CN108653297A - Application using nucleus inner tissue protease L as the ganoderma lucidum ketone glycol of target spot in pharmacy - Google Patents
Application using nucleus inner tissue protease L as the ganoderma lucidum ketone glycol of target spot in pharmacy Download PDFInfo
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Abstract
The invention discloses a kind of applications using nucleus inner tissue protease L as the ganoderma lucidum ketone glycol of target spot in pharmacy.By the way that ganoderma lucidum ketone glycol, inhibiting tumor cell proliferation experiment and Apoptosis show with cell cycle experiment in vitro, ganoderma lucidum ketone glycol is by CL activity in inhibition colorectal cancer cells core to reduce the truncated CUX1 factors (activated form), then the expression of cyclin Cyclin E2 is lowered, finally colorectal cancer cells is made to rest on phase cell cycle S, to realize the effect for inhibiting growth of cancer cells.Therefore, ganoderma lucidum ketone glycol can be used for preventing or treat and the relevant disease of colorectal cancer, including the carcinoma of the rectum, colon cancer and the constipation caused by the carcinoma of the rectum or colon cancer, have blood in stool, abdominal pain, abdominal cramps, colon-cancer cell infiltration and transfer etc. relevant diseases, be suitable for popularization and application.
Description
Invention field
The invention belongs to field of medicaments, and in particular to a kind of application of ganoderma lucidum ketone glycol in anti-colorectal cancer.
Background technology
Colorectal cancer includes colon cancer and the carcinoma of the rectum, refers to being happened at caecum, the colon ascendens, transverse colon, colon descendens, second shape knot
The malignant tumour of intestines, rectum is one of most common malignant tumor of digestive tract.World Health Organization's latest data shows colorectal cancer
Death toll is located at number of cancer deaths's third position, and the annual whole world has more than 1,200,000 patients and is diagnosed as colorectal cancer, and has super
It crosses 700,000 patients and directly or indirectly dies of colorectal cancer.There were significant differences for its incidence in each department, usual male's colorectal cancer
Incidence be higher than women.In addition, the incidence of colorectal cancer can increase with the increase at age.Colorectal cancer is also most in China
One of common malignant tumour, in 2012 in 1,360,000, the whole world colorectal cancer of diagnosis, Chinese neopathy number of cases reaches 250,000
Example, accounts for the 18.6% of global colorectal cancer new cases, is the most country of the annual new cases of global colorectal cancer.
Colorectal cancer originating from mesenchymal tissue under colorectal mucosa epithelium or mucous membrane, Molecular pathogenesis be heredity or environment because
Gene mutation caused by element.There is epidemiological study discovery：Social Development State, life style and diet structure and colorectal cancer are close
Cut phase is closed.The treatment of colorectal cancer at present is based on excision of performing the operation, in conjunction with the complex treatment of radiotherapy, chemotherapy or targeted therapy.Art
5 years survival rates and colorectal cancer pathological staging are closely related afterwards, the postoperative 5 years survival rates of Early cancer up to 80% or more, and in
Late period person is only 40%-10%.The main purpose of chemotherapy is to prevent and reduce recurrence and transfer, to improve operative treatment
Late result, can be divided into operation consent, operation and postoperative chemotherapy.Main chemotherapeutics is with 5-fluor-uracil (5- at this stage
Fu further include thering is Calciumlevofolinate (LV), capecitabine, oxaliplatin, iritican and Biological target therapy medicine shellfish to cut down pearl based on)
Monoclonal antibody etc..5-Fu is only 20% or so to the effective percentage of colorectal cancer, and certain effective percentage can be improved in scheme of combination drug therapy.It is anti-at present
The weakness of colorectal cancer chemotherapeutics is efficient not high, and selectivity is low, and toxic side effect is big.It is big to be such as combined 5-FU and LV treatment late periods
The chemotherapy regimen of intestinal cancer can cause the toxic side effects such as gastrointestinal reaction and bone marrow suppression, and nervous system can be caused using oxaliplatin
Toxicity, the treatment advanced CRC standard scheme of 5-FU and LV is combined using iritican can cause cholinergic syndrome and tardy
Property diarrhea etc..Using the bioactivity of vegf blocker (VEGF) or epidermal growth factor (EGF) as the list of target spot
Clonal antibody class drug is other than toxicity problem, and there is also the threats failed because downstream signaling pathway molecule mutates.It seeks
It looks for new drug target and develops the anti-large intestine cancer drug of high efficiency low toxicity side effect, be still very urgent in this field
It needs.
Cathepsin L (Cathepsin L, CL) is one of cysteine proteinase family member, and size is 333 ammonia
Base acid, is widely present in the various normal structures of human body and tumour cell, is the kDa major secretory protein of lysosome.Its main work(
It can be protein degradation, hydrolyze certain precursor proteins (proenzyme and prohormone) and generate its active form, or activate other proteolysis
Enzyme system, to participate in a variety of physiological activities of body.Such as：The submission of antigen, the generation of sperm and ovum, bone matrix drop
Solution, the maturation of central nervous system and development etc..Under pathological state, due to transcription, translation, modification, positioning, maturation, pH variations
And the exception that interacts between inhibitor and a variety of disease developments of wide participation, such as infiltration and transfer, the joint of tumour
Inflammation, osteoporosis, alzheimer ' nurse disease, multiple sclerosis and other chronic inflammation diseases etc..Recent domestic is many
Scholar studies CL, finds it in Several Kinds of Malignancy such as prostate cancer, melanoma, gastric cancer, colorectal cancer, cancers of pancreas
The high expression of middle presentation.
Research finds that CL has many different forms in the cell, and it was found that CL can enter in nucleus by cutting
The DNA binding abilities of nuclear factor CUX1 precursors enhancing CUX1 are cut to promote the cell cycle progression of cell.Also there is research
It was found that the CL enzymatic activitys in the cell cycle progression dependent cells core of colorectal cancer cells.It is related to 186 patients with colorectal cancer at one
Histotomy research in find, the process of CL levels and colorectal cancer in colorectal cancer cells core and be proportionate more afterwards.To the greatest extent
Managing these phenomenons prompts endonuclear CL and colorectal cancer closely related, using CL in nucleus as the anti-large intestine cancer drug of target spot still
It has not been reported.Thus, it is found that the exploitation of CL inhibitor treatment new drug highly selective to colorectal cancer has significantly in colorectal cell core
Meaning.
Ganoderma lucidum is Basidiomycetes Polyporaceae Ganoderma fungi, is traditional Chinese medicine treasure, has long medicinal history in China.
Ganoderma lucidum ketone glycol (Ganodermanondiol, GMD) is resisted big from a kind of isolated triterpenoid of ganoderma lucidum fruitbody
The activity of intestinal cancer has not been reported.Once there is research and utilization mtt assay to stablize (MSS) type colon cancer cell in gene microsatellite many years ago
The anti-colorectal cancer activity research of ganoderma lucidum ketone glycol is carried out on SW480 and SW1116 cells, but it is bright not find that ganoderma lucidum ketone glycol has
Aobvious activity.In our study, we use CCK-8 cytotoxicities that are sensitiveer and stablizing with proliferation assay to including base
Because the colorectal cancer cells HCT116 of microsatellite instability (MSI) type has found into anti-colorectal cancer activity research and action target spot analysis,
Ganoderma lucidum ketone glycol has colorectal cancer cells the inhibiting effect of highly significant, and evidence shows that the mechanism that it plays a role is to pass through
Cathepsin C L in selectively targeted colorectal cancer cells core is to inhibit the cell cycle of cancer cell to reach anticancer effect.This
A discovery has the shortcomings that overcome existing drug specificity not high and the anti-colorectal cancer of the big potentiality of toxic side effect for further researching and developing
New drug is of great significance.
Invention content
The present invention provide a kind of ganoderma lucidum ketone glycol prepare prevent or drug of the treatment with the relevant disease of colorectal cancer in
Using being preferably operable to the carcinoma of the rectum, colon cancer and the constipation caused by the carcinoma of the rectum or colon cancer, have blood in stool, abdominal pain, abdomen
A variety of relevant diseases such as spasm, colon-cancer cell infiltration and transfer.
Further, the present invention also provides a kind of for preventing or treating the pharmaceutical composition with the relevant disease of colorectal cancer
Object, it includes ganoderma lucidum ketone glycol or its pharmaceutically acceptable salt and at least one pharmaceutically acceptable carrier and/or dilutions
Agent, wherein ganoderma lucidum ketone glycol or its pharmaceutically acceptable salt inhibit the IC50 of colorectal cancer cells less than 10 μM, preferably shorter than 2.5
μM。
The pharmaceutical composition of the present invention can be used as administered orally or parentally (such as through intravenous, subcutaneous or intramuscular
Administration) dosage form.The dosage form of the oral medication be tablet, sustained release tablets, controlled release tablet, pastille, hard or soft capsule,
Aqueous or oil suspension, emulsion, dispersible powder or granule, syrup or elixir, dripping pill, pellet or oral administration solution, institute
The dosage form for the parenterai administration stated is the aqueous or oily solution of sterilizing, aseptic powdery, liposome, emulsion, microemulsion, receives
Rice milk agent or micro-capsule.
The pharmaceutical composition of the present invention can be obtained using customary pharmaceutical excipients well known in the art by conventional method
.So composition for oral use can contain for example one or more colorants, sweetener, corrigent and/or prevent
Rotten agent.
Suitable pharmaceutically acceptable excipient for tablet formulation includes for example, inert diluent such as lactose, sodium carbonate, phosphorus
Sour calcium or calcium carbonate；Granulation and disintegrant such as cornstarch and alginic acid；Adhesive such as starch；Lubricant such as stearic acid
Magnesium, stearic acid or talcum powder；Preservative such as ethyl-para-hydroxybenzoate or propyl ester and antioxidant, such as ascorbic acid.Tablet
Preparation can be uncoated, and coating can also be used follow-up in gastrointestinal tract to change its calving disaggregation and active constituent
Absorption, or its stability and/or appearance are improved, in any case, conventional coating agents known to the field can be used
And method.
The composition being administered orally can be the form of hard gelatin capsule, wherein active constituent and inert solid diluent example
Such as the mixing of calcium carbonate, calcium phosphate or kaolin or soft gelatin capsules, wherein active constituent and water or oily such as peanut
Oil, atoleine or olive oil mixing.
Aqueous suspension typically contains the active constituent of micronized form and one or more suspending agents, and the suspending agent is for example
For sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methyl cellulose, mosanom, Polyvinyl-pyrrolidone, tragacanth
And Arabic gum；Dispersion or wetting agent, such as (such as polyoxyethylene is hard for the condensation product of lecithin or alkylene oxide and aliphatic acid
Resin acid ester) or ethylene oxide and long-chain fatty alcohol condensation product, such as 17 oxy ethylene cetanols or ethylene oxide
With the condensation product derived from aliphatic acid and the partial ester of hexitol, such as polyoxyethylene sorbitan monooleate or epoxy
Ethane and the condensation product derived from aliphatic acid and the partial ester of hexitan, such as polyethylene sorbitan monooleate.
It is (such as anti-that aqueous suspension can also contain one or more preservatives (such as ethyl-para-hydroxybenzoate or propyl ester), antioxidant
Bad hematic acid), colorant, corrigent, and/or sweetener (such as sucrose, saccharin and aspartame).
It can be by the way that active constituent be suspended in vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or mineral
Oil suspensions are prepared in oily (such as atoleine).Oil suspensions can also contain thickener such as beeswax, solid paraffin
Or cetanol.Sweetener and corrigent as described above can be added to provide palatable oral preparation.These compositions can be with
By the way that antioxidant such as ascorbic acid is added come anti-corrosion.
Fit through be added water be made the dispersible powder of aqueous suspension and granule typically contain active constituent and
Dispersant or wetting agent, suspending agent and one or more preservatives.Dispersion appropriate or wetting agent and suspending agent are as described above.
There are other excipient such as sweetener, corrigent and colorants.
Pharmaceutical composition of the present invention can also use the form of oil-in-water emulsion.Oil phase can be vegetable oil, such as olive
Olive oil or peanut oil either mineral oil such as atoleine or their mixture.Emulsifier appropriate can be for example,
Natural gum such as Arabic gum or tragacanth, natural phospholipid such as soybean lecithin, and it is derived from aliphatic acid and hexitol
The ester or partial ester (such as E494) of acid anhydride and the condensation product of the partial ester and ethylene oxide, such as
Polyoxyethylene sorbitan monooleate.Emulsion can also contain sweetener, corrigent and preservative.
Syrup and elixir can be prepared with sweetener (such as glycerine, propylene glycol, D-sorbite, aspartame or sucrose),
And it can also contain moderator, preservative, corrigent and/or colorant.
Described pharmaceutical composition can also be that Injectable sterile is aqueous or the form of Oil suspensions, can be according to known
Method is prepared using one or more suitable dispersions or wetting agent and suspending agent, these reagents are as described above.Aseptic injection
Preparation can also be that the Injectable sterile in the acceptable diluent of nontoxic parenteral or solvent is aqueous or Oil suspensions, example
Solution such as in 1,3-BDO.
Can according to the difference of treated host and specific administration route, come determine mixed with one or more excipient with
Prepare the amount of single dose form active constituent.For example, for typically containing such as 0.5mg-2g to the preparation of oral administration in human
Activating agent and appropriate and convention amount excipient (5-98% for accounting for about composition gross weight).In unit formulation generally containing about
There is the active constituent of 1mg-500mg.
When ganoderma lucidum ketone glycol of the present invention is used to prepare treatment or prevents the purposes of colorectal cancer, for cancer cell strain
Effective dose is 0.01-10 μM, is further preferably 0.8-5 μM.
Ganoderma lucidum ketone glycol of the present invention can be applied in monotherapy, or with other one or more substances and/or
Indication treatment joint.Such combination therapy be can by each therapeutic component while, sequence or separate administration side
What formula reached.Treatment simultaneously may be used single tablet or use separated tablet form.For example, in the treatment of colorectal cancer,
It can also include the treatment of following main species：Antitumor agent, immunopotentiator.
Description of the drawings
Fig. 1 ganoderma lucidum ketone diol structure figures；
Fig. 2 ganoderma lucidum ketone glycol (GMD) carries out external inhibition growth of cancer cells activity rating；
Fig. 3 ganoderma lucidum ketone glycol (GMD) makes colorectal cancer cells cycle arrest in the S phases；
Colorectal cancer cells changes in gene expression situation map after Fig. 4 gene microarray analysis ganoderma lucidum ketone glycol (GMD) processing；
The active effect of Fig. 5 ganoderma lucidum ketone glycol (GMD) inhibiting cathepsin L (Cathepsin L)；
Fig. 6 difference nucleus inner tissue protease L (Cathepsin L) expression；
Fig. 7 ganoderma lucidum ketone glycol (GMD) reduces the level of the truncation CUX1 transcription factor expressions in colorectal cell core.
Specific implementation mode
Embodiment 1：Anti- colorectal cancer activity experiment and cytotoxicity experiment based on cell growth detection
1.1 experimental subjects：Colorectal cancer (HCT116 cell strains, Caco2 cell strains), breast cancer (MCF-7 cell strains), lung cancer
(HLC-1 cell strains), liver cancer (HepG2 cell strains), gastric cancer (HGC-27 cell strains), prostate cancer (LNCaP cell strains), uterus
Cancer (Hela cell strains) and leukaemia (HL60 cell strains), 4 kinds of normal human cells (normal skin fibrocyte NHDF, normally
Umbilical cord cells HUC-F2, lung normal fiber cell TIG-1, lung fibroblast HF19).
1.2 experimental drug：Ganoderma lucidum ketone glycol (is purchased from Wuhan City, Hubei Province ChemFaces Co., Ltds, HPLC >=98%).
1.3 experiment reagents and equipment：CCK-8 kits (purchased from Japanese colleague's chemical industry), (5-Fu is purchased from 5-fluor-uracil
InvivoGen companies of the U.S.), super microplate reader (MD, Flexstation 3).
1.4 experimental method：Various cells are with 1 × 104A/density per hole is inoculated in 96 porocyte culture plates, is waited for
Night culture cell grows up to the ganoderma lucidum diketone alcoholic solution of addition cell culture medium gradient dilution after single layer, while experimental setup is positive
Drug (5-FU) control group, normal cell controls group and sample toxicity control group.After culture plate is cultivated 3 days at 37 DEG C, use
CCK8 cell Proliferations are detected the proliferation and toxicity of cell with detection method of toxicity, and detection device used is MD companies of the U.S.
Super microplate reader (Flexstation 3).Then the Probit Regression function calculating halves of software SPSS is utilized to press down
Concentration (IC50) processed.The selection index (SI) of sample is determined eventually by formula IC50 (cancer cell)/IC50 (normal cell).
The result shows that ganoderma lucidum ketone glycol to the IC50 of colorectal cancer cells HCT116 and Caco2 be respectively 0.78 ± 0.5 μM and
2.25 ± 2.0 μM, far below 72.08 μM of breast cancer cell and other cancer cells and normal cell are more than 200 μM, show
The activity (Fig. 2) of significant selective depression colorectal cancer cells.
Embodiment 2：Colon cancer cells apoptosis and Cell cycle influences are tested：
2.1 experimental subjects：Colorectal cancer HCT116 cell strains.
2.2 experimental drug：Ganoderma lucidum ketone glycol (is purchased from Wuhan City, Hubei Province ChemFaces Co., Ltds, HPLC >=98%),
Ganoderma lucidum horse ketone (is purchased from Wuhan City, Hubei Province ChemFaces Co., Ltds, HPLC >=98%), and thymidine (is purchased from Nanjing Sen Beijiasheng
Object Science and Technology Ltd.).
2.3 experiment reagents and equipment：Fluorescent dye PI (propidium iodide is purchased from Shenzhen bio tech ltd Lai Bai),
Apoptosis annexin V-FITC kits (are purchased from abcam Co., Ltds of Britain), flow cytometer SH800 (Japan
Sony)。
2.4 experimental method：Colorectal cancer cells HCT116 is with 3 × 105The density in a/hole is inoculated in 6 orifice plates, is incubated overnight
Grow up to after cell monolayer and the culture solution containing 10% fetal calf serum is changed to the culture solution containing 1% fetal calf serum.Low serum free culture system
After liquid culture 16h, cell is added into 100 μM of sample solution, and structure is similar but inhibits proliferation of colorectal cancer cells activity without apparent
Triterpenoid ganoderma lucidum horse ketone (Ganodermanontriol, GMT) with and cell S Cycle block agent thymidines (thymidine)
Respectively as negative and positive control.37 DEG C are continued to cultivate, and cell are collected respectively at for 24 hours, after 48h and 72h, after ethyl alcohol is fixed
Carry out PI dyeing, finally by flow cytometer into the cell cycle detect.Apoptic effects are tested, annexin is used
V-FITC reagents are detected.
The results show that GMD has no significant effect the apoptosis of colorectal cancer cells, but the cell week of colorectal cancer cells can be made
It is stagnated in the S phases, to inhibit growth (Fig. 3).
Embodiment 3：Gene microarray analysis is tested：
3.1 experimental subjects：Colorectal cancer HCT116 cell strains.
3.2 experimental drug：Ganoderma lucidum ketone glycol (is purchased from Wuhan City, Hubei Province ChemFaces Co., Ltds, HPLC >=98%),
Ganoderma lucidum horse ketone (is purchased from Wuhan City, Hubei Province ChemFaces Co., Ltds, HPLC >=98%).
3.3 experiment reagent and equipment：Agilent people expression profiles of gene chip SurePrint G3Human GE
Microarray 8x60Kv2.0 (are purchased from Agilent companies of the U.S.), and RNA extracts kits are (public purchased from U.S. InvivoGen
Department).
3.4 experimental method：HCT116 cells are inoculated in 10cm diameter Petri dishes and cultivate to cell monolayer and cover first
Then 90% surface is added 100 μM of ganoderma lucidum ketone glycol (GMD), while setting solvent DMSO solvent control groups and ganoderma lucidum horse ketone
(GMT) negative control group.After 37 DEG C of culture 48h, collects cell and extract RNA, then use Agilent people's express spectra gene core
Piece carries out human genome expression pattern analysis (Japanese Cell Innovator companies).
As a result it shows that GMD lowers the expression of the cyclin Cyclin E2 of colorectal cancer cells, further demonstrates
The proliferation (Fig. 4) that GMD passes through adjusting Cyclin-dependent kinase colorectal cancer cells.
Embodiment 4：Inhibit cell tissue protease L (cathepsin L, CL) activity experiment：
4.1 experiment material ganoderma lucidum ketone glycol and other triterpene compounds are (limited purchased from Wuhan City, Hubei Province ChemFaces
Company, HPLC >=98%), cathepsin L's inhibitor screening kit (is purchased from Biovision companies of the U.S.), super enzyme mark
Instrument (MD, Flexstation 3).
4.2 experimental method：Cell tissue protease L inhibition of enzyme activity is carried out to ganoderma lucidum ketone glycol using commercial kit
Experiment.Analysis buffer (kit is included), the 1 μ L of 49 μ L are sequentially added in each hole of 96- porocyte culture plates
(0.2mU/ μ L) cell tissue protease L and 1 μ LDTT, is sufficiently mixed and is made into enzyme reaction solution.Then analysis buffer is used
Each sample to be analysed is diluted, and is added in enzyme reaction solution with the amount of 10 μ L of every hole, mixing, is reacted at room temperature 15 minutes.It is real
Test the setting positive and negative control group, 6 multiple holes of each concentration.After room temperature reaction, the bottom that 40 μ L have diluted is added per hole
Object solution and mixing.Finally, 37 DEG C of progress, 30 minutes Dynamic Fluorescence signal detections, wavelength used are in super microplate reader
Ex/Em=400/500nm.
The results show that ganoderma lucidum ketone glycol can significantly inhibit cell tissue protease L activity, IC50Value is 15.56 μM, compared with
Other triterpenoids are (Fig. 5) with obvious effects.
Embodiment 5：Confirm the expression experiment of colorectal cancer cells core cell tissue protease L (cathepsin L, CL)
5.1 experimental subjects：Colorectal cancer cells HCT116, Caco2, breast cancer cell (MCF-7), cervical cancer cell
(Hela), Normal human fibroblast HFLI and normal colorectal cell CCD841.
5.2 experiment reagent：Rabbit-anti Cathepsin L monoclonal antibodies (ab58991 is purchased from abcam Co., Ltds of Britain),
The goat anti-rabbit igg H＆L monoclonal antibodies (ab6717 is purchased from abcam Co., Ltds of Britain) of FITC labels, core dyestuff Hoechst
33528 (being purchased from MCE companies of the U.S.), laser confocal microscope (Leca Leica TCS SP8STED).
5.3 experimental method：Cell is with 0.5 × 104The density in a/hole is seeded to 96 porocyte culture plates and to contain 1% tire
The cell culture fluid of cow's serum is cultivated, and the cell culture fluid containing 10% fetal calf serum is changed to after 24 hours and continues to train
It supports 24 hours.Then culture solution is removed, and cell washes the methanol after one time using precooling with PBS and (100 holes μ L/) is fixed,
Room temperature 5 minutes.After fixation, removes methanol and wash cell three times with PBS, 5 minutes every time.Then, 100 μ L are added per hole to contain
The cell-penetrating liquid of 0.25%Trito X-100, room temperature are handled 10 minutes.After PBS washings three times, using containing 10% lowlenthal serum
It is closed with the PBS solution of 0.1% polysorbas20, room temperature 30 minutes.Primary antibody rabbit-anti Cathepsin L monoclonals are added after closing
Antibody (1/100 dilution) is simultaneously incubated overnight in 4 DEG C.The goat anti-rabbit igg H＆L that secondary antibody HRP labels are added in PBST washings afterwards three times is mono-
Clonal antibody (1/250 dilution), room temperature is protected from light incubation 1 hour.After PBST washings three times, Hoechst nuclear stainings (0.2 μ g/ are carried out
ML), room temperature dyes 10 minutes.Finally, it carries out laser confocal fluorescence microscope observation after removing dyeing liquor PBS washings and takes pictures
(FITC:Ex/Em=495/528nm, Hoechst:), Ex/Em=352/461nm and using software (CellProfiler) to figure
Piece carries out the ratio that processing analysis calculates the CL expressions in the nucleus of various cells and outside core.
The results show that CL expressions are all than the high (figure of other cells in the core of colorectal cancer cells and normal colorectal cell
6)。
Embodiment 6：Reduce the experiment of the truncation CUX1 transcription factor levels in the downstreams CL in nucleus
Then endonuclear cathepsin C L can be adjusted by carrying out the truncation CUX1 that cutting generates activation to CUX1
Ganglion cell cyclin Cyclin E2 are to promote cell cycle progression.In order to further confirm that CL is ganoderma lucidum ketone two in nucleus
The action target spot of alcohol, tests it.
6.1 experimental subjects：Colorectal cancer cells HCT116, normal colorectal cell CCD841.
6.2 experimental drug：Ganoderma lucidum ketone glycol (is purchased from Wuhan City, Hubei Province ChemFaces Co., Ltds, HPLC >=98%).
6.3 experiment reagent：Rabbit-anti CUX1 monoclonal antibodies (PA5-25788 is purchased from Invitrogen Co., Ltds of the U.S.),
Rabbit-anti HDAC1 monoclonal antibodies (ab32103 is purchased from abcam Co., Ltds of Britain), the goat anti-rabbit igg H＆L Dan Ke of HRP labels
Grand antibody (ab6721 is purchased from abcam Co., Ltds of Britain), subcellular proteome extraction agent box (is purchased from the U.S.
EMDBiosciences companies), ECL chemiluminescent substrates (are purchased from Thermo companies of the U.S.).
6.4 experimental method：The ganoderma lucidum of various concentration is added in 10cm diameter Petri dishes in cell culture after growing up to single layer
Ketone glycol (GMD), while drug solvent (DMSO) control group.After continuing culture 48 hours, cell is collected, EMD kits are used
Subcellular proteome component separation is carried out, nucleus and cytoplasm protein group sample are obtained.The Asia protein groups sample is carried out
SDS-PAGE electrophoresis, then western blotting analyze, closed using 5% milk soln after transferring film, then distinguished
Carry out the incubation of anti-CUX1 antibody or anti-HDAC1 antibody.Last washed rear upper secondary antibody is simultaneously detected using ECL substrates.
The results show that 50 μM of GMD processing, which can significantly reduce, truncates CUX1's in colorectal cancer or large intestine normal cell
Level confirms the fact that endonuclear cathepsin C L is GMD target molecules (Fig. 7) indirectly.
From external inhibiting tumor cell proliferation experiment and Apoptosis in terms of cell cycle experimental result, ganoderma lucidum ketone glycol can be special
Strange land inhibits the proliferation of colorectal cancer cells, can be the cell-cycle arrests of colorectal cancer cells in the S phases.Mechanism of action analysis shows
Endonuclear cathepsin L (CL) is its action target spot, and GMD is by CL activity in inhibition colorectal cancer cells core to reduce
The truncated CUX1 factors (activated form) then lower the expression of cyclin Cyclin E2, finally make colorectal cancer cells
Phase cell cycle S is rested on, to realize the effect for inhibiting growth of cancer cells.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (8)
1. the application of a kind of ganoderma lucidum ketone glycol in medicine preparation, wherein the drug is for treating colorectal cancer and and colorectal cancer
Relevant disease.
2. application according to claim 1, it is characterised in that：The colorectal cancer is colon and rectum carcinoma.
3. application according to claim 1, it is characterised in that：It is described be selected from the relevant disease of colorectal cancer by the carcinoma of the rectum or
Constipation caused by colon cancer, have blood in stool, abdominal pain, abdominal cramps, colon-cancer cell infiltration and transfer.
4. according to claim 1-3 any one of them applications, which is characterized in that the drug is by inhibiting colorectal cancer cells
The activity of core cell tissue protease L, to realize the effect for the treatment of colorectal cancer.
5. application according to claim 1, it is characterised in that：The effective dose of ganoderma lucidum ketone glycol is 0.01-10 μM.
6. application according to claim 5, it is characterised in that：The effective dose of ganoderma lucidum ketone glycol is 0.8-5 μM.
7. according to claim 1-6 any one of them applications, which is characterized in that the dosage form of the drug is：Suitable for oral administration
Or the dosage form of drug administration by injection.
8. application according to claim 7, it is characterised in that：The dosage form of the oral medication is tablet, sustained release
Piece, controlled release tablet, pastille, hard or soft capsule, dripping pill, pellet, aqueous or oil suspension, emulsion, dispersible powder or granule,
The dosage form of oral administration solution, syrup or elixir, the drug administration by injection is the aqueous or oily solution of sterilizing, aseptic powder
End, liposome, emulsion, microemulsion, nanoemulsion or micro-capsule.
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| CN109919924A (en) * | 2019-02-28 | 2019-06-21 | 江南大学 | A method of suitable for high-volume HE dyeing picture cell number wordization processing |
| CN115778901A (en) * | 2022-12-15 | 2023-03-14 | 广东医科大学附属医院 | Nano micelle and suppository for resisting colon cancer |
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| CN109919924B (en) * | 2019-02-28 | 2021-11-02 | 江南大学 | Method suitable for cell digital processing of large-batch HE staining pictures |
| CN115778901A (en) * | 2022-12-15 | 2023-03-14 | 广东医科大学附属医院 | Nano micelle and suppository for resisting colon cancer |
| CN115778901B (en) * | 2022-12-15 | 2024-04-30 | 广东医科大学附属医院 | Anti-colon cancer nano micelle and suppository |
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