Fig 1.
Bergamottin blocks prostate cancer cell growth.
(A) Cells were treated with bergamottin or vehicle control (DMSO) for 120 hours; 48 hours after plating. MTT assay was performed as described in methods at the end of the treatment, the OD540 values were converted to relative percentage growth and plotted. (B) Clonogenic assay showing number of colonies formed after 3 weeks of bergamottin and vehicle treatment in both the cell lines. The colonies were fixed and stained with crystal violet for visualization. Both the experiments were repeated three times and data from a representative experiment is shown.
Fig 2.
Bergamottin downregulated total AR expression, AR activation and downstream signaling: (A) Western blotting showed that bergamottin reduces total AR. GAPDH was used as an internal control for equal loading. Cells were plated in complete media, replaced with charcoal stripped serum and phenol red free media (CSSM) 24 hours after plating. Bergamottin (10μM) and vehicle control (DMSO) was added at the time of replacing with CSSM. DHT was added either 48 or 72 hours after replacing with CSSM depending on the treatment duration. (B) Analysis of AR nuclear localization shows that bergamottin reduces AR in nuclear fraction with or without DHT induction. Lamin and tubulin have been used as internal controls. The plating and bergamottin treatment time point was similar as (A) the DHT tretment was given 95 hours after replacement with CSSM. (C&D) Confocal microscopy showing effect of bergamottin on AR nuclear localization. Cell plating and treatment was similar to (B). Size bar 20μm. (E) Western blotting showing reduction in PSA production with bergamottin treatment (10μM, 96 hours).
Fig 3.
Bergamottin treatment causes accumulation of G0/G1 cells similar to CYP3A5 siRNA treated cells.
Watson Pragmatic cell cycle analysis showing frequency of G0/G1, S, and G2/M population in the vehicle control (DMSO), bergamottin and siRNA treated cells. (A and B) Cells were treated with indicated concentration of bergamottin for indicated time points. The cell cycle analysis was performed as described in methods and the analysis was done using FlowJo software, a representative experiment is shown with Watson pragmatic analysis. The average frequency of each population and SD values (±) were calculated after analysing the replicates (minimum of three) and is indicated next to each treatment conditions. (*)- indicates that the P values was less than 0.05 when compare with the respective controls. (C)- Cells were treated with either non target (NT) pool siRNA or CYP3A5 siRNA pool. The analysis method is same as in A and B.
Table 1.
Cell cycle analysis showing effect of bergamottin and CYP3A5 siRNA treatment in LNCaP and MDAPCa2b cells.
Fig 4.
Differential regulation of cell cycle regulating protein after bergamottin treatment.
(A &B) Western blot analysis of control and bergamottin treated cells depicting changes in cell cycle regulating proteins. Treatment was either given for 48 hrs. (high dose, 20 and 30μM) or 96 hrs. (low dose, 5 and 10μM) as described in Fig 3A and 3B. (C) the effect of CYP3A5 siRNA on cell cycle regulating proteins as compared to NT (non-target) control siRNA pool treatment (96 hours). GAPDH has been used as a loading control. The experiment was repeated three times, a representative replicate is shown.
Table 2.
Fold changes in cell cycle regulating proteins after bergamottin treatment.
Fig 5.
Bergamottin causes apoptosis in LNCaP and MDAPCa2b cells.
(A & B) LNCaP and MDAPCa2b cells were treated with bergamottin (10μM) or DMSO (control) for 4 days. Cells were then fixed and stained for tunnel assay reagents as described in methods. Experiment was repeated three times and a representative experiment is shown. Size bar 50μm. DAPI was used to stain the nucleus. (C) Western blot analysis showing cleaved PARP after bergamottin treatment (5 and 10 μM for 96 hrs. and 20 μM for 48 hrs.), GAPDH was used as an internal control. (D) Western blot showing PARP cleavage after CYP3A5 siRNA and NT (non-target) siRNA treatment (96 hrs.).