Discovery of cancer-preventive juices reactivating RB functions

Abstract

Background: Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired.

Methods: We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo.

Results: Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs.

Conclusions: RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

Keywords: Anti-inflammatory effects; Antioxidant effects; Cancer prevention; Juice; Kakadu plum; Lactic acid bacteria; Pomegranate; Retinoblastoma gene; Tumor necrosis factor-related apoptosis-inducing ligand.

Fig. 1

Effects of Kakadu plum juice and pomegranate juice on colony formation by LIM1215 cells. A. Clonogenic assay of LIM1215 cells treated with each juice (1.56 µL/mL) for 2 weeks. B. Cells were cultured with each concentration of each juice for 2 weeks. Data represent the means ± SD of three measurements. **: p < 0.01, ***: p < 0.001

Fig. 2

Effects of Kakadu plum juice and pomegranate juice on the cell cycle. LIM1215 cells were inoculated (1 × 10⁵ cells in 2 mL medium/well) and incubated in fresh medium with Kakadu plum juice or pomegranate juice at the indicated volumes for 24 hours after the synchronization of the cell cycle by serum-free medium for 2 days. The cell cycle was analyzed by flow cytometry. Each value is an average of two samples.

Fig. 3

Western blot analysis of LIM1215 cells treated with juices. Mixed juice samples were prepared using the 2 juices at each ratio indicated. Each sample (12.5 µL) was added to LIM1215 cells at 2 mL medium/well and incubated for 72 hours. The expression levels of phosphorylated RB, total RB, and p21 were measured by Western blotting. GAPDH was used as a loading control. A semi-quantitative analysis of the intensities of target bands was performed after normalization based on GAPDH levels.

Fig. 4

RB phosphorylation in LIM1215 cells treated with juice components. LIM1215 cells were treated with corilagin or punicalagin at the indicated concentrations for 72 hours. The expression levels of phosphorylated RB (Ser780) and total RB were evaluated by Western blotting. A semi-quantitative analysis of the intensities of target bands was performed after normalization based on α-tubulin levels.

Fig. 5

Kakadu plum and pomegranate juices inhibit TNF-α-induced IL-6 expression. LIM1215 cells were cultured with each sample for 1 hour. Cells were then treated with 10 ng/mL TNF-α for 1 hour. The expression level of IL-6 mRNA was measured by quantitative RT-PCR. The expression level of GAPDH was used for normalization. Each value is shown as a relative value against the control. A. Kakadu plum juice or pomegranate juice at the indicated doses. B. Mixture of 50% of each juice at the indicated doses. Data represent the means ± SD of three measurements. ***: p < 0.001

Fig. 6

Kakadu plum and pomegranate juices inhibit the induction of reactive oxygen species by hydrogen peroxide. LIM1215 cells were cultured in medium containing 6.25 µL/mL of each juice for 24 hours. To induce reactive oxygen species, cells were treated with 500 µM H₂O₂ for the last 1 hour. Intracellular reactive oxygen species levels were measured using FACSCalibur with 10 µM CM-H₂DCFDA as an indicator. Data were analyzed using Cell Quest software (n = 3). **: p < 0.01

Fig. 7

Induction of TRAIL protein levels in PBMC from healthy volunteers by the intake of S-PT84. Blood samples were collected from healthy volunteers who took placebo or S-PT84 tablets. Sampling was performed 1 week before the intake of tablets, 4 weeks after the start of the once-daily intake, and 4 weeks after the cessation of intake. A. Fold changes in TRAIL expression in the S-PT84 group (n = 16) and control group (n = 5) from before intake to 4 weeks after the start of the once-daily intake. B. Fold changes in TRAIL expression in the S-PT84 group (n = 16) and control group (n = 5) from before intake to 4 weeks after the cessation of intake. The quantity of TRAIL was normalized by GAPDH.