Abstract
BackgroundDoxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC).ResultsBoth cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis.ConclusionsDOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population.
Highlights
Doxorubicin (DOX) is widely used in both human and veterinary oncology the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure
The aims of the present study were: (1) to investigate the MDR mechanism associated with DOX treatment on two canine mammary tumours (CMTs) cell lines, comparing the expression of P-gp, Breast Cancer Resistance Protein (BCRP), tumour protein p53 (p53), the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) and the proliferation index Ki67 between standard condition and exposure to DOX treatment, and (2) to establish a repeatable in vitro model that allows to evaluate the in vivo chemotherapeutic drugs effects
The present study investigated the relationship between cancer cells chemosensitivity, the neoplastic phenotype and the subcellular distribution of the chemotherapeutic drug DOX in two CMT cell lines CIPp, originated from a primary mammary tumour, and CIPm, initiated from the corresponding lymph node metastases
Summary
Doxorubicin (DOX) is widely used in both human and veterinary oncology the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were evaluated by immunocytochemistry (ICC). Modifications of the expression and activity of ABC proteins can lead to an increased pumping out of drugs from the intracellular compartment and to a reduction of the effective concentration of the drug in cancer cells [5, 6]. Cancer cell lines are successfully used in many studies as an in vitro model to study cancer biology, molecular pathways and test the efficacy of anticancer drugs [17]
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