Recombinant Proteins Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran.
Cell Biochem Funct. 2020 Jul;38(5):651-659. doi: 10.1002/cbf.3525. Epub 2020 Mar 20.
It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.
已证实，激活白细胞细胞黏附分子（ALCAM）的过表达与他莫昔芬治疗耐药的发展以及 ER+乳腺癌细胞中细胞侵袭、迁移和转移的促进有关。因此，我们假设，用抗体阻断 ALCAM 的连接可能是一种有效的方法，可以逆转与乳腺癌细胞中 ALCAM 过表达相关的上述负面事件。在此，我们重组表达了一种抗 ALCAM scFv，并在整个研究过程中使用它来检查 ALCAM 阻断的潜在抗癌作用。抗 ALCAM scFv 的编码序列从 GenBank 数据库中获得，在添加 6×His 标签部分、信号肽和侧翼序列后，整个构建体在大肠杆菌中表达。然后，用纯化的重组抗 ALCAM scFv 预处理他莫昔芬耐药 MCF7 细胞 24 小时，再给予他莫昔芬。同时，还使用 CompuSyn 软件评估了抗 ALCAM scFv 与他莫昔芬联合治疗对他莫昔芬耐药和敏感 MCF7 细胞系的细胞毒性特征。还评估了抗 ALCAM scFv 对 MDA-MB-231 细胞的侵袭/迁移抑制作用。用抗 ALCAM scFv 预处理可以成功增强他莫昔芬对耐药 MCF-7 细胞系的增殖抑制作用。此外，19.2:1 的他莫昔芬与抗 ALCAM scFv 联合显示出对他莫昔芬耐药 MCF7 细胞系的协同细胞抑制作用。另外，用抗 ALCAM scFv 孵育 MDA-MB-231 细胞系可分别使侵袭和迁移的细胞数量减少 30%和 25%。总的来说，应用抗 ALCAM scFv 可显著抑制体外癌细胞转移，并调节他莫昔芬耐药 ER+ MCF7 细胞系对他莫昔芬的敏感性。研究的意义：对他莫昔芬治疗的耐药性的获得是与癌症化疗相关的主要挑战之一，它逐渐将一个有反应的肿瘤转变为一个更具侵袭性的难治性肿瘤，最终导致疾病进展和复发。在这里，我们报告了一种抗 ALCAM scFv 的表达，它能够在 24 小时预处理后增加他莫昔芬耐药 ER+ MCF-7 细胞对他莫昔芬治疗的敏感性。此外，我们还证明，抗 ALCAM scFv 单药治疗也能够抑制 Boyden 室测定中 MDA-MB-231 细胞的侵袭和迁移。
