Extracellular cholesterol oxidase production by Streptomyces aegyptia, in vitro anticancer activities against rhabdomyosarcoma, breast cancer cell-lines and in vivo apoptosis

Abstract

In recent years, microbial cholesterol oxidases have gained great attention due to its widespread use in medical applications for serum cholesterol determination. Streptomyces aegyptia strain NEAE-102 exhibited high level of extracellular cholesterol oxidase production using a minimum medium containing cholesterol as the sole source of carbon. Fifteen variables were screened using Plackett-Burman design for the enhanced cholesterol oxidase production. The most significant variables affecting enzyme production were further optimized by using the face-centered central composite design. The statistical optimization resulted in an overall 4.97-fold increase (15.631 UmL^-1) in cholesterol oxidase production in the optimized medium as compared with the unoptimized medium before applying Plackett Burman design (3.1 UmL^-1). The purified cholesterol oxidase was evaluated for its in vitro anticancer activities against five human cancer cell lines. The selectivity index values on rhabdomyosarcoma and breast cancer cell lines were 3.26 and 2.56; respectively. The in vivo anticancer activity of cholesterol oxidase was evaluated against Ehrlich solid tumor model. Compared with control mice, tumors growth was significantly inhibited in the mice injected with cholesterol oxidase alone, doxorubicin alone and cholesterol oxidase/doxorubicin combination by 60.97%, 72.99% and 97.04%; respectively. These results demonstrated that cholesterol oxidase can be used as a promising natural anticancer drug.

Figure 1

Estimated effects of independent variables on the enzyme production by the selected strain “The red color represents the most significant independent variables affecting enzyme production”.

Figure 2

Pareto chart for Plackett-Burman design rationalizing the order and the impact of each factor on the enzyme production by the selected strain (orange and blue color represent the positive and negative effects; respectively).

Figure 3

(A) Normal probability plot; (B) Box-Cox plot for model power transformation.

Figure 4

3D response surface and contour plots of the influences of cholesterol concentration (X₁), time of incubation (X₂) and pH (X₃) and their mutual effect on the cholesterol oxidase activity.

Figure 5

Cytotoxicity of five human tumor cell lines namely; rhabdomyosarcoma (RD), breast cancer (MCF-7), hepatocellular carcinoma (HepG-2), cervical epithelioid carcinoma (Hela), colon carcinoma (HCT-116) and non-cancerous cell line (human lung fibroblast, WI-38).

Figure 6

Effect of cholesterol oxidase treatment alone or in combination with doxorubicin on tumor volume of EAC bearing mice revealed cholesterol oxidase-induced apoptosis.

Figure 7

Effect of cholesterol oxidase treatment alone or in combination with doxorubicin on tumor weight of EAC bearing mice revealed cholesterol oxidase-induced apoptosis.

Figure 8

Effect of cholesterol oxidase treatment alone or in combination with doxorubicin on histopathological analysis of tumor sections: microphotographs showing (A) untreated mice (control/EAC), (B) EAC bearing mice treated with cholesterol oxidase (C) EAC bearing mice treated with doxorubicin and (D) EAC bearing mice treated with both doxorubicin and cholesterol oxidase.