Leptin induces cell invasion and the upregulation of matrilysin in human colon cancer cells
Meng-Chiu Lin,1 Shu-Yao Tsai,2 Fu-Yu Wang,1 Fu-Hsuan Liu 1, Jia-Ning Syu1 and Feng-Yao Tang1,*
1 Biomedical Science Laboratory, Department of Nutrition, China Medical University, Taichung 40402, Taiwan;
2 Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan
*Corresponding author: Dr. Feng-Yao Tang
Biomedical Science Laboratory Department of Nutrition
China Medical University 91 Hsueh-Shih Road, Taichung, 40402
Taiwan, Republic of China Telephone: (886-4) 22060643
Facsimile: (886-4) 22062891
E-mail: vincenttang@mail.cmu.edu.tw
Financial support: This material is based upon work funded in part by National Science Council grant, under agreement No. NSC-100-2320-B-039-003, 101-2320-B-039-054-MY3, 101-2811-B-039-024, 102-2811-B-039-027 and China Medical University (CMU) grant under Agreement No. CMU100- Award-10, CMU100-ASIA-11, CMU101-ASIA-3, CMU101-S-25. Opinions, findings, conclusions, or recommendations expressed herein are those of the author(s) and do not necessarily reflect views of the National Science Council, Taipei Medical University, Asia University or China Medical University.
Highlights
Leptin induced cell invasion through upregulation of MMP-7 in human colon cancer cells.
Abstract
Objective: To investigate effects of leptin on MMP-7 expression and cellular invasion of human colon cancer cells. Methods: Human colon cancer cells were stimulated with leptin at various concentrations (0, 1.2, 6, 25 and 100 nM), timulatory effects of leptin on the invasion of human colon cancer cells tested by transwell invasion assay. Effect of leptin on signaling cascade in colon cancer cells were examined by Western blot assay and zymogram analysis. Results: This study demonstrated leptin dose-dependently inducing MMP-7 expression and cellular invasion of human colon cancer HT-29 cells. Treatment of MMP-7 siRNA significantly blocked leptin-mediated cellular invasion. Wortmannin (PI3K specific inhibitor), PD098059 (MEK specific inhibitor) or GM-6001 (specific
inhibitor of MMPs) could suppress leptin-mediated cellular invasion markedly. These aver leptin inducing MMP-7 expression and/or cellular invasion, partly by modulation of PI3K/Akt and MAPK/ERK signaling pathway in human colon cancer HT-29 cells. Conclusion: Results suggest novel mechanism of leptin involved in MMP-7 expression and cellular invasion in human colon cancer HT-29 cells.
Keywords: leptin, matrilysin, invasion, siRNA, Akt, MAPK, human colon cancer cells
1. Introduction
Colorectal cancer is one of the leading causes of cancer death in many countries, including North America. In the United States alone, nearly thousands of deaths are attributed to this cancer annually [1]. Epidemiological study points to obesity as a risk factor in certain types of cancer, including colorectal [2-7]. Prior studies link increased blood leptin with malignant progression of colon cancer, suggesting that greater secretion of adipocyte-derived growth factors and hormones contribute to cellular transformation and tumorigenesis [8]. Leptin, a biologically active polypeptide secreted from mature adipocytes and other epithelial cells, circulates as 16 kDa protein partially bound to plasma proteins and exerts influence via its specific receptors by endocrine, paracrine or autocrine processes [9, 10]. In physiological status, leptin controls balance of normal body weight by regulation of food intake and other biological activities [11]. It has been identified as a mitogen associated with gastro-intestinal
malignancy [12-14]. In pathophysiological status, leptin induces tumor angiogenesis and malignant progression of several cancers [12, 15-17]. Overexpression of leptin and its receptor (ObR) has been elucidated in human colon cancer cells [18, 19]. With blood levels of leptin elevated in obese patients and excess body weight strongly correlated with risk of colon cancer, studies address the key role of leptin in its etiology [20, 21].
Some in vitro studies demonstrate leptin stimulating proliferation and migration of human colon cancer cells [12, 14], definitely activating PI3K and Src kinase pathways in metastatic colon cancer LS174T and HM7 cells [22]. Invasion of such cells into surrounding stroma occurs via activation of MMPs that degrade extracellular matrix and create a microenvironment for tumor angiogenesis, growth, and metastasis [23-26]. Increased expression of MMPs is associated with tumor invasion and metastasis [27-29]. MMP gene family divides into subgroups: collagenase, stromelysin, gelatinase, membrane-type MMPs and other MMPs. Unlike 2, 3, 9 and MMP-11 (produced by stromal cells) [30, 31], MMP-7 is expressed in colorectal carcinoma [32-34]. Expression of MMP-2 and MMP-9 is frequently observed in malignant tumors [35], overexpression of MMP-7 proven for colorectal tumor genicity [36]. High MMP-7 expression correlates with low survival rate of colon cancer cases [37, 38]; clinical studies cite matrix metalloproteinase-7 (MMP-7; matrilysin) as a key malignant biomarker in human colon cancer. Promoter regions of inducible MMP-2, MMP-7, MMP-9, et al. contain regulatory elements and are tightly modulated in mammalian cells by nuclear transcription factors. Gene expression is regulated by extracellular growth factor, proinflammatory cytokines, etc.[39]
Extracellular stimuli activating Wnt pathways and downstream signal molecules induce nuclear accumulation of -catenin transcription factor and regulate expression of MMP-7 [40]. Prior studies show strong linkage between -catenin and MMP-7 overexpression in several types of cancer [41]. While earlier studies examined roles of leptin in cell proliferation and migration, major matrix metalloproteinase (MMP) required to degrade surrounding stroma and cellular invasion in human colon cancer has not been demonstrated yet. We assess effect of leptin on MMP-7 expression and cellular invasion of human colon cancer HT-29 cells.
2. Materials and Methods
2.1 Reagents and Antibodies
Anti-MMP-7 antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA); McCoy’s medium and Calcein AM from Invitrogen Inc. (Carlsbad, CA); anti--actin antibody, AG 490 (JAK inhibitor), SP600125 (JNK inhibitor), PD098059 (MEK inhibitor), Wortmannin (PI3K inhibitor), SB 203580 (p38 inhibitor), and Ipegal CA-630 from Sigma (St. Louis, MO). GM-6001 (broad-spectrum inhibitor of MMPs) was purchased from Calbiochem (USA); human leptin recombinant protein from R & D Systems, Inc. (Minneapolis, MN); human colon cancer HT-29 cell line from American Type Culture Collection (Walkersville, MD); NE-PER Nuclear and Cytoplasmic Extract reagent Kit from Pierce Biotechnology (Lackford, IL).
2.2 Cell culture
Briefly, human colon cancer HT-29 cells were cultured in a 37oC humidified incubator with 5% CO2 and grown to confluency with fetal bovine serum (FBS) supplemented McCoy’s media. McCoy’s medium was supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and 1.5 g/L sodium bicarbonate. Cells used in different experiments have similar passage number.
Preparation of cell lysates
Human colon cancer HT-29 cells were cultured in McCoy’s media in the presence or absence of leptin at diverse concentrations. After the experiment, cells were washed with phosphate buffered saline (PBS) twice and lysed in ice-cold lysis buffer. Lysis buffer contains 1X PBS, 1% Ipegal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) with 100 M of phenylmethylsulfonyl fluoride (PMSF), aprotinin and specific phosphatase inhibitors, sodium orthovanadate. Lysates were sonicated and centrifuged at 10,000 xg for 15 minutes at 4oC to remove cell debris, supernatants labelled whole cell lysates. Nuclear and cytoplasma fractions were prepared with a NE-PER Nuclear and Cytoplasmic Extract reagent Kit containing protease inhibitor and phoshpatase inhibitors. Cross contamination between nuclear and cytoplasma fractions were barely found (data not shown).
Cellular proteins were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, then blotted with anti-MMP-7 monoclonal antibody, as per manufacturer’s instructions. Blots were stripped and reprobed with -actin antibody as loading control.
2.4 Assessment of cellular invasion
Invasion of tumor cells was analyzed in transwell boyden chambers with a polyvinyl/pyrrolidone-free polycarbonate filter of 8-m pore size. Each filter was coated with 50 L of a 1:5 diluted matrigel in cold McCoy medium to form a thin continuous film on the top of the filter. HT-29 cells pre-stained with Calcein AM and stimulated with leptin were added to each triplicate well in McCoy medium (5,000 cells/well). Following 18 h incubation, cells in 10 randomly selective fields were counted by Olympus IX-71 Inverted Fluorescence Microscope, those invading the lower side of the filter measured as invasion index.
2.5 Small interfering RNAs
HT-29 cells (6x104 cells in 12-well plates or 1.5x 105 cells in 6-well plate) were transfected with a small interfering RNA (siRNA) oligonucleotide (Dharmacon Inc., Lafayette, CO) designed to interrupt expression of human matrilysin gene. Scrambled siGENOME non-targeting siRNA served as negative control.
Casein zymography was performed on both control and treatment groups to measure MMP-7 activation, gelatin zymography on both groups to rate MMP-2 and MMP-9 activation. Gel was co-polymerised with either 0.1% casein or 0.1% gelatin. For each sample, an equal amount of protein was loaded. Electrophoresis used minigel slab apparatus Mini Protean 2 (Bio-Rad) at constant 150 V, until the dye reached the bottom of the gel. After electrophoresis, gel was washed in buffer (2.5% Triton X-100 in 50mM Tris-HCl (pH 7.5)) for 1 h in an orbital shaker and zymograms incubated for 24 h at 37oC in buffer (0.15 M NaCl, 10mM CaCl2, 0.02% NaN3 in 50mM Tris-HCl [pH 7.5]), stained with Coomassie blue, then destained with 7% methanol and 5% acetic acid. Areas of enzymatic activity appeared as clear bands over the dark background.
2.7 Statistical analysis
Biostatistic methodology differentiated cellular invasion among colon cancer cells. In brief, statistical analysis of invasive capacity among triplicate sets of experimental conditions used SPSS. Confirmation of different invasion index as statistically significant requires rejection of the null hypothesis: i.e., no difference between mean invasion indices obtained from replicate sets at the P=0.05 level with one-way ANOVA model. Post hoc test ascertained differences among groups.
3. Results
Leptin has emerged as a chief risk factor for many types of cancer. We examined whether leptin could induce cellular invasion. Figure 1A depictss leptin significantly inducing cellular invasion of human colon cancer HT-29 cells in a dose-dependent manner. In comparison to unstimulated cells, leptin (at concentrations of 1.2, 6, 25 and 100 nM) significantly induced cellular invasion up to 4.2 folds, respectively (Fig. 1B). These suggest leptin playing a prominent role in cellular invasion of human colon cancer HT-29 cells.
3.2 Dose-dependently induced MMP-7 expression in HT-29 cells To probe molecular actions of leptin, we measured protein expression levels of MMPs in human colon cancer HT-29 cells. Figure 2A shows zymogram analysis of leptin inducing expression of MMP-7 strongly, expression of MMP-2 slightly, but MMP-9 expression barely (data not shown). Western blot also demonstrated leptin inducing expression of MMP-7 in a dose-dependent manner (Fig. 2B). Results suggest leptin definitely inducing MMP-7 expression and activation in human colon cancer HT-29 cells.
3.3 Treatment of MMP-7 siRNA inhibited leptin- mediated cellular invasion of human colon cancer HT-29 cells
Since leptin induces cellular invasion and MMP-7 expression in human colon cancer HT-29 cells, it is plausible that MMP-7 plays a vital role in leptin-mediated cellular invasion of such cells. To evaluate the role of MMP-7 in leptin-mediated invasion of human colon cancer HT-29 cells, we used specific siRNA oligonucleotide
designed to interrupt MMP-7expression. Figure 3 plots treatment of specific siRNA against MMP-7 gene significantly suppressing cellular invasion even in the presence of leptin. These suggest inducible expression of MMP-7 molecule modulating cellular invasion and hence acting as a chief therapeutic target in human colon cancer.
3.4 PI3K/Akt and MAPK/ERK signaling pathway played key roles in leptin-mediated invasion of human colon cancer HT-29 cells To pinpoint molecular actions of leptin, we treated human colon cancer HT-29 cells with specific inhibitors under stimulation by leptin. Figure 4 illustrates wortmannin (specific inhibitor of PI3K), PD098059 (specific inhibitor of MEK)or GM-6001 (specific inhibitor of MMPs) significantly suppressing leptin-mediated cellular invasion in human colon cancer HT-29 cells. This suggests PI3K/Akt and MAPK/ERK signaling pathway taking pivotal roles in leptin-mediated MMP-7 expression and cellular invasion in human colon cancer HT-29 cells.
4. Discussion
Colorectal cancer is one of the leading causes of cancer death in many countries. Previous studies suggest adipocyte-derived growth factors such as leptin could contribute to cellular transformation and tumorigeneisis [8]. High circulating leptin level could be associated with malignancy of gastro-intestinal cancer [13]. Overexpression of MMP-7 has likewise been demonstrated for tumorgenicity of colon cancer and biomarkers of colorectal carcinoma. Higher circulating level of MMP-7 is associated with lower survival rate of colon cancer
patients. Still, effect of leptin on MMP-7 expression and the molecular mechanism of cellular invasion in human colon cancer cells have not been demonstrated. This study investigated molecular actions of leptin on cellular invasion of human colon cancer HT-29 cells in vitro.
Our results demonstrate leptin dose-dependently inducing cellular invasion and expression of MMP-7 molecules in human colon cancer HT-29 cells. However, leptin barely influenced expression of other types: e.g., MMP-2, MMP9. It is plausible that leptin induces MMP-7 expression and cellular invasion in human colon cancer HT-29 cells. To investigate whether the inducible form of MMP-7 is responsible for leptin-mediated cellular invasion, we used specific siRNA designed to interfere with MMP-7 gene expression. Figure 3 proves treatment of siRNA can significantly inhibit cellular invasion, suggesting MMP-7 molecule as the major molecule involved in leptin-mediated cellular invasion in human colon cancer cells. These suggest MMP-7 as an inducible molecule playing a critical role in cellular invasion under leptin stimulation. Prior studies indicated strong immunohistochemical staining of MMP-7 in various types of colonic adenomas [36]. Our results concur with earlier findings and provide a novel mechanism of leptin in tumorigenesis of human colon cancer. Studies also indicate expression of MMP-7 as significantly correlated with the presence of nodal or distant metastases [42, 43].
To examine the molecular mechanism of leptin in cellular invasion, we investigated major signalling pathways responsible for cellular invasion of human colon cancer cells. Using variant specific inhibitors against signalling pathways, we observed treatment of wortmannin (specific inhibitor of PI3K), PD098059 (specific inhibitor
of MEK) and GM-6001 (specific inhibitor of MMPs) significantly blocking cellular invasion. Results hint PI3K/Akt and MAPK/ERK signalling pathways playing key roles in leptin-mediated cellular invasion in human colon cancer HT-29 cells.
In sum, a mechanism by which leptin may exert malignant effect is partly through modulation of MMP-7 expression, as needed for cellular invasion during tumor development. Findings yield novel mechanistic insight into tumorogenic effects of leptin on the malignant progression of human colorectal cancer. We only tested effects of leptin on human colon cancer HT-29 cell lines, yet our in
vitro results are consistent with other findings that high circulating
level of leptin might be a risk factor associated with tumor malignancy, as evident from population studies and animal experiments showing enhanced tumor progression and cancer development. Our results also suggest a novel hypothesis that MMP-7 might act as a malignant biomarker in autocrine regulation manner in human colon cancer cells.
Acknowledgements
This material is based upon work supported, in part, by the National Science Council grant, under agreement No. NSC-100-2320-B-039-003, 101-2320-B-039-054-MY3, 101-2811-B-039-024, 102-2811-B-039-027 and China Medical University (CMU) grant under Agreement No. CMU100- Award -10, CMU100-ASIA-11, CMU101-ASIA-3, CMU101-S-25. Opinions, findings, conclusions, or recommendations expressed herein are those of the author(s) and do not necessarily
reflect the views of the National Science Council, Taipei Medical University, Asia University and China Medical University.
Conflict of interest
The authors have no competing financial interests.
Figure legends
Figure 1
Leptin dose-dependently induced cellular invasion of human colon cancer HT-29 cells
Invasion of tumor cells was analyzed in transwell boyden chambers with a polyvinyl/pyrrolidone-free polycarbonate filter of 8-m pore size. Each filter was coated with 50 L of 1:5 diluted matrigel in cold McCoy’s medium to form a thin continuous layer on the top of the filter. Confluent human colon cancer cells were cultured in McCoy’s medium with 10% fetal bovine serum at 37oC. After washing out the media, colon cancer cells were trypsinized, pre-stained with Calcein AM and transferred to matrigel coated transwell boyden chambers. Human colon cancer cells (5,000 cells/well) were added to each triplicate well in McCoy’s medium containing variant concentrations of leptin (0, 1.2, 6, 25 and 100 nM). After incubation for 18 h, cells were counted as described above; number of cells invading the lower side of the filter was measured as invasive activity. (A) represents the microphotograph of invasive colon cancer cells; (B) represents level
of invasion index. Letters represent statistically significant difference, p<0.05. Data shown represent three independent experiments.
Figure 2
Leptin dose-dependently induced expression of MMP-7 in human colon cancer HT-29 cells
Post confluent colon cancer cells cultured on 24 well plates were incubated in McCoy’s medium with 10% FBS at 37oC. After washing out media, cells were incubated in serum-free (conditioned medium) McCoy’s medium in the presence of different leptin concentrations (0, 1.2, 6 and 25 nM) at 37 oC for 24 hr. Conditioned medium was collected and loaded into casein- or gelatin-containing zymogram gel stained with coomassie blue stains (see Materials and Methods). The levels of detection represent zymogram expression of different MMPs in human colon cancer cells, MMP-7 and MMP-2 noted with arrow (A). Human colon cancer cells, cultured in McCoy’s medium with 10% FBS in a tissue culture dish, were lifted off by trypsinization, pelleted by centrifuge and resuspended in the same medium. After washing out media, cells were incubated in McCoy’s medium with 10% FBS in a tissue culture dish with variant concentrations of leptin (0, 0.6, 1.2, 6, 25 and 100 nM) for 24 hr. Total cell lysates were blotted with anti- MMP-7 antibody (see Materials and Methods). Levels of detection in cell lysates represent amount of MMP-7 in human colon cancer cells. Blots were stripped and reprobed with anti-actin antibody as loading control, immunoreactive bands noted with arrow (B).
Figure 3.
Treatment of MMP-7 siRNA inhibited leptin-mediated invasion of human colon cancer HT-29 cells
Post-confluent colon cancer cells cultured on 12-well or 6-well plates were cultured in McCoy’s medium with 10% FBS at 37oC. After washing out media, HT-29 cells (6x104 cells in 12-well plates) were transfected with a small interfering RNA (siRNA) oligonucleotide designed to interrupt expression of human matrilysin gene. Cells were incubated in serum-free (conditioned medium) McCoy’s medium for 24 hours; scrambled siGENOME non-targeting siRNA served as negative control. Invasion of tumor cells was analyzed in transwell boyden chambers with polyvinyl/pyrrolidone-free polycarbonate filter of 8-m pore size. Each filter was coated with 50 L of a 1:5 diluted matrigel in cold McCoy’s medium to form thin continuous layer on top of the filter. Human colon cancer cells transfected with siRNA were cultured in McCoy’s medium at 37oC. After washing out media, cells were trypsinized, pre-stained with Calcein AM, then transferred to matrigel coated transwell boyden chambers. Human colon cancer cells (5000 cells/well) were added to each of triplicate wells in serum-free McCoy’s medium in the presence or absence of 1.2 nM leptin for 18hr. Asterisk represents statistical difference compared to controls without treatment in inter-group (leptin vs. untreated control), p<0.05. Pound or cross sign represents statistical difference compared to control set in intra-group (MMP-7 siRNA vs. respective control), p<0.05.
PI3K/Akt signaling pathway played key roles in leptin-mediated invasion of human colon cancer HT-29 cells
Invasion of tumor cells was analyzed in transwell boyden chambers with a polyvinyl/pyrrolidone-free polycarbonate filter of 8-m pore size. Each filter was coated with 50 L of a 1:5 diluted matrigel in cold McCoy’s medium to form thin continuous layer on top of the filter. Confluent human colon cancer cells were cultured in McCoy’s medium with 10% fetal bovine serum at 37oC. After washing out media, colon cancer cells were trypsinized, pre-stained with Calcein AM, then transferred to matrigel coated transwell boyden chambers. Human colon cancer cells (5,000 cells/well) were treated with different inhibitor and stimulated with leptin (1.2 nM) in serum-free McCoy’s medium. After incubation for 18 h, cells were counted as described above; number of cells invading the lower side of filter was gauged as invasive activity. Letters represent statistical significance, p<0.05. Data shown represent three independent experiments.
References
[1] Siegel, R., Naishadham, D., Jemal, A., Cancer statistics, 2013. CA: a cancer journal for clinicians 2013, 63, 11-30.
[2] Abu-Abid, S., Szold, A., Klausner, J., Obesity and cancer. Journal
of Medicine 2002, 33, 73-86.
[3] Calle, E. E., Teras, L. R., Thun, M. J., Obesity and mortality. New
England Journal of Medicine 2005, 353, 2197-2199.
[4] Wang, Y., Jacobs, E. J., Patel, A. V., Rodriguez, C., et al., A prospective study of waist circumference and body mass index in relation to colorectal cancer incidence. Cancer causes & control:
[5] Percik, R., Stumvoll, M., Obesity and cancer. Experimental and
clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2009, 117,
563-566.
[6] Calle, E. E., Thun, M. J., Obesity and cancer. Oncogene 2004, 23, 6365-6378.
[7] Calle, E. E., Rodriguez, C., Walker-Thurmond, K., Thun, M. J., Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. New England Journal of
Medicine 2003,348, 1625-1638.
[8] Garofalo, C., Surmacz, E., Leptin and cancer. Journal of Cellular
Physiology 2006, 207, 12-22.
[9] Fruhbeck, G., Aguado, M., Martinez, J. A., In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/ paracrine role of leptin. Biochemical and Biophysical Research
Communications 1997, 240, 590-594.
[10] Muoio, D. M., Dohm, G. L., Fiedorek, F. T., Jr., Tapscott, E. B., Coleman, R. A., Leptin directly alters lipid partitioning in skeletal muscle. Diabetes 1997, 46, 1360-1363.
[11] Muoio, D. M., Lynis Dohm, G., Peripheral metabolic actions of leptin. Best practice & research. Clinical endocrinology &
metabolism 2002, 16, 653-666.
[12] Hoda, M. R., Keely, S. J., Bertelsen, L. S., Junger, W. G., et al., Leptin acts as a mitogenic and antiapoptotic factor for colonic cancer cells. The British journal of surgery 2007, 94, 346-354. [13] Howard, J. M., Pidgeon, G. P., Reynolds, J. V., Leptin and
gastro-intestinal malignancies. Obesity Reviews: an Official
Journal of the International Association for the Study of Obesity 2010, 11, 863-874.
[14] Ratke, J., Entschladen, F., Niggemann, B., Zanker, K. S., Lang, K., Leptin stimulates the migration of colon carcinoma cells by multiple signaling pathways. Endocrine-related cancer 2010, 17, 179-189.
[15] Fruhbeck, G., Jebb, S. A., Prentice, A. M., Leptin: physiology and pathophysiology. Clin Physiol 1998, 18, 399-419.
[16] Saxena, N. K., Sharma, D., Ding, X., Lin, S., et al., Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved in leptin-mediated promotion of invasion and migration of hepatocellular carcinoma cells. Cancer research 2007, 67, 2497-2507.
[17] Saxena, N. K., Titus, M. A., Ding, X., Floyd, J., et al., Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB journal : official
publication of the Federation of American Societies for Experimental Biology 2004, 18, 1612-1614.
[18] Uddin, S., Bavi, P. P., Hussain, A. R., Alsbeih, G., et al., Leptin receptor expression in Middle Eastern colorectal cancer and its potential clinical implication. Carcinogenesis 2009, 30, 1832-1840.
[19] Uddin, S., Bu, R., Ahmed, M., Abubaker, J., et al., O
verexpression of leptin receptor predicts an unfavorable outcome in Middle Eastern ovarian cancer. Molecular cancer 2009, 8, 74. [20] Huang, X. F., Chen, J. Z., Obesity, the PI3K/Akt signal pathway
and colon cancer. Obesity reviews : an official journal of the
International Association for the Study of Obesity 2009, 10,
610-616.
[21] Birmingham, J. M., Busik, J. V., Hansen-Smith, F. M., Fenton, J. I., Novel mechanism for obesity-induced colon cancer
progression. Carcinogenesis 2009, 30, 690-697. [22] Jaffe, T., Schwartz, B., Leptin promotes motility and
invasiveness in human colon cancer cells by activating multiple signal- transduction pathways. International Journal of Cancer.
Journal International du Cancer 2008, 123, 2543-2556.
[23] Westermarck, J., Kahari, V. M., Regulation of matrix
metalloproteinase expression in tumor invasion. FASEB Journal:
Official Publication of the Federation of American Societies for Experimental Biology 1999, 13, 781-792.
[24] Zhang, J. L., Chen, G. W., Liu, Y. C., Wang, P. Y., et al.,
Secreted protein acidic and rich in cysteine (SPARC) suppresses angiogenesis by down-regulating the expression of VEGF and MMP-7 in gastric cancer. PloS one 2012, 7, e44618.
[25] Jiang, Z. Q., Zhu, F. C., Qu, J. Y., Zheng, X., You, C. L., [Relationship between expression of matrix metalloproteinase (MMP-9) and tumor angiogenesis, cancer cell proliferation, invasion, and metastasis in invasive carcinoma of cervix]. Ai
zheng = Aizheng = Chinese journal of cancer 2003, 22, 178-184.
[26] Hawinkels, L. J., Zuidwijk, K., Verspaget, H. W., de Jonge-Muller, E. S., et al., VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis. Eur J
Cancer 2008, 44, 1904-1913.
[27] Zheng, S. Q., Huang, R. Q., Zhang, Y. J., [Role of matrix metalloproteinase (MMP)-2 and -9 and vascular endothelial growth factor C in lymph node metastasis of breast
cancer]. Zhonghua bing li xue za zhi Chinese Journal of
Pathology 2010, 39, 240-244.
[28] Li, Y. J., Wei, Z. M., Meng, Y. X., Ji, X. R., Beta-catenin up- regulates the expression of cyclinD1, c-myc and MMP-7 in human pancreatic cancer: relationships with carcinogenesis and
metastasis. World journal of gastroenterology : WJG 2005, 11, 2117-2123.
[29] Ogata, Y., Matono, K., Nakajima, M., Sasatomi, T., et al., Efficacy of the MMP inhibitor MMI270 against lung metastasis following removal of orthotopically transplanted human colon cancer in rat.International journal of cancer. Journal international
du cancer 2006, 118, 215-221.
[30] Kenny, H. A., Lengyel, E., MMP-2 functions as an early response protein in ovarian cancer metastasis. Cell Cycle 2009, 8, 683-688. [31] Eiro, N., Fernandez-Garcia, B., Gonzalez, L. O., Vizoso, F. J.,
Cytokines related to MMP-11 expression by inflammatory cells and breast cancer metastasis. Oncoimmunology 2013, 2, e24010. [32] Cheng, K., Xie, G., Raufman, J. P., Matrix metalloproteinase-7-
catalyzed release of HB-EGF mediates deoxycholyltaurine- induced proliferation of a human colon cancer cell
line. Biochemical pharmacology 2007, 73, 1001-1012.
[33] Kitamura, T., Biyajima, K., Aoki, M., Oshima, M., Taketo, M. M., Matrix metalloproteinase 7 is required for tumor formation, but dispensable for invasion and fibrosis in SMAD4-deficient intestinal
adenocarcinomas. Laboratory Investigation; a Journal of
Technical Methods and Pathology 2009, 89, 98-105.
[34] Horiuchi, S., Yamamoto, H., Min, Y., Adachi, Y., et al.,
Association of ets-related transcriptional factor E1AF expression with tumour progression and overexpression of MMP-1 and matrilysin in human colorectal cancer. Journal of Pathology 2003, 200, 568-576.
[35] Johnsen, M., Lund, L. R., Romer, J., Almholt, K., Dano, K., Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation. Current Opinion in Cell
Biology 1998, 10, 667-671.
[36] Kirimlioglu, H., Kirimlioglu, V., Yilmaz, S., Sagir, V., et al., Role of matrix metalloproteinase-7 in colorectal adenomas. Digestive
Diseases and Sciences 2006, 51, 2068-2072.
[37] Oshima, T., Akaike, M., Yoshihara, K., Shiozawa, M., et al., Clinicopathological significance of the gene expression of matrix metalloproteinase-7, insulin-like growth factor-1, insulin-like growth factor-2 and insulin-like growth factor-1 receptor in patients with colorectal cancer: insulin-like growth factor-1
receptor gene expression is a useful predictor of liver metastasis from colorectal cancer. Oncology Reports 2008, 20, 359-364. [38] Fang, Y. J., Lu, Z. H., Wang, G. Q., Pan, Z. Z., et al., Elevated
expressions of MMP7, TROP2, and survivin are associated with survival, disease recurrence, and liver metastasis of colon
cancer.International journal of colorectal disease 2009, 24, 875-884.
[39] Benbow, U., Brinckerhoff, C. E., The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix biology : journal of
the International Society for Matrix Biology 1997, 15, 519-526.
[40] Brabletz, T., Jung, A., Dag, S., Hlubek, F., Kirchner, T., beta- catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. American Journal of Pathology
1999,155, 1033-1038.
[41] Matono, H., Oda, Y., Nakamori, M., Tamiya, S., et al.,
Correlation between beta-catenin widespread nuclear expression and matrix metalloproteinase-7 overexpression in sporadic
[42] Adachi, Y., Yamamoto, H., Itoh, F., Arimura, Y., et al., Clinicopathologic and prognostic significance of matrilysin expression at the invasive front in human colorectal cancers.
International Journal of Cancer. Journal International du Cancer 2001, 95, 290-294.
[43] Adachi, Y., Yamamoto, H., Itoh, F., Hinoda, Y., et al.,
Contribution of matrilysin (MMP-7) to the metastatic pathway of human colorectal cancers. Gut 1999, 45, 252-258.
