Synergetic downregulation of 67 kDa laminin receptor by the green tea (Camellia sinensis) secondary plant compound epigallocatechin gallate: a new gateway in metastasis prevention?

Abstract

Background: In traditional Chinese medicine, green tea is considered to have a life-prolonging effect, possibly as a result of its rich content of antioxidant tea polyphenols, and hence has the potential to prevent cancer. This study investigated the role of the major tea secondary plant compound epigallocatechin gallate (EGCG) for its inhibitory effects on the metastasis-associated 67 kDa laminin receptor (67LR).

Methods: To clarify the impact of EGCG on siRNA-silenced expression of 67LR, we applied an adenoviral-based intestinal in vitro knockdown model, porcine IPEC-J2 cells. Quantitative real-time polymerase chain reaction was performed to analyze 67LR gene expression following treatment with physiological and pharmacological concentrations of EGCG (1.0 g/l, 0.1 g/l, 0.02 g/l and 0.002 g/l).

Results: We report co-regulation of EGCG and 67LR, which is known to be an EGCG receptor. siRNA selectively and highly significantly suppressed expression of 67LR under the impact of EGCG in a synergetic manner.

Conclusions: Our findings suggest that 67LR expression is regulated by EGCG via a negative feedback loop. The explicit occurrence of this effect in synergy with a small RNA pathway and a plant-derived drug reveals a new mode of action. Our findings may help to provide insights into the many unsolved health-promoting activities of other natural pharmaceuticals.

Figure 1

Knockdown assays comparing 67lr-KD and control-KD under EGCG treatment. a) Pooled data from seven assays normalized to the non-targeting virus samples. This normalization revealed net knockdown effects. b) Pooled data from ten assays normalized to the media control samples. This normalization revealed the net regulation induced by the drug treatment. (Legend: EGCG = EGCG treatment, MEDIA = media control, NV = non-targeting virus, TV = targeting virus, 67lr-KD = 67LR knockdown, control-KD = control knockdown).

Figure 2

Principal component analysis comparing the influence of EGCG on 67LR versus the control gene. a) For the control gene, the impact of the drug treatment was dependent on whether a gene knockdown was applied (I) or not (II). b) For the 67LR gene, siRNA-induced downregulation altered the extent of its synergy with EGCG (I) and a new data cluster was separated from the samples solely treated with the 67lr-KD (II). When no knockdown was applied, EGCG did not affect the gene regulation (III) in accordance with Figure  3a. (Color code: black labels indicate samples containing EGCG, grey labels indicate no EGCG; Legend: EGCG = EGCG treatment, MEDIA = media control, NV = non-targeting virus, TV = targeting virus, 67lr-KD = 67LR knockdown, control-KD = control knockdown).

Figure 3

67LR/EGCG-synergy when the secondary plant metabolite was added at potencies below the actual EGCG-content of freshly brewed green tea. (Legend: EGCG = EGCG treatment, MEDIA = media control, NV = non-targeting virus, TV = targeting virus, 67lr-KD = 67LR knockdown, control-KD = control knockdown).