Abstract
Background/Aim: AZD8055 is an inhibitor of mammalian target of rapamycin (mTOR) that can suppress both mTOR complex 1 (mTORC1) and mTORC2. This study investigated the antitumor effects of AZD8055 on colon cancer. Materials and Methods: The effects of AZD8055 on proliferation, apoptosis, and cell cycle of colon cancer cells, and tumor growth in a mouse colon cancer model were studied. Results: AZD8055 significantly inhibited proliferation and induced apoptosis of colon cancer cells (p<0.05). The phosphorylation of both AKT and S6 kinase 1 (S6K1) was suppressed by AZD8055. AZD8055 also induced G0/G1 cell-cycle arrest, reduced cyclin D1 and increased p27 expression, and suppressed the levels of phospho-cyclin-dependent kinase 2 and phospho-retinoblastoma. Compared to the control, oral administration of AZD8055 significantly suppressed tumor growth in mice (p<0.05). Conclusion: AZD8055 induces cytotoxicity, apoptosis, and cell-cycle arrest of colon cancer cells, and exerts an antitumor effect in mice. It also inhibits the mTOR signaling pathway and mTOR-dependent cell-cycle progression.
Colon cancer is one of the leading causes of cancer death in the world (1, 2). Many genetic alterations have been noted in colon cancer, including Ras activation, phosphatidylinositol 3-kinase (PI3K) pathway hyperactivation, p53 mutation, and dysregulation of the Wnt pathway (2, 3). Among these genetic changes, dysregulation of the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway occurs in about 60-70% of human colon cancer cases (4). Because mTOR activation is an early and common event in colon cancer, mTOR has become a therapeutic target for colon cancer therapy (2, 5-7). The mTOR kinase forms two complexes, known as mTOR complex 1 (mTORC1) and mTORC2, with the regulatory-associated protein of mTOR (RAPTOR) or rapamycin-insensitive companion of mTOR (RICTOR), respectively (8-10). mTORC1 functions through phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1) (8-10). mTORC2 activation results in the phosphorylation of AKT, which in turn increases the activity of mTORC1 (8-10). Although mTOR inhibitors have undergone clinical trials for the treatment of various malignancies, rapamycin and its analogs (rapalogs) have been found to have only modest antitumor effects in single-agent therapy trials (8, 10). The limited clinical effect of rapalogs is considered to be related to the enhanced AKT activity that occurs due to a lack of mTORC2 inhibition, which may then cause drug resistance in tumor cells (8, 11).
AZD8055, an ATP-competitive mTOR kinase inhibitor, can suppress the phosphorylation of the mTORC1 substrates S6K and 4E-BP1, as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins (8, 12, 13). AZD8055 shows excellent selectivity (approximately 1,000-fold) against all class I PI3K isoforms and other members of the PI3K-related kinase family, and had no significant activity against a panel of 260 kinases at concentrations up to 10 μM (8). It is orally bioavailable and rapidly absorbed from the gastrointestinal tract (14, 15). The inhibition of both mTORC1 and mTORC2 by AZD8055 may overcome the rapamycin-insensitivity of mTOR resulting from incomplete inhibition of mTOR activity [i.e. inhibition of mTORC1, but not mTORC2) by rapamycin (10, 16, 17)]. AZD8055 has been shown to have antiproliferative effects on different cancer types, including acute myeloid leukemia, neuroblastoma, pancreatic cancer, hepatocellular carcinoma, renal cell carcinoma, glioma, breast cancer, non-small cell lung cancer, prostate cancer, and uterine sarcoma (8, 11, 12, 14, 15, 18-24). Most studies have reported that AZD8055 achieved these antitumor effects at nanomolar drug levels (8, 11-13, 18, 25-27).
In this study, we investigated the effects of AZD8055 on AZD8055-resistant colon cancer cells in vitro. The effects of AZD8055 on cytotoxicity, apoptosis, and cell-cycle distribution, and the related mechanisms were explored. We also studied the in vivo effect of AZD8055 on tumor growth in a syngeneic mouse colon cancer model.
Materials and Methods
Cell lines. Human colon cancer cells HCT-15 and HCT-116 were gifts from Dr. J-H Chuang at Kaohsiung Chang Gung Memorial Hospital. Mouse CT-26, a syngeneic murine colon adenocarcinoma cell line induced in BALB/c mice by N-nitroso-N-methylurethane, was purchased from the American Tissue Culture Collection, (Rockville, MD, USA) (28).
AZD8055 preparation. AZD8055 was purchased from AstraZeneca (Wilmington, DE, USA). For in vitro experiments, AZD8055 powder was dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 mM as the stock solution. The stock solution aliquots were protected from light and stored at −20°C. For in vivo use, AZD8055 powder was mixed with 30% captisol solution (Captisol, San Diego, CA, USA) to reach a final concentration of 2 mg/ml. The AZD8055-captisol solution was stored at 4°C in small aliquots. For administration, the aliquots needed for daily dosing were pre-warmed in a 37°C water-bath for 10 min and gently vortexed immediately before use.
Cell viability. To elucidate the cytotoxic effects of AZD8055 on colon cancer cells, cells were seeded in a 24-well plate, with 5×104 cells per well, and cultured for 24 h. Cells were then separated into different treatment groups with six replicates in each group and incubated with different concentrations of AZD8055 for 24 or 48 h. The media were then replaced with fresh media without drugs and cells were cultured for an additional 72 h. Cell viability with different treatment regimens was analyzed using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay. The drug concentration at which 50% of cells were killed was designated as the LC50 value. Differences in the levels of cytotoxicity induced by various regimens were then compared.
Apoptosis analysis. The fraction of cells undergoing apoptosis in response to AZD8055 treatment was quantified by fluorescence-activated cell sorter (FACS) flow cytometry using a fluorescein isothiocyanate (FITC)-annexin V Apoptosis Detection Kit (BD, San Jose, CA, USA). All procedures were conducted according to the manufacturer's instructions. Briefly, the cells were treated with AZD8055 or DMSO for 48 h, were washed twice with cold phosphate-buffered saline (PBS), and then resuspended in Binding Buffer at a density of 1×106 cells/ml. FITC-annexin V and propidium iodide (PI) were added to the cell suspension, and the mixture was incubated for 15 min at room temperature in the dark. Cell staining was analyzed using a FACScan flow cytometer (FACSCalibur; Becton Dickinson Immunocytometry System, San Jose, CA, USA).
Cell-cycle analysis. The cell-cycle stage of cells treated with AZD8055 was determined using a NucleoCounter® NC-3000™ Imaging Cytometer system and a 2-step cell cycle assay kit (ChemoMetec A/S, Allerod, Denmark). All procedures were conducted according to the manufacturer's instructions. Briefly, the cells treated with AZD8055 or DMSO for 48 h were harvested and washed once with cold PBS, and then suspended in 4’,6-diamidino-2-phenylindole (DAPI)-containing Lysis Buffer at a concentration of 2×106 cells/ml. After incubation at 37°C for 5 min, the cell suspension was mixed with Stabilizing Buffer. The mixture was then imaged and analyzed using NucleoView® NC-3000 software.
Western blot analysis. Total cell lysates were prepared with RIPA buffer (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) containing 1 × protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) and cleared by centrifugation at 20,000 × g for 15 min at 4°C. The lysate was mixed with sodium dodecyl sulfate (SDS) sample buffer (250 mM Tris-HCl (pH 6.8), 8% (w/v) SDS, 20% (v/v) 2-mercaptoethanol, 40% (v/v) glycerol, 0.04% (w/v) bromophenol blue) and boiled for 5 min. After 3 min incubation on ice, followed by centrifugation, proteins in the supernatant were separated by SDS polyacrylamide gel (SDS-PAGE). The separated proteins were then transferred to a Hypond™-P hydrophobic polyvinylidene difluoride membrane (GE Healthcare, NJ, USA) in transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol) at 100 V for 100 min. Following this, the membrane was soaked in HyBlock Blocking Buffer (Goal Bio, Tao Yuan, Taiwan) with gentle shaking for 5 min. The membrane was then incubated with the appropriate primary antibody in blocking buffer at 4°C overnight. The following day, the membrane was washed five times (5 min each time) with Tris-buffered saline with Tween-20 [20 mM Tris (pH 7.6), 0.15 M NaCl, and 0.1% w/v Tween-20] on an orbital shaker, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After the membrane was washed, the horseradish peroxidase conjugated secondary antibodies were detected using an Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA). The antibodies used in this study were as follows: rabbit anti-AKT (Abcam, Cambridge, UK), rabbit anti-phospho-AKT (anti-p-AKT, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-mTOR (Cell Signaling Technology), rabbit anti-p-mTOR (Cell Signaling Technology), rabbit anti-S6K1 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-p-S6K1 (Cell Signaling Technology), rabbit anti-retinoblastoma (Rb) (Santa Cruz Biotechnology), rabbit anti-p-Rb (Cell Signaling Technology), rabbit anti-cyclin D1 (Abcam), rabbit anti-E2F transcription factor 1 (E2F1) (Abcam), rabbit anti- cyclin-dependent kinase 2 (CDK2) (Abcam), rabbit-anti-p-CDK2 (Abcam), rabbit anti-p21 (Abcam), rabbit anti-p27 (Abcam), and mouse anti-β-actin (Millipore, Billerica, MA, USA).
Animal studies. The animal experiments in this study were approved by the Committee on Laboratory Animal Research of the Far Eastern Memorial Hospital, Taiwan (103-02-20-A), and conducted according to the guidelines of the Laboratory Animal Center of the Far Eastern Memorial Hospital and adhered to the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society. The use of animals in this research was under the careful supervision of a person adequately trained in this field and the animals were treated humanely at all times. Male Balb/C mice (4 to 5 weeks old, 20-35 g) were purchased from the National Laboratory Animal Center, Taiwan. The mice were housed in a sterile environment and kept on a 12 h light/dark cycle (lights on from 6 AM to 6 PM) at 20°C. Food and water were provided ad libitum.
To study the antitumor effects of AZD8055 on CT-26 colon cancer in mice, subcutaneous tumors were induced by injecting 2×105 CT-26 cells (in 10 μl of PBS) into the right flank of each mouse. The experiment consisted of three groups with seven mice in each group. The day of tumor cell inoculation was designated as Day 0. The control group received oral gavage of vehicle from day 1 to day 28. The early treatment group received oral gavage of AZD8055 treatment (20 mg/kg per day) (9, 25) from day 1 to day 28. The delayed treatment group received oral gavage of AZD8055 treatment (20 mg/kg per day) from day 11 to day 28. The general appearance and activity of the mice were observed during the treatment period. Tumor size was measured twice a week. Tumor volume was calculated from the formula V=1/2 (d1×d2×d3), where d1, d2, and d3 were tumor diameters as measured with calipers in mutually perpendicular directions. Average daily tumor volumes from each group were compared. All animals were sacrificed after completion of the experiment, and the tumors were harvested.
Statistical analysis. A one-way ANOVA was used for statistical analyses of the extent of cell viability, apoptosis, cell cycle, and the difference in tumor sizes among various groups. Statistical significance was accepted only when p<0.05.
Results
AZD8055 induces cytotoxicity and apoptosis in colon cancer cells. In this study, the antitumor effect of AZD8055 on colon cancer cells was studied. The human HT-15 and HCT-116 colon cancer cell lines, and the mouse CT-26 colon cancer cell line, were treated with different concentrations of AZD8055 for 24 h or 48 h. Following this, cell viability was measured using an MTT assay (Figure 1a). AZD8055 was found to inhibit the proliferation of these three colon cancer cell lines in a time- and concentration-dependent manner (p<0.05). The LC50s for AZD8055 at 24 h and 48 h of treatment were 107.8 μM and 9.8 μM for HCT-15 cells, 124.6 μM and 21.5 μM for HCT-116 cells, and 3.0 μM and 0.43 μM for CT-26 cells, respectively. The LC50s for AZD8055 against the human HCT-15 and HCT-116 cells were significantly higher than for the mouse CT-26 cells. In addition, the 48 h LC50s were 9.1% to 17.3% of the 24 h LC50s in all three of these cell lines. To further investigate the effect on cell viability, apoptosis induction was examined in these colon cancer cells treated with different concentrations of AZD8055 for 48 h. As shown in Figure 1b, AZD8055 treatment caused a significant, concentration-dependent increase in apoptosis of these colon cancer cells (p<0.05). Taken together, these data indicate that AZD8055 induces both cytotoxicity in and apoptosis of these three colon cancer cell lines, and that these cells exhibit different sensitivities to AZD8055.
AZD8055 regulates the AKT/mTOR signaling pathway in colon cancer cells. To explore the mechanism by which AZD8055 inhibits the growth of colon cancer cells, a western blot analysis was used to assess the expression of different proteins in the AKT/mTOR pathway following treatment of colon cancer cells with different concentrations of AZD8055 for 48 h (Figure 2). While the expression of AKT was not affected by AZD8055, the level of p-AKT was significantly suppressed in a concentration-dependent manner in all three cell lines. In addition, AZD8055 suppressed the expression of mTOR and the levels of p-mTOR in HCH-15 and HCT-116 cells, whereas there was no effect on mTOR and p-mTOR in CT-26 cells. AZD8055 also suppressed the expression of S6K1, and the level of p-S6K1, in a concentration-dependent manner in HCT-116 and CT-26 cells. In HCT-15 cells, AZD8055 did not affect S6K1 expression, but it did inhibit the production of p-S6K1. All these data suggest that the AKT-mTOR pathway was inhibited by AZD8055 in these three colon cancer cell lines.
AZD8055 induces cell-cycle arrest in the G0/G1 phase in colon cancer cells. Since AZD8055 can down-regulate the activity of S6K1, which is an effector for the mTOR-dependent control of cell growth and cell-cycle progression (29, 30), we next investigated the effects of AZD8055 on cell-cycle progression in these colon cancer cells (Figure 3). After treatment with different concentrations of AZD8055 for 48 h, all three colon cancer cell lines showed accumulation of cells in the G0/G1 phase, and a decreased level of cells in the S and G2/M phases (p<0.05). After treatment with 10 μM AZD8055 for 48 h, the mean G0/G1 fraction was increased from 59.2% (control) to 90.6% for HCT-15 cells; from 56.4% (control) to 80.4% for HCT-116 cells; and from 54.6% (control) to 86.6% for CT-26 cells.
Effects of AZD8055 on the proliferation and apoptosis of colon cancer cells. A: HCT-15, HCT-116, and CT-26 cells were treated with different concentrations of AZD8055 for 24 or 48 h. Cell viability was quantified by the MTT assay, and the relative viability was calculated. B: HCT-15, HCT-116, and CT-26 cells were treated with different concentrations of AZD8055 for 48 h, after which the cells were stained with propidium iodide/annexin V, and analyzed by flow cytometry. The percentage of cells in early apoptosis (right lower phase) and late apoptosis (right upper phase) are shown.
AZD8055 affects the expression of cell-cycle regulators in colon cancer cells. We further examined the expression of the regulatory proteins involved in the transition from G0/G1 phase to S phase (Figure 4). In these three colon cancer cell lines, AZD8055 did not affect Rb expression, whereas it suppressed the level of p-Rb in a concentration-dependent manner. AZD8055 also inhibited the expression of CDK2, and of p-CDK2 in these colon cancer cell lines, with the exception that there was no effect on CDK2 expression in HCT-15 cells. Furthermore, AZD8055 increased the expression of p27, but did not affect the expression of p21. In addition, cyclin D1 expression was also suppressed by AZD8055 in these colon cancer cell lines. These data suggest that AZD8055 affects the expression of proteins that regulate the G0/G1-S transition, and thus induce G0/G1 arrest.
Effect of AZD8055 on the expression of mammalian target of rapamycin (mTOR) signaling proteins in colon cancer cells. HCT-15, HCT-116, and CT-26 colon cancer cells were treated with different concentrations of AZD8055 for 48 h. The levels of protein kinase B (AKT), phospho-AKT (p-AKT), mTOR, p-mTOR, S6 kinase 1 (S6K1), p-S6K1, and β-actin were analyzed by western blotting.
AZD8055 inhibits the tumor growth of subcutaneous CT-26 colon cancer in mice. As the in vitro studies showed that AZD8055 induced significant cytotoxicity, apoptosis, and cell-cycle arrest at the G0/G1 phase in all three studied colon cancer cells, we further investigated the in vivo effect of AZD8055 on tumor growth in a CT-26 syngeneic tumor model (Figure 5). The AZD8055 treatment was given from day 1 to 28 in the early treatment group, and from day 11 to 28 in the delayed treatment group. The administration of AZD8055 did not induce a significant change in the activity or body weight of mice throughout the treatment course, and all animals survived after completion of the treatment. The early-treatment group showed a significantly slower tumor growth rate than the delayed-treatment group (tumor size on day 28: 198.3±343.8 mm3 vs. 479.6±380.9 mm3; p=0.0067) or the control group (tumor size on Day 28, 198.3±343.8 mm3 vs. 1156.8±579.1 mm3; p=0.003). In addition, the tumor growth rate also significantly differed between the delayed-treatment group and the control group was (tumor size on day 28: 479.6±380.9 mm3 vs. 1156.8±579.1 mm3; p=0.017). These data suggest that AZD8055 treatment exerts an antitumor effect on the subcutaneous CT-26 tumor in vivo, and early treatment has a better effect than delayed treatment.
Discussion
In this study, we found that AZD8055 induced both cytotoxicity and apoptosis in colon cancer cells. The human HT-15 and HCT-116, and mouse CT-26 colon cancer cells were all inhibited by AZD8055 in a concentration- and time-dependent manner. The LC50s for AZD8055 at 24 h of treatment were greater than 100 μM for both HCT-15 and HCT-116 cells, and were much higher than the LC50 for CT-26 cells. The LC50s for HCT-15 and HCT-116 cells were similar to those in a previous study, which showed that 10 μM AZD8055 treatment for 24 h only reduced cell viability by 18.5% in HCT-15 cells and 35.1% in HCT-116 cells (31). In addition, the LC50s for AZD8055 treatment of the three cell lines studied here were much higher than those reported for most colon cancer cells, which were typically in the range of 30-50 nM at 24 h of AZD8055 treatment (12). In other cancer types, the LC50 for AZD8055 treatment has been found to be in the nanomolar to low micromolar levels (13, 18, 26, 27). For example, the LC50 for 24 h AZD8055 treatment in ovarian cancer cells has been reported to be 0.25-2 μM (13). Ten nanomoles of AZD8055 treatment for 24, 48, and 72 h has also been reported to inhibit both proliferation and glycolysis, and induce apoptosis of cervical cancer HeLa cells (26). These data suggest that there are considerable variations in the sensitivity to AZD8055 in different cancer cell types, such that the LC50s can range widely (over a 1,000-fold) in various cell lines derived from one cancer cell type, such as colon cancer. From this viewpoint, it may therefore be important to differentiate the cancer cells into AZD8055-sensitive and AZD8055-resistant before initiating AZD8055 treatment. However, it also appears that prolonged AZD8055 treatment may enhance the anticancer effect to a certain extent, since the LC50s with 48 h of AZD8055 treatment were markedly reduced to 9.1% to 17.3% of the LC50s at 24 h treatment in all three of the cell lines studied here. With respect to apoptosis, the fraction of AZD8055-treated colon cancer cells undergoing apoptosis was lower than the percentage of cell death induced by the same concentration of AZD8055. These data indicate that AZD8055 may cause cell death through induction of apoptosis, as well as through other mechanisms.
Effect of AZD8055 on cell-cycle distribution in colon cancer cells. HCT-15, HCT-116, and CT-26 cells were treated with different concentrations of AZD8055 for 48 h. The cells were then fixed and the DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI). The intensity of DAPI fluorescein was analyzed using an NC-3000 cytometer and the histograms show the percentage of cells in the different phases of the cell cycle. The stacked bar plot represents the collective data from the histograms.
Effect of AZD8055 on the expression of cell-cycle regulators in colon cancer cells. HCT-15, HCT-116, and CT-26 cells were treated with different concentrations of AZD8055 for 48 h. The levels of retinoblastoma (Rb), phospho-Rb (p-Rb), cyclin-dependent kinase 2 (CDK2), p-CDK2, p21, p27, cyclin D1, and β-actin were analyzed by western blotting.
Effect of AZD8055 on the growth of subcutaneous CT-26 tumors in mice. Mice bearing subcutaneous CT-26 tumors were treated with AZD8055. The control group was treated with vehicle from day 1 to day 28 after tumor cell inoculation. The early-treatment group (Early Tx) received AZD8055 treatment from day 1 to day 28 after tumor cell inoculation, and the delayed-treatment group (Delayed Tx) received AZD8055 treatment from day 11 to day 28 after tumor cell inoculation. A: Representative tumor specimens were harvested on day 29 after tumor cell inoculation. B: Tumor growth curves. Data are shown as mean±standard deviation. Each point represents the mean volume of tumors for each group. *Significantly different at p<0.05.
The high LC50s for AZD8055 observed for the HCT-15 and HCT-116 cells indicate that they were relatively AZD8055-resistant. There is a study showing that the coexistence of mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) and phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α isoform (PIK3CA) can attenuate the sensitivity to PI3K/mTOR inhibition in colorectal cancer cell lines (2). Because both HCT-15 and HCT-116 cells have PIK3CA and KRAS mutations (2), the drug resistance to AZD8055 seen in these two cell lines may be related to these mutations. CT-26 cells have only the KRAS mutation (2), which may partly explain why these cells were more sensitive to AZD8055 and had a lower LC50 than HCT-15 and HCT-116 cells. AZD8055 is a dual mTOR inhibitor, which can inhibit both mTORC1 and mTORC2 (12). The suppression of mTORC1 and mTORC2 by AZD8055 has been found to occur within 1 h, and such suppression can last for at least 48 h in colorectal cancer cells (12). In addition, the median relative IC50 for AZD8055 (i.e. the AZD8055 concentration that reduces cell survival by 50% of the maximum AZD8055 effect) against various Pediatric Preclinical Testing Program cell lines is 24.7 nM, and AZD8055 at levels around this concentration significantly suppressed the levels of both p-AKT and p-S6 from 1 to 24 h after treatment (11). These data indicate that AZD8055 can indeed suppress the mTOR pathway at a low concentration and in a short time. However, the LC50s for the colon cancer cells in this study were relatively high; therefore, we further investigated the effects of AZD8055 on the mTOR pathway. We found the levels of p-AKT, p-mTOR, and p-S6K1 were suppressed by AZD8055 in a concentration-dependent manner, indicating that both the mTORC1 and mTORC2 pathways were inhibited. The concentrations needed to suppress the levels of these phosphoproteins were in the range of 1-10 μM, which are higher than those in a previous study (11).
mTOR has been noted to regulate cell-cycle progression, whereas S6K1 is known to be an effector for mTOR-dependent cell-cycle progression (29, 30, 32). AZD8055 can also suppress the level of p-S6K1; therefore, it may affect cell-cycle progression. We found that AZD8055 treatment induced a concentration-dependent accumulation of colon cancer cells in the G0/G1 phase. These results were consistent with previous studies, which showed that AZD8055 causes G0/G1 arrest in both SW620 colorectal cancer cells and ovarian cancer cells (12, 13). However, the cell lines described in the previous studies are more sensitive to AZD8055 than HCT-15 and HCT-116 cells, with 24 h LC50s for AZD8055 treatment in the nanomolar to low micromolar range. For example, treatment of SW620 cells with 1 μM AZD8055 for 24 h has been reported to induce a loss of cyclin D1 and cause G1 cell-cycle arrest (12, 13). In contrast, in AZD8055-resistant SW620 cells, induced by repeated AZD8055 challenge, treatment with 2 μM AZD8055 for 24 h showed only a mild decrease in the expression of cyclin D1, which was different from that of the parental SW620 cells (12). These data suggest AZD8055 might not necessarily be able to affect cell-cycle progression in AZD8055-resistant cells. However, our study showed that AZD8055 induced significant G0/G1 arrest, in a concentration-dependent manner, in these drug-resistant colon cancer cells.
Progression through the cell cycle is regulated by CDKs, whose activity is inhibited by CDK inhibitors (33, 34). Cyclin D binds to CDK4/6 to form the cyclin D-CDK4/6 complex, which in turn phosphorylates Rb and allows it to dissociate from the E2F/Rb complex, thus, activating E2F (33, 34). Activation of E2F results in an increase in the transcription of various genes such as cyclin E. Cyclin E then binds to CDK2, forming the cyclin E–CDK2 complex, which pushes the cell from the G1 to the S phase (33, 34). CDK inhibitors, such as p21 and p27, may halt the cell cycle in the G1 phase, by binding to and inactivating cyclin-CDK complexes (33, 34). PI3K/AKT/mTOR/S6K1 signaling mediates G1 progression and cyclin expression, increases the translation of positive cell-cycle regulators, such as cyclin D1, and reduces the expression of p27 (33, 35). Therefore, mTOR inhibitors can inhibit the G1-S transition. The mTOR inhibitor rapamycin has been found to inhibit the G1-S progression and the expression of cyclin D1, CDK4, and Rb phosphorylation in prostate cancer cells (32-34). In addition, AZD8055 can increase Rb expression and the levels of pRb in breast cancer cells (36). To confirm the inhibition of cell cycle progression by AZD8055 in colon cancer cells, we further investigated the effect of AZD8055 on the expression of regulatory proteins involved in the phase transition between the G1 and S phases in AZD8055-resistant HCT-15 and HCT-116 cells, and CT-26 cells. In these three cell lines, the levels of p-Rb and p-CDK2 decreased, and the expression of p27 increased, although the expression of p21 was not significantly changed. These data suggest that AZD8055 can modulate the activity of mTOR-dependent cell cycle regulators and cause G1 arrest in colon cancer cells, even in AZD8055-resistant cells.
As the in vitro studies showed that AZD8055 induced significant cytotoxicity and apoptosis in colon cancer cells, we further investigated the in vivo antitumor effects of AZD8055 in a CT-26 syngeneic mouse model. We found that both early and delayed AZD8055 treatments exerted antitumor effects on the subcutaneous CT-26 tumors, with early treatment having a stronger antitumor effect than delayed treatment. Previously, AZD8055 had been shown to exert a tumor reducing effect in a cis-Apc/Smad4 mouse model of locally invasive intestinal adenocarcinoma (9). In this mouse tumor model, the intestinal adenomas begin to progress into invasive adenocarcinomas when the mice are around 12 weeks of age, and 55% of the tumors show an invasive pattern when the mice were 20 weeks of age (37). In the previous study, the cis-Apc/Smad4 mice were treated with AZD8055 from 12 to 20 weeks of age (9). Because the AZD8055 treatment was started in the beginning of tumor progression (9), such treatment is more likely an early or prophylactic treatment. In addition, this intestinal adenomas-tumor model is relatively heterogeneous (37). The mice had increased polyp sizes and numbers in both the small and large intestines; some mice developed carcinomas in the duodenal papilla of Vater, and more than half had epidermoid cysts of the skin (37). Therefore, this animal model is significantly different from the colon cancer model. Another xenograft model used LoVo or SW620 colon cancer cells to induce subcutaneous tumors, and 10 mg/kg AZD8055 was shown to inhibit about 70% of tumor growth in these LoVo and SW620 colon cancer cell xenografts (8, 25). However, these two cell lines are AZD8055-sensitive, with LC50s at the nanomolar level. AZD8055 thus has a significant antitumor effect on the xenografts derived from these two cell lines (12). The LC50 after 24 h AZD8055 treatment of CT-26 cells was 3 μM, being about 100 fold-higher than the LC50s of LoVo and SW620 cells. Therefore, the CT-26 tumor model differs significantly from both the cis-Apc/Smad4 mouse model of locally invasive intestinal adenocarcinoma and the LoVo and SW620 colon cancer cell xenograft models. The data from the CT-26 tumor model more likely represent colon cancer with moderate resistance to AZD8055.
From the above data, we found that AZD8055 can suppress the mTOR pathway and modulate mTOR-dependent cell-cycle regulators to induce cytotoxicity, apoptosis, and G0/G1 cell cycle arrest in colon cancer cells, even in the AZD8055-resistant HCT-15 and HCT-116 cells. In addition, an in vivo study showed that AZD8055 exerted an antitumor effect on the CT-26 subcutaneous tumors in mice. The LC50 for AZD8055 in the HCT-15 and HCT-116 cells was greater than 100 μM, which indicates that single drug treatment using AZD8055 might not be effective to treat these drug-resistant colon cancers. In addition, the dose of AZD8055 is limited by the side-effects of elevated serum transaminase levels noted in the clinical trial for the treatment of advanced solid tumors and lymphoma (15). Therefore, modification of the dosing schedule, or combination treatment with a lower dose of AZD8055 and other anticancer drugs may be necessary to mitigate the adverse effects and enhance the therapeutic efficacy (18). Treatment of Apc/Smad4 mice with AZD8055 has been found to increase epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling in tumor epithelial and stromal cells (9). Further, a combination of MEK1/2 inhibitors or checkpoint kinase 1 (CHK1) inhibitors with mTOR inhibitors can have a synergistic effect on cancers (12, 38). Therefore, co-administration of AZD8055 and an EGFR inhibitor or MEK inhibitor may induce synergistic antitumor effects on colon cancer (9). Further studies will be required to clarify these issues.
Acknowledgements
This work was supported by the Far Eastern Memorial Hospital, Taiwan, R.O.C. [grant numbers FEMH-2015- 039 and FEMH-2017-C-041].
Footnotes
↵* These Authors contributed equally to this study.
Conflicts of Interest
The Authors do not have any conflicts of interest to declare in regard to this study.
- Received December 3, 2017.
- Revision received January 14, 2018.
- Accepted January 15, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
