Solid state fermentation process with Aspergillus kawachii enhances the cancer-suppressive potential of silkworm larva in hepatocellular carcinoma cells

Abstract

Background: Mulberry silkworm larvae (Bombyx mori) are known as the oldest resource of food and traditional medicine. Although silkworm larvae have been reported to treat various chronic diseases, the effect of fermentation by microorganisms improving the biological activities of silkworm larvae was not reported. In the present study, fermented silkworm larvae was developed via solid-state fermentation with Aspergillus kawachii and investigated its anti-cancer activity in human hepatocellular carcinoma cells.

Methods: We investigated the anti-cancer effects of unfermented (SEE) and fermented silkworm larva ethanol extract (FSEE) on HepG2 human hepatocellular carcinoma cells as well as compared changes in free amino acid, fatty acid, and mineral contents. Anti-cancer activities were evaluated by SRB staining, cell cycle analysis, Annexin V staining, Hoechst staining, DNA fragmentation analysis and western blot analysis. Fatty acid, free amino acid and mineral contents of SEE and FSEE were determined by gas chromatography, amino acid analyzer and flame atomic absorption spectrophotometer, respectively.

Results: Compared with SEE, treatment with FSEE resulted in apoptotic cell death in HepG2 cells characterized by G0/G1 phase cell cycle arrest, DNA fragmentation, and formation of apoptotic bodies. Furthermore, FSEE significantly up-regulated pro-apoptotic as well as down-regulated anti-apoptotic proteins in HepG2 cells. However, an equivalent concentration of SEE did not induce cell cycle arrest or apoptosis in HepG2 cells. Moreover, fermentation process by Aspergillus kawachii resulted in enhancement of fatty acid contents in silkworm larvae, whereas amino acid and mineral contents were decreased.

Conclusion: Collectively, this study demonstrates that silkworm larvae solid state-fermented by Aspergillus kawachii strongly potentiates caspase-dependent and -independent apoptosis pathways in human hepatocellular carcinoma cells by regulating secondary metabolites.

Keywords: Apoptosis; Cell cycle arrest; Fermentation; Human hepatocellular carcinoma; Silkworm larvae.

Fig. 1

Cell growth inhibitory effects on HepG2 hepatocellular carcinoma cells treated with silkworm larva ethanol extract (SEE), and fermented silkworm larva ethanol extract (FSEE) for 24 h. a Cell viability was measured by SRB assay. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared with the control at *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA. b After 24 h incubation with SEE, and FSEE, cell morphology was visualized by inverted microscopy (× 200)

Fig. 2

Effect of SEE, and FSEE on G0/G1 cell cycle arrest in HepG2 hepatocellular carcinoma cells. Cells were treated with SEE, and FSEE for 24 h. a Cell cycle population was analyzed by flow cytometry. b After 24 h, total cell lysates were subjected to detect expression of cell cycle arrest-related proteins in HepG2 cells. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared with control at *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA

Fig. 3

Effect of SEE, and FSEE on induction of apoptotic cell death in HepG2 hepatocellular carcinoma. Cells were treated with SEE, and FSEE for 24 h. a Early and late apoptosis population was analyzed by Annexin V staining assay. b DNA fragmentation was observed by 2% agarose gel electrophoresis. c Nuclear condensation in response to SEE, and FSEE treatment as detected by Hoechst staining assay. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared with control at *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA

Fig. 4

Effect of SEE, and FSEE on caspase-dependent apoptosis by activating the mitochondrial apoptotic pathways in HepG2 cells. Cells were pretreated with 10 μM z-vad-fmk for 2 h, and then incubated with 300 μg/mL of SEE, and 200–300 μg/mL of FSEE for 24 h. Effects of the caspase inhibitor on (a) SEE, and FSEE-induced morphological changes, and (b) cell death in HepG2 cells were examined. After 24 h treatment, (c) total cell lysates were subjected to detect expression of apoptosis-related proteins in HepG2 cells. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared with control at *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA

Fig. 5

Effect of SEE, and FSEE on induction of caspase-independent apoptosis in HepG2 cells. Cells were treated with SEE, and FSEE for 24 h. a After 24 h, total cell lysates were subjected to detect expression of apoptosis-related proteins in HepG2 cells. Cells were pretreated with 2 μM AIF inhibitor (N-PM) for 2 h, and then incubated with 300 μg/mL of SEE, and 200–300 μg/mL of FSEE for 24 h. b Effect of the AIF inhibitor on SEE, and FSEE-induced cell death in HepG2 cells was examined by SRB assay. Data values were expressed as mean ± SD of triplicate determinations. Significant differences were compared with control at *p < 0.05, **p < 0.01, and ***p < 0.001 using one-way ANOVA