Stressing the ubiquitin-proteasome system without 20S proteolytic inhibition selectively kills cervical cancer cells

Abstract

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

Figure 1. Summary of the structure of compounds RAMB1–4 and their IC₅₀ values in HeLa cervical cancer cell line.

IC₅₀ values were determined by XXT assay. The IC₅₀ value reported are average of three independent determinations.

Figure 2. Effect of RAMB treatment upon cervical cancer cell lines and primary human keratinocytes.

Cultures of HPV-transformed cervical cancer cells (SiHa, CaSki and ME180) or primary human keratinocytes were treated with the indicated concentrations of RAMB1 (left panel) or RAMB4 (right panel) over a period of 48 hours. Cell viability was determined by XTT assay and plotted as a fraction of the untreated control cultures.

Figure 3. Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells.

Left panel: immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 µM RAMBs. 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 Ubiquitin/GAPDH ratios.

Figure 4. RAMB treatment fails to inhibit the 20S proteasome proteolytic activities.

Purified 20S proteasomes were treated for 30 min with or without RAMB compounds or Bortezomib, here used as positive control, at the indicated concentrations and the specific fluorogenic substrates for chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolyzing-like hydrolytic proteasome capacities were subsequently added. A representative example of two independent experiments is shown.

Figure 5. RAMB treatment fails to inhibit proteasomal activity in living cells.

Left panel: Luciferase activity in lysates of Ub-FL or FL-vector transfected cells either with or without treatment with RAMB compounds or Bortezomib were quantified in Relative Luminescence Units (RLU) and expressed as % of control. Error bars are Standard Deviation (SD) for three independent experiments. Middle panel: Quantification of the Ub-FL/FL ratio. Right panel: Luminescence activity in cell lysates derived from CaSki cervical cancer cells with or without RAMB or Botezomib treatment quantified in Relative Luminescence Units (RLUs). *, P<0.05, **, P<0.02.

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 µM 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 µM RAMB1 or 10 nM Bortezomib for 18 hours before fixation and immuno-fluorescent staining of DNA (blue), ubiquitin (green) and vimentin (red) before imaging (60×).

Figure 7. Stabilization of p53, reduction in cyclin D1 and onset of apoptosis following RAMB1 treatment.

A. Immunoblot analysis of p53 expression level in CaSki cells treated with the indicated concentration of RAMB1 over a period of 8 hours. Equal loading is each lane was verified by amido black staining. B. Top left panel: Immunoblot analysis of cyclin D1 expression level in CaSki cells with or without 10 µM RAMB1 for a period up to 8 hours. Protein loading was examined using an antibody against β-actin. Top right panel: Quantification of the cyclin D1/β-actin ratio. Bottom left panel: Immunoblot analysis of cyclin D1 expression level in CaSki cells with or without RAMB1, at the indicated concentration for a period of 8 hours. Equal protein loading in each lane was verified by using an antibody against β-actin. Bottom right panel: Quantification of the cyclin D1/β-actin ratio. C. Left panel. HeLa cells were treated with 10 µM of RAMB1 for 8 hours and analyzed by flow cytometry after staining for annexin V binding and 7-AAD incorporation. Middle panel: Immunoblot analysis of full length and cleaved PARP in HeLa cells treated 10 µM of RAMBs over a period of 8 hours. Protein loading was assessed using an antibody against β-actin. Right panel: Quantification of cleaved PARP/β-actin ratio.

Figure 8. RAMB1 treatment prevents anchorage-dependent colony formation and synergizes with Chloroquine to kill cervical cancer cells.

A. Equal numbers of SiHa and Caski cells (10³) were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentrations over a period of 10 days. Colonies were visualized by crystal violet staining. B. Left panel: Quantification of colony number in mock versus RAMB1 treated HeLa cells. Middle panel: Quantification of colony size in mock versus RAMB1 treated cervical cancer cells. Right panel: Representative experiment of HeLa cells (10²) which were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentration over a period of 10 days. Colonies were visualized by crystal violet staining and manually counted using an inverted microscope. Inserts: Representative example of colony size in mock versus RAMB1-treated cells. Middle panel: Quantification of colony number. Right panel: Quantification of colony size, representative of three independent experiments. **, P<0.02. C. CaSki cells were treated with checkerboard dilution series of RAMB1 and Chloroquine over a period of 24 hours. Cell viability was measured by XTT assay and calculated as percent of control untreated cultures. Synergy is expressed as combination index (CI).