Mechanism of Action and Interaction of Garlic Extract and Established Therapeutics in Prostate Cancer

Abstract

A detailed characterization of the mechanism of action of garlic extract (GE) on prostate cancer (PCa) cells is essential to ensure its safe use as a complementary therapy, particularly when combined with established treatments. A case report highlighted the potential benefits of GE in PCa management. A patient diagnosed with PCa, presenting an initial prostate-specific antigen (PSA) of 11.8 ng/mL, maintained PSA levels between 3.5 and 6 ng/mL for over 14 years with daily GE intake. To study GE's anti-proliferative effects and interactions with established therapeutics, healthy prostate epithelial cells (PNT2) and PCa cells (LNCaP, PC3, VCaP) were treated with GE. Proliferation, Integrin β1 pattern, DNA-damage, as well as androgen receptor (AR) and Cytochrome P450 (CYP450) expression were investigated. GE reduced the proliferation of LNCaP and PC3 cells compared to healthy PNT2 cells but had contrary effects on VCaP cells. The combination of GE with standard therapies, including chemotherapy, androgen deprivation therapy (ADT), and Poly-(ADP-ribose)-Polymerase inhibitors (PARPi), reduced the efficacy of these treatments in tumor cells, potentially due to the GE-induced upregulation of the metabolic enzyme CYP2C9 in PCa cell lines. These findings indicate that while GE has anti-proliferative effects, the use of highly concentrated natural extracts must be carefully assessed by expert physicians on a case-by-case basis, especially when combined with established therapies.

Keywords: CYP450; androgen receptor; drug-interaction; garlic; natural products; prostate cancer.

Figure 1

Influence of GE on cell proliferation. The relative proliferation after GE treatment over 48 h was analyzed for GE concentrations GE1, GE2, and GE3 using MTS for PNT2 (A) and LNCaP (B). The relative proliferation of PC3 (C) and VCaP (D) after 2 h of GE2 treatment are compared to untreated controls. Additionally, live/dead-staining was performed for VCaP cells to quantify viability after GE2 treatment (E). Selectivity of GE for PCa cells is characterized by the relative proliferation of LNCaP, PC3, and VCaP compared to the healthy cell line PNT2 (F). The statistical significances are marked by asterisks (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

Figure 2

Changes in DNA integrity by GE treatment. Fluorescence microscopy of the Comet assay of LNCaP (A), PC3 (B), and VCaP (C) cells without GE (CTRL) and with GE2 treatment for 2 h (2 h GE2) (D). Percentage of DNA-damaged cells from controls (CTRL) and treated (2 h GE2) samples in LNCaP, PC3, and VCaP (D). The statistical significances are indicated by asterisks (*: p < 0.05; **: p < 0.01; ***: p < 0.001). Scalebar = 100 µm.

Figure 3

Changes of Integrin β1 and Caspase-8 expression and localization. Fluorescence microscopy of Integrin β1 immunefluorescence (IF) in control samples ((CTRL; (A)) and 24 h GE-treated LNCaP cells (B) with zoom in images (red boxes). Cellular profiles indicate signal localization within cellular plasma membrane (Mem) and cytoplasm (Cyt) (C,D). Overlay images of Integrin β1 (red) and Caspase-8 (green) with zoom in images (red boxes) and colocalization plots (white boxes) (E,F). The Western blot image (G) indicates protein bands of anti-proliferative protein Bcl-2 (28 kDa) and a relative quantification in (H). The statistical significances are indicated by asterisks (***: p < 0.001). Scalebar = 100 µm/zoom in images 10 µm.

Figure 4

Influence of GE-treatment on PCa-Androgen receptors (AR). Immunefluorescence of AR ((A–C), green) and fluorescence intensity quantification (D) indicate reduced AR-membrane localization after GE2-treatment for LNCaP and VCaP. The AR-negative cell line PC3 shows no significant differences. A quantification of AR mRNA amount by qRT-PCR showed no significant differences for all cell types after GE treatment (E). The statistical significances are indicated by asterisks (*: p < 0.05; **: p < 0.01). Scalebar = 100 µm.

Figure 5

Combination therapy of GE2 and established therapeutics against PCa. Increasing concentrations (C1–C4) of the therapeutics Docetaxel (Doc—chemotherapeutic agent), Enzalutamide (Enza—ADT), and Olaparib (Ola—PARPi) were investigated by MTS assay regarding their anti-proliferative and combined effects with GE (+GE) for healthy prostate epithelial cells (PNT2; (A)) and PCa cells (LNCaP; (B)). Cells were treated with increasing concentrations of therapeutic agents (C1–C4) for 46 h, and in combination with GE, the incubation of GE2 was carried out for 2 h (treatment (46 h) + GE2 (2)). The resulting quantification of effects is shown in (C), where synergistic effects were assigned up to a value of 0.8, additive effects at values from 0.8 to 1.6, and antagonistic effects for values higher than 1.6. The analysis for PC3 cells was performed using the combination of the most effective Doc, Enza, and Ola concentrations with GE (D). The characterization of CYP2C9 with qRT-PCR (E) and activity assay (F) was used to investigate the reduced efficacy of the therapeutic agents under GE administration in PCa cells. The statistical significances are indicated by asterisks (*: p < 0.05; **: p < 0.01; ***: p < 0.001). Scalebar = 100 µm.