Mechanisms of doxorubicin-induced drug resistance and drug resistant tumour growth in a murine breast tumour model

Abstract

Background: Doxorubicin is currently the most effective chemotherapeutic drug used to treat breast cancer. It has, however, been shown that doxorubicin can induce drug resistance resulting in poor patient prognosis and survival. Studies reported that the interaction between signalling pathways can promote drug resistance through the induction of proliferation, cell cycle progression and prevention of apoptosis. The aim of this study was therefore to determine the effects of doxorubicin on apoptosis signalling, autophagy, the mitogen-activated protein kinase (MAPK)- and phosphoinositide 3-kinase (PI3K)/Akt signalling pathway, cell cycle control, and regulators of the epithelial-mesenchymal transition (EMT) process in murine breast cancer tumours.

Methods: A tumour-bearing mouse model was established by injecting murine E0771 breast cancer cells, suspended in Hank's Balances Salt Solution and Corning® Matrigel® Basement Membrane Matrix, into female C57BL/6 mice. Fourty-seven mice were randomly divided into three groups, namely tumour control (received Hank's Balances Salt Solution), low dose doxorubicin (received total of 6 mg/ml doxorubicin) and high dose doxorubicin (received total of 15 mg/ml doxorubicin) groups. A higher tumour growth rate was, however, observed in doxorubicin-treated mice compared to the untreated controls. We therefore compared the expression levels of markers involved in cell death and survival signalling pathways, by means of western blotting and fluorescence-based immunohistochemistry.

Results: Doxorubicin failed to induce cell death, by means of apoptosis or autophagy, and cell cycle arrest, indicating the occurrence of drug resistance and uncontrolled proliferation. Activation of the MAPK/ extracellular-signal-regulated kinase (ERK) pathway contributed to the resistance observed in treated mice, while no significant changes were found with the PI3K/Akt pathway and other MAPK pathways. Significant changes were also observed in cell cycle p21 and DNA replication minichromosome maintenance 2 proteins. No significant changes in EMT markers were observed after doxorubicin treatment.

Conclusions: Our results suggest that doxorubicin-induced drug resistance and tumour growth can occur through the adaptive role of the MAPK/ERK pathway in an effort to protect tumour cells. Previous studies have shown that the efficacy of doxorubicin can be improved by inhibition of the ERK signalling pathway and thereby treatment failure can be overcome.

Keywords: Breast cancer; Doxorubicin; Drug resistance; ERK; Signalling pathways; Tumour growth.

Fig. 1

Effects of DXR treatment on the average tumour volume (mm³). DXR treatment were initiated between day 6 and day 9. Three doses of DXR were administered three days apart. Error bars indicate the standard error of the mean (n = 15 (TC group), n = 16 (LD-DXR and HD-DXR groups)). The slope of the regression lines from the LD-DXR and HD-DXR groups were significantly different compared to the TC group. [31]

Fig. 2

a Protein expression of c-Casp 7 between the TC, LD-DXR and HD-DXR groups (n = 8). * - significantly different compared to TC group (p < 0.05). Representative images of Bcl-2, Casp 9, c-Casp 8, Casp 8, c-Casp 3 and Casp 3 protein expression between the TC, LD-DXR and HD-DXR groups (n = 8). b Representative images of p62 and LC3-I/−II protein expression between the TC, LD-DXR and HD-DXR groups (n = 8)

Fig. 3

a Protein expression of PDGFRα and p-ERK/ERK between the TC, LD-DXR and HD-DXR groups (n = 8). * - significantly different compared to TC group (p < 0.05). Representative images of p-cRaf, cRaf, p-p38, p38, p-JNK and JNK protein expressions between the TC, LD-DXR and HD-DXR groups (n = 8). b Representative images of p-PTEN, PTEN, p-PI3Kp85, PI3Kp85, p-PDK1, PDK1, p-Akt thr308, p-Akt ser473, Akt, p-mTOR and mTOR protein expressions between the TC, LD-DXR and HD-DXR groups (n = 8). c Representative images of p-ERK-FITC signal in tumour tissues following DXR treatment (n = 8). Hoechst 33342 – nuclei; FITC – p-ERK; solid arrows – localised areas of intense signal in cytoplasm; scale = 20 μm, 40x objective

Fig. 4

a Protein expression of p21 and MCM2 between the TC, LD-DXR and HD-DXR groups (n = 8). * - significantly different compared to TC group (p < 0.05). Representative images of p16 and p53 protein expressions between the TC, LD-DXR and HD-DXR groups (n = 8). b Representative images of p21-FITC signal and localisation in tumour tissues following DXR treatment (n = 8). Hoechst 33342 – nuclei; FITC – p21; solid arrows – localised areas of intense signal in nuclei; dashed arrows- localised areas of intense signal in cytoplasm; scale = 20 μm, 40x objective

Fig. 5

Representative images of α-SMA, E-cadherin, Snail and Vimentin protein expressions between the TC, LD-DXR and HD-DXR groups (n = 8)