in tumor-bearing mice
A recent study indicated that MMP-9 expression is associated with upregulation of COX-2 proteins in several types of cancer. Several clinical studies indicated that COX-2 expression is associated with the tumor inflammation and progression in CRC. Therefore, these evidences suggest
that COX-2 expression and its major metabolite, PGE2, could play crucial roles in tumor progression of CRC. In the present study, we further examined the inhibitory effects of lycopene and fish oil on the expression of COX-2 and PGE2 in these tumor-bearing mice. As shown in the Fig. 5A, the results indicated that the tumor control group had augmented the gene expression of COX-2. The immunohistochemical staining results also demonstrated that a
prevalent expression of COX-2 in the tumor control group (Fig. 4B). However, consumption of lycopene and fish oil significantly suppressed COX2 expression in these tumor-bearing mice (Fig. 4A & 4B). The results further demonstrated that mice inoculated with colon cancer HT-29 cells had increased serum PGE2 levels (Fig. 5C). Consumption of lycopene and fish oil significantly reduced PGE2 serum levels in vivo. These results suggested that consumption of lycopene and fish oil significantly suppressed the expression of COX-2 and its major metabolite, PGE2, in tumor-bearing mice. Taken together; it is probable that lycopene and fish oil could inhibit tumor inflammation and progression of CRC in part through suppression of COX-2 and PGE2 in these tumor-bearing mice.
4 Discussion
Many studies suggested that lycopene and fish oil might exert anti-cancer effects and inhibit the expression of oncoproteins associated with cancer prognosis. Our previous studies demonstrated that lycopene and fish oil inhibited the proliferation of human colon cancer HT-29 cells in vitro. The present study demonstrated that consumption of lycopene and fish oil inhibited tumor growth in significant or even synergistic ways. The current study demonstrated that concomitant consumption of lycopene and fish oil significantly inhibited the growth of colon cancer in a mouse xenograft model (Fig. 1). At low dosage (3 mg/kg BW), lycopene and fish oil (FO_Low_Lyc) effectively suppressed the growth of colon cancer up to 70 %. At a high dosage (6 mg/kg BW), lycopene and fish oil (FO_High_Lyc) effectively suppressed the proliferation of colon cancer cells up to 85 % in vivo. In our previous study, we already demonstrated that lycopene inhibited the proliferation of human colon cancer HT-29 cells through suppression of -catenin signaling pathways and cell cycle progression in a dose dependent manner (0, 2, 5 and 10 M) [30]. Our results further demonstrated that lycopene and EPA synergistically inhibited cell proliferation in human colon cancer HT-29 cells [31]. We further demonstrated that lycopene and fish oil
molecular actions of lycopene and fish oil on the suppression of tumor growth were associated with the upregulation of p21CIP1/WAF1 and p27Kip1 cell cycle inhibitor proteins. The results suggested that lycopene and fish oil effectively inhibited the cell cycle progression and cell proliferation in tumor-bearing mice. In the present study, we demonstrated that consumption of lycopene and fish oil significantly suppressed the level of malignant biomarker -catenin protein in vivo. Our novel evidences also demonstrated that lycopene and fish oil effectively inhibited the expression of cyclin D1 and c-Myc proteins in colon cancer cells in tumor-bearing mice. Previous studies indicated that MMP-7 was mostly overexpressed in human colon cancer patients. Here, we further demonstrated that consumption of lycopene and fish oil synergistically inhibited MMP-7 expression and the tumor progression of colon cancer cells in tumor bearing mice. Moreover, consumption lycopene and fish oil stabilize the adherent junction E-cadherin molecules through the suppression of MMP-7 proteins (Fig. 4). The -catenin has come onto to the scene and reached central status as an important regulator of cyclin D1, c-Myc and MMP-7 proteins during tumor development. Increasing evidence implicates -catenin is an important biomarker of malignant colon cancer. It is plausible that lycopene and fish oil synergistically inhibit tumor growth through the
modulation of -catenin signaling pathways. These results from the present study were consistent with our previous findings and suggested chemopreventive roles of lycopene and fish oil against human colon cancer.
Previous reports suggested that overexpression of biomarkers such as MMP-9, COX-2 and PGE2 maybe play crucial roles in the tumor progression and inflammation of colon cancer. Suppression of COX-2, MMP-9 and PGE2 would hinder the tumor progression of tumor. Recent study indicated that fish oil inhibited the expression of COX-2 and prostaglandin E2 (PGE2) in human colon cancer HT-29 cells [32] . We further examined whether consumption lycopene and fish oil could help prevent tumor inflammation and progression in tumor-bearing mice. The results demonstrated that lycopene and fish oil
significantly inhibit the expression of MMP-9, COX-2 and PGE2 in vivo.
In conclusion, lycopene and fish oil significantly inhibit the tumor growth, progression and inflammation in tumor-bearing mice. Here, we proposed a model of synergistic effects of lycopene and fish oil on the growth of human colon cancer HT-29 cells in vivo (Fig. 6). Lycopene and fish oil may potentially act as chemopreventive agents to suppress tumor growth in a mouse xenograft model of CRC.
Acknowledgements
This material is based upon work supported, in part, by the National Science Council grant, under agreement No. 97-2320-B-039-043-MY3, NSC-100-2320-B-039-003, and Taiwan Department of Health Clinical Trial and Research Center of Excellence, under Agreement No. DOH 100-TD-B-111-004, DOH 101-TD-B-111-100-TD-B-111-004, DOH-100-TD-C-111-005, DOH-101-TD-C-111-005. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the National Science Council and Department of Health.
Figure Legends
Figure 1. Lycopene and fish oil synergistically inhibited tumor growth of
colon cancer in a mouse xenograft model
(A) Xenograft nude mice (n=6 for each group) were divided into six groups (tumor, tumor with low lycopene, tumor with high lycopene) and given fish oil (0, 4% w/w) for 5 weeks. The extent of tumor growth was evaluated by using bioluminescent imaging system. Photos of primary tumors from different
groups were shown.
(B) Data of bioluminescent intensity represented the proliferation index in primary tumor tissues. Different letters represent statistically significant
difference, p<0.05.
(C) Data represent the change of tumor weight among tumor control group and experimental groups. Different letters represent statistically significant difference, p<0.05. (p<0.05 at week 5).
Figure 2. Lycopene and fish oil significantly modulated the expression of p21CIP1/WAF1, p27 Kip1 and PCNA proteins in the mouse xenograft tumor
model
(A) Cell lysates from animal tissues were prepared using Tissue Nuclear Extract Reagent Kit containing protease inhibitor and phosphatase inhibitors according to the manufacture’s instruction. After centrifugation for 10 minutes at 12,000 xg to remove cell debris, the supernatants were retained as a whole –tissue extract. Cross contamination between nuclear and cytoplasma
fractions were not found (data not shown). Cell lysates were blotted with either anti-cyclin D1, anti-PCNA, anti- p21CIP1/WAF1, or anti- p27 kip1 monoclonal antibody as described in Materials and Methods. The levels of detection in cell lysate represent the amount of cyclin D1, PCNA, p21CIP1/WAF1 or p27 kip1in human colon cancer cells. The blots were stripped and reprobed with anti-lamin A/C antibody as loading control. The results presented are representative of six different experiments. The immunoreactive bands are
noted with arrow.
(B) The integrated densities of cyclin D1, PCNA, p21CIP1/WAF1 and p27kip1 proteins adjusted with internal control protein (lamin A/C) were shown in the
panel. Different letters represent statistically significant difference, p<0.05.
(C)Tumor tissues were formalin-fixed, embedded in paraffin, sectioned, and subjected to H&E staining. Imaging was documented at 400X magnification.
Blue spots represented the nuclei stained with hematoxylin. Red spots represented cytoplasma stained with eosin.
Figure 3. Lycopene and fish oil suppressed the expression of -catenin
and c-Myc proteins in tumor-bearing mice
(A) Nuclear fractions of cell lysates were blotted with either anti--catenin or anti-c-Myc monoclonal antibodies as described in Materials and Methods.
The levels of detection in cell lysate represent the amount of -catenin or c-Myc in human colon cancer cells. The blots were stripped and reprobed with anti-lamin A/C antibody as loading control. The results presented are representative of six different experiments. The immunoreactive bands are noted with arrow. The integrated density of -catenin or c-Myc protein adjusted with internal control protein (lamin A/C) was shown in the bottom
panel. Different letters represent statistically significant difference, p<0.05.
(B) Tumor tissues were frozen, sectioned and subjected to anti--catenin antibody by immunohistochemical staining described in Material and Methods.
Imaging was documented at 200X magnification. Brown area represented distribution of -catenin proteins (indicated with red arrows) in HT-29 cells stained with monoclonal antibody. Blue spots represented the location of cell nuclei stained with hematoxylin. The results presented are representative of
six different experiments.
Figure 4.
Lycopene and fish oil significantly suppressed the expression of MMP-7
and MMP-9 in tumor-bearing mice
(A) The plasma levels of MMP-9 were determined by using ELISA Kit (R&D systems). Briefly, equal amount of diluted plasma sample (100 L) from each group was added to each well and reacted with MMP-9 antibody according to the manufacturer’s instructions. Upon completion of the ELISA process, fluorescence intensities were read using a wavelength of 450/570 nm. These results presented are representative of six different experiments and presented as plasma MMP-9 levels. Different letters represent statistically significant difference, p<0.05.
(B) Equal amount of plasma samples from each group were loaded into gelatin-containing gel. After incubation and staining of gelatin gel, the photographs of zymogram bands are noted with arrows. Areas of enzymatic activity appeared as clear bands over the dark background. These results presented are representative of six different experiments. Different letters represent statistically significant difference, p<0.05.
(C) Cytoplasma fractions of cell lysates were blotted with either anti-MMP7 or anti-E-cadherin monoclonal antibody as described in Materials and Methods.
The levels of detection in cell lysate represent the amount of MMP7 or E-cadherin in human colon cancer cells. The blots were stripped and reprobed with anti-actin antibody as loading control. The results presented are representative of six different experiments. The immunoreactive bands are noted with arrow. The integrated densities of MMP7 and E-cadherin proteins adjusted with internal control protein (actin) were shown in the bottom panel.
Different letters represent statistically significant difference, p<0.05.
Figure 5.
Lycopene and fish oil significantly suppressed tumor inflammation in
tumor-bearing mice
(A) The relative COX-2 mRNA expressions in tumor tissues were analyzed by using quantitative RT-PCR. Relative COX-2 mRNA levels in different experimental groups were compared to the corresponding negative control group (presented as folds of control). Different letters represent statistically significant difference, p<0.05.
(B) Tumor tissues were frozen, sectioned and subjected to anti-COX-2 antibody by immunohistochemical staining described in Material and Methods.
Imaging was documented at 400X magnification. Brown area represented distribution of COX-2 protein in HT-29 cells stained with monoclonal antibody.
Blue spots represented the location of cell nuclei stained with hematoxylin.
The results presented are representative of six different experiments.
(C) The serum levels of PGE2 were determined by using ELISA Kit (R&D systems). Briefly, equal amount of diluted serum sample (150 L) from each group was added to each well and reacted with PGE2 conjugate molecule according to the manufacturer’s instructions. Upon completion of the ELISA process, fluorescence intensities were read using a wavelength of 450/570 nm. These results presented are representative of six different experiments and presented as serum PGE2 levels. Different letters represent statistically significant difference, p<0.05.
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