Timely negative regulation of the immune system is critical to allow it to perform its duty while maintaining it under tight control to avoid overactivation. We previously reported that the neuronal receptor neuropilin‐1 (NP‐1) is expressed in human lymph nodes. However, the role of NP‐1 interaction with its physiological ligand semaphorin‐3A (Sema‐3A) on immune cells remains elusive. Here we show that Sema‐3A is expressed by activated DC and T cells, and that its secretion in DC/T cell cocultures is delayed. Sema‐3A/NP‐1 interaction down‐modulated T cell activation since addition of Sema‐3A in DC/T cell cocultures dramatically inhibited allogeneic T cell proliferation. More importantly, neutralization by blocking antibodies or by antagonist peptide of endogenous Sema‐3A produced by DC/T cell cocultures resulted in a 130% increase in T cell proliferation. Sema‐3A acted directly on T cells, since it could block anti‐CD3/CD28‐stimulated proliferation of T cells. Finally, immunomodulatory functions of Sema‐3A relied on the blockage of actin cytoskeleton reorganization, affecting TCR polarization and interfering with early TCR signal transduction events such as ZAP‐70 or focal adhesion kinase phosphorylation. Therefore, we propose that Sema‐3A secretion and the resulting NP‐1/Sema‐3A interaction are involved in a late negative feedback loop controlling DC‐induced T cell proliferation.

Background: There is ethnopharmacological evidence thatPetiveria alliaceacan have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiplein vitrobiological activities of an ethyl acetate soluble plant fraction over several tumor cell lines.Methods: Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS.Results: Petiveria alliaceafraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed.Conclusion: The results of this study indicate thatPetiveria alliaceaexerts multiple biological activitiesin vitroconsistent with cytotoxicity. Further studies in animal models are needed butPetiveria alliaceaappears to be a good candidate to be used as an antitumor agent.

Recent findings suggest that part of the anti-tumor effects of several chemotherapeutic agents require an intact immune system. This is in part due to the induction of immunogenic cell death. We have identified a gallotannin-rich fraction, obtained from Caesalpinia spinosa (P2Et) as an anti-tumor agent in both breast carcinoma and melanoma. Here, we report that P2Et treatment results in activation of caspase 3 and 9, mobilization of cytochrome c and externalization of annexin V in tumor cells, thus suggesting the induction of apoptosis. This was preceded by the onset of autophagy and the expression of immunogenic cell death markers. We further demonstrate that P2Et-treated tumor cells are highly immunogenic in vaccinated mice and induce immune system activation, clearly shown by the generation of interferon gamma (IFN-γ) producing tyrosine-related protein 2 antigen-specific CD8+ T cells. Moreover, the tumor protective effects of P2Et treatment were abolished in immunodeficient mice, and partially lost after CD4 and CD8 depletion, indicating that P2Et’s anti-tumor activity is highly dependent on immune system and at least in part of T cells. Altogether, these results support the hypothesis that the gallotannin-rich fraction P2Et’s anti-tumor effects are mediated to a great extent by the endogenous immune response following to the exposure to immunogenic dying tumor cells.

Background: Several treatment alternatives are available for primary breast cancer, although those for metastatic disease or inflammation associated with tumor progression are ineffective. Therefore, there is a great need for new therapeutic alternatives capable of generating an immune response against residual tumor cells, thus contributing to eradication of micrometastases and cancer stem cells. The use of complex natural products is an excellent therapeutic alternative widely used by Chinese, Hindu, Egyptian, and ancestral Latin-American Indian populations. Methods: The present study evaluated cytotoxic, antitumor, and tumor progression activities of a gallotannin-rich fraction derived from Caesalpinia spinosa (P2Et). The parameters evaluated in vitro were mitochondrial membrane depolarization, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and clonogenic activity. The parameters evaluated in vivo were tumor growth, leukocyte number, metastatic cell number, and cytokine production by flow cytometry. Results: The in vitro results showed that the P2Et fraction induced apoptosis with mitochondrial membrane potential loss, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and decreased clonogenic capacity of 4T1 cells. In vivo, the P2Et fraction induced primary tumor reduction in terms of diameter and weight in BALB/c mice transplanted with 4T1 cells and decreased numbers of metastatic cells, mainly in the spleen. Furthermore, decreases in the number of peripheral blood leukocytes (leukemoid reaction) and interleukin 6 (IL-6) serum levels were found, which are events associated with a poor prognosis. The P2Et fraction exerts its activity on the primary tumor, reduces cell migration to distant organs, and decreases IL-6 serum levels, implying tumor microenvironment mechanisms. Conclusions: Overall, the P2Et fraction lessens risk factors associated with tumor progression and diminishes primary tumor size, showing good potential for use as an adjuvant in breast cancer ER(+) treatment.

Cancer stem cells (CSC) are the primary cell type responsible for metastasis and relapse. ABC-transporters are integral membrane proteins involved in the translocation of substrates across membranes protecting CSC from chemotherapeutic agents. A plant extract derived from C. spinosa (P2Et) previously investigated for its antitumor activity has been shown to reduce lung and spleen metastasis in mice that have been transplanted with breast cancer cells, suggesting that P2Et has a significant activity against cancer stem cells (CSC). P2Et extract was thoroughly characterized by HPLC/MS. The cytotoxicity of P2Et extract was evaluated using a MTT assay in human and murine cell lines with different profiles of resistance, by Pgp overexpression or by enrichment in cancer stem cells. The synergistic effect of P2Et with doxorubicin was evaluated in vitro in several cell lines and in vivo in mice transplanted with TS/A cells, a highly resistant cell line and enriched in CD44${}^{\mathrm{\text{+}}}$CD24${}^{\mathrm{\text{low/-}}}$CSC. The chromatographic fingerprint of P2Et extract revealed 13 gallotannins. We also found that P2Et extract was cytotoxic to cells regardless of their resistant phenotype. Similarly, complementary activities were observed as drug efflux reversion and antioxidant activity. Short-treatment with P2Et extract, revealed a synergistic effect with doxorubicin in resistant cell lines. In vivo the P2Et increases mice survival in a TS/A breast cancer model associated with augmentation of calreticulin expression. Our results suggest that P2Et treatment could be used as adjuvant along with conventional chemotherapy to treat tumors with a MDR phenotype or with high frequency of CSC.