Abstract
Inhibition of mammalian target of rapamycin (m- TOR) is a potential method for cancer treatment. Effects of rapamycin (RAP) on the reversion of malignant breast epithelial cells were investigated on three-dimensional (3D) basement membrane extract (BME) cultures. Through continuous exposure to 20 nM of RAP, cell colony size was significantly reduced in 3D BME cultures of malignant breast epithelial cells, while normal cell colony size appeared unaffected. In unfixed 3D BME cultures of normal and RAP-treated malignant breast epithelial cells, the presence of luminal cell death was confirmed by ethidium bromide and propidium iodide labeling. Increased structural organization was observed by im- munofluorescence staining of F-actin and β-catenin in RAP-treated malignant breast epithelial cells. In monolayer cultures of normal and malignant breast epithelial cells, continuous exposure to 20 nM of RAP increased caspase 3/7 activity and decreased proliferation. Reverse transcriptase polymerase ch- ain reaction (RT-PCR) array analysis indicated a fold increase in the expression of a number of proteins related to polarity, cell-cell adhesion, and cell-matrix adhesion in the presence of RAP. Our data showed that phenotypic reversion of malignancy can be ach- ieved through RAP exposure on 3D BME cultures. This 3D BME culture system will provide correct microenvironments for observing the effects of other mTOR inhibitors on phenotypic reversion of malignant breast epithelial cells.
Highlights
The use of traditional two-dimensional (2D) cultures for breast cancer studies provides abundant fundamental information on the regulation of breast cancer, but its results often fail to properly translate into clinical applications [1]
basement membrane extract (BME) products are derived from the Engelbreth-Holm Swarm (EHS) tumor and are rich in laminin-1, with other extra-cellular matrix (ECM) components including procollagen IV, heparin sulfate proteoglycan, and entactin [8]
The effects of RAP exposure on the phenotypic reversion of malignant breast epithelial cells were investigated in 3D BME culture
Summary
The use of traditional two-dimensional (2D) cultures for breast cancer studies provides abundant fundamental information on the regulation of breast cancer, but its results often fail to properly translate into clinical applications [1]. Several signal molecules have previously been shown to affect phenotypic reversion through limited cell proliferation, increased multi-cellular organization, basolateral polarization, and programmed luminal cell death. These reversion factors include inhibitors of β1-integrin, epidermal growth factor receptor (EGFR), and the MAPK & PI3K kinases [10,11,12]. Such reversion studies have provided valuable insight for potential methods of cancer treatment
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