Oxidative stress and endoplasmic reticulum stress contribute to L. paracasei subsp. paracasei M5L exopolysaccharide‐induced apoptosis in HT‐29 cells

Abstract Colorectal cancer is the third most malignant cancer occurring around the world. Effective prevention and treatment have been increasingly the focus of global attention. Long‐term diet of fermented dairy inhibits proliferation of colon cancer cell, which is considered that not only live lactic acid bacteria but also the secreted exopolysaccharides exert the function. In this scenario, this study aimed to investigate the mechanism of growth inhibition on HT‐29 cells induced in vitro by exopolysaccharides isolated from Lactobacillus paracasei subsp. paracasei M5L (M5‐EPSs). HT‐29 cells which were treated by a set of concentrations of M5‐EPSs have been investigated of cell viability, characteristic changes, cell cycle distribution, and redox system. The results demonstrated that M5‐EPSs treatments induced HT‐29 cell apoptosis and resulted in upregulation of ROS levels and downregulation of antioxidant enzyme activities, leading to an imbalance in the oxidation system in HT‐29 cells. In response to M5‐EPSs, endogenous ER stress (ERS) markers, including GRP78, ATF4, and CHOP, were transcriptionally altered so that activating the ERS in HT‐29 cells. After NAC treatment, the oxidative stress was inhibited, and the expression of GRP78 and CHOP was significantly decreased, indicating that oxidative stress can significantly affect the ERS pathway. Furthermore, it suggested that the occurrence of apoptosis was associated with Bcl‐2 gene family. In conclusion, this study demonstrated that M5‐EPSs can induce HT‐29 cells apoptosis by destroying the redox system through activation of the ERS signaling pathway.


| INTRODUC TI ON
Colorectal cancer, the third most malignant cancer occurring around the world, is thought to be influenced by many factors, making this form of cancer a major health concern (Bray et al., 2018). Despite Conventional or complementary therapies, including chemotherapy, radiation, surgery, physical rehabilitation, and immunotherapy have been attempted to treat colorectal cancer, a successful treatment has not yet been found, and surgical resection is always used for colorectal cancer treatment (Adam et al.;Delaunoit et al., 2005;Zampino et al., 2016). However, the drug resistance of cancer cells has blocked their apoptosis; in addition, anticancer agents may have cytotoxic effects in normal cells (Alfarouk et al., 2015;Lichan Chen, 2018;Sun et al., 2012).
In recent years, an increasing number of natural products with anticancer compounds have had their pharmacological bioactivities confirmed and have been used to explain the mechanisms of cancer prevention in apoptosis.
The endoplasmic reticulum activates the unfolded protein response (UPR) when it undergoes stress. This response can protect cells from the damage caused by the endoplasmic reticulum stress (ERS) and restore cell function; however, when ERS is too strong or lasts too long, the endoplasmic reticulum homeostasis is seriously unbalanced and cannot be repaired, which will lead to cell apoptosis. The UPR normally activates three transcription factors, including inositol-requiring enzyme 1 (IRE1), PEK-like endoplasmic reticulum kinase (RERK/PEK), and activating transcription factor 6 (ATF6), which degrade deposited unfolded and misfolded proteins of these three transcription factors, ATF6, as a receptor protein in the endoplasmic reticulum, is one of the factors in the apoptosis and autophagy pathways induced by the ERS (Haque et al., 2015). ERS-induced death signaling pathways include the CHOP/GADDl53, JNK, and caspase pathways . Cells enhance ATF4 through the PERK pathway, and CHOP is also a transcription factor of the PERK pathway and the direct target of ATF4. CHOP and caspase expression are weak when homeostasis is balanced. When ERS occurs, CHOP and caspase expression will significantly increase.
Overexpression of CHOP and caspase can promote cell cycle stagnation or lead to apoptosis (Liu et al., 2015).
Another pathway that causes cell apoptosis is the oxidative stress pathway (Xiang et al., 2015). Including cancer, inflammation, diabetes, Parkinson's disease, Alzheimer's disease, atherosclerosis, and aging, various disorders and diseases have been considered to be related with massive production of reactive oxygen species (ROS) and oxygen-derived free radicals. Besides, dysfunction of cells, cell cycle arrest, and apoptosis were also involved in oxidative stress. Safety of synthetic antioxidants has recently been questioned, even though they have been shown to be fairly effective at the processes of slowing down the oxidation (Lu et al., 2017;Moloney & Cotter, 2018;Yuan et al., 2019).
Furthermore, severe side effects as well as increased tolerance to the treatments of cancer have been brought by constant use of medical treatment over time. As a result, exploring and utilizing natural antioxidants and cancer prevention agents including polysaccharides have been paying more attention for their lower cytotoxicity .
Bacterial exopolysaccharides (EPSs) have gradually attracted more attention from researchers because of their structural diversity as well as their versatile physicochemical and biological properties.
Among all beneficial bacteria, lactic acid bacteria (LAB) represent some of the best producers of EPSs due to their long history of safe use in food products (Durlu-Ozkaya et al., 2007;Wu et al., 2010).
Lactobacillus paracasei subsp. paracasei M5L was identified and obtained from our lab. According to our previous research, the cell wall and genomic DNA of L. paracasei M5L and even active or inactive strains display biological activity (Hu et al., 2015). Nevertheless, M5-EPSs mediate the effects in HT-29 cells by the mechanism, which has not yet been fully examined. Therefore, this study was focusing on the antitumor effects, as well as the apoptotic effects of M5-EPSs on HT-29 cells with disclosing molecular mechanisms.

| Bacterial strain
Bacterial strain of L. paracasei subsp. paracasei M5L (L. paracasei M5L) was isolated from kumiss which is a kind of conventional household food in Sinkiang. Lactobacillus paracasei M5L was identified by its conservative and polymorphous 16S rDNA. The broth of de Man, Rogosa, and Sharpe (MRS) was used to culture the bacteria at 37°C and stored at 4°C. After two-time subculture, a 10-fold serial dilution was plated on MRS medium to determine the colony-forming units (CFU/ml) of the strain.

| Cell line
HT-29, a human colonic cancer cell line, was provided by the Cancer Institute of the Chinese Academy of Medical Science. RPMI-1640 medium (Thermo Scientific HyClone) was used to culture the cells, which is supplemented with 10% fetal bovine serum (Thermo Scientific HyClone), penicillin (100 IU/ml), and streptomycin (100 lg/ ml), in a 5% carbon dioxide incubator with constant humidity system at 37°C.

| Preparation of M5-EPSs
After incubation at 37°C for 36 hr to denature enzymes, the L. para-caseiM5L cultures were heated for 15 min at 100°C and then cooled at room temperature. Lactobacilli were removed by centrifugation (10,000 ×g, 4°C) for 30 min, and the supernatant was retrieved for the extraction of M5-EPSs. Proteins were removed by adding 10% (w/v) trichloroacetic acid and incubating at 4°C overnight; the supernatant was collected after centrifugation (10,000 ×g, 20 min, 4°C).
After removing the protein, the product was precipitated with three volumes of ice-cold ethanol (95%) overnight at 4°C. Subsequently, M5-EPSs were collected by centrifugation and then dissolved in deionized water and dialyzed against water (4°C, 48 hr) to remove salts, and pure M5-EPSs were obtained after lyophilization. For further analysis, we used the phenol/sulfuric acid method to determine the total carbohydrate content (98%), and no protein or other components were found.

| Cell viability assay
The viability of HT-29 cells was investigated by an MTT assay. Cells were seeded in 96-well culture plates at a density of 1 × 10 5 cells/ ml and incubated for 24 hr. Then, a series of concentrations of M5-EPSs of 20, 250, 500, 500, and 1,000 μg/ml were added to the media for 24, 48, or 72 hr. MTT solution (0.5 mg/ml in media) was added to each well, and then, the plates were incubated for 4 hr at 37°C.
After washing, the formazan dye precipitates, which were proportional to the number of live cells, were dissolved in 150 μL DMSO.
The amount of formazan product was measured at 490 nm using an enzyme-linked immunosorbent assay plate reader (Bio-Rad-500, Bio-Rad Laboratories In).

| Microscopy observation
The morphological changes of HT-29 cells were observed using inverted light microscopy after adding 500 or 1,000 μg/ml M5-EPSs and incubating for 48 hr. Cells grown in the same manner without M5-EPSs were used as controls. After M5-EPSs treatment, HT-29 cells were examined under a microscope, and all photographs were taken with a digital camera.

| Transmission electron microscopy
After treatment with 500 and 1,000 μg/ml M5-EPSs for 48 hr, HT-29 cells (1 × 10 6 cells) were harvested by trypsinization. Then, the cells were washed twice with PBS, fixed in 2.5% glutaraldehyde for 90 min, and post fixed in 1% osmium tetroxide for 30 min at room temperature. After washing with PBS, the cells were gradually dehydrated in the upgrading of ethanol (50, 70, 95, and 100%) and embedded in Epon 812 resins. The blocks were cut into ultrathin sections with a microtome and were then stained with saturated uranyl acetate and lead citrate. Transmission electron microscope was used to examine the ultrastructure of the cells.

| Flow cytometry analysis
The effects of M5-EPSs on HT-29 cell cycle phase distribution were analyzed by flow cytometry. Briefly, HT-29 cells were treated with M5-EPSs at concentrations of 500 and 1,000 μg/ml for 48 hr after culture at a density of 5 × 10 5 cells/ml in 6-well plates. The cells were harvested by centrifugation at 10,000 rpm for 5 min, washed with 1 ml ice-cold PBS and fixed with 70% ethanol at 4°C overnight.
After centrifugation, the pellet was washed twice with cold PBS and incubated with a PI solution mixed with 100 μg/ml RNase and 0.2% Triton X-100 for 30 min at 37°C in the dark. Finally, the DNA content was measured via flow cytometry (Becton Dickinson) in triplicate for each experiment, and the percentage of cells in each phase of the cell cycle was analyzed.

| Determination of enzyme activities
Cells were incubated with 500 μg/ml and 1,000 μg/ml M5-EPSs for 24, 48, and 72 hr and lysed in cell lysis buffer. The supernatant was obtained after centrifugation at 12,000 g and collected for the determination of enzyme activities. The antioxidant activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH) were measured by spectrophotometry. The commercial kits used in these assays were supplied by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

| Real-time PCR
The effect of M5-EPSs on the gene expression of SOD, CAT, GSH-Px, caspase-3, Bax, and Bcl-2 was detected by real-time polymerase chain reaction. After NAC treatment, GRP78 and CHOP expression was also detected. Total RNA from HT-29 cells was isolated using the EasyPure TM RNA Kit (Sigma). Complimentary DNA (cDNA) synthesis from total RNA (2 μg) was performed using random primers and the TransScript ® One-Step gDNA Removal and cDNA Synthesis

| Statistical analysis
All experiments were performed in triplicate, and all results were expressed as the mean ± SEM. Differences observed were assessed using one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at a value of p < .05 in all of the comparisons.

| M5-EPSs induce cytotoxicity in a dose-and time-dependent manner
The

| Morphological alterations induced by M5-EPSs
As shown in Figure 1b

| Cell cycle arrest by M5-EPSs
The

M5-EPSs
In living organisms, a moderate increase of ROS is necessary for promoting cells proliferation and differentiation, whereas excessive amounts of ROS cause oxidative damage to cells and is even toxic

| Effects of M5-EPSs on CAT, SOD and GSH activity
As the major enzymes with antioxidant activity, antioxidants, in-

Cells
To investigate the possible mechanism by which M5-EPSs exert their effects on HT-29 cells, we further examined the levels of genes encoding GRP78, as well as ATF4, CHOP, which are the typical proteins involved in endoplasmic reticulum stress. Various mechanisms have been suggested to play a role in ER stress-induced apoptosis.
The UPR can reduce ER stress via inducing an initial decrease of general protein synthesis, promoting protein folding by inducing ER chaperones and preventing the accumulation of misfolded proteins.
However, if the stress is severe or prolonged, which may transduce distinct death signals during the UPR, and cells will undergo apoptosis. According to the TEM results, after treatment with M5-EPSs, the endoplasmic reticulum showed a damaged shape. To investigate the role of ER stress in the apoptotic effects of M5-EPSs treatment in HT-29 cells, several ER stress-related molecules were detected.
As shown in Figure 4a, M5-EPSs effectively increased the protein expression of GRP78, CHOP and ATF4, revealing that ER stress participated in the apoptosis triggered by M5-EPSs in HT-29 cells.
Importantly, the quantitative data showed a significant decrease in GRP78 expression in the 1,000 μg/ml group, and similar trends were also observed in the expression of CHOP and ATF4. All the results suggest that M5-EPSs can simultaneously induce oxidative stress and ER stress in HT-29 cells.

| Interaction between M5-EPSs-mediated modulation of the oxidative stress and ER stress pathway
The above results suggest that M5-EPSs can stimulate both oxidative stress and endoplasmic reticulum stress in HT-29 cells. We

| M5-EPSs-induced gene expression of caspase-3, Bax, Bcl-2 and Bcl-xl
The Bcl-2 protein family and caspases have been implicated in mediating apoptotic signals in response to oxidative stress and ER stress,

| D ISCUSS I ON
Tumor cells have a strong ability to proliferate and this ability plays a vital role in the growth and development of tumors (Menyhart et al., 2016). Apoptosis is a regulated and an organized process of programmed cell death (PCD), which plays a crucial role in controlling the development and homeostasis in multicellular organisms (Mondal et al., 2017). Any imbalance between proliferation and apoptosis can lead to carcinogenesis (Kutanzi et al., 2010). Because of their antitumor and immunomodulatory effects and known biological activities, natural polysaccharides have been the topic of much research. Recent evidence has demonstrated that many low-toxic natural polysaccharides can inhibit tumor cells proliferation and selectively induce apoptosis in various cell lines (Meng et al., 2016).
In this study, M5-EPSs treatment significantly inhibited HT-29 cells proliferation and growth in a dose-dependent manner. After treatment with 1,000 μg/ml EPSs for 48 hr, cell growth was severely inhibited with inhibition rate of 85.85% which was higher than 72 hr treatment. According to the mechanism of MTT assay Both oxidative stress and ER stress have been implicated in promoting cell death, tissue damage, and organ dysfunction. ROS generation could induce the release of the pro-apoptotic factors and modulate the apoptotic pathway (Song et al., 2014). Antioxidants, such as SOD and CAT, participate in the dismutation reaction of hydrogen peroxide by superoxide radical anions and then convert hydrogen peroxide to water (Ighodaro & Akinloye, 2018). GSH is involved in cell proliferation and promotes tumor metastasis potential. In addition, GSH maintains mitochondrial membrane integrity (Ortega et al., 2008 forming active caspase 8, an initiator caspase of the extrinsic pathway. The endogenous pathway is initiated when the mitochondrial membrane potential is lost, and then cytochrome c is released from mitochondria to the cytoplasm after the activation of caspase and Bcl-2 family members. In these two pathways, the activated initiator caspase leads to its own autoactivation, which further activates caspase-3, the effector caspase. Furthermore, the UPR in the ERS pathway can also activate Bcl-2 family and caspase proteins (Wong, 2011). In our studies, M5-EPSs upregulated caspase-3 expression in HT-29 cells. Furthermore, M5-EPSs also upregulated the expression of the pro-apoptotic protein Bax and downregulated the expression of the antiapoptotic protein Bcl-2 in HT-29 cells. These results suggest that M5-EPSs-induced apoptosis of HT-29 cell was associated with both the mitochondrial pathway and the death receptor pathway.
Therefore, EPSs from L. paracasei M5L could remarkably induce apoptosis through oxidative stress and the ERS pathway in HT-29 cells, and they have the potential to be developed into treatments with both health and economic benefits for cancer therapy.

| CON CLUS ION
In this study, we extracted M5-EPSs and cocultured them with HT-29 cells. After 500 μg/ml and 1,000 μg/ml M5-EPSs treatment for 48 hr, HT-29 cells showed obvious apoptosis: cell growth was significantly inhibited, cell size decreased, the structures of the endoplasmic reticulum and mitochondria were destroyed, and apoptotic bodies appeared. Intracellular ROS increased, while antioxidant enzymes decreased, and M5-EPSs disrupt the balance of the intracellular oxidation system. Moreover, endoplasmic reticulum-related factors increased significantly, causing the expression of caspase-3 and Bcl-2 family proteins. In future study, we will keep investigating the deep mechanism in mRNA level and protein level. In conclusion, M5-EPSs mediate HT-29 cell apoptosis through the oxidative stress and endoplasmic reticulum stress pathways.

ACK N OWLED G EM ENT
This study was supported by the projects of the National Natural

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly avail-