CN114010791B - Application of MyD88-IFN gamma R1 dimer as target in preparation of medicine for preventing colon cancer - Google Patents
Application of MyD88-IFN gamma R1 dimer as target in preparation of medicine for preventing colon cancer Download PDFInfo
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- CN114010791B CN114010791B CN202111369450.4A CN202111369450A CN114010791B CN 114010791 B CN114010791 B CN 114010791B CN 202111369450 A CN202111369450 A CN 202111369450A CN 114010791 B CN114010791 B CN 114010791B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57419—Specifically defined cancers of colon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of MyD88-IFN gamma R1 dimer serving as a target spot in preparation of a medicine for preventing colon cancer. The invention explores the mechanism of reversing the chronic colonitis to the colon cancer caused by the high-fat diet by using MyD88-IFN gamma R1 dimer from the cellular and overall level, and clearly shows that medicines such as myricetin inhibit the conversion of intestinal inflammation to the colon cancer by up-regulating the generation of Myd88-IFN gamma R1 dimer, and reverse the occurrence of the colon cancer, so that the Myd88-IFN gamma R1 dimer can be used as an action target for screening medicines for preventing the colon cancer.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of MyD88-IFN gamma R1 dimer serving as a target spot in preparation of a medicine for preventing colon cancer.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
High-fat diets can cause chronic inflammation of the colon, while chronic colitis is one of the important high-risk factors for colon cancer. Colon cancer induced by inflammation is a "inflammation-cancer" sequence of colon cancer which undergoes chronic inflammation-inflammatory hyperplasia-low grade, highly atypical hyperplasia, inhibiting colon inflammation, preventing inflammation-cancer transformation, and can greatly reduce incidence of colon cancer, which plays an important role in preventing and treating colon cancer. The high-fat diet is extremely easy to cause chronic colitis and colon canceration, and the prevention of inflammation is a key for preventing and treating colon cancer.
IFN-gamma and its receptor subunit (IFN gamma R1) have been found in the prior art to be closely related to colon cancer onset and prognosis, but the key node and specific mechanism of colitis-cancer transformation caused by high-fat diet are not clear, and there is no sufficient evidence and effective action targets for the development of medicines for preventing and treating colitis/cancer transformation.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides application of MyD88-IFN gamma R1 dimer serving as a target point in preparing a medicament for preventing colon cancer, the invention explores a mechanism of reversing chronic colitis into colon cancer caused by high-fat diet by using MyD88-IFN gamma R1 dimer from a cell and overall level, and makes clear that medicines such as myricetin inhibit the conversion of intestinal inflammation into colon cancer by up-regulating Myd88-IFN gamma R1 dimer generation, and reverse colon cancer, so that Myd88-IFN gamma R1 dimer can be used as an action target point for screening medicaments for preventing colon cancer.
The first aspect of the invention provides the use of MyD88-IFNγR1 dimer as a target in the manufacture of a medicament for the prevention of colon cancer.
In a second aspect, the invention provides the use of a kit for detecting ifnγr1—myd88 dimer in the manufacture of a product for the prevention of colon cancer.
In a third aspect the invention provides the use of MyD88 as a "molecular switch" to competitively bind IFNγR1 to form a dimer against tumour formation.
In a fourth aspect the invention provides a medicament for preventing colon cancer, which medicament is capable of upregulating ifnγr1—myd88 dimer levels.
The fifth aspect of the invention provides an application of a MyD88-IFNγR1 dimer small molecule promoter in preparing a medicine for preventing colon cancer.
In a sixth aspect the invention provides the use of a substance for increasing the level of ifnγr1—myd88 dimer in the manufacture of a product for inhibiting the conversion of colitis to colon cancer.
In a seventh aspect, the invention provides use of a substance that increases the level of ifnγr1—myd88 dimer in the preparation of an animal model of colon cancer.
One or more embodiments of the present invention have at least the following beneficial effects:
according to the invention, from the cellular and overall level, by discussing the mechanism of reversing chronic colitis cancer transformation caused by high-fat diet and inhibiting colon cancer progression of myricetin, the cause and key nodes of colitis/cancer transformation caused by high-fat diet are searched, and MyD 88-IFNgamma R1 dimer is determined to be an action target point of a medicament for preventing and treating colitis/cancer transformation, so that a new target point is provided for screening a medicament for preventing colon cancer chemistry, and experimental and theoretical support is provided for in-depth study of pathogenesis of colon cancer.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows histopathological staining of mice at various stages of colitis carcinoma transformation and MyD88/IFNγR1 dimer levels.
FIG. 2 shows that anti-colon cancer drugs such as IFN-gamma (100 ng/mL), myricetin (50. Mu. nol/mL) and the like increase the interaction between MyD88 and IFN-gamma R1;
FIG. 3 shows that anti-colon cancer drugs such as IFN-gamma (100 ng/mL), myricetin (50. Mu. nol/mL) and the like increase the physical association between MyD88 and IFN-gamma R1 (magnification of 200);
FIG. 4 is a graph showing that interfering with MyD88/IFN- γR1 dimer formation can reduce IFN- γ anti-colon cancer effects. (A) MyD88 associates with IFN- γR1 via small molecule TIRAP to form dimers, and TIRAP-siRNA is used to reduce TIRAP expression, which reduces MyD88/IFN- γR1 dimer levels. (B) Inhibiting the down regulation of MyD 88/IFN-gamma R1 dimer level caused by TIRAP can reduce the anti-colon cancer effect of IFN-gamma.
FIG. 5MyD88/IFNγR1 dimer is involved in the modulation of IFN- γ/STAT1 signaling in colon cancer cells. (A) IFN-gamma treatment increased the physical link between MyD88 and IFN-gamma R1 in HT-29 cells by Co-IP analysis. Cell lysates were immunoprecipitated with anti-ifnγr1 antibodies and immunoprecipitates were immunoblotted with anti-MyD 88 and anti-TIRAP antibodies. Cell lysates were analyzed with anti-MyD 88, anti-ifnγr1 and anti-GADPH antibodies. (B) Westernblotting showed that TIRAP-siRNA was used to down-regulate levels of TIRAP in cells (< 0.01). (C) TIRAP knockdown reduced IFN-gamma induced increases in phosphorylated STAT.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As described in the background art, the key nodes and specific mechanisms of the prior art for inducing the colitis/cancer transformation by high-fat diet are not clear, and sufficient evidence and effective acting targets are not yet available for the development of medicines for preventing and treating the colitis/cancer transformation.
In order to solve the technical problems as above, the first aspect of the invention provides an application of MyD88-IFNγR1 dimer as a target in preparing a medicament for preventing colon cancer.
The invention discovers the molecular switch function of MyD88 for the first time, proposes IFN gamma R1-MyD88 dimer to participate in reversing the process of colonitis conversion caused by high-fat diet to colonic cancer, and simultaneously, the invention also proves that medicines such as myricetin can inhibit the process of colonitis conversion to colonic cancer by up-regulating the level of the IFN gamma R1-MyD88 dimer and inhibiting the downstream inflammatory signal of TLR/MyD88 and the Wnt/beta-catenin signal, thus the invention has important theoretical significance for deeply knowing the colonitis/cancerous conversion incidence essence caused by high-fat diet, and the MyD88-IFN gamma R1 dimer can be used as an effective new target for screening colon cancer chemopreventive medicines.
Further, the prevention of colon cancer is prevention of the transition from colitis to colon cancer.
Further, the use includes screening for agents that prevent colon cancer.
In a second aspect, the invention provides the use of a kit for detecting ifnγr1—myd88 dimer in the manufacture of a product for the prevention of colon cancer.
In a third aspect the invention provides the use of MyD88 as a "molecular switch" to competitively bind IFNγR1 to form a dimer against tumour formation.
In a fourth aspect the invention provides a medicament for preventing colon cancer, which medicament is capable of upregulating ifnγr1—myd88 dimer levels.
The fifth aspect of the invention provides an application of a MyD88-IFNγR1 dimer small molecule promoter in preparing a medicine for preventing colon cancer.
In a sixth aspect the invention provides the use of a substance for increasing the level of ifnγr1—myd88 dimer in the manufacture of a product for inhibiting the conversion of colitis to colon cancer.
Preferably, the product is an experimental reagent.
Further, the agents that increase the level of ifnγr1—myd88 dimer are agents that include RNA interfering molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, and that effect lentiviral infection or gene knockout against ifnγr1—myd88 dimer.
In a seventh aspect, the invention provides use of a substance that increases the level of ifnγr1—myd88 dimer in the preparation of an animal model of colon cancer.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Experimental protocol:
(1) Cell lines and drugs
HT-29 and HCT116 cells were obtained from a cell bank of the national academy of sciences of China (Shanghai, china) and cultured in RPMI-1640 medium (containing 10% (v/v) fetal bovine serum (Solarbio technologies Co., beijing) at 37℃in a humidified incubator of 5% CO2, human interferon-gamma (cyt-206) was purchased from Prospec (Isonensin, ind.) Myricetin (analytically pure) was purchased from sigma.
(2) Hematoxylin-eosin staining method (HE staining)
Sequentially placing paraffin sections into xylene/10 min-absolute ethanol/5 min-95% alcohol/5 min-90% alcohol/5 min-80% alcohol/5 min-70% alcohol/5 min-distilled water for washing, dewaxing to water. Placing the slices into Hematoxylin (Hematoxylin) for dyeing for 3-8min, washing with tap water, differentiating 1% hydrochloric acid alcohol for several seconds, washing with tap water, returning blue with 0.6% ammonia water, washing with running water, and dyeing cell nuclei. The sections were stained in Eosin (Eosin) stain for 1-3min and the cytoplasm was stained. Dewatering and sealing the slice, sequentially placing the slice into 95% alcohol/5 min-absolute alcohol/5 min-xylene/5 min for dewatering and transparency, taking out the slice from the xylene, slightly airing, sealing the slice with neutral resin, microscopic examination, and image acquisition and analysis.
(3) Multiple immunohistochemistry (mhic)
For mIHC, anti-MyD 88 (ab 133739, abcam), anti-IFNγR1 (ab 134070, abcam), anti-TIRAP (sc-166149,Santa Cruz) antibodies and opal four-color fluorescent IHC kit (NEL 810001KT, perkinemer, USA) were used. On the basis of single stained slides we optimized the order and concentration of the three antibodies and established a spectral library. Slides were dewaxed with xylene and ethanol and then extracted with microwaves for antigen. Tissues were incubated with 3% h2o2 (fresh) for 10 minutes at room temperature, blocked in blocking buffer for 10 minutes, then incubated with primary antibody, secondary HRP, and opal working solution (perkinemer, usa). Slides used DAPI (san crus, california) extended gold fade prevention reagent. The slide was scanned using a Nikon C1 confocal microscope (Nikon Japan). Positive cell numbers and average intensities were analyzed using ImageJ software (national institutes of health). The total number of positive cells was counted using the analytical particle tool in ImageJ. Details are described in a previous study.
(4) Cell transfection of siRNA
HT-29 cells were maintained in RPMI-1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum. The siRNA target sequence of TIRAP (integrated biotechnology solution, shanghai, china) was resuspended to prepare a 20mM solution according to the manufacturer's instructions. Cells were transfected by siRNA using Lipofectamine 2000 (damshittat Invitrogen, germany) according to the manufacturer's protocol.
(5) Co-immunoprecipitation (Co-IP)
HT-29 cells were plated at 4X 10 5 cell/mL density was seeded into 6-well plates and IFN-gamma (50 or 100 ng/mL) was added the next day. Cells were incubated with the medium and an equal amount of sterile distilled water was used as a control. Cells were collected after 72 hours of culture. Cells were suspended in 0.5ml of lysis buffer and lysates were separated by centrifugation at 4 ℃. Aliquots of the lysate (500. Mu.g protein) were mixed with 20. Mu.l of protein g-Sepharose (Amersham, piscataway, N.J.) for 2 hours and stirred intermittently at 4 ℃. After centrifugation, the supernatant was incubated with anti-ifnγr1 antibodies overnight at 4 ℃. anti-MyD 88 (sc-136970) antibodies were purchased from St.Joule, calif. U.S. Anti-interferon gamma R1 (10808-1-AP) antibodies were purchased from the Proteintech group (USA). The complex was adsorbed on protein G-agarose and then washed three times with lysis buffer. Immunoblot analysis was performed as described in immunoblot analysis. Details are described in a previous study.
(6) Immunoblot analysis
The total cellular protein was extracted, and the protein lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (merck milbo, bir card, ma). The membrane was then blocked with 5% milk and incubated with primary antibody overnight at 4 ℃ and then with secondary antibody for 1 hour at room temperature. Anti-phospho (phospho) -STAT1 (9167; cell signaling), anti-STAT 1 (9172; cell signaling) and anti-GADPH antibody (sc-365062,Santa Cruz Biotechnology) were used. Data were normalized to the previously reported GADPH levels. Antibodies bound to the membrane were visualized using enhanced chemiluminescence reagents (merck microwells) and densitometry was performed using a ChemiDoc xrs+ image analyzer (Bio-Rad Laboratories inc., hercules, CA, USA) and ImageJ software.
(7) Statistical analysis
Data are expressed as mean ± Standard Deviation (SD). Statistical analysis was performed on the data using Student t-test and one-way analysis of variance (ANOVA). All experiments were repeated at least three times, p <0.05 was considered statistically significant, including p <0.05, p <0.01, and p <0.001.
Experimental results:
1. confirm that high-fat diet inhibits IFNγR1-MyD88 dimer formation, activates TLR/Myd88 downstream inflammatory signals and Wnt/beta-catenin signals, and promotes inflammatory/cancerous transformation;
the method comprises the following steps:
(1) Building an animal model: feeding C57BL/6J-Apc with high fat feed (containing 69.85% basal feed, 20% lard, 0.15% cholesterol and 10% yolk powder per 100g high fat feed) Min/+ Mice promote Apc Min/+ Adenoma progression induced by intestinal inflammation in mice.
(2) Histopathological analysis: apc for normal feeding and high fat diet feeding Min/+ Histopathological analysis of the colonic adenoma and adenocarcinoma tissues of mice;
(3) Co-immunoprecipitation and Western blotting: apc for detecting normal feeding and high fat diet feeding Min/+ Analyzing the effect of high-fat diet on IFN gamma R1-MyD88 dimer level in normal colon tissue, colitis hyperplasia tissue and canceration tissue of the mice;
results:
specific results are shown in FIG. 1, which shows that high-fat diet can lead to transformation of mouse colitis, and that MyD88/IFN- γR1 dimer level decreases with increasing inflammatory and malignant levels during transformation of mouse colitis caused by high-fat diet.
2. IFN-gamma and myricetin up-regulating MyD 88/IFN-gamma R1 dimer
The levels of MyD88/IFNγR1 protein complex in IFN- γ treated HT-29 cells were studied using Co-IP and immunoblot analysis. Immunoblot analysis showed a faint band of MyD88 in ifnγr1 pellet of untreated control cells. However, higher amounts of MyD88 were detected in IFN- γ or myricetin treated cells (fig. 2). Thus, in HT-29 cells, the physical link between MyD88 and IFNγR1 increases with IFN- γ or myricetin stimulation. Consistent with the co-immunoprecipitation results, IFN-gamma or myricetin increased MyD 88/IFN-gamma R1 dimer levels.
3. Anti-colon cancer drugs such as IFN-gamma (100 ng/mL), myricetin (50. Mu. nol/mL) and the like increase the physical association (yellow) between MyD88 and IFN-gamma R1 (magnification. Times.200).
The method comprises the following steps: multiple immunohistochemistry (mhic)
For mIHC, myD88 (ab 133739), IFNγR1 (ab 134070), TIRAP (sc-166149) antibodies and the four-color fluorescent IHC kit (NEL 810001 KT) were used. On the basis of single stained slides we optimized the order and concentration of the three antibodies and established a spectral library. Slides were dewaxed with xylene and ethanol and then extracted with microwaves for antigen. At room temperature, the tissue was combined with 3%H 2 O 2 (fresh) incubation for 10 minutes, blocking in blocking buffer for 10 minutes, then incubation with primary antibody, secondary HRP and working solution, slide prolonged fading with DAPI. The slide was scanned using a Nikon C1 confocal microscope (Nikon Japan). Positive cell numbers and average intensities were analyzed using ImageJ software and counted.
FIG. 3 shows that IFN-gamma (100 ng/mL) or myricetin (50. Mu. nol/mL) increases MyD88 levels around cell membranes and increases co-localization of IFN-gamma R1 and MyD88 in cell membranes in fusion images, indicating that anti-colon cancer drugs such as IFN-gamma (100 ng/mL), myricetin (50. Mu. nol/mL) increase the physical association (yellow) between MyD88 and IFN-gamma R1 (magnification of 200).
4. Interfering with the small molecule TIRAP linked in the middle of MyD88/IFN- γR1 dimer can inhibit the formation of MyD88/IFN- γR1 dimer (IP group), thereby reducing the anti-colon cancer effect of IFN- γ (100 ng/mL) and myricetin (50 mu nol/mL).
The method comprises the following steps: using siRNA to down regulate small molecule TIRAP level in colon cancer cells, using Co-immunoprecipitation method (Co-IP method) and western blotting (WB method) to detect the effect of TIRAP to knock out MyD 88/IFN-gamma R1 dimer level in colon cancer cells, using CCK8 method to detect the effect of IFN-gamma (100 ng/mL), myricetin (50 mu nol/mL) and other anti-colon cancer drugs and TIRAP inhibitor combined with proliferation of colon cancer cells;
as shown in FIG. 4, inhibiting TIRAP levels inhibited MyD88/IFN- γR1 dimer formation and reduced the anti-colon cancer effects of IFN- γ (100 ng/mL), myricetin (50 μ nol/mL).
5. MyD88/IFNγR1 protein complex involved in the modulation of CLC cell IFN- γ/STAT1 signaling
The present invention found that IFN-gamma treatment upregulated the physical link between MyD88 and IFN-gamma R1 (FIG. 5A). To reduce IFN-gamma-induced MyD 88/IFN-gamma R1 protein complexes, TIRAP siRNA was used to down-regulate TIRAP expression. TIRAP siRNA reduced TIRAP levels in HT-29 cells compared to negative control (FIG. 5B). Next, we examined the effect of IFN- γ and IFN- γ in combination with TIRAP siRNA on classical signal protein STAT1 and phosphorylated STAT. We found that IFN-gamma treatment induced STAT activation with no effect on STAT1 expression. Furthermore, TIRAP gene knockout reduced IFN-gamma-induced increases in phosphorylated STAT and inhibited the effect of IFN-gamma on STAT activation (FIG. 5C). Thus, we can conclude that the MyD88/IFNγR1 protein complex is involved in the modulation of IFN- γ/STAT1 signaling in colon cancer cells. The MyD 88/IFN-gamma R1 protein complex related signaling has significant synergistic regulation with IFN-gamma.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
- Use of IFN- γ or myricetin to promote up-regulation of MyD88 expression in colon cancer cells in an in vitro environment, to increase the physical association between MyD88 and ifnγr1, said use not comprising diagnosis and treatment of a disease.
- 2. The use according to claim 1, wherein the effective dose of IFN- γ is 100 ng/mL; the effective dose of the myricetin is 50 mu mol/mL.
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| WO1987005518A1 (en) * | 1986-03-17 | 1987-09-24 | Schering Corporation | Treatment of cancers with gamma interferon |
| CN106075453A (en) * | 2016-07-12 | 2016-11-09 | 中国医学科学院基础医学研究所 | A kind of anti-tumor medicinal preparation combination |
| CN107721959A (en) * | 2016-08-12 | 2018-02-23 | 青岛海洋生物医药研究院股份有限公司 | Myricetin derivative and preparation method, and its application in treatment colitis, the conversion of preventing and treating colitis cancer and treatment colorectal cancer |
| CN113368217A (en) * | 2020-03-09 | 2021-09-10 | 四川大学华西医院 | Application of IFN-gamma in preparation of anti-tumor auxiliary medicine |
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| WO1987005518A1 (en) * | 1986-03-17 | 1987-09-24 | Schering Corporation | Treatment of cancers with gamma interferon |
| CN106075453A (en) * | 2016-07-12 | 2016-11-09 | 中国医学科学院基础医学研究所 | A kind of anti-tumor medicinal preparation combination |
| CN107721959A (en) * | 2016-08-12 | 2018-02-23 | 青岛海洋生物医药研究院股份有限公司 | Myricetin derivative and preparation method, and its application in treatment colitis, the conversion of preventing and treating colitis cancer and treatment colorectal cancer |
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