There is increasing interest in the development of oncostatic compounds that prevent the growth and progression of cancers without cytotoxicity. This strategy may increase patient survival while minimizing treatment side-effects and chemoresistance in prostate and other cancers. The induction of cellular senescence is one mechanism by which this effect may be achieved(1
). Cellular senescence is a general program of persistent growth arrest in response to sub-lethal stresses in both normal non-transformed and immortalized transformed cells. Senescent cells cease dividing, become insensitive to mitogenic and certain apoptotic stimuli, and develop a phenotype similar to replicatively exhausted cells, exhibiting a characteristic enlarged and flattened morphology and increased senescence-associated β-galactosidase (SA-β-gal) staining activity ()(1
). While ongoing studies seek to identify universal markers and regulators of senescence, SA-β-gal staining remains a standard and accepted marker used to identify senescent cells.
Fig 1 Screen for the identification of senescence in cancer cells. A. Senescent morphology and SA-β-gal activity. Phase contrast microscopy of DU145 cells cultured with DMSO (control) or 250nM AZQ, identified by this study, for 3 days, fixed and stained (more ...)
Agents that generate oxidative stress, DNA damage and/or stress-related signaling induce cellular senescence. These include both endogenous processes including telomere loss, accumulated oxidative damage, dysregulated oncogene activity, and exogenous factors such as chemicals, viral oncogenes, UV light, and ionic radiation. In aging organisms, cellular senescence represents an in vivo
tumor suppressor mechanism that limits the proliferation of damaged cells(1
). This frequently involves the activity of tumor suppressors p53 and pRb, and increased protein expression of cyclin-dependent kinase (CDK) inhibitors p21waf1/cip1
). Cells exhibiting SA-β-gal staining and other senescence characteristics have been observed in benign lesions including lung adenomas(3
), melanocytic naevi(4
), and prostatic intraepithelial neoplasia(5
). A similar senescent state can be chemically induced in prostate and other cancer cell lines in vitro,
independent of p53, Rb and other tumor suppressor pathways(6
). In humans, SA-β-gal staining has been observed in lung tumors(8
) and breast tumors after treatment with genotoxic drugs(9
). Evidence in some studies suggest that the induction of senescence as a cancer treatment may benefit patients, including decreased incidence and severity of toxic side-effects, stimulation of immune responses and prolonged survival (1
). However, the investigation of drug-induced senescence in tumor models has been hampered by the lack of identified compounds that effectively induce this response.
Toward this end, we have developed a rapid semi-automated high-throughput method to screen libraries for novel compounds that induce senescence in prostate cancer cells. Cells are stained concurrently with DNA-binding Hoechst 33342 and for SA-β-gal activity, and compounds are selected on the basis of both growth inhibition associated with senescence and the phenotypic changes that result from its induction. Candidate compounds can then be further validated for induction of persistent growth arrest and expression of senescence marker genes. Using this assay, we screened a library of 4160 known bioactive compounds and natural products at a 10μM dose, identifying 4 lead compounds not previously associated with senescence induction and demonstrating the utility of these methods.