Recent epidemiological studies have suggested a link between cancer and pathophysiological conditions associated with hyperinsulinemia. In this report, we address the possible role of insulin exposure in melanocyte transformation. To this aim, normal melanocytes were exposed to chronic insulin and glucose supplementation (twice the standard medium concentration) for at least 3 wk. After 3-wk treatment, melanocytes increased proliferation (doubling time: 2.7 vs. 5.6 days, P < 0.01). After 3-wk treatment or after 3-wk treatment followed by 4-wk reculture in standard medium, melanocytes were able to grow in soft agar colonies. Treated melanocytes had increased DNA content (+8%, P < 0.05), chromosomal aberrations, and modified oncoprotein profile: p-Akt expression increased (+32%, P < 0.01), Akt decreased, and c-Myc increased (+40%, P < 0.05). PP2A protein expression increased (+42, P < 0.05), while PP2A methylation decreased (−42%, P < 0.05), and PP2A activity was reduced (−27%, P < 0.05). PP2A transcription level was increased (ppp2r1a, PP2A subunit A, +44%, P < 0.05). Also, transcriptomic data revealed modifications in insr (insulin receptors, +10%, P < 0.05) and Il8 (inflammation protein, +99%, P < 0.01). Glycolysis was modified with increased transcription of Pgk1 and Hif1a (P < 0.05), decreased transcription of Pfkfb3 (P < 0.05), decreased activity of pyruvate kinase (P < 0.01), and decreased pyruvate cell content as assessed by 1H-NMR spectroscopy. In addition, methyl group metabolism was altered with decreased global DNA methylation (−51%, P < 0.01), increased cytosolic protein methylation (+18%, P < 0.05), and consistent changes in methylated species on 1H-NMR spectra. In conclusion, exposure to chronic insulin and glucose supplementation induces oncogenic changes and methyl group metabolism redistribution, which may be a biomarker of transformation.
- protein phosphatase 2A
- nuclear magnetic resonance spectroscopy
cancer is a multistep process in which genetic alterations have a cumulative effect on the control of cell proliferation, division and growth control. Genetic instability has been prominently implicated in tumor formation. The main evidence comes from the discovery of chromosomal aberrations and mutations of oncogenes or tumor suppressor genes (25). There is experimental evidence that gene alterations can result from viral infection or carcinogenic insult (25).
In vivo, among normal epidermis cells, Langerhans cells and melanocytes are not able to proliferate, to the contrary of keratinocytes. In vitro, melanocytes can be cultured under highly specific conditions (11). Transformation capabilities of melanocytes are well known in relation to exposure to UV (9). However, a recent study raised doubt about the exclusive role of UV in melanocyte carcinogenesis (26). In vitro studies on human melanocytes and skin, and mouse models continue to refine knowledge on the specific effects of UV on normal and genetically susceptible melanocytes. Epidemiological data on the effects of UV on melanocyte transformation remain controversial (26, 38).
Recent studies have suggested a link between certain cancer types and pathophysiological conditions associated with hyperinsulinemia, including type 2 diabetes, metabolic syndrome, obesity, and chronic inflammation, all conditions whose incidence is rising in Western countries (22). Recent in vivo studies in mice have demonstrated the effect leptin, a product of the obese (ob) gene, secreted by adipocytes, as a promoter of tumor growth (3). Chronic hyperinsulinemia was shown to support melanoma tumor growth by inhibiting apoptosis and stimulating cell proliferation in the so-called insulin cancer (14). In addition, it has been reported that insulin resistance may be an independent risk factor for melanoma (4).
In this article, we sought whether exposure to chronic insulin and glucose supplementation could promote melanocyte transformation. To evaluate carcinogenesis, classical criteria were investigated, including cell proliferation rate, soft agar colony formation, DNA content, karyotype, oncoprotein expression (PP2A, Akt/p-Akt, c-Myc, PTEN, PP1, and Ras), and PP2A activity, which has been shown to be decreased in tumor cells (19). Metabolic alterations were explored including glycolysis and methyl group metabolism.
We found that normal human melanocytes (NHM) exposed to chronic insulin and glucose supplementation display increased proliferation and form colonies on soft agar. Also, these cells exhibit increased DNA content, alteration of karyotype, and oncoprotein profile changes, including increased expression of PP2A, p-Akt, and c-Myc. In contrast, PP2A methylation and activity were decreased. Transcriptomic data revealed consistent changes in PP2A regulation, Il8 inflammation mediator, and glycolysis. In addition, treated cells exhibited methyl group metabolism redistribution between nucleus and cytoplasm.
In conclusion, our data establish that chronic exposure to chronic insulin and glucose supplementation induces oncogenic changes and methyl group metabolism redistribution, which raises the hypothesis of a relationship between cellular oncogenic signaling and methylations. These findings are, to our knowledge, the first demonstration of the potential of chronic insulin and glucose supplementation to promote a precancerous state in human melanocytes.
MATERIALS AND METHODS
Glucose and insulin were purchased from Sigma. Glucose was used after dissolution in distilled water. Okadaic acid (Sigma, Saint Quentin Fallavier, France) was prepared as a 50 nM stock in DMSO. All these reagents were used in vitro in cell cultures: l-[14CH3]S-adenosyl methionine (l-[14CH3]SAM) (55 mCi/mmol specific activity) was purchased from Amersham Bioscience (Buckinghamshire, UK). TRIzol reagent was purchased from GIBCO.
Cell culture and cell treatments.
NHM were obtained from foreskin of kids aged 5–7 yr. After trypsinization, cells were cultured and maintained in a specific medium (Tebu); this medium contained insulin at 5 μg/ml and glucose at 1.081 g/ml. Others cell lines were used, including murine B16 melanoma, murine fibroblast L929, and human tumor ocular melanoma IPC 227F. These cell lines were maintained as monolayers in culture flasks in Eagle's MEM-glutaMAX medium (GIBCO) supplemented with 10% fetal calf serum (Sigma), 1 mM sodium pyruvate, 4 μg/ml gentamicin, 200 mM glutamine, 1× nonessential amino-acid solution (GIBCO), and vitamins (GIBCO). To determine growth curves, cells were cultured in triplicate wells in 12-well plates and counted every 2 days.
The dose-proliferation relationship of insulin and glucose supplementation was performed at various concentrations including 1-, 2-, 5-, 7-, 10-, and 50-fold the concentration of the standard medium (insulin, 5 μg/ml, glucose, 6 mM). For the rest of the study, the insulin and glucose supplementation was twice the insulin-glucose concentration of the standard medium. Cell culture was followed for 3–7 wk. Treated human melanocytes were separated into two groups, one exposed to 2× insulin-glucose supplementation with the medium changed twice a week and followed over more than 3 wk (ING), and the other treated for 3 wk and then recultured into standard medium without treatment for 4 wk (ING*).
Cell cycle analysis.
Melanocyte pellets were snap-frozen in liquid nitrogen for 10 min before use. The cells were incubated with R5125 RNAse A (Sigma-Aldrich, St-Quentin Fallavier, France) and 50 μg/μl propidium iodide (Sigma-Aldrich) for 15 min at +4°C in the dark. The stained cells were run in an EPICS XL flow cytometer (Beckman Coulter, Roissy CDG, France), and data were analyzed with MultiCycle software (Phoenix Flow Systems, San Diego, CA).
Colony formation in soft agar.
Colony-forming efficiency was determined using a double-layer soft agar method. Cells (2 × 103 per dish) were suspended in 1.5 ml of 0.3% Noble agar supplemented with complete culture medium containing 2× insulin and glucose. This suspension was layered over 1.5 ml of a 0.6% agar medium base layer containing 2× insulin and glucose in 35-mm culture dishes (Nuclon). After 20 days, cells colonies were counted.
Untreated and insulin-glucose-treated NHM were cultured as monolayers in culture flasks until 70% confluence and then treated with colchicine at 0.01 μg/ml for 17–57 h.
Radiolabeling study of cellular methylations.
For investigation of cellular methylations, melanocytes were incubated with 0.8 μCi/ml of l-[14CH3]S-adenosyl methionine (SAM) for 5 days before collection to achieve a steady-state level of radioactivity into the cells. Cells were divided into two groups; one was left untreated, and the other one was treated with insulin and glucose. At different times cells were harvested for analysis.
Total cell protein extracts and protein concentration.
Intact cells in a lysis buffer (50 mM Tris·HCl, pH 8, 100 mM NaCl) containing protease inhibitor mixture (Roche, Mannheim, Germany) were lysed by ultrasonication (3 × 15 s in ice). After centrifugation (14,000 g for 10 min at 4°C), the supernatant was kept at −80°C until analysis. Protein concentration was determined with Commassie blue (Pierce) at λ 595 nm with bovine albumin serum as a standard.
All of the extraction procedure was done in ice. Cells were Dounce homogenized with pestle “B” in fractionation buffer (10 mM Tris·HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA) containing protease inhibitor mixture. Complete cell lysis was checked by microscopy with trypan blue. Then, lysed cells were centrifuged (10,000 g for 10 min at 4°C). The supernatant was kept as the cytosolic fraction. The pellet was carefully washed with lysis buffer and centrifuged (10,000 g for 10 min at 4°C). The supernatant was added to the cytosolic fraction. The pellet containing nuclei was further disrupted by ultrasonication in fractionation buffer (3 15 s in ice) and centrifuged (10,000 g for 10 min at 4°C). The supernatant was kept as the nuclear fraction.
Radiolabel incorporation in proteins.
For the study of methyl group incorporation in cytoplasmic and nuclear proteins, cells were Dounce homogenized with pestle “B” in a fractionation buffer (10 mM Tris·HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA) containing protease inhibitor mixture. Complete cell lysis was checked by microscopy with trypan blue. Then, lysed cells were centrifuged (10,000 g for 10 min at 4°C). the supernatant was kept as the cytosolic fraction. The pellet was carefully washed with lysis buffer and centrifuged (10,000 g for 10 min at 4°C). The supernatant was added to the cytosolic fraction. The pellet containing nuclei was further disrupted by ultrasonication in fractionation buffer (3 × 15 s in ice) and centrifuged (10,000 g for 10 min at 4°C). The supernatant was kept as the nuclear fraction. Proteins from the cytoplasmic and the nuclear compartments were precipitated by adding 4 volumes of cold acetone to the cytoplasmic and nuclear fractions. The homogenates were vortexed, stored at −20°C for 2 h, and then centrifuged (14,000 g for 30 min at 4°C). The pellet containing the precipitated proteins was washed twice in acetone. Then acetone was evaporated under a nitrogen stream. The pellets were mixed with 1 M NaOH, and protein concentration was determined. An aliquot of the protein solution was mixed with liquid scintillation cocktail (Packard, Rungis, France), the radioactivity was measured in a scintillation counter (Winspectral Wallac 1414). Radioactivity incorporation was expressed as the percentage of dpm in insulin- and glucose-treated cell proteins compared with untreated cell dpm values. Three independent experiments were performed.
Radiolabel incorporation in DNA.
DNA was extracted by the TRIzol method according to the manufacturer's instructions, and its concentration was determined at λ 260 nm. Then, 5 μg of the dissolved DNA was mixed with 4.5 ml of a liquid scintillation cocktail (Ultima Gold, Packard), and the radioactivity was measured in a scintillation counter. Radioactivity incorporation was expressed as dpm per microgram of DNA. Data represent three independent experiments.
Twenty micrograms of proteins from total cells and from the cytoplasmic and nuclear fractions was subjected to SDS-PAGE on 10% SDS-polyacrylamide gels and transferred onto Immobilon-NC membrane (Millipore). The membrane was blocked in 4% nonfat milk-TBST (25 mM Tris·HCl, pH 8, 125 mM NaCl, 0.1% Tween 20) at 4°C overnight and probed with antibodies against preotein phosphatase (PP)2Ac subunit (1:2,500), methylated PP2Ac subunit (1:250), PP1 (1:2,000; Upstate, Lake Placid, NY), Akt (1:1,000), phosphorylated (p-)Akt (Ser473; 1:1,000), phosphorylated PTEN (Ser380/Thr382/383; 1:1,000), PTEN (1:1,000; Cell Signaling), c-Myc (1:200; Santa Cruz Biotechnology), Ras (1: 2,000), and β-tubulin (1:2,500; Sigma) overnight at 4°C and washed three times with TBST. Bound antibodies were detected with horseradish peroxidase-conjugated secondary antibody IgG (Upstate, Lake Placid, NY) using enhanced chemiluminescence Western blotting detection reagents (Amersham Bioscience, Buckinghamshire, UK). Equal loading of proteins was checked by Ponceau S staining of the membranes. Densitometric measurement of the band of interest was done using the Quantity One software (Bio-Rad). Normalization was done using β-tubulin densitometric values. Data are representative of three independent experiments.
As a control for PP2A methylation on Western blots, okadaic acid (OA), an antagonist of PP2A methylation, was added to the culture medium of untreated NHM at 5 nM for 48 h. Following treatment by OA, cells were rinsed in PBS and maintained in fresh culture medium for 5–7 days. Then, Western blots were performed to evaluate PP2A expression. The time exposure of OA was based on Ref. 19.
Metabolic profiling by 1H-NMR spectroscopy.
Proton NMR spectroscopy of intact cells was performed at 500 MHz using a high-resolution magic angle spinning coil (Bruker). The NMR sequence was a saturation recovery sequence with acquisition and processing parameters reported in Ref. 25. 1H-NMR spectra were line-broadened and phased. Signal attributions have been reported in Refs. 6 and 29.
PP2A phosphatase activity.
A PP2A immunoprecipitation phosphatase assay kit (Upstate) was used according to the manufacturer's instructions to detect PP2A activity. PP2A was immunoprecipitated with a monoclonal anti-PP2A antibody and protein A-Sepharose beads in lysis buffer. PP2A-bound beads were washed with phosphatase assay buffer and then with pNPP serine/threonine assay buffer (50 mM Tris·HCl, 100 mM CaCl2, pH 7.0; Upstate). Diluted phosphopeptide (K-R-pT-I-R-R) in serine/threonine assay buffer (250 μM) was added and then incubated for 15 min at 30°C. After centrifugation, 25 μl of supernatant was transferred to an assay plate, and 100 μl of Malachite green phosphate detection solution was added for 15 min of incubation at 30°C. The relative absorbance was measured at 630 nm wavelength. Data are representative of two independent experiments that were performed in duplicate.
Pyruvate kinase activity.
An aliquot of total cell protein extracts was incubated in 50 mmol/l Tris·HCl, pH 7.4, 100 mmol/l KCl, 5 mmol/l MgCl2, 0.6 mmol/l ADP, 0.9 mmol/l phosphoenolpyruvate, 0.3 mmol/l NADH, and 2.5 IU l-lactate dehydrogenase. Pyruvate kinase activity was measured at 25°C for 5 min by recording NADH oxidation at 340 nm and was calculated using A340/min obtained from the initial linear portion of the curve, with one unit of activity defined as that required for the oxidation of 1 μmol NADH·min−1·mg−1 protein at 25°C and pH 7.4.
To study mechanisms of melanocyte transformation, we used the TaqMan low-density array technique (Applied Biosystems) to screen 89 genes associated with cell cycle and signaling, oncoproteins and PP2A, bioenergetic metabolism, lipid metabolism, methyl group metabolism, and other metabolic pathways and oxidative stress.
In all experiments, data are given as means ± SD. Comparison between groups was performed using Student's t-test.
Chronic insulin and glucose supplementation promotes melanocyte proliferation.
Exposure of NHM to chronic insulin and glucose supplementation (ING and ING* cells) increased cell proliferation (Fig. 1A). ING cells exposed to concentrations of 2× to 10× exhibited shortened doubling time (Table 1). At 2× insulin-glucose supplementation, the doubling time was 2.7 vs. 5.6 days (P < 0.01); at 5× to 10×, the doubling time was collectively 3.4 days. In contrast, at 50×, cell proliferation strongly decreased (doubling time 15 days), showing that treatment had become overtly toxic.
As shown in Fig. 1A, the proliferation curve of ING* cells (treated for 3 wk with 2× insulin-glucose supplementation and then recultured for 4 wk without treatment) was superposed to that of ING cells.
ING* cells had modified morphology, with a large cytoplasm and a long extension of cytoplamic membrane, without any signs of blebbing or indentations of the nuclear membranes (Fig. 1B). ING* cells displayed decreased proportion in the G1 phase of the cell cycle and increased proportion in phase G2 [22 vs. 14%, ING* vs. untreated (UN)] and in phase S (25 vs. 11%, ING* vs. UN) (Fig. 1C).
Chronic insulin and glucose supplementation induces colonies in soft agar.
The ability of insulin-glucose-treated melanocytes to form colonies in soft agar is a classical marker for cell transformation (i.e., these cells display anchorage-independent growth). Several types of cells were tested: untreated human melanocytes (UN), insulin- and glucose-treated human melanocytes (ING) and (ING*), B16 murine melanoma cells, and L929 transformed fibroblasts. Among these cell types, only untreated melanocytes were unable to form colonies in soft agar (Fig. 2A). Quantification of the number of colonies of the different cell lines is shown in Fig. 2B.
Chronic insulin and glucose supplementation provokes DNA and chromosomal alterations.
Insulin and glucose exposure increased DNA content of treated cells (+8%, P < 0.05), as shown in Fig. 3A. The karyotype of untreated and insulin-glucose-treated melanocytes was performed. ING* cells exhibited an increase in the amount of chromosomes (46 vs. 94, ING* vs. UN) even if the number of concerned chromosomes could not be specified. In addition, chromosome arms were extended, suggesting activated telomerases (arrows, Fig. 3B).
Chronic insulin and glucose supplementation induces changes in oncoprotein and PP2A expression.
In treated cells, Akt expression was decreased, p-Akt expression increased (+32 and +35%, P < 0.05, ING and ING* vs. UN, respectively), c-Myc expression increased (+36 and +54%, P < 0.05, ING and ING* vs. UN, respectively), and Ras expression increased (Fig. 4A). PP1 expression varied poorly (Fig. 4, A and B). PTEN and p-PTEN, an oncosuppressor activated by phosphorylation, varied poorly (Fig. 4A). The ocular melanoma cell line IPC displayed a similar oncoprotein profile to ING and ING* cells (Fig. 4B), supporting that ING and ING* cells underwent oncogenic changes. Because ING* cells maintained the oncogenic profile despite their being recultured in standard medium for 4 wk, it may be concluded that the transformed phenotype was acquired during the first 3 wk of treatment.
As shown in Fig. 4, A and B, NHM expressed high levels of PP2AC subunits, both methylated C and nonmethylated. During exposure to chronic insulin and glucose supplementation, PP2AC expression was increased (+60 and +47%, P < 0.05, ING and ING* vs, UN, respectively); the methylated C subunit of PP2A was decreased (−42% P < 0.05).
As a control for PP2A methylation and its consequences on oncoprotein regulation, NHM were treated with OA, an antagonist of PP2A. OA inhibited methylation of PP2A (−50%), which upregulated p-Akt (+30%) and c-Myc (+35%) (Fig. 4C), showing that inhibiting the activity of PP2A promoted oncogenic changes in melanocytes.
The activity of PP2A phosphatase activity was assayed and found to be decreased (−27 and −30%, P < 0.05, ING and ING* vs. UN, respectively), in agreement with decreased methylation of PP2A. As a matter of comparison, PP2A activity was markedly diminished in ocular melanoma IPC tumor cells (−42%, IPC vs. UN, P < 0.01; Fig. 4D).
The regulation of oncoproteins and PP2A was investigated by transcriptomics (Table 2). Only some oncoproteins and oncosuppressors had altered transcription. In ING* cells, Nras was downregulated (−38%, P = 0.044) and Pink1 upregulated (+71%, P = 0.038). Myc and Kras decreased, although their variations were less significant (P < 0.10). Akt1 did not vary significantly. Data showed upregulation of ppp2r1a (subunit A of PP2A, +44%, P < 0.05), downregulation of Ppp1ca (P < 0.05) but a lack of variation of Pppc1b, and upregulation of lcmt1 (leucine carboxymethyltransferase, +26%, P < 0.05).
As could be expected, insulin-glucose treatment upregulated Insr, insulin receptor (+10%, P = 0.017), and Ccnd1 (+52%, P = 0.037), in relation with increased proliferation. Interestingly, Il8, a marker of inflammation, was markedly upregulated (+99%, P = 0.007); inflammation is a condition that favors carcinogenesis.
Chronic insulin and glucose supplementation alters glycolysis.
In ING* cells, bioenergetics-associated transcriptomic data showed the following alterations (Fig. 5A): Slc2a3 (+26%, P = 0.075), Pfkfb3, which encodes for 6-phosphofructo-2-kinase/fructose-2,6-biphosphate 3, (−49%, P = 0.033), pgk1, which encodes for phosphoglycerate kinase (+33%, P = 0.007), and Hif1a (+59%, P = 0.041). In addition, Pdk1 and Pkm2 moderately increased, although their variations were less significant (P < 0.10). Pyruvate kinase activity was markedly reduced in ING and ING* cells (−42 and −39%, P < 0.01, ING and ING* vs. UN, respectively), which compared with that of ocular melanoma IPC 227F tumor cells (−48%, IPC vs. UN) (Fig. 5B). As shown by 1H-NMR spectra, pyruvate (Pyr, signal at 2.37 ppm) and oxaloacetate (OAA, signal at 2.38 ppm), an intermediate of the TCA cycle, were decreased in ING* cells compared with NHM. In addition, alanine (Ala, signal at 1.47 ppm) and glutamate (Glu, signal at 2.35 ppm) levels were increased in IPC melanoma tumor cells compared with ING* cells. Ala and Glu are derivatives of the transamination of Pyr and OAA (Fig. 6A). Taken together, these data support that the phenotype of transformed human melanocytes has become more glycolytic, similarly to tumor cells.
Insulin and glucose supplementation induce cellular redistribution of methyl group metabolism.
In ING* cells, methyl group metabolism-related transcriptomic data showed the following alterations (Table 2): Lcmt1, which encodes for PP2A methyltransferase (+26%, P < 0.05), Pemt, which encodes for phosphatidylethanolamine methyltransferase (+31%, P < 0.05), and Mgmt, which encodes for O-(6)-alkylguanine methyltransferase (+32%, P < 0.05).
The distribution of methyl group metabolism was investigated in cellular and molecular compartments of insulin-glucose-treated cells. Incorporation of radioactivity from l-[14CH3]SAM was decreased in nucleic proteins (−51%, ING* vs. UN, P < 0.01) and increased in cytoplasmic proteins (+18%, ING* vs. UN, P < 0.05) (Fig. 6B). DNA methylation was decreased (−8%, ING and ING* vs. UN, P < 0.05; Fig. 6C).
Methyl group metabolism alterations were visible in 1H-NMR spectra of ING* cells, compared with UN and IPC cells (Fig. 6A). 1H-NMR spectra, which mostly reflect cytoplasmic content, showed, in ING* cells compared with NHM, decreased levels of methyl acceptors: glycine (Gly; signal at 3.56 ppm) and guanidino-acetate (GA; signal at 3.75 ppm) and increased levels of sarcosine (Sar; methyl signal at 2.73 ppm), which is both the methylated form of Gly and a methyl donor. In IPC tumor cells, compared with ING*, 1H-NMR spectra showed increased levels of creatine (Cr; methyl signal at 3.03 ppm), the methylated form of GA, and decreased levels of glycerophosphocholine (GPC; methyl signal at 3.23 ppm), a methyl donor.
Taken together, these data show that chronic insulin and glucose supplementation shifts cellular methyl group metabolism from the nucleus to the cytoplasm.
Other metabolic changes induced by chronic insulin and glucose supplementation.
Transcriptional data showed changes in lipid and phospholipid metabolism (Table 2), including Acly (−33%, P = 0.023), Fasn (−70%, P < 0.05), Pcyt1b (+%140.46, P < 0.05), Chpt1 (+31%, P = 0.032), Pla2r1 (+103%, P < 0.05), and Plcb4 (+24%, P = 0.042). Proton-NMR spectra showed a decrease in glycerophosphocholine (a product of GPC hydrolysis) in IPC tumor cells compared with ING* cells. Some of these data may appear controversial or indicate that in ING* cells lipid metabolism has not yet passed the oncogenic transition.
Also, transcriptomics showed alteration in oxidative stress pathways in insulin-glucose-treated cells, including Txnrd1 (+72%, P = 0.034), Gss (+19%, P = 0.045), and Gsr (−9%, P = 0.007). In agreement with these data, 1H-NMR spectra showed an increased content in GSH in IPC melanoma tumor cells compared with ING* cells. These data support that oxidative stress took place early in the process of transformation.
This study establishes that normal human melanocytes exposed to chronic insulin and glucose supplementation undergo oncogenic changes including proliferation, ability to grow in colonies, chromosomal abnormalities, increased oncoprotein expression, and decreased PP2A activity. These disorders are associated with metabolic alterations of oncogenic type, including increased glycolysis and oxidative stress response, and to cellular redistribution of methyl group metabolism.
In vivo, exposure to chemical carcinogens or radiation is considered a major factor of normal cell transformation or carcinogenesis in human cancers (25). However, in vitro, carcinogens alone have not successfully transformed normal human cells in culture (15, 27, 35). To undergo transformation, normal cells need prior exposure to a carcinogen to be immortalized by transfection with a cancer-associated virus (10).
During carcinogenesis, tumor promotion is usually preceded by an intense inflammatory reaction, itself resulting from the action of a tumor initiator. The effect of genotoxic carcinogenic compounds is not limited to epithelial cells; it causes extensive tissue damage resulting in the release of free radicals and chemicals through cell killing and replacement. Forty percent of chemicals that are carcinogenic in chronic animal tests are not mutagenic (8); these nongenotoxic compounds do not cause DNA damage.
As a genotoxic agent, ionizing radiations induce cancers in humans and in animals (12, 16). However, like for chemical carcinogenesis, numerous attempts to achieve transformation of normal human cells in vitro have been generally unsuccessful (34).
Genetic instability has been implicated in tumor formation. It is possible that the stress secondary to massive glucose influx increases the mutation rate (1). Nongenotoxic stress such as heat or serum starvation can induce a mutator phenotype with persistent and pronounced genetic instability. Perhaps triggered by exposure to carcinogens or abnormal physiological conditions, this hypermutable state could cause mutations in many genomic loci in a normal cell (25).
In this paper, we used normal human melanocytes and demonstrated that chronic insulin and glucose supplementation (2× the standard medium concentration) without any other carcinogen can induce a mutator phenotype with upregulation of several oncoproteins.
The combination of insulin and glucose was used to prevent a possible nutritional cause for impeding proliferation over the long term and because the phenotype of human melanocytes was expected to become more glycolytic, which was confirmed by transcriptional analysis. Glucose at 2× (∼12 mM) did not cause an osmotic shock because of rapid transport of glucose, further supported by administration of insulin, and metabolism of glucose (glycolysis). Increased transport of glucose was verified in insulin-glucose-supplemented melanocytes by transcriptional data.
The relationship between insulin and glucose uptake and oncogene activation is a complex one (18). In this study, without genetic manipulation, we found that insulin-glucose-treated normal melanocytes develop several criteria of transformation, commonly found in melanoma tumor cells, including increased rate proliferation, formation of colonies in soft agar, karyotype and DNA alterations, increased oncoprotein expression, decreased PP2A activity, and metabolic alterations commonly reported in tumors, increased glycolysis, and oxidative stress response.
Several studies have reported that oncogene transfection or virus introduction such as SV40, results in malignant transformation of normal cells (10). In this study, chronic insulin and glucose supplementation induced an upregulation of oncoproteins, (p-ATK and c-Myc and Ras), which signal the precancerous state of treated melanocytes.
PP2A regulates cell proliferation through the activation of oncoproteins including Akt and c-Myc (5, 19, 32). The Akt/PKB pathway is involved in cell proliferation, protein synthesis, and resistance to apoptosis (2, 7, 34). This pathway is often hyperactivated in cancer (37). It has been shown that glucose metabolites (citrate and phosphoenolpyruvate) inhibit PP2A carboxymethylation (31). In this study, glycolysis was upregulated, which may provide a link with decreased methylation and activity of PP2A.
At the molecular level, PP2A activity is regulated by the carboxymethylation of its catalytic subunit (23, 24). According to Fig. 7, insulin-glucose supplementation may provoke inhibition of LCMT1, activation of PME-1, and/or rerouting of SAM metabolism. Given the fact that ppme1 transcript, encoding for PME-1, did not vary, and that Lcmt1 was positively regulated, it is possible that SAM rerouting accounted for PP2A demethylation. Indeed, insulin-glucose-treated cells may be the place for intense competition for the substrate SAM, required by most transmethylations. Lcmt1 may be increased as a means for PP2A to dampen its demethylation.
Among genes differentially regulated in cells treated with insulin and glucose, Il8 (interleukin-8) was increased twofold. Il8 upregulation may indicate chronic inflammation, a condition favoring carcinogenesis. This may allow initiation and/or promotion during early carcinogenesis. Interleukin-8 (IL-8/CXCL8) has been described as a key effector in cancer progression and metastases (20). It has been underlined that, in vitro, the phenotype of melanoma cells is defined by features of high proliferation and noninvasiveness, in contrast to weak proliferation and high invasiveness (17). These phenotypes are correlated to increased gene expression of microphthlalmia-associated transcription factor (Mitf) (17). Mitf is critical for the regulation of melanocyte development and survival and is closely related to the proliferative phenotype, whereas the wingless-type MMTV integration site family 5A (Wnt5A) expression is mostly related to invasiveness capability.
In our study, insulin-glucose-treated melanocytes have switched on Mitf under the effect of microenvironment loading in insulin and glucose. The increased expression of Mitf suggests that these cells acquired a proliferative phenotype rather than an invasive one, despite our not having evaluated the Wnt5A expression level. In addition, Mitf-positive melanocytes (ING cells) have a high expression of Cdkn2a, which is implicated in cell proliferation. (21).
Glycolysis was upregulated in cells exposed to insulin-glucose, as evidenced by transcriptional, enzymatic, and metabolic changes in this study. pgk1 and hif1a increased expression and PK activity inhibition have been reported as markers of the so-called “aerobic glycolysis”, a major biochemical trait of tumor cells (13) (1). Also, glucose transporters were moderately upregulated (Slc2a1, Slc2a3). The latter change, which accompanies aerobic glycolysis, accounts for strong glucose uptake of melanoma (and other) tumors on FDG scans (33).
Methyl group metabolism alterations have been associated with cancer. Global DNA hypomethylation but hypermethylation of specific tumor suppressor genes is a hallmark of cancer. A number of epidemiological and clinical studies have shown a link between methyl group or folate deficiency and tumorogenesis. In addition, DNA methylating agents have proved efficient in the treatment of some tumor types including melanoma. However, the exact mechanism by which hypomethylation promotes neoplastic transformation remains unelucidated. Consistently with these data, chronic exposure to insulin and glucose supplementation induced a global demethylation of DNA. In addition, the transcription of Mgmt, which encodes for an enzyme demethylating DNA, was upregulated.
Besides genomic methylation abnormalities, tumor cells have been shown to have cytoplasmic methyl group metabolism alterations including a high content in methylated acceptors creatine, and phosphatidylcholine and other methylated acceptors found in proteins, like asymmetric dimethylarginine and trimethyllysine (29). In insulin-glucose-treated vs. untreated melanocytes, metabolic profiling based on 1H-NMR spectra, which mostly reflect cytoplasmic changes, showed decreased levels of methyl acceptors (GA, Gly) and Sar transient increase. Sar is a methylated form of Gly and was shown to increase in prostate tumor tissue due to increased activity of glycine-N-methyltransferase (GNMT) in relation with invasiveness (36).
GNMT is a major regulator of methyl group metabolism, also involved in diabetes, that has been shown to be associated with liver methyl group metabolism abnormalities, including increased activity of GNMT (30). In addition, in insulin-glucose-treated melanocytes, Pemt, which encodes for a cytoplasmic enzyme methylating phosphadylethanolamine into phosphatidylcholine, was upregulated, and PP2A was demethylated. It has been reported that methylation of the PP2A catalytic subunit was the most important methyl group consumer among the cellular proteins (19) (23). Thus, not only GNMT but PP2A may be important actors of methyl group metabolism redistribution.
Taken together, our findings raise the question whether global DNA demethylation and methyl group metabolism redistribution within the cell could result from competition between methyltransferases for the substrate SAM. In addition, methyl group metabolism redistribution could be an early event in the process of transformation besides upregulated glycolysis and oxidative stress response pathways.
In conclusion, human normal melanocytes exposed to chronic insulin and glucose supplementation undergo oncogenic changes and cellular redistribution methyl group metabolism. This model may serve to increase understanding of the relationship between cellular oncogenic signaling and methylations. These findings are, to our knowledge, the first demonstration of the potential of chronic insulin and glucose supplementation to promote cell transformation.
Institut National de la Santé et de la Recherche Médicale (INSERM), and Biorebus Society.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: D.M. and A.D. conception and design of research; D.M. and A.D. performed experiments; D.M. and A.D. analyzed data; D.M. and A.D. interpreted results of experiments; D.M. and A.D. prepared figures; D.M. and A.D. drafted manuscript; D.M., J.M.S., L.S., M.I., and A.D. edited and revised manuscript; D.M., J.M.S., L.S., M.I., and A.D. approved final version of manuscript.
We thank Mathilde Bonnet Duquennoy for technical assistance with the transcriptomic analysis and Samuel Guenin for technical assistance with the cell cultures.
- Copyright © 2012 the American Physiological Society