Introduction

Colorectal carcinoma, one of the most common fatal cancers, is the second leading cause of cancer-related deaths worldwide1. The primary method currently used to treat all stages of colon cancer is surgery. After tumor excision, chemotherapeutic drugs, such as irinotecan (CPT-11), 5-fluorouracil (5-FU), and oxaliplatin, are typically used to further eradicate any residual tumor cells2,3. However, in the majority of cancer patients, currently available chemotherapies have had only partial success, owing to chemoresistance and side effects4. Therefore, there is an urgent need for highly efficacious alternative chemotherapeutic agents that can selectively induce apoptosis in cancer cells. Elaboration of the molecular mechanisms responsible for colon cancer progression should facilitate the development of effective chemotherapeutic agents.

Apoptosis, an intracellular suicide program possessing specific morphologic characteristics and biochemical features5, is a mechanism exploited by many anticancer drugs to effect cancer cell death. The extrinsic apoptotic pathway involves two important pro-apoptotic molecules that belong to the tumor necrosis factor (TNF) receptor superfamily, death receptor 4 (DR4) and death receptor 5 (DR5). DR4 and DR5 can be assembled into a death-inducing signaling complex (DISC) after binding to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Upon association with the adaptor molecule Fas-associated protein with death domain (FADD), DISC leads to the cleavage and activation of an apoptosis initiator, caspase-8, which activates the downstream caspases-3, -6 and -7 to culminate in the extrinsic apoptosis of malignant cells6,7. Currently, a number of studies have suggested that many therapeutic agents, such as bioymifi, lapatinib, and etoposide, up-regulate DR5, thereby acting as key triggers of apoptosis in various cancer cell types in vitro and in vivo8,9,10. Crosstalk exists between the intrinsic and extrinsic apoptosis pathways: activated caspase-8 in the extrinsic pathway cleaves Bid, after which the cleaved Bid translocates to mitochondria to cause the release of cytochrome c and the subsequent initiation of the intrinsic apoptosis pathway11.

By triggering a variety of cellular responses leading to cell growth, differentiation, or cell death, reactive oxygen species (ROS) have recently been proposed to be involved in tumor metastasis as well as apoptosis induced by various compounds12,13,14. Cancer-induced ROS is controlled by antioxidants such as N-acetyl cysteine (NAC) and glutathione (GSH) in cancer15,16. GSH is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the etiology of various diseases, including cancer17,18. As a sulfhydryl donor, NAC not only contributes to the regeneration of GSH but also reacts directly with free ROS as its free thiol group is capable of interacting with electrophilic groups of ROS19. Two of the downstream molecules regulated by ROS are p-21 kinase (PAK) and mitogen-activated protein kinase (MAPK)20. MAPKs include p38 MAPK, stress-activated protein kinase/c-Jun NH2 terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). ERK is primarily involved in growth and survival, whereas JNK and p38 are generally associated with pro-apoptotic activities in many cell types21. JNK includes at least ten isoforms that are encoded by three genes, JNK1, JNK2, and JNK3. JNK1 and JNK2 are ubiquitously expressed, whereas expression of JNK3 is limited to the brain and heart22. Accumulating evidence suggests that JNK1 and JNK2 have different functions in the regulation of apoptosis and cell proliferation. Because of its importance in regulating apoptosis23,24, the JNK1 signal transduction pathway is of particular relevance to the work discussed here.

Substituted benzimidazole derivatives exhibit various bioactivities, such as anti-ulcerative25, anti-inflammatory26, anti-bacterial27, and anti-carcinogenic properties28. Acridine and its derivatives are types of polycyclic aromatic compounds with π-conjugated structures that possess the ability to intercalate into DNA, subsequently inhibiting topoisomerases to elicit anticancer effects29,30. Many investigations have reported the relationship between benzimidazole or acridine derivatives and ROS- or JNK-mediated apoptosis31,32,33. However, apoptosis induced by a benzimidazole acridine derivative through the ROS-JNK1 pathway has never been studied. Our recent work has demonstrated that an analog of a benzimidazole acridine derivative (8m) possess cytotoxic activity against a variety of cancer cell lines and can induce apoptosis of K562 human leukemia cells34. However, the effect of 8m on human colon cancer cells and the mechanism by which it induces apoptosis is largely unknown. In this work, we investigated the molecular events responsible for 8m-induced apoptosis in the HCT116 human colon cancer cell line. Our data provided sufficient evidence that N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine (8m)-induced apoptosis in HCT116 cells was strongly associated with activation of the ROS-JNK1 pathway.

Materials and methods

Materials

8m was obtained from Associate Professor Chun-mei GAO of the Graduate School at Shenzhen, Tsinghua University (Shenzhen, China). Its molecular structure is shown in Figure 1A. Modified RPMI-1640 medium was purchased from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) and Opti-MEM I reduced serum media were purchased from Gibco (Grand Island, NY, USA). Lipofectamine 2000, TRIzol Reagent and SlowFade® gold antifade mountant with DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) were purchased from eBioscience (San Diego, CA, USA). PrimeScript™ RT Master Mix (Perfect Real Time) kit and SYBR® Premix Ex Taq™ (Tli RNaseH Plus) kit were purchased from TaKaRa (Dalian, China). Short-interfering RNA (siRNA) was synthesized by GenePharma (Shanghai, China). Phosphor-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, phosphor-p38, p38, phosphor-ERK, ERK, Bcl-2, Bid, cleaved caspase-9, cleaved caspase-8, cleaved caspase-7, cleaved caspase-3, and cleaved PARP were purchased from Cell Signaling Technology (Danvers, MA, USA). DR5 was obtained from Abcam (Cambridge, UK). β-Actin antibody, HRP-labeled Goat Anti-Rabbit IgG (H±L), HRP-labeled Goat Anti-Mouse IgG (H±L) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Beyotime (Haimen, China). PVDF membrane, protease and phosphatase inhibitor cocktails were purchased from Roche (Mannheim, Germany). N-acetylcysteine (NAC), L-glutathione reduced (GSH) and 2′,7′-Dichloro-dihydrofluorescein diacetate (H2-DCFDA) were purchased from Sigma (St Louis, MO, USA).

Figure 1
figure 1

The effects of 8m on the viability of human colon cancer cells. (A) Chemical structure of 8m (N-((1H-benzo[d]imidazol-2-yl)methyl)-2-butylacridin-9-amine, C25N4H24). HCT116 cells (B) or SW480 cells (C) were incubated with the indicated concentrations of 8m for 48 h in 96-well plates, and cell viability was examined by the MTT assay. (D) HCT116 cells were treated with the indicated concentrations of 8m for 24 h, and apoptosis was detected by flow cytometry. (E) HCT116 cells were stained with DAPI and assessed by fluorescence microscopy. Apoptotic cells exhibited apoptotic bodies and chromatin condensation. Scale bar, 20 μm.

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Cell culture

All cells used in this study were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT116 and SW480 were grown in RPMI-1640 supplemented with 10% FBS. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

Cell viability assay

The MTT assay was used to examine the growth inhibitory effect of 8m. Cells were plated in 96-well culture plates at a cell density of 5000 cells per well in 100 μL of complete medium and incubated under standard cell culture conditions for 12 h. The medium was then removed and replaced with fresh medium containing the vehicle control or different concentrations of 8m (1, 2.5, 5, 10, 25, and 50 μmol/L) for 48 h. After agent treatment, the cells were incubated for 4 h in a medium containing 15 μL of MTT (5 mg/mL) solution. The formazan precipitate was dissolved in 100 μL of DMSO, and the absorbance at 490 nm was measured by a Multimode Detector DTX880 (Beckman Coulter, Atlanta, GA, USA).

Colony formation assay

HCT116 cells were seeded into 6-well plates (1×103 cells/well) and cultured overnight. Then, the culture medium was replaced with fresh medium containing the vehicle control or the indicated concentrations of 8m (or co-cultured with 5 mmol/L NAC) for 14 d. The culture medium was changed every 3 to 4 d. When colonies formed, the medium was then discarded, and each well was washed twice with PBS. After that, colonies were fixed with 100% cold ethanol for 30 min and then stained with 0.05% crystal violet.

Apoptotic nuclear measurement

Cells were seeded onto cover slides for 12 h and then treated with the vehicle control or various concentrations (5, 7.5, and 10 μmol/L) of 8m for 24 h. After the cells were washed with cold PBS, the cells were fixed in 4% paraformaldehyde for 10 min. The cells were subsequently washed with PBS and stained with 4,6-diamidino-2-phenylindole (DAPI) for 30 min in the dark at room temperature. Cover slips containing the cells were then washed with PBS-TX (10 mL PBS+10 μL 10% Tritonx-100) three times, and images were taken using a fluorescent microscope (Olympus, Tokyo, Japan).

ROS generation assay

Oxidative stimuli present in the cytoplasm may be transmitted to the nucleus through cellular signal transduction pathways to regulate cell division and survival. 2′,7′-Dichloro-dihydrofluorescein diacetate (H2-DCFDA, Molecular Probes) is metabolized by nonspecific esterases to the non-fluorescent product, 2′,7′-dichloro-dihydrofluoresceine, which is then oxidized by ROS to the fluorescent product DCF. HCT116 cells were incubated in 60 mm culture dishes under standard cell culture conditions for 12 h and then treated with 0, 5, 7.5, or 10 μmol/L 8m for 24 h. After incubation and washing with PBS, the cells were loaded with 10 μmol/L H2-DCFDA in complete medium for 1 h at 37 °C. After the removal of excessive H2-DCFDA, the cells were analyzed by a fluorescent microscope at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Visualization of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was assessed using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1). HCT116 cells were incubated in 60-mm culture dishes under standard cell culture conditions for 12 h and then treated with 0, 5, 7.5, or 10 μmol/L 8m for 24 h. After treatment with 8m, the cells were incubated for 30 min at 37 °C with 1 μg/mL of JC-1 in growth medium. MMP depolarization was visualized under a fluorescent microscope by measuring fluorescence at an excitation wavelength of 485 nm and emission wavelengths of 530 nm and 590 nm.

Flow cytometric quantification of apoptosis

Apoptosis was determined by Annexin V-FITC and PI double staining. HCT116 cells were cultured in 60-mm dishes in a humidified 5% CO2 atmosphere at 37 °C overnight and then were treated with the vehicle control or various concentrations (5 or 7.5 μmol/L) of 8m for 24 h. After trypsinization at the indicated time points, the cells were centrifuged at 400×g for 10 min at 4 °C, washed twice with cold PBS at the same centrifugation speed, and re-suspended in 400 μL of Annexin V-FITC binding buffer. The cells were then incubated with 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide (PI) for 15 min at room temperature protected from light, as per the manufacturer's instructions. The harvested cells, both adherent and floating cells, were analyzed by fluorescence activated cell sorting (FACS) using a flow cytometer (Beckman Coulter, Atlanta, GA, USA).

Transfection with siRNA

For RNA interference, cells were seeded at a density of 2×105/well in 60-mm dishes. Lipofectamine 2000 transfection reagent was used to silence JNK1, JNK2, and p38. A nonspecific siRNA was used as a control. A final concentration of 75 nmol/L siRNA was transfected into cells by standard protocols according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were washed with PBS, the culture medium was replaced, and the cells were stimulated with the vehicle control or 5 μmol/L 8m. The siRNA sequences of the target genes are as follows: JNK1 (sense: 5′-GGCAUGGGCUACAAAGAAAtt-3′ and antisense: 5′-UUUCUUUGUAGCCCAUGCCtt-3′); JNK2 (sense: 5′-GACUCAACCUUCACUGUCCUAtt-3′ and antisense: 5′-UAGGACAGUGAAGGUUGAGUCtt-3′); p38 (sense: 5′-GAACUGCGGUUACUUAAACtt-3′ and antisense: 5′-GUUUAAGUAACCGCAGUUCtt-3′); and nonspecific (sense: 5′-UUCUCCGAACGUGUCACGUtt-3′ and antisense: 5′-ACGUGACACGUUCGGAGAAtt-3′). The efficiency of JNK and p38 protein silencing was analyzed by Western blot at 24 and 48 h.

RNA isolation and real-time PCR analysis

Total RNA from tumor cells was isolated using the TRIzol reagent according to the manufacturer's instructions. The purity of RNA samples was determined by their OD260/280 (>1.8). First-strand cDNA was synthesized with 500 ng of total RNA using a PrimeScript™ RT Master Mix (Perfect Real Time) kit. Gene expression was quantified using a SYBR® Premix Ex Taq™ (Tli RNaseH Plus) kit on an ABI 7500 (Applied Biosystems, Carlsbad, CA, USA). The expression level of each mRNA was normalized with respect to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for PCR were DR4 (sense: 5′-GCAGCTGGACCTCACGAAAA-3′ and antisense: 5′-CCTGGGCCTGCTGTACCA-3′); DR5 (sense: 5′-GGCCACAGGGACACCTTGTA-3′ and antisense: 5′-TCGCCCGGTTTTGTTGA-3′); c-Fos (sense: 5′-GAGGGGCAAGGTGGAACAGT-3′ and antisense: 5′-CTTGCAGGCAGGTCGGTGAG-3′); and GAPDH (sense: 5′-TGGCAAATTCCATGGCACCG-3′ and antisense: 5′-CGCCCCACTTGATTTTGGAGG-3′).

Western blot

After treatment, the cells were washed twice with ice-cold PBS, and the reaction was terminated by the addition of ice-cold lysis buffer (10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1 mmol/L EDTA, 0.1% NP-40) including freshly added protease and phosphatase inhibitor cocktails. Lysates were cleared by centrifugation at 20 000×g, 4 °C for 10 min, and the supernatant was snap frozen at -80 °C. Thirty micrograms of total protein was separated in 12% SDS–polyacrylamide gels and electrophoretically transferred onto PVDF membranes. After blocking with 5% (w/v) BSA in TBST buffer (10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% v/v Tween-20, pH 7.8) for 2 h at room temperature, the membranes were incubated with the specific primary antibody (1:1000) overnight at 4 °C. Following incubation with an HRP-conjugated secondary antibody (1:3000) and after incubating the membrane in SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA), protein bands were visualized on an imaging system (Bio-Rad, Munich, Germany).

Statistical analysis

All values are presented as the mean±SEM from experiments performed in triplicates. Significance was determined using Student's t-test; P<0.05 was considered statistically significant.

Results

Cytotoxicity and apoptosis induced by 8m against human colon cancer cells

The potential of 8m (Figure 1A) to induce cell death in human colon cancer cells was examined using the MTT assay. Treatment of SW480 and HCT116 cells with 8m induced cell death in a concentration-dependent manner, and the 50% inhibitory concentrations (IC50) of 8m against SW480 and HCT116 cells were 6.77±0.19 μmol/L and 3.33±0.02 μmol/L, respectively (Figure 1B, 1C). To confirm that the enhanced cytotoxic effects by 8m in HCT116 cells were due to increased apoptosis, we examined the apoptotic response using FITC-annexin V/PI double staining flow cytometric analysis. After 24 h of treatment, we found that 5 and 7.5 μmol/L 8m induced apoptosis in 21.09% and 27.95% of HCT116 cells, respectively(Figure 1D). Using DAPI staining, we then examined cells for morphological features of apoptosis, such as condensed chromatin and apoptotic bodies (Figure 1E). Collectively, these results suggested that 8m exhibited potential antitumor activity, at least in part, by inducing apoptosis in colon cancer cells.

8m-induced apoptosis involves both the extrinsic and intrinsic apoptotic pathways

To ascertain the underlying mechanisms responsible for enhancement of apoptotic cell death, we examined the effects of 8m on the expression of apoptosis-related genes and proteins. To determine whether 8m acts through the extrinsic apoptotic pathway, the expression levels of two members of the tumor necrosis factor (TNF) receptor superfamily, DR4 and DR5, which are upstream mediators of the extrinsic apoptosis pathway6, were examined after 8m treatment. Real-time PCR analysis revealed increased DR4 and DR5 mRNA levels, with DR5 increasing more significantly than DR4, suggesting that DR5 may play a more important role than DR4 in mediating 8m-induced apoptosis (P<0.05, Figure 2A). We then examined the expression of DR5 at the protein level and found that DR5 protein expression was induced by 8m in a concentration- and time-dependent manner. The cleavage and truncation of Bid and caspase-8, two downstream effectors, were induced by 8m, as indicated by western blotting (Figure 2B, 2C). These results suggested that 8m induced apoptosis through the extrinsic apoptotic pathway.

Figure 2
figure 2

8m induction of apoptotic gene and protein expression. (A) HCT116 cells were treated with 8m at 5 μmol/L for 4 h before real-time PCR was performed. Gene expression of three indicated genes as shown relative to control cells (bP<0.05 vs control). (B) HCT116 cells were treated with the specified concentration of 8m for 24 h, and whole cell extracts were analyzed by Western blot using the indicated antibodies. β-Actin protein was used as an internal control. (C) HCT116 cells were treated with 5 μmol/L 8m for 4, 8, 12, or 24 h. Whole cell extracts were analyzed by Western blot using the indicated antibodies.

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Bcl-2 tightly regulates the release of cytochrome c from the mitochondria, thus controlling the initiation of intrinsic apoptotic pathways35. Following 8m treatment, Bcl-2 and caspase-9, two hallmarks of the intrinsic apoptotic pathway, were down-regulated and cleaved, respectively, in a concentration- and time-dependent manner (Figure 2B, 2C). These results suggested that 8m also induced apoptosis through the intrinsic apoptosis pathway. Furthermore, the subsequent activation of caspase-7, caspase-3 and poly (ADP-ribose) polymerase (PARP) were observed (Figure 2B, 2C). Collectively, these data suggested that 8m-induced apoptosis involved both the extrinsic and the intrinsic apoptotic pathways.

8m activated mitogen-activated protein kinase signaling

MAPK signaling can be activated in response to a variety of extracellular stimuli, including mitogens, heat shock and osmotic stress, and can regulate the levels of DRs, Bcl-2, and other apoptosis-related proteins36,37,38. To determine whether MAPK is involved in apoptosis after 8m treatment, we examined the effects of 8m on the activation of MAPK pathways. Treatment of HCT116 cells with 8m increased the amounts of phosphorylated JNK and p38, which reflect the activated states of these two kinases, in a dose-dependent manner. However, the phosphorylation of ERK was minimally affected (Figure 3A). Our subsequent analysis of the time course of JNK and p38 activation showed that the phosphorylation of JNK and p38 were time-dependent: both were induced at 4 h after exposure to 8m, reaching a maximum level at 12 h, with activity persisting until 24 h. Fluctuations in levels of phosphorylated ERK were barely apparent during this time period (Figure 3B). In addition, we noted that c-Fos, a well-known JNK substrate, was also up-regulated by 8m treatment in the HCT116 colon cancer cell line (P<0.05, Figure 2A).

Figure 3
figure 3

Effects of 8m on MAPK activation. Phosphorylation of JNK, p38 MAP kinase, and ERK in cell lysates was detected by immunoblot analysis using the indicated antibodies. (A) HCT116 cells were treated with the indicated concentrations of 8m for 24 h. (B) HCT116 cells were treated with 5 μmol/L 8m for 4, 8, 12, or 24 h.

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JNK1 played an important role in 8m-induced apoptosis

To further investigate the role of MAPK activation in 8m-induced apoptosis, we treated HCT116 cells with 8m after inhibiting the expression of JNK1, JNK2, and p38 via the addition of the indicated oligonucleotides. The effects of JNK1, JNK2, and p38 siRNA were still observable 48 h post-treatment, outlasting the duration of 8m treatment in the subsequent detection assays (Figure 4A, 4C). Compared with control siRNA, Western blotting analyses showed that JNK1 siRNA completely abolished not only the 8m-induced increase in phosphorylated-JNK (phosphor-JNK) but also partially blocked the 8m-induced up-regulation of DR5, down-regulation of Bcl-2 as well as cleavage of Bid, caspase-8, caspase-7, caspase-3, and PARP. However, other than the decrease in the intensity of the Bcl-2 band, transfection with JNK2 siRNA appeared to elicit opposite results (Figure 4B). We then examined the effects of p38 siRNA, which, unexpectedly, reduced the level of phosphorylated p38; however, p38 siRNA did not affect the levels of other apoptosis-related proteins (Figure 4D). These results suggested that JNK1 played an important role in mediating both the extrinsic and intrinsic apoptotic pathways induced by 8m. This result was further supported by our flow cytometry data, which demonstrated that JNK1 siRNA, compared with the control siRNA, rescued 8m-induced apoptosis (Figure 4E).

Figure 4
figure 4

Effects of JNK1 on 8m-induced apoptosis. (A) Effects of JNK1 and JNK2 knockdown for 24 and 48 h. (B) After transfection with control, JNK1, or JNK2 oligonucleotides for 24 h, HCT116 cells were treated with 5 μmol/L 8m for 12 h. Whole cell extracts were analyzed by Western blotting using the indicated antibodies. (C) Effects of p38 knockdown at 24 and 48 h. (D) HCT116 cells were treated with or without 5 μmol/L 8m at 12 h after transfection with control or p38 oligonucleotides. Whole cell extracts were analyzed by Western blotting with the indicated antibodies at 24 h. (E) After transfection with control or JNK1 siRNA for 24 h, cells were treated with 5 μmol/L 8m for another 24 h, and apoptosis was detected by flow cytometry.

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8m-induced apoptosis was ROS-dependent

Because oxidative stress and mitochondrial membrane depolarization can each induce cancer cell apoptosis, we examined these two indicators after 8m treatment. Using a fluorescent microscope, we determined the intracellular ROS level by measuring the oxidation of non-fluorescent H2-DCFDA to its highly fluorescent derivative 2′,7′-dichlorofluorescein (DCF). As shown in Figure 5A, 8m stimulated ROS formation in a concentration-dependent manner. JC-1 is a membrane-permeable dye whose maximal fluorescence emission changes from ∼590 nm to ∼530 nm when the mitochondrial membrane depolarizes. By measuring the shift in the fluorescence emission peak using fluorescence microscopy, we found that 8m treatment induced mitochondrial depolarization in a concentration-dependent manner (Figure 5B). Because both the DR and the mitochondrial apoptotic pathways mediated by JNK could be ROS-dependent13,39, two antioxidative ROS scavengers, GSH and NAC, were used to ascertain the relationship between 8m-induced cell death and ROS. Both scavengers reduced phosphor-JNK and the levels of other 8m-induced apoptotic proteins as well as the degree of cleavage of specific apoptotic mediators. The cleavage of caspase-7 and PARP were completely abrogated at a GSH concentration of 20 mmol/L (Figure 5C) as well as at NAC concentrations of 10 and 20 mmol/L (Figure 5D). By using the colony formation assay, we found that the numbers of colonies formed from cells co-cultured with 5 mmol/L NAC were higher and that the sizes of the colonies were also considerably larger than those in the 8m-alone culture group (Figure 5E).

Figure 5
figure 5

ROS mediation of 8m-induced cell death. HCT116 cells were treated with the indicated concentrations of 8m for 24 h. (A) ROS was detected using a fluorescent microscope and the H2-DCFDA fluorescent probe. The arrows point to cells producing ROS. Scale bar, 20 μm. (B) Mitochondrial transmembrane potential dissipation was measured using the JC-1 stain. Depolarization of mitochondrial transmembrane potential is specifically indicated by a decrease in the red-to-green fluorescence intensity ratio. Scale bar, 20 μm. (C) After a 12-h incubation in standard culture conditions, HCT116 cells were pretreated with GSH (0, 1, 10, or 20 mmol/L) for 1 h and then treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (D) After a 12-h incubation under standard culture conditions, HCT116 cells were pretreated with NAC (0, 1, 10, or 20 mmol/L) for 1 h and then were treated with 5 μmol/L 8m for 12 h. Whole-cell extracts were prepared and analyzed by Western blotting using the specified antibodies. (E) Cells were treated with 0, 2.5, or 5 μmol/L 8m and/or co-treated with 5 mmol/L NAC for the colony formation assay. Culture medium was changed every 3 to 4 d, and colony formation was inactivated after 14 d. (F) Schematic representation of signaling pathways through which 8m induces the apoptosis of HCT116 cells.

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Discussion

Our previous work demonstrated that treatment using a novel series of benzimidazole derivatives, 2-aryl benzimidazole compounds, can result in the pronounced apoptosis of HepG-2 cancer cells28. In this study, we used an MTT assay to demonstrate that 8m, a novel benzimidazole acridine derivative, potently reduced the viability of the human colon cancer cell lines SW480 and HCT116 with IC50 values of 6.77 and 3.33 μmol/L, respectively. HCT116 cells were selected for further study to determine the distinct mechanisms of 8m-induced cytotoxicity. DAPI staining of 8m-treated HCT116 cells demonstrated the presence of condensed chromatin and apoptotic bodies. Profound increases in externalized phosphatidylserine, a hallmark of early apoptosis, was also detected via flow cytometric analysis of 8m-treated cancer cells. These results suggested that 8m decreased cell viability by inducing the apoptosis of HCT116 cells.

To examine the mechanisms underlying 8m-induced apoptosis, we analyzed the changes in apoptotic proteins after 8m treatment. An apparent induction of cleaved caspase-8 and caspase-9 was detected, indicating that 8m-induced apoptosis involved both the intrinsic mitochondrial-initiated pathway and the extrinsic death receptor-mediated pathway. This result suggests that HCT116 cells are type II cells capable of amplifying apoptotic signaling initiated by the extrinsic pathway through the recruitment of the intrinsic pathway40, whereby activated caspase-9 (cleaved caspase-9) subsequently cleaves caspase-7 and caspase-3 to effect downstream apoptotic events5. PARP, a family of proteins involved in DNA repair in response to environmental stress, can be cleaved by the caspase-3 executioner protein. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis41. In the current study, we found that 8m treatment increased the cleavage of caspase-7, caspase-3, and PARP in a concentration- and time-dependent manner. Bcl-2 provides a distinct survival signal to cancer cells to support neoplastic growth; therefore, we conducted an intense mechanistic study to assess the ability of Bcl-2 to suppress 8m-induced apoptosis42. 8m-induced down-regulation of Bcl-2 can cause the release of cytochrome c from the mitochondria to the cytoplasm, thereby initiating the intrinsic apoptotic signaling pathway35. However, it is not known whether the initiation of the intrinsic pathway is more greatly influenced by Bcl-2 down-regulation or by crosstalk with the extrinsic pathway. Further studies are warranted to elucidate the mechanism underlying the activation of the intrinsic pathway.

Given its ability to trigger specific apoptotic cancer cell death without targeting normal cells, TRAIL is a promising target for cancer therapy. Nevertheless, resistance to TRAIL, resulting from decreased levels of DR4 and DR5 and/or mutations in these proteins, has been a treatment issue43. Furthermore, accumulating evidence indicates that the down-regulation or mutation of death receptors (DRs) may allow malignant cells to avoid destruction by the immune system44. Therefore, agents that increase the levels of DRs in cancer cells may prevent certain types of drug resistance and should therefore be investigated. In our study, 8m induced the expression of DR5 in a concentration- and time-dependent manner. This result is even more noteworthy because real-time PCR analysis suggested that after 8m treatment, DR5 may play a more important role than DR4 in apoptosis and that DR5 can transmit apoptotic signals to caspase-8 to initiate the extrinsic apoptotic pathway6. This evidence demonstrated the potential of 8m as an antitumor small molecule that can up-regulate the expression of DR5, thereby promoting cancer cell apoptosis and decreasing drug resistance.

A large body of evidence has shown that certain anticancer agents trigger apoptosis through the modulation of MAPK14,21,32, which can play an important role in the mediation of DR59,13. In this work, 8m treatment induced p38 and JNK phosphorylation, whereas JNK1 inhibition (but not inhibition of JNK2 or p38) abrogated 8m-induced JNK phosphorylation as well as the cleavage of caspases and PARP. However, the function of 8m-induced p38 phosphorylation remains unclear. Figures 3A, 3B, 4B, 5C, and 5D show the JNK proteins, which have distinct molecular masses of 46 kDa (p46 JNK, the lower lane) and 54 kDa (p54 JNK, the upper lane) and are largely, but not exclusively, composed of the JNK1 and JNK2 isoforms, respectively23. Integrating the results from the siRNA knockdown experiments, we have confirmed that 8m-induced JNK phosphorylation largely (but not absolutely) results from the production of phosphor-JNK1. The JNK1 signaling pathway tends to have a specific role in mediating apoptosis in several types of cancer cells, whereas JNK2 is involved particularly in cell survival signaling 23,24. The activation of JNK1 can lead to the phosphorylation of Bcl-2, resulting in Bcl-2 degradation and an inhibition of its anti-apoptotic properties45,46. However, 8m-induced JNK1 phosphorylation was only barely detectable after transfection with JNK1 siRNA. Knocking down JNK1, but not JNK2, rescued the 8m-induced down-regulation of DR5, Bcl-2, and other apoptotic proteins. Apoptosis studies via flow cytometry also demonstrated a pro-apoptotic role for JNK1 in our system. However, inhibiting JNK1 completely abolish 8m-induced cell death, as shown by Western blotting and flow cytometric assays. Thus, it is likely that JNK1-independent mechanisms also participate in 8m-induced apoptosis.

Environmental stimuli, especially drug stress, are known to damage mitochondrial dynamics and affect mitochondrial function, and vice versa. Mitochondrial dysfunctions include ROS overproduction and mitochondrial membrane depolarization, both of which can induce apoptosis47. In this study, using the fluorescent stains H2-DCFDA and JC-1, we detected 8m-induced ROS production and mitochondrial membrane depolarization in an 8m-concentration-dependent manner. ROS, as an upstream regulator of JNK, plays an important role in various cellular responses, such as proliferation, differentiation, necrosis, and especially cell apoptosis13,20. To examine the effect of ROS on JNK and other pathways, two potent antioxidants, NAC and GSH, were employed to detect the changes resulting from 8m-induced pathway activation or inhibition in antioxidant-pretreated cells. We observed that pretreatment with NAC or GSH prevented both 8m-induced JNK phosphorylation and caspase cleavage. The colony formation assay revealed that HCT116 cells co-cultured with NAC generated much larger colonies than the 8m-alone control. This indicated that ROS signaling was involved in the 8m-induced apoptosis of HCT116 cells and the phosphorylation of JNK1. Combined with the conclusion that JNK1 played a pro-apoptotic role in our system, we can conclude that 8m-induced apoptosis is ROS-JNK1 dependent (Figure 5F). In accordance with our results, many anticancer drugs induce the apoptosis of cancer cells via this pathway. For example, selenite-induced apoptosis in Chang liver cells involved ROS-JNK1 signaling48, and an ROS-JNK1-mediated process affected shikonin-induced apoptosis in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells49.

In conclusion, our study demonstrated that a new benzimidazole acridine derivative (8m) had cytotoxic activity in human colon cancer cell lines. Both the intrinsic and extrinsic death pathways were activated by 8m in a concentration- and time-dependent manner. Further investigation into its mechanism of action confirmed that the ROS-JNK1 pathway played an important role in mediating 8m-induced apoptosis. Furthermore, we demonstrated that 8m can upregulate DR5, a hallmark of effective anticancer agents. Therefore, our findings provide a basis for future investigations aimed at elucidating the role of apoptosis in colon cancer therapy.

Author contribution

Kang CHEN performed the research and drafted the manuscript; Bi-zhu CHU designed the research; Lu-lu LI and Bin LI performed some experiments; Chun-mei GAO provided the 8m compound; and Qin-sheng SUN, Zhi-fa SHEN, Feng LIU, and Yu-yang JIANG provided supervision and support in the project.