Preparation, Characterization Of A Novel Molecule ITIP And Its Anti-tumor Effect And Mechanisms

A common devastating disease is malignant tumor, which causing great insult to the health and the life quality of human. In recent years many different approaches to anticancer therapies have been developed. Some of them rely on the enhancement of an antitumor immune response through the attraction and activation of immune cells into the surrounding area of a developing tumor. Others are focused on disturbing proper energy supply through inhibition of angiogenesis. Chemokines are small, secreted proteins that activate G protein-coupled receptors, resulting in the activation of several distinct signaling cascades and leading most importantly, to directed migration of cells along the chemokine gradient. Based on either of these two mechanisms, several chemokines have been shown to elicit strong antitumor responses.ITAC and IP 10, members of the CXC chemokine family, have the same receptor CXCR3 which is predominantly expressed on activated T lymphocytes. Increasing evidence suggests that the ITAC and IP10 may be important in the development of type 1 cytokine-induced cell-mediated antitumor immunity, and at the same time inhibit tumor associated angiogenesis leading to suppression of tumor growth. ITAC is the most potent agonist of CXCR3 compared to the other ligands of CXCR3. Relative to ITAC, IP10 exert more effective angiogenesis activity. Previous studies of structure-activity relations have shown that both the NH2 terminus and N-loop region contribute to the higher receptor-binding affinity and activity of ITAC compared with IP10 and suggest that these two domains are the major functional determinants to interact with CXCR3. While other studies have suggested that the chemokines exert angiostatic effects directly on endothelial cells via the C-terminal domain. Chemokines adopt a remarkably conserved structure. The regions of chemokines with similar structure are interchangeable. On the basis of the functional differences of ITAC and IP10, combination of the two chemokines may result in a marked antitumor effect. However, they that act at the common receptor CXCR3 block the other activity. Thus ITAC and IP 10 in combination could not exhibit enhanced antitumor activity compared with either chemokine alone. Our previous study designed a novel molecular which was constructed by substituting the N-terminal and N-Loop region of mouse IP10 with those of ITAC, named ITIP. Analyzed with Insight II work station, ITIP could form a stable dimensional structure. The homogeneity of amino acid with IP 10 is 66%. We hypothesis that ITIP may exert chemokine like activity, moreover it retain the function superiority of both chemokines. And the new molecule ITIP would be expected to lead to enhanced antitumor responses as compared to either chemokine alone.In the present study, the novel protein was expressed and purified in Escherichia coli. The biological properties of that were assessed in vitro and in vivo. It was demonstrated that the novel protein displayed more potent antitumor activity than ITAC or IP10 in vivo in three tumor models. Further, the mechanisms of ITIP-mediated anti-tumor effects were explored on the CT26 tumor model.Part I Preparation and purification ITIP protein and its specific polyclonal antibodyThe nucleotide sequence encoding the mature protein region of ITIP cDNA was amplified with PCR and then cloned into an expression plasmid pET-32a. The final recombinant expression plasmid (pET32a-ITIP) was sequenced to ensure that the insert was in the correct reading frame and subsequently the protein was expressed in Escherichia coli BL21(DE3), under 0.1 mM ITPG. This system produces a recombinant thioredoxin fusion protein. Compared with before induction, a new protein band in SDS-PAGE appeared in the expected site with a predicted molecular mass of 27 KDa. To determine the location of the fusion protein in the cellular fractions of E. coli, the supernatant and inclusion body fractions were tested. The recombinant protein was found mainly in the soluble fraction. The recombinant fusion protein was purified by immobilized metal affinity chromatography (IMAC) using a nickel-chelating column according to the manufacturer's recommendations. The N-terminal fusion partner was cleaved from the fusion protein with enterokinase. Analysis of purity by Tricine-SDS-PAGE showed a single band with a predicted molecular mass of 8.7 KDa. The novel molecular ITIP was also examined by Western blot using both N-terminal Ab of ITAC and C-terminal Ab of IP10. The protein concentration was determined by the BCA protein assay with bovine serum albumin as standard. The pAb were prepared against ITIP and preliminarily purified. The titer was 1:100 000 assayed by ELISA. The specificity of pAb was identified by Western blot. For the need of the control protein in the animal study, we also constructed pET32a-ITAC and pET32a-IP10 recombinant expression plasmid. And the ITAC and IP10 protein were prepared with similar process as ITIP.Partâ…¡Functional characterization of ITIPThe functional activities of ITIP were first assessed by measuring chemotactic migration of Con A/IL-2-treated lymphocytes expressing CXCR3. Commercially available recombinant murine ITAC, IP10 were used as standard control. A dose-dependent chemotactic effect was obtained with ITIP on Con A/IL-2-treated T cells, which was significantly blocked by mCXCR3 antibody at 100ng/ml (p<0.001) but not by isotype IgG (p>0.05). As intended, similar capacity of chemotaxis was observed with ITIP compared to ITAC. Importantly, the chemotactic activity of ITIP was more potent than IP10 alone or in combination with ITAC at 10,100, and 1000ng/ml (p<0.05). These results suggested that the new molecule ITIP containing the N-terminal and N-Loop regions of ITAC retained its potent lymphocyte chemotactic activity against activated lymphocytes. To further dissect whether ITIP contained similar features of ITAC, the receptor internalization assay was performed by FACS before and after agent exposure. We initially used Con A/IL-2-treated lymphocytes to investigate the down-regulation of CXCR3 following incubation with different agent for 30 min. At higher concentrations, ITIP as ITAC was much more potent than IP 10 or ITAC plus IP10. To confirm this, the CHO/mCXCR3 cell line was established. The expression of CXCR3 was detected by RT-PCR, and FACS. A similar attenuated pattern was also found with CHO/mCXCR3 cells. For both cell types, ITIP as ITAC was more potent than IP10 or ITAC plus IP10, and the maximum level of receptor down-regulation observed was reduced to<50% of starting levels at 100ng/ml. Further, the Con A/IL-2-treated lymphocytes and CHO/mCXCR3 cells were used to investigate whether ITIP could desensitize calcium signaling through CXCR3. Upon stimulation with ITIP, ITAC, IP10, or ITAC plus IP10, there were evidences of calcium mobilization on Con A/IL-2-treated lymphocytes and CHO/mCXCR3 cells. Importantly, ITIP and ITAC were more efficient to induce an [Ca+]i compared with IP10, or ITAC plus IP 10.We postulated that ITIP generated from IP10 may also be an inhibitor of angiogenesis. To test this hypothesis, an in vitro wound-healing assay was performed. The capacity of ITIP to block bEND-3 cell regrowth after wounding was assessed by quantifying cell numbers in the denuded zone. The results suggested ITIP was a more-potent inhibitor compared with ITAC (P<0.01) or ITAC plus IP 10 (P<0.05), whereas no statistically significant difference was found between the inhibitory potency of ITIP and IP10 (P=0.5347). This in vitro potency of ITIP to inhibit endothelial cell migration was finally confirmed using the in vivo Matrigel plug assay for angiogenesis. Addition of ITIP to Matrigel plus bFGF resulted in marked reduction in the number of endothelial cells invading the plug and the absence of blood vessels. In this assay, ITIP was also more effective than ITAC or ITAC plus IP10 (P<0.05) but has similar potency as IP10. These results indicated that the novel molecular ITIP retained the angiostatic effect of IP 10.Part III Anti-tumor effects of ITIPThe antitumor efficacy of ITIP was evaluated in BALB/c mice with established CT26 tumors,4T1 tumors and in C57BL/6 mice with established 3LL tumors. Mice were intratumoral administrated of ITIP, while the control groups were injected with PBS solution, ITAC, IP10, or in combination of both at the same time and points. In the three tumor models, tumor volume and life span of mice assay showed that both ITAC and IP 10 individually or in combination resulted in reduction of tumor growth and led to tumor stasis. Remarkably, ITIP treatment had a superior antitumor effect, versus either chemokine alone or in combination with both (P<0.05). More importantly, in the CT26 and 4T1 tumor models, ITIP resulted in complete tumor eradication and the mice survived 90 days.ITIP-treated animals without tumor burden were particularly investigated for potential toxicity of ITIP for>2 months. None of pathologic changes in liver, lung, kidney, etc. were found by microscopic examination after administration of ITIP. No adverse consequences were showed in gross measures, such as weight loss, ruffling of fur, life span, behavior, or feeding. No autoreactive antibody in serum can be determined by ELISA. Part IV The anti-tumor mechanisms of ITIPThe mechanisms of ITIP-mediated anti-tumor effects were explored in the CT26 tumor model. Firstly, to analyze distant effects of ITIP intratumoral treatment, we injected CT26 tumor cells into mice at two separate sites, and treated one of the resultant tumors. For mice that had received ITIP injections in the right tumors, the reduction was observed for the untreated left tumors through day 15. These data suggest that ITIP treatment initiates immune responses capable of acting both locally against the treated primary tumor and systemically against a spatially distant tumor.The lymphocyte subsets of splenocytes from tumor-bearing mice that had been treated intratumorally with ITIP or other control were analyzed by FACS. The results showed that the CXCR3-expressing lymphocytes of ITIP treated mice increased versus IP10, ITAC plus IP10 group (P<0.05). The increased cells were mainly CD3+ T cells, including both CD4+ and CD8+ lymphocytes. Histological examination of tumor sections by H&E staining showed that there were remarkable response with necrosis and lymphocyte infiltration in ITIP and ITAC treated group. To identify tumor-infiltrating leukocytes, we performed immunofluorescent (IF) on cryosections from treated tumors. IP10 alone or combine with ITAC-treated tumor sections had few infiltrating CD3+ T cells. In contrast, ITIP and ITAC treated tumor sections had increased numbers of CD3+ T cells spread diffusely throughout the entire section, including both CD4+ and CD8+ lymphocytes.We further quantified vessel density as measures of angiogenesis by immunolabeling of CD31 in tissue sections. The ITIP apparently reduced the number of vessels compared with control groups, including ITAC alone or in combination with IP 10 (P<0.05). No statistically significant differences were obtained between ITIP and IP 10 group.The cellular immune response was examined in tumor bearing mice. After stimulation with CT26 cells in vitro, splenocytes from the ITIP and ITAC mice exhibited vigorous specific proliferation and cytotoxic activity.In summary, the novel molecule ITIP constructed by combining the N-terminal, N-LOOP region of ITAC with the C-terminal of IP10 was demonstrated to have anti-tumor effects. Recruiting CXCR3-expressing lymphocytes, inducing enhanced cellular immune response and anti-angiogenesis might be involved in ITIP-mediated anti-tumor effects.