Antitumor and genotoxic effects of lactoferrin in Walker-256 tumor-bearing rats

Aim: To investigate the influence of exogenous lactoferrin (LF) on tumor growth, energy and lipid metabolism of Walker-256 carcinosarcoma and to assess genotoxic effects of LF. Materials and Methods: The study was performed on Walker-256 tumor-bearing rats. Total lipids and phospholipids were determined by thin-layer chromatography. Comet assay was used to investigate the genotoxic effects of LF. Results: Daily i.p. administrations of exogenous LF at concentrations of 1 mg/kg and 10 mg/kg starting from the 4th day after tumor transplantation suppressed growth of Walker-256 carcinosarcoma by almost 44%. After treatment with recombinant LF in both doses, the phospholipid composition of Walker-256 carcinosarcoma cells was changed (3-fold increase of phosphatidylethanolamine, 3.4-fold increase of phosphatidylcholine, and 1.8-fold increase of sphingomyelin, while the cardiolipin content decreased by 67%. Exogenous LF was not genotoxic for bone marrow cells (as assessed by the ratio of PCE/NCE, number of micronuclei) and peripheral blood lymphocytes (percentage of DNA in the tail of a comet) in Walker-256 carcinosarcoma-bearing rats. Conclusion: Exogenous LF caused the inhibition of Walker-256 carcinosarcoma growth and a decrease in the microviscosity of plasma cell membranes, and exerted no genotoxicity toward bone marrow cells and peripheral blood of experimental animals. Key Words: lactoferrin, breast cancer, Walker-256 carcinosarcoma, phospholipid content, genotoxicity

Lactoferrin expression in breast cancer in relation to biologic properties of tumors and clinical features of disease

Aim: To determine the patterns of lactoferrin (LF) expression in breast cancer (BC) in relation to biologic properties of the neoplasms and clinical features of the disease course. Materials and Methods: Clinical specimens of 266 BC patients (115 patients with BC of stages I–II — retrospective study, and 151 BC patients — prospective study) were analyzed. Morphological, immunohistochemical and statistical methods were used. Results: The number of LF-positive tumors in retrospective and prospective groups was similar (52.1 and 52.8%, respectively). Among common clinical criteria for prognosis of the disease outcome in BC patients (patient’s age; stage of the disease; histological type, differentiation grade, receptor status; presence of metastases), a strong correlation was found only between expression indexes of LF and estrogen receptors (ER). In ER-positive tumors expression of LF was significantly higher than in ER-negative tumors (35 vs 18%). 5-Year survival rate of BC patients was higher in LF-positive group (70 vs 52% in LF-negative group). The presence of regional metastasis tended to correlate with an increased number of LF-positive tumors. In the patients with invasive ductal carcinoma, expression level of LF moderately correlated with occurrence of luminal A subtype (r = 0.43), while in the patients with invasive lobular carcinoma this index strongly correlated with occurrence of luminal B subtype (r = 0.71). LF expression correlated positively with low and moderate differentiation grade of luminal B or basal tumors, and negatively with luminal B or basal tumors of high differentiation grade (r = −0.57 and −0.63, respectively). Conclusion: It has been shown that LF expression in breast tumors correlated with life expectancy of BC patients and important physiologic and clinical features of the disease, while the character of such relation strongly depended on molecular phenotype of tumor, i.e. luminal A, luminal B or basal

Influence of exogenous lactoferrin on the oxidant/ antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro

Aim: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. Materials and Methods: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Results: Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of “free” iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4–1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14–24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G₂/M phase, especially in receptor-negative cell lines. Conclusion: The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, “free” iron and NO generation rate, resalting in the blocking of cell cycle at G₂/M-phase and apoptosis of malignant cells

ANTITUMOR AND GENOTOXIC EFFECTS OF LACTOFERRIN IN WALKER-256 TUMOR-BEARING RATS

Aim: To investigate the influence of exogenous lactoferrin (LF) on tumor growth, energy and lipid metabolism of Walker-256 carcinosarcoma and to assess genotoxic effects of LF. Materials and Methods: The study was performed on Walker-256 tumor-bearing rats. Total lipids and phospholipids were determined by thin-layer chromatography. Comet assay was used to investigate the genotoxic effects of LF. Results: Daily i.p. administrations of exogenous LF at concentrations of 1 mg/kg and 10 mg/kg starting from the 4th day after tumor transplantation suppressed growth of Walker-256 carcinosarcoma by almost 44%. After treatment with recombinant LF in both doses, the phospholipid composition of Walker-256 carcinosarcoma cells was changed (3-fold increase of phosphatidylethanolamine, 3.4-fold increase of phosphatidylcholine, and 1.8-fold increase of sphingomyelin, while the cardiolipin content decreased by 67%. Exogenous LF was not genotoxic for bone marrow cells (as assessed by the ratio of PCE/NCE, number of micronuclei) and peripheral blood lymphocytes (percentage of DNA in the tail of a comet) in Walker-256 carcinosarcoma-bearing rats. Conclusion: Exogenous LF caused the inhibition of Walker-256 carcinosarcoma growth and a decrease in the microviscosity of plasma cell membranes, and exerted no genotoxicity toward bone marrow cells and peripheral blood of experimental animals. Key Words: lactoferrin, breast cancer, Walker-256 carcinosarcoma, phospholipid content, genotoxicity

LACTOFERRIN EXPRESSION IN BREAST CANCER IN RELATION TO BIOLOGIC PROPERTIES OF TUMORS AND CLINICAL FEATURES OF DISEASE

Aim: To determine the patterns of lactoferrin (LF) expression in breast cancer (BC) in relation to biologic properties of the neoplasms and clinical features of the disease course. Materials and Methods: Clinical specimens of 266 BC patients (115 patients with BC of stages I–II — retrospective study, and 151 BC patients — prospective study) were analyzed. Morphological, immunohistochemical and statistical methods were used. Results: The number of LF-positive tumors in retrospective and prospective groups was similar (52.1 and 52.8%, respectively). Among common clinical criteria for prognosis of the disease outcome in BC patients (patient’s age; stage of the disease; histological type, differentiation grade, receptor status; presence of metastases), a strong correlation was found only between expression indexes of LF and estrogen receptors (ER). In ER-positive tumors expression of LF was significantly higher than in ER-negative tumors (35 vs 18%). 5-Year survival rate of BC patients was higher in LF-positive group (70 vs 52% in LF-negative group). The presence of regional metastasis tended to correlate with an increased number of LF-positive tumors. In the patients with invasive ductal carcinoma, expression level of LF moderately correlated with occurrence of luminal A subtype (r = 0.43), while in the patients with invasive lobular carcinoma this index strongly correlated with occurrence of luminal B subtype (r = 0.71). LF expression correlated positively with low and moderate differentiation grade of luminal B or basal tumors, and negatively with luminal B or basal tumors of high differentiation grade (r = −0.57 and −0.63, respectively). Conclusion: It has been shown that LF expression in breast tumors correlated with life expectancy of BC patients and important physiologic and clinical features of the disease, while the character of such relation strongly depended on molecular phenotype of tumor, i.e. luminal A, luminal B or basal

INFLUENCE OF EXOGENOUS LACTOFERRIN ON THE OXIDANT/ ANTIOXIDANT BALANCE AND MOLECULAR PROFILE OF HORMONE RECEPTOR-POSITIvE AND -NEGATIvE HUMAN BREAST CANCER CELLS IN VITRO

Aim: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. Materials and Methods: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Results: Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of “free” iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4–1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14–24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G₂/M phase, especially in receptor-negative cell lines. Conclusion: The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, “free” iron and NO generation rate, resalting in the blocking of cell cycle at G₂/M-phase and apoptosis of malignant cells