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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cancer stem cells (CSCs) are resistant to radiotherapy and chemotherapy and play a significant role in cancer recurrence. Design of better treatment strategies that can eliminate or otherwise control CSC populations in tumors is necessary. In this study, the sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity and the effect of amurensin G, a novel sirtuin 1 (SIRT1) inhibitor, were examined using the CSC-enriched fraction of HCT-15 human colon cancer cells. Cancer stem cell-enriched HCT-15 colony cells were paradoxically less sensitive to doxorubicin, and more sensitive to TRAIL-induced cytotoxicity, than their parental cells. Also, CD44+ HCT-15 cells were more susceptible to TRAIL-mediated cytotoxicity than CD44 HCT-15 cells, possibly due to increased levels of death receptors DR4 and DR5 as well as c-Myc, and decreased levels of c-FLIPL/S in CD44+ cells compared with CD44 HCT-15 cells. The combination effect of amurensin G on TRAIL-mediated cytotoxicity was much more apparent in CD44+ cells than in CD44 HCT-15 cells, and this was associated with more prominent downregulation of c-FLIPL/S in CD44+ cells than in CD44 HCT-15 cells. These results indicate that HCT-15 colony or CD44+ cells, which may have CSC properties, are more sensitive to TRAIL than parental or CD44 HCT-15 cells. Amurensin G may be effective in eliminating colon CSCs and be applicable to potentiate the sensitivity of colon CSCs to TRAIL.

Cancer stem cells (CSCs) are cancer cells that possess characteristics associated with normal stem cells and generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors.[1] Tumors rich in CSCs are associated with higher rates of metastasis and poor patient prognosis.[2] It has been reported that as many as 25% of the cancer cells within certain tumors have the properties of CSCs.[3] In cancer, a lack of a curative treatment for metastatic disease and the high level of disease recurrence following standard therapies support increasing evidence of the importance of CSCs in disease progression.[4, 5] As CSCs are refractory to conventional radiotherapy and chemotherapy,[6-8] development of specific therapies targeting CSCs holds hope for improvement of survival and quality of life for cancer patients, especially for sufferers of metastatic disease.[1]

Much of the advance in CSCs comes from FACS analysis, which identifies CSCs from primary tumors, colony forming assay, and tumorigenic assay in immune-deficient mice.[9, 10] One of the characteristics of CSCs is that they can be grown to form spherical colonies in vitro, when plated in limited numbers under anchorage-independent conditions in a serum-free defined media supplemented with growth factors.[11] It is possible to use cell-surface marker profiles to isolate cancer cell subpopulations that are enriched for or depleted of CSCs. Colorectal CSCs can be identified by specific surface markers, such as CD133, CD44, and CD166.[11, 12] More recently, Lgr5, Musashi-1, and OCT-4 have been added to the list of stem cell markers for colon cancer.[13-15] Conventional radiotherapy and chemotherapy destroy the proliferating and differentiated cells that form the bulk of the tumor, but are largely ineffective against the relatively quiescent/dormant CSCs that form a very small proportion of the tumor and have protective mechanisms for repairing DNA and counteracting cytotoxic drugs.[16] Therefore, to overcome radiation/drug resistance, which leads to patient relapse, CSCs should be targeted.

Colon cancer is thought to arise from premalignant polyps that can give rise to malignant tumors through a stepwise series of genetic mutations in adenomatous polyposis coli (APC), p53, K-Ras, and Smad, the so-called adenoma–carcinoma sequence.[17, 18] Colorectal CSCs arise from the transformation of normal stem cells that acquire a transformed phenotype.[19] Currently, no colorectal CSC-directed therapies are available for colon cancer patients. Conventional chemotherapy is limited by its inability to eradicate colorectal CSCs, known to be often resistant to therapies. Therefore, a comprehensive knowledge of the molecular mechanisms regulating colorectal CSCs maintenance and renewal may lead to the development of curative therapies. The most relevant functional activity in colorectal CSCs is likely to be recognized in the Wnt signaling pathway,[20] and its deregulation is known to strongly affect transition from normal colon mucosa to adenoma, whereas its activation in established cancers is pivotal to the expression of a stem cell-like phenotype.

Proposed to be deregulated in a broad range of human cancers, the overexpression of c-Myc frequently correlates with aggressive, poorly differentiated tumors and poor prognosis.[21, 22] Colorectal cancers are crucially associated with the activation of the Wnt/β-catenin signaling pathway that leads to the transcriptional induction of the c-Myc oncogene.[23] It has been reported that c-Myc sensitizes human cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by upregulating the cell surface level of the DR5 death receptor and stimulation of caspase-8 activity.[24] Tumor necrosis factor-related apoptosis-inducing ligand has received much attention as a potential cancer therapeutic, because it is capable of inducing apoptosis in a wide variety of tumor cells while sparing most normal cell types.[25, 26] Binding of TRAIL to death receptors DR4 and DR5 causes receptor trimerization, followed by recruitment of Fas associated with death domain protein and initiator caspases (caspase-8 and -10) to form the death-inducing signaling complex, which promotes autoactivation of caspase-8/10 and the subsequent activation of effector caspases (primarily caspase-3, -6 and -7), which induce apoptosis finally. Previously, it has been shown that the suppression of sirtuin 1 (SIRT1) with siRNA or amurensin G, a SIRT1 inhibitor, sensitizes TRAIL-resistant K562 cells to TRAIL-induced apoptosis, possibly by the upregulation of c-Myc and DR5 surface expression.[27]

Therefore, in this study, the sensitivity to TRAIL-induced cytotoxicity and the effect of amurensin G on TRAIL-induced apoptosis were compared between CD44+ and CD44 fractions of human colon cancer cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cell culture

The HCT-15 human colon cancer cell line was purchased from ATCC (Rockville, MD, USA). Cells were maintained in RPMI medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) heat-inactivated FBS (Gibco BRL, Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a 5% CO2 humidified incubator at 37°C. Cell number and viability were assessed using a standard assay of the exclusion of Trypan blue. Recombinant human soluble TRAIL was obtained from R&D Systems (Minneapolis, MN, USA), and amurensin G, which has an inhibitory effect on SIRT1 enzymatic activity, was isolated from the stem of Vitis amurensis.[28] For soft agar culture, 5000 single cells were seeded in 1 mL 0.35% top agar mixture in 2 × RPMI-1640 medium containing 20% FBS onto a 6-well culture plate prepared with a 0.5% bottom agar mixture in 2 × RPMI-1640 medium containing 20% FBS in a 5% CO2 humidified incubator at 37°C for 14 days.

Cell proliferation assay

Cell proliferation was measured by counting viable cells by using the MTT colorimetric dye-reduction method. Exponentially growing cells (2 × 104 cells/well) were plated in a 96-well plate and incubated in growth medium treated with the indicated concentration of TRAIL, doxorubicin, and/or amurensin G at 37°C. After 96 h, the medium was aspirated using centrifugation and MTT-formazan crystals solubilized in 100 μL DMSO. The optical density of each sample at 570 nm was measured using an ELISA reader. The optical density of the medium was proportional to the number of viable cells. Inhibition of proliferation was evaluated as a percentage of control growth (no drug in the medium). All experiments were repeated in at least two experiments in triplicate. The IC50 (50% inhibitory concentration) values of TRAIL and amurensin G were calculated using the ED50plus version 1.0 Excel (Microsoft) add-in program (Dr. M.H. Vargas, Institute of Nuclear Energy Research, Tlalpan, Mexico). Interaction between amurensin G and TRAIL was assessed using CompuSyn Software (ComboSyn, Paramus, NJ, USA). A combination index (CI) < 0.9 represents drug synergism, 0.9 < CI <1.1 implies nearly additive interactions, and CI > 1.1 indicates antagonism. All experiments were carried out in triplicate.

Western blot analysis

Cells were washed with ice-cold phosphate buffer, lysed in lysis buffer consisting of 1% (w/v) SDS, 1 mM sodium orthovanadate, and 10 mM Tris (pH 7.4) and sonicated for 5 s. Lysate-containing proteins were quantified using a Bradford protein assay kit (Pierce, Rockford, IL, USA). Protein samples were separated by 10% SDS-PAGE using a mini gel apparatus (Bio-Rad, Hercules, CA, USA) and were transferred onto nitrocellulose membranes (Hybond-ECL; GE Healthcare, Piscataway, NJ, USA). Each membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20. Protein bands were probed with primary antibody followed by labeling with HRP-conjugated anti-mouse, anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The antibodies used were: SIRT1, caspase-8, capase-9 (all Cell Signaling Technology), c-Myc, caspase-7 (both Epitomics, Burlingame, CA, USA), caspase-3, poly(ADP-ribose) polymerase (PARP) (both Santa Cruz Biotechnology, Santa Cruz, CA, USA), FLICE-inhibitory proteinL/S (c-FLIPL/S; Enzo Life Sciences, Farmingdale, NY, USA), and β-actin antibody (Sigma-Aldrich). Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer's instructions. The band intensities were quantified with MultiGauge version 2.0 software (Fujifilm, Tokyo, Japan).

Flow cytometric analysis of TRAIL receptors

The profile of cell surface molecule expression was carried out on unsorted HCT-15 cells and gated using mouse anti-human CD44-FITC (BD Biosciences, San Jose, CA, USA) and mouse anti-human CD133-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). HCT-15 cells (5 × 105 cells/well) were centrifuged at 500g and resuspended in 500 mL PBS. Cells were then incubated for 2 h on ice with 4 μL mouse IgG, anti-DR5 monoclonal mouse antibody (1:100; R&D Systems) for 30 min. After washing with PBS, FITC-conjugated rabbit anti-mouse IgG (1:200; Sigma Chemical Co., St. Louis, MO, USA) was added to cell suspensions, incubated for 30 min on ice, and washed with PBS. After rinsing, samples were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using CellQuest software.

Conventional and real-time RT-PCR

Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and the RNA concentration was determined from the absorbance at 260 nm. First-strand DNA was reverse transcribed from 1 μg total RNA in a final volume of 20 μL. The DNA was added to a 20 μL PCR reaction mixture with each set of gene-specific primers: the 5′-forward and 3′-reverse-complement PCR primers for amplification of each gene were as follows: CD44 (forward) 5′-AGAAGGTGTGGGCAGAAGAA-3′ and (reverse), 5′-AAATGCACCATTTCCTGAGA-3′; FLIPL (forward) 5′-TTCCAG GCTTT CGGTTTCTT-3′ and (reverse) 5′-GTCCGAAACAAGGTCAGGGT-3′; FLIPS (forward) 5′-ACCCTCACCTTGT TTCGGAC-3′ and (reverse) 5′-CTTTTGGATTGCTGCTTGGA-3′; c-Myc (forward) 5′-GCAGCCGTATTTCTACTGCG-3′ and (reverse) 5′-GTCGTTGAGA GGGTAGGGGA-3′; DR4 (forward) 5′-AGGGATGGTCAAGGTCAAGG-3′ and (reverse) 5′-ATGAGCTGGTCCCAGGAGTC-3′; DR5 (forward) 5′-CGAGATGCCTCTGTCCACAC-3′ and (reverse) 5′-GCACAAACGGAATGATCCAG-3′, 5′-CCGCAGCTTACACATGTTCT-3′; ABCC1 (forward) 5′-TCTACCTCCTGTGGCTGAATCTG-3′ and (reverse) 5′-CCGATTGTCTTTGCTCTTCATG-3′; ABCC2 (forward) 5′-TCCTTGCGCAGCTGGATTACAT-3′ and (reverse) 5′-TCGCTGAAGTGAGAGTAGATTG; SIRT1 (forward) 5′-CTAGGTGCCCAGCTGATGAA and (reverse) 5′-GTGGGTGGCAACTCTGACAA-3′ 3; β-actin (forward), 5′-ACCAACTGGGACGACATGGAG-3′ and (reverse), 5′-GTGAGGATCTTCATGAGGTAGTC-3′. The thermal cycling conditions used consisted of initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 60 s, 58°C for 60 s, and 72°C for 90 s, and a final extension for 10 min at 72°C. The final PCR products were electrophoresed on 1% agarose gel and photographed under UV illumination. In all experiments, GAPDH was used as the endogenous control. Results were analyzed as a relative quantity based on vehicle-treated samples.

Statistical analysis

Results are given as the mean ± SE of at least three independent experiments. The statistical significance of differences was assessed using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant in all experiments.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Comparison of sensitivity to doxorubicin and TRAIL between HCT-15 parental and colony cells

To compare sensitivity to doxorubicin and TRAIL between bulk tumor cells and CSCs, colony cells were isolated to enrich CSCs, as CSCs can for colonies in soft agar.[29, 30] The expression of CD133 and CD44 has been used to identify colon CSCs.[9, 10] Therefore, the expression of CD44 and CD133 was analyzed in HCT-15 parental and colony cells using flow cytometry (Fig. 1a). The proportion of CD44+ cells was higher in colony cells (89.26%) compared with their parental HCT-15 cells (48.19%), and there was no difference in the expression of CD133 between the parental (1.50%) and their colony cells (2.24%). This result suggests that the colony cells contain more CSCs than their parental HCT-15 cells, and CD44 may be an important determinant for CSCs.

image

Figure 1. (a) After labeling HCT-15 parental (P) and their colony (C) cells with anti-CD44 and anti-CD133 antibody (1:10), the cell surface expression of CD44 and CD133 was quantified by flow cytometry. (b) P and C cells were treated with various concentrations of doxorubicin (DOX) for 96 h, and cell survival was determined using MTT assay. Each bar represents the mean ± SD of triplicate experiments. ***< 0.001.

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One characteristic of CSCs is drug resistance, possibly due to enrichment of ABC transporter proteins in CSCs and resultant rapid and effective efflux of drugs out of the cells.[31] Therefore, to determine the chemoresistant property of colony cells, the cells were treated with different concentrations of doxorubicin (Fig. 1b). HCT-15 colony cells (IC50 = 0.031 μg/mL) showed resistance to doxorubicin, compared with parental HCT-15 cells (IC50 = 0.078 μg/mL), suggesting that HCT-15 colony cells may contain more CSCs with chemoresistance than their parental bulk tumor cells.

As a targeted cancer therapy, TRAIL preferentially kills cancer versus normal cells,[25, 26] therefore TRAIL-mediated cytotoxicity was compared between HCT-15 parental and colony cells to measure the TRAIL effectiveness for CSCs. In contrast with resistance to doxorubicin, HCT-15 colony cells (IC50 = 7.2 ng/mL) were more sensitive to TRAIL-induced cytotoxicity than their parental cells (IC50 = 13.23 ng/mL) (Fig. 2a). These results suggest that TRAIL may be useful to eradicate CSCs with therapy resistance. As TRAIL acts through the TRAIL receptors (DR5 and DR4) to induce apoptosis, the protein levels of DR4/5 were compared between HCT-15 parental and colony cells (Fig. 2b). The DR4/5 level of colony cells was increased compared with that of parental cells. To further understand the factors that contribute to the increased sensitivity of colony cells to TRAIL, the expression of cellular c-FLIPL/S, an important downstream regulator of death receptor-mediated apoptosis, were determined. In contrast to DR5, the c-FLIPL/S levels of colony cells were significantly decreased as compared with those of parental cells. As c-Myc regulates, positively and negatively, the expression of DR5 and c-FLIP, respectively,[25, 26] the level of c-Myc was compared. The c-Myc level of colony cells was significantly higher than that of parental cells.

image

Figure 2. (a) HCT-15 parental (P) and colony cells (C) were treated with increasing concentrations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for 96 h, and the cell survival was determined by MTT assay. Each bar represents the mean ± SD of triplicate experiments. *< 0.05 and **< 0.01. (b) Western blot analysis was carried out to determine the levels of death receptor (DR) 4 and 5, FLICE-inhibitory protein (FLIPL/S), c-Myc, and sirtuin 1 (SIRT1) in P and C cells. Actin was used as a loading control.

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It has been reported that upregulation of c-Myc is associated with downregulation of SIRT1 expression.[32] Therefore, the expression of SIRT1 was compared in both cells. The SIRT1 expression was reduced in colony cells compared with parental cells. Therefore, our data indicate that the increased susceptibility of colony cells to TRAIL was closely associated with upregulated DR5 and downregulated c-FLIP.

Effects of amurensin G on TRAIL-induced cytotoxicity of HCT-15 parental and colony cells

Amurensin G, a new potent SIRT1 inhibitor, enhanced TRAIL-induced caspase-dependent apoptosis by upregulation of DR5 and downregulation of c-FLIP in human leukemic K562 cells,[27] so the cytotoxic effect of amurensin G was compared between HCT-15 parental and colony cells. Colony cells (IC50 = 2.37 ng/mL) were more sensitive to amurensin G compared with parental HCT-15 cells (IC50 = 9.51 μg/mL) (Fig. 3a). When the colony cells were treated with gradually increasing doses of amurensin G, the levels of c-FLIPL/S as well as SIRT1 were decreased and, inversely, the levels of c-Myc and DR4/5 were increased in a dose-dependent manner (Fig. 3b). Moreover, amurensin G significantly potentiated the sensitivity of colony cells to TRAIL when compared to parental cells (Fig. 3c). These results suggest the possibility that combined treatment with TRAIL and amurensin G might be effective in eliminating colon CSCs.

image

Figure 3. (a) HCT-15 parental (P) and colony (C) cells were treated with amurensin G (2.5 and 5 μg/mL) for 96 h, and the cell survival was determined by MTT assay. (b) C cells were treated with various concentrations of amurensin G for 12 h, and levels of death receptor (DR) 4 and 5, cellular FLICE-inhibitory protein (FLIPL/S), c-Myc, and sirtuin 1 (SIRT1) were determined by Western blotting. (c) C cells were treated with various concentrations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the presence (+) or absence (−) of amurensin G (2.5 μg/mL) for 96 h, and cell survival was determined using MTT assay. Each bar represents the mean ± SD of triplicate experiments. *< 0.05; ***< 0.001.

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Comparison of sensitivity to TRAIL between CD44 and CD44+ HCT-15 cells

Cancer stem cells can have colony-forming ability in soft agar,[33, 34] and the proportion of CD44+ cells was higher in colony cells compared with their parental HCT-15 cells (Fig. 1a), CD44+ and CD44 fractions were sorted with FACS to investigate the sensitivity to TRAIL of CD44+ cells, which may have cancer stem-like properties (Fig. 4a, left). As the Wnt/β-catenin pathway plays a critical role in growth and maintenance of colonospheres, which are highly enriched in CSCs,[35] the mRNA and protein levels of β-catenin were compared between CD44+ and CD44 HCT-15 cells. The mRNA and protein levels of β-catenin were higher in CD44+ cells than CD44 cells (Fig. 4a, right). Also, CD44+ cells showed higher mRNA expressions of multidrug resistance-associated proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) and OCT-4, important stem cell-related genes, compared with those of CD44 cells (Fig. 4b). These results suggest that CD44+ HCT-15 cells would be a fraction containing CSCs.

image

Figure 4. (a) After sorting the CD44+ and CD44 HCT-15 cells with FACS, cell surface expression of CD44 in both groups was confirmed by flow cytometry, and β-catenin mRNA and protein expression in both cells was determined by RT-PCR and Western blotting, respectively. (b) The mRNA levels of CD44, OCT-4, ABCC1/2, c-Myc, SIRT1, DR4/5, and c-FLIPL/S were determined by RT-PCR. Bands were quantified using the software MultiGauge. (c) After labeling with control IgG, death receptor (DR) 4 or 5 antibody (1:50) and subsequently staining with FITC-conjugated secondary antibodies (1:50), cell surface expressions of both DR4 and DR5 were quantified by flow cytometry.

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In addition, consistent with the molecular changes in colony cells, the mRNA levels of DR4/DR5 and c-Myc increased and that of c-FLIPL/S decreased in CD44+ cells compared with CD44 cells (Fig. 4b). Analysis with FACS also showed higher cell surface expression of DR4 and DR5 in CD44+ cells than in CD44 cells (Fig. 4c). Therefore, these data suggest that, like colony cells, CD44+ cells, which may be a CSC-enriched fraction, would be more susceptible to TRAIL-induced cytotoxicity.

The molecules involved in the TRAIL pathway were differentially expressed between CD44+ and CD44 HCT-15 cells, therefore, the susceptibility to TRAIL was compared between CD44+ and CD44 HCT-15 cells. CD44+ HCT-15 cells were more susceptible to TRAIL-mediated cytotoxicity than CD44 HCT-15 cells (Fig. 5a). The increased susceptibility of CD44+ cells to TRAIL was followed by an increased cleavage of initiator (caspase-8 and -9) and effector (caspase-3 and -7) caspases, and PARP, a hallmark of caspase-dependent apoptosis, after treatment with TRAIL in CD44+ cells compared with those of CD44 HCT-15 cells (Fig. 5b). These results suggest that CD44+ HCT-15 cells are more susceptible to TRAIL-mediated cytotoxicity than CD44 HCT-15 cells, possibly due to the increased levels of DR4/DR5 and c-Myc and the decreased levels of c-FLIPL/S in CD44+ cells compared with CD44 cells.

image

Figure 5. (a) CD44+ and CD44 cells and unsorted HCT-15 cells (as control) were treated with increasing concentrations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) for 96 h before MTT assay. Each column represents the mean ± SD of triplicate experiments. **< 0.01; ***< 0.001. (b) CD44+ and CD44 HCT-15 cells were treated with TRAIL (5 or 10 ng/mL) for 12 h. Western blot analysis was carried out to determine the cleavage of pro-caspases (Pro-C3 and Pro-C7–9) and poly(ADP-ribose) polymerase (PARP).

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Effects of amurensin G on TRAIL-mediated cytotoxicity of CD44+ and CD44 HCT-15 cells

To compare the cytotoxic effect of amurensin G between CD44+ and CD44 HCT-15 cells, cells were treated with increasing concentrations of amurensin G and the cytotoxicity was determined by MTT assay (Fig. 6a). Amurensin G was found to induce higher cytotoxicity in CD44+ HCT-15 cells compared with CD44 HCT-15 cells. This result was followed by higher activation of caspase-8, -9, and -3 in CD44+ HCT-15 cells compared with CD44 HCT-15 cells after treatment with amurensin G. Like the colony cells, the basal level of c-FLIPL/S was significantly lower in CD44+ cells compared with CD44 cells (Fig. 6b). Treatment with amurensin G also resulted in decreases in the levels of c-FLIPL/S in both CD44+ and CD44 HCT-15 cells. These findings suggest that CD44+ HCT-15 cells are more susceptible to amurensin G, possibly due to the lower levels of c-FLIPL/S compared with CD44 HCT-15 cells.

image

Figure 6. (a) CD44+ and CD44 HCT-15 cells were treated with increasing concentrations of amurensin G for 96 h, and the cell survival was determined by MTT assay. Each column represents the mean ± SD of triplicate experiments. *< 0.05; ***< 0.001. (b) CD44+ and CD44 HCT-15 cells were treated with amurensin G (1 or 5 μg/mL) for 12 h. Western blot analysis was carried out to determine the levels of c-Myc and FLICE-inhibitory protein (FLIPL/S), and cleavage of pro-caspases (Pro-C3, -C8, and -C9).

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To determine whether amurensin G can enhance TRAIL-mediated cytotoxicity, the cytotoxic effect of TRAIL was compared between CD44+ and CD44 HCT-15 cells in the presence or absence of amurensin G. Our data showed that amurensin G enhanced TRAIL cytotoxicity synergistically in CD44+ cells (CI = 0.56, 0.20, and 0.13 for 1.0, 5.0, and 10.0 ng/mL TRAIL, respectively), and additively in CD44 HCT-15 cells (Fig. 7a).

image

Figure 7. (a) CD44+ and CD44 HCT-15 cells were treated with various concentrations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the presence (+) or absence (−) of amurensin G (2.5 μg/mL) for 96 h, and the cell survival was determined by MTT assay. Each column represents the mean ± SD of triplicate experiments. *< 0.05; ***< 0.001. (b) CD44+ and CD44 HCT-15 cells were treated with TRAIL (5 or 10 ng/mL) in the presence (+) or absence (−) of amurensin G (2.5 μg/mL) for 12 h, and Western blot analysis was carried out to determine changed levels of cellular FLICE-inhibitory protein (c-FLIPL/S), c-Myc, and sirtuin 1 (SIRT1) and activation of pro-caspases (Pro-C8 and -C9) and poly(ADP-ribose) polymerase (PARP) cleavage. Bands of cleaved pro-caspases and PARP (active form) in the Western blots were quantified by densitometry using the software MultiGauge.

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Next, the modulation of molecules associated with the TRAIL pathway by amurensin G was compared between CD44+ cells and CD44 HCT-15 cells (Fig. 7b). The cleavage of PARP and pro-caspase-8 and -9 by treatment with TRAIL was increased by combined treatment with amurensin G, and the combination effect of TRAIL and amurensin G on activation of pro-caspase-8 and -9 and cleavage of PARP was more prominent in CD44+ cells than in CD44 HCT-15 cells. These results were followed by the downregulation of SIRT1 and c-FLIPL/S and upregulation of c-Myc after combined treatment with TRAIL and amurensin G, and the modulation of these molecules seemed to be more prominent in CD44+ HCT-15 cells than CD44 HCT-15 cells. These results suggest that the combined treatment with TRAIL and amurensin G would be effective to eradicate the CD44+ HCT-15 cells, which may be a CSC-enriched fraction.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cancer stem cell populations are intrinsically resistant to chemotherapy due to their expression of ABC efflux pumps, which afford protection to CSCs from the adverse effects of chemotherapeutic insult,[36, 37] therefore, it is necessary to discover new therapeutic targets and improve current anticancer strategies. A previous study showed that the MDR variant of CEM cells was hypersensitive to TRAIL due to upregulation of DR5 and concomitant down-regulation of c-FLIP, and degradation of P-gp,[38] and amurensin G sensitized TRAIL-resistant human leukemic K562 cells to TRAIL-induced apoptosis by SIRT1 inhibition.[27]

In the present study, we showed that CD44+ HCT-15 cells, which may be a CSC-enriched fraction, were more sensitive to combined treatment with TRAIL and amurensin G than CD44 HCT-15 cells. As colony cells had many more CD44+ HCT-15 cells than the parental HCT-15 cells, colony cells and CD44+ cells, as CSC-enriched fractions, were used to study the sensitivity of colon CSCs to anticancer drug and TRAIL. HCT-15 colony cells responded paradoxically to doxorubicin and TRAIL compared with parental cells, that is, HCT-15 colony cells showed less and more sensitivity to doxorubicin- and TRAIL-induced cytotoxicity than their parental cells, respectively. It was reported that side population (SP) cells, defined to possess stem cell characteristics, from human colon SW480 cell lines, which are most often resistant to chemotherapeutic agents such as etoposide, cisplatin, and 5-fluorouracil, are more sensitive to TRAIL than non-SP cells.[39] In addition, similar results were observed in leukemia-initiating cells derived from precursor B-cell acute lymphoblastic leukemia,[40] a CD44(+)CD24(−/low) subpopulation of cells within the B6 PyMT-MMTV transgenic mouse-derived AT-3 mammary carcinoma cell line with CSC-like characteristics,[40] and basal-like breast cancer stem cells.[41] However, CD133-high Jurkat cells were more resistant to TRAIL-induced apoptosis than their CD133-low counterparts,[42] and glioma cells with stem cell features, such as CD133 expression and neurosphere formation, were more resistant to TRAIL as well as cytotoxic drugs than glioma cells lacking stem-cell like features.[43] Therefore, it could be suggested that, despite the refractory nature of CSCs to conventional therapies, TRAIL may be useful to eradicate CSC-like cells of some cancer types.

Tumor necrosis factor-related apoptosis-inducing ligand acts through the TRAIL receptors (DR5 and DR4) to induce apoptosis, so molecules associated with the TRAIL pathway would be involved in hypersensitivity of CSC-like cells to TRAIL. Indeed, HCT-15 colony and CD44+ populations showed higher levels of DR4/DR5 and c-Myc and lower levels of c-FLIPL/S compared with their counterparts. Colonospheres show increased levels of total β-catenin, cyclin-D1, and c-Myc, and upregulation of c-Myc greatly enhances the formation of colonospheres.[35] c-Myc can upregulate and downregulate the expression of DR5 and c-FLIP, respectively,[25, 26] and knockdown of the c-Myc gene reduces the responsiveness to TRAIL and the expression of DR5 in PC3-MM2 cells.[44] Therefore, c-Myc, which is required for properties of CSCs, may be responsible for hypersensitivity of HCT-15 colony and CD44+ cells to TRAIL. This hypothesis was supported by a previous report indicating that SP-cells with higher levels of c-Myc, which activates DR4 transcription, have a higher sensitivity to TRAIL than non-SP cells.[39]

The potentiating effect of amurensin G on TRAIL-mediated cytotoxicity in CD44+ HCT-15 cells was higher than that in CD44 HCT-15 cells. These results were associated with the more prominent downregulation of c-FLIPL/S and upregulation of c-Myc after combined treatment with TRAIL and amurensin G in CD44+ HCT-15 cells than in CD44 HCT-15 cells. Previously, it has been shown that amurensin G, a novel SIRT1 inhibitor, sensitizes TRAIL-resistant human leukemic K562 cells to TRAIL-induced apoptosis, possibly by the upregulation of c-Myc and DR5 surface expression, and the downregulation of c-FLIP and Mcl-1.[27] Therefore, amurensin G would be used as a sensitizer of TRAIL. However, we do not know why amurensin G preferentially sensitized the colony cells and CD44+ cells to TRAIL compared with the parent or CD44 cells. It has been known that an increase of drug targets is one of many ways in which cancer cells became resistant to anticancer drugs.[45] As the level of SIRT1, which is a target of amurensin G, was significantly lower in colony and CD44+ cells compared with parent cells and CD44 cells, respectively, the colony and CD44+ cells might be more sensitive to amurensin G. However, the molecular mechanisms remain to be fully elucidated.

In conclusion, these results indicate that HCT-15 colony or CD44+ cells, which may have CSC properties, are more sensitive to TRAIL than HCT-15 parental or CD44 cells. Amurensin G may be effective in eliminating colon CSCs and be applicable to potentiate the sensitivity of colon CSCs to TRAIL.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This research was supported by the Basic Science Research Program (No. 2012R1A1A2005016) through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology and the National R&D Program for Cancer Control, Ministry for Health, Welfare, and Family Affairs (No. 0920050), Korea.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References