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

Abstract

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.

Keywords: CGA-N46; Chromogranin A; additive combination; anti-proliferation; antifungal peptide; physicochemical stability.

Figure 1.

Hemolytic activity of CGA-N46. The minimal hemolytic concentration was determined against human red blood cells. Values are presented as the mean ± standard error of at least three independent experiments.

Figure 2.

Effect of CGA-N46 on the growth of normal primary CEF cells. The proliferative activity of primary CEF cells was assessed using MTT assay. Values are presented as the mean ± standard error of at least three independent experiments. *P<0.01 compared with control (one-way analysis of variance and subsequent Least Significant Difference and Duncan's tests). CEF, chicken embryo fibroblast.

Figure 3.

Effect of CGA-N46 on the growth of lung cancer cells. The proliferative activity of A549 cells was assessed using MTT assay. Values are presented as the mean ± standard error of at least three independent experiments. *P<0.01 compared with control (one-way analysis of variance and subsequent Least Significant Difference and Duncan's tests).

Figure 4.

Effect of physicochemical factors on the stability of the antifungal activity of CGA-N46 against C. krusei. (A) The heat stability of CGA-N46 was assessed by comparing the MICs of CGA-N46 following treatment at different temperatures for 30 min. (B) The pH stability of CGA-N46 was indicated by the MICs of CGA-N46 following treatment in different pH environments. MIC, minimum inhibitory concentration.

Figure 5.

Checkerboard analysis of the activity of CGA-N46 plus antibiotics against C. krusei. The lowest concentration of each drug combination with growth inhibitory activity (minimum inhibitory concentration) is shown for (A) CGA-N46 combined with terbinafine and (B) CGA-N46 combined with fluconazole. An FIC index of >0.5 and ≤1 denotes an additive combination. FIC, fractional inhibitory concentration.