Turkish coffee has an antitumor effect on breast cancer cells in vitro and in vivo

Abstract

Background: Breast cancer is the most diagnosed cancer in women. Its pathogenesis includes several pathways in cancer proliferation, apoptosis, and metastasis. Some clinical data have indicated the association between coffee consumption and decreased cancer risk. However, little data is available on the effect of coffee on breast cancer cells in vitro and in vivo.

Methods: In our study, we assessed the effect of Turkish coffee and Fridamycin-H on different pathways in breast cancer, including apoptosis, proliferation, and oxidative stress. A human breast cancer cell line (MCF-7) was treated for 48 h with either coffee extract (5% or 10 v/v) or Fridamycin-H (10 ng/ml). Ehrlich solid tumors were induced in mice for in vivo modeling of breast cancer. Mice with Ehrlich solid tumors were treated orally with coffee extract in drinking water at a final concentration (v/v) of either 3%, 5%, or 10% daily for 21 days. Protein expression levels of Caspase-8 were determined in both in vitro and in vivo models using ELISA assay. Moreover, P-glycoprotein and peroxisome proliferator-activated receptor gamma (PPAR-γ) protein expression levels were analyzed in the in vitro model. β-catenin protein expression was analyzed in tumor sections using immunohistochemical analysis. In addition, malondialdehyde (MDA) serum levels were analyzed using colorimetry.

Results: Both coffee extract and Fridamycin-H significantly increased Caspase-8, P-glycoprotein, and PPAR-γ protein levels in MCF-7 cells. Consistently, all doses of in vivo coffee treatment induced a significant increase in Caspase-8 and necrotic zones and a significant decrease in β- catenin, MDA, tumor volume, tumor weight, and viable tumor cell density.

Conclusion: These findings suggest that coffee extract and Fridamycin-H warrant further exploration as potential therapies for breast cancer.

Keywords: Breast cancer; Caspase-8; Coffee extract; Fridamycin-H; PPAR-γ; Β-catenin.

Fig. 1

Protein expression levels of Caspase-8 (A) and P-glycoprotein (B) in cell lysate of untreated breast cancer cell line (MCF-7), MCF-7 treated for 48 h with 5% (V/V) coffee extract (MCF-7 + 5%), MCF-7 treated for 48 h with 10% (V/V) coffee extract (MCF-7 + 10%), and MCF-7 treated for 48 h with Fridamycin-H (MCF-7 + Frida.-H). (_**): very significant difference (P˂0.01); (_***): highly significant difference (P˂0.001)

Fig. 2

Protein expression levels of insulin-like growth factor binding protein-3 (IGFBP-3) in cell supernatant (A) and peroxisome proliferator-activated receptor gamma (PPAR-γ) in cell lysate (B) of untreated breast cancer cell line (MCF-7), MCF-7 treated for 48 h with 5% (V/V) coffee extract (MCF-7 + 5%), MCF-7 treated for 48 h with 10% (V/V) coffee extract (MCF-7 + 10%), and MCF-7 treated for 48 h with Fridamycin-H (MCF-7 + Frida.-H). (_**): very significant difference (P˂0.01); (_***): highly significant difference (P˂0.001)

Fig. 3

Protein expression levels of Caspase-8 (A) and insulin-like growth factor binding protein-3 (IGFBP-3) (B) in right hind limb skeletal muscle tissue lysate of wild type mice (Control), wild type mice treated daily for 21 days with 10% (V/V) coffee extract (Control + 10%), untreated mice with solid Ehrlich ascites carcinoma (EAC), and EAC mice treated daily for 21 days with either 3% (V/V) coffee extract (EAC + 3%), 5% (V/V) coffee extract (EAC + 5%), or 10% (V/V) coffee extract (EAC + 10%). (*): significant difference (P˂0.05); (**): very significant difference (P˂0.01); (***): highly significant difference (P˂0.001)

Fig. 4

Microscopic images of hematoxylin and eosin stained sections (A) showing normal skeletal muscles in the untreated mice control group (Control) and wild mice treated with 10% (V/V) coffee extract (Control + 10%). Sections from mice with solid Ehrlich ascites carcinoma (EAC) tumor masses show wide viable areas (V) and mitotic figures interspaced by small necrotic zones (N). In comparison, EAC mice treated daily for 21 days with coffee extract at a final concentration (V/V) of either 3% (EAC + 3%), 5% (EAC + 5%), or 10% (EAC + 10%) showed an increase in necrotic zones with a decrease in the density of viable tumor cells in a dose-dependent manner. Microscopic images of immunohistochemistry-stained sections against β- catenin (B) showing negative staining in the Control and Control + 10% groups. EAC group showed a positive β- catenin signal. β- catenin protein expression level gradually decreased by increasing the coffee extract daily dose, as seen in the EAC + 3%, EAC + 5%, and EAC + 10% groups. Scale Bar: 100 μm

Fig. 5

Tumor volume (A) in solid Ehrlich tumor-bearing mice. Ehrlich ascites carcinoma cells were injected into mice. Day 5 after injection was considered Day 0. The solid tumors’ volume was measured every fifth day till the end of the experiment in untreated mice bearing solid Ehrlich tumor (EAC), and EAC treated daily for 21 days with coffee extract at a final concentration (V/V) of either 3% (EAC + 3%), 5% (EAC + 5%), or 10% (EAC + 10%). (!): highly significant difference as compared to the EAC group (P < 0.001). (#): highly significant difference as compared to EAC + 3% group (P < 0.001). Tumor weight (B) at the end of the experiment in EAC, EAC + 3%, EAC + 5%, and EAC + 10%. (**): very significant difference (P˂0.01). (***): highly significant difference (P˂0.001)

Fig. 6

Malondialdehyde serum levels at the end of experiment in untreated mice (Control) group, control group treated daily for 21 days with 10% (V/V) coffee extract (Control + 10%), untreated mice bearing solid Ehrlich tumor (EAC), and EAC treated daily for 21 days with coffee extract at a final concentration (V/V) of either 3% (EAC + 3%), 5% (EAC + 5%), or 10% (EAC + 10%). (**): very significant difference (P ˂ 0.01). (***): highly significant difference (P ˂ 0.001)