Causing UPS Stress for Cervical Cancer Treatment Figure 6. RAMB treatment induces accumulation of Hsp90 and aggresomes. A. Left panel: immunoblot analysis of Hsp90 expression levels in HeLa cervical cancer cells after 8 hours exposure with or without 10 mM RAMBs treatment. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. Right panel: Quantification of the Hsp90/GAPDH ratio. B. HeLa cells were incubated with or without 5 mM RAMB1 or 10 nM Bortezomib for 18 hours before fixation and immuno-fluorescent staining of DNA (blue), ubiquitin (green) and vimentin (red) before imaging (606). doi:10.1371/journal.pone.0023888.g006 shown in Figure 7A, 8 hours RAMB1 exposure is associated with dose-dependent accumulation of p53 in CaSki cervical cancer cells as compared to mock control. Importantly, p53 stabilization causes suppression of cyclin D1 promoter resulting in active repression of cyclin D1 transcription [40]. To test whether this results in reduction of cyclin D1 expression levels, CaSki cervical cancer cells were exposed to increasing doses of RAMB1 over a period up to 8 hours. As shown in Figure 7B (left panel) RAMB1 treatment causes time-dependent (top) and dose-dependent (bottom) decrease of cyclin D1 levels suggesting failure of cervical cancer to enter the S- phase of the cell cycle as a cause of cell toxicity [41]. A semi quantitative analysis of b-actin-normalized cyclin D1 levels is given in Figure 7B (right panel top and bottom). To test whether this results in reduction of cyclin D1 expression levels, CaSki cervical cancer cells were exposed to increasing doses of RAMB1 over a period up to 8 hours. A semi quantitative analysis of b-actin-normalized cyclin D1 levels is given in Figure 7B (right panel top and bottom). As shown in Figure 7B (left panel) RAMB1 treatment causes very rapid (top) and dose-dependent (bottom) decrease of cyclin D1 levels suggesting failure of cervical cancer to enter the S-phase of the cell cycle may contribute to the loss of cell viability [41]. Stabilization of p53 steady-state levels and destabilization of cyclin D1 levels suggest that the reduction in cell viability observed in the panel of cervical cancer cells following RAMBs exposure might trigger the onset of apoptosis. To test this hypothesis, HeLa cervical cancer cells were exposed to 5 mM of RAMB1 and analyzed by flow cytometry after staining for annexin V binding and 7-AAD incorporation. Annexin V and 7-AAD staining allows to discriminate viable (annexin Vneg/7-AADneg), apoptotic (an- nexin Vpos/7-AADneg), and dead (annexin Vpos/7-AADpos) cells. As shown in Figure 7C (left panel), 24 hr exposure to RAMB1 caused an increase in both the early (annexin V positive population) and late (annexin V and 7-AAD double positive population) and apoptotic population in treated cultures versus controls. To evaluate whether this is due to caspase activation, we measured the levels of PARP cleavage in HeLa cancer cells following exposure to RAMB1. As shown in Figure 7C (middle panel), higher levels of cleaved PARP are apparent in treated versus untreated cultures indicating caspase-3 activation. Quantification of the cleaved PARP/GAPDH is provided in Figure 7C (right panel). These findings support our hypothesis that RAMBs treatment triggers apoptosis in cervical cancer cells. RAMB1 treatment prevents anchorage-dependent tumor-colony formation of cervical cancer cells and synergizes with the lysosome inhibitor Chloroquine A hallmark of cancer cells is their capacity to form colonies in vitro that is indicative of the loss of contact inhibition typical of normal cells. To test the potential of RAMB1 as a therapeutic agent for cervical cancer, we have examined the effects of RAMB1 treatment on the anchorage-dependent growth of SiHa, CaSki and HeLa cervical cancer cell lines. Specifically, SiHa and CaSki cancer cells were either mock treated or treated with escalating doses of RAMB1 over a period of 10 days and their ability to form colonies was evaluated by crystal violet staining. As shown in Figure 8A, RAMB1 exposure resulted in a dose-dependent inhibition of colony formation by SiHa and CaSki cervical cancer PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23888