Prophylactic role of Lactobacillus Paracasei exopolysaccharides on colon cancer cells through apoptosis not ferroptosis CURRENT

Nowadays despite conventional like exopolysaccharides (EPSs) are of great importance and are considered as potential therapeutic agents. This study aimed to investigate the inhibitory effect of the EPS of L. paracasei on different colon cancer cell lines (SW-480, HT-29, and HCT-116). For this purpose, several cellular and molecular experiments including MTS assay, DAPI staining, Annexin V/PI assay, quantitative real-time PCR (qPCR) and some important ferroptosis-related assays were performed. Based on the findings, L. paracasei EPS can induce apoptosis confirmed by all apoptosis-related assays and could not act through ferroptosis pathways. L. paracasei EPS could hinder the Akt1, mTOR, and Jak-1 mRNAs, and induces apoptosis through down-regulation of the anti-apoptotic gene (Bcl-2), up-regulation of pro-apoptotic genes (BAX, caspase-3, 8). The exploited EPS of an indigenous probiotic strain with anticancer potential with low/insignificant cytotoxicity to normal cells is proposed for future applications in molecular-targeted therapy of colon cancer treatment. Furthermore, in vivo and clinical trials should be performed to evaluate the applicability of this component besides conventional methods to increase the survival rate of colon cancer patients. compared to untreated control groups. The expression of pro-apoptotic genes (BAX, caspase-3, caspase-8) was increased and the anti-apoptotic gene (Bcl-2) was significantly decreased in L. paracasei EPS-treated cancer cells compared to untreated control cells. The upregulation in the mentioned genes by L. paracasei EPS in all treated groups was similar to the anticancer agent (5-FU) indicating the same inducing pathways of apoptosis. These study findings showed non-significant alternation on apoptotic genes expression in an epithelial normal cell line.

chemotherapy-resistant cells [9]. Therefore, there is an urgent need for the development of novel and safe anti-cancer agents [5] based on mechanisms underlying colon cancer cell growth, metastasis and invasion [3].
Probiotic microorganisms are defined as 'live microorganisms when administered in adequate amounts, confer a health benefit on the host'. Most probiotics are lactic acid bacteria (LAB), a group of gram-positive bacteria, and among them, Lactobacillus is one of the most common species [2,10].
They have recently acquired considerable importance because of their innumerable health beneficial properties [2,11,12]. Many concerns about consuming live probiotics especially in immunecompromised and immature immunity during childhood, provoked researchers to investigate the health benefits of probiotic derived components [13]. One of these important components is polysaccharides/glycans as natural biopolymers of carbohydrates. These so-called exopolysaccharides (EPSs) provide numerous health benefits including immunomodulatory properties, anti-cancer, antioxidant activity as well as blood glucose/cholesterol-lowering properties, and antihypertensive activity. Studies revealed that EPSs from probiotics like LAB exerts less adverse side effects and may act as a source of the new anticancer agent [13].
A deeper understanding of the consequences during colon cancer development and progression will facilitate the development of novel therapeutic strategies with lower or no side effects. The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway plays a critical role in proliferation, resistance to apoptosis, angiogenesis, and metastasis during colon cancer development [5,[13][14][15] Components of the Akt signaling pathway promotes cell survival, proliferation, invasion and chemo-resistance [16]. mTOR signaling controls cell survival and regulation of metabolism [17]. Another vital signaling axis is the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, which is involved in numerous physiological processes including cell growth, differentiation, and migration of tumor cells, especially in colon cancer [18,19].
Recent studies have been suggested that blocking components of PI3K/Akt/mTOR signaling pathways, are interesting targets for colon cancer therapy [5,12,13,[15][16][17]. Ever-growing evidence suggested that apoptosis -which encompasses extrinsic and intrinsic pathways -acts as a vital regulator in tumor regression [20,21]. Consequently, any defect in these pathways may result in tumor development and resistance to chemotherapeutics. Therefore, provoking apoptotic pathway seems to be a valuable target in cancer therapy [21]. The biological importance of apoptosis is highlighted in cancer research. However, apoptosis is not the only form of programmed cell death (PCD) [22].
Induction of PCD is an important goal of anticancer drugs [23]. Although, resistance to apoptosis occurs in most cases, investigating non-apoptotic forms of cell death is a promising field of research in the elimination of cancer cells [23]. The term Ferroptosis was coined in 2012 by Stockwell and is a non-apoptotic form of regulated cell death which is distinct from apoptosis, necroptosis and autophagic cell death. Ferroptosis is characterized by extensive lipid peroxidation and ROS production, which can be suppressed by iron chelators or lipophilic antioxidants [22,24]. The induction of ferroptosis and understanding the molecular mechanisms involved may provide new approaches in cancer treatment [23][24][25]. In this regard, we carried out this research to determine the underlying mechanisms by which exopolysaccharide of L. paracasei can modulate aforementioned signaling pathways on colon cancer cell lines (with different metastatic potential -non (SW-480), low (HT-29) and high metastatic (HCT-116)).

Growth condition and EPS isolation
A stock culture of the Lactobacillus paracasei strain isolated from dairy products was stored at -80 °C in sterile glycerol plus media [26]. The strain was thawed and pre-grown in MRS broth (containing 10 g/L peptone, 8 g/L meet extract, 4 g/L yeast extract, 20 g/L dextrose, 1 g/L polysorbate 80, 2 g/L triammonium citrate, 5 g/L sodium acetate trihydrate, 0.1 g/L magnesium sulphate, 0.05 g/L manganese sulphate, 2 g/L dipotassium phosphate) incubated overnight at 37 °C. To obtain sufficient glycosidic components, this procedure was repeated in 2000 mL Erlenmeyer flasks. In brief, after 24 h of incubation at 37 °C, the culture of L. paracasei was centrifuged at 8000 × g for 15 min at 4 °C to remove the bacterial cells, then the supernatant was collected. Then trichloroacetic acid (TCA) was added to the supernatant to a final concentration of 4% (w/v), mixed with agitation overnight at 4 °C, and precipitated proteins were separated by centrifugation (12,000 × g, 4 °C, 15 min). Next, 4 volumes of ethanol were added to the supernatant to precipitate EPS. After incubation for 12 h, EPS was gathered by centrifugation (12,000 × g, 15 min). The precipitates were dissolved in distilled water, then crude EPS was obtained by lyophilization and EPS was used for the next experiments [27] Here, the cells were seeded at the density of 3 × 10 5 cells per well on sterile coverslips in 6-well plates. After overnight incubation, the treated cells with L. paracasei EPS and 5FU were incubated at 37 °C for 24 h then, the cells were fixed with 4% formaldehyde and were permeated with 0.1% Triton X-100 for 5 min. Twice washed cells with PBS were finally stained with 250 ng/mL of DAPI for 3 min at room temperature. The stained cells were evaluated by an inverted fluorescent microscope (Olympus BX64, Olympus, Japan) equipped with a U-MWU2 fluorescence filter.

Flowcytometry
For evaluating the extent of apoptotic and necrotic cell death, annexin V-FITC (fluorescein isothiocyanate) apoptosis detection kit was used. For this purpose, the cells were seeded into 6-well plates (seeding density: 5 × 10 5 cells per well) and treated as above. Then, the cells were detached by trypsin-EDTA and washed by PBS. According to the annexin V-FITC/PI instructions the cells were resuspended in 100 µL 1x binding buffer and 5 µL of FITC-conjugated annexin V was added to cell suspensions. Afterward, 10 µL of PI (propidium iodide) staining solution was added to the cells and incubated at room temperature under the dark condition for 15 min. The cells were analyzed using FACS Calibur flowcytometry (Becton Dickinson, San Jose, CA). Data were analyzed using Cell Quest Pro software (BD Biosciences) and total events of 10,000 cells at a rate of 900 cells/s were acquired. 1.7. RNA extraction, cDNA synthesis, and Quantitative real-time PCR Total RNA from 5-FU and L. paracasei EPS treated and untreated control cells were extracted by using RNX plus reagent. The RNA pellet was dissolved in DEPC treated water, then, the yield and purity were determined using Nanodrop (Epoch™ Microplate Spectrophotometer, BioTek Instruments, USA).
One microgram of isolated RNA was used for the synthesis of cDNA using Prime Script RT Reagent kit according to the manufacturer's instructions. For performing Real-time PCR, specific primers (listed in Table 1) were designed using the OLIGO v. 7.56 software (Molecular Biology Insights, Inc, USA). All reactions were carried out in triplicate for each sample using SYBR green master mix (high ROX) on an ABI-step I plus (Applied Biosystems, CA, USA) detection system. Real-time PCR was performed using the following profiles: 1 cycle at 95 °C for 5 min, followed by 40 cycles at 95 °C for 20 sec, 60 °C for 35 sec and 72 °C for 10 sec. Then the melting curves were acquired by stepwise increases in the temperature from 55 °C to 95 °C to ensure the specific amplification. The 2 −ΔΔCT method [28] was used for interpretation of results and the threshold cycle values were normalized to the housekeeping gene (GAPDH).

Ferroptosis assays 1.8.1. Iron level
The iron assay kit was used to monitor the intracellular iron levels in treated and untreated control cells [24]. Cancer and normal cell lines were seeded in 96-well plates (1.2 × 10 4 cells per well). After overnight incubation, cells were treated with L. paracasei EPS and FIN56 (5 µM) as a positive control.
According to the manufacturer's instructions, iron assay buffer and iron reducer were added to each well. After 30 min of incubation at room temperature, 100 µL of the iron probe were added into wells and incubated for further 60 min under the dark condition at room temperature. Then, the iron level was measured at 593 nm by Nanodrop (Epoch™ Microplate Spectrophotometer, BioTek Instruments, USA).

Statistical Analysis
All data were expressed as mean values ± standard deviation (SD). Each experiment was repeated at least three times. Statistical analyses were performed using Graphpad Prism version 5 (Graphpad Software, San Diego, CA) using one-way ANOVA test considering p ≤ 0.05 as the significance level.

DAPI staining
For investigating the morphological changes as a result of induction of apoptosis on treated/untreated cell lines, DAPI staining was applied. As shown in Fig. 2, L. paracasei EPS treatment (15 µg/mL at 24 h) caused a significant fragmentation in the nucleus of the treated cells compared to untreated control cells, further normal cell line showed no changes. The L. paracasei EPS-treated cells demonstrated significant fragmentation in the chromatin within the nucleus of colon cancer cells but their morphology did not change in untreated control and normal cell line (Fig. 2). In cancerous cell lines, the nucleus seems to be separated and the formation of apoptotic bodies indicate the occurrence of apoptosis in these cells.

Flowcytometry
To distinguish early/late apoptosis and necrotic cell death, all cell lines were stained with Annexin V- Moreover, the cancerous cells treated with L. paracasei EPS showed a significant reduction in AKT-1, JAK-1, and mTOR genes expression levels in all cancerous cell lines compared to untreated control cells (Fig. 4) while the aforementioned genes expression levels in normal cells were non-significant.
For the molecular mechanism of ferroptosis determination, Nrf-2 and CoQ10 genes expression were evaluated and as shown in Fig. 4, L. paracasei EPS could up-regulate and FIN56 down-regulate significantly the Nrf-2 gene (suppresser the formation of superoxide as well as other reactive oxygen species) expression at mRNA level. In addition, significant upregulation was not detected in CoQ10 mRNA expression levels in all L. paracasei EPS-treated cell lines compared to untreated control.

Total and lipid ROS levels
To evaluate total and lipid ROS, all cell lines were stained with DCF-DA and C11-BODIPY probe, respectively and assessed by flowcytometry [24]. Treatment of cancerous and normal cells with L.
paracasei EPS could not generate ROS and should not be considered as a ferroptosis inducer while FIN56 (5 µM) as positive control, results in increase in lipid and total ROS levels in all treated cell lines.

Glutathione and Iron assays
To explore the glutathione and iron levels in all cell lines treated with L. paracasei EPS and FIN56, which are two important events in triggering ferroptosis, we applied glutathione and iron assay kits, respectively. Figure 6A showed the glutathione concentration (µM) (as compared to calibrator in glutathione assay kit) in cancerous and normal cell lines. As shown in Fig. 6A there is no significant depletion of glutathione level in all L. paracasei EPS treated-cell lines subjected to the assay. Ferrous iron (Fe 2+ ) is one of the essential factors for the execution of ferroptosis. The iron chelators can prevent cells from undergoing ferroptosis. Therefore, we investigated the effect of L. paracasei EPS on the changes in iron levels in human colon cancer and normal cell lines. As shown in Fig. 6B, there is no significant alternation in iron levels in L. paracasei EPS-treated colon cancer cells as well as normal cell line compared to untreated control cells.

Discussion
Colon cancer is one of the common malignant digestive tract tumors and the leading cause of cancer-related death worldwide. Its incidence is high in western countries as well as Iran ranked third among men and fourth in women [14,30]. Surgery, chemotherapy, and radiation therapy are among the main therapeutic modalities for the treatment of colon cancer, however, such treatments are frequently prohibited due to their numerous adverse side effects [3,6]. In most cases the majority of cancer cells might develop resistance to conventional therapeutic agents [4], therefore, targeting vital signaling pathways in colon cancer including those involved in growth signaling cascades, invasion, angiogenesis, and apoptosis is a prompt need which may exert selective cytotoxicity towards cancer cells [1]. Further, the development of new anticancer treatment modalities based on probiotics with less adverse effects is urgently needed [1]. Probiotics are known as safe microorganisms, which are thought to have low cytotoxicity, hence, applying probiotic-derived molecules as anticancer agents seem to have fewer adverse effects than the current approaches for colon cancer treatment [1,30]. The beneficial and antitumor effects of probiotics and their derivatives have been reported previously [2,6,8,9,11,13,[15][16][17]. However, it is noteworthy that these characteristics are completely strain-dependent [14]. and reduction of p21 expression [32]. Iyer et al. showed that L. reuteri downregulated nuclear factor-kB (NF-kB)-dependent gene products and promoted apoptosis by enhancing MAPK activities in myeloid leukemia-derived cells [33]. However, consuming live probiotics may cause concerns from the FAO and WHO, especially in immune-compromised patients [13,14,34,35]. Thus, recently exploiting probiotic bacteria's components has been interestingly increased [4,6,9,14,16]. EPSsone of these components produced by lactic acid bacteria -provides numerous health benefits like antioxidant, cholesterol-lowering properties, anti-ulcer, anti-tumor, and immunomodulating activities.
Researches revealed that these glycosidic components generally are less cytotoxic toward normal cells which makes them a promising candidate in cancer therapy [13,36,37]. In this regard, we carried out experimental assays for evaluating the possible modulatory role of L. paracasei EPS on the molecular targets involved in cancer cell growth, apoptosis, survival, and inflammation signaling pathways. Previous studies have shown that AKT, mTOR, JAK are among important molecules, which can modulate colon cancer cell proliferation, metabolism, metastasis, and survival [5,17,38]. Our data showed that L. paracasei EPS is able to significantly reduce the expression levels of AKT-1, JAK-1 and mTOR mRNAs which have been reported that over-activated in colon cancer. Further, induction of apoptosis in human colon cancer cell lines was correlated with our findings of real-time PCR that showed a significant reduction in Bcl-2 and increase in BAX mRNA expression levels in all cancerous cell lines same or even higher than 5-FU as positive control which indicates the effect of L. paracasei EPS on mitochondrial apoptosis pathway. Further, increased expression levels of both caspase-3 and caspase-8 mRNAs were detected in L. paracasei EPS treatment (Fig. 4), which is confirmed by DAPI staining (Fig. 2), Annexin V/PI (Fig. 3) assays. Regarding our data, the L. paracasei EPS could exert its apoptotic activity via Akt/ Jak1 and mTOR signaling pathway in the colon cancer cell lines. also it increases the expression levels of pro-apoptotic genes [27]. Ever-growing evidence demonstrated the vital role of induction of apoptosis in cancer research. Although resistance to apoptosis has been found in many types of cancers which in turn hinders anticancer therapeutics efficacy. Consequently, exploiting alternative regulated cell death mechanisms is of great importance in various diseases treatment especially cancer [41]. Ferroptosis described as a form of regulated cell death which is morphologically, biochemically and genetically distinct from other forms of regulated cell death and occurs as a result of high lipid peroxidation, production of iron-dependent reactive oxygen species (ROS) [29,42,43]. Ferroptosis cells exhibit morphological changes in mitochondrial structure without any changes in nucleus structural integrity [24]. However, typical features of apoptosis (mitochondrial cytochrome c release, caspase activation, and chromatin fragmentation) are not observed in ferroptosis cells [42]. Main regulators of ferroptosis are iron metabolism, lipid peroxidation signaling and activation of the mitogen-activated protein kinase (MAPK) pathway [24]. paracasei EPS has a promising future ahead in the field of targeted therapy in colon cancer.

Conclusion
Debilitating effects of cancer, especially colon cancer with the highest incidence rate and mortality which remains an uphill battle, prompted us to investigate safe treatment modalities based on probiotic-derived components. Based on our findings, the glycosidic components of the L. paracasei showed promising effects on key molecular targets which play an important role in colon cancer initiation, progression, metastasis, and chemo-resistance. In this research, we have exploited the EPS of an indigenous probiotic strain with anticancer potential with low or insignificant cytotoxicity to   Effects of L. paracasei EPS (15μg/mL), 5-FU (50mg/mL) and FIN56 (5μM) (positive control group) on human colon cancer and normal cell lines signaling pathways. Target genes were normalized with GAPDH as a reference gene. All experiments were performed in triplicate.
Data expressed as fold change.

Figure 5
Effects of L. paracasei EPS (15μg/mL) and FIN56 (5μM) (positive control group) on total and lipid ROS production on human colon cancer and normal cell lines assessed by flow cytometry.