Cytotoxicity and mechanism of action of a new ROS-generating microsphere formulation for circumventing multidrug resistance in breast cancer cells

Abstract

Multidrug resistance (MDR) is one of the main challenges in the treatment of breast cancer. A new microsphere formulation able to generate reactive oxygen species (ROS) locally was thus investigated for circumventing MDR in breast cancer cells in this work. Glucose oxidase (GOX) was encapsulated in alginate/chitosan hydrogel microspheres (ACMS-GOX). The in vitro cytotoxicity of ACMS-GOX to murine breast cancer EMT6/AR1.0 cells, which overexpress P-glycoprotein (P-gp), was evaluated by a clonogenic assay. The mechanism of the cytotoxicity of ACMS-GOX was investigated by using various extracellular and intracellular ROS scavengers and antioxidant enzyme inhibitors. The effect of lipid peroxidation and cellular uptake of GOX was also evaluated. ACMS-GOX exhibited similar dose and time-dependent cytotoxicity to EMT6/AR1.0 cells as to their wild-type EMT6/WT parent cells, in effect circumventing the MDR phenotype of EMT6/AR1.0 cells. Extracellular H(2)O(2) and intracellular hydroxyl radical were found to play critical roles in the cytotoxicity of ACMS-GOX. Cellular uptake of GOX was negligible and thus not responsible for intracellular ROS generation. Combining ACMS-GOX with intracellular antioxidant inhibitors-enhanced cytoxicity. This work demonstrates that the ACMS-GOX are effective in circumventing P-gp-mediated MDR in breast cancer cells.

Characterization of a Microsphere Formulation Containing Glucose Oxidase and its In Vivo Efficacy in a Murine Solid Tumor Model

“…Our mechanistic study, using various extracellular reactive oxygen metabolite scavengers and intracellular antioxidant enzyme inhibitors and iron chelators, indicated that extracellular H 2 O 2 and . O 2 − played a more important role in the cytotoxicity of ACMS-GOX than FR-GOX, and that ·OH is the major ROS accounting for intracellular cytotoxicity (14). As cellular uptake of GOX was not detectable, either upon incubation with FR-GOX or ACMS-GOX and OH has a very short half life (14), it is likely that the intracellular OH was produced inside the cells by H 2 O 2 that is generated outside and then diffuses into the cells.…”

“…glucose oxidase (GOX), have been investigated for treatment of tumors (11,12). GOX, which generates hydrogen peroxide in the presence of oxygen and glucose, has been shown to exhibit antitumor activity both in vitro and in vivo under physiological conditions (11)(12)(13)(14) and efficacy in overcoming MDR in breast cancer cells (13,14). Because GOX is susceptible to degradation and denaturation, daily intratumoral injection of GOX was applied to inhibit tumor growth in vivo (11).…”

“…This treatment approach could be used (1) before surgery to shrink a tumour to a smaller size (also called neoadjuvant therapy), allowing conservative surgery, such as a lumpectomy, rather than a mastectomy, or (2) after surgery to reduce the chance of metastasis or recurrence (also called adjuvant therapy) by eliminating remaining cancer cells. Our laboratory has successfully developed biodegradable microspheres and polymer-lipid hybrid nanoparticles (13,14,(22)(23)(24)(25)(26)(27)(28) for local delivery of doxorubicin, mitomycin C, paclitaxel and MDR reversing agents. Significant delays in tumor growth and even elimination of tumor recurrence and lymphatic metastasis have been achieved with these particulate formulations (22)(23)(24)(25)(26).…”

“…Significant delays in tumor growth and even elimination of tumor recurrence and lymphatic metastasis have been achieved with these particulate formulations (22)(23)(24)(25)(26). We have also developed a microsphere formulation of GOX using an emulsification-internal gelation and polyeletrolyte coating method for production of ROS locally (13,14,28). GOX encapsulated in alginate/chitosan microspheres (ACMS-GOX) has been shown to effectively circumvent MRD in vitro (14).…”

“…We have also developed a microsphere formulation of GOX using an emulsification-internal gelation and polyeletrolyte coating method for production of ROS locally (13,14,28). GOX encapsulated in alginate/chitosan microspheres (ACMS-GOX) has been shown to effectively circumvent MRD in vitro (14). The enzymatic activity of GOX was maintained as demonstrated by prolonged production of H 2 O 2 in glucose solutions (28).…”