| Background:Advanced pancreatic cancer (PC) is one of the most devastating malignancies. There is no satisfying chemotherapeutics to treat APC, even the promising anticancer agent-gemcitabine (GEM), which is currently the first line drug of PC, can’t improve the prognosis. Resistance to chemotherapy is the major impediment for advanced pancreatic cancer. Herb-derived structural compound triptolide can inhibit proliferation of pancreatic cancer cell lines with multiple drug resistance in vitro, which could be developed as a potential drug for advanced pancreatic cancer. However, Triptolide-induced toxicity due to non-specific exposure to helathy tissues (e.g. liver) is the bottleneck for its clinical translation. Aptamers are single-stranded oligonucleotides demonstrating high specificity and affinity to surface molecular of target cells. An aptamer against nucleolin (specifically over-expressed in surface of tumor cells, e.g. pancreatic cancer cells) has been proved to be safety in human studies, which would facilitate achieve tumor-specific delivery for triptolide. This research validated the selectivity and anticancer activity of TP-nucleolin aptamer conjugate in vitro and in vivo, which provided powerful evidences for translating TP-nucleolin aptamer conjugate into clinical application.Goals:The research, which acted as a part of new agent development of triptolide-nucleolin aptamer compoud for targeted therapy in pancreactic cancer, was amied at providing evidence for the parmacology and toxicology research and clinical translation. In this research,we tested the selectivity and anticancer activity of this compounds in vitro and in vivo. Methods:1) Evaluate the selectivity in vitroTo qualitatively examine the selectivity of TMPC001to tumor cells, normal liver cells and normal pancreatic cells, either the Cy-3labeled TMPC001or CRO-TP conjugate (TP-nonspecific nucleic acid sequence conjugate) was incubated with human pancreatic carcinoma cell line (MIA PaCa-2), normal human liver cell line (L-02) and normal human pancreatic cell (HPC-Y5), respectively. Then, the fluorescence intensity of the incubated cells was determined by flow cytometry and the compounds distribution in these three kinds of cells was visually showed by fluorescence microscopy.2) Evaluate the selectivity in vivoTen nude mice were injected with human pancreatic cancer cell line MIA PaCa-2on the right frank.Once the tumors reached100mm3, the mice were randomly divided into2groups (n=5for each group), and respectively subjected to Cy3-labeled CRO-TP conjugate and Cy3-labeled TP-AS1411conjugate, mice in each group were be detected the distribution of fluorescence singnal in vivo at4,12,24,48hour after the administration. Then,mice in each group were sacrificed and the major organs (heart, liver, spleen, lung, kidney and tumor tissue) will be collected. The fluorescence signal of those organs in each group will be detected using Xenogen I VIS imaging system.3) Evaluate the anticancer activity in vitroTo examine the antitumor activity of TMPC001in vitro, Gemcitabine-resistant human pancreatic cancer cell line (MIA PaCa-2) was incubated with either Gemcitabine or TP-AS1411conjugate at different concentrations for24/48hours, respectively. The cell viability was determined using methyl thiazolyl tetrazolium (MTT) assay. And the angiogenesis related factor (VEGF) was quatified by westren blot.4) Evaluate the anticancer activity in vivoTo evaluate the pharmacodynamics of TP-Nucleolin aptamer conjugate, fifty female BALB/c nude mice were subcutaneously injected with MIA PaCa-2cells into right flank. When the tumors reach100mm3, all mice were randomly divided into4groups (n=10for each group): (1) Normal saline (NS),(2) TP,(3) TP-Nucleolin aptamer conjugate,(4) CRO-TP conjugate (low dose) and (5) CRO-TP conjugate (high dose). For8weeks, the mice in each group were receive intravenous injections of each angent once a week, respectively. During this period, the mice were observed for body weight changes and tumor size every weeks. At the end of week4and week8, mice in each group were sacrificed, and the tumors were cut to calculate anti-sarcoma rate, while the blood were collected to obtain the serum to determine the level of chemoresistance-related cytokines by ELISA.Result:1. The fluorescence intensity in MIA PaCa-2was higher than that in other two normal cells incubated with TP-AS1411conjugate, whereas no differences were found in the fluorescence intensity among all the three cell types incubated with TP-CRO conjugate.2. The intensity of the tumor fluorescence signal of Cy3was always obvious in the mouse injected with TP-AS1411conjugate through the whole process, whereas, there was no fluorescence signal accumulated in the tumor of the mice injected with TP-CRO conjugate. On the other hand, the hepatic fluorescence signal of TP-AS1411conjugate Group was lower than that of TP-CRO Group.3. The cell viability decreased with the increased incubation concentration of all the three angents. Furthermore, the cell viability in TP-AS1411incubation group was always lower than that in Gemcitabine incubation group at the same concentration (from50nM to200nM). When the concentration range from50nM to200nM,the TP-AS1411can obviously downregulate the expression of VEGF in the Mia PaCa-2cells.4.After being treated with TP-AS1411conjugate in4or8weeks,the tumor inhibition rate of in TP-AS1411high dose group respectively were56.33%and74.29%, the tumor inhibition rate of in TP-AS1411low dose group respectively were48.27%a nd67.24%,and he tumor inhibition rate of in GEM group respectively were40.29%and44.35%. ELISA results showed that the level of VEGF was decreased in the periheral blood of tumor beared nude mices.Conclude: 1. The conjugated nucleolin aptamer could facilitate TP selectively targeting MIA PaCa-2rather than those normal cells in vitro.2. the AS1411aptamer facilitated delivering TP to tumor tissue and reducing exposure to helthy organs after systemic administration.3.the AS1411aptamer did not affect the anticancer activity and anti-angiogenesis activity of TP. |
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