SEARCH

SEARCH BY CITATION

Keywords:

  • Chloroquine;
  • colon cancer;
  • autophagy;
  • 5-fluorouracil;
  • cell cycle

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Autophagy is a conserved catabolic process that degrades cytoplasmic proteins and organelles for recycling. The role of autophagy in tumorigenesis is controversial because autophagy can be either protective or damaging to tumor cells, and its effects may change during tumor progression. A number of cancer cell lines have been exposed to chloroquine, an anti-malarial drug, with the aim of inhibiting cell growth and inducing cell death. In addition, chloroquine inhibits a late phase of autophagy. This study was conducted to investigate the anti-cancer effect of autophagy inhibition, using chloroquine together with 5-fluorouracil (5-FU) in a colon cancer cell line. Human colon cancer DLD-1 cells were treated with 5-FU (10 μΜ) or chloroquine (100 μΜ), or a combination of both. Autophagy was evaluated by western blot analysis of microtubule-associated protein light chain3 (LC3). Proliferative activity, alterations of the cell cycle, and apoptosis were measured by MTT assays, flow cytometry, and western blotting. LC3-II protein increased after treatment with 5-FU, and chloroquine potentiated the cytotoxicity of 5-FU. MTT assays showed that 5-FU inhibited proliferation of the DLD-1 cells and that chloroquine enhanced this inhibitory effect of 5-FU. The combination of 5-FU and chloroquine induced G1 arrest, up-regulation of p27 and p53, and down-regulation of CDK2 and cyclin D1. These results suggest that chloroquine may potentiate the anti-cancer effect of 5-FU via cell cycle inhibition. Chloroquine potentiates the anti-cancer effect of 5-FU in colon cancer cells. Supplementation of conventional chemotherapy with chloroquine may provide a new cancer therapy modality.

Autophagy, from the Greek, means ‘self-eating’, and is a conserved eukaryotic catabolic process involving the degradation and recycling of cytoplasmic proteins and organelles in response to stress or starvation. At the outset of autophagy, double-membrane structures called autophagosomes form in the cytosol and sequester cytoplasmic material. These autophagosomes fuse with lysosomes to form autolysosomes, and the sequestered material is degraded by lysosomal enzymes. Autophagy plays important roles in survival, development, and homeostasis, as well as in pathologic conditions such as neurodegenerative disease, infection, immune responses, and cancer [1-3]. However, the role of autophagy in tumorigenesis remains controversial, because it can both protect and destroy tumor cells, and its role may change during tumor progression [3, 4].

There are several autophagy inhibitors that can be used to study the role of autophagy. Chloroquine is one of the autophage inhibitors. It is lysosomotropic and inhibits the fusion of autophagosomes and lysosomes. Chloroquine is widely used as an anti-malarial, and also as an anti-inflammatory in the treatment of rheumatoid arthritis and lupus erythematosus. Recently, attention has focused on its potential as an anti-cancer agent and chemotherapy sensitizer [5, 6]. Although the precise basis of the anti-cancer effects of chloroquine is still under investigation, inhibition of autophagy most probably plays a part in anti-tumoral effect.

Colon cancer cell lines are resistant to nutritional deprivation due to activation of autophagy [7]. A previous report showed that activation of autophagy contributes to the survival of colon cancer cells, and that inhibition of autophagy augments 5-fluorouracil (5-FU)-induced apoptosis of colon cancer cells [8]. In addition, it was recently reported that chloroquine enhanced the effects of 5-FU chemotherapy in a human colon cancer cell line [6].

The aim of this study was to investigate the anti-cancer effect of combination therapy with chloroquine and 5-FU in a colon cancer cell line.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Cell and cell culture

The DLD-1 colon cancer cells were obtained from the Korean cell line bank. They were cultured in RPMI 1640 medium (Gibco-BRL, Gaithrsburg, MD, USA) containing 10% fetal bovine serum (Gibco-BRL) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Sigma Chemical, St Louis, MO, USA) in a humidified 5% CO2 incubator at 37 °C.

Reagents and antibodies

Chloroquine and 5-FU were purchased from Sigma (Sigma Chemical). Anti-LC3B, p-mTOR, p-PTEN, and p-PDK1 antibodies were obtained using Cell Signaling Technology (Cell signaling Technology, Beverly, MA, USA). Anti-p53, p27, cyclin A, cyclin D1, cyclin E, CDK2, and CDK4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). To determine the dose of 5-FU for 50% inhibition of proliferation (IC50), DLD-1 cells were treated with 0.1, 1, 5, 10, 50, 100 μM 5-FU for 24, 48, and 72 h. In addition, to establish the dose of chloroquine that inhibits autophagy without affecting proliferation, DLD-1 cells were treated with 0.1, 1, 10, 100 μM chloroquine for 12 h. For treatment with 5-FU and chloroquine, DLD-1 cells were pre-treated with the determined dose of chloroquine for 12 h, and after changing the medium to remove the chloroquine they were exposed to 5-FU [6].

Growth inhibition

Growth inhibition by chloroquine or 5-FU was measured with MTT (3-[4,5-dimetylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma Chemical) as described previously [9]. Briefly, 2 × 103 cells/well were seeded in 96-well microtiter plates (Nunc, Roskilde, Denmark) and incubated at 37 °C for 48 and 72 h. MTT solution (50 mL) from Sigma (2 μg/mL in PBS; Sigma Chemical) was added to each well, and the plates were incubated for an additional 4 h at 37 °C, and the MTT solution was aspirated off. To solubilize the formazan crystals formed in viable cells, 200 μL of DMSO was added to each well. The plates were shaken for 30 min at room temperature, and absorbances were read immediately at a wavelength of 540 nm using a scanning multiwell spectrophotometer (Titert Multiscan MC; Flow Laboratory, CA, USA).

Cell cycle analysis

Cells were washed twice with PBS, fixed with methanol for 1 h, washed with PBS (Gibco-BRL), and stained with 50 μg/mL of propidium iodide (PI; Sigma Chemical) containing 50 μg/mL of RNase A (Sigma Chemical). The DNA contents of the cells (10 000 cells/experimental group) were analyzed using a FACStar flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a ModFit LT program (LYSIS II, CELLFIT). The percentages of the cell population in each cell cycle phase (G1, S or G2/M) were calculated from the DNA content histograms.

Evaluation of apoptosis

Apoptosis was determined by staining cells with annexin V-FITC and PI because annexin V can identify the externalization of phosphatidylserine during the progression of apoptosis and therefore can detect early apoptotic cells [10]. To quantify apoptosis, cells were washed twice with cold PBS (Gibco-BRL) and resuspended in binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at 1 × 106 cells/mL. A quantity of 100 μL of this suspension was transferred to a 5 mL culture tube with 5 mL of annexin V-FITC (Pharmingen, San Diego, CA, USA) and 10 mL of 20 μg/mL PI, and analyzed using the FACStar flow cytometer.

Western blot analysis

Cells were washed with PBS, suspended in lysis buffer containing 50 mM Tris (pH 7.5), 1% NP-40, 2 mM EDTA, 10 mM NaCl, 20 μg/mL aprotinin, 20 μg/mL leupeptin, and 1 mM phenylmethylsulphonyl fluoride, and placed on ice for 20 min. Samples containing 20–30 μg of total protein were separated on 10–15% SDS-polyacrylamide denaturing gels and transferred to nitrocellulose membranes for 90–120 min. The blotted membranes were blocked with 5% skim milk for 1 h and incubated overnight with the following primary antibodies at 1:1000 dilutions: a rabbit monoclonal antibody against LC3B for autophagy, anti-p-mTOR, p-PDK1, and p-PTEN for the PI3K-mTOR pathway, and anti-p53, p27, cyclin A, cyclin D1, cyclin E, CDK2 and CDK4 for cell cycle proteins. The blots were developed with an ECL kit (Intron, Biotechnology, Seongnam, Korea).

Quantitative real-time PCR

Total RNAs were isolated with TRI reagent (Molecular Research Center, Inc. Cincinnati, OH, USA), and cDNAs were synthesized from 1 μg of total RNA using ImProm-II Reverse Transcriptase (Promega Corporation, Madison, WI, USA) using random hexamers.

Quantitative PCR was performed using an iCycler IQ detection system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green I as a double-strand DNA-specific binding dye. Thermocycling was performed in a final volume of 20 μL containing 4 μL cDNA sample, 10 pmol of each primer, 0.125 mM dNTP mixture, 0.25 mg/mL BSA, 0.05% Tween 20, 1× rTaq reaction buffer containing 1.5 mM MgCl2 (Takara, Japan), 1 unit rTaq DNA polymerase (Takara, Japan), and 1× SYBR Green 1 (Molecular probe). After an initial denaturation at 95 °C for 10 min, 35 cycles at 94 °C for 30 s, annealing at 62 °C for 30 s, and at 72 °C for 30 s were carried out. PCR amplification was carried with the following primers: mdm2 sense 5′-CGCGCCCCGRGAAGGAAACT-3′ and antisense 5′-TGCTCCTCACCATCCGGGGT-3′; GAPDH sense 5′- ACTGATTTGGTCTATTGGGCG-3′ and antisense 5′- CTCCTGGAAGATGGTGATGG-3′. All cDNA samples were synthesized in parallel, and PCR reactions were run in triplicate. The mRNA levels were derived from standard curves and are expressed as relative changes after normalization vs β-actin mRNA levels. The comparative threshold cycle (2−ΔΔCT) method was used to enable quantification of the mRNA of the gene. All samples were performed in triplets. The relative amount of target gene was calculated using 2−ΔΔCT method. The relative amplification efficiencies of those primers were tested and shown to be similar.

Immunofluorescence for microtubule-associated protein light chain3

Immunofluorsecence for microtubule-associated protein light chain3 (LC3) was performed as described previously [11, 12]. Cells on the cover slide were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. After fixation, the cells were permeabilized with 0.1% Triton X-100 for 15 min at room temperature and blocked with PBS containing 2% BSA (Sigma Chemical) for 1 h at room temperature. Cells were then incubated with anti-LC3 (1:200 diluted in PBS containing 2% BSA) antibody for overnight at 4 °C. After being washed with PBS, cells were incubated with Alexa Fluor 488 conjugated anti-rabbit antibody (1:1000 diluted in PBS containing 2% BSA; Molecular probe, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Cells were lysozyme stained with LysoTracker Red (Invitrogen). Slides were mounted and examined using a Zeiss LSM510 META confocal microscope and Zeiss LSM510 v.3.2 software (Carl Zeiss, Jena, Germany).

Statistical analysis

The data shown are means ± SEM (error bars). Differences were analyzed with Student's t-tests. The p-values <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Effect of 5-FU and chloroquine in autophagy

Autophagy was evaluated by using western blot analysis of microtubule-associated protein light chain3 (LC3). During autophagy, LC3 is converted from a soluble cytosolic form (LC3-I) to a membrane-bound form (LC3-II), which is conjugated with phosphatidylethanolamine for insertion into the autophagosome membrane. LC3-II is a measure of autophagy, because the amount of LC3-II is a function of the number of autophagosomes [13].

The level of LC3-II increased after treatment for 4 h with 5-FU alone, and it increases much more in response to chloroquine with or without 5-FU. The pattern of LC3 –II expression after 24 h treatment was similar, but the amounts of LC3-II protein were reduced (Fig. 1).

image

Figure 1. Western blot analysis of LC3B-II expression at 4 h and 24 h. LC3B-II protein increaseed after treatment with 5-FU alone for 4 h; there was a much greater increase in response to chloroquine with or without 5-FU.

Download figure to PowerPoint

These results showed that 5-FU induced the autophagy activation and autophagy process occurred within several hours after treatment with drug. Since chloroquine inhibits the fusion of the autophagosme and lysosome, and then, induces accumulation of autophagosome, the LC3-II expression level increased after chloroquine treatment.

Synergetic effect of 5-FU/chloroquine in growth inhibition of DLD-1 cell

The viability of DLD-1 cells was determined using MTT assay. Treatment with 5-FU for 72 h reduced the number of viable cells, with an IC50 of 5–10 μM (Fig. 2A). Chloroquine, at doses up to 100 μM for 12 h, had little effect on cell viability (Fig. 2B). Based on these results, we pretreated DLD-1 cells with 100 μM of chloroquine for 12 h, and then exposed them to 10 μM of 5-FU in subsequent experiments. As shown in Fig. 2C,D, chloroquine enhanced the 5-FU-induced growth inhibition of DLD-1 cells.

image

Figure 2. Effects of 5-FU and chloroquine on cell viability. (A) Viability of DLD-1 cells decreased in a dose-dependent manner after 5-FU treatment for 72 h, with an IC50 of 5–10 μM. (B) Chloroquine at doses below 100 μM for 12 h had almost no inhibitory effect on DLD-1. Chloroquine enhanced 5-FU induced growth inhibition of DLD-1 colon cancer cells at (C) 48 h and (D) 72 h.

Download figure to PowerPoint

Effect of 5-FU and chloroquine in cell cycle

The effects of chloroquine and/or 5-FU on the cell cycle were determined by fluorescence-activated cell sorting (FACS) using propidium iodide. As shown in Fig. 3, treatment of DLD-1 cells with 5-FU for 72 h led to an accumulation of cells in S-phase. Pre-treatment with chloroquine followed by 5-FU induced more G1 arrest than 5-FU alone (61.6% vs 43.0%). In addition, the combination of 5-FU and chloroquine induced down-regulation of CDK2 and cyclin D1 and up-regulation of p27 and p53 with little detectable change in CDK4, cyclin A and cyclin E protein (Fig. 4A). These results suggest that the anti-proliferative effect of chloroquine may be due to G1 cell cycle arrest.

image

Figure 3. Effects of 5-FU, chloroquine and the combination, on the cell cycle. Cell cycle parameters were determined by FACS analysis using propidium iodide. 5-FU treatment for 72 h leads to accumulation of cells in S-phase. Pre-treatment with chloroquine followed by 5-FU, but not 5-FU alone, induced a G1 arrest (p 0.05).

Download figure to PowerPoint

image

Figure 4. Western blot analysis of the cell cycle and mTOR pathway. (A) 5-FU and chloroquine together cause up-regulation of p27 and p53, and down-regulation of CDK2 and cyclin D1. (B) 5-FU treatment of DLD-1 cells increased p-PTEN, and decreased p-PDK1 and p-mTOR, but pre-treatment of cells with chloroquine opposed these changes.

Download figure to PowerPoint

The PI3K-mTOR pathway and apoptosis

Examination of the effect on PI3K-mTOR pathway showed that 5-FU treatment increased p-PTEN and decreased p-PDK1 and p-mTOR. This suggests that 5-FU inhibits the PI3K-mTOR pathway by up-regulating PTEN. However, pre-treatment with chloroquine reversed these changes (Fig. 4B).

Regarding the apoptosis, FACS analysis revealed that pre-treatment of DLD-1 cells with chloroquine did not increase the sub-G1 population (Fig. 3), and also did not increase the proportion of annexin V-staining cells (data not shown). These results suggest that apoptosis may not be the dominant mechanism by which pre-treatment with chloroquine followed by 5-FU shows the synergistic inhibitory effect of proliferation of DLD-1 cells.

p53 reactivation by 5-FU and accumulation of p53

DLD-1 cells used in this study are known to carry mutated-p53 transcription factor. Since 5-FU up-regulated p53 protein in this study, we examined the status of mdm2 and PUMA (p53 up-regulated modulator of apoptosis) molecule in 5-FU-treated DLD-1 cells to evaluate whether or not mutated-p53 protein in DLD-1 cells was reactivated by 5-FU. Mdm2 is a critical negative regulator of p53 molecule. When DLD-1 cells were treated with 10 μM of 5-FU for 72 h, mRNA level of mdm2 was down-regulated (Fig. 5A). In addition, treatment of chloroquine alone, 5-FU alone, or combination of both agents increased the protein level of PUMA which is a p53 down-stream molecule (Fig. 5B). These results indicated that 5-FU reactivated mutated p53 in DLD-1 cells.

image

Figure 5. Reactivation of mutated p53 in DLD-1 by 5-FU. (A) 5-FU-treated DLD-1 cells were down-regulated mdm2 for 72 h by real-time qPCR. (B) 5-FU, chloroquine and combination treatment of DLD-1 cells were much more up-regulated PUMA than control.

Download figure to PowerPoint

Synergetic effect of 5-FU/chloroquine in LC3 accumulation in DLD-1 cells

In the control cells, only few labeled LC3 were shown in the cytoplasm. However, chloroquine increased the autophagosome-associated LC3 protein in DLD-1 cells. Combined treatment of 5-FU/ chloroquine stained the LC3 protein more strongly compared to treatment of 5-FU only. More importantly, these autophagosomes co-localized with the lysosomotropic dye, LysoTracker RED (Fig. 6).

image

Figure 6. Detection of LC3 by confocal microscope. DLD-1 cells were treated chloroquine, 5-FU or combination for indicated time. DLD-1 cells were fixed and stained for anti-LC3 (Green) and with LysoTracker (Red).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

In this study, we found that chloroquine enhanced the anti-proliferative effect of 5-FU in DLD-1 colon cancer cells, and the combination of 5-FU and chloroquine induced G1 arrest, up-regulation of p27 and p53, and down-regulation of CDK2 and cyclin D1. However, chloroquine did not induce the apoptosis. These results suggest that the cell growth inhibitory effect of chloroquine may be associated with G1 arrest of cell cycle rather than apoptosis.

Before performing this study, we had hypothesized that pre-treatment of the colon cancer cells with chloroquine to inhibit the autophagy would enhance the anti-cancer activity of 5-FU by activating the apoptosis. A recent report showed that inhibition of autophagy using 3-methyladenine augmented 5-FU induced apoptosis in human colon cancer cell lines [8]. In addition, this synergistic effect was also seen in a DLD-1 xenograft tumor model. These results showed that 5-FU induced the autophagy activation and the autophagy process occurred within several hours after treatment of drug. Since chloroquine inhibits the fusion of the autophagosome and lysosome, and then, induces accumulation of autophagosome, the LC3-II expression level is increased after chloroquine treatment. In addition, LC3 (cytoplasmic vacuoles by confocal microscope) was detected in chloroquine or 5-FU/chloroquine treated DLD-1 cells.

There are several reports of the effects of chloroquine on the cell cycle. A recent study revealed that chloroquine protected against breast cancer in a p53-dependent manner [14]. In that study, chloroquine reduced the incidence of N-methyl-N-nitrosourea-induced mammary tumors and the growth rate of tumors in animal models. Chloroquine induced up-regulation of tumor suppressor p53 and the p53 downstream target gene p21, leading to G1 cell cycle arrest. In addition, p27 was reported to be up-regulated after chloroquine treatment, and p27 also played a role in cell cycle arrest in that study. On the contrary, in our study, neither chloroquine nor 5-FU alone induced G1 arrest, but the combination did, and the same was true for up-regulation of p27. The differences between our observations and those results may be resulted from the relatively short exposure time of chloroquine in our experiments or different cancer cell line. Our results suggest that chloroquine may enhance the anti-cancer effect of 5-FU in colon cancer cells by promoting p27-induced cell cycle arrest.

DLD-1 was known to be a mutated p53 transcription factor. Therefore, it might not be reasonable that synergistic anti-tumor effect of 5-FU and chloroquine was induced via up-regulation of p53 in our experiment. However, many evidences have been accumulated that small molecules could reactivate the p53 mutant, and 5-FU is one of the small molecules. Aizu et al. demonstrated that AJ02-MM cells were not mutated p53 gene, but lacked in p53 DNA-binding activity. However, treatment of AJ02-MM cells with 5-FU resulted in the reversal of p53 DNA-binding activity [15]. Endo et al. also reported that up-regulation of p53 protein could be seen in NUCG-1, mutated p53 gastric cell line, after 5-FU treatment for 24 h [16]. In our experiments, PUMA was up-regulated, and mdm2 was down-regulated after treatment of 5-FU and chloroquine, indicating that 5-FU might reactivate the mutated p53 in DLD-1 cells.

The mammalian class I phosphatidylinositol-3-kinase (PI3K)-target of rapamycin (mTOR) pathway plays a key role in regulation of cell growth, protein translation, metabolism, and autophagy, and alterations of the mTOR pathway are common in many types of cancer [3, 17, 18]. The class I PI3K generates PIP3, which binds to AKT and PDK1. AKT activates kinase-mTOR signaling, which suppresses autophagy. The tumor suppressor PTEN inhibits class I PI3K and allows the initiation of autophagy. Recently, p53 activation was shown to inhibit mTOR activity and affect its downstream targets, such as autophagy, by activating AMP kinase followed by the tuberous sclerosis (TSC) 1/TSC2 complex [19]. Other investigators have reported that 5-FU treatment increased p53 expression in colon cancer cells at the translational level [20]. We also found that 5-FU treatment caused up-regulation of p53 and p-PTEN, followed by reductions in p-PDK1 and p-mTOR, and the eventual initiation of autophagy. These results are consistent with previous studies [8], and suggest that 5-FU may suppress the mTOR pathway by up-regulating p53 and PTEN. Chloroquine is known to block the fusion of the autophagosome and lysosome. Therefore, chloroquine pre-treatment appeared to have the opposite effects to 5-FU on PTEN, PDK1, and mTOR expression in this study.

The present study had several limitations. We used only one colon cancer cell line, and did not perform any in vivo expression experiments. In addition, the expression of cell cycle proteins was assessed by western blotting only. More precise results could be obtained by immunoprecipitation and kinase assays. Nevertheless, we have shown that chloroquine potentiates the anti-cancer effects of 5-FU in DLD-1 colon cancer cells by affecting the cell cycle. Our results indicate that chloroquine may be an effective chemotherapy sensitizer, and supplementation of conventional chemotherapy with chloroquine may provide a novel therapeutic modality in colon cancer.

This work was supported by the research fund of Hanyang University (HY-2009-MC).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References