Yersinia pestis is a Gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS) to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC50 values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.

Current immunotherapies are limited by several factors, including the failure to recruit sufficient numbers of immune effector cells to tumors. The chemokine monokine induced by gamma-interferon (Mig; CXCL9) attracts activated T cells and natural killer (NK) cells bearing the chemokine receptor CXCR3. We investigated Mig as an immunotherapeutic agent in a syngeneic murine model of metastatic breast cancer. We transfected the highly malignant murine mammary tumor cell line 66.1 to stably express murine Mig cDNA. Immune-competent mice injected with Mig-expressing tumor cells developed smaller local tumors and fewer lung metastases, and they survived longer than mice injected with vector-control tumor cells. Mig-mediated inhibition of local tumor growth was lost in the absence of host T cells. Mig-transduced tumors had increased numbers of CD4 T cells compared with vector-control tumors, consistent with the T-cell chemoattractant property of Mig, and many tumor-infiltrating host cells expressed CXCR3. NK cells had not been examined previously as a possible effector cell in Mig-based therapies. Our studies now show that NK cells are critical to the mechanism by which Mig limits metastasis. Inhibition of angiogenesis was not implicated as a mechanism of Mig-mediated therapy in this model. These studies support the hypothesis that by manipulating the Mig-CXCR3 gradient, it is possible to direct host immune effector cells to tumors, curtailing both local tumor growth and metastasis. These studies also implicate host NK cells as an additional effector cell critical for Mig-mediated control of metastasis.

Elevated prostaglandin E 2 (PGE 2 ) production is a common feature of human malignancies. This activity has often been attributed to increased metabolic activity of the cyclooxygenase enzymes, although a direct comparison of these 2 parameters i.e., prostaglandin production and cox protein expression, is rarely performed in the same malignant tissue. Using a murine model of metastatic breast cancer, we show that PGE 2 levels are positively correlated with increased tumorigenic and metastatic potential. Because prostaglandin synthesis is a product of 2 isoforms of the cyclooxygenase enzyme, we examined the expression and activity of both isoforms. All tumor cell lines examined, regardless of phenotype, express both cox-1 and cox-2 proteins in vitro. In contrast to the uniform cox-2 expression in vitro, only tumors resulting from the transplantation of metastatic cell lines express cox-2 in vivo. Cox-1 is detected in both metastatic and nonmetastatic tumors. Thus, this is the first evidence that, in the tumor milieu, cox-2 expression can be regulated differently in metastatic vs. nonmetastatic lesions. Examination of PGE 2 synthesis in vitro reveals that nearly complete inhibition of prostaglandin synthesis occurs in the presence of either indomethacin, which inhibits both isoforms, or NS398, which is selective for the cox-2 isoform. Thus, even though cell lines express both isoforms, the majority of the prostaglandin synthesis stems from the activity of the inducible, cox-2 isoform. Likewise, cell growth is inhibited by both indomethacin and NS398 in a dose-dependent manner, albeit at higher drug concentrations than required to ablate PGE 2 synthesis. Despite the inhibition of prostaglandin synthesis, the cox-2 enzyme levels (protein and mRNA) were increased by either indomethacin or NS398. © 2001 Wiley-Liss, Inc. Key words: cyclooxygenase; prostaglandin; breast cancer; metastasisHigh cyclooxygenase activity is a common feature of human epithelial malignancies, 1,2 however, the biological significance of this metabolic activity has never been established. Recent epidemiologic studies have indicated that prolonged use of nonsteroidal antiinflammatory drugs (NSAIDs) is associated with a decreased risk of several malignancies, most notably colorectal cancers. 3 More recently, similar relationships have been observed in lung, breast and other cancers as well. 4 -6 Because NSAIDs have, as a principle action, inhibition of cyclooxygenases, these findings suggest that prostaglandin synthesis contributes to the risk of developing primary malignancies.We sought evidence for a role of prostaglandin synthesis in late tumor progression, i.e., tumor metastasis. Using a murine model of human breast cancer, we showed that higher prostaglandin E 2 (PGE 2 ) levels were observed in malignant mammary tissue in comparison to normal or premalignant gland. Furthermore, the highest PGE 2 levels were observed in the most malignant and metastatic tumors. 7 Two isoforms of cyclooxygenase (cox) are responsible for prostaglandin synthesis....

Transfection of cDNA for IL‐10 into line 66.1 murine mammary tumor cells results in marked suppression of tumor growth and metastasis. Others have reported that nitric oxide has potent antitumor activity and IL‐10 is known to regulate the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We identified nitric oxide production in mammary tumors as indicated by electron paramagnetic resonance detection of nitric oxide‐hemoglobin (NO‐Hb). IL‐10 expression resulted in elevated levels of NO‐Hb in mammary tumors. Immunohistochemical examination of mammary tumors for iNOS protein revealed few positively staining cells in parental or control neo‐transfected tumors but strong iNOS staining in all IL‐10 transfected tumors, consistent with the NO‐Hb data. To determine if mammary epithelial tumor cells themselves, express nitric oxide synthase activity, cultured tumor cells were treated with pro‐inflammatory cytokines and nitrite accumulation was assessed in the conditioned medium. All IL‐10 producing cell lines accumulated uM concentrations of nitrite in response to short term (24 hr) cytokine stimulation. Cells not expressing IL‐10 (parental and neo‐transfectants) accumulated no nitrite under similar culture conditions. After longer stimulation (48 hr), parental and 66‐neo cells accumulated lower amounts of nitrite. IL‐10 gene transfer is associated with increased iNOS protein expression and enzymatic activity detected both in vitro<0R> and in vivo<0R>. Our findings suggest that the antimetastatic and antitumor activity of IL‐10 is related to enhanced production of nitric oxide. Int. J. Cancer 76:713–719, 1998.© 1998 Wiley‐Liss, Inc.

Several laboratories have reported marked tumor inhibition when the cytokine interleukin-10 (IL-10) is overexpressed as a transgene in a variety of tumor cells. To identify critical effector molecules, we compared the expression of the chemokine crg-2, the murine homolog of human inducible protein 10 (human IP-10) in murine mammary tumors derived from the transplantation of six IL-10 expressing clones of tumor cell line 66.1, parental 66.1, or 66-neo-cells. We observed increased levels of IP-10 mRNA in all IL-10-expressing tumors examined in comparison to 66-neo. IP-10 mRNA was not detected in parental 66.1 tumors. The closely related chemokine Mig (monokine induced by interferon-gamma [IFN-gamma]) was also induced in all IL-10-expressing tumors. Studies of cultured tumor cells in vitro show that mammary epithelial tumor cells, in the absence of host elements, can express IP-10 and Mig in response to induction with either lipopolysaccharide (LPS) or IFN-gamma alone. The combination of LPS plus IFN-gamma resulted in even greater induction of IP-10 RNA. The kinetics of induction differ somewhat for the two chemokines, with IP-10 showing slower induction and less rapid decline. Because both Mig and IP-10 are chemotactic for tumor-infiltrating lymphocytes, we examined the presence of CD4+ and CD8+ lymphocytes in these tumors. Consistent with the upregulation of Mig and IP-10, we saw significantly increased numbers of CD8+ cells and a lesser increase in CD4+ cells in tumors with elevated levels of both chemokines.