The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5–15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10–15 µM) for 12 h. Moreover, 5–15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin β1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.

Graphical abstract

Protein-based drugs have increasingly become an important segment of cancer treatment. In comparison with chemotherapy, they offer high efficacy and fewer side effects due to specifically targeting only cancer cells. Monoclonal antibodies are currently the main protein-based drugs in the market but their complexity and limitations in tumour penetration led to the development of alternative protein therapeutics such as pore-forming toxins. Colicin N (ColN), a pore-forming protein produced by E. coli, was previously found to exhibit cytotoxicity and selectivity in human lung cancer cells with promising potential for further development. Here we aimed to screen for the cytotoxicity of ColN in breast (MCF-7 and MDA-MB-231), lung (A549) and colon cancer cells (HT-29 and HCT-116) by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay with various concentrations for 72 h and to investigate the cytotoxic effect of ColN domains on cancer cells. It showed that ColN mildly mediated the decrease in cell viability except for MCF-7. The highest effect was seen in A549 and HCT-116 cells which showed 31.9% and 31.5% decrease in cell viability, respectively. The mild inhibition or promotion of cancer cell proliferation by ColN tend to be based on the cell types. Furthermore, to search for the functional domain of ColN used for cytotoxicity, full-length ColN and truncated ColN with deletion of translocating, receptor binding and pore-forming domains were also tested on HCT-116 colon cancer cells. The findings indicated that HCT-116 cells were not significantly sensitive to ColN but full length ColN caused slight decrease in cancer cell viability. The data in this study will benefit the further development of ColN for alternative protein-based cancer therapy.

Cancer is one of the leading global causes of death. In 2018, WHO estimated that 9.6 million patients died from cancer, with 18.1 million new cases emerged. In recent years, immunotherapy is considered as a treatment improving cancer-related immune function by manipulating the immune checkpoints in the immune system. Cytotoxic-T-lymphocyte antigen 4 (CTLA-4) is a immune checkpoint transmembrane protein. The expression of CTLA-4 on the surface of activated T-cell could downregulate the production and proliferation of cytotoxic T-lymphocyte cells (CTLs). CTLs are the host-defended immune cells that could retard the growth of cancer cells by recognizing the tumor-associated antigens expressed on the membrane of cancer cells, then eliminate them before spreading and evading throughout the body. The overexpression of CTLA-4 founded in cancer has a suppressive effect on cytotoxic T-lymphocyte cells, encouraging the tumor cell's growth. Recently, the monoclonal antibodies targeting CTLA-4 have successfully proved to be an active agent in immunotherapy. However, the large amount and multiple dosing had been a major hindrance. In this study, we developed heat-labile enterotoxin B subunit (LTB) fused CTLA-4 or LTB-CTLA4 as a cancer vaccine, to reduce the disadvantage in term of quantity, price, and frequency of dosing. LTB-CTLA4 was produced in Nicotiana benthamiana plant as a bio-factory, a promising platform for the plant-derived biopharmaceutical production which has abilities to produce high value but cost-effective vaccines. The highest LTB-CTLA4 level reached 1.29 µg per gram plant fresh weight and induced BALB/c mice to produce specific IgG against CTLA-4. LTB-CTLA4 has a potential as a candidate agent for cancer vaccine either to prevent the development of cancer or to treat cancer in the future.

Bacteria, exposed to stress from the environment or competition, release toxins called bacteriocins. Bacteriocins are classified by their mode of cytotoxicity such as pore former, endonuclease, etc. Colicins are the member of bacteriocins produced by Escherichia coli (E.coli). Among several types of colicins cytotoxicity, Pore-forming colicins has been reported in both bacteria and various type of cancer cells. Colicin N is also a pore-forming colicin and the anticancer activity of colicin N has not been extensively reported. Here we examined the cytotoxic effects of colicin N and its domain on cancer cells. The expression and purification of recombinant full-length and truncated colicin N were performed in E. coli and an affinity chromatography. The physicochemical properties of purified proteins were then assessed by SDS-PAGE, western blot, Circular Dichroism and mass spectrometry. Then, HCT-116, HT-29, MCF-7, MDA-MB-231 and A549 cells were treated with full-length Colicin N for anticancer test by MTT assay. Furthermore, a series of truncated colicin N with a deletion of one or two domains were tested on HCT-116 cells. We show that, full-length and truncated colicin N were produced successfully and showed the expected physical properties such as molecular weight and secondary structures. Regarding the cytotoxicity against cancer cells, we found that HCT-116 cells (colon cancer cells) was the most sensitive to full length colicin N. Likewise, HT-29, MDA-MB-231 and A549 cells were also sensitive to full-length colicin N. In contrast to these cancer cells, the cell viability of MCF-7 was promoted in the presence of full-length colicin N. The effect of full-length colicin N on cancer cells seemed to be dependent on types of cancer cells. The truncated colicin N did not cause cytotoxicity to cancer cells hence the toxic domain of colicin N is not sufficient for anticancer activity. Furthermore, colicin N mutant with increased number of positive charges on its surface was constructed. The preliminary results demonstrated that this mutant was more cytotoxic than wild type colicin N. This could be due to the difference in charges or conformational changes of colicin N mutant. The results from this study can improve the basic knowledge about colicin N related cytotoxic activity on cancer cells and suggestions that colicin N may be considered for its promising application of therapeutic and natural antitumor drugs.