Related Experiment Video
Updated: Oct 28, 2025
Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells
Published on: October 27, 2020
ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells
Abdolamir Ghadaksaz1, Abbas Ali Imani Fooladi2, Hamideh Mahmoodzadeh Hosseini3
1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Abstract:
Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.
Insights
A novel recombinant protein, ARA-linker-TGFαL3, effectively targets EGFR-expressing breast cancer cells, demonstrating enhanced binding affinity and cytotoxicity. This targeted therapy shows promise in overcoming chemotherapy resistance by inducing apoptosis and reducing angiogenesis and metastasis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Chemotherapy resistance in cancer cells necessitates novel targeted therapies.
- Bacterial metabolites and immunotoxins offer potential strategies for overcoming resistance.
- Targeting overexpressed receptors on cancer cells is a key approach in drug design.
Purpose of the Study:
- To design and evaluate a novel recombinant protein, ARA-linker-TGFαL3, for targeting EGFR-expressing breast cancer cells.
- To assess the binding affinity, cytotoxicity, and apoptosis-inducing effects of ARA-linker-TGFαL3.
- To compare the therapeutic potential of ARA-linker-TGFαL3 with its components and a standard chemotherapeutic drug.
Main Methods:
- Cloning and expression of recombinant protein (ARA-linker-TGFαL3) in Escherichia coli.
- In vitro assays using EGFR-positive (MDA-MB-468) and EGFR-negative (MDA-MB-453) breast cancer cell lines.
- Assessment of binding affinity (Cell ELISA), cytotoxicity (MTT, LDH release), apoptosis induction (flow cytometry, caspase-3 assay), and gene expression (RT-PCR).
Main Results:
- ARA-linker-TGFαL3 exhibited higher binding affinity and cytotoxicity against EGFR-positive MDA-MB-468 cells compared to Arazyme alone.
- Both recombinant proteins induced early apoptosis in MDA-MB-468 cells, while Taxotere induced late apoptosis.
- Treatment with ARA-linker-TGFαL3 or Arazyme decreased the expression of angiogenesis and metastasis genes in both cell lines.
Conclusions:
- ARA-linker-TGFαL3 demonstrates significant therapeutic potential against EGFR-expressing breast cancer cells.
- The designed construct effectively targets cancer cells, induces apoptosis, and modulates key cancer-related gene expressions.
- This study provides in vitro evidence supporting the development of ARA-linker-TGFαL3 as a targeted cancer therapy.
