Apoptosis is one way of controlling immune responses, and a variety of immunosuppressive drugs suppress harmful immune responses by inducing apoptosis of lymphocytes. In this study we observed that rosmarinic acid, a secondary metabolite of herbal plants, induced apoptosis in an p56lck (Lck)-dependent manner; Lck+ Jurkat T cells undergo apoptosis in response to rosmarinic acid (RosA) treatment, whereas Lck− Jurkat subclone J.CaM1.6 cells do not. J.CaM1.6 cells with various Lck mutants indicated that Lck SH2 domain, but not Lck kinase activity, was required for RosA-induced apoptosis. RosA induced apoptosis in the absence of a TCR stimulus, and this was not prevented by interruption of the Fas/Fas ligand interaction. Instead, RosA-mediated apoptosis involved a mitochondrial pathway as indicated by cytochrome c release and the complete blockage of apoptosis by an inhibitor of mitochondrial membrane depolarization. Both caspase-3 and -8 were indispensable in RosA-induced apoptosis and work downstream of mitochondria and caspase-9 in the order of caspase-9/caspase-3/caspase-8. In freshly isolated human PBMC, RosA specifically induced apoptosis of Lck+ subsets such as T and NK cells, but not Lck-deficient cells, including B cells and monocytes. Moreover, RosA’s ability to kill T and NK cells was restricted to actively proliferating cells, but not to resting cells. In conclusion, Lck-dependent apoptotic activity may make RosA an attractive therapeutic tool for the treatment of diseases in which T cell apoptosis is beneficial.

The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC).

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Lck is a T cell-restricted Src family protein tyrosine kinase that plays pivotal roles in TCRmediated signaling. We aimed to identify novel agents that could disrupt the molecular interaction of the Src homology 2-domain of Lck (Lck SH2) with its binding partners, with the expectation that this would impair TCR signaling and generate immunosuppression. Largescale screening of plant extracts indicated that rosmarinic acid (RosA) in extracts of Prunella vulgaris consistently inhibits the interaction between Lck SH2 and a peptide containing its consensus binding sequence (pYEEI). The inhibitory effect of RosA was specific for SH2 domains of Src family protein tyrosine kinase. RosA inhibited TCR-induced-Ca 2+ mobilization and IL-2 promoter activation but not phorbol 12-myristate 13-acetate/ionomycininduced IL-2 promoter activation, indicating its point of inhibition at the membrane proximal site of TCR signaling. Furthermore, RosA inhibited TCR-induced splenocyte proliferation as well as one-way MLR at an IC 50 of 25-50 ? M and inhibited cytokine expression such as IL-2 and IFN-+ . Here, we first report RosA as an inhibitor of TCR-signaling and subsequent T cell proliferation.

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