Abstract
Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3+) and SkBr3 (GPC3−) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept.
Highlights
Out-of-control growth and proliferation of cells lead to tumor formation [1]
After codon adaptation of DT389-(GGGGS)2-YP7 IT based on the E. coli strand, the primary expression was optimized and prepared for continued investigation
Results showed that purification of proteins was accurate. 1: purified IT (69 kDa), 2: ladder, 3: purified truncated Diphtheria (DT389) (42 kDa), 4: purified YP7 single-chain fragment variable (scFv) (35 kDa), 5: total protein extraction of native E. coli BL21 without vector. (f)
Summary
Out-of-control growth and proliferation of cells lead to tumor formation [1]. Traditional approaches, such as surgery, radiation therapy, chemotherapy, or a combination of them, in treating malignancies are insufficient and accompanied by many side effects on normal tissues [2,3]. GPC3, as an oncofetal antigen, is overexpressed in HCC cells and involved in tumor development through Wnt [13,14], Yap [15], and TGFβ2 [16] signaling pathways. Specific binding of the antibody to the antigen causes the toxin to enter into the cancer cells, subsequently killing the cell by inhibiting protein synthesis. This specific binding diminishes the negative effects of immunotoxins on normal cells [25]
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