Effect of Different Molecular Weight Fractions Derived From Probiotic Cell-Free Supernatant on Colon Cancer Stem-Like CellInvasion and Apoptosis

Background and Aim: Cancer stem cells play an essential role in tumormetastasis. Decreased expression of E-cadherin and dysregulatedexpression and activity of matrix metalloproteinases (MMPs), especiallyMMP-9 can lead to tumor invasion and metastasis. Many studies havereported anti-cancer effects of Lactobacillus reuteri as a probioticbacterium. In this study, we investigated the effect of different molecularweight fractions derived from the L. reuteri supernatant and its heatkilled killedsonicated fraction (HK-SON) on invasion factors and apoptosis inpreviously established human colon cancer stem-like cells derived fromHT-29 cell line engineered by E-cadherin shRNA (HT29-CSC like).Methods: Cell-free supernatant (CFS) of L. reuteri was fractionated intodefined molecular weight ranges (<3 kD, 3-10 kD, 10-50 kD, 50-100kD, > 100 kD) using ultra-filtration tubes. Also, 109 CFU/mL of L. reuteriwas heat-killed at 100°C in DMEM medium, and then sonicated andcentrifuged. HT29-CSCs were treated with different molecular weightfractions of CFS, HK-SON, and uninoculated bacterial growth media(MRS) as a control. Subsequently, cell invasion assessed using matrigelcoatedinsert plates. The expression of MMP-9 and TIMP-1 mRNAs weredetermined using real-time PCR. The gelatinolytic activity of MMP-9 wasevaluated by Zymography and the apoptosis and necrosis percentage ofcells were determined using Annexin V-PE and 7AAD by flow cytometry.Results: Cell invasion was significantly decreased following treatmentwith HK-SON and different molecular weight fractions of CFS incomparison with an MRS-treated group (P<0.05). Also, HK-SON, 10-50kD, 50-100 kD, and > 100 kD fractions significantly decreased activityand expression level of MMP-9 (P<0.05). Furthermore, the expressionlevel of TIMP-1 was significantly up-regulated by crude CFS, and > 100kD fraction (P<0.05). Apoptosis was significantly decreased followingtreatment with HK-SON and > 100 kD fractions.Conclusion: Our results indicate that CFS of L. reuteri possesses antimetastaticproperties. Since fractions containing high molecular weightsecretory factors decreased cell invasion, it suggests that the inhibitorycompound(s) may fall into a macromolecule range such as a protein,nucleic acid, or a polysaccharide.

کلیدواژه ها:

Apoptosis ، Invasion ، Lactobacillus reuteri ، Matrix metalloproteinase ، Probiotic