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  • br The e ects of


    The effects of these 22 fractions on three different tumor Norfloxacin of digestive system and CIK cells were observed by microscope. Four ca-tegories of fractions were divided: (a) fractions promoted growth of tumor cells and CIK cells, namely F1; (b) fractions inhibited growth of tumor cells and CIK cells, namely F4–F17; (c) fractions inhibited growth of tumor cells but not obviously inhibited growth of CIK cells, namely F18 and F19; (d) unstable fractions susceptible to concentration, namely F2–3 and F20–22. Representative photomicrographs of each category from HCT116, BGC823, SW1990 and CIK cells co-cultured with three concentrations of 22 fractions were shown in Fig. 1.
    3.2. Screening of HELT fractions of bufadienolides
    Based on in vitro ATP-TCA experiments, the cell growth inhibition by 22 fractions was examined. As the definition we described pre-viously, fractions whose IR ≥70% to tumor cells and IR < 50% to CIK cells were considered as HELT fraction. The results indicated that cell growth inhibition of each fraction on tumor cells and CIK cells were
    Table 1
    Physical property of fractions from bufadienolides.
    Fraction Color Property Solubility
    1 Brown Meliceris Water-solubility 2 Puce Meliceris Water-solubility 3 Brown Meliceris Water-solubility 4 Brown Granular Water-solubility 5 Brown Granular Water-solubility 6 Beige Granular Liposolubility 7 Beige Granular Liposolubility 8 Beige Granular Liposolubility 9 Brown Granular Liposolubility 10 Beige Granular Liposolubility 11 Beige Granular Liposolubility 12 Brown Meliceris Liposolubility 13 Beige Granular Liposolubility 14 Beige Granular Liposolubility 15 Ecru Powder Liposolubility 16 Brown Meliceris Liposolubility 17 Brown Granular Liposolubility 18 Puce Meliceris Liposolubility 19 Puce Meliceris Liposolubility 20 Puce Meliceris Liposolubility 21 Puce Meliceris Liposolubility 22 Puce Meliceris Liposolubility
    Fig. 2. Assessment of the in vitro cell growth inhibition of treatment with representative factions. HCT116, BGC823, SW1990, and CIK cells were respectively treated with F1, F11, F18, and F19 at concentrations of 2, 0.5, and 0.1 μg/mL for 3 days. The cell growth inhibition rate was evaluated by ATP-TCA. The data are presented as the mean ± SD of three independent experiments. The red dashed line represents IR = 70%. The green dashed line represents IR = 50%. Negative value illustrates a promoter action to cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    3.3. Antitumor activity of lymphatic chemotherapy with HELT fraction emulsions in animal model
    We used CRC lymphatic metastatic model to investigate the anti-tumor effect of HELT fraction in vivo. The animal model was established
    Fig. 4. In vivo antitumor effects of the HELT fraction. A. Tumor size was mea-sured and gross tumor volume was calculated as (width2 × length) / 2. The data are presented as the mean ± SD (n = 8). ANOVA analysis results indicated that tumor volume in the mice treated with HD-E and MD-E were significant smaller than that treated with LD-E (P < 0.05), CE (P < 0.001), and CI (P < 0.05), respectively. The tumor volume of CI group was significant smaller than NS group (P < 0.001). B. Tumor weight was measured at the end of treatment. The data are presented as the mean ± SD (n = 8). HD-E and MD-E produced significantly greater antitumor effects than LD-E (P < 0.05), CE (P < 0.001), and CI (P < 0.05), respectively. The tumor weight of CI group was significant smaller than NS group (P = 0.004). C and D. H&E staining of footpad tumor sections. C. ×100 magnification. D. ×400 magnification, black arrow presents a pathological mitotic figure. NS: normal saline; CI: Cinobufacini Injection; CE: control emulsion; LD-E: low-dose emulsion; MD-E: middle-dose emulsion; HD-E: high-dose emulsion.
    by inoculating HCT116 cells into the right hind footpad of BALB/c nude mice. The in-situ tumor grew continuously into a palpable mass 3 days after tumor challenging and the tumor initiation rate was 100%. After  International Immunopharmacology 70 (2019) 241–251
    Fig. 3. The schedule of in vivo experiment. After es-tablishing colorectal cancer lymphatic metastasis model based on injecting 4 × 106 of HCT116 cells into the right hind footpad of nude mice, the mice were treated with normal saline (NS), Cinobufacini Injection (CI), control emulsion (CE), low-dose emulsion (LD-E), middle-dose emulsion (MD-E), and high-dose emulsion (HD-E). The treatment duration was 18 days in all. The status of each mouse was assessed every 3 days.