In the present article, we describe a mechanistic study of a novel derivative of N-amidino-substituted benzimidazo[1,2-α]quinoline in two human colorectal cancer cell lines differing in p53 gene status. We used a proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry to complement the results obtained by common molecular biology methods for analyzing cell proliferation, cell cycle, and apoptosis. Tested quinoline derivative inhibited colon cancer cell growth, whereby p53 gene status seemed to be critical for its differential response patterns. DNA damage and oxidative stress are likely to be the common triggers of molecular events underlying its antiproliferative effects. In HCT 116 (wild-type p53), this compound induced a p53-dependent response resulting in accumulation of the G1- and S-phase cells and induction of apoptosis via both caspase-3-dependent and caspase-independent pathways. Quinoline derivative triggered transient, p53-independent G2-M arrest in mutant p53 cells (SW620) and succeeding mitotic transition, whereby these cells underwent cell death probably due to aberrant mitosis (mitotic catastrophe). Proteomic approach used in this study proved to be a valuable tool for investigating cancer cell response to newly synthesized compound, as it specifically unraveled some molecular changes that would not have been otherwise detected (e.g., up-regulation of the p53-dependent chemotherapeutic response marker maspin in HCT 116 and impairment in ribosome biogenesis in SW620). Finally, antiproliferative effects of tested quinoline derivative on SW620 cells strongly support its possible role as an antimetastatic agent and encourage further in vivo studies on the chemotherapeutic potential of this compound against colorectal carcinoma. [Mol Cancer Ther 2008;7(7):2121–32]

Advances in understanding the neoplastic progression of colorectal cancer at the cellular and molecular levels have fostered development of new drugs, some of which have already been implemented into clinical practice, namely targeted therapy including monoclonal antibodies specifically designed to target growth factors and to prevent transduction of their signals within tumor cells. However, current therapeutic options for colorectal cancer have yielded partial success, as these drugs often prove to be ineffective, causing either adverse side effects or drug resistance. Therefore, the need for designing new agents against colorectal cancer still remains a priority.

In our previous study (1), we reported on the synthesis and DNA-binding properties of the novel cyclic amidino-substituted derivative of benzimidazo[1,2-a]quinoline (Supplementary Fig. S1).4

4

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

The cell-free in vitro topoisomerase II inhibitor screening assay showed this derivative to prevent formation of topoisomerase II relaxation products, which supports its role as a catalytic inhibitor of topoisomerase II. In addition, we established that this compound has an ability to intercalate into ct-DNA via its benzimidazo[1,2-a]quinoline moiety, pointing to its DNA-damaging property. Evaluation of the antitumor effects of quinoline derivative against a panel of five different human cancer cell lines showed this compound to exert highly selective cytostatic activity for SW620 colorectal adenocarcinoma cells (1). This finding has elicited the need to further investigate the chemotherapeutic potential of the aforementioned quinoline derivative for colorectal cancer.

As cancer cells are characterized by the ability to divide and multiply in an uncontrolled manner whereby a set of specific proteins modulate cell division processes, large-scale protein expression profiling technologies seem to be a suitable tool for seeking out molecular mediators of anticancer drug action and resistance (2). Proteomics holds great potential in monitoring therapeutic and toxic effects of newly developed antitumor compounds, as it might give a clue to which signaling pathways modulating particular molecular events are responsible for cancer cell response to a drug.

We hypothesized that response of colon cancer cells to quinoline derivative possessing DNA-damaging ability is predetermined by the functional status of their p53 gene. Therefore, our main goal was to investigate the molecular mechanisms by which this compound inhibits the growth of HCT 116 and SW620 colon cancer cells differing in p53 status and to determine the role of p53 in these mechanisms. Here, common molecular biology methods for analyzing cell proliferation rate, cell cycle progression, and apoptosis induction were complemented by proteomic analysis based on two-dimensional gel electrophoresis combined with mass spectrometric identification of selected proteins.

Test Compound

Novel amidino-substituted derivative of benzimidazo[1,2-a]quinoline was synthesized in the group of Professor Grace Karminski-Zamola (Department of Organic Chemistry, Faculty of Chemical Engineering and Technology in Zagreb) as described previously (1).

Cell Lines and Culture Conditions

The human colorectal adenocarcinoma SW620 (mutated p53) and carcinoma HCT 116 [wild-type p53 (wt-p53)] cell lines were obtained from the American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FCS (Invitrogen/Life Technologies), 2 mmol/L l-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen/Life Technologies) and maintained in a humidified atmosphere containing 5% CO2 at 37°C.

Cell Proliferation/Viability Assay

Effect of quinoline derivative on the proliferative capacity of the colon cancer cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described previously (3). Results were expressed as IC50 (the concentration that causes 50% growth inhibition) calculated from the dose-response curves using linear regression analysis (1).

DNA Cell Cycle Analysis

Flow cytometric analysis was used to measure the DNA content of the cells (4). Briefly, 2 × 105 cells were seeded per well in six-well plates. After an overnight incubation, quinoline derivative was added and the cells were cultured for 24, 48, and 72 h. The cells were then harvested by trypsinization, washed thrice with PBS, fixed with cold ethanol, and stored at -20°C. Immediately before analysis, cell suspension was centrifuged, the pellet was washed twice with PBS and incubated with 0.1 μg/μL RNase A at 37°C for 15 min followed by staining with propidium iodide (PI; 1 μg/mL final concentration) for 30 min in the dark. Analysis was carried out using FACSCalibur instrument (BD Biosciences), whereby ∼20,000 events were recorded for each sample. WinMDI 2.8 (The Scripps Institute) and Cylchred (Cardiff University) software were used to determine the percentage of the cells in different phases of the cell cycle.

Quantification of Apoptotic Cells

Induction of apoptosis was determined by flow cytometry using the Annexin V-FLUOS Staining Kit (Roche) following the instructions of the manufacturer. Analyses were done by FACSCalibur instrument (BD Biosciences). Summit V3.1 software (Cytomation) was used for offline fluorescence compensation.

Two-Dimensional Gel Electrophoresis

Cells were lysed in two-dimensional gel electrophoresis lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, and 1% DTT) supplemented with 0.2% Bio-Lyte ampholyte (pH 3-10; Bio-Rad), nuclease mix (GE Healthcare), and protease inhibitor cocktail (Roche). Total proteins (800 μg) were solubilized in 330 μL rehydration buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 1% DTT, and 0.2% Bio-Lyte ampholyte, pH 3-10), loaded onto 17-cm IPG strips, pH 3-10NL (Bio-Rad), and subjected to isoelectric focusing on PROTEAN IEF cell (Bio-Rad) for ∼100,000 V h. Subsequently, proteins were resolved on the vertical 12% polyacrylamide gels in the Laemmli Tris-glycine buffer system (5) by the PROTEAN II XL cell (Bio-Rad). Obtained gels were stained with Colloidal Coomassie Blue (Bio-Rad; ref. 6) and scanned by the VersaDoc Imaging System 4000 (Bio-Rad). Image analysis was carried out using PDQuest software version 7.0 (Bio-Rad), whereby total density in gel image was used as normalization method. Protein spots with >3-fold change in intensity level were selected for subsequent mass spectrometric identification. For each experimental condition, gels were run in tetraplicate.

Mass Spectrometry

Differentially expressed protein spots were excised from the gels, digested with trypsin, and spotted on matrix-assisted laser desorption/ionization (MALDI) plate as described previously (7). Cyano-4-hydroxycinnamic acid (4 mg) in 65% acetonitrile/35% H2O containing 0.1% trifluoroacetic acid was used as MALDI matrix solution. Samples were analyzed on the 4700 Proteomics Analyzer MALDI-time-of-flight (TOF)/TOF system (Applied Biosystems) equipped with Nd:YAG laser operating at 200 Hz. All mass spectra were recorded from 750 to 4,000 Da in a positive reflector mode and were generated by accumulating data from 5,000 laser pulses. Peptide fragmentation was done at collision energy of 1 kV and collision gas pressure of ∼2 × 10−7 Torr. Global Proteome Server Explorer software version 3.6 (Applied Biosystems) was used for submitting mass spectrometry (MS) and tandem MS data for database searching, and Mascot version 2.1.0 (Matrix Science) was used as the search engine. Database searching of MS and tandem MS spectra was done using a human protein sequence database downloaded from the EBI (40794 sequences; release date: 19th March2005; source: ftp.ebi.ac.uk/pub/databases/SPproteomes/fasta/proteomes/25.H_sapiens.fasta.gz).

Western Blot Analysis

Cells were lysed in the buffer containing 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.2 mmol/L EGTA, 10% glycerol, 1% Triton X-100, and protease inhibitor cocktail. Total proteins (30 μg) were resolved on 8% to 12% Tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Subsequently, membranes were blocked for 1 h at room temperature with 4% nonfat dry milk in TBST [50 mmol/L Tris base, 150 mmol/L NaCl, 0.1% Tween 20 (pH 7.5)] and probed overnight at 4°C with primary antibodies raised against cyclin B1, apoptosis-inducing factor (AIF), and caspase-3 (Santa Cruz Biotechnology; dilutions 1:200, 1:600, and 1:100, respectively), procaspase-3 and p21 (BD Biosciences; dilutions 1:400 and 1:250, respectively), and p53 (Calbiochem; dilution 1:200). Membranes were washed with TBST and incubated with an anti-mouse (Amersham Biosciences) or anti-rabbit (DakoCytomation) horseradish peroxidase–conjugated secondary antibody at room temperature for 1 h. Individual proteins were visualized by the Western Lightening Chemiluminescence Reagent Plus kit (Perkin-Elmer). Signal intensities of the particular bands were quantified by the Quantity One software (Bio-Rad). α-Tubulin (Sigma-Aldrich; dilution 1:1,000) was used as a loading control.

Statistical Analysis

All experiments were done in triplicate. Differences between control and treated cells were assessed using one-way ANOVA and a significance level of P < 0.05 was required.

Quinoline Derivative Suppresses the Proliferation of Colon Cancer Cells

To determine response profile of HCT 116 (p53+/+) cells, we measured viability of the cells treated with five different concentrations of quinoline derivative (0.01-100 μmol/L) for 72 h by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Similarly to SW620 (1), quinoline derivative caused a dose-dependent reduction in HCT 116 cell viability (Fig. 1). Interestingly, we observed a variation between the IC50 values for quinoline derivative in HCT 116 and SW620 (2.5 ± 0.9 and 1.2 ± 0.5 μmol/L, respectively), which is suggestive of differential colon cancer cell response to this compound.

Figure 1.

Tested amidino-substituted derivative of benzimidazo[1,2-a]quinoline inhibits the proliferation of colon cancer cells in a dose-dependent manner. The inhibitory effect on the cell viability of SW620 and HCT 116 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described in Materials and Methods. Points, mean of four replicates in three individual experiments.

Figure 1.

Tested amidino-substituted derivative of benzimidazo[1,2-a]quinoline inhibits the proliferation of colon cancer cells in a dose-dependent manner. The inhibitory effect on the cell viability of SW620 and HCT 116 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described in Materials and Methods. Points, mean of four replicates in three individual experiments.

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Quinoline Derivative Induces Cell Cycle Perturbations in Colon Cancer Cells

We did flow cytometric analysis of the cell cycle distributions of HCT 116 and SW620 cells treated with quinoline derivative at three different concentrations that were in the range of obtained IC50 values (0.5, 1, and 5 μmol/L) for 24, 48, and 72 h.

Treatment of HCT 116 cells with 5 μmol/L quinoline derivative for 24 h resulted in significant accumulation of the cells in G1 phase with concomitant reduction in the S-phase cells (Fig. 2; Supplementary Table S1).4 This pattern of the cell cycle distribution persisted over the next 24 h as well. However, 72-h treatment period revealed a significant increase in the S-phase cell population accompanied by reduced number of the cells that entered G2-M phase of the cell cycle. Interestingly, lower tested concentrations did not yield significant alterations in the cell cycle distribution in this cell line (Supplementary Table S1).4

Figure 2.

DNA histograms of colon cancer cells treated with amidino-substituted derivative of benzimidazo[1,2-a]quinoline. Cells were stained with PI and their relative DNA contents were measured by flow cytometry as described in Materials and Methods.

Figure 2.

DNA histograms of colon cancer cells treated with amidino-substituted derivative of benzimidazo[1,2-a]quinoline. Cells were stained with PI and their relative DNA contents were measured by flow cytometry as described in Materials and Methods.

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It is well known that the tumor suppressor gene p53 (TP53) plays a key role in activation of the G1-S cell cycle checkpoint through its downstream effector, the p21WAF1/Cip1 gene (8, 9). Obtained flow cytometric results opened the door to the possibility that TP53 and its transcriptional target p21WAF1/Cip1 gene might be involved in quinoline derivative-induced cell cycle alterations in HCT 116 cells. Indeed, Western blot analysis of the cells treated with 5 μmol/L quinoline derivative for 24 and 72 h clearly showed accumulation of wt-p53 protein and consequential induction of p21 protein expression (Fig. 3). Expectedly, we noticed an absence of p53 protein expression in nontreated HCT 116 cells (Fig. 3), as wt-p53 protein has short half-life and is rapidly ubiquitinated and degraded by the proteasome in unstressed mammalian cells.

Figure 3.

Western blot analysis of several key regulators of cell cycle and apoptosis in colon cancer cells on treatment with amidino-substituted derivative of benzimidazo[1,2-a]quinoline. HCT 116 and SW620 cells were incubated with 5 μmol/L quinoline derivative for 24 and 72 h. Whole-cell lysates were subjected to SDS-PAGE and probed with the indicated antibodies as described in Materials and Methods. HCT 116: lines 1 to 4, control 24 h, control 72 h, treated 24 h, and treated 72 h, respectively; SW620: lines 5 to 8, control 24 h, control 72 h, treated 24 h, and treated 72 h, respectively. α-Tubulin was used as a loading control. Representative blots from three independent experiments with identical observations.

Figure 3.

Western blot analysis of several key regulators of cell cycle and apoptosis in colon cancer cells on treatment with amidino-substituted derivative of benzimidazo[1,2-a]quinoline. HCT 116 and SW620 cells were incubated with 5 μmol/L quinoline derivative for 24 and 72 h. Whole-cell lysates were subjected to SDS-PAGE and probed with the indicated antibodies as described in Materials and Methods. HCT 116: lines 1 to 4, control 24 h, control 72 h, treated 24 h, and treated 72 h, respectively; SW620: lines 5 to 8, control 24 h, control 72 h, treated 24 h, and treated 72 h, respectively. α-Tubulin was used as a loading control. Representative blots from three independent experiments with identical observations.

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In SW620, treatment with 5 μmol/L quinoline derivative for 24 h induced significant accumulation of the cells in S phase, which was accompanied by marked reduction in G1 cells in comparison with nontreated cells (Fig. 2; Supplementary Table S1).4 However, the cells continued to progress through the cell cycle and arrested in G2-M after 48 and 72 h with concomitant decline in G1- and S-phase cells. Interestingly, although they did not exert significant influence on the cell cycle progression, lower concentrations showed tendency to increase the G2-M population as well (Supplementary Table S1).4

Western blot analysis showed abnormal expression of p53 protein in both treated (5 μmol/L) and nontreated SW620 cells after 24 and 72 h (Fig. 3), which results from the mutation in the DNA-binding domain of the p53 protein that inhibits its ubiquitination, allowing it to accumulate within the cell. This mutated protein is nonfunctional and fails to induce the expression of the p21 protein (Fig. 3). Despite the well-established role of p53 protein in G2-M checkpoint where it prevents the cell with damaged genome from entering mitosis, our findings provide an evidence that profound G2-M arrest in SW620 cells is triggered by the p53-independent mechanisms.

We next examined the levels of the cyclin B1 protein in the cells treated with quinoline derivative (5 μmol/L) for 24 and 72 h using Western blot. Cyclin B1 drives the progression from G2 to mitosis in complex with the cyclin-dependent kinase-1, also known as Cdc2 (10). In treated SW620 cells, we detected a noticeable increase in the intracellular level of cyclin B1 after 24 h (Fig. 3). However, in the same cells treated for 72 h that were undergoing G2-M arrest, we detected tremendous increase in the cyclin B1 level in comparison with nontreated cells. These findings strongly suggest that treated SW620 cells arrest initially in G2 but then escape and enter mitosis. This phenomenon was not observed in HCT 116 cells (Fig. 3).

Quinoline Derivative Triggers Colon Cancer Cell Death

Flow cytometric analysis also revealed statistically significant increase in the sub-G1 population in treated (5 μmol/L) cell lines at all time points tested (Supplementary Table S1),4 indicating cell death, which prompted us to further investigate the influence of quinoline derivative on induction of apoptosis at this concentration. For that purpose, we used Annexin V binding assay as described previously. Hereby, several cell populations were distinguished: normal intact cells (low Annexin V-FLUOS/low PI fluorescence), early apoptotic cells (high Annexin V-FLUOS/low PI fluorescence), and late apoptotic/early necrotic cells (high Annexin V-FLUOS/high PI fluorescence).

Treatment of HCT 116 cells for 24 h increased the percentage of early apoptotic and late apoptotic/early necrotic cells for 2% and 4.4%, respectively (Fig. 4). However, 72-h treatment period resulted in more dramatic changes in cell viability, as we observed significant raise by 15.6% in the proportion of the cells entering early stages of apoptosis. However, a certain portion of cell population proceeded further to terminal phases of apoptosis as evidenced by an increase in the fraction of late apoptotic cells for 5.6% (Fig. 4).

Figure 4.

Tested amidino-substituted derivative of benzimidazo[1,2-a]quinoline elicits apoptotic response in colon cancer cells. Cells were treated with 5 μmol/L quinoline derivative for 24 and 72 h, harvested for analysis of apoptosis using the Annexin V-FLUOS Staining Kit, and analyzed by flow cytometry as detailed in Materials and Methods. PI−/Annexin V−, viable cells; PI−/Annexin V+, early apoptotic cells; PI+/Annexin V+, late apoptotic and/or early necrotic cells. Mean of two replicates in three independent experiments; bars, SD. *, P < 0.05.

Figure 4.

Tested amidino-substituted derivative of benzimidazo[1,2-a]quinoline elicits apoptotic response in colon cancer cells. Cells were treated with 5 μmol/L quinoline derivative for 24 and 72 h, harvested for analysis of apoptosis using the Annexin V-FLUOS Staining Kit, and analyzed by flow cytometry as detailed in Materials and Methods. PI−/Annexin V−, viable cells; PI−/Annexin V+, early apoptotic cells; PI+/Annexin V+, late apoptotic and/or early necrotic cells. Mean of two replicates in three independent experiments; bars, SD. *, P < 0.05.

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We next investigated the role of caspase-3, known to be critically involved in the execution phase of apoptosis, in the quinoline derivative-induced cell death in HCT 116. Activation of caspase-3 is characterized by the cleavage of inactive precursor, 32-kDa procaspase-3 into the p17 and p11 subunits. As shown in Fig. 3, Western blot analysis revealed the appearance of the p17 subunit at 72 h after treatment with 5 μmol/L quinoline derivative, which was accompanied by a 2.5-fold decrease in the procaspase-3 level. These results clearly show that quinoline derivative triggers caspase-3-dependent apoptosis in HCT 116. However, this compound also induced caspase-independent response in HCT 116, as evidenced by the activation of the AIF protein after 72 h (Fig. 3), which is considered to play a central role in the regulation of caspase-independent apoptosis (11).

In SW620, 24-h treatment (5 μmol/L) interval resulted in 2.8% increase in the late apoptotic/necrotic cells (Fig. 4). Similar pattern of cell death was sustained after 72 h as well, because we detected a pronounced rise of 13.5% in the cell population undergoing final stages of apoptosis accompanied by significant increase in the percentage of early apoptotic cells for 2.3% (Fig. 4). Unlike HCT 116, quinoline derivative-induced cell death pathway in SW620 does not seem to involve caspase-3 activation as substantiated by the stable levels of procaspase-3 and an absence of the p17 subunit of the mature caspase-3 in treated cells (Fig. 3). As for caspase-independent pathway, absence of the AIF protein induction throughout the treatment timescale (Fig. 3) implies that the aforementioned cell death pathway is probably not responsible for antiproliferative effects of quinoline derivative in SW620.

Diverse cell death mechanisms between HCT 116 and SW620 induced by quinoline derivative reflect distinct morphologic features of these cells as can be seen in Supplementary Fig. S2.4

Proteomic Changes in Colon Cancer Cells in Response to Quinoline Derivative

To gain a comprehensive insight into molecular processes triggered by quinoline derivative, we carried out proteomic expression profiling of treated (5 μmol/L) versus nontreated cells after 24- and 72-h treatment periods. This experimental approach was based on the separation of protein mixture by two-dimensional gel electrophoresis (Supplementary Fig. S3)4 followed by MALDI-TOF/TOF mass spectrometric identification of differentially expressed protein spots. Biological functions of identified proteins were determined by literature mining using PubMed.5

Proteomic analysis showed the test compound to induce alterations in the expression of 58 and 64 protein species in HCT 116 (Table 1) and SW620 (Table 2), respectively. Importantly, proteomic-derived data substantiated previously observed cellular changes associated with cell proliferation, cell cycle progression, and apoptosis induction by revealing molecular players involved in these processes. In addition, the two-dimensional gel electrophoresis/MS approach uncovered a broad range of other cellular events on the global scale that would not have been otherwise detected as witnessed by alteration in the expression of proteins involved in cell signaling, antioxidant proteins, metabolic enzymes (glycolysis, fatty acid oxidation, and pentose-phosphate pathway), proteins involved in RNA synthesis and processing, regulators of protein synthesis, chaperons, proteins responsible for nucleotide synthesis, proteasomal components and proteases, cytoskeleton-related proteins, proteins indispensable for structural organization of organelles and their proper functioning, and, finally, proteins regulating intracellular sorting.

Table 1.

Proteins differentially expressed in response to amidino-substituted derivative of benzimidazo[1,2-α]quinoline in HCT 116 cells

ProteinAccession no.Expression levelTime (h)Prot. score CI (%)Molecular weight (Da)
Lamin A/C P02545 ↓ 24 100 74,379.8 
Heterogeneous nuclear ribonucleoprotein L P14866 ↓ 24/72 100 60,719.4 
DNA helicase V Q96AE4 ↓ 24 100 67,602.5 
GTP-binding protein 9 Q9NTK5 ↓ 24/72 100 44,943.4 
Heterogeneous nuclear ribonucleoprotein D0 Q14103 ↓ 24 100 38,581.4 
28-kDa heat- and acid-stable phosphoprotein Q13442 ↓ 24 100 20,617.6 
26S proteasome non-ATPase regulatory subunit 4 P55036 ↓ 24 100 40,939.3 
Nascent polypeptide-associated complex subunit α Q13765 ↓ 24/72 100 23,369.7 
Acidic leucine-rich nuclear phosphoprotein 32 family member A P39687 ↓ 24/72 100 28,682.3 
Proteasome subunit α type 3 P25788 ↓ 24/72 100 28,512.1 
Ran-specific GTPase-activating protein P43487 ↓ 24 100 23,466.6 
Heterochromatin protein 1 homologue γ Q13185 ↓ 24 100 20,981.4 
Cold shock domain-containing protein E1 O75534 ↑ 24 100 89,684.2 
Leukotriene A-4 hydrolase P09960 ↑ 24 100 69,737.4 
26S protease regulatory subunit 4 P62191 ↑ 24 100 49,324.8 
Fascin Q16658 ↑ 24/72 100 54,992.2 
Cytosolic nonspecific dipeptidase Q96KP4 ↑ 24 100 53,187 
Multifunctional protein ADE2 P22234 ↑ 24/72 100 47,659.4 
Pyruvate dehydrogenase E1 component subunit α P08559 ↑ 24 100 43,951.9 
26S proteasome non-ATPase regulatory subunit 7 P51665 ↑ 24/72 100 37,059.5 
Cytosolic acyl coenzyme A thioester hydrolase O00154 ↑ 24 100 42,435.6 
Maspin (serpin B5) P36952 ↑ 24/72 100 42,567.6 
Annexin A2 P07355 ↑ 24 100 38,676.9 
Proline synthetase cotranscribed bacterial homologue protein O94903 ↑ 24/72 99.94 30,609.7 
Nucleoside diphosphate kinase B P22392 ↑ 24 100 17,401 
G-rich sequence factor 1 Q12849 Control 24 100 50,651.3 
Protein phosphatase 1G O15355 Control 24 100 59,918.9 
Protein SEC13 homologue P55735 Control 24 100 35,900.3 
Y-box binding protein 1 P67809 ↓ 72 100 35,902.7 
Reticulocalbin-2 (precursor) Q14257 ↓ 72 100 36,910.7 
Thioredoxin-like protein p46 Q8NBS9 ↓ 72 100 48,282.9 
Elongation factor 2 P13639 ↓ 72 100 96,115.3 
Ribonucleoside diphosphate reductase subunit M1 P23921 ↓ 72 100 90,924.9 
Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase P13995 ↓ 72 100 37,468 
Transcription factor BTF3 P20290 ↓ 72 100 22,211.4 
Actin-interacting protein 1 O75083 ↑ 72 100 66,705.1 
Lamin A/C P02545 ↑ 72 100 74,379.8 
Elongation factor 1γ P26641 ↑ 72 100 50,298.2 
Nicotinamide phosphoribosyltransferase P43490 ↑ 72 100 55,771.7 
Cyclic AMP–dependent protein kinase type Iα regulatory subunit P10644 ↑ 72 100 43,183.1 
Annexin A1 P04083 ↑ 72 100 38,787 
Galectin-3 P17931 ↑ 72 100 26,098 
26S protease regulatory subunit 8 P62195 ↑ 72 100 45,768.1 
Leukocyte elastase inhibitor (serpin B1) P30740 ↑ 72 100 42,828.7 
ADP-sugar pyrophosphatase Q9UKK9 Control 72 100 24,597.3 
Glutathione S-transferase κ1 Q9Y2Q3 Control 72 100 25,463.3 
Zinc finger protein 9 P62633 Control 72 100 20,704 
EH domain-containing protein 1 Q9H4M9 Treated 72 100 60,645.7 
Guanine nucleotide-binding protein G(k) subunit α P08754 Treated 72 100 40,945.4 
Retinol-binding protein I, cellular P09455 Treated 72 100 15,879.9 
Ferritin heavy chain P02794 Treated 72 100 21,252.3 
DNAJ homologue subfamily C member 8 O75937 Treated 72 100 29,823.4 
Cofilin-1 P23528 Treated 72 100 18,587.7 
Calponin 3, acidic Q15417 Treated 72 100 36,561.9 
Ubiquitin-like domain-containing CTD phosphatase 1 Q8WVY7 Treated 72 100 36,838.2 
α2-Macroglobulin receptor-associated protein P30533 Treated 72 100 41,440.9 
Cold-inducible RNA-binding protein Q14011 Treated 72 100 18,636.7 
Adenylate kinase isoenzyme 1 P00568 Treated 72 100 21,735.3 
Cytosolic nonspecific dipeptidase Q96KP4 Treated 72 100 53,187 
ProteinAccession no.Expression levelTime (h)Prot. score CI (%)Molecular weight (Da)
Lamin A/C P02545 ↓ 24 100 74,379.8 
Heterogeneous nuclear ribonucleoprotein L P14866 ↓ 24/72 100 60,719.4 
DNA helicase V Q96AE4 ↓ 24 100 67,602.5 
GTP-binding protein 9 Q9NTK5 ↓ 24/72 100 44,943.4 
Heterogeneous nuclear ribonucleoprotein D0 Q14103 ↓ 24 100 38,581.4 
28-kDa heat- and acid-stable phosphoprotein Q13442 ↓ 24 100 20,617.6 
26S proteasome non-ATPase regulatory subunit 4 P55036 ↓ 24 100 40,939.3 
Nascent polypeptide-associated complex subunit α Q13765 ↓ 24/72 100 23,369.7 
Acidic leucine-rich nuclear phosphoprotein 32 family member A P39687 ↓ 24/72 100 28,682.3 
Proteasome subunit α type 3 P25788 ↓ 24/72 100 28,512.1 
Ran-specific GTPase-activating protein P43487 ↓ 24 100 23,466.6 
Heterochromatin protein 1 homologue γ Q13185 ↓ 24 100 20,981.4 
Cold shock domain-containing protein E1 O75534 ↑ 24 100 89,684.2 
Leukotriene A-4 hydrolase P09960 ↑ 24 100 69,737.4 
26S protease regulatory subunit 4 P62191 ↑ 24 100 49,324.8 
Fascin Q16658 ↑ 24/72 100 54,992.2 
Cytosolic nonspecific dipeptidase Q96KP4 ↑ 24 100 53,187 
Multifunctional protein ADE2 P22234 ↑ 24/72 100 47,659.4 
Pyruvate dehydrogenase E1 component subunit α P08559 ↑ 24 100 43,951.9 
26S proteasome non-ATPase regulatory subunit 7 P51665 ↑ 24/72 100 37,059.5 
Cytosolic acyl coenzyme A thioester hydrolase O00154 ↑ 24 100 42,435.6 
Maspin (serpin B5) P36952 ↑ 24/72 100 42,567.6 
Annexin A2 P07355 ↑ 24 100 38,676.9 
Proline synthetase cotranscribed bacterial homologue protein O94903 ↑ 24/72 99.94 30,609.7 
Nucleoside diphosphate kinase B P22392 ↑ 24 100 17,401 
G-rich sequence factor 1 Q12849 Control 24 100 50,651.3 
Protein phosphatase 1G O15355 Control 24 100 59,918.9 
Protein SEC13 homologue P55735 Control 24 100 35,900.3 
Y-box binding protein 1 P67809 ↓ 72 100 35,902.7 
Reticulocalbin-2 (precursor) Q14257 ↓ 72 100 36,910.7 
Thioredoxin-like protein p46 Q8NBS9 ↓ 72 100 48,282.9 
Elongation factor 2 P13639 ↓ 72 100 96,115.3 
Ribonucleoside diphosphate reductase subunit M1 P23921 ↓ 72 100 90,924.9 
Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase P13995 ↓ 72 100 37,468 
Transcription factor BTF3 P20290 ↓ 72 100 22,211.4 
Actin-interacting protein 1 O75083 ↑ 72 100 66,705.1 
Lamin A/C P02545 ↑ 72 100 74,379.8 
Elongation factor 1γ P26641 ↑ 72 100 50,298.2 
Nicotinamide phosphoribosyltransferase P43490 ↑ 72 100 55,771.7 
Cyclic AMP–dependent protein kinase type Iα regulatory subunit P10644 ↑ 72 100 43,183.1 
Annexin A1 P04083 ↑ 72 100 38,787 
Galectin-3 P17931 ↑ 72 100 26,098 
26S protease regulatory subunit 8 P62195 ↑ 72 100 45,768.1 
Leukocyte elastase inhibitor (serpin B1) P30740 ↑ 72 100 42,828.7 
ADP-sugar pyrophosphatase Q9UKK9 Control 72 100 24,597.3 
Glutathione S-transferase κ1 Q9Y2Q3 Control 72 100 25,463.3 
Zinc finger protein 9 P62633 Control 72 100 20,704 
EH domain-containing protein 1 Q9H4M9 Treated 72 100 60,645.7 
Guanine nucleotide-binding protein G(k) subunit α P08754 Treated 72 100 40,945.4 
Retinol-binding protein I, cellular P09455 Treated 72 100 15,879.9 
Ferritin heavy chain P02794 Treated 72 100 21,252.3 
DNAJ homologue subfamily C member 8 O75937 Treated 72 100 29,823.4 
Cofilin-1 P23528 Treated 72 100 18,587.7 
Calponin 3, acidic Q15417 Treated 72 100 36,561.9 
Ubiquitin-like domain-containing CTD phosphatase 1 Q8WVY7 Treated 72 100 36,838.2 
α2-Macroglobulin receptor-associated protein P30533 Treated 72 100 41,440.9 
Cold-inducible RNA-binding protein Q14011 Treated 72 100 18,636.7 
Adenylate kinase isoenzyme 1 P00568 Treated 72 100 21,735.3 
Cytosolic nonspecific dipeptidase Q96KP4 Treated 72 100 53,187 

NOTE: ↓, down-regulated in treated cells; ↑, up-regulated in treated cells; Control, expressed only in nontreated cells; Treated, expressed only in treated cells.

Cells were cultured in the presence of 5 μmol/L quinoline derivative for 24 and 72 h. Total protein extracts were separated by two-dimensional gel electrophoresis and resulting gels were stained by Colloidal Coomassie Blue. After image analysis, protein spots with >3-fold change in the expression level were digested with trypsin and identified by MALDI-TOF/TOF mass spectrometer.

Table 2.

Proteins differentially expressed in response to amidino-substituted derivative of benzimidazo[1,2-α]quinoline in SW620 cells

ProteinAccession no.Expression levelTime (h)Prot. score CI (%)Molecular weight (Da)
Calpactin I light chain P60903 ↓ 24 100 11,178.5 
Peptidyl-prolyl cis-trans isomerase A P62937 ↓ 24 100 18,097.9 
Mortalin (stress 70 protein, mitochondrial) P38646 ↓ 24/72 100 73,919.9 
Cofilin-1 P23528 ↓ 24 100 18,587.7 
40S ribosomal protein S12 Q76M58 ↓ 24 100 14,904.6 
Dihydrofolate reductase P00374 ↓ 24 100 21,365 
3-Hydroxyacyl-CoA dehydrogenase type II Q99714 ↓ 24 100 27,134.2 
Thioredoxin-dependent peroxide reductase, mitochondrial P30048 ↓ 24 100 28,017.3 
Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase P13995 ↓ 24 100 37,468 
S-formylglutathione hydrolase P10768 ↓ 24 100 31,955.7 
LIM and SH3 domain protein 1 Q14847 ↓ 24 100 30,097.4 
Heterogeneous nuclear ribonucleoprotein H3 P31942 ↓ 24 100 36,960.1 
Heterogeneous nuclear ribonucleoprotein A/B Q99729 ↓ 24 100 36,703.9 
α-Enolase P06733 ↓ 24 100 47,350.4 
Proliferation-associated protein 2G4 Q9UQ80 ↓ 24 100 44,101.3 
Acetyl-CoA acetyltransferase, cytosolic Q9BWD1 ↓ 24 99.64 41,837.6 
Actin-interacting protein 1 O75083 ↓ 24 100 66,705.1 
Neutral α-glucosidase AB Q14697 ↓ 24 100 107,262.8 
Heat shock 70-kDa protein 8 P11142 ↓ 24 100 71,082.3 
Oncogene DJ1 Q99497 ↑ 24/72 100 20,049.6 
L-plastin P13796 ↑ 24 100 70,815 
Transaldolase 1 Q96DB1 ↑ 24/72 100 35,534.5 
Chromatin assembly factor 1 subunit C Q09028 ↑ 24 100 47,780.1 
Eukaryotic initiation factor 4A-I P60842 ↑ 24/72 100 46,352.6 
Cytokeratin-18 P05783 ↑ 24/72 100 47,897.5 
Annexin IV P09525 Control 24 100 35,957.2 
Nucleolin P19338 Control 24 100 76,224.2 
γ-Actin P63261 Control 24 100 42,107.9 
Heterogeneous nuclear ribonucleoprotein F Q5T0N2 Treated 24 100 45,985 
Eukaryotic translation initiation factor 5 P55010 Treated 24 100 49,648 
Telomerase-binding protein p23 (Hsp90 cochaperone) Q15185 ↓ 72 100 18,970.5 
Eukaryotic translation initiation factor 3 subunit 12 Q9UBQ5 ↓ 72 100 25,328.6 
Acidic leucine-rich nuclear phosphoprotein 32 family member A P39687 ↓ 72 100 28,682.3 
Proteasome activator complex subunit 3 P61289 ↓ 72 100 29,601.6 
60S acidic ribosomal protein P0 P05388 ↓ 72 100 34,422.9 
Ubiquitin thioesterase OTUB1 Q96FW1 ↓ 72 100 31,492.5 
40S ribosomal protein SA Q6IPD2 ↓ 72 100 32,947.5 
Tubulin folding cofactor B Q99426 ↓ 72 100 27,593.6 
Histone-binding Q5T624 ↓ 72 100 45,936.2 
Glycyl-tRNA synthetase P41250 ↓ 72 100 85,335.7 
DNA helicase V Q96AE4 ↓ 72 100 67,602.5 
Elongation factor 2 P13639 ↓ 72 100 96,115.3 
γ-Actin P63261 ↓ 72 100 42,107.9 
Mitochondrial inner membrane protein Q16891 ↑ 72 100 84,025.5 
Lysyl-tRNA synthetase Q15046 ↑ 72 100 68,460.8 
Growth-inhibiting protein 12 Q5DSM0 ↑ 72 99.52 80,272.7 
Heterogeneous nuclear ribonucleoprotein Q O60506 ↑ 72 100 69,817.7 
Nicotinamide phosphoribosyltransferase P43490 ↑ 72 100 55,771.7 
α-Aminoadipic semialdehyde dehydrogenase P49419 ↑ 72 100 55,713.6 
Mitochondrial-processing peptidase subunit β O75439 ↑ 72 100 55,072.8 
TAR DNA-binding protein 43 Q13148 ↑ 72 100 45,053.4 
HSPC027 Q9Y6E3 ↑ 72 100 42,872.2 
Annexin I P04083 ↑ 72 100 38,787 
Cofilin-1 P23528 ↑ 72 100 18,587.7 
Putative RNA-binding protein 3 P98179 ↑ 72 100 17,160 
Galectin-1 P09382 ↑ 72 100 14,917.3 
Protein S100-A6 P06703 Control 72 100 10,230.4 
Nuclear transport factor 2 P61970 Control 72 100 14,640.1 
Proteasome subunit β type-6 P28072 Control 72 100 25,569.5 
Poly(rC)-binding protein 1 Q15365 Control 72 100 37,987.1 
Dihydropyrimidinase-related protein 2 Q16555 Control 72 100 62,710.7 
RNA helicase p68 P17844 Control 72 100 69,617.9 
Histone H2B type 1-O P23527 Control 72 100 13,766.5 
DNAJ homologue subfamily C member 8 O75937 Treated 72 100 29,823.4 
Heterogeneous nuclear ribonucleoprotein H3 P31942 Treated 72 100 36,960.1 
Actin-interacting protein 1 O75083 Treated 72 100 66,705.1 
ProteinAccession no.Expression levelTime (h)Prot. score CI (%)Molecular weight (Da)
Calpactin I light chain P60903 ↓ 24 100 11,178.5 
Peptidyl-prolyl cis-trans isomerase A P62937 ↓ 24 100 18,097.9 
Mortalin (stress 70 protein, mitochondrial) P38646 ↓ 24/72 100 73,919.9 
Cofilin-1 P23528 ↓ 24 100 18,587.7 
40S ribosomal protein S12 Q76M58 ↓ 24 100 14,904.6 
Dihydrofolate reductase P00374 ↓ 24 100 21,365 
3-Hydroxyacyl-CoA dehydrogenase type II Q99714 ↓ 24 100 27,134.2 
Thioredoxin-dependent peroxide reductase, mitochondrial P30048 ↓ 24 100 28,017.3 
Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase P13995 ↓ 24 100 37,468 
S-formylglutathione hydrolase P10768 ↓ 24 100 31,955.7 
LIM and SH3 domain protein 1 Q14847 ↓ 24 100 30,097.4 
Heterogeneous nuclear ribonucleoprotein H3 P31942 ↓ 24 100 36,960.1 
Heterogeneous nuclear ribonucleoprotein A/B Q99729 ↓ 24 100 36,703.9 
α-Enolase P06733 ↓ 24 100 47,350.4 
Proliferation-associated protein 2G4 Q9UQ80 ↓ 24 100 44,101.3 
Acetyl-CoA acetyltransferase, cytosolic Q9BWD1 ↓ 24 99.64 41,837.6 
Actin-interacting protein 1 O75083 ↓ 24 100 66,705.1 
Neutral α-glucosidase AB Q14697 ↓ 24 100 107,262.8 
Heat shock 70-kDa protein 8 P11142 ↓ 24 100 71,082.3 
Oncogene DJ1 Q99497 ↑ 24/72 100 20,049.6 
L-plastin P13796 ↑ 24 100 70,815 
Transaldolase 1 Q96DB1 ↑ 24/72 100 35,534.5 
Chromatin assembly factor 1 subunit C Q09028 ↑ 24 100 47,780.1 
Eukaryotic initiation factor 4A-I P60842 ↑ 24/72 100 46,352.6 
Cytokeratin-18 P05783 ↑ 24/72 100 47,897.5 
Annexin IV P09525 Control 24 100 35,957.2 
Nucleolin P19338 Control 24 100 76,224.2 
γ-Actin P63261 Control 24 100 42,107.9 
Heterogeneous nuclear ribonucleoprotein F Q5T0N2 Treated 24 100 45,985 
Eukaryotic translation initiation factor 5 P55010 Treated 24 100 49,648 
Telomerase-binding protein p23 (Hsp90 cochaperone) Q15185 ↓ 72 100 18,970.5 
Eukaryotic translation initiation factor 3 subunit 12 Q9UBQ5 ↓ 72 100 25,328.6 
Acidic leucine-rich nuclear phosphoprotein 32 family member A P39687 ↓ 72 100 28,682.3 
Proteasome activator complex subunit 3 P61289 ↓ 72 100 29,601.6 
60S acidic ribosomal protein P0 P05388 ↓ 72 100 34,422.9 
Ubiquitin thioesterase OTUB1 Q96FW1 ↓ 72 100 31,492.5 
40S ribosomal protein SA Q6IPD2 ↓ 72 100 32,947.5 
Tubulin folding cofactor B Q99426 ↓ 72 100 27,593.6 
Histone-binding Q5T624 ↓ 72 100 45,936.2 
Glycyl-tRNA synthetase P41250 ↓ 72 100 85,335.7 
DNA helicase V Q96AE4 ↓ 72 100 67,602.5 
Elongation factor 2 P13639 ↓ 72 100 96,115.3 
γ-Actin P63261 ↓ 72 100 42,107.9 
Mitochondrial inner membrane protein Q16891 ↑ 72 100 84,025.5 
Lysyl-tRNA synthetase Q15046 ↑ 72 100 68,460.8 
Growth-inhibiting protein 12 Q5DSM0 ↑ 72 99.52 80,272.7 
Heterogeneous nuclear ribonucleoprotein Q O60506 ↑ 72 100 69,817.7 
Nicotinamide phosphoribosyltransferase P43490 ↑ 72 100 55,771.7 
α-Aminoadipic semialdehyde dehydrogenase P49419 ↑ 72 100 55,713.6 
Mitochondrial-processing peptidase subunit β O75439 ↑ 72 100 55,072.8 
TAR DNA-binding protein 43 Q13148 ↑ 72 100 45,053.4 
HSPC027 Q9Y6E3 ↑ 72 100 42,872.2 
Annexin I P04083 ↑ 72 100 38,787 
Cofilin-1 P23528 ↑ 72 100 18,587.7 
Putative RNA-binding protein 3 P98179 ↑ 72 100 17,160 
Galectin-1 P09382 ↑ 72 100 14,917.3 
Protein S100-A6 P06703 Control 72 100 10,230.4 
Nuclear transport factor 2 P61970 Control 72 100 14,640.1 
Proteasome subunit β type-6 P28072 Control 72 100 25,569.5 
Poly(rC)-binding protein 1 Q15365 Control 72 100 37,987.1 
Dihydropyrimidinase-related protein 2 Q16555 Control 72 100 62,710.7 
RNA helicase p68 P17844 Control 72 100 69,617.9 
Histone H2B type 1-O P23527 Control 72 100 13,766.5 
DNAJ homologue subfamily C member 8 O75937 Treated 72 100 29,823.4 
Heterogeneous nuclear ribonucleoprotein H3 P31942 Treated 72 100 36,960.1 
Actin-interacting protein 1 O75083 Treated 72 100 66,705.1 

NOTE: ↓, down-regulated in treated cells; ↑, up-regulated in treated cells; Control, expressed only in nontreated cells; Treated, expressed only in treated cells.

Cells were cultured in the presence of 5 μmol/L quinoline derivative for 24 and 72 h. Total protein extracts were separated by two-dimensional gel electrophoresis and resulting gels were stained by Colloidal Coomassie Blue. After image analysis, protein spots with >3-fold change in the expression level were digested with trypsin and identified by MALDI-TOF/TOF mass spectrometer.

In the present article, we describe a mechanistic study of quinoline derivative-induced growth inhibition of two human colon cancer cell lines differing in the functional status of the TP53. This gene is activated in response to diverse cellular stresses, including DNA damage, hypoxia, and oncogene activation, whereby it can trigger cell cycle arrest, senescence, differentiation, DNA repair, apoptosis, and inhibition of angiogenesis (12). In wt-p53 cells (HCT 116), quinoline derivative induced p53-dependent response as evidenced by induction of wt-p53 protein and its downstream target, p21 protein. This resulted in prolonged p21-regulated cell cycle arrest at G1 phase followed by S-phase block. Cell cycle arrest at S phase corresponds well with reduced level of the ribonucleoside diphosphate reductase M1 protein detected by two-dimensional gel electrophoresis/MS approach. This protein is a large subunit of the enzyme catalyzing the rate-limiting step in DNA synthesis, the conversion of ribonucleotides to deoxyribonucleotides. Due to its persistence through the cell cycle in rapidly cycling cells and relatively long half-life, ribonucleoside diphosphate reductase M1 protein is considered a suitable marker for cellular proliferation (13). Decreased expression of this protein in treated HCT 116 cells conforms to their observed diminished proliferative capacities.

We identified some other proteins in HCT 116 besides p21 that are closely connected to p53, e.g. maspin (serpin B5) whose level was up-regulated in treated cells as revealed by proteomics analyses. This protein belongs to the serine protease inhibitor superfamily and has been identified as tumor suppressor that blocks tumor invasion, angiogenesis, and metastasis (14). In vitro studies on prostate and breast tumor cells have shown that wt-p53 specifically induces maspin expression by binding directly to the p53 consensus-binding site present in the maspin promoter (15). Dependence of maspin expression on the p53 functionality has also been substantiated in colorectal cancer by studies showing an inverse correlation between mutant p53 and maspin expression (16, 17). Furthermore, it has been established that DNA-damaging agents and cytotoxic drugs induce maspin expression in the cells containing endogenous wt-p53, whereas the cells harboring mutant p53 fail to induce maspin expression under the aforementioned conditions (15). In light of these facts, our study puts forward maspin as a possible marker for wt-p53-dependent chemotherapeutic response. Accordingly, treated SW620 cells with mutated p53 did not induce maspin expression.

TP53 can also carry out its cellular functions via specific interactions with other proteins, such as Y box binding protein 1, whose expression was found to be decreased in treated HCT 116 cells. Y box binding protein 1 protein directly associates with the p53 protein in vivo and in vitro, and this interaction reciprocally modulates the DNA-binding function of each protein (18). Expression of the Y box binding protein 1 protein is increased in proliferating normal and cancer cells (19). Its down-regulation in treated HCT 116 cells corresponds well with their reduced proliferation rate.

Proteomic results unveiled alterations in the expression of several proteins in treated HCT 116 cells known to regulate apoptosis, which was in line with the previous data provided by the Annexin V binding assay; for example, maspin, which has already been mentioned, is renowned for its ability to sensitize the apoptotic response of breast and prostate carcinoma cells to various drugs (20). Its expression was shown to correlate with increased activation of caspase-3 (20). We also found caspase-3 to be activated in treated HCT 116 cells. In addition, proteomic analysis revealed two known caspase-3 targets, namely heterogeneous nuclear ribonucleoprotein L and heterogeneous nuclear ribonucleoprotein D, whose decreased expression speaks in favor of caspase-mediated proteolytic processes typically occurring during apoptosis. Drastic reduction in the viability of treated HCT 116 cells was accompanied by an apparent induction of several other proapoptotic proteins as well, including cofilin 1, one of the key regulators of actin filament turnover and cytoskeleton reorganization. Several proteins functionally interact with cofilin to modulate the dynamics of actin filaments, such as actin interacting protein 1, whose level was also elevated in treated cells. Chua et al. (21) have found that, after induction of apoptosis, cofilin translocates from cytosol into mitochondria before release of cytochrome c and have suggested that cofilin has an important function during the initiation phase of apoptosis. It is tempting to believe that a similar process also occurs in HCT 116 cells treated with quinoline derivative, because the longest treatment period gave rise to significant increase in the fraction of early apoptotic cells.

Several reports dealing with identification of p53 targets in colon carcinoma cells by proteomic tools have given prominence to Annexin I in p53-mediated apoptosis (22, 23); for example, proteomic analysis of mitomycin C–treated HCT 116 cells carrying wt-p53 revealed Annexin I in the group of up-regulated proteins containing p53-binding site (23). Our proteomic analysis revealed an elevated expression of this protein in treated HCT 116 cells as well. Several lines of evidence indicate that an overexpression of Annexin I induces caspase-3-mediated apoptosis in monocytic (24), bronchoalveolar (25), and prostate cancer cells (26), which again brings attention to the role of caspase-3-dependent apoptotic pathway in HCT 116. However, we cannot rule out the possibility that other apoptosis pathways are also provoked in this cell line, as we found activation of the AIF protein in treated cells, which provides direct evidence for induction of caspase-independent cell death process. Altogether, obtained data suggest that quinoline derivative-induced apoptosis in HCT 116 cells is p53-regulated and results from the intricate involvement of multiple apoptotic processes, which are both caspase-3 dependent and caspase independent.

Opposite to HCT 116, quinoline derivative-triggered cellular events leading to cell cycle arrest in SW620 cells occur independently of p53 and p21 as witnessed by the aberrant expression of mutated p53 protein that failed to induce p21 protein. Quinoline derivative initially triggered accumulation of the S-phase cells, which was additionally confirmed at the proteome level by increased expression of chromatin assembly factor 1 subunit C in these cells, a nuclear protein involved in chromatin assembly following DNA replication and DNA repair (27). However, after prolonged treatment periods, SW620 cells transited through S phase into G2 but were unable to maintain G2 arrest and entered mitosis as revealed by the expression level of mitotic marker cyclin B1 in treated cells, which reached the maximum after 72 h. The level of this protein has been shown to rise during S phase and to peak in mitosis (28). G2-M progression of treated SW620 cells was further confirmed at the proteome level, revealing increased expression of TAR DNA-binding protein implicated in regulating the chromosomal movement along microtubules during mitosis (29). In addition, we observed decline in the expression of nuclear autoantigenic sperm protein (NASP) in G2-M-phase cells. NASP is H1 histone-binding protein involved in transporting histones into the nucleus of dividing cells. Richardson et al. (30) reported that NASP protein levels remained constant throughout the cell cycle in actively growing cells, whereas confluent cells that were not dividing showed little or no NASP present, indicating the eventual loss of the NASP protein in nondividing cells. These data lead us to believe that quinoline derivative impairs the cell division process in SW620.

Although apoptosis is considered to be the most prevalent form of cell death in response to chemotherapy, other types of cell death might also shape the ultimate therapeutic outcome, including mitotic catastrophe, which is characterized by defective chromosome segregation and cell division leading to the formation of large nonviable cells with multiple micronuclei (31). Inhibition or genetic suppression of several G2 checkpoint genes including p53, p21WAF1/Cip1, and 14-3-3σ is known to promote mitotic cell death on DNA damage (32, 33). For example, after γ-radiation, wt-p53 human colorectal cancer cells underwent sustained G2 arrest, whereas large fraction of the p53-mutant cells or cells with both p53 alleles disrupted entered mitosis (32). Similarly, γ-irradiated HCT 116/ p21−/− cells arrested at G2 but resumed their cell cycles and entered mitosis. However, both p21−/− and p53−/− cells never completed cytokinesis and eventually died (32). Analogously, we observed that treated SW620 cells undergoing G2-M transition were induced to die as illustrated by significant raise in the proportion of late apoptotic/early necrotic cells and subsequently confirmed at the proteome level disclosing an overexpression of several proteins possessing apoptogenic activities (e.g., Annexin I, cofilin 1, and galectin-1). Aforementioned results, along with the previously observed accumulation of cyclin B1 in G2-M cells, raise the possibility that defect in p53 and consequential lack of p21 in SW620 cells render them susceptible to the induction of mitotic catastrophe after quinoline derivative-induced DNA damage. Indeed, many cases of mitotic catastrophe induced by pharmacologic or genetic manipulations have been accompanied by increased nuclear cyclin B1 (10).

Growing body of evidence indicates that caspase activation is not a prerequisite for induction of cell death by mitotic catastrophe (34, 35). Thus, Eom et al. (34) treated human hepatoma cells carrying mutant p53 with low doses of doxorubicin and found that these cells underwent mitotic catastrophe, whereby caspase-3 and several other initiator and executor caspases were not activated. Similarly, we did not detect activation of caspase-3 in treated SW620 cells, which implies that mitotic cell death in this cell line is caspase-3 independent. In addition, an absence of the AIF protein expression in these cells rules out the involvement of the caspase-independent cell death pathway regulated by this protein. This result, however, seems feasible in light of the fact that AIF gene is a transcriptional target of p53 and caspase-independent death is compromised in cells lacking functional p53 (11).

Strong antiproliferative activity of quinoline derivative toward SW620 cells, which are metastasis of colorectal adenocarcinoma derived from the lymph node, underscores its potential antimetastatic properties. Our proteomic study revealed decline in the expression of several proteins in treated cells linked with the colon tumorigenesis, namely mitochondrial heat shock 70-kDa protein 9 (mortalin) and three ribosomal proteins including 40S ribosomal protein SA (34/67-kDa laminin receptor), 40S ribosomal protein S12, and 60S acidic ribosomal protein P0. Dundas et al. (36) found mortalin to be overexpressed in colorectal adenocarcinomas. Negative regulation of its expression was shown to induce growth arrest of transformed human cells through both activation of p53 and p53-independent pathways (37), the latter possibly being the case in SW620. Similarly, increased levels of the 40S ribosomal protein SA and 40S ribosomal protein S12 transcripts were found in human colon carcinoma in comparison with adjacent normal colonic epithelium (38, 39). Based on their study uncovering increased levels of mRNAs encoding several other ribosomal proteins (P0, S6, S8, and L5) in colorectal tumors and adenomatous polyps, Pogue-Geile et al. (39) hypothesized about an increased synthesis of ribosomes in colorectal tumors, which is probably an early event in colon neoplasia. Our findings revealing lowered levels of several ribosomal proteins in SW620 put forward the possibility that quinoline derivative interferes with ribosome biogenesis in these cells leading to their reduced proliferation ability.

Although there appears to be different pathways by which colon cancer cells undergo quinoline derivative-induced cell death, oxidative stress might be a common trigger for this event regardless of p53 status. This possibility was corroborated by proteomic profiling of treated cells revealing the expression of several proteins associated with the oxidative stress response and antioxidant activities in both cell lines. For example, maspin (40) and ferritin heavy chain (41) were induced in HCT 116, whereas transaldolase 1 (42), oncogene DJ1 (43), and lactotransferrin (44) were up-regulated in SW620. Similarly, the antitumor effect of another catalytic inhibitor of topoisomerase II, namely aclarubicin, which is strong DNA-intercalating agent as well, has been correlated with the generation of reactive oxygen species (45). These findings accentuate the role of quinoline derivative as potential oxidant or prooxidative chemotherapeutic agent.

Studied quinoline derivative has stronger cytostatic activity than the antimetabolite 5-fluorouracil (IC50, 10 ± 1.4 and 6.7 ± 1.1 μmol/L in HCT 116 and SW620, respectively)6

6

Unpublished data.

and quite similar to a topoisomerase I inhibitor irinotecan [IC50, 2.0 ± 0.9 μmol/L in SW620 (46) and 2.2 μmol/L in HCT 116 (47)], standard agents in colorectal cancer therapy. These comparison data shed new light on the role of quinoline derivative as promising, novel agent against colorectal cancer.

In summary, we have shown that quinoline derivative inhibits colon cancer cell growth in vitro and that p53 gene status seems to be a molecular determinant of its distinct antiproliferative mechanisms, which are likely to be commonly triggered by DNA damage and oxidative stress. In HCT 116, this compound induced p53-dependent apoptosis. On the contrary, mitotic cell death seemed to be the primary mode of death in SW620, which was apparently mediated by the p53-independent pathways. Proteomic approach used in this study proved to be a valuable tool for investigating cancer cell response to newly synthesized compound revealing some specific molecular changes related to the expression of the p53-dependent chemotherapeutic response marker maspin and cell proliferation markers Y box binding protein 1 and ribonucleoside diphosphate reductase M1 protein in wt-p53 cells and impairment in ribosome biogenesis in mutant p53 cells. Finally, antiproliferative effects of quinoline derivative on SW620 cells strongly support its possible role as an antimetastatic agent and encourage further in vivo studies on the chemotherapeutic potential of this compound against colorectal carcinoma.

No potential conflicts of interest were disclosed.

Grant support: Croatian Ministry of Science, Education and Sport grants 098-0982464-2393 and 125-0982464-1356 and Croatian JEZGRE-TEST grant 14M09800. Mass spectrometry analyses were done at and financed by the Functional Genomics Center Zurich.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Karlo Hock for assistance with preparation of the figures.

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Supplementary data