Abstract:
A pharmaceutical composition for treating or preventing peritoneal dissemination is provided. The pharmaceutical composition includes an effective dose of connective tissue growth factor (CTGF) and a pharmaceutically acceptable carrier thereof.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. patent application Ser. No. 12/220,332 filed Jul. 23, 2008, the disclosure of which application is hereby incorporated by reference into this application. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a pharmaceutical composition. In particular, the present invention relates to a pharmaceutical composition for inhibiting peritoneal dissemination. 
     BACKGROUND OF THE INVENTION 
     Peritoneal metastasis mostly originates from the tumors in the neighboring digestive system and organs, such as intestine, stomach, liver, pancreas, etc. Tumor cells accumulate and adhere abundantly on the peritoneum through blood or the lymph node after falling into peritoneal cavity, but do not invade other substantial organs (such as spleen, uterus and ovaries) directly. These tumor cells adhere on the surfaces of these organs, and will proliferate to invade these organs directly for a long time. This is a common phenomenon for cancers in the digestive tract of the peritoneal cavity. For instance, cancers include gastric cancer, small intestine cancer, colon cancer, rectal cancer, appendiceal cancer and pancreatic cancer. Certainly, there are few cancer cells originated from the peritoneum, and this situation is named primary peritoneal surface cancer. 
     Metastasis in the peritoneal cavity was deemed as an expression manner of the late-stage cancer in the past. Therefore, most of doctors in oncology only administrated an assistant or a remissive treatment, such as radiotherapy, anti-cancer chemotherapy and immunotherapy, etc. However, immunotherapy dose not yet have a concrete result acceptable for everybody. Most of patients appear the phenomena of ascites accumulation and intestinal obstruction, so as to lead to extreme pain and starvation. 
     If active treatment is not administrated, these patients diagnosed the peritoneal metastasis in the peritoneal cavity only have several months to live (average survival rate is 5 to 6 months). Among this, colorectal peritoneal carcinomatosis is deemed as a disease treated with relaxation therapy before death. 
     In the past decade, some new therapies, including active cyto-reductive surgery combining with hyperthermic intraperitoneal chemotherapy, have shown that these therapies can improve the average post-operative survival rate to 32.4 months (Glehen et al., J. Clin. Oncol., 2004). This strategy seems to bring a hope to the late-stage cancer patients with peritoneal metastasis. However, with regard to the researches of the active cyto-reductive surgery supplemented with hyperthermic intraperitoneal chemotherapy, more than 50% researches are still shown that the post-operative mortality of this strategy is 5% high (Glehen et al., Lancet Oncol., 2004; Koppe et al., 2006; Yan et al., 2006), and the incidence is ranged between 25% and 35% (Glehen et al., J. Clin. Oncol., 2004; Glehen et al., Lancet Oncol., 2004; Koppe et al., 2006; Yan et al., 2006; Verwaal et al., 2003; Glehen et al., 2003). This strategy only has effect on the early-stage cancer patients. Therefore, researching the mechanism of peritoneal metastasis and developing new therapies are urgently needed to the late-stage cancer patients with peritoneal metastasis. 
     In the progress of the peritoneal cavity cancer patients (including colorectal cancer, gastric cancer and ovary cancer, etc.), in order to absorb more nutrients, cancer cells usually proceed the peritoneal metastasis. At this time, cancer cells must adhere and invade the mesothelial layer, proliferate and begin angiogenesis. Therefore, the behavior that cancer cells adhere on the surface of peritoneum can be deemed as the key point in the beginning of peritoneal metastasis. 
     The adhesion ability of cancer cells can be regulated by the amount of connective tissue growth factor (CTGF). CTGF is an extracellular matrix-associated molecule (Rocnik et al., 2006; Bornstein et al., 2000; Bornstein et al., 2002), and CTGF has been proved to influence various important cellular functions, such as regulation of mitosis, apoptosis, generation of extracellular matrix, angiogenesis and metastasis (Lau et al., 1999; Bork et al., 1993; Moussad et al., 2000; Brigstock et al., 1999; Perbal 2001; Babic et al., 1999; Planque et al., 2003). Recently, many researches are also shown that CTGF can enhance the adhesion of different normal cells, such as fibroblasts, platelets, endothelia cells and rat hepatic stellate cells (Babic et al., 1999; Gao et al., 2004; Chen et al., 2001; Jedsadayanmata et al., 1999). However, the regulation mechanism of CTGF adhering to cancer cells are not completely clarified. 
     Taiwanese Patent No. 1282419 has been disclosed that the amount of CTGF represents the inverse correlation with the possibility of invasion or metastasis in the lung cancer patients. U.S. Patent Application No. 2005/0147986 is further disclosed that the truncation of the constructed CTGF plasmid is performed to identify the active fragment of CTGF, and is further founded that the inhibition effect of the C-terminal (CT) domain-lacked CTGF to the cancer cells will decrease enormously. 
     Therefore, in order to clarify whether CTGF can change the activity of peritoneal dissemination of cancer cells, the possible regulation mechanism of CTGF to the cancer cell adhesion is further researched in the present invention. Furthermore, the possible relationship between CTGF and peritoneal metastasis recurrence is further researched in the present invention. 
     It is therefore attempted by the applicant to deal with the above situation encountered in the prior art. 
     SUMMARY OF THE INVENTION 
     The issue that CTGF can inhibit cancer cells to adhere on the peritoneal tissue and further decreases the occurrence of peritoneal metastasis and peritoneal carcinoma recurrence is firstly provided in the present invention. 
     Therefore, a pharmaceutical composition having one of functions of treating peritoneal dissemination and preventing peritoneal dissemination is provided. The pharmaceutical composition includes at least one of CTGF and an active fragment thereof. 
     Preferably, the at least one of CTGF and the active fragment thereof inhibits at least one matter selected from the group consisting of a cancer cell adhering to a peritoneal cavity, a formation of a peritoneal nodule and an activity of a trigger of an immune response. 
     Preferably, CTGF is a recombinant CTGF, and the active fragment of CTGF is one selected from the group consisting of a C-terminal (CT) domain, a mutant of CT domain, an active fragment of the recombinant CTGF and a recombination thereof. 
     Preferably, peritoneal dissemination is caused by a cancer selected from the group consisting of colon cancer, gastric cancer, ovary cancer and a combination thereof. 
     Preferably, the at least one of CTGF and the active fragment thereof forms an effective dose. 
     Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier. 
     In accordance with another aspect of the present invention, a pharmaceutical composition having one of functions of treating peritoneal carcinoma recurrence and preventing peritoneal carcinoma recurrence is provided. The pharmaceutical composition includes at least one of CTGF and an active fragment of CTGF. Preferably, peritoneal carcinoma recurrence is occurred after surgery, and the pharmaceutical composition further includes a pharmaceutical acceptable carrier. 
     In accordance with another aspect of the present invention, a method for screening a inhibitor of peritoneal dissemination is provided. The method includes steps of: (a) causing a compound to bind with a cell expressing CTGF; (b) measuring a first amount of an expression of the CTGF of the cell; and (c) determining whether the first amount is higher than a reference amount. The inhibitor inhibits a cancer cell adhering to a peritoneal tissue. Preferably, the cell is selected from a peritoneal tissue, and the reference amount is determined by a second amount of the expression of CTGF of the cell without being bound to the compound. 
     In accordance with another aspect of the present invention, a method for measuring a possibility of peritoneal dissemination being occurring to a patient is provided. The method includes steps of: (a) providing a peritoneal tissue from the patient; (b) measuring a first amount of an expression of CTGF of the peritoneal tissue; and (c) comparing the first amount with a reference amount. Preferably, the reference amount is determined by a second amount of the expression of CTGF of a normal peritoneal tissue. When the first amount of the expression of CTGF of the peritoneal tissue is higher than the reference amount, the compound has potential for positively regulating CTGF. 
     In accordance with another aspect of the present invention, a method for measuring a possibility of peritoneal carcinoma recurrence occurring to a patient is provided. The method includes steps of: (a) providing a peritoneal tissue from the patient; (b) measuring a first amount of an expression of CTGF; and (c) comparing the first amount with a reference amount. When a first amount is lower than the reference amount of CTGF of the normal peritoneal tissue, the cancer patient is deemed as in the high risk of peritoneal dissemination. 
     In accordance with another aspect of the present invention, a method for inhibiting peritoneal dissemination of a patient is provided. The method includes a step of providing the patient with at least one of CTGF and an active fragment of CTGF. 
     In accordance with another aspect of the present invention, a method for inhibiting peritoneal carcinoma recurrence of a patient is provided. The method includes a step of administering the patient with at least one of CTGF and an active fragment of CTGF. 
     The above objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings, in which: 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. 
         FIG. 1  is an electrophoresis diagram of CTGF mRNA and CTGF protein expression in HCT116/CTGF-M transfectant and HCT116/Neo cell line of the present invention; 
         FIGS. 2-I  to  2 -IV are the peritoneal tumor distributions of dissection after injecting HCT116/CTGF-M and HCT116/Neo cell lines respectively into SCID mice for eight weeks; 
         FIG. 3  is a relationship diagram showing the proportion of survival and time (days) after injecting HCT 116/Neo and HCT116/CTGF-M transfectants respectively to the SCID mice; 
         FIGS. 4-I  to  4 -IX are the dissection diagrams showing the effect of peritoneal dissemination while injecting rCTGF into the SCID mice in the control, co-treatment and post-treatment groups; 
         FIG. 5(A)  is the quantitative result of the abdominal circumference of mice in the control, co-treatment and post-treatment groups; 
         FIG. 5(B)  is are the quantitative result of the body weight of mice in the control, co-treatment and post-treatment groups; 
         FIG. 6  is the quantitative result of peritoneal nodules of mice in the control, co-treatment and post-treatment groups; 
         FIG. 7  is the expressions of CTGF mRNA and CTGF protein, and the cellular adhesion ability of CTGF in HCT116, LoVo, HT-29 and Caco-2 cell lines; 
         FIG. 8  is the CTGF expressions and the cellular adhesion abilities of HCT116/Neo, HCT116/CTGF-M, HCT116/CTGF-4 and HCT116/CTGF-18 transfectants; 
         FIG. 9  is the influence of adhesion ability that different doses of the CTGF expression plasmid transiently transfect to HCT116 cells; 
         FIG. 10  is the function of different doses of CTGF neutralizing antibody antagonizing the stable CTGF expression in HT29 cell line; 
         FIG. 11  is the CTGF expression and the CTGF adhesion ability of HT29 cell lines transfected with different doses of anti-sense CTGF plasmid; 
         FIGS. 12-I  to  12 -IV are the most representative immunohistochemical staining in 136 colorectal cancer patient samples; 
         FIG. 13  is the relationship diagram showing time and the recurrence rate from post-operation to recurrence between the high CTGF expression group and the low CTGF expression group; and 
         FIG. 14  is the relationship diagram showing time and the survival rate between the high CTGF expression group and the low CTGF expression group. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed. 
     Pharmacological Experiments 
     1. Surgical Sampling and Immunohistological Staining 
     Subserosa (T3)- and perforating visceral peritoneum or peripheral organ (T4)-invasive colorectal cancer tissues from 136 consecutive patients were included at National Taiwan University Hospital during the period from December 1993 to July 1999. All patients underwent complete surgical resection, and their clinical and pathological data were available at the same time. Patients with histological proven peritoneal metastasis of colorectal cancer (CRC) diagnosed at the first presentation were defined as synchronous peritoneal seeding. All patients were followed up and this involved periodic examinations comprising serum blood-chemistry panels, carcinoembryonic antigen (CEA) level, endoscopy and abdominal ultrasonography and radiograms of the thorax. Patients with metachronous carcinomatosis were deemed to be clear of peritoneal disease at the initial curative colorectal resection, but subsequently became symptomatic on follow-up and were diagnosed with peritoneal recurrence on computed tomography or laparotomy. Immunohistological staining of CTGF was performed by the biotin-peroxidase complex using a polyclonal goat anti-human CTGF antibody (R&amp;D Systems, MN). The pathologist assessing immunostaining intensity was blinded to patients&#39; information and the results of immunohistological staining were classified using extent; these were level 0 (negative staining), level 1 (&lt;5% of tumor cell stained), level 2 (&lt;50% of tumor cells stained) and level 3 (&gt;50% of tumor cells stained). 
     2. Incubation of Cells 
     The cell lines, HCT116, Coca-2 and LoVo, were incubated in the DMEM medium (Life Technologies, Inc.) supplemented with 4 mM L-glutamine and 10 mM sodium pyruvate. The medium of Coca-2 was further supplemented with 10 mg/ml of transferrin. HT29 cells were incubated in the RPMI 1640 medium (Life technologies, Inc.). All media were supplemented with 10% fetal bovine serum and 1% penicillin (10,000 U/ml)-streptomycin (10,000 U/ml) (Life Technologies, Inc.). All incubating conditions of these cell lines were controlled at 37° C. in the air with 5% CO 2 . All cell lines should be refreshed the medium for every 2 to 3 days before fusion. 
     3. Recombination of CTGF 
     The Suspension FreeStyle™ 293-F cells (Invitrogen, San Diego, Calif.) were adopted to be the expression system. In accordance with the user&#39;s manual of FreeStyle 293-F expression system, the CTGF expression plasmid was transfected to the FreeStyle 293-F cell line, which then was incubated at 37° C. in the orbital shaker in the air with 8% CO 2  for 48 hours. The expression cells were microfiltrated and the diluted protein sample was concentrated. The recombinant protein was further separated speedy by Amicon Ultra-15 (Millipore Corp., Bedford, Mass.) so as to avoid the decrease of CTGF activity. 
     4. Western Blot Analysis 
     The abundantly-expressed cells were washed with phosphate buffered saline (PBS) containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM sodium orthovanadate. The cell pellet then was suspended in the lysis buffer (containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1% NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF), 20 μM leupeptin and 0.15 U/ml aprotinin) and was preserved on ice for 30 minutes. The tumor portions of CRC patients were also homogenized by the lysis buffer. The lysed cells were centrifuged at 4° C. in 14,500×g for 30 minutes, and the supernatant was collected. The protein in the supernatant was quantified by the spectrophotometer. After the electrophoresis was performed with 12% SDS-polyacrylamide gel, protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P membrane, Millpore Corp., Bedford, Mass.). After the blot was blocked in a solution of 3% bovine serum albumin (BSA), 0.1% Tween 20 and PBS, the membrane-bound protein was probed with primary antibodies against β-actin (Sigma Chemical Co., St. Louis, Mo.), -catenin (BD Transduction Laboratories, BD Biosciences, Woburn, Mass.) or CTGF (R&amp;D Systems, MN). The membrane was washed and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 30 minutes. The antibody-bound protein bands were detected with the enhanced chemical fluorescent reagent (Amersham Bioscience) and photographed with Kodak X-Omat Blue autoradiography film. 
     5. Preparation of CTGF Antibody 
     After the computed analysis, the amino acid fragment (aa243-263) of CTGF was adopted to synthesize the artificial peptide being the antigen for immunizing the rabbit. This amino acid fragment is similar with Fisp12 (mouse&#39;s CTGF) but different with Cyr61, Nov, WISP-1/Elm-1, WISP-2/rCop-1/CTGF-3 or WISP-3. The purification of CTGF antibody in the serum was processed in accordance with the literature published by Shimo et al (1999). 
     6. Analysis of Cell Attachment 
     Extracellular matrix (ECM)- or Matrigel-coated 24-well culture plate was adopted. Each well was coated 200 μl, and the culture plate was placed at 37° C. for 30 minutes. After 4×10 3  HCT116 cells were seeded in each well, the antibody was administrated so as to adhere on the well coated ECM or Metrigel. After incubating at 37° C. for 30 minutes, the wells were washed with 1× PBS twice, the attached cells were fixed with methanol and stained with crystal violet. 
     The excised rabbit peritoneum (about 1.6 centimeter square) was placed in a 6-well culture plate, then 1 ml of 1% BSA/RPMI 1640 medium was added in each well. Cells were labeled with the fluorescent substance, 1.77 mM 5-chloromethylfluorescein diacetate (CMFDA), at 37° C. for 30 minutes and then washed with 1% BSA/RPMI 1640 medium twice. The previous cellular supernatant (2×10 5  cells per 1% BSA/RPMI 1640 medium, and a total of 0.5 ml) was covered on the peritoneum in the 6-well culture plate and incubated at 37° C. for 40 minutes. After washing gently with PBS, the cells adhered on the peritoneum were observed and counted under the fluorescence microscope (Olympus IX70; Olympus, Tokyo, Japan), which was qualified an NIBA filter (Ex=470 to 490 nm, and Em=515 to 550 nm) for observing CMFDA. 
     7. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 
     Reverse transcription of RNA isolated from cells was performed in a final reaction volume of 20 μl containing 5 μg total RNA in the First Strand Buffer with 10 mM dithiothreitol (DTT), 2.5 mM dNTP, 1 μg Oligo (dT) 12-18 primer and 200 unit/μl Moloney murine leukemia virus (MMLV) reverse transcriptase. The reaction was carried out at 37° C. for 2 hours, and was terminated by heating at 70° C. for 10 minutes. One microliter of the reaction mixture was then amplified by PCR using either of the following pairs of primers: (1) sense SEQ ID NO. 1 and antisense SEQ ID NO. 2, to produce a 500-bp fragment of the CTGF gene; (2) sense SEQ ID NO. 3 and antisense SEQ ID NO. 4, to produce a 450-bp fragment of the Cyr61 gene; (3) sense SEQ ID NO. 5 and antisense SEQ ID NO. 6, to produce a 420-bp fragment of the DAPK gene; and (4) sense SEQ ID NO. 7 and antisense SEQ ID NO. 8, to produce a 320-fragment of the -actin gene. 
     The PCR amplification was conducted in a reaction buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 167 μM dNTPs, 2.5 units of Taq DNA polymerase and 0.1 μtM primers. The reactions were performed in the Biometra Thermoblock (Biometra Inc., Florida, USA) using the following program: denaturing at 95° C. for 1 minute, annealing at 58° C. for 1 minute, and elongating at 72° C. for 1 minute, for a total of 23 cycles; the final extension took place at 72° C. for 10 minutes. Equal volumes of each PCR sample were subjected to electrophoresis in a 1% agarose gel, which was then stained with ethidium bromide and photographed under UV illumination. 
     8. Construction of CTGF Expression Plasmid 
     The cloning process of CTGF expression plasmid was described previously. Briefly, total RNA was extracted from the lung adenocarcinoma cell line, CL1-0, and CTGF cDNA was cloned and amplified by RT-PCR with the primers of SEQ ID NO. 9 and SEQ ID NO. 10 (PubMed Accession number: XM-037056), and subcloned into a pcDNA3/V5-His TOPO TA vector (Invitrogen, San Diego, Calif.) in the sense or the antisense direction. The CTGF expression plasmid was transiently or stably transfected into the colorectal cancer cell lines in vitro. 
     9. Stable Transfection Screening 
     Three microgram of the purified plasmid DNA was transfected into HCT116 cells and 3 μg AS-CTGF plasmid was transfected into HT29 cells by the TransFast™ transfection reagent (Promega, Madison, Wis.). After 24 hours of transfection, gentamycin (G418; Life Technologies, Inc.) was used to screen the stable transfectants. Thereafter, the selection medium was refleshed for every three days. After a two-week selection in G418, clones of resistant cells were isolated and allowed to proliferate in 100 μg/ml G418-containing medium. Integration of the transfected plasmid DNA was confirmed by RT-PCR and western blotting analysis. 
     10. Research of Colon Cancer Metastasis of Severe Combined Immunodeficiency (SCID) Mice 
     HCT116 cells were harvested in 0.25% of trypsin-PBS-EDTA, washed with PBS and then resuspended in PBS at 1 million cells per 200 μl. One million HCT116 cells were injected intraperitoneally into 5 week-old female SCID mice, and the mice were divided into three groups. 
     Group I was the control group, and Group II was the co-treatment group. After injecting with HCT116 cells, mice in the Group I and Group II were immediately injected with the equal volumes of dimethyl sulfoxide (DMSO) and the recombinant CTGF (rCTGF, 1.5 mg/kg) respectively. Subsequently, the abovementioned step were continued once per two days for 14 days. 
     Group III was the post-treatment group. After the mice in the post-treatment group were injected with HCT116 cells for 3 days, rCTGF was injected therein every day for 7 days. 
     Mice were euthanized when they experienced a ˜10% loss in body weight or if they appeared ill. Postmortem examinations included sectioning of kidney, lung, and liver that were stained with hematoxylin and eosin (H&amp;E) followed by examination for tissue toxicity/damage by an experienced pathologist who was blinded to therapy. 
     11. Statistic Analysis 
     A comparison of the background data was carried out between the low-CTGF expression group and the high-CTGF expression group. The scale variables (expressed as mean±standard deviation) was proceeded by a Mann-Whitney test, and the nominal variables were proceeded by a Fisher&#39;s exact test. The analysis of the survival rate and recurrence data was done by the Kaplan-Meier method, and the Kaplan-Meier curves were compared by a log-rank test. 
     EXPERIMENTAL RESULTS 
     1. Suppression of Peritoneal Metastasis by CTGF in SCID Mice 
     It was hypothesized that CTGF, a metastasis-suppressor, in human colorectal cancer may act to cancer cell adhesion which is the key step of peritoneal metastasis in the present invention. To prove this hypothesis, the abundantly-CTGF-expressed HCT116/CTGF-M transfectant and HCT116/Neo cell line (the control group) for intra-abdominal cancer seeding were set up in the present invention. 
     Please refer to  FIG. 1 , which is an electrophoresis diagram of CTGF mRNA and CTGF protein expression in HCT116/CTGF-M transfectant and HCT116/Neo cell line of the present invention. From the result in  FIG. 1 , it was known that the CTGF-transfected HCT116/CTGF-M cell line really could abundantly express CTGF as comparing to HCT116/Neo cell line. 
     Please refer to  FIGS. 2-I  to  2 -IV, which are the peritoneal tumor distributions of dissection after injecting HCT116/CTGF-M and HCT116/Neo cell lines respectively into SCID mice for eight weeks. After injecting HCT116/CTGF-M and HCT116/Neo cell lines for five weeks (before dissection), the phenomenon that peritoneal tumor protrusion leads to the enlarged abdominal circumference was obvious (data not shown). In  FIGS. 2-I  and  2 -II, HCT116/Neo transfectant was injected into the SCID mice, and the distributions of hemo-peritoneum and many nodules could be found after dissection. In  FIGS. 2-III  and  2 -IV, HCT116/CTGF-M transfectant was injected into the SCID mice, and the phenomenon of hemo-peritoneum and the distribution of nodules were significantly decreased as comparing to the control group. 
     Table 1 is the result of peritoneal seeding of injecting HCT116/Neo and HCT116/CTGF-M transfectants respectively into SCID mice. Among this, the sample number of SCID mice was 10, and the diaphragm seeding distribution, the phenomenon of local bowel invasion and nodule number were determined respectively. The result in Table 1 is shown that the abundantly-expressed CTGF could substantially inhibit the diaphragm seeding distribution, bowel invasion and the number of tumor seeding. 
     
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results of peritoneal seeding of HCT116/Neo and 
               
               
                 HCT116/CTGF-M transfectants respectively in SCID mice 
               
             
          
           
               
                   
                 Peritoneal seeding 
               
             
          
           
               
                   
                 Diaphragm 
                 Local bowel 
                   
               
               
                   
                 seeding #   
                 invasion #   
                 Nodule count &amp;   
               
             
          
           
               
                   
                 Involved 
                 P- 
                 Involved 
                 P- 
                 Median No. 
                   
               
               
                   
                 No. 
                 value 
                 No. 
                 value 
                 (range) 
                 P-value 
               
               
                   
                   
               
             
          
           
               
                 HCT116/ 
                 6/10 
                 0.057 
                 7/10 
                 0.070 
                 29 (23-38) 
                 0.0001 
               
               
                 Neo 
               
               
                 HCT116/ 
                 1/10 
                   
                 2/10 
                   
                 10 (7-13)  
               
               
                 CTGF-M 
               
               
                   
               
               
                   # Fisher&#39;s exact test; 
               
               
                   &amp; Mann-Whitney test 
               
             
          
         
       
     
     Please refer to  FIG. 3 , which is a relationship diagram showing the proportion of survival and time (days) after injecting HCT 116/Neo and HCT116/CTGF-M transfectants respectively to the SCID mice. From the result in  FIG. 3 , it could be found that the survival rate of the HCT116/CTGF-M-injected SCID mice was higher than that of the HCT116/Neo-injected SCID mice. The average survival days of the HCT116/CTGF-M-injected SCID mice were 58 days; however, the average survival days of the HCT116/CTGF-M-injected SCID mice were 81.8 days (p=0.001). Therefore, in accordance with the above-mentioned data, it was supposed that the abundantly-expressed CTGF could effectively inhibit the peritoneal dissemination progress of colorectal cancer, and the abundantly-expressed CTGF was beneficial in increasing the survival rate in vivo. 
     Subsequently, in order to research whether the growth of HCT116 colon cancer cells could be inhibited by rCTGF in the present invention, the mice were divided into three groups. The first group was the control group. After one million HCT116 cells were injected into the peritoneal cavity of female SCID mice, DMSO was injected thereinto for every two days for 14 days. The second group was the co-treatment group. After one million HCT116 cells were injected into the peritoneal cavity of female SCID mice, 1.5 mg/kg rCTGF was injected thereinto immediately (once per two days for 14 days), wherein the purpose of co-treatment group was mainly in simulating the direct intraoperative situation with free cancer cells on the peritoneal surface. The third group was the post-treatment group. After one million HCT116 cells were injected into the peritoneal cavity of female SCID mice, rCTGF was injected thereinto everyday from days 3 to 9, wherein the purpose of the post-treatment group was to simulate the clinical situation of the early-stage microscopic-transplanted surgery patients. 
     Please refer to  FIGS. 4-I  to  4 -IX, which are the dissection diagrams showing the effect of peritoneal dissemination while injecting rCTGF into the SCID mice of control, co-treatment and post-treatment groups. In  FIGS. 4-I  to  4 -III, all SCID mice were moribund within 40 days, and the increasing abdominal circumference was measured obviously. Bloody ascites were noted during dissecting the abdominal cavity and numerous peritoneal nodules were found. In the co-treatment ( FIGS. 4-IV ,  4 -V and  4 -VI) and the post-treatment ( FIGS. 4-VII ,  4 -VIII and  4 -IX) groups, the abdominal circumference of mice did not increase obviously, and the situations of the ascites and the peritoneal nodules in the peritoneal cavity were improved significantly after the mice in co-treatment group and the post-treatment groups were dissected as comparing to the control group, wherein the peritoneal cavities of three mice in the co-treatment group and one mouse in the post-treatment group did not have tumor dissemination (data not shown). 
     Please refer to  FIGS. 5(A) and 5(B)  respectively, which are the quantitative results of the abdominal circumference and body weight of mice in the control, co-treatment and post-treatment groups. The quantitative results in  FIGS. 5(A) and 5(B)  were corresponding to the results in  FIGS. 4-I  to  4 -IX. 
     Please refer to  FIG. 6 , which is the quantitative result of peritoneal nodules of mice in the control, co-treatment and post-treatment groups. The number of the peritoneal nodules in the mice were significantly decreased in the co-treatment and post-treatment groups as comparing to the control group. 
     In summary, the expression of CTGF indeed plays an important regulation role in the mechanism of peritoneal metastasis. 
     2. Inhibition of CTGF to Adhesion of Colon Cancer Cells 
     In order to clarify the playing role of CTGF in the adhesion of colon cancer cells, firstly, the expressions of CTGF in four different human colon carcinoma cell line (HCT116, LoVo, HT-29 and Caco-2) are determined by RT-PCR and Western blot. 
     Please refer to  FIG. 7 , which is the expressions of CTGF mRNA and CTGF protein and the cellular adhesion ability of CTGF in HCT116, LoVo, HT-29 and Caco-2 cell lines. The result was shown that HCT116 cells had the lowest CTGF mRNA and protein expressions and the highest cellular adhesion ability. As comparing to Caco-2 cells, Caco-2 cells had the highest CTGF mRNA and protein expressions and the lowest cellular adhesion ability. 
     The aforementioned experimental results using HCT116/Neo and HCT116/CTGF-M transfectants have been proved that the CTGF expression indeed have the effect to influence the peritoneal metastasis. Furthermore, the HCT116 transfectants (HCT116/CTGF-4 and HCT116/CTGF-18) with different CTGF expressions were screened in the present invention. Please refer to  FIG. 8 , which is the CTGF expressions and the cellular adhesion abilities of HCT116/Neo, HCT116/CTGF-M, HCT116/CTGF-4 and HCT116/CTGF-18 transfectants. Comparing with the control group (HCT116/Neo), the result was proved that CTGF expression represented the inverse correlation with the adhesion ability. 
     Please refer to  FIG. 9 , which is the influence of adhesion ability that different doses of the CTGF expression plasmid transiently transfects to HCT116 cells. The result was shown that different doses of CTGF would influence the adhesion ability of HCT116 cells. The higher CTGF dose made the worse adhesion ability, and the lower CTGF dose made the better adhesion ability. 
     Please refer to  FIG. 10 , which is the function of different doses of CTGF neutralizing antibody antagonizing the stable CTGF expression in HT29 cell line. The result was shown that the adhesion of HT29 cells would be significantly increased along with the doses (1 to 3 μg) of neutralizing antibody. 
     Subsequently, the relationship between the CTGF expression and the cellular adhesion was discussed by transfecting with the antisense CTGF plasmid. Please refer to  FIG. 11 , which is the CTGF expression and the CTGF adhesion ability of HT29 cell lines transfected with different doses of anti-sense CTGF plasmid. The result was shown that the cells transfected with higher doses (0.5 to 3 μg) of anti-sense CTGF plasmid made the higher cellular adhesion. 
     3. CTGF Being a Predicted Marker of the Peritoneal Metastasis in Human&#39;s Colorectal Cancer (CRC) 
     The previous research (Lin et al., 2005) has been proved that the CTGF expression represents the inverse correlation with the lymph node metastasis of the colorectal cancer patients and the recurrence level of metastasis. Therefore, the possible relation between the CTGF contents of the primary tumor and the generated peritoneal carcinomatosis was further discussed in the present invention. 
     Please refer to  FIGS. 12-I  to  12 -IV, which are the most representative immunohistochemical staining in 136 colorectal cancer patient samples.  FIG. 12-I  was the representative diagram of Level 3, which had a very high CTGF expression in the basal membrane and cytoplasm of the well-differentiated epidermoid carcinoma.  FIGS. 12-II  and  12 -III were the representative diagrams of Level 0, and it could be found that the weak immune response existed in the poorly-differentiated colorectal cancer cells ( FIG. 12-II ) and their peritoneal dissemination nodules ( FIG. 12-III ).  FIG. 12-IV  was an immunohistochemical diagram of negative staining, which used IgG as the control group. Please refer to Table 2, which is the related pathological data of the clinical characteristics and CTGF expression diagrams of the immunohistochemical staining. In the analysis of 136 colorectal cancer patient samples, the low CTGF expression group occupied 55% (75/136) of the total samples and the high CTGF expression group occupied 45% (61/136) of the total samples. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Clinical and pathological characteristics for high and low CTGF 
               
               
                 expression in T3 and T4 colorectal cancer (CRC) 
               
             
          
           
               
                   
                 CTGF 
                   
               
               
                   
                 expression 
               
             
          
           
               
                   
                 Feature 
                 Number 
                 Low 
                 High 
                 P-value 
               
               
                   
                   
               
             
          
           
               
                   
                 Patients 
                 136 
                 75 
                 61 
                   
               
               
                   
                 Mean age (Year) 
                   
                 62.6 
                 62.1 
                 0.82 
               
               
                   
                 Sex 
                   
                   
                   
                 0.618 
               
               
                   
                 Male 
                 68 
                 33 
                 35 
               
               
                   
                 Female 
                 68 
                 42 
                 26 
               
               
                   
                 Tumor site 
                   
                   
                   
                 0.823 
               
               
                   
                 Right 
                 45 
                 23 
                 22 
               
               
                   
                 Left 
                 44 
                 25 
                 19 
               
               
                   
                 Rectum 
                 47 
                 27 
                 20 
               
               
                   
                 Tumor 
                   
                   
                   
                 0.007 
               
               
                   
                 Differentiation 
               
               
                   
                 Poor 
                 26 
                 21 
                 5 
               
               
                   
                 Moderate 
                 106 
                 52 
                 54 
               
               
                   
                 Well 
                 4 
                 2 
                 2 
               
               
                   
                 Stage 
                   
                   
                   
                 0.040 
               
               
                   
                 II 
                 47 
                 20 
                 27 
               
               
                   
                 III 
                 63 
                 36 
                 27 
               
               
                   
                 IV 
                 26 
                 19 
                 7 
               
               
                   
                 Lymph node 
                   
                   
                   
                 0.017 
               
               
                   
                 N0 
                 56 
                 24 
                 32 
               
               
                   
                 N1 
                 43 
                 24 
                 19 
               
               
                   
                 N2 
                 37 
                 27 
                 10 
               
               
                   
                 Intra-tumor 
                   
                   
                   
                 0.301 
               
               
                   
                 invasion 
               
               
                   
                 Present 
                 63 
                 38 
                 25 
               
               
                   
                 Absent 
                 73 
                 37 
                 36 
               
               
                   
                 CEA level (ng/ml) 
                   
                   
                   
                 0.582 
               
               
                   
                 3 
                 44 
                 23 
                 21 
               
               
                   
                 &gt;3 
                 92 
                 52 
                 40 
               
               
                   
                 Synchronous 
                   
                   
                   
                 0.030 
               
               
                   
                 peritoneal seeding 
               
               
                   
                 Present 
                 35 
                 25 
                 10 
               
               
                   
                 Absent 
                 101 
                 50 
                 51 
               
               
                   
                 Metachronous 
                   
                   
                   
                 0.001 
               
               
                   
                 peritoneal seeding 
               
               
                   
                 Present 
                 21 
                 17 
                 4 
               
               
                   
                 Absent 
                 80 
                 33 
                 47 
               
               
                   
                   
               
             
          
         
       
     
     In the low CTGF expression group, the CTGF expression had no significant relations with the age, sex, tumor location, pre-operative CEA content and intra-tumor invasion of the patients. In the high CTGF expression group, 16% (10/61) patients had synchronous peritoneal metastasis; however, 33% (25/75) patients had the phenomenon of synchronous peritoneal metastasis (p=0.030) in the low CTGF expression group. 
     From the result in Table 2, it could be known that the status of tumor differentiation, clinical level and lymph node metastasis significantly related to the CTGF expression. (p-values were 0.007, 0.040 and 0.017 respectively). 
     Furthermore, from the result in Table 2, it could be known that the patient samples without peritoneal metastasis diagnosed in the early surgery were 101, wherein 50 persons had low CTGF expression and 51 had high CTGF expression. The traces of the follow-up recurrence ( FIG. 13 ) and survival rate ( FIG. 14 ) were proceeded in accordance with this feature. From the comparison result in  FIG. 13 , it could be known that the time from post-operation to recurrence in the high CTGF expression group was significantly higher than that in the low CTGF expression group (p&lt;0.001). From the result in  FIG. 14 , the significant difference (p&lt;0.001) of the survival rate in the high and low CTGF expression patients could be known. Therefore, the above-mentioned clinical data have been proved that CTGF was the predictable marker of synchronous or non-synchronous peritoneal metastasis in the colorectal cancer patients. 
     In summary, adhesion ability of cancer cells on the peritoneum plays a considerable important key role in the peritoneal metastasis of the colorectal cancer. The present invention is first proved that CTGF not only can be the biomarker in predicting the recurrence of peritoneal metastasis, but also can be the another candidate therapy for the peritoneal cancer dissemination of the colorectal cancer patients. 
     In conclusion, the clinical data of immunohistological staining of the present invention have been proved that the probability of synchronous and non-synchronous peritoneal metastasis of the colorectal cancer which the lower CTGF content patients suffer is higher than that of which the higher CTGF content patients suffer. In addition, CTGF expressions in different colorectal cancer cell lines represents the inverse correlation with their adhesion ability, and the variances of CTGF expression affect the adhesion abilities of human colorectal cancer cell lines directly. Therefore, whether cancer patients possess the high risk of peritoneal metastasis is identified by determining the CTGF expression of primary colorectal cancer, and the phenomenon of peritoneal carcinomatosis of cancer patients is prevented and treated by administrating CTGF. 
     Furthermore, the abovementioned experimental results can be used in the present invention so as to provide a method for screening the inhibitor of peritoneal dissemination. First, a compound is bound with the cells expressing CTGF in the peritoneal tissue, and CTGF has inhibition ability of cancer cell adherence in the peritoneal tissue. Subsequently, the CTGF expression in the peritoneal tissue is determined. When the CTGF expression in the peritoneal tissue is higher than that in the cells without binding with the compound, the compound has potential for positively regulating CTGF. 
     While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.