signaling pathways in experimental animals
The results described above clearly show the inhibitory effects of CAPE and CAPPE on the growth of CRC cells in a mouse xenograft model. We also demonstrated the molecular mechanisms of action of the CA derivatives in vitro. To verify these mechanistic findings, we further examined the molecular
effects of CAPE and CAPPE in these tumor-bearing mice. As shown in Figure. 9A, CAPE and CAPPE consumption each significantly inhibited the expression of cyclin D1, Cdk4, cyclin E and c-myc proteins in vivo. Moreover, the in vivo chemopreventive effects of CAPE and CAPPE were associated
with the upregulation of the p21CIP1/WAF1 protein.
It is well known that the PI3-K/Akt and MAPK/ERK signaling cascades play an important role in tumor growth and progression [4,41]. Suppression of the PI3-K/Akt and MAPK/ERK signaling cascades leads to down-regulation of
downstream target proteins such as cyclin D1/Cdk4 and a blockade of the cell 533
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cycle [4,36,41-45]. Therefore, we further investigated the inhibitory effects of CAPE and CAPPE on the PI3-K/ Akt and MAPK/ ERK signaling pathways. As shown in Figure. 9B, consumption of CAPE or CAPPE effectively inhibited the activation of the Akt, mTOR and ERK 1/2 signaling molecules. CA derivative-mediated suppression of the Akt, mTOR and ERK 1/2 signaling cascades was associated with an up-regulation of E-cadherin as well as a suppression of N-cadherin. Moreover, CAPE and CAPPE -mediated suppression of FASN protein was associated with the augmentation of the AMPK cascade in tumor-bearing mice (Figure. 9B). These results show that CAPE or
CAPPE-mediated suppression of PI3-K/Akt and MAPK/ ERK signaling cascades, as well as an augmentation of the AMPK signaling pathway is associated with the suppression of tumor growth at least in small laboratory animals.
Discussion
Previous studies suggest that CAPE has potential as a chemopreventive and therapeutic agents [46-49]. Many studies demonstrated that CAPE could inhibit tumor angiogenesis and suppress the growth of several types of cancer [47-52]. The aberrant PI3K/Akt pathway has been shown to be the
predominant pathway in the tumorigenesis of many types of cancer including 552
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colon cancer [53]. Studies suggested that suppression of the PI3K/Akt and integrin-mediated signaling pathways by CAPE could effectively inhibit the tumor growth [50,54]. To date, the effects of CAPPE on the proliferation and survival of human CRC cells have not been convincingly demonstrated. In the current study, we demonstrate the inhibitory effects of CA derivatives (CAPE and CAPPE) on the proliferation of human colon cancer cells both in vitro and in vivo. The results show that CAPE and CAPPE each effectively suppressed
the proliferation of human colon cancer cells in a dose-dependent manner.
CAPE and CAPPE effectively suppressed the proliferation of human CRC cells through the induction of cell cycle arrest at the G0/G1 phase. Previous studies suggested that the overexpression of cell cycle-related proteins, such as D1 and Cdk4, is correlated with the proliferation of human cancer cells [33].
In this study, the results showed that CAPE or CAPPE significantly inhibited the expression of cyclin D1 protein. Recently, cyclin D1 was identified as a target of the PI3-K/Akt pathways in CRC cells [44]. We further confirmed that the molecular effects of CAPE and CAPPE were carried out through the inhibition of the PI3-K/Akt and mTOR signaling pathways in human CRC cells.
Moreover, CAPE and CAPPE inhibited the expression of FASN through an augmentation of the AMPK cascade. A recent study reports that the activation 571
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of AMPK is associated with an increased cellular AMP/ATP ratio [55]. A low energy status leads to the phosphorylation (i.e. activation ) of AMPK and the suppression of mTOR activity through the effect on the LKB1 protein [55]. The current study suggested than CAPPE may suppress the activity of mTOR protein in a LKB-independent manner. In contrast, CAPPE-mediated activation of the AMPK molecule was more significantly correlated with the decreased ATP levels in CRC cells. Therefore, it is probable that the
respective CAPE- and CAPPE- mediated augmentation of the AMPK cascade and suppression of mTOR protein are in part associated with a decreased level of ATP in these CRC cells. There are several possible scenarios to explain why CAPPE is a more effective anti-cancer compound than CAPE.
One explanation might be that CAPPE has a cell membrane solubility higher than that of CAPE. This possibility is consistent with the findings of an earlier toxicity study [56]. Previous studies demonstrated that the inhibitory effect of CA derivatives on nitric oxide (NO) production is correlated with the increasing length of the alkyl chain (i.e. CAPPE> CAPE) [56]. A recent study showed that the L-arginine -mediated NO reaction is also associated with AMPK activation [57]. These findings suggest that the upregulation of AMPK activation is dependent on the increasing length of the CA derivatives. It is to be expected, 590
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therefore, that CAPPE would be more effective in AMPK activation than CAPE. This may explain why CAPPE is a more effective regulator of AMPK activation and the suppression of cell proliferation than CAPE. The anti-proliferation effect of CAPPE could be achieved by increasing the dosage levels of CAPE (Figure. 2,3). The current study also showed an inverse correlation between AMPK and mTOR activity in vivo. These results are consistent with AMPK -mediated downregulation of mTOR activity [22,23].
This suggests that CAPE and CAPPE may act through this pathway as effective anti-cancer agents against human CRC cells. Moreover, the results suggested that CAPE and CAPPE mediated- suppression of cell proliferation
was independent of NF-κB pathway in human CRC cells.
To verify these in vitro findings, we further examined the respective inhibitory effects of CAPE and CAPPE on the growth of colorectal tumor in a xenograft mouse model. As shown in Figure. 8, consumption of CAPE or CAPPE significantly inhibited tumor growth in vivo. We also examined the actions of these bioactive compounds on multiple signaling pathways including PI3-K/Akt, MAPK/ERK and AMPK signaling cascades (Figure. 9). The results demonstrated that CAPE and CAPPE also effectively induced the activation of the AMPK cascade and suppressed the activation of both the PI3-K/Akt and 609
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MAPK/ERK signaling cascades. CAPE and CAPPE further significantly inhibited the expression of FASN , cyclin D1 , cyclin E, Cdk4 and c-myc proteins of tumor tissues in an in vivo animal study. We further examined whether the consumption of CAPE or CAPPE would help prevent tumor progression in tumor-bearing mice. The results demonstrated that CAPE or CAPPE significantly inhibited the expression of plasma MMP-9 in vivo (Figure.
8F). These results are consistent with the in vitro findings.
In conclusion, this is the first demonstration of the inhibitory effects of CA derivatives (CAPE and CAPPE) on the proliferation of human colon cancer cells both in vitro and in vivo. The directional changes in protein expression produced by CAPE and CAPPE are in relevant pathways and consistent with the properties of a chemopreventive agent. Whether CAPPE is a more potent chemopreventive agent than CAPE will require further preclinical studies.
Figure Legends
Figure 1 Chemical structure of the CA derivatives
The CA derivatives are depicted in Fig.1. (A) CAPE and (B) CAPPE differ in the elongation of the alkyl side chain of the caffeic acid ester.
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Figure 2 CA derivatives significantly inhibited the proliferation of human
CRC HCT-116 cells in vitro
(A) Human CRC HCT-116 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in the presence or absence of compound C (10 μM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were
conducted before the treatment of CA derivatives . The cell proliferation was 648
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measured by MTT assay as described in Materials and Methods. Data are the mean SD (standard deviation) of three independent experiments. The different symbols ( for CAPE and for CAPPE) represent a statistically ※ △ significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (＃ for
CAPE_Akt, § for CAPE_compound C, ▲ for CAPPE_Akt, and ■ for
CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage
subgroup, respectively, at P<0.05.
(B-C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα
Figure 3 CA derivatives significantly inhibited the proliferation of human
CRC SW-480 cells in vitro
(A) Human CRC SW-480 cells were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in the presence or absence of compound C (10 μM) for 24 h. Transfections of constitutively active Akt (Myr-Akt1) and empty vector (pcDNA3) were
conducted before the treatment of CA derivatives . The cell proliferation was 669
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measured by MTT assay as described in Materials and Methods. Data are the mean SD (standard deviation) of three independent experiments. The different symbols ( for CAPE and for CAPPE) represent a statistically ※ △ significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (＃ for
CAPE_Akt, § for CAPE_compound C, ▲ for CAPPE_Akt, and ■ for
CAPPE_compound C) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage
subgroup, respectively, at P<0.05.
(B-C) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against phosphorylation Akt (S473), total-Akt, anti-phosphorylation AMPKα (T172) and total-AMPKα
Figure 4 CAPE and CAPPE each induced G0/G1 cell cycle arrest in CRC
cells
Human CRC cells were synchronized in RPMI-1640 medium with 0.05 % FBS in tissue culture dishes overnight. To measure the distribution of the cell cycle, cell were cultured in the presence or absence of CAPE and CAPPE (0, 10, 50 and 100 μM) cultured in 10% FBS RPMI-1640 medium for another 24 h. (A) The measurement of the cell population at different cell cycle phases was 688
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performed using flow cytometry analysis, as described under Materials and Methods. The data indicate the (B) HCT-116 cell (C) SW-480 cell population percentage at different cell phases under the treatment of CAPE or CAPPE in
human CRC cells.
Human CRC (D) HCT-116 cells (E) SW-480 cells were treated with either CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10%
FBS RPMI-1640 for 24h. Nuclear proteins were prepared for Western blotting analysis using monoclonal antibodies against cyclin D1, Cdk4, PCNA, and lamin A antibodies, as described under Materials and Methods. The levels of detection represent the amounts of cyclin D1, Cdk4 and PCNA in the nuclei of human CRC cells. The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The immunoreactive bands are noted with an arrow. The mean integrated densities of these proteins adjusted with the internal control lamin A protein are shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively (P<0.05).
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Figure 5 CAPE and CAPPE inhibited the proliferation of human CRC
HCT-116 cells through the modulation of the PI3K/Akt, AMPK and mTOR
signaling pathways
Human CRC HCT-116 cells were treated with either CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10% FBS RPMI-1640 for
24h.
(A) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against N-cadherin, PTEN, anti-phosphorylation PDK1 (S241), total-PDK1, phosphorylation Akt (S473), total-Akt, anti-726
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phosphorylation GSK3α (S21), total- GSK3α, anti-phosphorylation GSK3β(S9), total- GSK3β, anti-phosphorylation FOXO3 (T32), total- FOXO3,
total- TSC1, total- TSC2, total- LKB1, total- 14-3-3, anti-phosphorylation AMPKα (T172), total-AMPKα, anti-phosphorylation m-TOR (S2448), total-m-TOR, anti-FASN and β-actin as described under Materials and Methods. The levels of detection represent the amounts of each protein in the cytoplasm of HCT-116 cells. The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The immunoreactive bands are noted with an arrow. The mean integrated densities of these proteins adjusted with the control protein are shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively
(P<0.05).
(B) The measurement of cellular ATP was performed as described under Materials and Methods. Data represent the percentage of cellular ATP levels in the CAPE- or CAPPE-treated human CRC HCT-116 cells. A single or double asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively (P<0.05).
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Figure 6 CAPE and CAPPE inhibited the proliferation of human CRC
SW-480 cells through the modulation of the PI3K/Akt, AMPK and mTOR
signaling pathways
Human CRC SW-480 cells were treated with either CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10% FBS RPMI-1640 for
24h.
(A) Cytoplasmic proteins were prepared for Western blotting analysis using monoclonal antibodies against N-cadherin, PTEN, anti-phosphorylation PDK1 (S241), total-PDK1, phosphorylation Akt (S473), total-Akt, anti-phosphorylation GSK3α (S21), total- GSK3α, anti-anti-phosphorylation 766
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GSK3β(S9), total- GSK3β, anti-phosphorylation FOXO3 (T32), total- FOXO3,
total- TSC1, total- TSC2, total- LKB1, total- 14-3-3, anti-phosphorylation AMPKα (T172), total-AMPKα, anti-phosphorylation m-TOR (S2448), total-m-TOR, anti-FASN and β-actin as described under Materials and Methods. The levels of detection represent the amounts of each protein in the cytoplasm of HCT-116 cells. The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The immunoreactive bands are noted with an arrow. The mean integrated densities of these proteins adjusted with the control protein are shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively
(P<0.05).
(B) The measurement of cellular ATP was performed as described under Materials and Methods. Data represent the percentage of cellular ATP levels in the CAPE- or CAPPE-treated human CRC SW-480 cells. A single or double asterisk indicates a significant difference compared to the CAPE- or CAPPE-untreated control group, respectively (P<0.05).
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Figure 7 CAPE and CAPPE inhibited the proliferation of CRC cells
independently of NF-B signaling pathway
(A-B) Human CRC cells were treated with either CAPE or CAPPE (at
concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10% FBS RPMI-1640 for 2h.
Nuclear proteins were prepared for Western blotting analysis using
monoclonal antibodies against NF-κB (p65) and lamin A as described under Materials and Methods. The levels of detection represent the amounts of each protein in the nuclei of HCT-116 cells (A) or SW-480 cells (B). The results (mean ± SD) represent the folds change of control group. The mean integrated densities of these proteins adjusted with the control protein are 806
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shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. Human CRC HCT-116 cells (C) or SW-480 cells (D) were transfected with NF-κB-RE plasmid and then treated with either CAPE or CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in 10%
FBS RPMI-1640 for 24h. The relative light units (R.L.U) were measured by the manufacturer's instruction as described under Materials and Methods. A single or double asterisk indicates a significant difference compared to the
CAPE- or CAPPE-untreated control group, respectively (P<0.05).
Human CRC HCT-116 cells (E) or SW-480 cells (F) were cultured in RPMI-1640 medium with CAPE and CAPPE (at concentrations of 0, 5, 10, 20, 50 and 100 μM) in the presence or absence of TNF-α (1 ng/mL) for 24 h. The cell proliferation was measured by MTT assay as described in Materials and Methods. Data are the mean SD (standard deviation) of three independent experiments. The different symbols ( for CAPE and for CAPPE) represent※ △ a statistically significant difference compared to the CA derivative -untreated control group in each group, respectively, at P<0.05. The different symbols (▲
for CAPE_TNF-α and ■ for CAPPE_TNF-α) represent a statistically significant difference compared to each corresponding CA derivative- treated control group in each dosage subgroup, respectively, at P<0.05.
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Figure 8 Consumption of CAPE or CAPPE suppressed the growth of
colorectal tumor in a mouse xenograft model
Xenograft nude mice (n=6 for each group) were divided into three groups (the tumor group, tumor with CAPE, tumor with CAPPE) and given CAPE or CAPPE (at a dosage of 50 nmol /kg of body weight (BW)/day) for 6 weeks.
Data (mean ± SD) represent the change in the tumor volume (A) or tumor weight (B) among the tumor group (i.e. the control group), tumor with CAPE and tumor with CAPPE. The different letters at the same time point represent
a statistically significant difference, (P<0.05).
Tumor tissues were formalin-fixed, embedded in paraffin, sectioned and subjected to hematoxylin-eosin (H&E) staining (C) as described under Materials and Methods. Blue spots represent the nuclei stained with 845
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hematoxylin. The red spots represent cytoplasm stained with eosin. For immunohistochemical (IHC) staining, tumor tissues (at week 6) were frozen , sectioned and subjected to either anti- PCNA (D) or anti-FASN (E) antibodies.
The intense dark brown color indicates the distribution of the PCNA or FASN proteins in HCT-116 cells stained with a monoclonal antibody. The blue area represents the localization of the cell nuclei. Imaging was documented at 200X magnification. (F) The plasma levels of MMP-9 were determined using an ELISA Kit (R&D systems). Upon completion of the ELISA process,
fluorescence intensities were read using a wavelength of 450/570 nm. The results presented are representative of six different experiments and are presented as plasma MMP-9 levels. The different letters represent a significant difference in a comparison of normal mice, tumor control mice, CAPE-treat mice and CAPPE-treated mice, P<0.05.
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Figure 9 CAPE- or CAPPE-mediated suppression of tumor growth was
associated with the modulation of the PI3-K/Akt, AMPK and mTOR
signaling pathways in the experimental animals
(A) Nuclear proteins from tumor tissues were prepared for Western blotting analysis using monoclonal antibodies against p21CIP1/WAF1, cyclin D1, cyclin E, Cdk4 and c-myc as described under Materials and Methods. The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The immunoreactive bands are noted with an arrow. The levels of detection represent the amount of these proteins in the nuclei of CRC cells in the experimental animals. The mean integrated densities of these proteins are adjusted with the control protein and shown in bottom row. The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk represent a statistically significant difference compared to the control group, P<0.05.
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(B) Cytoplasmic proteins from tumor tissues were prepared for Western blotting analysis using monoclonal antibodies against E-cadherin, N-cadherin, p-Akt, p-mTOR, p-ERK 1/2, p-AMPK, FASN and actin, as described under Materials and Methods. The results (mean ± SD) represent the folds change of control group and are representative of three different experiments. The levels of detection represent the amount of these proteins in the cytoplasm of CRC cells in the experimental animals. The mean integrated densities of these proteins are adjusted with the control protein and shown in bottom row.
The standard deviation (SD) of each measured protein was indicated in the parenthesis. A single asterisk represent a statistically significant difference compared to the control group, P<0.05.
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ReferencesX
1. Siegel R, Ma J, Zou Z, Jemal A (2014) Cancer statistics, 2014. CA Cancer J Clin 64: 9-29.
2. Siegel R, Desantis C, Jemal A (2014) Colorectal cancer statistics, 2014. CA Cancer J Clin 64: 104-117.
3. Vogt PK, Kang S, Elsliger MA, Gymnopoulos M (2007) Cancer-specific mutations in phosphatidylinositol 3-kinase. Trends Biochem Sci 32: 342-349.
4. Halilovic E, She QB, Ye Q, Pagliarini R, Sellers WR, et al. (2010) PIK3CA mutation uncouples tumor growth and cyclin D1 regulation from MEK/ERK and mutant KRAS signaling. Cancer Res 70: 6804-6814.
5. Gustin JP, Karakas B, Weiss MB, Abukhdeir AM, Lauring J, et al.
(2009) Knockin of mutant PIK3CA activates multiple oncogenic pathways. Proc Natl Acad Sci U S A 106: 2835-2840.
6. Kang S, Bader AG, Vogt PK (2005) Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci U S A 102: 802-807.
7. Wang QS, Papanikolaou A, Sabourin CL, Rosenberg DW (1998)
Altered expression of cyclin D1 and cyclin-dependent kinase 4 in azoxymethane-induced mouse colon tumorigenesis.
Carcinogenesis 19: 2001-2006.
8. Wolter F, Akoglu B, Clausnitzer A, Stein J (2001) Downregulation of the cyclin D1/Cdk4 complex occurs during resveratrol-induced cell cycle arrest in colon cancer cell lines. J Nutr 131: 2197-2203.
9. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646-674.
9. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646-674.