Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphologicalchanges were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

Baicalin,silver titanate,Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing

Objective This study tests the efficacy of Bletilla striata polysaccharide(BSP),carboxymethyl chitosan(CMC),baicalin(BA)and silver titanate(ST)in a wound dressings to fight infection,promote healing and provide superior biocompatibility.Methods The antibacterial activity of BA and ST was evaluated in vitro using the inhibition zone method.BA/ST/BSP/CMC porous sponge dressings were prepared and characterized.The biocompatibility of BA/ST/BSP/CMC was assessed using the cell counting kit-8 assay.The therapeutic effect of BA/ST/BSP/CMC was further investigated using the dorsal skin burn model in Sprague-Dawley rats.Results The wound dressing had good antibacterial activity against Escherichia coli and Staphylococcus aureus through BA and ST,while the combination of BSP and CMC played an important role in promoting wound healing.The BA/ST/BSP/CMC porous sponge dressings were prepared using a freeze-drying method with the concentrations of BA and ST at 20 and 0.83 mg/mL,respectively,and the optimal ratio of 5%BSP to 4%CMC was 1:3.The average porosity,water absorption and air permeability of BA/ST/BSP/CMC porous sponge dressings were measured to be 90.43%,746.1%and 66.60%,respectively.After treatment for 3 and 7 days,the healing rates of the BA/ST/BSP/CMC group and BA/BSP/CMC group were significantly higher than those of the normal saline(NS)group and silver sulfadiazine(SSD)group(P<0.05).Interleukin-1βexpression in the BA/ST/BSP/CMC group at 1 and 3 days was significantly lower than that in the other three groups(P<0.05).After being treated for 3 days,vascular endothelial growth factor expression in the BA/BSP/CMC group and BA/ST/BSP/CMC group was significantly higher than that in the NS group and SSD group(P<0.05).Inspection of histological sections showed that the BA/ST/BSP/CMC group and BA/BSP/CMC group began to develop scabbing and peeling of damaged skin after 3 days of treatment,indicating accelerated healing relative to the NS group and SSD group.Conclusion The optimized concentration of BA/ST/BSP/CMC dressing was as follows:6 mg BSP,14.4 mg CMC,0.5 mg ST and 12 mg BA.The BA/ST/BSP/CMC dressing,containing antibacterial constituents,was non-cytotoxic and effective in accelerating the healing of burn wounds,making it a promising candidate for wound healing.

Design of dual targeting immunomicelles loaded with bufalin and study of their anti-tumor effect on liver cancer

Objective: Bufalin is an effective drug for the treatment of liver cancer. But its high toxicity, poor watersolubility, fast metabolism and short elimination half-life limit its use in tumor treatment. How to make the drug accumulate in the tumor and reduce side effects while maintaining its efficacy are urgent problems to be solved. The goal of this study is to solve these problems.Methods: A copolymer with tunable poly-N-isopropylacrylamide and polylactic acid was designed and synthesized. The corresponding dual targeting immunomicelles(DTIs) loaded with bufalin(DTIs–BF)were synthesized by copolymer self-assembly in an aqueous solution. The size and structure of DTIs–BF were determined by ZetaSizer Nano-ZS and transmission electron microscopy. Then, its temperature sensitivity, serum stability, critical micelle concentration(CMC), entrapment efficiency(EE), drug release and non-cytotoxicity of blank block copolymer micelles(BCMs) were evaluated. Next, the effects of DTIs–BF on cellular uptake, cytotoxicity, and tumor cell inhibition were evaluated. Finally, the accumulation of DTIs–fluorescein isothiocyanate(FITC) and the in vivo anti-tumor effect were observed using an interactive video information system.Results: DTIs–BF had a small size, spherical shape, good temperature sensitivity, high serum stability, low CMC, high EE, and slow drug release. The blank BCMs had very low cytotoxicity. Compared with free bufalin, the in vitro cellular internalization and cytotoxicity of DTIs–BF against SMMC-7721 cells were significantly enhanced, and the effects were obviously better at 40 ℃ than 37 ℃. In addition, the therapeutic effect on SMMC-7721 cells was further enhanced by the programmed cell death specifically caused by bufalin. When DTIs–FITC were injected intravenously in BALB/c nude mice bearing liver cancer,the accumulation of FITC was significantly increased in tumors.Conclusion: DTIs–BF is a potentially effective nano-formulation and has broad prospects in the clinical treatment of liver cancer.