Methyl Donors Reduce Cell Proliferation by Diminishing Erk-Signaling and NFkB Levels, While Increasing E-Cadherin Expression in Panc-1 Cell Line

Pancreatic cancer is an aggressive malignancy with high metastatic potential. There are several lifestyle-related determinants in its etiology, including diet. Methyl donors are dietary micronutrients which play an important role in fueling vital metabolic pathways, and as bioactive food components provide methyl groups as substrates and cofactors. The imbalanced nutritional status of methyl donors has recently been linked to pathological conditions. Therefore, we hypothesized that dietary methyl donors may improve the physiology of cancer patients, including those with pancreatic cancer, and could be used for intervention therapy. In this study, methyl-donor treatment (L-methionine, choline chloride, folic acid and vitamin B12) of an aggressive pancreatic adenocarcinoma cell line (Panc-1) resulted in significantly increased p21WAF1/Cip1 cyclin-dependent kinase inhibitor levels, along with apoptotic SubG1 fractions. At the same time, phospho-Erk1/2 levels and proliferation rate were significantly reduced. Though methyl-donor treatments also increased the pro-apoptotic protein Bak, Puma and Caspase-9, it failed to elevate cleaved Caspase-3 levels. In addition, the treatment significantly reduced the production of the pro-inflammatory cytokine IL-17a and the transcription factor NFkB. Similarly, a significant decrease in VEGF and SDF-1a levels were detected, which may indicate reduced metastatic potential. As expected, E-cadherin expression was inversely associated with these changes, showing elevated expression after methyl-donor treatment. In summary, we found that methyl donors may have the potential to reduce aggressive and proliferative phenotype of Panc-1 cells. This suggests a promising role of dietary methyl donors for complementing relevant cancer therapies, even in treatment-resistant pancreatic adenocarcinomas.


Introduction
The incidence and mortality rate of pancreatic cancer is growing worldwide, and it is expected that it will be ranked the third leading cause of cancer death by 2025 [1]. Pancreatic cancer is an aggressive disease with poor prognosis because of the early presence of metastases, treatment resistance, multiple genetic and epigenetic changes, as well as the fact that it is usually diagnosed at advanced stages [2,3]. All of these contribute to the only 10% 5-year survival rate. In addition to non-modifiable risk factors, lifestyle habits such as smoking, alcohol usage, diet, obesity, and physical activity have been identified as determinant in the etiology of pancreatic cancer [4].
One-carbon metabolism-consisting of folate and methionine cycles-for its optimal function requires substrates and cofactors called methyl donors (such as folate, betaine, is associated with NFkB-related inflammation and high metastatic potential [26], as well as chemoresistance [34], we also aimed to examine whether methyl donors could affect: 1.
Some aspects of inflammatory processes influenced by pancreatic cancer cells; 2.
The expression levels of related metastatic markers in Panc-1 cancer cell line.

Methyl Donors Affect Tumor Cell Proliferation
MTS proliferation assay was used to detect the effect of methyl-donor treatments on the growth of Panc-1 cancer cell line. Both concentrations of methyl donors used significantly reduced the proliferation rate at 48 h (p < 0.01 at both concentrations) and 72 h (p < 0.001 and p < 0.01 at 1× and 20× concentrations, respectively) compared to the non-treated control group ( Figure 1A). development is associated with NFkB-related inflammation and high metastatic potential [26], as well as chemoresistance [34], we also aimed to examine whether methyl donors could affect: 1. Some aspects of inflammatory processes influenced by pancreatic cancer cells; 2. The expression levels of related metastatic markers in Panc-1 cancer cell line.

Methyl donors Affect Tumor Cell Proliferation
MTS proliferation assay was used to detect the effect of methyl-donor treatments on the growth of Panc-1 cancer cell line. Both concentrations of methyl donors used significantly reduced the proliferation rate at 48 h (p < 0.01 at both concentrations) and 72 h (p < 0.001 and p < 0.01 at 1× and 20× concentrations, respectively) compared to the non-treated control group ( Figure 1A).

The Effect of Methyl Donors on Cell Cycle
Changes in SubG1 fraction, indicating apoptotic cells as well, were measured by flow cytometry after methyl-donor treatments. A significantly increased SubG1 fraction of Panc-1 cells was detected after 20× methyl-donor treatments (p < 0.05) compared to control ( Figure 1B,C). Therefore, we investigated the level of p21 WAF1/Cip1 growth inhibitory protein, which is a mediator of p53-dependent G1 phase arrest, using Western blot analysis. We found significantly increased levels of p21 WAF1/Cip1 protein (p < 0.001) at the 20× methyl-donor concentration after 48 h treatment compared to control ( Figure 1D,E).
It is known that the activation of the MAPK/ERK signaling pathway is required to induce genes involved in cell cycle entry and to suppress those genes, which inhibit this process, such as CDKN1A, the gene of p21 WAF1/Cip1 protein. We found significantly decreased p-Erk 1/2 protein levels at 48 h in both the 1× and 20× methyl-donor treated groups (p < 0.05 and p < 0.001, respectively) measured by Western blot (Figure 1D,E).

Detection of Apoptosis and Related Pathway Elements after Methyl-Donor Treatments
Annexin V positive early apoptotic cell fraction was measured using flow cytometry after methyl-donor treatment, but only a tendency (p = 0.326) of increase was seen in Panc-1 tumor cells compared to control. However, a significantly decreased number of live cells was detected after 20× methyl-donor treatments compared to the non-treated control (p < 0.01) (Figure 2A,B).
is a mediator of p53-dependent G1 phase arrest, using Western blot analysis. We found significantly increased levels of p21 WAF1/Cip1 protein (p < 0.001) at the 20× methyl-donor concentration after 48 h treatment compared to control ( Figure 1D,E).
It is known that the activation of the MAPK/ERK signaling pathway is required to induce genes involved in cell cycle entry and to suppress those genes, which inhibit this process, such as CDKN1A, the gene of p21 WAF1/Cip1 protein. We found significantly decreased p-Erk 1/2 protein levels at 48 h in both the 1× and 20× methyl-donor treated groups (p < 0.05 and p < 0.001, respectively) measured by Western blot (Figure 1D,E).

Detection of Apoptosis and Related Pathway Elements after Methyl-Donor Treatments
Annexin V positive early apoptotic cell fraction was measured using flow cytometry after methyl-donor treatment, but only a tendency (p = 0.326) of increase was seen in Panc-1 tumor cells compared to control. However, a significantly decreased number of live cells was detected after 20× methyl-donor treatments compared to the non-treated control (p < 0.01) (Figure 2A,B). We investigated methyl-donor-induced changes in the expression of pro-and antiapoptotic proteins using Western blot. The pro-apoptotic Bak, Puma and Caspase-9 protein levels significantly increased at 72 h in the 20× methyl-donor-treated group compared to control (p < 0.01, Figure 3A-D). However, we could not detect cleaved Caspase-3. The anti-apoptotic proteins Bcl-2 and Mcl-1 did not change significantly after methyl-donor treatments in Panc-1 cells. We investigated methyl-donor-induced changes in the expression of pro-and antiapoptotic proteins using Western blot. The pro-apoptotic Bak, Puma and Caspase-9 protein levels significantly increased at 72 h in the 20× methyl-donor-treated group compared to control (p < 0.01, Figure 3A-D). However, we could not detect cleaved Caspase-3. The anti-apoptotic proteins Bcl-2 and Mcl-1 did not change significantly after methyl-donor treatments in Panc-1 cells.

The Effects of Methyl Donors on Metastatic Potential of Panc-1 Cell Line
Pancreatic ductal carcinoma is described as an aggressive tumor with invasive tumor growth and early metastasis; therefore, we checked the protein level of VEGF, SDF-1, MMP-9 by cytokine array ( Figure 4A,B) and SDF-1, MMP-9 and E-cadherin by Western blot ( Figure 4C,D). We found a significant decrease in the protein level of VEGF and SDF-1a (p < 0.05 and p < 0.01, respectively) after 48 h treatment with 20× methyl donors compared to the control by cytokine array ( Figure 4A,B) and SDF-1a level by Western blot ( Figure 4C,D).
Additionally, we found a significant increase in the protein level of E-cadherin after 48 h of treatment with both concentrations of methyl donors compared to the non-treated control group ( Figure 4C,D).

The Effect of Methyl Donors on Immune-Related Pathways and Molecules
We detected significantly decreased IL-17a cytokine levels (p < 0.01) at 48 h of 20× methyl-donor treatment compared to non-treated Panc-1 cells by cytokine array ( Figure 5A,B).
Additionally, we found significantly decreased NFkB protein levels (p < 0.05) at 48 h of 20× methyl-donor treatment in Panc-1 cells compared to the controls by Western blot ( Figure 5C,D). However, we could not reach significance in the decrease in the levels of CD30, either by cytokine array or by Western blot, nor of STAT3 protein measured by Western blot (Figure 5A,D).

The Effects of Methyl donors on Metastatic Potential of Panc-1 Cell Line
Pancreatic ductal carcinoma is described as an aggressive tumor with invasive tumor  . Expectedly, E-cadherin level changed inversely, and its significant increase was detected after 48 h of 20× methyl-donor treatment (C,D). Unexpectedly, we could not measure significantly decreased MMP-9 level after the methyl-donor treatments. Each bar represents the average normalized density from at least 3 repeats ± SD. Statistical significance are plotted as *: p < 0.05; **: p < 0.01. 1× and 20×: concentrations of methyl donors.
Additionally, we found a significant increase in the protein level of E-cadherin after 48 h of treatment with both concentrations of methyl donors compared to the non-treated control group ( Figure 4C,D).

The Effect of Methyl donors on Immune-Related Pathways and Molecules
We detected significantly decreased IL-17a cytokine levels (p < 0.01) at 48 h of 20× methyl-donor treatment compared to non-treated Panc-1 cells by cytokine array ( Figure  5A,B).

The Effect of Methyl donors on Immune-Related Pathways and Molecules
We detected significantly decreased IL-17a cytokine levels (p < 0.01) at 48 h of 20× methyl-donor treatment compared to non-treated Panc-1 cells by cytokine array ( Figure  5A,B).

The Level of IL-8 and IL-6 Cytokines in Plasma Samples from Pancreatic Cancer Patients
We collected 35 serum samples from pancreatic cancer patients and measured IL-8 and IL-6 pro-inflammatory cytokine levels in their plasma by ELISA. We found significantly increased IL-8 and IL-6 cytokine levels in the plasma of pancreatic cancer patients compared to the healthy control group (p < 0.01 and p < 0.0001, respectively) ( Figure 6A,B).
of 20× methyl-donor treatment in Panc-1 cells compared to the controls by Wester ( Figure 5C,D). However, we could not reach significance in the decrease in the le CD30, either by cytokine array or by Western blot, nor of STAT3 protein measu Western blot ( Figure 5A,D).

The Level of IL-8 and IL-6 Cytokines in Plasma Samples from Pancreatic Cancer Patie
We collected 35 serum samples from pancreatic cancer patients and measure and IL-6 pro-inflammatory cytokine levels in their plasma by ELISA. We found s cantly increased IL-8 and IL-6 cytokine levels in the plasma of pancreatic cancer p compared to the healthy control group (p < 0.01 and p < 0.0001, respectively) ( Figure   Figure 6. Measurement of plasma IL-8 and IL-6 cytokine levels of pancreatic cancer patients. icantly increased levels of IL-8 and IL-6 were detected by ELISA from 35 pancreatic cancer p compared to non-cancerous controls (A,B). Each dot represents a concentration of either IL8 cytokine level in a pancreatic cancer patient where boxes show the median value with the int tile range. Statistical significances are plotted as **: p < 0.01; ****: p < 0.0001.

Discussion
Previously, we described that methyl donors are able to induce apoptosis and uate proliferation pathways in breast and lung cancer cell lines [33]. In our present we aimed to explore if similar changes could be detectable in Panc-1 cancer cells. P atic cancer is considered a lethal disease with high mortality as a consequence of it metastatic potential, chemoresistance, and poor prognosis [2,3]. Recent studies s that this phenotype is closely related to both the constitutive and induced activa nuclear factor-kB (NFkB), which gives the mechanistic links of the chronic inflamm to cancer development in pancreatic cancer [34]. Therefore, we also aimed to e whether and how methyl-donor treatment can influence Panc-1-related inflamm processes, as well as its metastatic potential.
There are several approaches to overcome disease burden and progression, inc preventive and complementary therapies, such as diet, besides conventional cancer ments.
Altered methyl-donor metabolism is connected to pathological disturbances s heart disease, birth defects, aging, and pancreatic toxicity, but it is mostly studied i tion to cancer development [9]. In a meta-analysis, Bo et al. found that dietary folate

Discussion
Previously, we described that methyl donors are able to induce apoptosis and attenuate proliferation pathways in breast and lung cancer cell lines [33]. In our present study, we aimed to explore if similar changes could be detectable in Panc-1 cancer cells. Pancreatic cancer is considered a lethal disease with high mortality as a consequence of its high metastatic potential, chemoresistance, and poor prognosis [2,3]. Recent studies suggest that this phenotype is closely related to both the constitutive and induced activation of nuclear factor-kB (NFkB), which gives the mechanistic links of the chronic inflammation to cancer development in pancreatic cancer [34]. Therefore, we also aimed to explore whether and how methyl-donor treatment can influence Panc-1-related inflammation processes, as well as its metastatic potential.
There are several approaches to overcome disease burden and progression, including preventive and complementary therapies, such as diet, besides conventional cancer treatments.
Altered methyl-donor metabolism is connected to pathological disturbances such as heart disease, birth defects, aging, and pancreatic toxicity, but it is mostly studied in relation to cancer development [9]. In a meta-analysis, Bo et al. found that dietary folate intake is associated with decreased risk of all-cause mortality and a wide range of chronic diseases, including cancer [17]. Wei and Mao found that high vitamin B6 intake might be associated with a reduced risk of pancreatic cancer [19]. Moreover, another meta-analysis assessing only case-control studies observed an inverse association between the high intake of folate and pancreatic cancer [18]. Methyl donors such as B vitamins are required for energyyielding metabolism, oxygen transport, and neuronal functions, and these are essential in optimal cognitive and psychological states; therefore, they could support conventional therapy in cancer patients by overcoming mental and physical fatigue [35,36].
We found that methyl-donor treatment significantly decreased the proliferation of Panc-1 cells along with p-Erk 1/2 level, and significantly increased the p21 WAF1/Cip1 cyclindependent kinase inhibitor level as well. Although p21 WAF1/Cip1 is a growth inhibitory protein, which is a mediator of p53-dependent G1 phase arrest, the TP53 gene is mutated in Panc-1 cells; thus, we expected a p53-independent upregulation of this protein induced by methyl donors in this cell line [37]. It is also known that the moderate activation of the MAPK/ERK signaling pathway is required to induce genes involved in cell cycle entry and to suppress those genes that inhibit this process, such as p21 WAF1/Cip1 [38].
The p21 WAF1/Cip1 expression is inversely associated with tumor differentiation, clinical stages, and lymph node metastasis in pancreatic cancers [39]. Therefore, we suppose that the decreased p-Erk 1/2 expression opened the way to the increased p21 WAF1/Cip1 expression level, and resulted in a significantly decreased proliferation rate after methyldonor treatment.
We found significantly increased subG1 fraction in cell cycle analysis, which indicates apoptotic cells; therefore, we investigated the intrinsic apoptotic pathway based on our previous study [33]. We found significantly increased pro-apoptotic Bak, Puma and Caspase-9 protein levels after methyl-donor treatment, but not in the case of the cleaved Caspase-3 level. This, however, could explain why we could not detect significantly increased Annexin-V-positive cells, neither in early nor in late apoptotic events after methyl-donor treatment by flow cytometry, although there is caspase-independent pathway in mitochondria-initiated apoptosis through the activation of AIF as well [40]. It is also known that the apoptosis inhibitor cIAP-2, survivin, livin and XIAP are overexpressed in pancreatic adenocarcinoma and are implicated in resistance to chemotherapy, and NFkB has been described to upregulate these genes [41].
Inflammation contributes to the development of several diseases. Cancers, including pancreatic cancer, are also known as inflammation-related disorders; additionally, chronic pancreatitis has been shown as a predisposition to pancreatic cancer [23,34,42]. NFkB has significant role in the activation of pro-inflammatory immune response, in promoting metastasis and chemoresistance [34], and it is constitutively activated in around 70% of pancreatic cancers [23,24]. IL-17 can activate the canonical NFkB signaling pathway [27] and switches on the pro-tumorigenic program in pancreatic cancers [28]. Parallel with these findings, we also found significantly increased pro-inflammatory cytokine IL-6 and IL-8 from pancreatic cancer patients compared to control, indicating the high probability of an activated NFkB pathway in these patients.
Interestingly, after methyl-donor treatments in our in vitro experiments, we found significantly decreased NFkB as well as IL-17a cytokine levels. This suggests that an appropriate amount of methyl donors in the diet might be an effective complementary and also preventive therapy, from which pancreatic cancer patients with increased NFkB and IL17 level, as well as healthy people, could benefit to keep their inflammation level under control. Some researchers have investigated p-NFkB as well; however, this is a post-translational modification of the NFkB, and another layer of this signaling, which was out of scope of our project.
As it is known that the overexpression of STAT3 is a prognostic marker in several tumors, including PDAC [31], and that NFkB and STAT3 signaling are connected with each other at several levels, affecting cell cycle and apoptosis [29,30], we aimed to only measure the protein level of STAT3, itself a prognostic factor in PDAC, without its activation level (p-STAT3). Although we could not demonstrate significance in the decrease in the STAT3 level after a single dose of methyl-donor treatment in vitro, we have seen this in case of IL-17 and NFkB. As we also found significantly increased levels of IL-8 and IL-6 cytokine in the plasma of pancreatic cancer patients, we suppose this was probably through IL17/NFkB/STAT3 signaling. These findings suggest that PDAC patients could benefit from a well-balanced methyl-donor-containing diet reducing the activity of the above-mentioned signaling path, and thus the related inflammation level, with several physiological benefits.
CD30 is a rediscovered target of new immunotherapies in non-lymphomatous solid tumors [43]. It triggers anti-apoptotic and pro-survival functions [44] through partly activating the NFkB pathway as well as mitogen-activated protein kinase (MAPK) pathways, including ERK1 and ERK2 [45]. As part of our investigation of inflammation processes after methyl-donor treatment, we aimed to measure the changes of CD30. However, we could not reach significance regarding the decrease in CD30 after methyl-donor treatment, neither by cytokine array nor by Western blot.
As pancreatic cancers are described as a malignancy with high metastatic potential, we aimed to detect if methyl donors could change this phenotype. We found the methyldonor treatment could significantly decrease the SDF-1 and VEGF level, while significantly increasing the expression level of E-cadherin, a cell adhesion molecule, which is a marker of normal polarized epithelial cells as well as of well-differentiated tumor cells [46]. However, we could not detect a significant decrease in MMP-9 after methyl-donor treatment compared to controls. Stromal cell-derived factor 1 (SDF-1) is expressed both in stromal and cancer cells, and has a well-known role in pancreatic tumor cell migration and angiogenesis [47]. Its elevated level is responsible for poor prognosis [48]. Additionally, SDF-1 can protect cancer cells from drug-induced apoptosis acting on NFkB or, indirectly, modulating tumor cell adherence [49].
Some researchers have questioned the relevance of the application of methyl donors even from a dietary source, implying that, in Nicotinamid N-methyl-transferase (NNMT)overexpressing tumors, methyl donors might fuel the NNMT enzyme causing a worse prognosis. They refer to studies where silenced NNMT has resulted in decreased migration, invasion, proliferation, and increased chemosensitivity [50], and also that NNMT expression is enhanced in cancer stem cells [51]. The NNMT enzyme takes a methyl group from S-adenosyl-L-methionine (SAM) and adds it to nicotinamide (NA), generating methyl-nicotinamid (MNA) and S-adenosyl-L-homocysteine as a result [52]. It is believed that it would decrease the methylation capacity of the methionine cycle towards the DNA/histone methylation path, causing aberrant methylation, which could lead to cancer progression in the case of the overexpression of NNMT. However, an earlier study from 2013 described that NNMT-overexpressed and silenced cells showed enhanced or reduced migration/invasion, respectively, if the cells were grown in methionine-low conditions, while those in methionine-high medium did not show differences in these phenotypes. This group also reported that NNMT, which is overexpressed in a diverse set of cancers, regulates the protein methylation state of tumor cells through a distinct mechanism that involves altering ratios of SAM:SAH [53]. These results also support our hypothesis, and our findings as well, suggesting that balanced nutritional status including methyl donors can revert healthy states of the cells, even with altered, overexpressed protein conditions, such as NNMT overexpression.
In conclusion, we demonstrated that methyl-donor treatments are able to diminish the proliferation of Panc-1 cells, possibly by decreasing the MAPK/ERK pathway and through increased levels of p21 WAF1/Cip1 growth inhibitory protein, along with significantly increased SubG1 fractions. Moreover, methyl-donor treatments were able to induce the intrinsic apoptotic pathway, and reduce the level of IL-17a with related NFkB. Consequently, this attenuated several unfavorable phenotypes of the Panc-1 cell line, such as loss of cell polarization by significantly increased E-cadherin expression, and high VEGF and SDF-1 expression by their significantly decreased expression. Therefore, we can conclude that adequate amounts of methyl donors could initiate the restoration of normal celllike phenotype and function in Panc-1 cancer cells, and thus might be a reasonable tool in a dietary intervention or alternative treatment applied in pancreatic cancer patients. However, additional and well-designed studies are needed to confirm its application in clinical settings.

Human Samples
Anticoagulated (K3-EDTA) whole blood samples were taken from 35 pancreatic cancer patients and 9 non-cancerous controls based on the research project approved by the Hungarian Ethical Committee of Scientific Research (No. 28123-6/2019/EÜIG). Plasma samples were collected after centrifugation to detect the levels of IL-6 and IL-8 cytokines by ELISA.

Cell Cycle Measurement
Cells were plated onto 6-well plates at a cell density of 3 × 104 cells/mL. After 24, 48, and 72 h treatments of methyl donors, cells were washed with PBS then trypsinized using 1× trypsin-EDTA solution (XC-T1717; Biosera, Nuaille, France). After washing steps, cells were fixed in ice-cold 70% ethanol at room temperature for 20 min, then were kept at −20 • C for an additional 30 min. Cells then were washed twice in PBS. After resuspension in PBS containing 1% RNaseA (R5503; Sigma Aldrich, 10 mg/mL) and 20 µL propidium iodide solution (P3566, Thermo Fischer Scientific Inc, Waltham, MA, USA; 1 mg/mL), samples were incubated for 1h at 4 • C. Cell cycles were detected by the CytoFLEX flow cytometer using CytExpert software (Beckman Coulter, Indianapolis, IN, USA).

Detection of Apoptosis
Panc-1 cells were plated and treated as described above in the section of 'Cell Cycle Measurement'. After centrifugation (1500 rpm, 5 min), apoptotic cell fraction was determined by FITC Annexin V Apoptosis Detection Kit with PI (640914; BioLegend, San Diego, CA, USA). Annexin V and/or PI positive cell fractions were detected by the CytoFLEX flow cytometer using CytExpert software (Beckman Coulter, Indianapolis, IN, USA).