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 Table of Contents  
Year : 2016  |  Volume : 2  |  Issue : 6  |  Page : 163-174

Choline kinase inhibitors synergize with TRAIL in the treatment of colorectal tumors and overcomes TRAIL resistance

1 Instituto de Investigación Sanitaria IdiPAZ, Madrid, Spain
2 Laboratory of Cell Signaling and Apoptosis, Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic

Date of Submission17-Oct-2016
Date of Acceptance15-Nov-2016
Date of Web Publication28-Dec-2016

Correspondence Address:
Prof. Juan Carlos Lacal
Instituto de Investigación Sanitaria IdiPAZ, Paseo de la Castellana, 261, 28046 Madrid
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2395-3977.196910

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Aim: The aim of this study was to investigate the effects of the combination of choline kinase inhibitor MN58b and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) against colon cancer cells.
Methods: TRAIL-sensitive (DLD-1) and TRAIL-resistant (SW620) cells were treated with MN58b and/or TRAIL. Cell viability and induction of apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide and flow cytometry. Posttreatment expression levels of different proteins (PARP, caspase-3, X-linked inhibitor of apoptosis protein [XIAP], CHOP, DR5, DR4, CHOP) were analyzed by quantitative reverse transcription polymerase chain reaction, Western blot, and flow cytometry.In vivo antitumoral activity was assessed by xenograft models.
Results: A strong synergistic effect of TRAIL and MN58b was observed in both TRAIL-sensitive and resistant cells. The combinatory treatment induced an increase in PARP and active-caspase 3 fragments along with a decrease in XIAP, enhancing TRAIL sensitivity. Reduced cellular viability and increased cell death correlated with increased DR5 expression and membrane surface recruitment, an effect that was concomitant with CHOP expression.
Conclusion: MN58b, which alone exhibits anticancer activities against a wide variety of tumor-derived cell lines, synergizes with TRAIL through a mechanism that involves DR5 upregulation. This study supports the use of MN58b in combination with TRAIL on colorectal tumors, including those that develop TRAIL resistance.

Keywords: Choline kinase inhibitors, colon cancer, combinatorial chemotherapy, DR5, tumor necrosis factor-related apoptosis inducing ligand, tumor necrosis factor-related apoptosis inducing ligand resistance

How to cite this article:
Lacal JC, Andera L. Choline kinase inhibitors synergize with TRAIL in the treatment of colorectal tumors and overcomes TRAIL resistance. Cancer Transl Med 2016;2:163-74

How to cite this URL:
Lacal JC, Andera L. Choline kinase inhibitors synergize with TRAIL in the treatment of colorectal tumors and overcomes TRAIL resistance. Cancer Transl Med [serial online] 2016 [cited 2020 Aug 5];2:163-74. Available from: http://www.cancertm.com/text.asp?2016/2/6/163/196910

  Introduction Top

Despite continuous efforts in cancer treatment over the past decades, the prognosis of most tumors remains poor. Many tumors are intrinsically resistant to chemotherapy, and many others that recur, though initially sensitive, become resistant not only to the initial therapeutic agents but also to other drugs that were not used in the prior treatment. Thus, inherent and acquired resistance of tumor cells to chemotherapeutic agents is one of the principal reasons for the need to devise novel therapeutic approaches. Attempts to overcome such drug resistance mainly involve the use of combination drug therapy, using different classes of drugs, with the aim of reducing toxicities and to allow maximal dose with narrowest cycle intervals necessary for bone marrow recovery.[1],[2]

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL), also known as Apo2 L, is a member of the TNF superfamily that has been implicated in cancer treatment.[3],[4] TRAIL induces extrinsic apoptosis of the cancer cell through interacting with its membrane receptors. Five different receptors have been identified: TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and a soluble receptor termed osteoprotegerin.[5],[6],[7],[8],[9],[10] Both DR4 and DR5 amino acid sequences contain a conserved death domain (DD) motif, which mediates their role as proapoptotic receptors.[11] On the other hand, DcR1 lacks an intracellular domain and DcR2 has a truncated DD acting as decoy receptors and antagonizes TRAIL-induced apoptosis by competing for ligand binding.[8] TRAIL, when bound to DR4 and/or DR5, triggers receptor trimerization and apoptosis through the extrinsic pathway.[12]

In recent years, TRAIL has emerged as a promising target for cancer therapy due to its remarkable selectivity to tumor cells in inducing apoptosis, without causing toxicity to normal cells.[3],[4],[13],[14],[15],[16] Because of this selectivity toward tumors cells and excellent preclinical results, TRAIL and agonistic antibodies against its receptors were conveyed to Phase I and II clinical trials for cancer treatment either alone or in combination with various chemotherapeutic agents.[17],[18],[19],[20],[21] However, although TRAIL selectively and actively kills cancer cells under in vitro experiments, most human tumors do not respond or are resistant to TRAIL.[22],[23],[24] The mechanism of TRAIL resistance is not fully understood, but is believed to occur at multiple levels, such as loss of death receptors, enhanced expression of molecules that can interfere with caspase-8 activation such as cFLIP, high expression levels of the inhibitor of apoptosis (IAP) family members, alterations in expression of the Bcl-2 family proteins or MADD, or in cell metabolism.[25],[26],[27],[28],[29] Thus, agents that can upregulate TRAIL death receptors, downregulate antiapoptotic proteins, or interfere with cell metabolism have the potential to enhance the apoptotic effects of TRAIL.

Choline kinase alpha (ChoKα), the first enzyme of the Kennedy Pathway for the synthesis of phosphatidylcholine, is overexpressed in different human tumors including breast, lung, prostate, colorectal, ovary, and endometrial cancers and T-cell lymphomas.[30] In addition to ChoKα overexpression, an increased enzymatic activity has been observed in human tumors such as breast [31] and colon cancer [32] and found overexpressed in a large panel of tumor-derived cell lines including osteosarcoma, glioblastoma, breast, colon, liver, and pancreatic tumors.[30],[31],[32] Furthermore, ChoKα has been described as an independent prognostic factor to predict patient outcome in early-stage nonsmall-cell lung cancer patients, and hepatocellular carcinoma in which tumor ChoKα expression was associated with increased mortality.[33] Tumor aggressiveness and metastasis have been associated with increased levels of ChoKα in breast, bladder, liver, and ovarian tumors or have been associated to ER-status in breast cancer.[30] Therefore, ChoKα inhibition constitutes an efficient antitumor strategy which has demonstrated antiproliferative activity in vitro and antitumor activity in vivo.[34],[35],[36] Similar to what has been reported for TRAIL, ChoKα inhibitors are specific toward tumor cells, without affecting normal cells. Thus, ChoKα inhibition constitutes an efficient and new strategy for cancer treatment, with demonstrated activity both in vitro and in vivo.[30],[34],[35],[36],[37],[38],[39],[40] This hypothesis has been supported by the recent introduction of the first ChoKα inhibitor into Phase I clinical trials (http://clinicaltrials.gov/ct2/show/NCT01215864).

Tumor resistance to chemotherapy is one of the major challenges in treating solid tumors, and new approaches to overcome this problem are needed for a better management of cancer patients. TRAIL has recently emerged as a promising therapy due to its selectivity toward tumor cells and has been evaluated in clinical trials with a poor outcome as a single therapy. ChoKα inhibition has also recently proven to be an efficient strategy to specifically induce tumor cell death which have reached Phase I clinical trials. Here, we propose a specific ChoKα inhibitor, MN58b, and TRAIL as a novel combination treatment for both regular and TRAIL-resistant colorectal cancer.

  Methods Top

Cell culture and reagents

Two colon cancer cell lines were used: DLD-1 (CCL-221, ATCC, Manassas VA, USA) and SW620 (CCL-227, ATCC, Manassas VA, USA). DLD-1 cells were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) and SW620 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA), both supplemented with 10% (v/v) fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 2 mM glutamine (Invitrogen, Carlsbad, CA, USA) at standard conditions of temperature (37°C), CO2 (5%), and humidity (95%). TRAIL was provided by Apronex Ltd, Czech Republic. MN58b, produced by the group, has been previously described.[35],[36],[37],[38],[39],[40] Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Invitrogen, Carlsbad, CA, USA) under indicated conditions.

Interaction analysis: combination index

The CalcuSyn software (Biosoft, Cambridge, UK) was used to calculate the synergistic, additive, or antagonistic effects of the drug combinations, according to the method of Chou and Talalay.[41] Combination index (CI) = 1 indicates an additive effect, < 1 indicates synergy, > 1 indicates antagonism. MN58b and TRAIL were applied in different combinations of dose/time as indicated. Quadruplicate data points were used for each concentration; each experiment was repeated at least three times.

Western blot analysis

Protein determinations were performed by standard Western blot technique as previously described.[37],[38],[39] Equal amounts of total protein, after determination by Bradford method,[42] were loaded into SDS-PAGE 10% acrylamide gels and resolved proteins were transferred onto nitrocellulose membranes. The following antibodies were used: anti-PARP (sc-7150, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3, CHOP antibody, DR5, X-linked inhibitor of apoptosis protein (XIAP) from cell signaling technology, and as loading control GAPDH was used (Chemicon International, Inc., Temecula, CA, USA). After washing, membranes were incubated with a peroxidase-conjugated secondary antibody and developed using an ECL chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Autoradiographs were scanned and Quantity One Software was used for densitometric analyses (Bio-Rad, Spain).

Apoptosis detection by flow cytometry

Cell death was analyzed after treatment with indicated compounds by flow cytometry using ApoScreen Annexin V Apoptosis Kit (Southern Biotech Associates, Inc., Birmingham, AL, USA), according to manufacturer procedures.

RNA extraction and real-time quantitative polymerase chain reaction

DR4 and DR5 mRNA levels were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using TaqMan probes (Applied Biosystems, Foster City, CA, USA). Total RNA extraction was performed using the TRizol Reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Valencia, CA, USA), as per manufacturer suggested protocol. Reverse transcription of 1 µg of total RNA was performed by High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA), for 2 h at 37°C. Gene expression assays of cDNAs were done in triplicate using ABI PRISM 7900 Sequence Detector (Applied Biosystems, Foster, CA, USA). The 18S ribosomal RNA was amplified as an internal control. Probes, from Applied Biosystems, used for amplification are as follows: DR5 (TNFRSF10B) Assay ID-Hs00187196_m1, DR4 (TNFRSF10A) Assay ID-Hs00269492_m1, CHOP (Hs_01090850_m1), 18S ribosomal RNA Assay ID-Hs99999901_s1. The mRNA copy numbers were calculated for each sample by the instrument software using Ct value (arithmetic fit point analysis for the lightcycler). Results were expressed in copy numbers, calculated relative to unstimulated cells, after normalization against 18S.

Cell surface staining of death receptors and flow cytometry analysis

Cells surface staining was performed as previously reported.[43] After treatments, cells were harvested, washed, and incubated in ice-cold blocking solution (phosphate buffered saline [PBS] containing 20% normal human AB serum, 0.2% gelatin, 0.1% sodium azide) for 10 min. Cells were then washed once with wash buffer (PBS containing 0.2% gelatin, 0.1% sodium azide), incubated with appropriate monoclonal antibodies, washed twice with ice-cold wash buffer, and finally incubated with the secondary goat anti-mouse antibody coupled to phycoerythrin (IgG1-PE). All incubations were performed on ice. After two final washes, the surface expression of the receptors was assessed using an XL-MCL flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA). The following antibodies were used: mouse IgG1 (0571 Immunotech, Marseille, France) as isotypic control, goat anti-mouse IgG1-PE (731914 Beckman Coulter, Inc., Fullerton, CA, USA), anti-DR4 and anti-DR5 mouse monoclonal antibodies, which were reported previously.[44]

In vivo xenograft model in nude mice

All experiments concerning living laboratory animals were performed after obtaining an approval of the protocol by the local ethics committee, following the Spanish Laboratory Animal Handling laws. 1 × 106 DLD-1 or SW620 cells were mixed with Matrigel (354234 BD Biosciences, San Jose, CA, USA) 1:1 and then injected subcutaneously in female BALB/c nude mice. Combinatorial treatments, administered through intraperitoneal injection, were started when tumors reached a volume of 0.15 cm [3]. Ten mice per group were included and were injected in both flanks (n = 20). Mice were treated for 3 weeks with both MN58b and TRAIL as follows: Monday, Wednesday, and Friday with 2 mg/kg of MN58b, and on Tuesday and Thursday with 20 mg/kg or 40 mg/kg of TRAIL. Tumors were measured twice a week at their greatest length and width, and volume was calculated as (tumor width [2] × tumor length)/2. After 3 weeks of treatment, mice were fed for one more week to continue measuring tumor volume.

Statistical analysis

Results are expressed as mean ± standard error mean fold change over control. One-way ANOVA was used to compare gene and/or protein expression levels between the groups. When statistical significance was found, Bonferroni post hoc comparison test was used to identify differences between groups. Differences were considered significant at P < 0.05. Statistical analyses were performed using the SPSS statistical software, version 11.0. For in vivo studies, Mann–Whitney U-test analysis was performed using SPSS software, version 13.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as P < 0.05.

  Results Top

SW620 cell line is resistant to TRAIL but not to MN58b

To evaluate the potential effect of MN58b and TRAIL combination, two different colon cancer-derived cell lines were used: DLD-1 and SW620. Cell viability was studied to determine the sensitivity of these cells to individual drugs. Cells were treated with different doses of TRAIL and MN58b, followed by MTT cell viability assay at 24 and 48 h posttreatment. Dose response experiments show that DLD-1 cells are sensitive to TRAIL (LC50 around 15–25 ng/mL) while SW620 cells are resistant to it (LC50 > 250 ng/mL) [Figure 1]a. Both cell lines demonstrated very similar sensitivity to MN58b [Figure 1]b, suggesting that the resistance phenotype of SW620 is specific for TRAIL treatment, and both compounds follow different mechanisms of action. Moreover, other cell lines tested (HCT-116 and SW480) were sensitive to both drugs (data not shown). These results allow us to consider SW620 cell line to be of particular interest as in vitro and in vivo TRAIL-resistant colon cancer model and thus in testing combinatorial treatments with MN58b.
Figure 1: Synergistic effect of MN58b and TRAIL combinatorial treatment. (a) Cell viability after 24 h and exposure to TRAIL (left) or 48 h to MN58b (right). (b and c) Effect of combinatorial treatment of TRAIL and MN58b following simultaneous or sequential schedules. Combination index is indicated. (d) In vivo cooperation of MN58b and TRAIL in nude mice inoculated with DLD-1 or SW620 xenografts. Data shown are mean ± standard error (n = 20); asterisk denotes statistically significant differences between cells cotreated with TRAIL and MN58b, versus control or single-agent treated cells (P < 0.05). TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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MN58b and TRAIL combined treatment elicits a synergistic effect on cell viability

The availability of TRAIL resistant cell line, SW620, prompted us to test whether combined therapy of TRAIL and MN58b could display an efficient antiproliferative effect in TRAIL-resistant cells. To identify the most effective schedule of combinations, different concentrations of both drugs were used in a simultaneous, sequential, or concomitant manner, in both cell lines: DLD-1 and SW620. For sequential treatments, cells were first treated with MN58b for 2 or 6 h, then medium was completely removed and treated with TRAIL containing cell culture medium for 24 h, or vice versa; treating first with TRAIL for 2 or 6 h, and then with MN58b for 24 h. For concomitant treatments, cells were treated first with MN58b for 2 or 6 h, and then TRAIL was added without removing MN58b, and incubated for 24 h, or vice versa; treating first with TRAIL for 2 or 6 h, and then with MN58b for 24 h, without removing TRAIL. For simultaneous treatment, both drugs were added at the same time and incubated for 24 h. This schedule of treatments allowed us to establish the best strategy of combined treatment. The cell response to concomitant treatment was qualitatively similar, but not as potent as in the case of simultaneous treatment (data not shown). Thus, after treating DLD-1 cells with 20 µM of MN58b or 10 ng/mL of TRAIL alone, the observed cytotoxicity was 34% and 37%, respectively. However, when both drugs were combined simultaneously, cytotoxicity was found increased to 85% (CI = 0.196), indicating a strong synergism between the two [Figure 1]b. In the TRAIL-resistant SW620 cell line, MN58b (20 µM)-induced cytotoxicity was 45% while TRAIL, at a dose as high as 100 ng/mL, induced cytotoxicity was 12%. However, when the drugs were combined simultaneously, the cytotoxicity of SW620 cells was increased to 85%, with a CI = 0.155, which indicates a strong and effective synergism between the drugs even under TRAIL resistance conditions [Figure 1]c.

Thus, a combined treatment using ChoKα inhibitors and TRAIL can potentiate the cytotoxic effect not only in TRAIL-sensitive cells, such as DLD-1 cells, but also in TRAIL-resistant cells, such as SW620 cells.

Combinatorial treatment of choline kinase alpha inhibitors and TRAIL induces a synergistic antitumor effect in tumor xenografts

The synergistic effect observed in vitro was tested in both DLD-1 and SW620 xenograft models. The mice were subcutaneously injected with DLD-1 or SW620 cells, and when tumors reached 0.15 cm [3] volume, they were treated with each drug independently or in combination. The drugs were delivered as intraperitoneal injections, three times per week with 2 mg/kg MN58b and twice a week with 10, 20, or 40 mg/kg of TRAIL, for 3 weeks. At the end of the study, no weight loss was observed in any treatment group. Tumors induced by DLD-1 cells were inhibited by 42% or 51% when treated with MN58b or with TRAIL (20 mg/kg), respectively [Figure 1]d. When both drugs were combined, the effect was much stronger, reaching a 78% inhibition, demonstrating a synergistic effect in suppressing tumor growth in DLD-1 xenograft model. Similar results were obtained when 10 mg/kg of TRAIL was used (data not shown). SW620 xenograft response to MN58b was weaker, with only 26% inhibition [Figure 1]d, while no significant response was found to TRAIL treatment, as expected of a cell line resistant to TRAIL. However, when MN58b and TRAIL were combined, a marked improvement in the tumor growth inhibition (71%) was observed. The results were consistent with the in vitro data, demonstrating the ability of MN58b to sensitize SW620 cells to TRAIL-induced apoptosis in vivo.

MN58b and TRAIL combined treatment elicits a synergistic effect on cell death

The pharmacological synergistic effect between MN58b and TRAIL was confirmed in both cell lines by analyzing apoptotic proteins, through Western blot technique. Both cleaved PARP and active caspase-3 were clearly higher in the SW620 cells receiving combination treatment [Figure 2]a and [Figure 2]b. In the case of DLD-1 cells, cleaved PARP was increased in the combinatorial treatment, compared to the drugs applied separately [Figure 2]a. At the assessed time (24 h of treatment), TRAIL, as a single treatment, induced an increase in active caspase-3 in DLD-1 cells, not observed by MN58b, and this increase was maintained in combination treatment, compared to control (untreated cells). Finally, XIAP protein levels were reduced after combinatorial treatment in both DLD1 and SW620 cell lines [Figure 2]c.
Figure 2: Analysis of apoptosis-related proteins after MN58b and TRAIL treatment. Both DLD-1 and SW620 cells were treated with MN58b and TRAIL simultaneously for 24 h with indicated concentrations of MN58b and TRAIL. Apoptosis markers were analyzed by Western blot. Increased PARP degradation (a), procaspase-3 cleavage (b), and X-linked inhibitor of apoptosis degradation (c) is observed when MN58b and TRAIL are combined. Figures shown are representative gels and the graph bar shows data as mean ± standard error mean of three independent experiments. Asterisk denotes statistically significant differences between cells cotreated with TRAIL and MN58b, versus control cells (P < 0.05). TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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The effect of combinatorial treatment of MN58b and TRAIL on both DLD-1 and SW620 cell lines was also analyzed by flow cytometry. After the designated treatments, the cells were stained with Annexin V-propidium iodide to analyze the phosphoserine externalization, an early sensitive marker of apoptosis. Treatment of DLD-1 cells for 12 h with either MN58b (20 µM) or TRAIL (10 ng/mL), or both, induced an increase in both Annexin V positive and double positive cells. The proportion of cell death reached 50% when MN58b and TRAIL were combined [Figure 3]a and [Figure 3]b. With respect to SW620 cell line, 12 h of treatment with MN58b (20 µM) or TRAIL (100 ng/mL) produced negligible increases in the proportion of cell death. However, when both drugs were combined, cell death was increased to more than 90% as reflected by double positive cells [Figure 3]c and [Figure 3]d. The simultaneous treatment induced an increase in apoptosis in both cell lines, with SW620 cells being more sensitive than DLD-1 cells. Furthermore, a reduction in the time of exposure to MN58b, to trigger cell death, was achieved. Thus, not only a synergistic mechanism was elicited by the combination of both drugs but also an accelerated process in the response was observed.
Figure 3: Simultaneous treatment with MN58b and TRAIL increases cell death. DLD-1 (a and b) and SW620 (c and d) cells were treated with MN58b or TRAIL alone or with both simultaneously and analyzed by flow cytometry. Figures show a representative flow cytometry experiment (b and d) or data as mean ± standard deviation of three independent assays performed (a and c). #P < 0.05 vs. MN58b alone; $P < 0.05 vs. TRAIL alone; TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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MN58b increases CHOP which remains elevated in MN58b and TRAIL combined treatment

Previous studies in our group have demonstrated that ChoKα inhibitors, MN58b and RSM932A, markedly increased endoplasmic reticulum stress proteins including CHOP.[40] We also demonstrated that CHOP is a key mediator in apoptosis induced by ChoKα inhibitors. To evaluate the mechanism involved in the synergistic effect observed after MN58b and TRAIL combination, we assessed whether MN58b increases CHOP in DLD-1 and SW620 colorectal cancer cells. Consistent with our previous data, MN58b induced CHOP gene expression as soon as 9 h and remained elevated after 24 h. Moreover, elevated CHOP level was retained when MN58b and TRAIL were added together [Figure 4]a and [Figure 4]b.
Figure 4: CHOP expression after treatment with MN58b or TRAIL. DLD-1 (a) and SW620 (b) cells were treated with MN58b and TRAIL for 24 h. mRNA levels of CHOP were analyzed by quantitative reverse transcription polymerase chain reaction. Mean ± standard deviation of three independent assays is shown as log10of the relative quantity of CHOP expression relative to unstimulated cells and normalization against 18S (left). Protein levels were determined by Western blot, and a representative experiment is shown (right). Asterisk denotes statistically significant differences between cells treated with MN58b alone or with combined TRAIL and MN58b, versus control cells and cells treated with TRAIL alone (P < 0.05). TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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MN58b upregulates TRAIL receptor DR5 gene expression, localized to the plasma membrane

To get an insight in the cellular mechanisms underlying MN58b potentiating TRAIL-induced apoptosis, the effect of MN58b on TRAIL receptors DR4 and DR5 gene expression was investigated. Both DLD-1 and SW620 cells were treated for different durations with MN58b or TRAIL, or both, and mRNA levels were analyzed by qRT-PCR. Basal levels of DR4 and DR5 in DLD-1 cells were higher relative to SW620 cells, which might explain the differential sensitivity to TRAIL in these cell lines [Figure 5]a.
Figure 5: MN58b treatment upregulates DR5 expression. (a) Basal mRNA levels of DR5 were analyzed in DLD-1 and SW620 cell lines. (b) Both SW620 and DLD-1 were treated with MN58b, TRAIL, or both, and mRNA levels were analyzed after 9 or 24 h. The results are shown as log10of the relative quantity of DR5 expression. (c and d) SW620 and DLD-1 cell lines were treated with 15 μM MN58b for 24 h and then harvested for analysis of cell surface DR5 by immunofluorescent staining and subsequent flow cytometry. (e) Levels of DR5 protein were analyzed after TRAIL and MN58b treatment in SW620 cells. Asterisk denotes statistically significant differences between cells treated with MN58b or TRAIL, versus control cells (P < 0.05). TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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DR5 expression was specifically increased in MN58b-treated cells in both cell lines [Figure 5]b. After 9 and 24 h of MN58b treatment, the DR5 gene expression was increased by 2- and 3-fold increase in SW620 cell line and by 3- and 5-fold increase in DLD-1 cells, respectively. TRAIL treatment alone increased the levels of DR5 only in DLD1 cells, an effect that was reduced at 24 h of treatment. The expression level of DR4 was significantly affected in the DLD-1 cells under all treatment conditions while no such effect was observed in SW620 cells [Figure 5]b.

Combined MN58b and TRAIL did not further increase the DR5 expression in SW620 cells [Figure 5]b. In DLD-1 cells, the combination treatment induced a further increase in DR5 expression, compared to each drug alone, only at 9 h but not at 24 h suggesting a transient effect on DR5 expression.

As a proof of functional activation, the effect of MN58b treatment on the expression of DR5 on the cell surface was also analyzed by flow cytometry in both cell lines. MN58b increased DR5 cell surface expression in both SW620 and DLD-1 cell lines [Figure 5]c and [Figure 5]d. No changes in DR4 cell surface expression were observed (data not shown). Finally, the level of DR5 protein was also increased in MN58b-treated SW620 cells [Figure 5]e. Thus, MN58b upregulates DR5 gene expression in a dose-dependent manner resulting in an increased total DR5 protein expression and its membrane localization. This is consistent with an augmented sensitivity to TRAIL induced by MN58b.

DR5-silenced cells failed to elicit the synergistic effect observed after MN58b and TRAIL combined treatment

Combined treatment of TRAIL and MN58b induced apoptosis of SW620 cells as determined by annexin V staining. This effect coincides with increased expression of DR5 [Figure 6]a. Furthermore, cell death induced by MN58b and TRAIL in SW620 cells, with downregulated expression of DR5, was dependent on DR5 expression [Figure 6]b and [Figure 6]c. These results suggest that at least partially, the synergistic effect of combinatorial TRAIL and MN58b in tumorigenic colon cancer cells depends on DR5 induction.
Figure 6: DR5 is required for cell death induction by MN58b and TRAIL in SW620 cells. SW620 cells were treated with MN58b or TRAIL alone or with both simultaneously and analyzed by flow cytometry for DR5 expression. (a) A representative flow cytometry experiment is shown (left), and data as mean ± standard deviation of three independent assays performed (right). (b) DR5 protein levels determined by Western blot after downregulation by control (left) or DR5-specific siRNA (right). (c) Effects of DR5 downregulation were correlated with induction of cell death after treatment with MN58b or TRAIL alone and in combination. Asterisk denotes statistically significant differences between cells treated with TRAIL versus control cells (P < 0.05). TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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  Discussion Top

Targeting TRAIL as a therapeutic agent in colon cancer has attracted much interest in recent years.[3],[4],[13],[14],[15],[16],[17],[18],[19],[20],[21] However, despite the promising in vitro results, demonstrating effective antitumor activity with low side effects, lack of a therapeutic effect, and resistance to the treatment has been observed in clinical trials.[22],[24] Since many tumors are found resistant to TRAIL,[45],[46] a big effort has been made to search for appropriate combinatorial treatments to overcome this effect.

ChoKα is overexpressed in different tumor-derived cell lines as well as in different tumors, including colorectal tumors.[30] ChoKα inhibitors have been found to exert an efficient antitumor effect against colon xenografts.[35],[40] In the present work, we have explored the use of ChoKα inhibitors to overcome TRAIL resistance in this type of tumors, suggesting an additional use to this novel class of molecules.

A strong synergistic effect was observed when combining these two drugs with remarkable activity and specificity toward tumor cells. The colon cancer-derived cell line DLD-1, which is the most sensitive to TRAIL among the analyzed cell lines, was used for single and combinatorial treatments with MN58b and TRAIL. Our data show a strong synergistic effect to induce tumor cell death. The results were further verified with the metastasis-derived TRAIL-resistant cell line, SW620 [Figure 1]a.[44] As both cell lines are sensitive to MN58b, we hypothesized that both drugs follow different mechanisms of action. The synergistic effect observed with MN58b and TRAIL is of major importance as the SW620 cells, which are intrinsically resistant to TRAIL, could be sensitized to this cancer-specific drug. The synergistic effect observed in vitro was also verified in vivo using xenograft models of both cell lines. MN58b at suboptimal concentrations had no significant effect in SW620 xenografts. However, a strong synergistic effect was observed when both compounds were combined. With these results, we can conclude that MN58b synergizes with TRAIL to induce tumor growth inhibition in DLD-1 xenograft and also sensitizes TRAIL-resistant SW620-induced tumors to TRAIL.

The mechanism by which MN58b enhances TRAIL-induced apoptosis may involve the induction of TRAIL receptor DR5. These results fit in the framework of strategies focused on the discovery of new drugs targeting the TRAIL death receptors with the aim of either to potentiate TRAIL effect or to increase DR4 and DR5 receptors expression.[44] MN58b increased the expression and cell surface exposure of DR5 in both SW620 and DLD-1 cells. This increase might allow cooperation between MN58b and TRAIL to induce tumor cell death as previous studies have shown that DR4 and DR5 levels contribute to TRAIL sensitivity in some tumor cell lines and DRs overexpression can sensitize different cell lines to TRAIL.[47],[48],[49] DR5 upregulation is a possible mechanism that would explain the observed synergism, but other pathways to sensitize SW620 cells to TRAIL-induced apoptosis have been reported. Thus, when SW620 cells are treated with TRAIL, caspase-8 is recruited and activated at the death-inducing-signaling complex and Bid is cleaved, but there is no caspase-9 activation. Experimental evidence points to the fact that resistance of SW620 cells is based on the mitochondrial ability to withstand large amounts of Bid.[50] Moreover, SW620 cell line expresses high amounts of XIAP and downregulation of XIAP is sufficient to restore the sensitivity of SW620 cells to TRAIL-induced apoptosis.[50] Our data show that XIAP protein levels were decreased after MN58b and TRAIL simultaneous treatment as soon as 9 h, supporting the fact that XIAP reduction promotes TRAIL sensitivity. Therefore, other pathways might also be involved in the mechanism of synergism between MN58b and TRAIL.

Some cancer cells may simultaneously exhibit more than one mechanism of resistance; thus, it is a priority to study the mechanism of TRAIL sensitization by ChoKα inhibition not only in relation to death receptors expression but also considering the specific effects of ChoKα inhibitors on lipid metabolism. It has been proposed that TRAIL resistance in SW620 is caused by a defective ceramide signaling since the combination of C6-ceramide with TRAIL can overcome the resistance of SW620 cells to TRAIL-induced apoptosis.[45] Furthermore, SW620 cells express low levels of Ceramide Synthase 6 (CerS6, also known as longevity assurance homolog 6) and elevation of CerS6 expression was sufficient to overcome their TRAIL resistance.[51] As ceramide levels increase after MN58b treatment,[38] this might contribute to the synergism observed between MN58b and TRAIL. Whether MN58b is altering CerS6 gene expression, or other sphingolipid metabolism genes, deserves special attention and should be clarified in future experiments.

It has been recently reported that CHOP plays an important role in TRAIL sensitivity, through the induction of DR5.[52],[53] This is in agreement with our previous findings that MN58b promotes CHOP overexpression in several cell lines,[40] and we show here that this includes DLD-1 and SW620 cells [Figure 4]. Since our results clearly indicate that MN58b-dependent induction of cell death relies at least in part on an increase in functional DR5, a plausible explanation is that DR5 induction depends on CHOP. Although further studies will be required to demonstrate that the functional relationship between inhibition of ChoKα and induction of DR5 is indeed mediated by CHOP, we propose this as a plausible mechanism that deserves to be explored further [Figure 7].
Figure 7: Putative mechanism of action of ChoKα inhibitors for the sensitization of TRAIL resistant cells. Based on the result shown, sensitivity to TRAIL achieved by MN58b treatment is due to an increase in DR5 expression. MN58b could mediate this effect by upregulation of CHOP. TRAIL: Tumor necrosis factor-related apoptosis inducing ligand

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Taking together, our results have an implication in the treatment of colorectal tumors by a combination of TRAIL and ChoKα inhibitors, and this is especially relevant in those tumors that develop TRAIL resistance. ChoKα inhibition enhances TRAIL-induced apoptosis and can abrogate TRAIL resistance. Considering that MN58b when used alone exhibits anticancer activities in vitro and in vivo against a wide variety of tumor-derived cell lines, it can also have an additional use in combination with TRAIL in a large diversity of tumors. This may represent an interesting approach for the clinical development of both drugs since they have reached clinical trials.


The authors wish to thank Elsa Sánchez López, Maria Alvarez, Ma Angeles Ramos, and Mercedes Berra for technical assistance and performance of some of the experiments.

Financial support and sponsorship

This work was supported by grants to JCL and LA from UE (ONCODEATH LSHG-CT-2006-037278) and to JCL: Comunidad de Madrid (S-BIO/0280/2006), Ministerio de Ciencia e Innovación (SAF2008-03750 and RD06-0020-0016).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

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