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Keywords:

  • guttiferone K;
  • anti-tumor;
  • colon cancer;
  • apoptosis;
  • cell cycle

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration- and time-dependently reduced the viability of human colon cancer HT-29 cells (IC50 value 5.39 ± 0.22 μM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G0/G1 cell cycle arrest in HT-29 cells by down-regulating cyclins D1, D3 and cyclin-dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 μM) induced cleavage of PARP, caspases-3, -8 and -9 and chromatin condensation to stimulate caspase-3-mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK-induced caspase-3 activity, indicating the possible involvement of JNK in GUTK-induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5-fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase-3 and a decrease in cell proliferation marker Ki-67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.

Colorectal cancer is the third leading cause of cancer-related mortality in the United States1 and has the highest incidence rate amongst cancers in Australia, Europe and North America.2 Currently, the standard treatment for colorectal cancer is surgical resection followed by chemotherapy or radiation therapy.3 Patients are usually administered combinations of 5-fluorouracil, leucovorin, oxaliplatin, irinotecan and bevacizumab. However, problems including low response rates, drug resistance and adverse effects, together with high recurrence and metastasis of the disease, have limited the success of treatment to 10 to 60%.4, 5 To date, all cytotoxic anticancer drugs show adverse effects when used alone or in combination and their lack of selectivity for cancer cells continues to be the largest barrier for anticancer drug discovery. Therefore, there is a high demand for new antitumor drugs that are potent and selective toward cancers. Since many clinically used chemotherapeutic drugs originate from plants,6 there is a growing interest in the anticancer potential of compounds isolated from herbs.

Garcinia species (Family Guttiferae) are tropical evergreen trees and shrubs that are widely distributed in Southeastern Asia and their phytochemistry has been widely studied.7 Classic and caged xanthones have been isolated from various parts of these plants, and identified as their major bioactive components.8 Traditionally, Garcinia extract (called gamboge) has been used in folk and Chinese medicine to promote detoxification and treat inflammation and wounds,8, 9 and recently xanthones isolated from various Garcinia species also showed antibacterial, antioxidant, antiviral and neuroprotective effects.7, 10-12 In the last decade, most of the research on Garcinia species has focused on the anticancer activity of gambogic acid, a caged xanthone found at high concentrations in gamboge.8

Gambogic acid and its derivatives are cytotoxic in a wide spectrum of cancer cell lines by binding to the transferrin receptor and induction of G2/M cell cycle arrest and mitochondrial and death receptor-mediated apoptosis.13-18 Gambogic acid also reduces invasion and angiogenesis,19, 20 telomerase mRNA expression and activity21 and tumor volume in vivo.13, 16, 20 However, the antitumor effect of gambogic acid is not selective and it induces toxicity to the liver and kidney, which hinders its development into a clinically useful anticancer drug.22, 23

Due to the toxicity of gambogic acid, our recent research has focused on the discovery of novel and more selective compounds isolated from various Garcinia species. We have screened various components using different cancer cells, and found that several polyprenylated acylphloroglucinol (PPAP) compounds had potent cytotoxic effects on human colorectal cancer cell lines without affecting the normal human colon fibroblasts, yet their mechanisms of action are unknown.24 Therefore, in this study, we investigated the selective cytotoxic effects and mechanisms of guttiferone K (GUTK) (Fig. 1a), the most potent PPAP derivative isolated from Garcinia cowa from our previous screen, on human colon cancer HT-29 cell line. Moreover, a murine subcutaneous Colon-26 tumor model was used to investigate the antitumor potential of GUTK administered alone or concurrently with 5-fluorouracil, a first-line anticancer drug used for colon cancer, for potential development of a potent and selective chemotherapeutic drug against colon cancer.

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Figure 1. GUTK exhibited selective cytotoxicity toward colon cancer cells. (a) Chemical structure of GUTK. (b) Effects of GUTK (5.0 μM) on the viability of Colon-26, HCT116 and HT-29 cells after 24 hr and 48 hr treatment. (c) Effects of GUTK (5.0–20.0 μM) on the viability of CCD 841 CoN cells after 24 hr and 48 hr treatment (**p < 0.01, ***p < 0.001 compared with solvent control of respective time point). Effects of GUTK on the viability of Colon-26 and HT-29 cells after (d) 24 hr and (e) 48 hr treatment. (f) Effects of GUTK on the release of lactate dehydrogenase in HT-29 cells after 24 hr treatment (*p < 0.05, **p < 0.01, ***p < 0.001 compared with solvent control). (n = 3 for all experiments).

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Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Other materials and methods not stated in the main text are shown in the Supporting Information Materials and Methods section.

Chemicals and reagents

All drugs, chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. The twigs of G. cowa Roxb. were collected in Xishuangbanna, Yunnan Province, People's Republic of China, and authenticated by Professor Wang Hong, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. The isolation and structural elucidation of GUTK (purity >98%) have been previously described.24

Cell lines and cell culture

Human colorectal carcinoma HCT116, HT-29 and human normal colon epithelial CCD 841 CoN cell lines were purchased from American Type Culture Collection (Rockville, MD). The murine colorectal adenocarcinoma Colon-26 cell line was a gift from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer at Tohoku University (Sendai, Japan). The cancer and CCD 841 CoN cell lines were grown in RPMI 1640 medium and MEM, respectively, supplemented with 10% fetal bovine serum and penicillin-streptomycin. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Cell viability was assessed by MTT assay as previously described.24 The absorbance of untreated cells in medium (negative control) was considered 100%.

Lactate dehydrogenase (LDH) activity assay

Cytotoxicity was assessed by the release of cytoplasmic lactate dehydrogenase into culture medium using Cytotoxicity Detection Kit (LDH) (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol.

4′,6-Diamidino-2-phenylindole (DAPI) staining assay

HT-29 cells were treated with various concentrations of GUTK for 24 hr and adherent cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were washed once with PBS and then incubated with DAPI solution (1 μg/ml in PBS) for 10 min at room temperature. Cells were washed three times with PBS and their nuclear morphology was viewed using Zeiss Akioskop II Plus fluorescent microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada) with 330 to 380 nm excitation filter.

Flow cytometry assay

HT-29 cells were treated with various concentrations of GUTK for 6, 12 and 24 hr after overnight serum starvation. After harvesting, cells were resuspended in PBS, fixed with 75% ethanol and stored at −20°C until analysis. Cells were washed twice with PBS and stained with propidium iodide solution (50 μg/ml propidium iodide, 10 μg/ml RNase A and 3.8 mM sodium citrate) at room temperature in the dark for 30 min. Then, stained cells were passed through a 40 to 70 μm filter and 10,000 events were analyzed per sample using BD FACSAria II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ). Forward scatter and side scatter were measured to identify single cells and gating was performed using pulse width versus pulse area in the single cell population and in the PI scatter plot to gate out the debris. Cell cycle phase histogram analysis was performed using BD FACSDiva™ software. Representative scatter plots and histograms along with gating strategy and number of events are shown in Supporting Information Figure 1.

Protein extraction and Western blot analysis

HT-29 cells were treated with various concentrations of GUTK for 12 hr and 24 hr. Floating and adherent cells were harvested and treated with ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5% cholic acid, 2 mM EDTA, 10% glycerol, 150 mM NaCl, 0.1% SDS and 1% Triton X-100) containing complete mini protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in washing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5 and 0.1% Tween-20) for 1 hr at room temperature with shaking and incubated with the respective primary antibodies in 5% nonfat milk or BSA in washing buffer overnight at 4°C with shaking. The primary antibodies used included anti-PARP, anticaspase-3, anticaspase-8, anticaspase-9, anticyclinD1, anticyclinD3, anti-CDK4, anti-CDK6, anti-p15INK4B, anti-p16INK4A, anti-p21Waf1/Cip1, anti-p27Kip1, antiphospho-p44/p42, anti-p44/p42, antiphospho-SAPK/JNK, anti-SAPK/JNK, antiphospho-p38 and anti-p38, anti-p53 anti-p-Rb (Ser780) and anti-β-actin (Cell Signaling Technology, Inc., Danvers, MA and Santa Cruz Biotechnology Inc., Santa Cruz, CA). Membranes were washed and incubated with horseradish peroxidase-conjugated antimouse or antirabbit secondary antibodies diluted in 5% nonfat dry milk in washing buffer for 1 hr at room temperature with shaking. Protein bands were detected using ChemiDoc XRS Molecular Imager system.

Caspase-3 activity assay

Caspase-3 activity was measured using a commercially available kit according to the manufacturer's protocol. The role of the JNK pathway in GUTK-induced apoptosis was determined using 20.0 μM JNK inhibitor, SP600125 (Calbiochem, Merck KGaA, Darmstadt, Germany).

Animals

Male BALB/c mice (age 6–8 weeks) were provided by Laboratory Animal Services Center of The Chinese University of Hong Kong. All animals were maintained in a pathogen-free environment, air-conditioned at 24 ± 2°C with a standard 12-hr light/12-hr dark cycle, allowed access to tap water and standard pellet diet ad libitum. Care of animals and all experimental procedures were approved by the Animal Ethics Committee of The Chinese University of Hong Kong.

Effects of GUTK on Colon-26 tumor-bearing mice

Colon-26 murine colorectal adenocarcinoma cells (2.5 × 106 cells per mouse) were subcutaneously inoculated into the backs of BALB/c mice. When tumor dimensions reached 200 to 400 mm3, the mice were randomly divided into four groups (four mice per group) for intraperitoneal treatment with vehicle control (5% Tween-80 in saline) (Group 1); 5-fluorouracil, 25.0 mg/kg (Group 2); GUTK, 5.0 and 10.0 mg/kg (Group 3 and Group 4), after recording of tumor volume and body weight once every other day for 14 days. Mice were sacrificed at any point of the experiment if they showed signs of distress, greater than 20% of body weight loss, excessive tumor burden or tumors that were ulcerated, necrotic or infected. Tumor size was measured with vernier calipers and tumor volumes were calculated according to the formula: [(shortest diameter)2 × (longest diameter)]/2. The effects of a combination of 5-fluorouracil and GUTK on tumor growth were examined with five mice randomly divided into each group. Group 1: vehicle control; Group 2: 5-fluorouracil, 25.0 mg/kg; Group 3: GUTK, 10.0 mg/kg; and Group 4: 5-fluorouracil, 25.0 mg/kg plus GUTK, 10.0 mg/kg. Mice were treated once every other day for 14 days. On day 16, the mice were sacrificed by cervical dislocation and tumors were removed, embedded in OCT compound and cryosectioned at 10 μm for immunohistochemical staining.

Immunohistochemistry

Tumor sections were incubated with 10% normal goat serum as blocking buffer for 1 hr at room temperature after washing in PBS. The cleaved caspase-3 (Cell Signaling Technology, Inc., Danvers, MA) and Ki-67 (Abcam, Cambridge, UK) antibodies, diluted in 10% goat serum at 1:100, were added to the sections and incubated overnight in a humidified chamber at 4°C. The sections were washed three times with PBS before Alexa-Fluor goat-anti-rabbit secondary antibody (Invitrogen), diluted in 10% goat serum at 1:800, was added to the sections, and slides were incubated in a humidified chamber for 1 hr in the dark. Slides were washed three times with PBS followed by incubation with DAPI (1 μg/ml in PBS) in the dark for 5 to 10 min. Sections were rinsed twice with PBS and cover slips were mounted using 30% glycerol. Sections were stored at 4°C in the dark until viewed with a fluorescent microscope.

Statistical analysis

For the in vitro experiments, data are expressed as mean values ± SEM from at least three independent experiments conducted in at least duplicate. The data were analyzed by one-way or two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison post hoc test, using GraphPad Prism 5.0. For the in vivo experiments, repeated-measures ANOVA was used to compare tumor volumes and body weight among different treatment groups on different treatment days. Differences were considered statistically significant at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

GUTK selectively reduced the viability of colon cancer cells

The cytotoxic effects of GUTK at 5.0 μM, which equates to the IC50 value obtained from our previous viability study in HT-29 cells,24 were evaluated in two human colorectal cancer HCT116 and HT-29 cell lines and murine Colon-26 colorectal cancer cell line after 24 hr and 48 hr treatment using MTT assay. GUTK significantly reduced the viability of all colorectal cancer cell lines in a time-dependent manner (Fig. 1b), but did not significantly alter the viability of normal human colon epithelial CCD 841 CoN cells up to 20.0 μM and 15.0 μM after 24 hr and 48 hr incubation, respectively (Fig. 1c, p < 0.01). The results demonstrated that GUTK had great selective cytotoxicity toward all colorectal cancer cells tested without affecting normal cells. Thus, human HT-29 cells and murine Colon-26 syngeneic tumor-bearing mice were selected for further in vitro mechanism and in vivo anticancer activity studies, respectively. The subsequent concentration–response study indicated that GUTK reduced the viability of both colon cancer cell lines in a concentration- and time-dependent manner with similar IC50 values (Figs. 1d and 1e), which were lower than IC50 value of first-line antitumor drugs, cisplatin (25.51 ± 2.57 μM) and 5-fluorouracil (93.53 ± 16.09 μM), in HT-29 cells after 24 hr and 48 hr treatment, respectively (Supporting Information Table 1). The IC50 values of GUTK in Colon-26 and HT-29 cells were 4.95 ± 0.64 μM and 5.39 ± 0.22 μM after 24 hr, respectively, and 3.19 ± 0.15 μM and 3.29 ± 0.25 μM after 48 hr, respectively. Furthermore, GUTK significantly and concentration-dependently increased cytoplasmic lactate dehydrogenase release starting from 10.0 μM (p < 0.01, Fig. 1f). The results indicated that GUTK exhibited potent and selective cytotoxicity toward colon cancer cells, and thus its mechanism was further studied.

GUTK induced G0/G1 cell cycle arrest

The mechanisms underlying the cytotoxic effects of GUTK were investigated using cell cycle distribution analysis in HT-29 cells. Cells were treated with GUTK at IC50, 2 × IC50, 3 × IC50 and 4 × IC50 values for 24 hr and results showed that GUTK concentration-dependently reduced number of cells in S and G2/M phases, with a significant decrease of cells in S phase from 23.1% to 10.8% at concentration =15.0 μM (p < 0.01, Fig. 2a and Supporting Information Fig. 1d). A concentration-dependent accumulation of cells in apoptotic sub-G1 phase was also observed with significantly increased cell numbers from 3.8% to 12.8% at 10.0 μM (p < 0.05). The time-course of cell cycle distribution change caused by 10.0 μM GUTK were further examined. After 6 hr incubation, there was a significant reduction of HT-29 cells in S phase with no changes in other phases (p < 0.05, Fig. 2b). After 12 hr treatment, GUTK significantly increased the number of cells in G0/G1 phase from 49.0 to 68.5% to induce a G0/G1 cell cycle arrest and prevent entry of cells into the subsequent S and G2/M phases (p < 0.001, Fig. 2c). Hence, the number of cells in S and G2/M phases decreased by 30.4 and 4.4%, respectively, accompanied with a simultaneous significant increase by 15.5% in the sub-G1 cell population, and this trend continued up to 24 hr (p < 0.001, Figs. 2c and 2d).

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Figure 2. GUTK induced G0/G1 cell cycle arrest in HT-29 cells. Effects of GUTK on the cell cycle distribution after (a) 24 hr treatment at various concentrations (n = 4), (b) 6 hr treatment (n = 3), (c) 12 hr treatment (n = 3) and (d) 24 hr treatment (n = 3) at 10.0 μM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the solvent control in each respective cell cycle phase.

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GUTK down-regulated cyclins and up-regulated p21Waf1/Cip1 and p27Kip1

Since GUTK-induced G0/G1 arrest was most prominent after 12 hr, the effect of GUTK on expression of cell cycle regulatory proteins associated with G1/S phase transition was then examined. GUTK reduced the protein expression of cyclins D1 and D3 and CDKs 4 and 6 in a concentration- and time-dependent manner (Fig. 3a) in colon cancer HT-29 cells, but not in the normal colon CCD 841 CoN cells (Fig. 3d). After GUTK treatment, the retinoblastoma tumor suppressor protein p-Rb, which is crucial for G1 to S phase progression, was also down-regulated in a concentration- and time-dependent manner in cancer cells (Fig. 3a), but was not apparently expressed and not altered in normal cells (Fig. 3d). Furthermore, the effects of GUTK on protein expression of INK4 (p15INK4B and p16INK4A) and CIP/KIP (p21Waf1/Cip1 and p27Kip1) family of CDK inhibitors were studied since they are negative regulators of G1/S phase progression. Interestingly, GUTK at both concentrations (10.0 and 15.0 μM) and times (12 and 24 hr) tested, significantly up-regulated protein expression of p21Waf1/Cip1 and p27Kip1, but did not alter the expression of p15INK4B and p16INK4A in colon cancer cells (Fig. 3b). However, the expressions of all these proteins in normal colon cells were not affected by GUTK (Fig. 3d). While there was no significant difference between the expression of p15INK4B and p16INK4A in human colon cancer HT-29 and normal human colon CCD 841 CoN cells, there was almost no p21Waf1/Cip1 and low p27Kip1 expression in HT-29 cells when compared with CCD 841 CoN cells (Fig. 3c), indicating the lack of tumor suppressing regulators in the cancer cells. Therefore, the selective down-regulation of cyclins D1 and D3 and CDK4 and CDK6 and up-regulation of p21Waf1/Cip1 and p27Kip1 in HT-29 cells contributed to the G0/G1 cell cycle arrest induced by GUTK.

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Figure 3. GUTK specifically decreased the expression of cyclins and CDKs associated with G1/S phase progression and increased the expression of p21Waf1/Cip1 and p27Kip1 in HT-29 cells. (a) Effects of GUTK on protein expression of cyclin D1, cyclin D3, CDK4, CDK6 and p-Rb (Ser 780) in HT-29 cells. (b) Effects of GUTK on protein expression of p15INK4B, p16INK4A, p21Waf1/Cip1, p27Kip1 and p53 in HT-29 cells. (c) The basal protein expression of the INK4 and CIP/KIP families of tumor suppressor proteins in HT-29 and CCD 841 CoN cells. (d) Effects of GUTK (10.0 μM) on protein expression of cyclin D1, cyclin D3, CDK4, CDK6, p15INK4B, p16INK4A, p21Waf1/Cip1, p27Kip1, p-Rb and p53 in CCD 841 CoN cells after 12 hr and 24 hr treatment. (n = 3 for all experiments).

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GUTK induced apoptosis in HT-29 cells

Since GUTK increased number of cells in sub-G1 phase indicated potential apoptosis, nuclear staining with DAPI was used to determine whether GUTK induced morphological characteristics of apoptosis. As shown in Figure 4a, there was a gradual loss of adherent HT-29 cells along with the increase in concentration of GUTK after 24 hr treatment. GUTK at 10.0 and 15.0 μM produced condensed chromatin in the periphery of the nuclei and fragmented nuclei (indicated by red arrows in bottom panel in Fig. 4a), which are morphological hallmarks of apoptotic cell death. At the highest concentration (15.0 μM) tested, severe fragmentation of nuclei was detected, accompanied with the formation of apoptotic bodies (Fig. 4a).

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Figure 4. GUTK induced caspase-3-dependent apoptosis. (a) Effects of GUTK on nuclear morphology of HT-29 cells after 24 hr treatment using phase contrast microscopy at ×100 (top row), ×400 (middle row) and fluorescence microscopy at ×400 (bottom row) after DAPI staining. Scale bars = 80 μm and 20 μm at ×100 and ×400, respectively. The images are representative of results obtained from three independent experiments. Effects of GUTK on (b) cleavage and protein expression of caspase-3 and PARP in HT-29 cells after 24 hr treatment, (c) caspase-3 activity in HT-29 cells after 24 hr treatment as determined by the rate of pNa formation and (d) in the presence of caspase-3 inhibitor, Ac-DEVD-CHO. (e) Effects of GUTK on cleavage and protein expression of caspase-8 and caspase-9 in HT-29 cells after 24 hr treatment. **p < 0.01, ***p < 0.001 compared with solvent control; ˆˆp < 0.01 compared with GUTK treatment at 5.0 μM; ###p < 0.001 compared with GUTK treatment at 10.0 μM (n = 3 for all experiments). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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To further study the apoptotic mechanisms underlying the cytotoxic effects of GUTK, the effects of GUTK on the cleavage of pro-caspases-3, -8 and -9 in the caspase cascade and poly (ADP-ribose) polymerase (PARP) were investigated after 24 hr treatment. GUTK at 10.0 and 15.0 μM induced the cleavage of pro-caspase-3 into its two active fragments (17 and 19 kDa), as exemplified by the reduction of intact pro-caspase-3 expression, and a simultaneous rise in cleaved caspase-3 expression (Fig. 4b). GUTK also stimulated the cleavage of PARP, a DNA repair enzyme that is one of the cleavage targets of activated caspase-3 (Fig. 4b). As shown in Figure 4c, GUTK-stimulated cleavage of caspase-3 was associated with increased caspase-3 activity in a concentration-dependent manner, with a 3.7-fold increase in cells treated with 10.0 μM GUTK (p < 0.001). The caspase-3 inhibitor, Ac-DEVD-CHO, significantly diminished the elevated caspase-3 activity stimulated by GUTK (p < 0.001, Fig. 4d), suggesting that GUTK stimulated caspase-3-dependent apoptosis, resulting in the cleavage of important nuclear and cytoplasmic proteins, such as PARP. After 24 hr GUTK treatment, the active and cleaved fragments of pro-caspases-8 and -9 appeared with concomitant decrease of their corresponding intact forms in a concentration-dependent manner (Fig. 4e). These results suggested that GUTK activated the caspase cascade (both caspase-8 and -9) to produce active caspase-3 to cleave important cellular proteins during apoptosis.

JNK was partially responsible for GUTK-induced apoptosis

Since JNK, ERK and p38 pathways are crucial in apoptosis, cell survival and proliferation, the effects of GUTK on activation of the MAPK pathways were studied. GUTK at 10.0 μM increased protein expression of phosphorylated JNK, without changing the amount of total JNK (Fig. 5a). However, GUTK did not change the phosphorylation status of ERK and p-38. To acquire a better understanding of the connection between JNK pathway and apoptosis, caspase-3 activity was also determined in HT-29 cells treated with JNK inhibitor, SP600125 before GUTK treatment. Pretreatment with SP600125 for 1 hr significantly reduced the elevation of caspase-3 activity induced by GUTK (GUTK alone: 262.3; 148.6 pmol pNa/min·mg protein vs. SP600125 before GUTK: 148.6 pmol pNa/min·mg protein, p < 0.001, Fig. 5b), indicating that the apoptosis induced by GUTK was partially mediated by JNK pathway.

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Figure 5. GUTK-induced apoptosis was partially mediated by JNK. Effects of (a) GUTK (10.0 μM) on phosphorylation status of ERK, JNK and p38 after 3, 6 and 12 hr treatment in HT-29 cells and (b) 1 hr pretreatment with JNK inhibitor, SP600125 (20.0 μM), on caspase-3 activity induced by GUTK (10.0 μM) after 24 hr treatment. ***p < 0.001 compared with solvent control; ˆˆˆp < 0.001 compared with GUTK treatment at 10.0 μM (n = 3 for all experiments).

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GUTK had antitumor effects alone and in combination with 5-fluorouracil

The antitumor effects of GUTK were further investigated using Colon-26 subcutaneous tumor model in immunocompetent mice and the safety of GUTK in the mice was evaluated before antitumor study. Using the same dosage regimen as gambogic acid reported in previous studies,25 mice were treated with GUTK (10.0 mg/kg, i.p.) every other day for 14 days. No significant differences in body weights between the vehicle and GUTK treatment groups were observed (Supporting Information Fig. 2a). GUTK did not alter cellular morphology nor induce hemorrhage in the brain, heart, lung, stomach, small intestine, colon, kidney and liver (Supporting Information Fig. 2b). Moreover, no changes in serum ALT and creatinine levels were observed (Supporting Information Figs. 2b–2d). The results demonstrated that GUTK did not exhibit any toxicity in the treated mice.

Treatment with 5-fluorouracil, (25.0 mg/kg, i.p.) every other day for 14 days significantly reduced tumor volume in mice compared with the vehicle control group on days 14 and 16. GUTK also exhibited a dose-dependent inhibitory effect on tumor growth (Fig. 6a). The mean tumor volumes measured on day 16 for vehicle control, 5-fluorouracil, GUTK 5.0 and 10.0 mg/kg groups were 4,199.2, 1,669.5, 3,253.4 and 2,689.7 mm3, respectively. However, a significant decrease in body weight was observed in the mice treated with vehicle control and 5-fluorouracil, but not in GUTK groups (Supporting Information Fig. 3).

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Figure 6. GUTK had antitumor effects in Colon-26 tumor bearing mice alone and in combination with 5-fluorouracil. (a) Effects of GUTK on tumor volume. Mice were treated with 5-fluorouracil (positive control, 25.0 mg/kg, i.p.) or GUTK (5.0 or 10.0 mg/kg, i.p.) every other day for 14 days (n = 4). (b) Effects of GUTK (10.0 mg/kg, i.p.) in combination with 5-fluorouracil (25.0 mg/kg, i.p.) on tumor volume. *p < 0.05 and ***p < 0.001compared with vehicle control (5% Tween-80 in saline) on the respective treatment day (n = 5). Representative images of immunohistochemical staining of (c) cleaved caspase-3 and (d) Ki-67 in tumors isolated on day 16 from the mice treated with combination treatment. The nuclei of tumor cells are shown by counter-staining with DAPI and images were viewed using a fluorescent microscope at ×200. Scale bar = 100 μm. The images are representative of results obtained from five mice per treatment group (n = 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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In addition, the antitumor effects of GUTK were also studied in its concurrent administration with 5-fluorouracil. The results demonstrated that starting from day 8, GUTK potentiated the antitumor effects of 5-fluorouracil, and from day 14 the tumor volume was significantly reduced compared with the vehicle control group (p < 0.05, Fig. 6b). The mean tumor volumes of vehicle control, 5-fluorouracil, GUTK 10.0 mg/kg and combination treatment groups measured on day 16 were 3,920.7, 2,182.7, 2,900.0 and 1,226.8 mm3, respectively. Furthermore, immunohistochemical staining of tumor sections showed that both 5-fluorouracil and GUTK alone increased the amount of apoptotic marker, cleaved caspase-3, while the elevation of cleaved caspase-3 level was much more significant in the combination treatment (Fig. 6c). The levels of cell proliferation marker Ki-67 in tumor sections obtained from GUTK and combination treatment groups were markedly lower than that in vehicle and 5-fluorouracil treated groups (Fig. 6d). The results demonstrated that antitumor effect of GUTK alone and in combination with 5-fluorouracil was due to the induction of apoptosis and reduction of cell proliferation in the tumor, which further confirmed the mechanisms delineated from the aforementioned in vitro studies.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

In this study, the in vitro and in vivo antitumor activity and mechanism of GUTK, a novel PAPP derivative isolated from G. cowa, was investigated for potential development as an anticancer drug for colon cancer. Several studies have demonstrated the potent anticancer effects of gambogic acid isolated from Garcinia hanburyi in various types of cancer.13-16, 20 However, chronic toxicological studies using beagle dogs injected with gambogic acid (i.p.) every other day for 13 weeks showed cellular damage in the kidney and liver.22 Similarly, rats fed orally with gambogic acid at 30, 60 and 120 mg/kg every other day for 13 weeks had necrotic damage to the liver and kidney, and a decreased leukocyte count and increased serum ALT and creatinine levels at the highest dose, revealing that gambogic acid induced adverse effects due to its lack of selectivity.23 Although GUTK was not as potent as gambogic acid in HT-29 cells (IC50 for 48 hr: 0.41 ± 0.13 μM24), GUTK did not have cytotoxic effects in normal human colon epithelial cells up to 15.0 μM after 48 hr incubation, while gambogic acid significantly reduced the viability of normal human colon fibroblasts at a concentration equal to its IC50 value.24 Our in vivo studies further demonstrated that GUTK did not induce toxicity to any organs at a dose, which exerted antitumor effects. These results strongly suggested that GUTK with high potency and selectivity toward colon cancer cells had advantages over gambogic acid and other anticancer drugs, which lack selectivity. Moreover, GUTK induced G0/G1 cell cycle arrest, demonstrating that GUTK acted through a different mechanism from gambogic acid and other caged xanthones, which induced microtubule depolymerization and G2/M cell cycle arrest in cancer cells.26 Therefore, GUTK may be useful in the treatment of cancer patients who have developed resistance to gambogic acid, other caged xanthones and conventional antitumor agents that disrupt the microtubules.

The subsequent study for understanding the underlying mechanism of its selective anticancer action revealed that GUTK down-regulated cyclins and CDKs, resulting in less cyclin and CDK available in cancer cells, and thus reduced the formation of the activated cyclin/CDK complex and inhibited the downstream transcription of genes needed for G1/S phase transition.27 Similarly, garcinol, a structurally related benzophenone derivative, has been found to reduce nicotine-induced cell proliferation in human breast cancer cells by down-regulation of cyclin D3.28 More interestingly, for the first time, we have found that GUTK specifically restored p21Waf1/Cip1 and p27Kip1 levels in colon cancer cells to the levels in normal colon cells. Previous studies have shown that lack of p21Waf1/Cip1 expression in tumors was correlated with higher proliferation and advanced tumor grade in human tumor biopsies and p21Waf1/Cip1 expression was correlated with prolonged disease-free survival and more favorable outcomes in patients.29, 30 On the other hand, maxillary sinus squamous cell carcinoma and high-grade breast cancer tumors with high p21Waf1/Cip1 or p27Kip1 levels demonstrated higher apoptotic indices.29, 31 Although p21Waf1/Cip1 is a CDK inhibitor, recent evidence suggested that it may play a role in apoptosis induced by TNF-family of death receptors and may also interact with or regulate the DNA repair mechanisms to induce apoptosis, especially in cells with mutated or nonfunctional p53.32 Therefore, the selective up-regulation of p21Waf1/Cip1 by GUTK in p53-mutated HT-29 cells may not only induce G0/G1 cell cycle arrest, but may also be responsible for apoptosis. Our findings of specific up-regulation of p21Waf1/Cip1 and p27Kip1 levels and down-regulation of various CDKs and cyclin Ds, including D3, by GUTK in colon cancer cells suggested that such protein modifications may be the possible mechanism to account for the selective cytotoxic and antiproliferative effects of GUTK on cancer cells. In addition, GUTK did not alter p53 levels in p53-mutated HT-29 colon cancer33 and normal colon cells. This suggested that GUTK-induced apoptosis might be p53-independent, and GUTK may also induce apoptosis in other p53-mutant cancer cells considering that p53 is mutated in almost half of all human cancers.34 Therefore, it is warranted to further investigate in this regard and also whether GUTK exhibits cytotoxicity in p53-null and/or p53-wild-type tumors.

Apoptosis is an energy-dependent genetically programmed cell death mechanism that is of importance in cancer research since abnormal cells that do not undergo apoptosis may become malignant, reduced apoptosis has been associated with resistance to chemotherapy and progression of cancer, and most of the current anticancer drugs induce apoptosis as their mechanism of action.35 One of the main biochemical hallmarks is the activation of the caspase cascade, in which a series of cysteinyl aspartic acid-specific proteases are activated by cleavage of the zymogen form to activate the two main pathways of apoptosis: the extrinsic death receptor-mediated and intrinsic mitochondrial-mediated pathways.36 Since GUTK induced the cleavage and activation of both caspases-8 and -9, both death receptor and mitochondrial pathways may be involved in GUTK-induced apoptosis. GUTK may activate the death receptor pathway by up-regulation of death ligands and receptors since many antitumor agents have been shown to up-regulate FasL and FasR.37 It has been reported that garcinol selectively up-regulated TRAIL-R1 and TRAIL-R2 and potentiated TRAIL-induced apoptosis in many cancer cell lines.38 Although not investigated in this study, the effects of GUTK on the mRNA and protein expression of death ligands and receptors, which also play a large role in drug resistance, may hint at its use in multidrug-resistant cancers and are worthy for further study.

Our results also demonstrated the involvement of MAPK pathway in the GUTK-induced apoptosis as evidenced by its concentration-dependent JNK activation. It has been suggested that there is a causal link between activation of MAPK pathway and apoptosis induction.39 The phosphorylation of antiapoptotic Bcl-2 by JNK has been hypothesized to prevent Bcl-2 from sequestering proapoptotic Bcl-2 family members.40 In addition, JNK activation may lead to the transcription of TNF-α and Bak, leading to the induction of apoptosis through extrinsic and intrinsic pathways.41 Studies have shown that kinases in the JNK pathway are often mutated in many cancers, including colon cancer.42, 43 Our results showed that JNK inhibition could not fully reverse GUTK-induced apoptosis (Fig. 5b) suggesting that (i) JNK might play an important, but not the only role in GUTK-induced apoptosis; (ii) other signaling pathways (such as NF-κB and PI3K/AKT) or molecular targets might be involved; and (iii) GUTK could still induce apoptosis even if JNK was not functional. Therefore, if potential resistance to GUTK were to be developed, down-regulation of JNK signaling in tumor may contribute to, but not be the predominant pathway. Further studies on the apoptotic effects of GUTK in the colon cancer cells with dominant negative constructs of JNK1 and JNK2 will shed more light on role of JNK in GUTK-induced apoptosis and developed resistance to GUTK.

It is interestingly noted that a slight increase in GUTK concentration caused significant increase in its anticancer activity (Figs. 1d–1f and 4c), which suggests the multitarget action of GUTK. It may be advantageous if GUTK acts on multiple targets, because GUTK did not affect normal epithelial cell up to 20.0 μM, but a slight increase in its concentration caused significant increase in its anticancer activity. This implies that GUTK has potential to be developed as a potent and selective anticancer drug even in a relatively narrow but effective concentration range to provide a wider therapeutic index for its safe use. However, the specific and multiple targets of GUTK are unknown and worthy for further study.

The in vivo antitumor effect of GUTK was studied using a syngeneic Colon-26 mouse tumor model in healthy, immune-competent mice, which ensures tumor compatibility with the host animal and mimics the clinical situation of cancer initiation and progression. Our results demonstrated that GUTK reduced tumor volume by approximately 36% and the combination of GUTK and 5-fluorouracil exhibited more significant reduction of tumor volume by approximately 69%. Not only does GUTK have selective antitumor effects when used alone, it can also potentiate the antitumor effects of 5-fluorouracil, without toxicity to the animals. Yet, 5-fluorouracil alone reduced the body weight of tumor-bearing mice with potential toxicity. This finding supports the advantages of the concurrent usage of GUTK with other chemotherapeutic drugs to minimize their toxicity/adverse effects via the reduction of their dosages.

In conclusion, for the first time, we have revealed the possible mechanisms of the anticancer activity and selectivity of GUTK, a novel polyprenylated acylphloroglucinol derivative isolated from G. cowa, on colon cancer and demonstrated its antitumor effects in vivo. This study provides promising results supporting GUTK as a novel antitumor drug candidate with high potency and selectivity that has potential for further development as a single agent or in combinational use in anticancer therapy.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_27694_sm_SuppFig1A.TIF271KSupporting Information Figure 1A.
IJC_27694_sm_SuppFig1B.TIF241KSupporting Information Figure 1B.
IJC_27694_sm_SuppFig1C.TIF121KSupporting Information Figure 1C.
IJC_27694_sm_SuppFig1D.TIF161KSupporting Information Figure 1D.
IJC_27694_sm_SuppFig2A.tif418KSupporting Information Figure 2A.
IJC_27694_sm_SuppFig2B.TIF1164KSupporting Information Figure 2B.
IJC_27694_sm_SuppFig2C.tif596KSupporting Information Figure 2C.
IJC_27694_sm_SuppFig2D.tif570KSupporting Information Figure 2D.
IJC_27694_sm_SuppFig3.tif590KSupporting Information Figure 3.

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