Abstract
Recently, we reported a novel therapeutic probiotic-derived protein, p8, which has anti-colorectal cancer (anti-CRC) properties. In vitro experiments using a CRC cell line (DLD-1), anti-proliferation activity (about 20%) did not improve after increasing the dose of recombinant-p8 (r-p8) to >10 μM. Here, we show that this was due to the low penetrative efficiency of r-p8 exogenous treatment. Furthermore, we found that r-p8 entered the cytosol through endocytosis, which might be a reason for the low penetration efficiency. Therefore, to improve the therapeutic efficacy of p8, we tried to improve delivery to CRC cells. This resulted in endogenous expression of p8 and increased the anti-proliferative effects by up to 2-fold compared with the exogenous treatment (40 μM). Anti-migration activity also increased markedly. Furthermore, we found that the anti-proliferation activity of p8 was mediated by inhibition of the p53-p21-Cyclin B1/Cdk1 signal pathway, resulting in growth arrest at the G2 phase of the cell cycle. Taken together, these results suggest that p8 is toxic to cancer cells, shows stable expression within cells, and shows strong cancer suppressive activity by inducing cell cycle arrest. Therefore, p8 is a strong candidate for gene therapy if it can be loaded onto cancer-specific viruses.
Highlights
In 2018, an estimated 145,600 adults in the United States were diagnosed with colorectal cancer (CRC) and there were an estimated 51,020 deaths
P8 protein was derived from Lactobacillus rhamnosus (LR) KCTC 12202BP, which was isolated from the human feces
Human CRC cell line DLD-1 was purchased from the Korean Cell Line Bank (KCLB; Seoul, Korea) and maintained under 5% CO2 /37 ◦ C in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco)
Summary
In 2018, an estimated 145,600 adults in the United States were diagnosed with colorectal cancer (CRC) and there were an estimated 51,020 deaths. Because human intestinal microbes and probiotics are generally regarded as safe, isolated proteins may have anti-CRC effects but may show reduced systemic toxicity [8,9,10]. The second approach is based on a viral-based delivery system for biopharmaceuticals such as proteins, peptides, and non-coding small RNAs. Most viruses can be engineered to express anti-cancer protein-coding genes; many recombinant viruses infect specific target cells and express anti-cancer proteins [23,24,25]. Most viruses can be engineered to express anti-cancer protein-coding genes; many recombinant viruses infect specific target cells and express anti-cancer proteins [23,24,25] These viral delivery systems are easier to develop and use than other immunotherapy strategies such as whole tumor cells [26]. Endogenous p8 expression may be a powerful form of gene therapy if it can be loaded onto cancer-specific viruses such as adenoviruses, adeno-associated viruses, or retroviruses
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