Dipteran Carboxymethyl Chitosan as an Inexhaustible Derivative with a Potential Antiproliferative Activity in Hepatocellular Carcinoma Cells

Abstract

Traditional folk therapies indicate that insects have diverse medicinal potentials. However, the therapeutic application of insect chitosan and its derivatives has not been explored. To investigate the application of chitosan and its derivatives, the carboxymethyl derivative of chitosan (CM-Ch) was extracted from two dipteran larvae species, Chrysomya albiceps and Sarcophaga aegyptiaca. The degree of deacetylation (DD) and CM-Ch functional groups were validated using Fourier-transform infrared (FTIR) spectroscopy analysis and proton nuclear magnetic resonance spectroscopy (¹H NMR), respectively. The molecular weight was estimated using MALDI-TOF MS analysis. The effect of CM-Ch on the morphology and proliferation of human liver HepG2 cancer cells was assessed. IC₅₀ of CM-Ch induced significant growth-inhibitory effects in HepG2 cells. CM-Ch treatment altered the morphology of HepG2 in a dose-dependent manner and induced apoptosis in a caspase-dependent manner. CM-Ch treatment showed no signs of toxicity, and no alterations in liver and kidney biochemical markers were observed in albino rats. A CM-Ch derivative from commercial crustacean chitosan was used to assess the efficacy of the insect-derived CM-Ch. The data presented here introduce insect CM-Ch as a promising, inexhaustible, safe derivative of chitosan with antitumor potential in liver cancer. This is the first report highlighting the anticancer activity of insect CM-Ch in hepatocellular carcinoma cells.

Figure 1

FTIR spectra of chitosan and carboxymethyl chitosan (CM-Ch). Spectra of chitosan extracted from Chrysomya albiceps (a) and Sarcophaga aegyptiaca (b). Spectra of the CM-Ch prepared from C. albiceps (c), S. aegyptiaca (d), and commercial crustacean chitosan (e).

Figure 2

¹H NMR spectra and molecular weights of carboxymethyl chitosan (CM-Ch). H¹NMR spectra of Chrysomya albiceps CM-Ch (Ca-CM-Ch) (a), Sarcophaga aegyptiaca CM-Ch (Sa-CM-Ch) (b), and commercial chitosan CM-Ch (sy-CM-Ch) (c). Molecular weights of Ca-CM-Ch (d), Sa-CM-Ch (e), and sy-CM-Ch (f).

Figure 3

Effects of carboxymethyl chitosan (CM-Ch) on the viability of HepG2 cells. Cells were treated with CM-Ch (1–1200 µg/mL) for 48 h. Controls were treated with DMSO only. (a) CM-Ch reduced HepG2 cell viability. (b) Semilogarithmic plotting of HepG2 cell viability at increasing CM-Ch concentrations was used to calculate IC₅₀. Ca-CM-Ch, Chrysomya albiceps CM-Ch; Sa-CM-Ch, Sarcophaga aegyptiaca CM-Ch; sy-CM-Ch, CM-Ch of commercial chitosan. Data (n = 3) were presented as means ± SEM. p value was calculated vs. control cells: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, and ^∗∗∗∗p < 0.001.

Figure 4

Carboxymethyl chitosan (CM-Ch) induced morphological changes in HepG2 cells. Cells were treated with the indicated concentrations of CM-Ch for 48 or 72 h (sy-CM-Ch). Representative images were shown from three independent experiments. Ca-CM-Ch, Chrysomya albiceps CM-Ch; Sa-CM-Ch, Sarcophaga aegyptiaca CM-Ch; sy-CM-Ch, commercial chitosan CM-Ch. 400×.

Figure 5

Carboxymethyl chitosan (CM-Ch) induced caspase-dependent apoptosis in HepG2 cells. Cells were either untreated or treated with the indicated CM-Ch (300 µg/mL) for 48 h. Lysed cells were subjected to (a) enzyme-linked immunosorbent apoptosis assay to measure histone release as an indication of apoptosis; (b) caspase-3 activity assay. Each assay was performed in triplicate, and the standard error of the mean (SEM) was calculated. Data were presented as mean ± SEM. p value was calculated vs. control cells: ^∗∗∗p < 0.005 and ^∗∗∗∗p < 0.001. Ca-CM-Ch, Chrysomya albiceps CM-Ch; Sa-CM-Ch, Sarcophaga aegyptiaca CM-Ch; sy-CM-Ch, CM-Ch of commercial chitosan.

Figure 6

CM-Ch treatment did not affect body weight, organ weight, and percentage of organ weight relative to body weight in rats. Animals were treated with a single oral dose of either Ca-CM-Ch (1.5 g/kg), Sa-CM-Ch (1.1 g/kg), sy-CM-Ch (1.1 g/kg), or saline (1 mL/kg) as a control. Body weight, organ weight, and percentage of organ weight relative to body weight were measured after 15 days of treatment. Data were represented as mean ± SEM of five animals. Ca-CM-Ch, Chrysomya albiceps CM-Ch; Sa-CM-Ch, Sarcophaga aegyptiaca CM-Ch; sy-CM-Ch, CM-Ch of commercial chitosan. Body and organ weights and percentage of kidney, liver, and spleen organ weight relative to body weight did not significantly differ between treated groups and controls.

Figure 7

Kidney and liver biochemical parameters in rats treated with CM-Ch. Animals were treated with a single oral dose of either Ca-CM-Ch (1.5 g/kg), Sa-CM-Ch (1.1 g/kg), sy-CM-Ch (1.1 g/kg), or saline (1 mL/kg) as a control. Biochemical parameters of the kidney (serum urea and creatinine) and liver (AST and ALT) were measured after 15 days of treatment. Data were presented as mean ± SEM of five animals. Ca-CM-Ch, Chrysomya albiceps CM-Ch; Sa-CM-Ch, Sarcophaga aegyptiaca CM-Ch; sy-CM-Ch, CM-Ch of commercial chitosan. No significant difference in liver and kidney parameters was observed in treated groups and controls.