The Mechanism Of Exopolysaccharide-induced Apoptosis In HT-29 Cancer Cell

Colorectal cancer is one of the most common human malignant gastrointestinal cancers, and it continues to be a major healthcare concern worldwide with its incidence increasing annually, effective prevention of it has become a hot spot nowadays. Many causes can induce colon cancer, especially dietary habits. A great deal of data confirmed that exopolysaccharide(EPS) has unique physical properties and physiological functions such as antitumor activity, however, the mechanism was obscure. Our reasearch adopts EPS isolated from Lactobacillus paracasei supsp. paracasei M5(L. paracasei M5) to co-culture with colon cancer HT-29 cell to study the mechanism of apoptosis.We extracted the EPS of Lactobacillus paracasei supsp. paracasei M5, Lactobacillus paracasei supsp. paracasei X12, Lactobacillus coryniformis supsp. torquens T3 which identified by our lab, using MTT to determine the inhibition of three EPS on colon cancer HT-29 cells, results showed that 500 μg/m L and 1000 μg/m L M5-EPS treatment for 48 h is the best, can be used for the subsequent research; The yield of M5-EPS is the largest when the fermentation conditions is p H: 6.5, bacteria concertration: 108,temperature: 37 °C and time:12 h; Comparing with the control group, the cell number in M5-EPS treatment group decreased significantly observed by inverted light microscope, illustrating that M5-EPS can obviously inhibit the growth of HT-29 cells; Hoechst 33258 fluorescent staining demonstrated that apoptosis was probable following M5- EPS treatment of HT-29 cells; Transmission electron microscopy(TEM) showed that M5-EPS induced apoptosis in the HT-29 cells and resulted in the disappearance of cell surface villus, mitochondria degeneration, endoplasmic reticulum(ER) swelling, functional decline, and damage to organelles; Flow cytometry was used to analyze cell-cycle distribution, results revealed that M5-EPS could influence cell cycle distribution with inhibition of G0/G1 phase.M5-EPS can destroy the balance of internal oxidatve system: Useing the fluorescent dye DCF-DA to determine intracellular ROS levels, we can see that M5-EPS significantly induced ROS production in the HT-29 cells compared with the control group; Real- time PCR detection of antioxidant system transcription regulatory protein SOD, CAT and GSH-PX expression, results showed that after M5-EPS treatment for 48 h, SOD, CAT and GSH-PX m RNA expression decreased significantly compared with the control group in a concentration-dependent manner; Furthermore, the antioxidant activities of SOD, CAT and GSH-PX were measured by spectrophotometrical methods, the activities of these enzymes in HT-29 cells were significantly lower than the control group following treatment with M5-EPS; O xidative stress was suppressed after adding N-acetyl- L-cysteine, the HT-29 cell number obviously increased and cell morphology was better than that not treated with N-acetyl-L-cysteine. In summary, oxidative stress participates in the process of HT-29 apoptosis induced by M5-EPS.M5-EPS can activate the endoplasmic reticulum stress in HT-29 cell: As a Ca2+-binding protein, CRT has a high capacity to buffer Ca2+ located in the lumen of the ER, we found that M5-EPS have the capacity to induce CRT translocation from the ER to the surface of the cytomembrane, demonstrating that may activate endoplasmic reticulum stress in HT-29 cell; In response to M5-EPS, induction of GRP78, activation of CHOP were obvserved, M5-EPS also triggered activation of UPR components, including ATF4 in a concentration-dependent manner; Determination of apoptosis related factors, the results showed the M5-EPS can significantly activate caspase-3, Bax and Bad expression, also dramaticlly reducing Bcl-xl expression; N-acetyl-L-cysteine could decrease the level of ROS in HT-29 cell, and also transcriptionally inhibited induction of GRP78 and CHOP by M5-EPS, illustrating that oxidative stress can significantly affect the endoplasmic reticulum stress pathway.