Trichosanthin Promotes Anti-Tumor Immunity through Mediating Chemokines and Granzyme B Secretion in Hepatocellular Carcinoma.

Trichosanthin (TCS) is a type I ribosome-inactivating protein extracted from the tuberous root of the plant Trichosanthes. TCS shows promising potential in clinical drug abortion, anti-tumor and immunological regulation. However, the molecular mechanisms of its anti-tumor and immune regulation properties are still not well discovered. In the present study, we investigated the anti-tumor activity of TCS in hepatocellular carcinoma (HCC), both in vitro and in vivo. Both HCC cell lines and xenograft tumor tissues showed considerable growth inhibition after they were treated with TCS. TCS provoked caspase-mediated apoptosis in HCC cells and xenograft tumor tissues. The recruitment of CD8+ T cells to HCC tissues and the expression of chemokines, CCL2 and CCL22, were promoted upon TCS treatment. In addition, TCS induced an upregulation of Granzyme B (GrzB), TNF-α and IFN-γ in HCC tissues, which are the major cytotoxic mediators produced by T cells. Furthermore, TCS also resulted in an increase of mannose-6-phosphate receptor (M6PR), the major receptor of GrzB, in HCC tissues. In summary, these results suggest that TCS perhaps increases T-cell immunity via promoting the secretion of chemokines and accelerating the entry of GrzB to HCC cells, which highlights the potential role of TCS in anti-tumor immunotherapy.

Trichosanthin cooperates with Granzyme B to restrain tumor formation in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common type of oral cancer, with a relatively poor prognosis and low post-treatment survival rate. Various strategies and novel drugs to treat TSCC are emerging and under investigation. Trichosanthin (TCS), extracted from the root tubers of Tian-Hua-Fen, has been found to have multiple biological and pharmacological functions, including inhibiting the growth of cancer cells. Granzyme B (GrzB) is a common toxic protein secreted by natural killer cells and cytotoxic T cells. Our group has reported that TCS combined with GrzB might be a superior approach to inhibit liver tumor progression, but data relating to the use of this combination to treat TSCC remain limited. The aim of this study was to examine the effectiveness of TCS on TSCC processes and underlying mechanisms. METHODS: First, we screened the potential antitumor activity of TCS using two types of SCC cell lines. Subsequently, a subcutaneous squamous cell carcinoma xenograft model in nude mice was established. These model mice were randomly divided into four groups and treated as follows: control group, TCS treatment group, GrzB treatment group, and TCS/GrzB combination treatment group. Various tumorigenesis parameters, such as Ki67, PCNA, caspase-3, Bcl-2 and VEGFA, et al., were performed to determine the effects of these treatments on tumor development. RESULTS: Screening confirmed that the SCC25 line exhibited greater sensitivity than the SCC15 line to TCS in vitro studies. TCS or GrzB treatment significantly inhibited tumor growth compared with the inhibition seen in the control group. The TCS/GrzB combination inhibited tumor growth more than either drug alone. TCS treatment inhibited tumor proliferation by downregulating Ki67 and Bcl2 protein expression while accelerating tumor apoptosis. In the TCS/GrzB-treated group, expression of Ki67 was further downregulated, while the level of activated caspase-3 was increased, compared with their expression in either of the single drug treatment groups. CONCLUSION: These results suggest that the TCS/GrzB combination could represent an effective immunotherapy for TSCC.

Neoadjuvant radiochemotherapy decreases the total amount of tumor infiltrating lymphocytes, but increases the number of CD8+/Granzyme B+ (GrzB) cytotoxic T-cells in rectal cancer.

Although neoadjuvant radiochemotherapy (nRCTx) is an established oncological treatment in patients with advanced rectal cancer, little is known about its effects on the tumor microenvironment. Quantity and composition of tumor infiltrating lymphocytes (TILs) are known to influence patients' prognosis but nRCTx-induced modifications are still unclear. We determined the composition of the immune cell infiltrate in rectal cancer after nRCTx and its influence on tumor regression, local recurrence rate and survival. We investigated density and composition of tumor infiltrating CD3+ and CD8+ T-cells and the quantity and ratio of CD8+/GrzB+ T-cells to CD8+ T-cells in 130 rectal cancers after nRCTx compared to a cohort of 30 primarily resected rectal cancers. Furthermore, we analyzed 22 pretherapeutic rectal cancer biopsies, later treated with nRCTx and surgery to evaluate nRCTx-induced modifications of the tumor microenvironment. The total numbers of CD3+ and CD8+ T-cells in tumor stroma (p < 0.001) and tumor epithelium (p < 0.001 CD3; 0.002 CD8) were significantly lower in rectal cancers after nRCTx compared to primarily resected cases, while the ratio of CD8+/GrzB+ T-cells to CD8+ T-cells was significantly increased in the nRCTx cohort (p < 0.001). In multivariate analyses, CD8+/GrzB+ T-cells in the tumor stroma were significantly associated with high regression grade and a lower likelihood of local recurrence (p = 0.029). nRCTx modifies the tumor microenvironment of rectal cancer leading to a total decrease of TILs, but a relative increase in CD8+/GrzB+ T-cells in the tumor stroma. CD8+/GrzB+ T-cells may contribute to local tumor control and the better outcome.

Trichosanthin increases Granzyme B penetration into tumor cells by upregulation of CI-MPR on the cell surface.

Trichosanthin is a plant toxin belonging to the family of ribosome-inactivating proteins. It has various biological and pharmacological activities, including anti-tumor and immunoregulatory effects. In this study, we explored the potential medicinal applications of trichosanthin in cancer immunotherapy. We found that trichosanthin and cation-independent mannose-6-phosphate receptor competitively bind to the Golgi-localized, γ-ear containing and Arf-binding proteins. It in turn promotes the translocation of cation-independent mannose-6-phosphate receptor from the cytosol to the plasma membrane, which is a receptor of Granzyme B. The upregulation of this receptor on the tumor cell surface increased the cell permeability to Granzyme B, and the latter is one of the major factors of cytotoxic T lymphocyte-mediated tumor cell apoptosis. These results suggest a novel potential application of trichosanthin and shed light on its anti-tumor immunotherapy.

Human plasma cells express granzyme B.

While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.