Antiproliferative effect of ascorbic acid is associated with the inhibition of g

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Antiproliferative Effect of Ascorbic Acid Is Associated with the Inhibition of

Genes Necessary to Cell Cycle Progression

Sophie Belin1, Ferdinand Kaya1, Ghislaine Duisit1, Giacometti2, ph

Ciccolini2, Michel Fontés1*

1 EA 4263, Therapy of Genetic Disorder, Faculté de Médecine de la Timone,

Marseille, France, 2 UPRES EA 3286, Laboratory of Pharmacokinetic and

Toxicokinetic, Faculté de Pharmacie, Marseille, France

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0004409

This is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution, and

reproduction in any medium, provided the original author and source are

credited.

Funding: This work has been funded by French ANR maladies rares program. S.Belin

is supported by INSERM and PACA region. The funders had no role in study design,

data collection and analysis, decision to publish, or preparation of the

manuscript.

Introduction

For a long time, ascorbic acid (AA) has been described as a molecule absolutely

required for the integrity and normal life span in mammalians. Most of princeps

studies have been derived from the observation on patients suffering from

scurvy. Numerous articles have been published demonstrating the necessity of

supplement human nutrition with AA, either originating from foods or from

nutrition complements. Different biochemical properties have been attributed to

AA, essentially in relation with its well-documented antioxidant activity.

However, little is known about the effects of AA treatment on gene expression.

In previous studies we showed that high concentrations of AA down-regulate the

cAMP-dependent expression of PMP22, partially correcting Charcot-Marie Tooth

disease phenotype in mouse models [1]. In additional experiments, we

demonstrated this action relies on the AA-induced reduction of intracellular

pool of cAMP [2]. Importantly, other antioxidants like retinol and

& #945;-tocopherol were unable to modulate the pool [3], strongly suggesting the

inhibitory effect of AA was unrelated to its antioxidant properties. Indeed, we

have recently reported that AA is a competitive inhibitor of adenylate cyclase,

probably due to partial three-dimensional structures similitude between AA and

ATP [4].

Because of the critical role of cAMP in the modulation of gene expression, we

sought to determine whether other genes could be sensitive to AA treatment. We

conducted a series of in vitro and in vivo experiments using varying

concentrations of AA. We first analyzed the impact on gene expression using

human pangenomic microarrays. As we found that several genes implicated in cell

proliferation were underexpressed in presence of AA, we further analyzed the

effect of AA administration on cell division and tumor progression. Data

presented here strongly indicate that AA has an antiproliferative activity,

potentially due to the inhibition of expression of genes involved in cell

division progression and may be related to its action as a “global regulator” of

intracellular cAMP pool.

Materials and Methods

Cells and culture conditions

The human skin fibroblast cell line was obtained from the Coriell Cell

Repository. All others cells were purchased from the American type Culture

Collection. Cells were grown according to the manufacturer’s instructions in

RPMI 1640 with 25 mM HEPES supplemented with 15% fetal bovine serum (GIBCO) at

37°C in 5% CO2/95% air. Cells were counted on a microscope (10× objective) using

a Neubauer hemocytometer. Cell viability was estimated using trypan blue

staining.

AA Treatment

L(+) ascorbic acid (99.7%, Riedel-de-Haën) was dissolved as needed in 1× PBS and

then filtered. It was added to a final concentration of 50 mg/ml in pH 7 medium

supplemented with 25 mM HEPES.

RNA extraction and analysis

Following the incubation period, total RNA was extracted from cells using TRIZOL

reagent (Invitrogen) in accordance with the manufacturer’s instructions. To

ensure the integrity of the RNA prior to use, the quality and concentration of

each sample was tested using an Agilent 2100 Bioanalyzer.

Hybridization and microarray scanning

The following materials were purchased from Agilent Technologies: Low RNA Input

Fluorescent Linear Amplification Kit, in situ hybridization kit, and 60-mer

human oligomicroarray kit. The reverse transcription labelling reactions and

hybridizations essentially followed the protocol recommended by Agilent

Technologies version 4.1 (2004).

Briefly, a 500-ng aliquot of each total RNA sample was reverse transcribed into

cDNA using an oligo(dT) primer.

The reaction was carried out in a solution containing 500 & #956;M dNTP and 400 U

MMLV reverse transcriptase at 40°C for 2 h, and then terminated by incubation at

65°C for 15 min. Reverse transcription and incorporation were performed at 40°C

for 2 h in a mixture containing either 400 & #956;M cyanine 5-carbamylated

protein (Cy3 for the untreated samples) or 400 & #956;M cyanine 5-carbamylated

protein (Cy5 for the treated samples), the transcription mix, and T7 RNA

polymerase. RNeasy spin columns (Qiagen) were used according to the

manufacturer’s protocol to purify the amplified labelled cDNA samples.

Hybridization was carried out in 440 & #956;l of mixture containing 750 ng of

Cy3- and Cy5-labeled cDNA probes, control targets, and the fragmented DNA

supplied by the manufacturer at 60°C for 17 h. The microarrays were then washed

according to the manufacturer’s protocol, dried, and scanned using an Agilent

microarray scanner with 532 nm laser for Cy3 measurement and a 635 nm laser for

Cy5 measurement. The results were analyzed using the Luminator Bioinformatics

platform (Rosetta Biosoftware). Results have been deposited in MIAMExpress data

base (accession number E-MEXP-1861).

Reverse transcriptase-PCR analysis

To confirm the gene expression patterns observed on the microarrays we verified

gene expression levels following AA treatment at each concentration by

quantitative reverse transcriptase-PCR using the Light Cycler 480 Real-Time PCR

System (Roche) with universal probe library (UPL). Two micrograms of total RNA

from each sample were reverse transcribed using Superscript II reverse

transcriptase (Invitrogen) to produce cDNA. Human & #946;-actin (ACTB) and 18S

human ribosomal DNA were used for data normalization. The results were treated

using the comparative CT method, where the amount of the target, normalized to

the endogenous reference and relative to a calibrator, is given by

2 & #8722; & #916; & #916;CT [5]. (An asterisk indicates a P value<0,05).

Cell cycle and death analysis—flow cytometry

To determine the cell cycle status after treatment, we stained cells (106

cells/condition) with 50 & #956;g of propidium iodide and subsequently analyzed

the DNA content by fluorescence-activated cell sorting (FACS). All cell cycle

results were analyzed using the ModFit Software provided with the FACSCalibur

flow cytometer. The relative cell cycle distribution was calculated using the

FIT option. The same process was repeated for all samples.

To determine cell death, we used the Vybrant Apoptosis Assay Kit #2 (Invitrogen)

(105 cells/condition). The cell cycle distribution and the apoptosis assay were

analyzed using ELITE Flow Cytometry (Beckman Coulter) and Cell Quest software.

Duplicate assays were performed in all cases.

Animal studies

The antiproliferative action of ascorbic acid was investigated in the HT29

xenograft mouse model. Mouse care followed the animal welfare guidelines of our

institution, and local animal ethics committee approval was obtained prior to

starting the experiments. Four-week-old female BalbC nude mice ( River,

Lyon, France) (n = 7 per group) were subcutaneously inoculated with 1×106 HT29

cells on the right flank. Ten days after implant, and once tumors had reached

sizes that could be measured accurately, mice were treated with AA at the

following doses: 15 mg/kg, 100 mg/kg, or 1,000 mg/kg. Drugs were administered

intraperitoneally on a daily basis for 1 month. Tumor size was measured every 2

days in three dimensions using Vernier calipers, and tumor weight (g) was

calculated using an ellipsoid shape to approximate the tumor mass: m =

& #960;/6×length×width×height. Studies were terminated after 30 days of

treatment. Surviving mice have been sacrificed, and tumor weight evaluated. In

addition, the date and number of surviving animals were recorded for surviving

analysis.

Statistical analysis

We used Prism v5 software to perform the statistical analysis. For tumor weight

analysis we used the Mann-Whitney two-tailed statistical significance test, with

a confidence interval of 95%. For the survival study the Kaplan-Meier method and

log-rank tests were used. We considered a P value lower than 0.05 as

significant.

Results

AA and gene expression

Primary cultures of normal human fibroblasts were treated for 24 h with three

different concentrations of AA, ranging from 0.3 mM to 0.8 mM. RNAs were then

extracted, reverse transcribed, and hybridized on AGILENT human pangenomic

microarrays. RNA samples from untreated cells were labelled with Cy3, and those

from treated cells were labelled with Cy5. In a reciprocal experiment, the dye

labels were switched, so that untreated cells were labelled with Cy5 and treated

cells with Cy3. Genes that were over- or under expressed in the initial screen

and behaved in a reciprocal manner in the second screen (e.g., under expressed

in the first screen and over expressed in the second screen) were selected and

included in a minilibrary. Data were analyzed using Rosetta bioinformatics

software. We considered only genes that varied in the same direction (up or

down-expression) on all three chips with a P value of <0.05. Using these

stringent criteria, we found that only a few genes were over expressed in cells

incubated with AA, without an obvious biological function. In contrast, 31 genes

appeared to be down regulated (Table S1). Of these 31 genes, 12 code for members

of two protein families: tRNA synthetases and translation initiation factor

subunits (Fig. 1a). These data were confirmed using qPCR (Fig. 1b).

Figure 1. Human pangenomic microarrays and qPCR analysis.

(a) Primary cultures of human skin fibroblasts were incubated in medium

containing 0, 0.3, 0.6, and 0.8 mM AA. After 24 h, RNA was extracted; reverse

transcribed, and hybridized on AGILENT human pangenomic microarrays. Dye swap

experiments were also conducted. Data were analyzed using the Rosetta Luminator

software package. And only those genes that were up- or downregulated in

AA-treated cells were analyzed further. Among the downregulated genes, 40%

belong to two classes (tRNA synthetases and translation initiation factor

subunits) involved in protein synthesis. ( Validation of microarray results

using qPCR and Roche UPL-specific probes.

doi:10.1371/journal.pone.0004409.g001

AA and in vitro cell division

Both tRNA synthetases and translation initiation factor subunits are involved in

protein synthesis and cell cycle progression in prokaryotes, basic eukaryotes,

and mammals [6]-[11]. To determine whether AA had an impact on cell

proliferation, we treated the same type of fibroblasts used in microarray assays

with increasing concentrations of the drug and monitored the growth curves (Fig.

2a). At moderate concentration (0,3 mM), AA partially inhibited cell

proliferation. Higher concentrations (0.6 and 2mM) respectively resulted in a

cell proliferation arrest or cellular death. We then tested the effect of AA on

cell lines derived from human cancers. Cell growth was affected by AA treatment

in all of the cell lines, although sensitivity varied (Fig. 2b, Tables S2-S3).

This difference is not understood at present. It may be related, at least in

part, to varying AA uptake between cell lines since Raji cells, which are the

most sensitive cells to AA treatment, also express the highest level of

sodium-dependent vitamin C transporter 2 (SVCT2) mRNA (data not shown). In

addition to the experiments above, we confirmed the effect of AA on cell

division using a BrdU incorporation test (data not shown).

Figure 2. Impact of AA treatment on cell growth.

Growth curves of healthy human fibroblasts (a) or Raji cells ( incubated with

various AA concentrations. Cell density estimation of the reversal of AA

treatment on Raji cells incubated with 0.6 mM ©, 2 mM (d), or 3 mM (e) AA.

doi:10.1371/journal.pone.0004409.g002

To evaluate the potential reversibility of the effect, cells incubated with

increasing AA concentrations were either collected 24 h later, washed and

cultured in AA-free medium, or let in presence of the drug for the next three

days. We observed that the partial inhibition of cell division obtained with 0.6

mM of AA could be reversed (Fig. 2c). However, with AA doses enable to induce

cell death (2 mM and 3 mM), cells continued to die even after the AA-containing

medium was removed (Fig. 2d,e).

Cell cycle profiles were analyzed by FACS to determine at which stage of cell

division the cells treated with AA were blocked. We noted that 3 mM AA caused a

growth arrest during S phase in healthy fibroblasts cells (Fig. 3a) as well as

in Raji cells (Fig. 3b, c), with no cells detected in G2/M. In addition, we

observed a significant increase in cell death, which we confirmed by trepan blue

uptake (Fig. 4a). To determine the type of cell death induced by AA treatment,

we used flow cytometry on Raji cells, either double labelling with Annexin V (to

identify the early apoptotic phase characterized by the external membrane

phosphatidylserine translocation) and propidium iodide labelling (to identify

plasma membrane permeabilisation related to cell necrosis) (Fig. 4b) or using a

TUNEL enzymatic labelling assay of apoptosis (Fig. S1a). Nearly all of the cells

treated with increasing concentrations of AA became necrotic, without an

increase in apoptosis. This was confirmed by the typical swelling and bubbling

morphology of the necrotic cells (Fig. 4c, d). When we treated quiescent cells

(human primary fibroblasts at confluence) with the same concentration of AA,

there was no effect (Fig. S1b). This data indicates that only actively

proliferating cells are affected by AA treatment, and that cells are not dying

from toxicity induced by high AA concentrations.

Figure 3. Cell cycle analysis.

Cell cycles in control and treated human primary fibroblasts (a) or Raji cells

(, were analyzed using propidium iodide. DNA content was analyzed using FACS

to determine the cell cycle distribution. All cell cycle results were analyzed

using the ModFit software provided with the FACSCalibur flow cytometer. The FACS

profile is shown in ©.

doi:10.1371/journal.pone.0004409.g003Figure 4. Cell viability analysis.

(a) Cell viability of Raji cells treated with varying concentrations of AA was

evaluated using trypan blue staining. ( The same cells were analyzed using a

propidium iodide (PI) /Annexin-V double label (see Methods). ELITE Flow

Cytometry (Beckman Coulter) and Cell Quest software was used to analyze the

different cell populations. Morphology of untreated © and treated (3mM AA) (d)

colon adenocarcinoma (HT29) cells. Cells were observed with a light microscope

using the phase contrast setting.

doi:10.1371/journal.pone.0004409.g004

AA and tumor progression

To test the potential effect of AA treatment in vivo, human tumor cells were

implanted by subcutaneous injection in nude mice. Colon adenocarcinoma cells

(HT29) have been used since they were sensitive to AA treatment in vitro (Fig.

S1c). Four groups of 7 grafted mice presenting visible tumors, 10 days

post-implantation, were randomly separated. Animals were given a daily

intraperitoneal injection during 1 month, containing either a placebo solution

or AA in concentrations ranging from 15 mg/kg/d to 1000 mg/kg/d. Tumor size was

evaluated every two days for 1 month (Fig. 5a). After 1 month, surviving mice

were sacrificed and tumor weight was evaluated (Fig. 5b and Table S4).

Figure 5. Tumor growth and weight evaluation.

Kaplan Meier survival curves. Gene expression in tumor. HT29 cells were injected

into nude male mice. Mice presenting with a tumor after 10 days were treated

every day with either a placebo (physiological serum) or one of three AA

concentrations. (a) Tumor growth in placebo-treated mice and mice treated with a

daily intraperitoneal injection of AA (1000 mg/kg/d). ( Mice were sacrificed

after 1 month of a daily AA treatment and tumors were weighted to determine the

effect of AA treatment. An asterisk indicates a P value<0,05. © Kaplan-Meier

survival curves of mice treated with placebo or with 15 mg/kg/d, 100 mg/kg/d, or

1000 mg/kg/d AA. Once visible tumors were established (10 days after injection)

mice were randomized and treated either with ascorbic acid or with placebo.

Comparison of placebo treated curve and 1000 mg/kg/d treated animals, using a

long rank statistical test, indicates a high statistical significance (P value =

0.0022). (d) Relative quantification of mRNA coding for tRNA synthetase and a

translation initiation factor subunit using qPCR technology and Universal Probe

Library (Roche). An asterisk indicates a P value<0,05.

doi:10.1371/journal.pone.0004409.g005

Tumor growth was clearly reduced in animals receiving the highest concentration

of AA (1000 mg/kg/d) when compared to the placebo group (Fig. 5a, . All of the

seven grafted mice in the 1000 mg/kg/d group were alive at the end of 1 month

(Fig. 5c). On the contrary, tumor growth was not affected in the 15 mg/kg/d

group (Fig. 5 . Moreover, only three of the seven grafted mice were still

alive at the end of the assay the others having died abruptly during the

experiment (Fig. 5c). An intermediate situation was observed in animal treated

with 100 mg/kg/d (Fig. 5b and c). A summary is presented in Table S4. The exact

cause of death in animal cohorts treated either with the lower AA concentration

or placebo is not easy to define. However, autopsies of animals sacrificed after

30 days or from spontaneous dead animals, revealed a massive metastatic spread

with peritoneal (Fig. S2a and S2b) as well as peri hepatic carcinosis (Fig.

S2c). Therefore, we propose that spontaneous death of animals that have not been

treated with the highest dose of AA is due to a highly invasive carcinogenic

process that is not observed in animals treated with 1000 mg/days. This suggests

that AA could also protect from metastatic invasion, although this hypothesis

should be confirmed in further experiments.

At the end of the treatment period, total RNAs were extracted after tumor

dissection, and subjected to RT-qPCR to evaluate the deregulation of the gene

expression. Consistently with the data obtained in vitro, tRNA synthetase and

translation initiation factor subunits genes were under expressed in grafted

tumors after AA treatment, in a dose-dependent manner (Fig. 5d). Hence we

demonstrated that AA inhibits the expression of the same genes in vitro as well

as in vivo.

Discussion

Ascorbic acid is most well known as Vitamin C, the nutritional supplement

essential for preventing scurvy. The recommended daily dose has varied over

time, but it is presently about 75 to 90 mg/day. However, several authors, Linus

ing among them, have suggested that higher daily doses might prevent cancer

[12]. This has been a hotly debated topic in the scientific community. Although

several papers have described the effect of AA on cell proliferation [13]-[15],

the antiproliferative effects were always ascribed to the antioxidant properties

of AA [13].

Based on the experiments described in this paper, we propose that AA inhibits

cell division and further promotes necrosis by down-modulating the expression of

genes necessary for S-phase progression. We found that actively proliferating

but not quiescent cells are susceptible to AA treatment, excluding an

non-specific toxicity of the highest AA concentrations. It is tempting to

speculate the inhibited expression of tRNA synthetases and translation

initiation elongation factor subunits leads to the rapid cessation of energy

production in proliferating cells, resulting in necrotic cell death.

We have recently demonstrated that ascorbic acid is a competitive inhibitor of

adenylate cyclase activity [4], resulting in a decrease of intracellular cAMP

concentration. However, the mechanism underlying the regulation of tRNA

synthetase and ieF subunits expression in mammalian cells are still unknown. In

particular, the role of cAMP remains to be clarified.

Few experiments using animal models, with spontaneous tumors, have been

performed. These studies, using an oral AA administration, report a decreased

mortality for treated animals [16]. In humans, clinical trials results are

mixed; some studies indicate a benefit to patients treated with AA [17], while

others do not reveal any beneficial effect following AA treatment [18], [19]. We

may note that positive trials involved IV injection, in contrast with negative

results that involved oral administration. From data presented in this

manuscript, it is obvious that treatment with increasing doses of AA induces a

specific down regulation of expression of a selected set of genes, resulting in

an arrest of cell proliferation and, at higher doses, in cell death.

Treatment of xenografted animals, either with a placebo or with increasing doses

of AA, allowed us to draw some conclusions:

- Treatment with high doses of AA results in a lowering of tumor progression in

terms of tumor weight.

- Xenografted mice treated with the highest AA concentration survive after 40

days (30 days of treatment plus 10 days of grafting). We have no explanation but

we observed numerous carcinogenic invasion, in placebo treated animals and in

animals treated with lower doses of AA. In contrast, animals treated with 1000

mg/days did not present carcinogenic invasion. It has been described in the

literature [20]-[22] that HT29 cells are able to form metastatic carcinogenic

colonies (in colon and liver). In addition, Tremblay et al demonstrate that HT29

are able to perform diapedesis.

- Genes that have been demonstrated to be down regulated in cellular in vitro

experiments have also been found to be down regulated in vivo, and more

precisely in tumors.

In conclusion, in terms of anticancer therapy, it appears that AA treatment will

only be effective if a high enough concentration of AA can be reached (probably

higher than 1mM). According to published data from healthy volunteers [23], high

concentrations may only be reached by intravenous injection. These high doses

have been found not to be toxic in animal as well as in human [24], [25].

Clinical trials in which patients, with advanced cancer, receiving injections of

high AA doses would shed some light on the therapeutic utility of AA.

Supporting Information

Figure S1.

Tunnel enzymatic labelling assay and cell density analysis in cellular junction

conditions. (a) Detection and quantification of apoptotic cells by a TUNEL assay

using the In-Situ Cell Death Detection Kit, TMR red (Roche Applied Science) in

accordance with the manufacturer's instructions. Analyses of apoptosis following

treatment with two different AA concentrations (0.6 mM and 2 mM) were conducted

on human primary fibroblasts. ( Analysis the effect of increasing amounts of

AA on confluent cultures of human primary fibroblasts. © Growth curves of HT29

cells (evaluated using a Neubauer hemocytometer) incubated with either 3 mM of

AA or medium containing no AA.

(0.85 MB EPS)

Figure S2.

Animals treated with a placebo, have been sacrificed after 30 days of treatment

(plus 10 days of grafting) and autopsied. All animals present either peritoneal

carcinogenic invasion (a and and/or peri hepatic carcinogenesis ©. Animals

treated with the highest dose did not present any invasion.

(4.47 MB EPS)

Table S1.

(0.05 MB DOC)

Table S2.

(0.02 MB DOC)

Table S3.

(0.02 MB DOC)

Table S4.

(0.02 MB DOC)

Acknowledgments

We would like to thank C.Chabannon and A.M.Imbert for providing cell lines and

for excellent technical counselling. We thank Dr Prevot for his advise

during the FACS experiments.

Author Contributions

Conceived and designed the experiments: SB MF. Performed the experiments: SB FK

SG. Analyzed the data: SB FK GD JC MF. Contributed reagents/materials/analysis

tools: FK GD SG JC. Wrote the paper: MF.

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