The Anti-Tumor Effect of Lactococcus lactis Bacteria-Secreting Human Soluble TRAIL Can Be Enhanced by Metformin Both In Vitro and In Vivo in a Mouse Model of Human Colorectal Cancer.

Katarzyna Kaczmarek,Kazimierz Węglarczyk,Maciej Siedlar,Jerzy Więckiewicz,Jarek Baran

doi:10.3390/cancers13123004

Abstract

Simple SummaryColorectal cancer (CRC) is a major cause of morbidity and mortality in Europe, and accounts for over 10% of all cancer-related deaths worldwide. These indicate an urgent need for novel therapeutic options in CRC. Here, we analysed if genetically modified non-pathogenic Lactococcus lactis bacteria can be used for local delivery of human recombinant Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and induction of tumor cells death in vitro and in vivo in CRC mouse model. We showed that modified L. lactis bacteria were able to secrete biologically active human soluble TRAIL (L. lactis(hsTRAIL+)), which selectively eliminated human CRC cells in vitro, and was further strengthened by metformin (MetF). Our results from in vitro studies were confirmed in vivo using subcutaneous NOD-SCID mouse model of human CRC. The data showed a significant reduction of the tumor growth by intratumor injection of L. lactis(hsTRAIL+) bacteria producing hsTRAIL. This effect could be further enhanced by oral administration of MetF.Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) induces apoptosis of many cancer cells, including CRC cells, being non-harmful for normal ones. However, recombinant form of human TRAIL failed in clinical trial when administered intravenously. To assess the importance of TRAIL in CRC patients, new form of TRAIL delivery would be required. Here we used genetically modified, non-pathogenic Lactococcus lactis bacteria as a vehicle for local delivery of human soluble TRAIL (hsTRAIL) in CRC. Operating under the Nisin Controlled Gene Expression System (NICE), the modified bacteria (L. lactis(hsTRAIL+)) were able to induce cell death of HCT116 and SW480 human cancer cells and reduce the growth of HCT116-tumor spheres in vitro. This effect was cancer cell specific as the cells of normal colon epithelium (FHC cells) were not affected by hsTRAIL-producing bacteria. Metformin (MetF), 5-fluorouracil (5-FU) and irinotecan (CPT-11) enhanced the anti-tumor actions of hsTRAIL in vitro. In the NOD-SCID mouse model, treatment of subcutaneous HCT116-tumors with L. lactis(hsTRAIL+) bacteria given intratumorally, significantly reduced the tumor growth. This anti-tumor activity of hsTRAIL in vivo was further enhanced by oral administration of MetF. These findings indicate that L. lactis bacteria could be suitable for local delivery of biologically active human proteins. At the same time, we documented that anti-tumor activity of hsTRAIL in experimental therapy of CRC can be further enhanced by MetF given orally, opening a venue for alternative CRC-treatment strategies.

Highlights

Colorectal cancer (CRC) represents the group of gastrointestinal cancers (GI) and includes malignant tumors of the colon and rectum
We have documented the biological activity of L. lactis-derived human soluble TRAIL (hsTRAIL), when HCT116 cells were incubated in the presence of supernatant from the broth culture of L. lactis(hsTRAIL+) bacteria [41]
We assessed the potential anti-tumor effect of hsTRAIL-producing bacteria in a co-culture with human CRC cells. For this purpose L. lactis(hsTRAIL+) or corresponding control (L. lactis(“empty” vector)) bacteria were added to the culture of HCT116 human CRC cells, or cells of the normal colon epithelium (FHC cell line) and after 48 h of co-culture, viability of the cells was assesed by MTS assay

Summary

Introduction

Colorectal cancer (CRC) represents the group of gastrointestinal cancers (GI) and includes malignant tumors of the colon and rectum. It has a significant contribution in the global cancer incidence (10.2% CRC from 26.3% for all GI) and cancer-related mortality (9.2% CRC from 35.4% for all GI) in 2018 [1]. Alterations in the composition of gut microbiome and its metabolites are considered as risk factor for CRC development [11] and recently gained clinical interest as potential biomarkers for CRC screening and prognosis [12,13]. The most important are anaerobic Fusobacterium sp. and Porphyromonas sp. with proven pro-tumorigenic activity [14,15], while supplementation of diet with probiotics of Lactobacillus or Bifidobacterium sp., has been shown to reduce the risk of CRC [16,17,18]

Methods

Results

Conclusion