Background
Colorectal cancer (CRC) is the third most common cancer in men (663,000 cases, 10.0% of the total) and the second in women (571,000 cases, 9.4% of the total) worldwide (Globocan, an international agency for research on cancer). American cancer Society estimates that in 2013, approximately 142,820 new cases of colorectal cancer will be diagnosed with 50,830 (26,300 men and 24,530 women) in United States alone. Overall, the lifetime risk of developing colorectal cancer is about 1 in 20 (5.1%) [
1,
2]. A number of different drugs have shown significant antitumor activity in CRC, including the systemic drugs 5-fluorouracil (5-FU), irinotecan, oxaliplatin, bevacizumab, cetuximab and panitumumab, and the oral drug capecitabine. Different regimens of these drugs, such as the FOLFOX (leucovorin, 5-FU and oxaliplatin), FOLFIRI (leucovorin, 5-FU and irinotecan) and XELOX (oxaliplatin and capecitabine), with or without a monoclonal antibody agent have shown improved outcomes in CRC [
3‐
10]. The efficacy of chemotherapy has reached a plateau and a 5-year survival rate of patients with advanced CRC still remains < 8% [
11] with the underlying molecular basis still not clearly defined.
With the advancement of genomic technology and availability of various genetic animal models, it has been proposed that the progression of CRC is from cumulative changes in key genes controlling cell proliferation, apoptosis and invasion [
12‐
17]. Abnormally high activation of multiple signaling pathways such as RAS-RAF, and WNT/APC/β-Catenin has been demonstrated to be required for initiation and progression of colorectal carcinoma [
18‐
20]. Some of these pathways are regulated by key enzymes known as tyrosine kinases which phosphorylate tyrosine residues in protein that are associated with either transmembrane receptor-linked proteins or non-receptor cytoplasmic proteins [
21]. Activated forms of these enzymes are known to increase tumor cell proliferation and growth, induce antiapoptotic effects and promote angiogenesis and metastasis [
22]. In addition to activation by growth factors, kinase activation by somatic mutations is also a common mechanism for tumorigenesis [
23]. Mutations in
kRAS (31%) and
BRAF (9.6%) are both thought to occur early in colorectal carcinogenesis and are associated with significantly poor survival [
24,
25]. Although majority studies show that these two mutations are rarely observed together, a recent study in Chinese patients with CRC showed approximately 25% of the population harboring both kRAS and bRAF mutations [
26]. The presence of multiple mutations has always posed potential limitations to the inhibitors. Since receptor tyrosine kinase activation initiates these effects, they are the key targets for inhibitors [
22,
27]. The majority of currently available tyrosine kinase inhibitors has provided a new approach for cancer therapy and has the potential for avoiding some of the drawbacks of cytotoxic chemotherapy [
22]. Targeted agents have also offered an opportunity to reverse chemotherapy resistance and enhance response in patients with localized or advanced cancer [
28]. Along with holding a great promise, these inhibitors have also posed drawbacks, being beneficial to only certain subpopulations of patients and limiting resistance in patients who initially responded [
29‐
31].
Dovitinib, or TKI258 (4-amino-5-fluoro-3-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2 (1H)-one; formerly known as CHIR-258), is a small molecule adenosine 5′-triphosphate–competitive inhibitor of class III, IV, and V receptor tyrosine kinases (RTKs), which include fibroblast growth factor receptor (FGFR), vascular endothelial growth factor (VEGFR), Tyrosine-protein kinase kit (c-KIT), and FMS-like tyrosine kinase 3 (FLT3) [
32‐
35]. According to previous studies, dovitinib exhibits potent tumor growth inhibition
in vitro and in a broad range of preclinical animal models [
32,
36‐
38]. For example, dovitinib induced apoptosis in Fibroblast growth factor receptor (FGFR) expressing mammary cells via inhibition of Phosphoinositide-3-kinase (PI3K)/Akt signaling pathway [
39]. In addition, dovitinib specifically inhibited proliferation and survival of primary cells and cell lines with FGFR1 fusion genes associated with the 8p11 myeloproliferative syndrome [
40].
There remains a need for not only novel regimens but also refinement of existing regimens to improve and extend survival and decrease treatment related toxicities. In the present study, we hypothesized that Dovitinib may attempt to boost therapeutic kill by employing combination regimen with oxaliplatin. Our results reveal that co- treatment of Dovitinib and Oxaliplatin in colon cancer cell lines induced superior cell killing in comparison to either of these drugs alone in all colon cancer cell lines regardless of their mutation status. The significantly enhanced antitumor activity that results from the combination of Oxaliplatin and Dovitinib offers promise as a novel treatment for patients with colon cancer. This combination will achieve a greater anticancer effect at a lower efficacious dose with a less chance of a cell developing resistance along with reduced injury to normal cells.
Discussion
In this study, we evaluated the growth inhibitory effects of dovitinib and oxaliplatin combination in cell culture and xenograft models of colon cancer, and our goal for this investigation was to elucidate potential molecular mechanisms of action for the compounds contributing to the antiproliferative and anticancer capacity of human colon cancer cells. We found that both oxaliplatin and dovitinib (in the low micromolar range) effectively diminished the growth of colon cancer cell lines regardless of their RAS-RAF mutation status (Figure
1). Of greater interest, we found that this combination showed a synergistic antiproliferative activity and inhibition of angiogenesis in a colon cancer xenograft model with a bRAF mutation and multi drug resistant phenotype (Figure
4).
Initiation and progression of colorectal carcinoma is defined by abnormally high activation of RAS-RAF signaling pathway controlled by tyrosine kinases [
18‐
20]. Activated forms of tyrosine kinases such as VEGFR, FGFR and PDGFR are known to play role in tumor angiogenesis, a process essential for growth of tumors. [
22]. These receptors are activated by their corresponding growth factors secreted by tumor cells resulting in proliferation, migration and survival of tumor endothelial cells [
49]. Although VEGF RTKs are the major targets for dovitinib, preclinical studies have also shown that FGF signaling is a possible mechanism of escape from and resistance to anti-VEGF therapy [
50]. Therefore, dovitinib’s uniqueness in inhibiting growth factor receptors including FGFR and VEGFR makes it stand out among other RTKs inhibitors. A high percentage of colorectal carcinomas over-express a lot of growth factors and their receptors, including fibroblast growth factor (FGF) and FGF receptor (FGFR) [
51]. Takayama
et al. have shown that over-expression of FGFR correlates with liver metastasis in CRC [
52]. Our results showed a decrease in phosphorylation of VEGFR and FGFR in two colon cancer cell lines tested.
In vitro data in HCT116, HT-29 and SW-480 cell lines showed decreases in expression of all proteins in MAP kinase pathway such as kRAS, bRAF and pERK. Previous studies have shown that use of MEK inhibitors impaired proliferation thereby impacting a diverse array of cellular events, including differentiation, apoptosis, and angiogenesis [
53]. However, Turke et al. have shown that MEK inhibitor led to activation of a parallel PI3K/AKT signaling pathway involving several feedback systems [
54]. The
vice versa has also been shown true in which inhibition of PI3K pathway activated MAP kinase pathway [
55], thereby decreasing the effectiveness of single-agent targeted therapies. This suggests that concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and shrink the tumor. Since inhibition by dovitinib in these cell lines was an upstream of kRAS, a parallel inhibition of both RAS-RAF-MAPK and PI3K-AKT suggest a synergistic effect of both pathways on downstream effectors of growth and proliferation. Our data also showed an inhibition of expression of pAKT in all three cell lines. Our results are in agreement with a recent report showing a concomitant down-regulation of PI3K and MEK induced regression of kRAS mutant cancers
in vivo[
56].
Our results with wound healing assay showed a significant decrease in wound repair with the use of combination of two as compared to either of the drugs alone confirmed a simultaneous inhibition of both signaling pathways (MAP Kinase and PI3Kinase) which are known to contribute to the inhibition of protein synthesis, cell growth, proliferation and survival. Lee et al. have shown that inhibition of FGFR and PDGFR starts as early as 4 h in the presence of Dovitinib [
32].
A phase 1 pharmacokinetic and pharmacodynamic study of dovitinib in patients with advanced solid tumors showed a dose limiting toxicity of grade 3 hypertension and fatigue [
57]. The strategy to improve the efficacy of the therapy and alleviate the symptom burden without increasing the toxicity is to add chemotherapeutic drug. In clinical studies, Oxaliplatin by itself has shown modest activity against advanced colorectal cancer. It has been extensively studied in combination with 5-FU and Folinic Acid. Our results demonstrate that when combined with dovitinib it showed a synergistic cytotoxicity by inducing apoptosis in colon cancer cell lines tested. There is compelling evidence that defects in apoptosis contributes to cancer. The molecular mechanism showed an increase in phosphorylation of histone H2AX at serine 139 in response to DNA double strand break by oxaliplatin. This DNA damage activated and stabilized p53, in turn, regulating the apoptotic pathway. It has been demonstrated many times that activation of p53 by DNA damage can lead to apoptosis by transcriptional activation of pro-apoptotic members of Bcl-2 family (Bax and Bak) and inhibition of anti-apoptotic (Puma, Noxa, Bcl2, Bclxl, Mcl-1 etc.) proteins, which together regulate mitochondrial permeability [
58‐
60]. Also, it has been reported that AKT directly regulates members of the Bcl-2 super family and indirectly regulates apoptosis through the transcriptional factors that control apoptotic events [
61]. Our results demonstrated an up-regulation of Bax and down-regulation of Bcl2 and Bclxl after treatment with the combination of oxaliplatin and dovitinib. The combination showed a more pronounced effect than either of the drugs alone. Mcl-1 a member of Bcl2 family and an inhibitor of apoptosis, showed a significantly higher expression in colon adenoma and carcinoma patient compared to healthy colonic epithelium [
62]. Also, it has been shown that sustained activation of Akt resulted in increased expression of the antiapoptotic protein, Mcl-1 [
63]. Our results showed a significant decrease in the expression of Mcl-1 after treatment with dovitinib possibly through the inactivation of AKT kinase. The expression was further reduced in the combination group in all three cell lines tested. Survivin is another molecule described to be involved in both the control of cell survival and regulation of cell cycle. Dramatic over-expression of Survivin compared with normal tissue has been shown in different kinds of cancer (reviewed in [
64]). Survivin is also known to inhibit apoptosis mainly through targeting terminal effector caspase 3 activity in the apoptotic protease cascade [
65]. Caspases are proteins known to be involved in the cascade of initiation and execution of apoptosis. Our results showed a decrease in Survivin after treatment with dovitinib and/or oxaliplatin in all cell lines. The combination treatment also showed a decrease in expression of procaspase 3, 8 and 9 with a subsequent increase in cleaved caspases. Our data also show a decrease and increase in expression of PARP and cleaved PARP respectively, a downstream target of activated caspase-3.
In parallel,
in vivo results showed an inhibition of tumor growth in HT-29 tumor model with coordinating decrease in the expression of Ki-67 (biomarker for proliferation) and CD31 (biomarker for angiogenesis). The decrease was more pronounced in the combination group as compared to either of the groups alone. These results confirm that the combination inhibited angiogenesis which correlates to slow tumor growth supposedly because of lack of factors that are supplied through blood, thereby inhibiting the tumor growth. Our results are in agreement with previous reports showing inhibition of expression of Ki-67 and CD31 correlating to the shrinking of tumors and overall disease free survival [
66,
67].
Methods
Materials
Human colon cancer cell lines HCT-116, HT-29, SW-480, Caco2 and LS-174 T were purchased from American Type Culture Collection (Manassas, VA). Cell culture media and serum were obtained from Invitrogen Life Technologies (Carlsbad, CA). Dovitinib (TKI-258) was obtained from Novartis (East Hanover, NJ) and Oxaliplatin was obtained from Sigma-Aldrich (St. Louis, MO). Antibodies against different proteins were obtained from Santacruz Biotechnologies Inc. (Santacruz, CA) or Cell signaling technology Inc. (Beverley, MA). HRP Conjugated anti-mouse IgG and Enhanced chemiluminescence plus (ECL plus) western blotting detection reagent were purchased from Amersham Bioscience (Arlington Heights, IL), X-OMAT AR films (Eastman Kodak, Rochester, NY). All other reagents were obtained from Fisher Scientific (Pittsburg, PA).
Cell culture
The tumor cell lines were maintained in culture as adherent cells in a monolayer in humidified atmosphere at 37°C and 5% CO2 in McCoy’s 5A (HCT-116 and HT-29), Leibovitz’s L-15 Medium (SW-480), and Eagle’s Minimum Essential Medium (Caco2 and LS-174 T) and supplemented with 10% (20% for Caco2) heat-inactivated fetal calf serum. The cells were passaged twice a week and discarded after 20 passages.
Cell viability assay
The cell viability was measured using MTS assay as described earlier [
68]. IC
50 values were calculated from the dose response curve generated from the colon cancer cell lines in the absence or presence of the drug(s). A real-time cell electronic sensing (RT-CES, ACEA Biosciences Inc., San Diego, CA) system was also used for measurement of cell response for combination of dovitinib and oxaliplatin in HCT-116, HT-29 and SW-480 cell lines. Briefly, 5000 cells were grown onto the surface of microelectronic sensors in a 16-well plate supplied by the manufacturer. After 24 h, a wide range of concentrations of drugs were added and the cells were continuously monitored by the system. The experiments were repeated with comparison of simultaneous and sequential addition of the two drugs. These results were further confirmed using MTS assay. Briefly, for combination experiments the drugs were mixed in 1:1 ratio of IC
50 concentration or maximum achievable dosage and diluted to ½ and ¼ concentrations before the addition to the cells. Data from cell viability assay (MTS) were expressed as the fraction of cells killed by the individual drugs or the combination of drugs and compared to untreated cells.
Determination of Synergism
The interaction between drug combinations was analyzed using Calcusyn software program (Biosoft, Cambridge, UK) to determine whether the combination was additive or synergistic. This program is based on Chou-Talalay method and calculates a combination index (CI), when CI =1, it indicates an additive effect and when below 1.0, it indicates synergism.
Wound-healing assay
Cells were plated in 24-well plates and grown to confluence. The monolayer was wounded using the tip of a sterile 200 μl pipette. Cell debris was removed by washing twice with serum-free medium and replacing with medium containing serum and Dovitinib and/or Oxaliplatin. Cells were then allowed to migrate into the denuded areas for 24 hr. Photographs were taken immediately after wounding (t0) and 24 hr later (t24) using the Leica DMI3000 B inverted microscope. The results were quantified as a percentage of the wound width closed by the cells at time 24 hr (T24 = (100/t0)/t24). The mean of three experiments was graphed with standard deviations represented as error bars.
Western blot analysis
Cells were collected after 24 h treatment with dovitinib and oxaliplatin and washed once with PBS and second time with cold PBS containing 0.1 mM orthovanadate. The whole cell lysates were prepared according to the procedures described previously [
68]. Protein was measured using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and. The proteins were transferred to PVDF membrane (Amersham, Arlington Heights, IL) after resolving by (25 μg protein per lane) 4-12% gel electrophoresis (SDS-PAGE) and probed with one of the following: RAS, RAF, p-ERK, ERK, p-AKT, AKT, Survivin, Caspase 3, Caspase 9 (Cell signaling technology, Boston, MA), p53, Anti-phosphotyrosine (4G10), γH2AX and pATM, β-actin (EMD Millipore, Billerica, MA ), p21, Bax, Bcl-2, Bcl
xl, Mcl-1, cytochrome C, c-Myc (Santa Cruz biotechnology, Santacruz, CA), GAPDH (GeneTex, Irvine, CA) and Cleaved PARP (Promega, Madison, WI) antibodies and HRP conjugated secondary antibody (Amersham). The optical density for each band was determined using Image Quant software (GE Healthcare Biosciences, Pittsburg, PA).
Apoptotic cell death assay
To quantify apoptosis, HCT-116, HT-29 and SW-480 cells were stained with annexin V and PI using FITC Annexin V Apoptosis detection kit 1 from BD Pharmingen following the step by step protocol as provided by the manufacturer and analyzed by flow cytometry (BD Bioscience, San Diego, CA). Briefly, at the end of treatment with dovitinib and oxaliplatin either alone or in combination for 48 h, both floating and attached cells were collected, washed twice with cold PBS and subjected to annexin V-FITC and PI staining and analyzed using flow cytometry.
Subcutaneous human tumor xenograft
In vivo evaluation of Dovitinib and/or Oxaliplatin in HT-29 human colorectal cancer model was performed at Institute of Translational Medicine, Taipai Medical University (TMU), Taipei, Taiwan.
Animal
Forty female athymic nude mice (BALB/cAnN.Cg-Foxn1
nu
/CrlNarl; 5 weeks of age) were purchased from the NAR Labs National Laboratory Animal Center (Taipei, Taiwan). Mice were housed in TMU Laboratory Animal Center (Taipei, Taiwan) around a specific pathogen-free animal facility at constant temperature (20 ± 3°C) and humidity (50 ± 20%). The animals had free access to irradiation-sterilized dry granule food and water during the study period. Animal care and the treatment were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) based on guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Cell culture
HT-29 tumor cells were maintained in vitro in McCoy’s 5A medium supplemented with 10% FBS and 0.1 mM NEAA. The cells growing in exponential growth phase were harvested and counted for tumor inoculation.
Tumor inoculation
Each mouse was inoculated subcutaneously at the rear right flank with HT29 tumor cells (3 × 105) in 0.1 ml of PBS for tumor development. After 10 days of tumor inoculation, the animals were weighed and measured for tumor volume and randomly divided into 4 groups of 10 animals each based on the randomized block design method for homogeneous group formation when the mean tumor size reached approximately 80–125 mm3 (10 Days).
Drug treatment
The treatment was started intravenously (i.v.) on the 11th day post tumor inoculation with 0.9% saline (Group 1), 6.7 mg/Kg Oxaliplatin (Group 2), 60 mg/Kg Dovitinib (Group 3) and 6.7 and 60 mg/Kg Oxaliplatin and Dovitinib (Group 4) respectively. The treatment was continued for 3 weeks with a regimen of once per week (i.v.) for Oxaliplatin and every two days (oral) for Dovitinib.
Tumor measurement
The animals were visually monitored for food and water consumption everyday and once/week for body weight (gain/loss) and tumor size. Tumor volumes were calculated using formula: V = 0.5 × a × b2 where a and b are the long and short tumor diameters respectively and euthanized when the tumor volume reached a predetermined size of approximately 3000 mm3. This end-point tumor size was chosen to maximize the number of tumor doublings within the exponential growth phase in the untreated group. All the tumors were harvested a week after the last treatment and fast frozen for immunohistochemistry.
Tissue preparation and immunohistochemical staining
The immunohisto-chemical assays were performed using a Dako Autostainer Plus (Dako Colorado Inc., CO) with fresh sections of vehicle control and treated tissue stained at the same time with the help of research pathology core facility at the City of Hope as described in [
69]. Primary rabbit Ki67 (Novus Biologicals) or mouse monoclonal CD31 antibody (Cell Marque, CA) were used for IHC at a final concentration of 1:100(Ki-67) or 1:75 (CD31). The sections were counterstained with Meyer’s haematoxylin and each run also included phosphate buffered solution (PBS) used as the primary antibody for the negative controls while samples known to express Ki-67 or CD31 strongly served as positive controls. Photomicrographs were taken on a Nikon microscope equipped with a CCD camera.
Statistical analysis
Data points for cell proliferation and apoptosis were presented as mean ± standard error mean of at least three independent cell populations. Results were compared using two-tailed student’s t-test using Microsoft Excel Program 2003. A p-value <0.05 was considered statistically significant. Animal data results were compared using student’s t-test. Animal study data were evaluated using one-way ANOVA followed by Dunnett’s post-test if significance was observed. The data were analyzed using SPSS version 16.0. A p-value <0.05 was considered statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SG and YY designed the study. SG, LC, VA, WL and YW performed the experiments. VC, NYH and HSS executed the animal experiments. SG and YY analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.