Production and evaluation of cytotoxic effects of DT386-BR2 fusion protein as a novel anti-cancer agent

Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

doi:10.1016/j.mimet.2016.09.004

Cited by 25 publications

(17 citation statements)

References 30 publications

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“…Whereas DT386-BR2 protein showed cytotoxic effects on HeLa and MCF-7 cancer cell lines with IC 50 s of 0.55 and 2.08 μg/mL, respectively, it did not have any toxic effects on HUVEC and HEK 293 normal cell lines. The evaluation of cytotoxic effects of BR2 on cancer and normal cells showed no inhibitory effects on both cancer and normal cells (21).…”

Section: Discussionmentioning

confidence: 99%

“…The IC 50 of rIL24 was 0.8 μg/mL (11). According to this study and others studies, such as GST-MDA-7 (14) RGD-IL24 (1), and DT386-BR2 (21) that express IL24 or BR2 in E. coli successfully, we used E. coli BL21(DE3) as the host for the expression of IL24-BR2. However, one of the problems that have always been posed with E. coli expression system is the production of insoluble protein, which is mis-folding aggregates called inclusion bodies (22).…”

Section: Discussionmentioning

confidence: 99%

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Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein

et al. 2019

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Interleukin (IL) 24 is a pro-inflammatory and tumor suppressor cytokine capable of inducing selective apoptosis in various cancer cells. BR2, on the other hand, is an anti-microbial peptide with selective penetrability to the cancer cells. In this study, we aimed to produce and purify a fusion protein containing IL24 as the toxic moiety fused to BR2, as targeting moiety, and then to evaluate its cytotoxic activities. For this purpose, the coding sequence of IL24-BR2 fusion protein and IL24 were cloned into the pET28a vector and used to transform E. coli BL21 (DE3) cells. Following induction of expression, protein purification performed using Ni-NTA chromatography. SDS-PAGE and western blotting were performed to confirm the expression and purification. Finally, cytotoxic effects of the purified proteins were evaluated on MCF-7 and HUVEC cell lines. Analysis of crude lysate of induced recombinant E. coli BL21 (DE3) bacteria and also purified proteins showed a band of approximately 22 and 18 KDa on SDS-PAGE and western blotting for IL24-BR2 and IL24, respectively. Finally, statistical analysis showed significant cytotoxic effects of IL24-BR2 on MCF-7 cells at 10, 20, and 40 µg/mL concentrations compared to IL24 alone, which showed no significant cytotoxic effects on cancer cells except in the highest concentration. In conclusion, production and purification of IL24-BR2 fusion protein with potential specific toxicity toward cancer cells was successfully achieved. However, further investigation of the cytotoxic effects of this fusion protein on other cell lines and in vivo cancer models must be performed.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein

et al. 2019

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“…DT386-BR2 is a 47 KDa fusion protein containing the first 386 residues of DT fused via a rigid peptide linker, (AP) 4 , to the antimicrobial peptide, BR2 (Shafiee et al, 2016, 2017c). BR2 is derived from bufoin IIb (RAGLQFPVG[RLLR] 3 ), a potent antimicrobial peptide with cell-penetrating and anti-cancer properties (Lee et al, 2008; Cho et al, 2009).…”

Section: Diphtheria Toxin-based Immunotoxinsmentioning

confidence: 99%

“…BR2 has only two terminal RLLR repeats and is selective in its ability to penetrate and kill cancer cells without affecting normal cells (Lim et al, 2013; Shafiee et al, 2017b). In vitro studies showed that DT386-BR2 was cytotoxic against HeLa (cervical carcinoma) and MCF-7 (breast cancer) cells, with little toxicity to HEK 293 and HUVEC cells (Shafiee et al, 2016). Compared to other immunotoxins, however, the reported IC 50 s between 10 –8 and 4 × 10 –8 M against HeLa and MCF-7 are relatively high.…”

Section: Diphtheria Toxin-based Immunotoxinsmentioning

confidence: 99%

Targeted Diphtheria Toxin-Based Therapy: A Review Article

Shafiee¹,

Aucoin²,

Jahanian-Najafabadi³

2019

Front. Microbiol.

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Cancer remains one of the leading causes of death worldwide. Conventional therapeutic strategies usually offer limited specificity, resulting in severe side effects and toxicity to normal tissues. Targeted cancer therapy, on the other hand, can improve the therapeutic potential of anti-cancer agents and decrease unwanted side effects. Targeted applications of cytolethal bacterial toxins have been found to be especially useful for the specific eradication of cancer cells. Targeting is either mediated by peptides or by protein-targeting moieties, such as antibodies, antibody fragments, cell-penetrating peptides (CPPs), growth factors, or cytokines. Together with a toxin domain, these molecules are more commonly referred to as immunotoxins. Targeting can also be achieved through gene delivery and cell-specific expression of a toxin. Of the available cytolethal toxins, diphtheria toxin (DT) is one of the most frequently used for these strategies. Of the many DT-based therapeutic strategies investigated to date, two immunotoxins, OntakTM and TagraxofuspTM, have gained FDA approval for clinical application. Despite some success with immunotoxins, suicide-gene therapy strategies, whereby controlled tumor-specific expression of DT is used for the eradication of malignant cells, are gaining prominence. The first part of this review focuses on DT-based immunotoxins, and it then discusses recent developments in tumor-specific expression of DT.

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“…Several PCR-based methods have been developed for the identification of the N. meningitidis based on the polysialyltransferase siaD gene or other genes including crgA , 16S rRNA and porA 25. Additionally, real-time and multiplex PCR assays for identification of N. meningitidis have also been reported 5,26,27. However, these methods are expensive due to the requirement of specific equipment and they are complicated to perform in resource-poor laboratories in many developing countries 8…”

Section: Introductionmentioning

confidence: 99%

<p>Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay to detect <em>Neisseria meningitidis</em></p>

Li¹,

Liu²,

Liu³

et al. 2019

IDR

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Background Neisseria meningitidis is a leading pathogen of meningococcal disease in humans worldwide. Multiple cross displacement mplification (MCDA) combined with nanoparticles-based lateral flow biosensor (MCDA-LFB) has been reported for the rapid detection of several bacterial pathogens in recent years. Here, therefore we developed an MCDA-LFB assay for the rapid detection of N. meningitis . Methods A set of 10 primers specifically to recognize 10 different regions of the ctrA gene of N. meningitidis were designed. MCDA was developed and combined with a LFB to detect the ctrA gene of N. meningitidis . The reaction time and temperature condition for the MCDA-LFB were optimized and then the MCDA-LFB was applied to detect the DNA from clinical samples. Results MCDA-LFB assay was successfully established for the detection of N. meningitidis based on the ctrA gene. The MCDA assay was optimized at 64°C for only 35 mins and the products of amplification were directly sensed by LFB. The whole operation, including DNA template preparation (~20 mins), MCDA reaction (35 mins) and results interpretation (~2 mins) could be finished in no more than 60 mins. The detection limit was as low as 10 fg/reaction (around 3 CFUs/reaction) of pure N. meningitidis DNA, with no cross-reaction with other bacterial DNA. Conclusion The MCDA-LFB techniques developed in the present study are an effective tool for the rapid detection of N. meningitidis , especially in resource-poor countries in meningococcal disease epidemic period.

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Production and evaluation of cytotoxic effects of DT386-BR2 fusion protein as a novel anti-cancer agent

Cited by 25 publications

References 30 publications

Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein

Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein

Targeted Diphtheria Toxin-Based Therapy: A Review Article

<p>Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay to detect <em>Neisseria meningitidis</em></p>

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