S-Adenosyl-l-Methionine Overcomes uL3-Mediated Drug Resistance in p53 Deleted Colon Cancer Cells

Purpose: In order to study novel therapeutic approaches taking advantage of natural compounds showing anticancer and anti-proliferative effects, we focused our interest on S-adenosyl-l-methionine, a naturally occurring sulfur-containing nucleoside synthesized from adenosine triphosphate and methionine by methionine adenosyltransferase, and its potential in overcoming drug resistance in colon cancer cells devoid of p53. Results: In the present study, we demonstrated that S-adenosyl-l-methionine overcomes uL3-mediated drug resistance in p53 deleted colon cancer cells. In particular, we demonstrated that S-adenosyl-l-methionine causes cell cycle arrest at the S phase; inhibits autophagy; augments reactive oxygen species; and induces apoptosis in these cancer cells. Conclusions: Results reported in this paper led us to propose S-adenosyl-l-methionine as a potential promising agent for cancer therapy by examining p53 and uL3 profiles in tumors to yield a better clinical outcomes.


Introduction
Colon cancer is one of the most common causes of cancer-related death worldwide and its incidence is steadily rising [1]. Surgery, chemotherapy, and radiotherapy, either individually or in combination, are recognized as key treatments for all stages of colon cancer and are crucial in determining patient survival and quality of life. Although, chemotherapy is very effective after surgery, the clinical outcome of this therapeutic approach can be unsatisfactory, especially in patients with advanced disease or in the presence of metastasis [2]. In cancer therapy, the development of intrinsic or acquired drug resistance and/or multidrug resistance (MDR) is the leading cause of colon tumor recurrence [3]. Thus, recent studies have yielded important information on how to overcome the resistance improving cancer chemotherapy and this field remains one of the major challenges.
Over the past decades, several studies have reported a role for ribosomal proteins (RPs) in multidrug resistance. To date, the upregulation of uL4 and uL18 was associated with doxorubicin resistance in human colon cancer cells [4]; higher intracellular levels of eS28 was associated with acquired cisplatin resistance in human head and neck carcinoma cell lines [5]. Other studies have reported the high expression of eL8 and eL6 in adriamycinresistant gastric cancer cells [6]. Shi et al. have demonstrated the involvement of uS15 and uL14 in the development of resistance to vincristine, adriamycin, and 5-fluorouracil (5-FU) in a multidrug-resistant gastric cancer cell line SGC790 [7].

AdoMet Inhibits Proliferation in HCT 116 p53−/− and uL3∆HCT 116 p53−/− Colon Cancer Cells
With the aim to investigate the role of AdoMet in uL3-mediated drug resistance, we first evaluated its effects on cell proliferation in our model of colon cancer cells deleted of p53 [17]. To this aim, sensitive HCT116 p53−/− cells and drug-resistant uL3∆HCT116 p53−/− cells were incubated with different concentrations (from 72 to 1000 µM) of AdoMet. The 24, 48, and 72 h later cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. As shown in Figure 1A, HCT 116 p53−/− cells showed sensitivity to AdoMet and cell viability was significantly decreased in a doseand time-dependent manner. These data demonstrated that AdoMet exerted cytotoxic effects in HCT 116 p53−/− colon cancer cells. Of interest, AdoMet also showed cytotoxic activity in resistant uL3∆HCT 116 p53−/− cells ( Figure 1B), although with lower efficacy as demonstrated by an IC 50 value of 750 µM compared to an IC 50 value of 500 µM in HCT 116 p53−/− cells at 72 h. These data suggest that AdoMet could restore the sensitivity of resistant uL3∆HCT 116 p53−/− cells. We have previously demonstrated that in these cells, the silencing of uL3 completely abolished the cytotoxicity of 5-FU treatment [17]. To demonstrate the crucial role of AdoMet in overcoming uL3-mediated drug resistance, we investigated the role of AdoMet alone or in combination with 5-FU on cell viability. To this purpose, uL3∆HCT 116 p53−/− cells were treated with AdoMet (from 125 to 1000 µM), 5-FU (from 12.5 to 100 µM), or with a combination of 5-FU and AdoMet. For the 72 h later, cell viability was estimated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. As expected, the percentage of cell viability decreased after AdoMet treatment while the treatment with 5-FU had no effect on cell viability. Of note, in cells treated with 5-FU plus AdoMet, we found that the cell viability was strongly reduced compared to that of cells exposed to AdoMet alone ( Figure 1C). Altogether, these data show that AdoMet re-sensitizes drug resistant colon cancer cells to  with lower efficacy as demonstrated by an IC50 value of 750 µM compared to an IC50 value of 500 µM in HCT 116 p53−/− cells at 72 h. These data suggest that AdoMet could restore the sensitivity of resistant uL3ΔHCT 116 p53−/− cells. We have previously demonstrated that in these cells, the silencing of uL3 completely abolished the cytotoxicity of 5-FU treatment [17]. To demonstrate the crucial role of AdoMet in overcoming uL3-mediated drug resistance, we investigated the role of AdoMet alone or in combination with 5-FU on cell viability. To this purpose, uL3ΔHCT 116 p53−/− cells were treated with AdoMet (from 125 to 1000 µM), 5-FU (from 12.5 to 100 µM), or with a combination of 5-FU and AdoMet. For the 72 h later, cell viability was estimated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. As expected, the percentage of cell viability decreased after AdoMet treatment while the treatment with 5-FU had no effect on cell viability. Of note, in cells treated with 5-FU plus AdoMet, we found that the cell viability was strongly reduced compared to that of cells exposed to AdoMet alone ( Figure 1C). Altogether, these data show that AdoMet re-sensitizes drug resistant colon cancer cells to 5-FU treatment. were treated with increasing amounts of AdoMet (72-1000 µM) from 24 to 72 h, then cell viability was measured by the MTT assay. Results are presented as a percentage of the control cells. * p < 0.05 versus untreated cells (C) uL3ΔHCT 116 p53−/− were treated with AdoMet (from 125 to 1000 µM), and 5-FU (from 12.5 to 100 µM), alone or in combination, for 72 h. Cell viability was assayed using the MTT assay. Data represent the average of three independent experiments; error bars represent the standard deviation. * p < 0.05 versus untreated cells. were treated with increasing amounts of AdoMet (72-1000 µM) from 24 to 72 h, then cell viability was measured by the MTT assay. Results are presented as a percentage of the control cells. * p < 0.05 versus untreated cells (C) uL3∆HCT 116 p53−/− were treated with AdoMet (from 125 to 1000 µM), and 5-FU (from 12.5 to 100 µM), alone or in combination, for 72 h. Cell viability was assayed using the MTT assay. Data represent the average of three independent experiments; error bars represent the standard deviation. * p < 0.05 versus untreated cells.

AdoMet Causes Cell Cycle Arrest in S Phase in uL3∆HCT 116 p53−/−
In order to investigate the mechanism by which AdoMet exerts its cytotoxic activity in p53 deleted colon cancer cells, alterations in the cell cycle distribution were analyzed. To this aim, HCT 116 p53−/− and uL3∆HCT 116 p53−/− cells were treated with 500 µM of AdoMet. Seventy-two hours later, cell cycle distribution was monitored by flow cytometry. Analysis of cell cycle profile of treated HCT 116 p53−/− cells showed that AdoMet had no effect (Figure 2A,C). To confirm this result, the expression levels of key regulators of the cell cycle phases were assessed. Consistently, the expression profile of cyclin B, D, and E resulted in being unaltered upon AdoMet treatment, while the expression levels of cyclin A were increased ( Figure 3A). Interestingly, the observed upregulation of cyclin A could be involved in mechanisms of cell death as already reported [34]. In the absence of uL3, the exposure of cells to AdoMet caused an increased percentage of cell population in the S phase (from about 13% to about 40%) with a relative decreased cell population in the G2/M phase (from about 20% to about 11%) and G1 phase (from about 65% to about 41%) ( Figure 2B,C). Analysis of cyclin level in these cells showed that AdoMet treatment caused an upregulation of the G1 phase-related protein as cyclin E and G2/M phase-related cyclin B. Furthermore, as observed in HCT 116 p53−/− , the levels of cyclin A increased following AdoMet treatment ( Figure 3B). More importantly, the treatment of uL3∆HCT 116 p53−/− cells with AdoMet was associated with a marked downregulation of cyclin D that, as we have previously demonstrated [10], is upregulated in these cells as aa consequence of uL3 silencing.
All these data indicate that the inhibitory effects of AdoMet on uL3∆HCT 116 p53−/− cell proliferation were associated with the alteration in cell cycle progression, downregulation of cyclin D1, and upregulation of Cyclin A.

AdoMet Induces Apoptosis in HCT 116 p53−/− and uL3∆HCT 116 p53−/− Colon Cancer Cells
To further investigate the underlying mechanism that contributes to AdoMet-mediated growth inhibition in our model of colon cancer cells, we determined whether AdoMet decreased cell survival through the induction of apoptosis. To this aim, Annexin V analysis was performed. HCT 116 p53−/− cells and uL3∆HCT 116 p53−/− cells were treated with 500 µM AdoMet and 72 h after were assessed with Annexin V-FITC. As shown in Figure  4A, the percentage of Annexin-V positive cells increased after AdoMet treatment compared to untreated cells in both cell lines. Specifically, we found that in HCT 116 p53−/− cells, AdoMet significantly increased the percentage of late apoptotic cells (Annexin V+ and PI+) from 6% in the untreated cells to 13% in the treated cells ( Figure 4A). Upon exposure to AdoMet in the uL3∆HCT 116 p53−/− cell line, apoptotic cells proportion raised from    All these data indicate that the inhibitory effects of AdoMet on uL3ΔHCT 116 p cell proliferation were associated with the alteration in cell cycle progression, downr ulation of cyclin D1, and upregulation of Cyclin A.

AdoMet Induces Apoptosis in HCT 116 p53−/− and uL3ΔHCT 116 p53−/− Colon Cancer Cells
To further investigate the underlying mechanism that contributes to Ado et-mediated growth inhibition in our model of colon cancer cells, we determined whet AdoMet decreased cell survival through the induction of apoptosis. To this aim, Anne V analysis was performed. HCT 116 p53−/− cells and uL3ΔHCT 116 p53−/− cells were trea with 500 µM AdoMet and 72 h after were assessed with Annexin V-FITC. As shown Figure 4A, the percentage of Annexin-V positive cells increased after AdoMet treatm  Figure S1. play pivotal roles in the initiation and execution of apoptosis, the expression of thes apoptosis-related proteins was assessed by western blot analysis. As expected, Figure 4 showed the downregulation of PARP-1, pro-caspase 3, 9, 8, and an increase in the rati between pro-apoptotic Bad and anti-apoptotic Bcl-2 in both cell lines [35].

AdoMet Increases ROS Production in HCT 116 p53−/− and uL3∆HCT 116 p53−/− Colon Cancer Cells
Since many anticancer drugs and natural compounds have been highlighted to exert a pro-apoptotic effect in neoplastic cells due to their pro-oxidant activity, we were interested in evaluating the levels of reactive oxygen species (ROS) in colon cancer cells in the presence and absence of uL3. Therefore, HCT 116 p53−/− cells and uL3∆HCT 116 p53−/− cells were treated with 500 µM AdoMet for 72 h and the intracellular ROS levels were quantitatively evaluated by the 2 ,7 -dichlorofluorescein diacetate (DCF-DA) flow cytometric assay.
Results in Figure 5A showed an increase in ROS production after AdoMet treatment in both cell lines, as evidenced by the reported median intensity fluorescence (MFI) value. Specifically, an increase of 2-and 3-fold in ROS levels was observed in uL3∆HCT 116 p53−/− and HCT 116 p53−/− cells, respectively, compared to the control cells. Overall, these results indicate that the activation of ROS signaling could represent one of the mechanisms underlying AdoMet-induced apoptosis in these cell lines.

AdoMet Inhibits Autophagy in HCT 116 p53−/− and uL3∆HCT 116 p53−/− Colon Cancer Cells
Autophagy plays a role as a pro-survival mechanism for drug resistance [36]. We have recently demonstrated that the chemoresistance observed in uL3∆HCT 116 p53−/− cells might depend on the higher autophagic flux displayed by this cell population [11]. To investigate if the responsivity of uL3∆HCT 116 p53−/− cells to AdoMet exposure may be correlated with autophagy modulation, we were interested in evaluating the autophagy occurrence in treated cells. To this aim, HCT 116 p53−/− and uL3∆HCT 116 p53−/− cells were treated with 500 µM AdoMet and 48 h later were stained with vital dye LysoTracker (LTR), a fluorescent dye for labeling and tracking cellular acidic compartments, lysosomes, and autophagolysosomes. As shown in Figure 5B, fluorescence microscopy analysis revealed a decreased formation of red dotted acidic vacuoles in comparison with untreated cells (CTR), providing evidence of autophagic flux inhibition caused by AdoMet treatment in both cell lines. To further characterize the effect of AdoMet, flow cytometry analysis was performed in the same cells. Results in Figure 5C showed a decrease in acidic vesicles after treatment with AdoMet as assessed by measuring median fluorescence in both cell lines.
One important step in autophagosome formation involves the cleavage of LC3B-I to LC3B-II by the coordinated activity of several proteins including Atg7 and subsequent association of LC3B-II with autophagosomal membranes. Thus, p62 binds directly to ubiquitinated proteins and gets degraded during active autophagy [37].
To further demonstrate the involvement of autophagy in the AdoMet response of colon cancer cells, and in particular, the ability of AdoMet to overcome the drug resistance of uL3∆HCT 116 p53−/− cells, we evaluated the protein expression levels of Atg7, p62, and LC3B by western blot analysis in both cell lines. Figure 6A,B showed that AdoMet treatment caused a reduction of Atg7 and an increase in p62 levels in both cell lines, which was associated with a decrease in LC3BII/I ratio ( Figure 6C,D).

Discussion
The failure of conventional anticancer therapy is mainly due to the development of drug resistance that represents a serious problem in the treatment of cancer. Although many mechanisms of tumor chemoresistance have been discovered and elucidated, it remains the leading cause of chemotherapy failure, especially in relapsed or metastatic tumors. Re-sensitizing cancer cells to the drugs by using novel effective strategies is a promising approach to overcome the chemoresistance and improve clinical treatment [38,39]. Recently, we evaluated uL3 clinical significance in colon cancer and demonstrated that uL3 expression in colon tumor tissues is downregulated; in particular, the decrease in uL3 mRNA levels associated with malignance progression and tumor grade and were inversely proportional to the ratio Bcl-2/Bax [15]. Loss of uL3 in p53-deleted colon cancer cells was associated with characteristic invasive phenotype with EMT and correlated with the development of resistance to different chemotherapeutic agents commonly used in clinic for the treatment of colon cancer as 5-FU [9, 10,15].
Depletion of uL3 confers drug resistance through the alteration of various signaling pathways regulating cell cycle, cell migration, apoptosis, and autophagy [8,40]. Different approaches have been proposed to come up with effective drugs to overcome drug resistance. Combination of different drugs attacking multiple pathways simultaneously presents some limitations including deleterious effects from drug-drug interactions, overlapped toxicity, and costs of combination therapy [38]. Moreover, accumulating evidence suggests that simultaneous inhibition of multiple signaling pathways by natural compounds could represent a better therapeutic approach than that of individual inhibitors.
Over the past two decades, the in vitro and in vivo impacts of AdoMet against the development and the progression of many types of human cancer has been demonstrated through its involvement in different biological pathways [26][27][28][29][30][31][32][33]. In this regard, AdoMet has become particularly attractive, taking advantage of its pleiotropic effects on different survival pathways known to be important in colon carcinogenesis and could represent a promising strategy to overcome uL3-mediated drug resistance.
AdoMet has been shown to induce the apoptotic process and reduce the mechanism of inflammation and invasion in colon cancer cells [41][42][43][44][45]. In RKO and HT-29 colon cancer cells, AdoMet treatment reduced the expression of several anti-apoptotic genes such as the gene encoding FLICE-like inhibitory protein and caused the activation of caspase 8 with the consequent release of cytochrome from the mitochondria [41].
Luo et al. reported that in colon cancer cells, AdoMet inhibited the tumor cell growth by reversing the DNA hypomethylation on promoters of c-Myc and H-ras oncogenes, thus downregulating their expression [42]. Furthermore, AdoMet was able to prevent the inflammation-induced colon cancer by azoxymethane and dextran sulfate sodium in Balb/c mice through inhibition of β-catenin, IL-6 signaling, pro-inflammatory, pro-growth, and proliferation pathways [43].
In the highly invasive SW-620 colorectal cancer cell line, the sulfonium compound reduces the metastatic process by the hypermethylation of specific genes like the matrix metalloproteinase-2 and the membrane type 1 matrix metalloproteinase [44]. Recently, in colon SW480 and HCT116 cancer cells with constitutively active β-catenin signaling, AdoMet treatment inhibited β-catenin activity, preventing its access to the nuclear compartment. Moreover, in colon RKO cancer cells expressing wild-type Wnt/β-catenin, AdoMet enhanced GSK3β-mediated degradation of β-catenin by elevating the activity of protein phosphatase 2A [45]. In this regard, thanks to its high safety profile, AdoMet represents a hopeful strategy to overcome uL3-mediated drug resistance, without the common side effects of chemotherapy drugs [33].
In this study, we focused our study on examining the therapeutical potential of AdoMet to overcome drug resistance in the uL3∆HCT 116 p53−/− cell line, a model of colon cancer cells resistant to conventional anticancer drugs including 5-FU, based on the absence of p53 and low levels of uL3 [10]. Results of cytotoxicity experiments demonstrated that AdoMet caused cell death in uL3∆HCT 116 p53−/− cells in a dose-and time-dependent manner. Of note, the combined treatment of resistant cells with AdoMet plus 5-FU was able to rescue the cytotoxic effects of 5-FU. These data strongly demonstrate the key role of AdoMet in overcoming uL3 mediated drug resistance. To our knowledge, the present study demonstrates, for the first time, evidence that AdoMet treatment re-sensitizes drug resistant colon cancer cells. These data raise the possibility that AdoMet might be a potential therapeutic agent for resistant colon cancer cells showing a low expression profile of p53 and uL3. In light of these observations, we were interested to exploring the mechanism underlying AdoMet-induced cell death.
Cell cycle progression and its regulation are the key events of cell proliferation. In HCT 116 p53−/− cells, AdoMet treatment did not affect cell cycle profile, as confirmed by the analysis of the expression profile of cell cycle related proteins, indicating that the observed cytotoxic effects were not due to the deregulation of cell cycle checkpoints. Although no apparent modifications of the expression profile of most cyclins was evidenced, an increase of cyclin A level was observed upon AdoMet treatment. Recent findings highlighted cyclin A involvement in the triggering of apoptosis, suggesting that the observed activation of the apoptotic process by AdoMet in HCT 116 p53−/− cells could be due to the alteration of cyclin A expression [34].
Of interest, in uL3∆HCT 116 p53−/− cells, AdoMet treatment affected cell cycle, causing a marked arrest of cell cycle at the S phase, where this effect was accompanied by a strong increase of cyclin E. Furthermore, a downregulation of cyclin D expression was also observed in these cells. It is known that the overexpression of cyclin D results in drug resistance in different cancers [46]. We have previously demonstrated a role of cyclin D in uL3-mediated drug resistance showing that its increased expression may contribute to cellular chemoresistance in uL3∆HCT 116 p53−/− cells [10]. We believe that the downregulation of cyclin D upon AdoMet treatment observed only in uL3∆HCT 116 p53−/− cells is responsible for the resensitizing of the cells to anticancer treatments. This is in accordance with the view that targeting and inhibiting cyclin D is an effective strategy to overcome drug resistance.
In addition to its role in cell cycle regulation, cyclin D is also involved in the regulation of apoptosis. Impairing apoptosis is a critical point in the development of drug resistance [47]. Our data demonstrated that the exposure of uL3∆HCT 116 p53−/− cells to AdoMet induced apoptosis and this was in accordance with the ability of AdoMet to downregulate cyclin D1 level.
Caspases are aspartate-specific cysteine proteases that play crucial roles in apoptosis [48]. Activation of caspases results in the cleavage and the inactivation of key cellular proteins including the DNA repair enzyme poly(ADP ribose) polymerase 1 (PARP-1). Our results showed that AdoMet caused a downregulation of PARP-1 associated to the cleavage of pro-caspase 3, which was specifically mediated by caspase 9. In addition, apoptosis was also evaluated by measuring the ratio between specific anti-apoptotic and pro-apoptotic proteins. We found that AdoMet treatment caused a strong reduction in anti-apoptotic Bcl-2 levels, favoring the expression of pro-apoptotic Bad. In addition to its canonical anti-apoptotic activity, Bcl-2 has also been implicated in the regulation of ROS levels in the mitochondrion [49]. Several studies suggest a reciprocal relationship between ROS and Bcl-2 levels within cells; increases in ROS correlate with decreases in Bcl-2 levels and vice versa [49]. In this study, we reported that AdoMet treatment is associated with an increase in ROS production and a downregulation of Bcl-2 expression in both cell lines.
It has been largely demonstrated that defects of autophagy are associated with an increase of ROS, which, in turn, can activate the intrinsic apoptotic pathway [50].
Autophagy has been defined as a 'double-edged sword' in cancer progression; it may act as a tumor suppressor by clearing damaged organelle, preventing oxidative stress and genomic instability, particularly during malignant transformation and carcinogenesis. However, in established tumors, it may act as a pro-survival pathway during stress conditions like nutrient deprivation, hypoxia, and cancer therapeutic treatment. Moreover, the inhibition of autophagy has been shown to prevent resistance of cancer cells to chemother-apy [36]. Recently, we have shown that resistance to drugs caused by uL3 depletion in p53-deleted colon cancer cells was due to autophagy induction [11].
An involvement of AdoMet in autophagy regulation has already been demonstrated in breast cancer cells. In MCF-7 cells, AdoMet showed a synergistic effect with chloroquine (CQ) in the inhibition of autophagy through the targeting of the AKT/mTOR pathway [29]. Our results demonstrated that AdoMet treatment inhibited the autophagy flux in uL3∆HCT 116 p53−/− cells, characterized by a high basal protective autophagy. Consistently, a reduction in LC3BII/I ratio and Atg7 levels, and an increase in p62 amount were found. Since it has been demonstrated that p62 overexpression leads to caspase-8-dependent apoptosis [51], our results suggest that inhibition of autophagy caused by AdoMet leads to an increase of apoptosis, promoting tumor cell death.

Cell Viability Assays
HCT 116 p53−/− cells and uL3∆HCT 116 p53−/− cells were plated in serum-containing media in 96-well plates at the proper density. After 24 h incubation, the cells were treated with increasing concentrations of AdoMet (from 72 to 1000 µM) for 24, 48, and 72 h. Cell viability was assessed by adding MTT as previously reported [52]. The absorbance values of the solution in each well were detected at 570 nm using a Bio-Rad IMark microplate reader (Bio-Rad Laboratories, Milan, Italy). All MTT experiments were performed in quadruplicate. Cell viability was expressed as the percentage of absorbance values in treated samples with respect to that of the control (100%). For the treatments, AdoMet plus 5-FU, uL3∆HCT 116 p53−/− cells were seeded at proper density in 96-multiwell plates. After 24 h incubation, the cells were treated with different concentrations of AdoMet (from 125 to 1000 µM) and 5-FU (12.5 to 100 µM), alone or in combination, for 72 h and cell viability was detected by the MTT assay.

Cell Cycle Analysis
HCT 116 p53−/− cells and uL3∆HCT 116 p53−/− cells were seeded into 35 mm tissue culture plates at a confluency about 50-60%. Then, cells were starved overnight and treated with 500 µM AdoMet for 72 h. After treatment, the cells were harvested and centrifuged at 400× g for 5 min, washed once with cold PBS and stained in a PI solution as previously reported [53]. Flow cytometric analysis was performed using a BD Accuri™ C6 flow cytometer (BD Biosciences). To evaluate cell cycle progression, PI fluorescence was collected as FL3-A (linear scale) using ModFIT software (Verity Software House). For each sample, at least 20,000 events were analyzed in at least three different experiments, giving a standard deviation (SD) < 5%.

Cell Death Assay
HCT 116 p53−/− and uL3∆HCT 116 p53−/− cells were seeded in 6-well plate, starved overnight, and treated with 500 µM AdoMet for 72 h. The cells were harvested by trypsinization and washed twice with PBS. Annexin VFITC was used in conjunction with the vital dye PI to distinguish apoptotic (Annexin VFITC positive, PI positive) from necrotic (Annexin VFITC negative, PI positive) cells as previously reported [54]. Briefly, cells were re-suspended in 200 µL of Binding Buffer 1X and incubated with 5 µL Annexin V-FITC and 10 µL PI (20 µg/mL) for 30 min at room temperature, as recommended by the manufacturers. The detection of viable cells, early apoptotic, late apoptotic, and necrotic cells was performed by BD Accuri™ C6 flow cytometer (BD Biosciences). For each sample, 20,000 events were recorded. Analysis was carried out by triplicate determination on at least three separate experiments.

Determination of ROS by the 2 ,7 -dichlorofluorescein Diacetate (DCFH-DA) Assay
Colon cancer cells were seeded in 6-well plates at the proper density, and after 24 h of incubation, the cells were treated with AdoMet 500 µM for 72 h. The positive control was represented by the cells treated with menadione at the final concentration of 100 µM for 1 h at 37 • C. After treatment, cells were stained with 10 µM DCF-DA for 30 min at 37 • C in the dark [55]. DCFH-DA can freely pass the cell membrane where it is trapped after being hydrolyzed by intracellular esterases in a non-fluorescent compound (DCFH). Then, DCFH can be oxidized to the fluorescent DCF by reacting with intracellular ROS. Following incubation, cells were washed twice with PBS, trypsinized, resuspended in PBS, and immediately analyzed with a BD Accuri™ C6 flow cytometer (BD Biosciences). For each sample, 20,000 events were recorded. Analysis was carried out by triplicate determination on at least three separate experiments.

LysoTracker-Red Staining
HCT 116 p53−/− and uL3∆HCT 116 p53−/− cells were seeded in a 6-well plate and treated with 500 µM AdoMet for 48 h, using CQ as the positive control. LTR was added to each well for 20 min at 37 • C at a final concentration of 0.1 µM in medium. The cells were then washed with PBS and observed by fluorescence microscopy. The fluorescence intensity of the cells was then analyzed by flow cytometry. Briefly, the cells were detached by incubation with EDTA-trypsin, washed twice with PBS, and collected by centrifugation. For the quantitative evaluation of LTR, FlowJo software was used to calculate the median fluorescence intensities (MFI) by the formula (MFI-treated/MFI-control), where MFI-treated is the fluorescence intensity of cells treated with the various compounds and MFI-control is the fluorescence intensity of the untreated cells. For each sample, 20,000 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.

Western Blot Analysis
After the treatment time, cells were harvested and protein extracts were prepared as previously described [56]. A western blot analysis was performed as previously re-ported [57]. All primary antibodies were used at a dilution of 1:1000; all secondary antibodies were used at a dilution of 1:5000. Blots were then developed using enhanced chemiluminescence detection reagents ECL (Cyanagen, Bologna, Italy) and exposed to X-ray film. All films were scanned by using Image J software (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis
Experiments were performed at least three times with replicate samples. Data are expressed as mean ± standard deviation (SD). p-values were determined using unpaired two-tailed Student's t-test. * p < 0.05 was considered significant, ** p < 0.01 was considered highly significant.

Conclusions
All these data led us to propose the model shown in Figure 7 in which AdoMet, by targeting multiple pathways, overcomes the drug resistance of p53-deleted colon cancer cells expressing low levels of uL3. In particular, AdoMet blocks cell cycle at the S phase, inhibits autophagy, augments ROS species, and finally triggers the activation of the apoptotic pathway.  Overall, our findings highlighted AdoMet, a naturally-occurring multifunctional sulfonium compound, as a potential candidate for novel therapeutic approaches to overcome the drug resistance of colon cancer depending on p53 and uL3 status.

Conflicts of Interest:
The authors declare no conflict of interest.