Abstract
BackgroundIn normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery.ResultsAAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis.ConclusionAAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.
Highlights
In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis
We investigated the ability of Associated Virus Type 2 (AAV2) to infect and induce apoptosis in a variety of human breast cancer derived cell lines, representing a range of breast cancer types graded upon hormone dependency and aggressiveness: the luminal MCF-7 (ERa-positive) are weakly invasive and do not normally metastasize to bone [41]; MDA-MB-468 (ERa-negative) are weakly invasive and non-metastatic and were derived from human breast adenocarcinoma [42]; and MDAMB-231 (ERa-negative) are highly invasive, basal cell line [43] derived from metastatic, infiltrating ductal breast carcinoma [44]
The MCF-7, MDA-MB-468, MDA-MB-231 and normal human mammary epithelial cells (nHMECs) cells were synchronized as described in Materials and Methods, when approximately two-thirds or more of the cells were in the G1 phase of the cell cycle [13,48]
Summary
In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. We examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. Routine screening and early detection have reduced the incidence of breast cancer, but despite optimal treatment, about 30% of women with recurrent disease develop distant metastases [3]. There is a clinical need for breast cancer therapeutics which potently target malignant cells resultant with identifiable biomarkers, independent of the type of breast cancer profile presented by the patient
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