Therapeutic Potential of Lactobacillus rhamnosus DS3316 via Cell Apoptosis in Colorectal Cancer

Abstract

Colorectal cancer (CRC) has a very high mortality rate worldwide. Although various therapies have been developed to treat CRC, the need for novel therapeutic approaches has been increasing due to severe side effects and limited efficacy of current treatments. Recently, although research on the gut microbiome and its association with colon cancer has been growing, the mechanisms of gut microbiome inhibition in CRC remain insufficiently understood. Thus, in this study, we investigated the growth-inhibitory effects of the culture supernatant of Lactobacillus rhamnosus DS3316, isolated from infant feces, on CRC cell lines (HCT116 and SNUC5). And RNA-seq analysis revealed an increase in apoptosis-related terms induced by L. rhamnosus DS3316 treatment. Also, we found the non-toxicity of L. rhamnosus DS3316 in human iPSC-derived intenstine organoid. Thus, we suggested that L. rhamnosus DS3316 inhibits the growth of colorectal cancer cell lines without affecting normal cells. And L. rhamnosus DS3316 is expected to be a promising candidate for the development of microbiome-based colorectal cancer therapies. Furthermore, its combined use with various colorectal cancer treatment methods could lead to the proposal of more effective therapeutic approaches.

Keywords: Lactobacillus rhamnosus; apoptosis; colorectal cancer.

Fig. 1. L. rhamnosus DS3316 treatment inhibited cell growth in HCT116 and SNUC5 cell lines.

(A) Cell growth assay after L. rhamnosus DS3316 treatment for 72 h. HCT116 and SNUC5 cells were fixed in 100% methanol and stained with crystal violet solution. Scale bars 200 μm. (B) CCK-8 assay after L. rhamnosus DS3316 treatment for 72 h. HCT116 and SNUC5 cells were incubated for 5 min at 37°C after addition of CCK-8 solution. Cell viability was measured using a microplate reader (450 nm). The mean ± of three independent experiments is shown. P values were calculated using Student’s t-test (***p < 0.001).

Fig. 2. GO term analysis demonstrate apoptosis induction by L. rhamnosus DS3316 treatment in HCT116 and SNUC5 cell lines.

(A) and (B), DAVID (A) and ClueGO (B)-based GO analysis of the RNA-seq results, among the 349 upregulated genes. Enriched GO terms are shown. (C) Human intestinal organoids after L. rhamnosus DS3316 treatment for 72 h. Scale bars 200 μm.

Fig. 3. L. rhamnosus DS3316 treatment induced cell apoptosis.

(A) FACS analysis of Annexin V staining was conducted after L. rhamnosus DS3316 treatment for 72 h. The lower right and upper right quadrants indicate early apoptosis and late apoptosis (upper), respectively. The quantification of apoptosis. Mean ± SD of three independent experiments. The P values were calculated using Student’s t‐test (***P < 0.001). (B) FACS analysis using Muse Caspase 3/7 working solution was conducted after L. rhamnosus DS3316 treatment for 72 h. The upper right panel indicates the apoptotic and dead cell proportions (upper). The quantification of caspase‐3/7 activity. Mean ± SD of three independent experiments. The P values were calculated using Student’s t‐test (***P < 0.001). (C) Western blot analysis after L. rhamnosus DS3316 treatment using anti- PARP antibody. ACTB was used as the internal control in HCT116 and SNUC5 cells. The signal intensities were quantified using ImageJ software.

Fig. 4. L. rhamnosus DS3316 treatment reduced aggregation of 3D spheroid culture system.

(A) 3D spheroid formation assay. L. rhamnosus DS3316 treatment was performed in HCT116 cells for 72 h. Cells were loaded onto ULA plates. (B) 3D spheroid formation assay. L. rhamnosus DS3316 treatment was performed in SNUC5 cells for 72 h. Cells were loaded onto ULA plates. Scale bars 200 μm.

Fig. 5. L. rhamnosus DS3316 treatment induced cell apoptosis in 3D spheroid culture system.

(A) After 72 h of L. rhamnosus DS3316 treatment, PI staining was conducted. Scale bars 200 μm. (B) Western blot analysis after L. rhamnosus DS3316 treatment using anti-PARP antibody. ACTB was used as the internal control in HCT116 and SNUC5 cells. The signal intensities were quantified using ImageJ software.