Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl. 9, 1113 Sofia , Bulgaria.
Department of Medical Genetics, Medical University of Sofia, Zdrave str. 2, 1431Sofia, Bulgaria.
Curr Med Chem. 2022;29(42):6479-6498. doi: 10.2174/0929867329666220820095122.
Some molluscan hemocyanins (Hcs) have significant immunological and antitumor potential, enabling their application in oncology. The antitumor activity of Hcs from marine snails Rapana venosa (RvH), giant keyhole limpet Megathura crenulata (KLH) and garden snails Helix lucorum (HlH), as well as their different derivatives, were studied in vitro on a permanent T24 cell line of bladder cancer and normal urothelial cell line HL 10/29 compared to doxorubicin.
The antiproliferative activity of the tested Hcs was determined using the WST-1 assay and BrdU ELISA assay. Morphological changes in both urothelial cell lines were confirmed by fluorescence microscopy. The proteomic analysis of a bladder cancer cell line before and after treatment with functional unit (FU) βc-HlH-h using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry revealed differences in the expression of some proteins.
Studies prove that the T24 tumor cell line is dose- and time-dependent, sensitive to the action of the tested isoforms, and it glycosylated FU of these hemocyanins. Selective inhibition of T24 cell growth was observed after incubation with structural subunits (βc-HlH, RvHI and RvHII) and FUs (βc-HlH-h and RvHII-e). Additionally, fluorescent microphotographs did not show apoptotic or necrotic alterations in the normal urothelial cell line HL 10/29. The FU βc-HlH-h demonstrated the highest antiproliferative effect (similarly to doxorubicin), in which predominantly apoptotic and less late apoptotic or necrotic changes in the tumor cells were observed. Several downand up-regulated proteins identified by proteome analysis may be associated with the apoptosis pathway.
The present study illustrated the selectivity of the cytotoxic effect of Hcs against the Т24 cancer cell line. This is the first report of protein expression in T24 human bladder cancer cells under the influence of FU βc-HlH-h. That is probably due to the specific oligosaccharide structures rich in methylated hexoses exposed on the surface of βc-HlH-h.
一些软体动物血蓝蛋白（Hcs）具有显著的免疫和抗肿瘤潜力，使其能够应用于肿瘤学领域。本研究比较了海洋蜗牛芋螺（RvH）、巨石房蛤（KLH）和花园蜗牛亮大蜗牛（HlH）的血蓝蛋白及其不同衍生物在体外对膀胱癌永久 T24 细胞系和正常尿路上皮细胞系 HL10/29 的抗肿瘤活性，以及与阿霉素相比的作用。
采用 WST-1 法和 BrdU ELISA 法测定受试 Hcs 的增殖抑制活性。荧光显微镜观察两种尿路上皮细胞系的形态变化。采用二维聚丙烯酰胺凝胶电泳（2D-PAGE）和质谱法对经功能单位（FU）βc-HlH-h 处理前后的膀胱癌细胞系进行蛋白质组分析，揭示了一些蛋白质表达的差异。
研究证明，T24 肿瘤细胞系对所测试的同工型呈剂量和时间依赖性敏感，并且能够对这些血蓝蛋白进行糖基化。在用结构亚基（βc-HlH、RvHI 和 RvHII）和 FUs（βc-HlH-h 和 RvHII-e）孵育后，观察到 T24 细胞生长的选择性抑制。此外，荧光显微镜照片未显示正常尿路上皮细胞系 HL10/29 发生凋亡或坏死改变。FUβc-HlH-h 表现出最高的增殖抑制作用（与阿霉素相似），其中观察到肿瘤细胞中主要为凋亡，较少为晚期凋亡或坏死改变。通过蛋白质组分析鉴定的几个下调和上调蛋白可能与凋亡途径有关。
本研究说明了 Hcs 对 T24 癌细胞系的细胞毒性作用具有选择性。这是首次报道 FUβc-HlH-h 对人膀胱癌 T24 细胞的蛋白表达影响。这可能是由于βc-HlH-h 表面暴露的富含甲基化己糖的特定寡糖结构所致。
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