Figure 1.
Standardization of Rosemary extract.
A), Representative chromatogram of carnosic acid (analyte) and rosmarinic acid (internal standard) B), Representative chromatogram of carnosic acid at mass 331/287 C), Chemical structures of carnosic acid and rosmarinic acid.
Figure 2.
Rosemary extract modulates cell growth and induces cell cycle arrest in prostate cancer cell lines.
A) 22Rv1 and LNCaP cells were treated with 0 and 50 µg/ml of Rosemary Extract standardized to 40% carnosic acid (RE-40) for 24 hrs. Cell proliferation was assessed by BrdU assay. B) Cell viability of 22Rv1 and LNCaP cells was determined using MTT assay. Cells were treated with increasing concentrations of RE-40 (0–70 µM) for 48 hrs. C) Effect of RE-40 treatment on cell cycle arrest in 22Rv1 and LNCaP cells. Cells were treated with RE-40 at 50 µg/ml for 24 hrs followed by labeling using the APODIRECT kit for flow cytometric analysis. Columns, results are representative of experiments performed in minimum of three replicates; bars, SD. *P<0.05, **P<0.01, ***P<0.001, RE-40 treated samples versus controls.
Figure 3.
Rosemary extract promotes apoptosis in prostate cancer cells.
A) 22Rv1 and LNCaP cells were treated with RE-40 for 24 hrs after which whole cell lysates were prepared and analyzed for expression of pro apoptotic protein, Bax, by Western blotting. Equal loading of protein was confirmed by probing with β-actin antibody. B) Apoptosis induction after 24 hrs of 0 and 50 µg/ml RE-40 treatments in 22Rv1 and LNCaP cells was assessed by TUNEL assay. For both cell lines the first column shows: DAPI-stained nuclei appear blue, second column: FITC-stained nuclei appear green showing induction of apoptosis, third column: merged picture of DAPI and FITC. Cells were also treated with etoposide as a positive control for apoptosis. C) Cleaved caspase-3 levels were detected by ELISA in 22Rv1 and LNCaP cells after treatment with 0, 20, 40 and 50 µg/ml RE-40 for 24hrs. Columns, results are mean of two different experiments performed in duplicate; bars, SD. *P<0.05, ***P<0.001, RE-40 treated samples versus controls.
Figure 4.
Modulation of AR and PSA proteins with Rosemary extract.
A) 22Rv1 and LNCaP cells were treated with Rosemary Extract standardized to 40% carnosic acid (RE-40) for 24 hrs and cell lysates used to evaluate protein expression of AR by Western blot. B) Effect of RE-40 on PSA production in LNCaP cells after 24hrs was detected by ELISA. Columns, results are representative of experiments performed in six replicates; bars, SD. ***P<0.001, RE-40 treated sample versus control.
Figure 5.
Rosemary extract modulates ER stress proteins.
A) Cell lysates of 22Rv1 and LNCaP cells were prepared after treatment with RE-40 for 24hrs. Cell lysates were used to evaluate expression of ER stress proteins PERK, IRE1α, BiP and CHOP using Western blotting. Equal loading of protein was confirmed by probing the blot with β-actin. B) Splicing of XBP1 mRNA as an indicator of ER stress activation in both 22Rv1 and LNCaP treated cells. GAPDH was used as an internal control for RT-PCR. C) 22Rv1 and LNCaP cell lysates were tested for caspase-4 activation during ER stress by western blotting. D) Immunofluorescence staining for CHOP expression in 22Rv1 and LNCaP prostate cancer cells after treatment with 50 µg/ml of RE-40. For both cell lines the first column shows: DAPI-stained nuclei appear blue, second column: Tx-red stained nuclei appear red displaying expression of CHOP protein, third column: merged picture of DAPI and Tx-red. E) To determine the effect of RE-40 on human cells, prostate epithelial cells were treated with RE-40 and lysates confirmed for expression of ER stress protein, PERK and CHOP, by Western blotting
Figure 6.
Rosemary extract modulates BiP and CHOP resulting in decreased AR expression.
A). BiP siRNA treated cell lysates were prepared and checked for AR and BiP protein expression using Western blotting. Equal loading of protein was confirmed by probing the blot with β-actin. B) 22Rv1 cell lysates were used to detect protein levels of cleaved caspase 3 protein by ELISA as a marker for apoptosis. C) LNCaP cell lysates were used to detect protein levels of cleaved caspase 3 by ELISA as a marker for apoptosis. D) 22Rv1 and LNCaP cell lysates transfected with CHOP siRNA were prepared and checked for AR and CHOP protein expression using Western blotting. Equal loading of protein was confirmed by probing the blot with β-actin. E) 22Rv1 cell lysates were used to detect protein levels of cleaved caspase 3 protein by ELISA as a marker for apoptosis. F) LNCaP cell lysates were used to detect protein levels of cleaved caspase 3 by ELISA as a marker for apoptosis. Columns, results are representative of experiments performed in duplicate; bars, SD. Each experiment was repeated two independent times.
Figure 7.
Rosemary extract decreases prostate cancer xenograft tumor growth in athymic nude mice.
A) Graph of body weight measurements in grams for control and RE-40 treated animals against number of days of 22Rv1 tumor inoculation. B) Average tumor volume (mm3) of both control and RE-40 treated mice plotted over days of tumor cell inoculation. C) Representative pictures of control and RE-40 treated mice and tumors removed from them. D) Mouse tissue lysates were prepared from control and rosemary extract treated groups and analyzed for expression of AR, PSA, CHOP and β-actin by western blotting.