Especially, the migrations of tumor cells and their aggressive phenotypes is associated with αvβ3 related signaling (Hamidi et al. 2016). Furthermore, enhanced αvβ3 expression have been showed to promote the growth of breast cancer cells and metastasis to bone (Takayama et al. 2005). So, these receptor, over-expressed on the several cancer cells not normal body cells, and involved in invading the tumor cells, are attractive approach to targeted delivery of therapeutic agents to cancer cells. In the design of fusion protein, because the iRGD moiety must have a free CendR terminus, we had to put it at the C-terminal of DFF40 protein. On the other hand, because of the greater effect of β3 in the binding of RGD peptide than the other sub-unit (Garrigues et al. 2008), β3 was used for molecular docking. The results of protein docking with the receptor in HADDOCK indicate that binding of the DFF40 protein to the iRGD peptide not only prevents exposure and binds to the αVβ3 integrin receptor, but also results in a more stable structure.
In the expression stage, it is an established remark that post-induction temperature and inducer concentration are the most important factors influencing the expression level of proteins. In general, lowering the post-induction temperature can reduce inclusion body formation by reducing the expression of the proteins (Vasina et al. 1997). In a study by San-Miguel et al. E. coli BL21 (DE3) was used for the expression of Progesterone 5β-reductase, showed that only proteins expressed at 15°C and below are in active form (San-Miguel et al. 2013). So, lowering the temperature as well as inducer concentration led to reduce the speed of protein expression and finally, increased the soluble protein level (de Groot et al. 2006).
The results of Design-Expert software showed a significant effect of temperature on the soluble expression of both proteins; this factor has the greatest effect on the soluble expression of DFF40 and DFF40-iRGD; a few increasing in temperature, led to a significant decrease in the expression in protein soluble form. In addition, the results indicated that the incubation period had the least effect compared to other independent variables according to the Design-Expert software results.
In general, the addition of inducer in low amounts (IPTG used in our study) results in insufficient induction and consequently low expression of the recombinant protein; while adding extra amounts of these expensive inducers, in addition to wasting costs, can lead to toxic effects on cell growth (Ramirez et al. 1994).
The results of this study showed that increasing the concentration of IPTG in the induction of DFF40 protein increases the expression of soluble and insoluble proteins; while the highest expression of DFF40-iRGD protein in soluble form was observed in the lowest inducer concentration (0.1 mM) and increasing IPTG concentration led to the increased insoluble expression and consequently increased inactive protein content.
The purification technique using the intein system has been used to produce various recombinant proteins. For example, human GM-CSF was produced and purified by its fusing with the intein in the pTYB11 vector (Goffe et al. 2003). In a study conducted by Wang, Parathyroid hormone was also cloned into the pTXB1 vector and purified using the IMPACT system by adding DTT (Wang et al. 2007). In two these studies, reducing agents, especially dithiotritol, have been used to induce the self-cleavage properties of intein. The addition of this chemical, in addition to increasing the cost of large-scale purification, allows the three-dimensional structure of disulfide-containing proteins to be modified by reducing these bonds, while the use of intein-1 in pTWIN1 vector and induction the self-cleavage property of intein by pH change, is more logical and convenient.
For the first fusion protein containing DDF40, GM-CSF-DFF40, cytotoxicity was confirmed at 200 nM of this protein against THP-1 cells, indicating a high potency of this protein in inducing cell apoptosis. Also, the rate of apoptosis of cells treated with 200 nM of GM-CSF-DFF40 by Annexin V-FITC staining kit showed 30–35% of apoptosis in this cell line (Mosrati et al. 1993). However, in our study, the calculated IC50 of DFF40-iRGD was determined as 15 nM after 72 hours of treatment can be attributed to the more efficient internalizing efficacy of this fusion protein than GM-CSF-DFF40. Furthermore, the rate of apoptosis due to treatment of MDA-MB-231 cells with a concentration of 0.5 µg/ml (near to the concentration of IC50) of DFF40-iRGD was equal to 76%, which well Confirms the apoptosis inducing effects of this protein on integrin receptor positive tumor cells. These apoptosis inducing effects of DFF40 was also confirmed in our pervious study on B16 cell transfected with a recombinant pCDNA vector containing the DFF40 gene under the control of Survivin promoter (minayian et al. 2021).
For GnRH-DFF40 fusion protein, on the other hand, apoptosis induction was established by the DNA fragmentation assay and increasing in the caspase 3 and 9 activity, especially after 24 hours of the treatment of colo 205 colon carcinoma cell line (Ben-Yehudah et al. 2003). These results indicated that treatment for 24 hours can successfully induce apoptosis in the tumor cells.
For iRGD peptide, used in the present as the targeting moiety, there are several similar designed molecules. In the study of Zhang et al. (2016), the concomitant use of the iRGD peptide with cetuximab (Erbitux®) was proposed as a new strategy for the treatment of Non-Small-Cell Lung Cancer (NSCLC). In this study, A549 cells were used with the high expression levels of αVβ3, αVβ5 and NRP-1 receptors. The results of this study showed a significant increase in the apoptotic index of cells treated with co-administration of Erbitux® and iRGD peptide due to more cell penetration. Also, this simultaneous use in xenograft mice reduced the weight and size of the induced tumor. A 2019 study by Yang et al. (2019) for IL-24-iRGD fusion protein on the same cell line showed similar results in the treatment of NSCLC lung cancer. In the mentioned study, IL24-iRGD induced apoptosis after 48 hours of exposure to the A549 cells. While in the same concentrations, the apoptotic effect of native IL-24 was negligible. Again, these data confirmed that more cytotoxic effects of DFF40-iRGD in comparison to the native DFF-40, refer to the presence of iRGD in its structure led to more penetration ability to αVβ3 positive cells (MDA-MB-231). Furthermore, it may be possible to observe more apoptotic effects by increasing the treatment time.
For the MCF-7 cells, we used as a negative control cell line and assumed with low level of αVβ3 integrin receptor, we observed some significant cytotoxicity especially with increasing the time of incubation. This toxicity may be related to the non-receptor mediated endocytosis for native form of DFF40, as shown for the treated MDA-MB-231 cells, as well as interaction of iRGD with other related integrin receptor sub-types presented on the surface of this cell line (Yan et al. 2016) .