A Phase II trial of a combination herbal supplement for men with biochemically recurrent prostate cancer

Abstract

Background: Men with biochemical recurrence (BCR) of prostate cancer are typically observed or treated with androgen-deprivation therapy. Non-hormonal, non-toxic treatments to slow the rise of PSA are desirable. We studied a combination herbal supplement, Prostate Health Cocktail (PHC), in prostate cancer cell lines and in a population of men with BCR.

Methods: PC3, LAPC3 and LNCaP cells were incubated with increasing concentrations of PHC suspension. Men previously treated for prostate cancer with surgery, radiation or both with rising PSA but no radiographic metastases were treated with three capsules of PHC daily; the primary end point was 50% PSA decline. Circulating tumor cells (CTCs) were identified using parylene membrane filters.

Results: PHC showed a strong dose-dependent anti-proliferative effect in androgen-sensitive and independent cell lines in vitro and suppression of androgen receptor expression. Forty eligible patients were enrolled in the clinical trial. Median baseline PSA was 2.8 ng ml(-1) (1.1-84.1) and 15 men (38%) had a PSA decline on study (1-55% reduction); 25 (62%) had rising PSA on study. The median duration of PSA stability was 6.4 months. Two patients had grade 2/3 transaminitis; the only other grade 2 toxicities were hyperglycemia, hypercalcemia and flatulence. There were no significant changes in testosterone or dihydrotestosterone. CTCs were identified in 19 men (47%).

Conclusions: Although the primary end point was not met, PHC was well tolerated and was associated with PSA declines and stabilization in a significant number of patients. We believe this is the first report of detecting CTCs in men with BCR prostate cancer. Randomized studies are needed to better define the effect of PHC in men with BCR.

Figure 1. Prostate Health Cocktail Suppresses the Proliferation of Prostate Cancer Cell Lines Dose-dependently and suppresses AR expression

Human prostate cancer LNCaP, PC3 and LAPC3 cells were seeded into 96-well plates (3000 cells/well) to grow to 80% confluency. The dry PHC powder was dissolved in serum-free RPMI 1640 (SF-RPMI) and added to the cells at the final concentrations of 0 (SF-RPMI as the vehicle control), 0.01, 0.1 and 1 mg/ml, respectively. Following 72 hour incubation at 37°C in a 5% CO2 atmosphere, the cells were washed and subjected to the MTS assay. The absorbance was detected at 490 nm with a Microplate Reader. Optical density (OD) decreased in a dose-dependent manner, representing suppression of proliferation (A). AR expression was measured via Western Blot and also showed that PHC reduced expression in a dose-dependent pattern (B). βactin is used as control, representing protein expression of a “housekeeping” gene.

Figure 1. Prostate Health Cocktail Suppresses the Proliferation of Prostate Cancer Cell Lines Dose-dependently and suppresses AR expression

Human prostate cancer LNCaP, PC3 and LAPC3 cells were seeded into 96-well plates (3000 cells/well) to grow to 80% confluency. The dry PHC powder was dissolved in serum-free RPMI 1640 (SF-RPMI) and added to the cells at the final concentrations of 0 (SF-RPMI as the vehicle control), 0.01, 0.1 and 1 mg/ml, respectively. Following 72 hour incubation at 37°C in a 5% CO2 atmosphere, the cells were washed and subjected to the MTS assay. The absorbance was detected at 490 nm with a Microplate Reader. Optical density (OD) decreased in a dose-dependent manner, representing suppression of proliferation (A). AR expression was measured via Western Blot and also showed that PHC reduced expression in a dose-dependent pattern (B). βactin is used as control, representing protein expression of a “housekeeping” gene.

Figure 2. Waterfall plot of PSA changes during treatment with PHC in men with biochemically recurrent prostate cancer

A) “best” change, which excludes a patient who came off study before completing one month due to intercurrent illness, and so was not evaluable and B) PSA change at 12 weeks. Changes are noted in percentage compared to baseline, not absolute PSA level changes.

Figure 2. Waterfall plot of PSA changes during treatment with PHC in men with biochemically recurrent prostate cancer

A) “best” change, which excludes a patient who came off study before completing one month due to intercurrent illness, and so was not evaluable and B) PSA change at 12 weeks. Changes are noted in percentage compared to baseline, not absolute PSA level changes.

Figure 3. PHC Induces Apoptosis in Prostate Cancer Cells

Three cells lines, LNCaP, LAPC3 and 22RV1, of prostate cancer were cultured on cover slips and treated with PHC (10 μg/ml) for 4 days. The PHC-induced apoptosis was examined by identifying DNA fragmentation with fluorescent TUNEL assay and counter-staining with DAPI. All apoptotic (TUNEL-positive) cell nuclei had bright green fluorescence under a fluorescence microscope. The result was expressed as an apoptotic index defined as the mean of TUNEL-positive cells counted per 400x field. P<0.05 indicated significant difference. Representative photomicrographs demonstrated the DNA fragmentation and dysmorphic nuclear condensation (Figure 3A), which were significantly increased in response to PHC treatment in all three cell lines (Figure 3A, d-f, j-l, p-r, Figure 3B).

Figure 3. PHC Induces Apoptosis in Prostate Cancer Cells

Three cells lines, LNCaP, LAPC3 and 22RV1, of prostate cancer were cultured on cover slips and treated with PHC (10 μg/ml) for 4 days. The PHC-induced apoptosis was examined by identifying DNA fragmentation with fluorescent TUNEL assay and counter-staining with DAPI. All apoptotic (TUNEL-positive) cell nuclei had bright green fluorescence under a fluorescence microscope. The result was expressed as an apoptotic index defined as the mean of TUNEL-positive cells counted per 400x field. P<0.05 indicated significant difference. Representative photomicrographs demonstrated the DNA fragmentation and dysmorphic nuclear condensation (Figure 3A), which were significantly increased in response to PHC treatment in all three cell lines (Figure 3A, d-f, j-l, p-r, Figure 3B).