantly in treated HCT-116 cells compared to untreated. In conclusion, the rhCGA-N46 peptide was produced successfully in the native form with promising anti-colon cancer activity.

Internalization and membrane activity of the antimicrobial peptide CGA-N12.

Antimicrobial peptides (AMPs) are conventional antibiotic alternatives due to their broad-spectrum antimicrobial activities and special mechanisms of action against pathogens. The antifungal peptide CGA-N12 was originally derived from human chromogranin A (CGA) and consists of the 65th to 76th amino acids of the CGA N-terminal region. In the present study, we found that CGA-N12 had fungicidal activity and exhibited time-dependent inhibition activity against Candida tropicalis. CGA-N12 entered the cells to exert its antagonist activity. The internalization of CGA-N12 was energy-dependent and accompanied by actin cytoskeleton-, clathrin-, sulfate proteoglycan-, endosome-, and lipid-depleting agent-mediated endocytosis. Moreover, the CGA-N12 internalization pathway was related to the peptide concentration. The effects of CGA-N12 on the cell membrane were investigated. CGA-N12 at a low concentration less than 4 × MIC100 did not destroy the cell membrane. While with increasing concentration, the damage to the cell membrane caused by CGA-N12 became more serious. At concentrations greater than 4 × MIC100, CGA-N12 destroyed the cell membrane integrity. Therefore, the membrane activity of CGA-N12 is concentration dependant.

Chromogranin A and chromogranin B in pancreatic neuroendocrine tumors

For the first time in Russia a comparative study of chromogranin A (CgA) and chromogranin B (CgB) in neuroendocrine tumors (NETs) of the pancreas was performed. We examined 50 primary patients with pancreatic NETs and 42 healthy people. The determination of CgA and CgB was performed in blood serum using standard enzyme-linked immunoassay test-systems (Chromogranin A NEOLISA, Eurodiagnostica; Human Chromogranin B, USCN). The levels of CgA and CgB in pancreatic NETs significantly differed from the control group. There was found the association between CgA levels and the dissemination of the process, while CgB demonstrated the properties of a marker independent from the tumor dissemination. The diagnostic sensitivity of CgA was 76 %, CgB – 68 %. Complex determination of CgA and CgB enhanced the diagnostic sensitivity to 84 %. Our data indicate the potential usefulness of complex CgA and CgB in the diagnosis of pancreatic NETs.

Expression of chromogranin A-derived antifungal peptide CGA-N12 in Pichia pastoris

ABSTRACT The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.

Synthesis and molecular cloning of antimicrobial peptide chromogranin A N-46 gene using conventional PCR

BackgroundChromogranin A N-46 (CGA-N46) is an antimicrobial peptide (AMP) consisting of partial N terminus of human chromogranin A. ObjectiveSynthesis and molecular cloning the gene encoding CGA-N46. Materials and methodsThe coding sequence of CGA-N46 gene was synthesized using free bioinformatics tool (Gene2Oligo) that allows the development of in vitro gene synthesis. In this study two methods have been established, stepwise PCR and One Step Simplified Gene Synthesis (SGS) methods. The gene was obtained in stepwise PCR by three steps: 1st PCR to generate short DNA fragments using specific oligonucleotides, 2nd step was the overlap extension PCR (OE-PCR) to bond the short DNA fragments and the final step was the whole gene assembly using amplification primers. Nevertheless, the SGS method was achieved by only one step PCR with various concentrations of oligonucleotides (1000, 500, 250, 100, 50 and 25 nM) and amplification primers (400 nM). In order to achieve a DNA library for future studies, the obtained CGA-N46 gene was cloned into pCR-II TOPO TA cloning vector to create a recombinant vector which was preserved in Escherichia coli (E. coli) TOP10 bacterial stock using transformation process. ResultsThe stepwise PCR method generated a sharp band at expected MW (150 bp). In addition, the SGS method gave one faint band at 1 μM of oligonucleotides. Digestion and PCR analysis revealed that the CGA-N46 gene was successfully cloned in pCR-II TOPO TA cloning vector.

The chromogranin A-derived antifungal peptide CGA-N9 induces apoptosis in Candida tropicalis.

CGA-N9, a peptide derived from human chromogranin A (CGA), was found to have antimicrobial activity in our previous investigation, but its mechanism of action remains unclear. Herein, the mechanism of action of CGA-N9 was investigated. We found that CGA-N9 induced the depolarization of the cell membrane and uptake of calcium ions into the cytosol and mitochondria. With the disruption of the mitochondrial membrane potential, the generation of intracellular reactive oxygen species (ROS) increased. Accordingly, we assessed apoptotic processes in Candida tropicalis cells post-treatment with CGA-N9 and found cytochrome c leakage, chromatin condensation and DNA degradation. The interaction of CGA-N9 with DNA in vitro showed that CGA-N9 did not degrade DNA but bound to DNA via an electrostatic interaction. In conclusion, CGA-N9 exhibits antifungal activity by inducing apoptosis in C. tropicalis.

CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis

CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47–55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells, even at 4°C and in the presence of sodium azide (NaN3), both of which block all energy-dependent transport mechanisms. In addition, peptide internalization was affected by the endocytic inhibitors 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), cytochalasin D (CyD) and heparin; chlorpromazine (CPZ) also had some effect on CGA-N9 internalization. Similar results were obtained in the MIC assays, whereby the anticandidal activity of CGA-N9 was blocked to different degrees in the presence of EIPA, CyD, heparin or CPZ. Therefore, most CGA-N9 passes through the C. tropicalis cell membrane via direct cell penetration, whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis, with a slight contribution from clathrin-mediated endocytosis.

Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis

BackgroundHuman chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker.ResultsThe synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG2b, and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 109 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively.ConclusionsA sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis.

CGA-N12, a peptide derived from chromogranin A, promotes apoptosis of Candida tropicalis by attenuating mitochondrial functions.

CGA-N12 (the amino acid sequence from the 65th to the 76th residue of the N-terminus of chromagranin A) is an antifungal peptide derived from human chromogranin A (CGA). In our previous investigation, CGA-N12 was found to have specific anti-candidal activity, though the mechanism of action remained unclear. Here, we investigated the effects of CGA-N12 on mitochondria. We found that CGA-N12 induced an over-generation of intracellular reactive oxygen species and dissipation in mitochondrial membrane potential, in which the former plays key roles in the initiation of apoptosis and the latter is a sign of the cell apoptosis. Accordingly, we assessed the apoptosis features of Candida tropicalis cells after treatment with CGA-N12 and found the following: leakage of cytochrome c and uptake of calcium ions into mitochondria and the cytosol; metacaspase activation; and apoptotic phenotypes, such as chromatin condensation and DNA degradation. In conclusion, CGA-N12 is capable of inducing apoptosis in C. tropicalis cells through mitochondrial dysfunction and metacaspase activation. Antifungal peptide CGA-N12 from human CGA exhibits a novel apoptotic mechanism as an antifungal agent.

A haplotype variant of the human chromogranin A gene (CHGA) promoter increases CHGA expression and the risk for cardiometabolic disorders

The acidic glycoprotein chromogranin A (CHGA) is co-stored/co-secreted with catecholamines and crucial for secretory vesicle biogenesis in neuronal/neuroendocrine cells. CHGA is dysregulated in several cardiovascular diseases, but the underlying mechanisms are not well established. Here, we sought to identify common polymorphisms in the CHGA promoter and to explore the mechanistic basis of their plausible contribution to regulating CHGA protein levels in circulation. Resequencing of the CHGA promoter in an Indian population (n = 769) yielded nine single-nucleotide polymorphisms (SNPs): G-1106A, A-1018T, T-1014C, T-988G, G-513A, G-462A, T-415C, C-89A, and C-57T. Linkage disequilibrium (LD) analysis indicated strong LD among SNPs at the -1014, -988, -462, and -89 bp positions and between the -1018 and -57 bp positions. Haplotype analysis predicted five major promoter haplotypes that displayed differential promoter activities in neuronal cells; specifically, haplotype 2 (containing variant T alleles at -1018 and -57 bp) exhibited the highest promoter activity. Systematic computational and experimental analyses revealed that transcription factor c-Rel has a role in activating the CHGA promoter haplotype 2 under basal and pathophysiological conditions (viz. inflammation and hypoxia). Consistent with the higher in vitro CHGA promoter activity of haplotype 2, individuals carrying this haplotype had higher plasma CHGA levels, plasma glucose levels, diastolic blood pressure, and body mass index. In conclusion, these results suggest a functional role of the CHGA promoter haplotype 2 (occurring in a large proportion of the world population) in enhancing CHGA expression in haplotype 2 carriers who may be at higher risk for cardiovascular/metabolic disorders.

Protective effect of a novel antifungal peptide derived from human chromogranin a on the immunity of mice infected with Candida krusei.

Invasive fungal infections threat the life of immunocompromised patients. Chromogranin A N-46 (CGA-N46), corresponding to the 31st to 76th amino acids of the N-terminus of human chromogranin A, is an antifungal peptide. In order to elucidate the antifungal effects of CGA-N46 in vivo, we studied its effects on cell-mediated immunity in Candida krusei-infected mice. The results showed that the treatment with CGA-N46 increased the average body weight and decreased the mortality of the immunocompromised mice model infected with Candida krusei. The spleen and thymus indices of treated mice has markedly increased compared with that of the control group (P<0.05), and the immune cell levels in peripheral blood also increased significantly (P<0.05). The immuno-modulatory effect of CGA-N46 (60 mg/kg/day) was found to be comparable to that of terbinafine. Additionally, CGA-N46 could alleviate or eliminate histopathological symptoms in the liver, spleen, kidney, and lung tissues. In conclusion, the present study suggests that CGA-N46 may offer a new strategy for antifungal therapeutic option. This study is an essential step in elucidating the effect of CGA-N46 in vivo.

Label-Free Detection of Human Glycoprotein (CgA) Using an Extended-Gated Organic Transistor-Based Immunosensor.

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

Preclinical Evaluation of AdVince, an Oncolytic Adenovirus Adapted for Treatment of Liver Metastases from Neuroendocrine Cancer

Cancer immunotherapy is becoming a cornerstone in the clinical care of cancer patients due to the breakthrough trials with immune checkpoint blockade antibodies and chimeric antigen receptor T cells. The next breakthrough in cancer immunotherapy is likely to be oncolytic viruses engineered to selectively kill tumor cells and deceive the immune system to believe that the tumor is a foreign entity that needs to be eradicated. We have developed AdVince, an oncolytic adenovirus for treatment of liver metastases from neuroendocrine tumor (NET). AdVince includes the gene promoter from human chromogranin A for selective replication in neuroendocrine cells, miR122 target sequences for reduced liver toxicity, and a cell-penetrating peptide in the capsid for increased infectivity of tumor cells and optimized spread within tumors. This paper describes the preclinical evaluation of AdVince on freshly isolated human gastrointestinal NET cells resected from liver metastases and freshly isolated human hepatocytes as well as in fresh human blood. AdVince selectively replicates in and kills NET cells. Approximately 73-fold higher concentration of AdVince is needed to induce a similar level of cytotoxicity in NET cells as in hepatocytes. AdVince did not activate complement or induce considerable amount of proinflammatory cytokines or chemokines in human blood. The data presented herein indicate that AdVince can be safely evaluated in a phase I/IIa clinical trial for patients with liver-dominant NET.

Molecular Design, Structural Analysis and Antifungal Activity of Derivatives of Peptide CGA-N46.

Chromogranin A (CGA)-N46, a derived peptide of human chromogranin A, has antifungal activity. To further research the active domain of CGA-N46, a series of derivatives were designed by successively deleting amino acid from both terminus of CGA-N46, and the amino acid sequence of each derivative was analyzed by bioinformatic software. Based on the predicted physicochemical properties of the peptides, including half-life time in mammalian reticulocytes (in vitro), yeast (in vivo) and E. coli (in vivo), instability index, aliphatic index and grand average of hydropathicity (GRAVY), the secondary structure, net charge, the distribution of hydrophobic residues and hydrophilic residues, the final derivatives CGA-N15, CGA-N16, CGA-N12 and CGA-N8 were synthesized by solid-phase peptide synthesis. The results of bioinformatic analysis showed that CGA-N46 and its derivatives were α-helix, neutral or weak positive charge, hydrophilic, and CGA-N12 and CGA-N8 were more stable than the other derivatives. The results of circular dichroism confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecylsulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities than their mother peptide CGA-N46. Among of the derived peptides, CGA-N12 showed the least hemolytic activity. In conclusion, we have successfully identified the active domain of CGA-N46 with strong antifungal activity and weak hemolytic activity, which provides the possibility to develop a new class of antibiotics.

Antiproliferative effect and characterization of a novel antifungal peptide derived from human Chromogranin A.

CGA-N46 is a novel antifungal peptide derived from the N-terminus of human Chromogranin A, corresponding to the 31st to 76th amino acids. Further research on its activities and characteristics may be helpful for the application of CGA-N46 in medical or other situations. In the present study, the antifungal spectrum and physicochemical characteristics of CGA-N46 were investigated using an antifungal assay, its antiproliferative effects on cancer and normal cells were assessed using MTT assay and its combinatorial effect with other antibiotics was analyzed using checkerboard analysis. The results showed that CGA-N46 exhibited antifungal activity against the tested Candidas (C. glabrata, C. parapsilosis, C. krusei, C. tropicalis and C. albicans) at a concentration of <0.8 mM, but had no effect on the growth of filamentous fungi or other types of fungi (Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Fusarium moniliforme, Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton mentagrophytes), even at a concentration of 3.2 mM. CGA-N46 had an inhibitory effect on the proliferation of lung cancer A549 cells and a reversible effect on the growth of normal primary chicken embryo fibroblast cells, but no hemolytic activity on human erythrocytes at the minimum inhibitory concentration of CGA-N46 against yeasts. The antifungal activity of CGA-N46 was stable at a temperature <40°C or within a broad pH range (pH 5.0-7.0). Its antifungal activity was enhanced when the peptide was used in combination with fluconazole and terbinafine. The present results indicate that CGA-N46 is a safe, physicochemically stable, antifungal peptide with anticancer cell activity that exhibits an additive effect with conventional antibiotics.

Anti-candidal activity of a novel peptide derived from human chromogranin A and its mechanism of action against Candida krusei.

Candida species (Candida spp.) are important fungal pathogens, which cause numerous clinical diseases associated with significant mortality and morbidity in healthcare settings. In our previous study, we identified a recombinant peptide, chromogranin A (CGA)-N46, corresponding to the N-terminal Pro31-Gln76 sequence of human CGA, that exhibited antifungal activity against Candida albicans. The present study investigated the antifungal activity of CGA-N46, and its underlying mechanism, against numerous Candida spp. CGA-N46 inhibited the growth of all of the tested Candida spp., of which Candida krusei exhibited the greatest sensitivity. CGA-N46 was able to disrupt the stability of the phospholipid monolayer without damaging the integrity and permeability of the outer membrane of C. krusei cells, and induced cytoplasm vacuolization and mitochondrial damage. In addition, treatment of C. krusei with CGA-N46 was associated with decreased levels of intracellular reactive oxygen species, a reduction in the mitochondrial membrane potential, and DNA synthesis inhibition. The results of the present study suggested that CGA-N46 was able to pass through the cell membrane of Candida spp. by temporarily destabilizing the phospholipid membrane, which in turn led to mitochondrial dysfunction and inhibition of DNA synthesis. Therefore, CGA-N46 may be considered a novel antifungal compound for the treatment of patients with C. krusei infections.