Our targeted genes were all multifold overexpressed. (Fig. 1) SLC2A2 (GLUT2) increased by > 200-fold. GLUT2 is a glucose transporter that is expressed in pancreatic β-cells and plays a key role in glucose-stimulated insulin secretion by sensing changes in blood glucose levels.20 PANC-1 cells showed a multifold increase in GLUT2 expression when treated with metformin, a drug that lowers blood glucose levels by inhibiting hepatic gluconeogenesis.21 The relevance of this increase could be related to the role of GLUT2 in regulating intracellular glucose metabolism and the sensitivity to apoptosis in PANC-1 cells. Metformin induces GLUT2 expression through AMPK activation, and GLUT2 mediates the antiproliferative and proapoptotic effects of metformin on PANC-1 cells. 22
NKX2-2 expression, regulated in PANC-1 cells by PDX1,25 showed a 40-fold increase. PDX1 binds and activates the transcription of the NKX2-2 promoter and can cooperate with NEUROD1, a basic helix–loop–helix transcription factor that is critical for β-cell maturation and maintenance, to enhance NKX2-2 expression.26 This suggests that PDX1 and NEUROD1 overexpression in PANC-1 cells leads to increased NKX2-2 expression and induces a more β-cell-like phenotype.
Pancreatic cell interconversions depend on NGN3, a key endocrine progenitor transcription factor necessary for the specification of endocrine cells, and lead to differentiation into insulin-producing cells in ductal progenitor cells.27,28 Our results indicate a 13-fold increase in NGN3 expression in PANC-1 cells. Notch signaling is a conserved pathway that controls cell fate decisions and maintains progenitor cells in an undifferentiated state. It also represses NGN3 expression by inhibiting the activity of PDX1.29
PANC-1 cells show low expression of PAX6 compared with normal pancreatic ductal cells; therefore, a 10-fold expression of PAX6 is significant. Consistent with our findings, a study found that PAX6 overexpression in PANC-1 cells inhibits their growth, migration, and invasion by inducing apoptosis and cell cycle arrest.30 The significance of increased PAX6 expression in PANC-1 cells could be related to the role of PAX6 in regulating the differentiation and function of pancreatic endocrine cells, including alpha (α) cells that produce glucagon and β-cells that produce insulin. It is likely that PAX6 may act as a tumor suppressor in pancreatic cancer by restoring the normal phenotype and function of pancreatic ductal cells.31
MAFA and NEUROD1 increased by 20- and 4-fold, respectively, and they synergistically activated the SLC2A2 (GLUT2) gene in β-cells.32 MAFA regulates insulin gene expression and β-cell function. It is one of the key factors that defines the mature β-cell phenotype, along with PDX1.33 MAFA expression in PANC-1 cells is very low or absent. Under normal conditions, PANC-1 cells do not express insulin or other β-cell markers. However, MAFA expression can be induced in PANC-1 cells by various factors, such as PDX1, a transcription factor that is essential for pancreatic development and β-cell function. PDX1 can activate the MAFA promoter and increase the mRNA levels in PANC-1 cells.34
SIRT1 expression is increased by 10-fold. Notch signaling represses SIRT1 expression by inhibiting the activity of NGN3, which can bind to the promoter of SIRT1 and activate its transcription.
PANC-1 cells show low expression of FOXO1 compared with normal pancreatic ductal cells. Our results show that overexpression of FOXO1 in PANC-1 cells suppresses their proliferation, migration, and invasion by inducing apoptosis and cell cycle arrest.35 FOXO1 is known to respond to glucose levels and insulin receptor activation by modulating the expression of genes involved in glucose uptake, glycolysis, gluconeogenesis, and glycogen synthesis. The significance of increased FOXO1 expression in PANC-1 cells could be related to the role of FOXO1 in regulating the differentiation and function of pancreatic endocrine cells, including α-cells that produce glucagon and β-cells that produce insulin.36 Thus, FOXO1 may act as a tumor suppressor in pancreatic cancer by restoring the normal phenotype and function of pancreatic ductal cells.
Alkaline phosphatase (ALP) activity (Fig. 2) was significantly increased when 100 pg/mL Metadichol® was used. ALP activity has been upregulated in pluripotent stem cells, including undifferentiated embryonic stem and germ cells and induced pluripotent stem cells.37 ALP activity is often used to distinguish stem cells from feeder cells as well as from parental cells in reprogramming experiments. This confirms its pluripotency through the presence of islet-like structures on day 8 (Fig. 3, 4).
Upfield genes are likely to be activated by binding with Metadichol® in a cascade of signaling events that lead to the 12 expressed gene sets. We recently showed that Metadichol® can activate all 48 nuclear receptors (NRs) in human mesenchymal stem cells (HMSCs) at concentrations ranging between 1 pg/mL and 100 ng/mL.38 Therefore, downfield gene expression could result from NR activation.
For example, retinoic acid, a metabolite of vitamin A, can bind to nuclear retinoic acid receptors (RARs) and modulate the expression of PAX6 and other genes involved in eye development.39 RAR-NRs can also regulate the expression of PDX1, NKX6.1, and MAFA transcription factors that are crucial for β-cell development and maturation by interacting with their promoters.40
Another example is NR4A1, which can bind to the MAFA promoter and enhance its expression in pancreatic β-cells. 41 Additionally, NR4A1 can bind to the CA9 promoter and enhance its expression under hypoxic conditions, and it can activate the expression of insulin and other genes involved in glucose homeostasis. Conversely, NR4A2 can bind to the GCG promoter and enhance its expression in pancreatic α-cells42 and to the NEUROD1 promoter and enhance its expression in dopaminergic neurons.
NR5A2 can bind to the PAX6 promoter and enhance its expression in pancreatic β-cells. 43 It can also activate the expression of insulin and other genes involved in glucose homeostasis in pancreatic β-cells. Similarly, NR5A2 can bind to the NKX2-2 promoter, enhance its expression in pancreatic endocrine progenitor cells, and activate the expression of MAFA and other genes involved in β-cell maturation.44 Furthermore, NR5A2 activates the expression of PDX1, NKX2-2, and NGN3, transcription factors that are involved in endocrine cell differentiation.45 The liver X receptor (LXR) is an NR that regulates cholesterol homeostasis, lipid metabolism, and inflammation. LXR can activate SIRT1 by increasing its expression or by directly interacting with it and stimulating its deacetylase activity. SIRT1 can also deacetylate and activate LXR, creating a positive feedback loop.46,47,48
Thyroid hormone (TH), a metabolite of the thyroid gland, can bind to nuclear TH receptors (THRA and THRB) and modulate and regulate the expression of PDX1, another transcription factor that is crucial for pancreatic β-cell development and function 49, by interacting with MAFA on the PDX1 promoter.50
Table 5; NR expression in Stem cells
Nomenclature name
|
Common name
|
1 pg
|
100 pg
|
1 ng
|
100 ng
|
Control
|
NR1A1
|
THRA
|
16.16
|
12.24
|
7.7
|
5.32
|
1
|
NR1A2
|
THRB
|
7.71
|
1.94
|
15.11
|
8.71
|
1
|
NR1B1
|
RARA
|
1.27
|
0.79
|
0.52
|
0.44
|
1
|
NR1B2
|
RARB
|
1.67
|
1.39
|
0.48
|
0.73
|
1
|
NR1B3
|
RARG
|
2.52
|
1.04
|
0.96
|
0.82
|
1
|
NR1H3
|
LXRA
|
1.28
|
0.97
|
0.55
|
0.19
|
1
|
NR1H2
|
LXRB
|
1.28
|
1.17
|
0.84
|
0.18
|
1
|
NR4A1
|
NGFIB
|
1.82
|
0.67
|
1.16
|
0.61
|
1
|
NR5A2
|
LRH1
|
1.3
|
0.72
|
0.29
|
0.15
|
1
|
NR4A2
|
NURR1
|
0.48
|
4.55
|
5.24
|
7.44
|
1
|
All these relevevant NRs (nuclear receptors) are expressed by stem cells treated with Metadichol®, as shown in Table 5. Our study adds some additional information and shows for the first time that all the genes needed for differentiation are directly activated by upstream genes involved in transcription, namely, NRs. An increased number of involved NRs leads to a high degree of regulation. These NRs express all the downstream genes needed for differentiation. The key is the activation of NRs by Metadichol®, leading to a tight set of highly connected genes. These genes have more interactions among themselves than expected for a gene set of the same size and distribution degree randomly selected from the genome. This enrichment indicates that this set of genes shares a significant biological connection. The analysis of gene networks Pathway Studio and protein–protein interaction maps 51,52,53 indicated the formation of a loop feedback network, as shown in Fig. 5.
Metadichol® simultaneously increased the expression of insulin, glucagon, and SLC2A2 in PANC-1 cells, which has potential implications in the following.
Blood sugar regulation
Increasing the expression of insulin and glucagon, along with SLC2A2, in PANC-1 cells regulates blood sugar levels. Insulin helps lower blood sugar, glucagon raises it, and SLC2A2 facilitates glucose uptake into cells. This could have implications for the metabolism of both cancer cells and the surrounding tissues.
Cancer cells
Manipulating the expression of insulin and glucagon in cancer cells could have complex effects on the tumor’s biology. Insulin has been shown to have growth-promoting effects, which might raise concerns about increased cancer cell proliferation. Conversely, the role of glucagon in stimulating the release of glucose from the liver might have implications on tumor metabolism. Metadichol®, as previously shown, expresses Klotho, an antitumor molecule that controls the growth of cancer cells.54,55 We also measured Klotho expression and showed a seventy-fold increase after 8 days (Fig. 6).
Therapeutic strategy
This induction of the expression of both insulin and glucagon in cancer cells might be explored as a novel therapeutic strategy because Metadichol® is a nontoxic molecule and safe for human use.56–58
Metabolic control
Manipulating the hormonal environment within cancer cells might have broader metabolic effects beyond blood sugar regulation. This will affect other cellular processes and signaling pathways.
An analysis of gene networks was performed using Pathway Studio software and showed a closed loop feedback network (Table 5). All the important processes are related to β-cells, α-cells, and insulin synthesis. A complete list of cellular processes regulated by expressed genes is available in the Supplementary Material.
NEUROD1, NEUROG3, NKX2-2, PAX6, PDX1, and MAFA are all transcription factors that play critical roles in the development and function of pancreatic β-cells, which produce and secrete insulin. These factors can activate or repress the expression of each gene, and their interaction is essential for the proper development and function of β-cells.
-
SLC2A2 (GLUT2) is a glucose transporter protein expressed in pancreatic β-cells and plays a role in regulating insulin secretion. The expression of SLC2A2 is regulated by transcription factors such as PDX1 and FOXO1.
-
INS and GCG are hormones produced by pancreatic islet cells and play opposing roles in the regulation of glucose metabolism. Insulin promotes the uptake and storage of glucose, whereas glucagon promotes the release of glucose into the bloodstream. Various transcription factors, including PDX1, MAFA, and FOXO1, regulate INS and GCG expression.
-
CA9 regulates pH in pancreatic β-cells, which is essential for proper insulin secretion. CA9 expression is regulated by various signaling pathways, including HIF-1α, which is activated in response to hypoxia.
Both SIRT1 and FOXO1 are involved in regulating glucose metabolism and insulin sensitivity. SIRT1 deacetylates and activates FOXO1, which can promote the expression of genes involved in glucogenesis and insulin resistance. However, under certain conditions, such as calorie restriction, when intracellular NAD + levels are high, SIRT1 can deacetylate and inhibit FOXO1, improving glucose homeostasis and insulin sensitivity. This effect is thought to be mediated through several mechanisms, including the promotion of mitochondrial biogenesis, the activation of AMPK, and the repression of genes involved in gluconeogenesis and inflammation. Some studies have suggested that SIRT1-mediated inhibition of FOXO1 plays a role in the beneficial effects of caloric restriction and fasting on glucose metabolism and lifespan.
Table 5
Top 10 list of significantly enriched pathways. (The complete list is in the Supplementary Material)
Name
|
Overlap
|
Overlap %
|
p value
|
Genes from the list
|
Transcription factors in β-cell neogenesis (rodent model)
|
7
|
28
|
1.25 (×) 10− 16
|
NKX2-2 PAX6, PDX1,NEUROG3,MAFA, INS, NEUROD1.
|
α-Cell to β-cell interconversion (hypothesis)
|
6
|
27
|
3.64 (×) 10− 14
|
PAX6, PDX1, GCG, NEUROG3, INS, NEUROD.1
|
Neonatal diabetes mellitus
|
6
|
19
|
3.58 (×) 10− 13
|
PAX6, PDX1, NEUROG3; SLC2A2; INS; NEUROD1
|
FOXO1 and SREBP-1C roles in β-cell suppression (rodent model)
|
6
|
14
|
2.54 (×) 10− 12
|
SIRT1, PDX1, MAFA; INS; NEUROD1, FOXO1
|
Insulin synthesis in β-cell
|
6
|
11
|
9.79 (× )10− 12
|
SLC2A2, PAX6; PDX1; MAFA; INS; NEUROD1
|
β-Cell death in diabetes mellitus type 2
|
5
|
13
|
2.84 (×) 10− 10
|
PDX1, MAFA, SLC2A2, INS; NEUROD1
|
Insulin secretion
|
5
|
13
|
3.78 (×) 10− 10
|
PDX1, GCG, SLC2A2, INS; NEUROD1
|
Autoimmune polyglandular syndrome progression (hypothesis)
|
3
|
21
|
4.35 (×)10− 7
|
PDX1, INS, MAFA
|
Maturity-onset diabetes of the young (MODY)
|
3
|
16
|
9.72 (×) 10− 7
|
PDX1, INS;,NEUROD1
|
GLIS3 targets in thyroid dysgenesis (hypothesis)
|
3
|
13
|
2.1 (× )10− 6
|
PAX6, MAFA, INS
|
This research shows that diseases, pathways and biological processes are closely connected through related gene networks, and this is an approach that can be exploited to modulate multiple targets to enhance therapeutic effects, as ligands today are focused on a single target and limited by their efficacy. Metadichol® represents the first of a safe class of therapeutics that target multiple genes, pathways 59–62 and multiple diseases, which points to importanceof a relevance of the network-based approach.63