CN114099519A - Application of cucurbitacin I - Google Patents
Application of cucurbitacin I Download PDFInfo
- Publication number
- CN114099519A CN114099519A CN202111508173.0A CN202111508173A CN114099519A CN 114099519 A CN114099519 A CN 114099519A CN 202111508173 A CN202111508173 A CN 202111508173A CN 114099519 A CN114099519 A CN 114099519A
- Authority
- CN
- China
- Prior art keywords
- cucurbitacin
- macrophage
- macrophages
- inhibitor
- cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of cucurbitacin I, and belongs to the technical field of biomedicine. Experiments prove that cucurbitacin I in THP-1 derived macrophages can stimulate the polarization of macrophages to M1 type and inhibit the polarization of macrophages to M2 type to a certain extent. Meanwhile, cucurbitacin I can also up-regulate the expression of factors such as TNF, iNOS and the like and down-regulate the expression of factors such as TGF-beta, CD206 and the like. The cucurbitacin I can be used as a novel M1 type macrophage activator/M2 type macrophage inhibitor, is a potential novel anti-cancer drug aiming at macrophages in a tumor microenvironment, can be used as an effective active ingredient for preparing medicaments for various cancers such as liver cancer, kidney cancer, breast cancer, colorectal cancer and the like, is favorable for inhibiting the immunosuppressive action generated by various factors in the tumor microenvironment, and further develops the possibility of cancer treatment from two aspects of tumor killing and immune activation.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of cucurbitacin I.
Background
Solid tumors include not only malignant cells, but also extracellular matrix and many other non-malignant cell types, including fibroblasts, endothelial cells, and inflammatory cells, such as macrophages, neutrophils, mast cells, and lymphocytes, among others. Tumors promote the development of the tumors by various mechanisms such as malignant proliferation, invasion and metastasis, immune evasion and the like, and a Tumor Microenvironment (TME) is created for the tumors to further resist the immunity of the organism and maintain the development, so that three conventional treatment methods such as surgery, radiotherapy and chemotherapy still cannot achieve effective treatment on many cancers. It is now clear that most malignant tumors contain a large number of macrophages as a major component of host leukocyte infiltration, a key member of stromal cells. It is now generally recognized that tumor-associated macrophages (TAMs) are an integral part of most malignancies and may even account for 50% of tumors in some cases. TAMs, when activated by cancer cells, release a variety of growth factors, proteolytic enzymes, cytokines, and inflammatory mediators, most of which are critical factors in cancer metastasis, and promote cancer metastasis through a variety of mechanisms, including tumor angiogenesis, tumor growth, and tumor cell migration and invasion.
Macrophages are double-edged sword, and have a positive or negative dual effect on the immune system. In terms of positive effects, it is not only the cells associated with tumor antigen presentation, but also exerts direct antitumor cytotoxic effects, promoting specific immunity by inducing T cell recruitment and activation. In the aspect of negative effects, macrophages can generate negative effects on the immune system by promoting the growth of tumor cells, inhibit the response reaction of T cells and the anti-tumor reaction of natural killer cells through a certain biological process, and show the effects of promoting the growth, diffusion, angiogenesis and immunosuppression of cancer cells. The health and the diseases are balanced by controlling the regulation mechanism of macrophage functions in the tumor growth process, and cytokines and proinflammatory substances produced in the tumor environment can play roles in changing the macrophage functions and destroying the anti-tumor functions of the macrophages, thereby being beneficial to the growth of the tumors. The existing clinical and experimental evidence shows that the macrophage can promote the generation and malignant progression of cancer, in the process of tumorigenesis, the macrophage can mutate and evolve into an inflammatory environment for promoting growth, and along with the development of tumors, the macrophage stimulates angiogenesis, so that the migration and invasion of tumor cells are enhanced, and the anti-tumor immunity is inhibited.
Macrophage polarization means that macrophages are activated at a given point in space and time, and the polarization is not fixed because macrophages are plastic enough to integrate multiple signals. Multifaceted differentiated cells, which are of the mononuclear phagocyte lineage, have specific markers expressing specific markers, which can be divided into subgroups involved in specific immune processes according to their origin, ancestry and growth factors of expression. Polarized macrophages are largely divided into two categories: selectively activated macrophages and classical activated macrophages. It has been found that macrophages activated with lipopolysaccharide (type M1), with or without interferon gamma, are pro-inflammatory; while macrophages activated with interleukins (type M2) are more involved in the resolution of inflammation and resistance to various pathogenic microorganisms, IL-4 and IL-13 polarize macrophages to the M2 phenotype by activation of STAT6 by IL-4 ra, while the IL-10 receptor promotes the M2 phenotype by activation of STAT 3. The M1 and M2 types describe two opposite biological characteristics of macrophages, M1 type macrophage can inhibit cell proliferation and cause tissue damage, and is a key effector cell for eliminating pathogens, virus infection and cancer cells, while M2 type macrophage can promote cell proliferation and tissue repair, and in this mode can promote tumor growth to some extent, and in the microenvironment of the tumor, the tumor-associated macrophage is considered as polarized M2 phenotype, so that tumor progression is enhanced and patient prognosis is poor. Macrophages find an explanation in functional plasticity due to their paradoxical effects in cancer, which in part leads to the development of polarized expression of pro-or anti-tumor functions. Key players in the phenotypic setting are the microenvironment signals to which macrophages are exposed, which selectively modulate their function within a spectrum of functions encompassing the extremes of M1 and M2.
Therefore, macrophage disordering is a novel means in tumor therapy and is receiving wide attention from researchers all over the world. Cucurbitacin I is originally identified as a potent selective inhibitor of a JAK2/STAT3 signal pathway, has the characteristic of antiproliferative effect, and can meet the clinical requirements of combined precise targeting and immunotherapy by utilizing the cancer promotion function of the existing anticancer drugs for pertinently reversing macrophages.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of cucurbitacin I. The new functional application of cucurbitacin I provided by the invention can construct a double-effect platform for synergistic cancer killing and immune activation aiming at macrophages in a tumor microenvironment, and more efficient cancer treatment is realized.
The purpose of the invention is realized by the following technical scheme:
applications of cucurbitacin I include, but are not limited to:
cucurbitacin I can be used as an M1 type macrophage agonist for novel anticancer application.
Cucurbitacin I can be used as an M2 type macrophage inhibitor for new anticancer application.
The present invention discloses a new function of anticancer medicine.
In one embodiment, the macrophage agonist of M1 type, i.e., the agonist that stimulates the polarization of macrophage M1, is an agent that upregulates the expression of the genes of the M1-related molecules CD86, CD80, CXCL2, IL1B, iNOS, TNF.
In one embodiment, the M2-type macrophage inhibitor, i.e., an inhibitor that inhibits the polarization of macrophage M2, is an agent that down-regulates the expression of the M2-associated molecules CD163, CD206, FIZZ1, TGF- β, Ym1, ARG1 gene.
Application of cucurbitacin I in preparation of M1 type macrophage agonist is provided.
Application of cucurbitacin I in preparing M2 type macrophage inhibitor is provided.
Application of cucurbitacin I in preparing medicine for treating renal cancer is provided.
Preferably, the renal cancer is renal clear cell carcinoma.
Further, in the above-mentioned case,
application of cucurbitacin I in preparing growth proliferation inhibitor of renal cancer cells is provided.
Application of cucurbitacin I in preparation of renal cancer cell invasion and metastasis inhibitor is provided.
Application of cucurbitacin I in preparation of clone formation inhibitor of renal cancer cells is provided.
In one embodiment, the renal cancer cell is a renal clear cell carcinoma cell; further, renal clear cell carcinoma cell 786-O.
Preferably, cucurbitacin I can be any pharmaceutically acceptable cucurbitacin I salt.
Cucurbitacin I may be in any pharmaceutically acceptable dosage form.
Cucurbitacin I may be in any pharmaceutically acceptable dosage.
In one embodiment, cucurbitacin I is formulated with a pharmaceutically acceptable carrier into a tablet, powder, granule, capsule, oral liquid, injection, or corrosion inhibitor, but not limited thereto.
According to the invention, the cucurbitacin I is found to be capable of up-regulating the expression of M1 related genes and down-regulating the expression of M2 related genes in tumor-induced M2 polarized macrophages by research. Meanwhile, the research finds that the cucurbitacin I has the effects of inhibiting proliferation, metastasis and colony formation of a 786-O cell line of the renal clear cell carcinoma. The cucurbitacin I can be used as a novel M1 type macrophage activating agent and a novel M2 type macrophage inhibiting agent, is a novel anticancer drug with potential combined tumor killing and immune activation, can be used as a novel active ingredient in drugs for cancers such as renal clear cell carcinoma and the like, and is favorable for improving the treatment effect of the drugs on cancer patients.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention aims at the research on the specific problem of how to utilize the existing medicines to simultaneously kill the tumor and regulate and control the immunity so as to further explore the treatment mechanism of the tumor; explores the possibility of new application of old drugs and expands the possibility of using some auxiliary drugs as anti-tumor and immune regulation drugs.
(2) The invention combines the methods of tumor killing and immune regulation, jointly discusses the influence of the medicament on the tumor immune microenvironment, and further develops a new method for anti-tumor treatment.
(3) Experiments prove that cucurbitacin I in THP-1 derived macrophages can stimulate the polarization of macrophages to M1 type and inhibit the polarization of macrophages to M2 type to a certain extent. Meanwhile, cucurbitacin I can also up-regulate the expression of factors such as TNF, iNOS and the like and down-regulate the expression of factors such as TGF-beta, CD206 and the like. The cucurbitacin I can be used as a novel M1 type macrophage activator/M2 type macrophage inhibitor, is a potential novel anti-cancer drug aiming at macrophages in a tumor microenvironment, can be used as an effective active ingredient for preparing medicaments for various cancers such as liver cancer, kidney cancer, breast cancer, colorectal cancer and the like, is favorable for inhibiting the immunosuppressive action generated by various factors in the tumor microenvironment, and further develops the possibility of cancer treatment from two aspects of tumor killing and immune activation.
Drawings
FIG. 1 is the result of PMA stimulation of macrophage differentiation in example 1; the left panel shows untreated THP-1 monocytes, now in suspension and round in morphology; the right panel shows THP-1 macrophages after PMA treatment, at which time the cells are in an adherent state and the morphology gradually appears irregular.
FIG. 2 is a graph showing the expression level of M1-related factor after macrophage cell treatment with cucurbitacin I in example 1; the abscissa represents monocyte THP-1 and macrophage
And the macrophage after the cucurbitacin I (CuI) treatment, and the ordinate is the relative expression quantity of each factor.
FIG. 3 is a graph showing the expression level of M2-related factor after macrophage cell treatment with cucurbitacin I in example 1; the abscissa is macrophage
And the macrophage after the cucurbitacin I (CuI) treatment, and the ordinate is the relative expression quantity of each factor.
FIG. 4 is the effect of tumor cells on macrophage polarization after general differentiation and treatment with cucurbitacin I in example 2; from top to bottomRespectively cucurbitacin I treatment group (CuI), kupffer and tumor cell co-culture group
Cucurbitacin I treatment macrophage and tumor cell co-culture group
General differentiation macrophage group
From left to right, DAPI nuclear staining, CD 86M 1 marker staining, and staining overlap, respectively.
FIG. 5 is the result of examining the killing effect of cucurbitacin I on renal clear cell carcinoma cells in example 3; the abscissa represents the concentration of the drug, the ordinate represents the cell activity, and the weaker the cell activity represents the stronger the killing ability of the drug on renal clear cell carcinoma cells; the round broken line is the cell activity after 24h of cucurbitacin I treatment, and the square broken line is the cell activity after 48h of cucurbitacin I treatment.
FIG. 6 is the result of measurement of wound healing inhibitory effect of cucurbitacin I on renal clear cell carcinoma cells in example 4; the concentrations of the drugs added from left to right are respectively 0, 1/10IC50, 1/4IC50 and 1/2IC50, the incubation times are respectively 0h and 12h from top to bottom, and the wound healing capacity of the renal clear cell carcinoma cells is judged according to the size of the wound area or the length of the section distance.
FIG. 7 is the result of measurement of the inhibitory effect of cucurbitacin I on renal clear cell carcinoma colony formation in example 4; the concentrations of the added drugs are respectively 0, 1/10IC50, 1/4IC50 and 1/2IC50, the blue-violet is the cell stained by the crystal violet solution, and the colony forming capability of the renal clear cell carcinoma is judged according to the number of the cells.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The main materials used in the following examples are as follows:
cucurbitacin I (Cucurbitacin I, CuI): cucurbitacin I was purchased from shanghai pottery biotechnology limited (TQ0196) and prepared at 96.69% purity using DMSO at a concentration of 50 μ M, and stored in a refrigerator at-80 ℃.
Antibody: antibodies to CD86 were purchased from Thermofish Scientific as CD86(B7-2) Monoclonal Antibody (IT2.2) Alexa Fluor 488eBioscienceTM(53-0869-42)。
Cell line: the macrophage line THP-1 and the cancer cell line 786-O were obtained from the cell resource center of Shanghai Life sciences institute of Chinese academy of sciences or ATCC.
Example 1 cucurbitacin I promotes the increase of M1-related factor and the decrease of M2-related factor in tumor-induced macrophage
Treatment with PMA (phorbol ester-12-myristate-13-acetate) successfully induced differentiation of THP-1 cells: given that the amount of CD68 in differentiated macrophages is increased, RNA samples were collected and QCPR-tested for success in differentiation. 1 x 10 of6The THP-1 mononuclear cells are inoculated in a six-hole plate, treated by 100ng/mL PMA for 48 hours to stimulate differentiation, continuously cultured for 24-48 hours to remove the influence of the PMA, and the state and the morphology of the cells are observed, as shown in figure 1; collecting RNA samples from untreated macrophages and treated macrophages, and carrying out QPCR detection on a differentiation related factor CD68, wherein the specific operation steps are as follows:
the method for RNA extraction is as follows:
(1) adding Trizol, standing at room temperature for 5min to fully crack, centrifuging at 12000rpm for 5min, and removing precipitate;
(2) adding chloroform into 200 μ L chloroform/mL Trizol, shaking, mixing, standing at room temperature for 10min, and centrifuging at 4 deg.C and 12000rpm for 15 min;
(3) sucking the upper layer of water phase into another centrifugal tube, generally 200-400 μ L. Adding isopropanol with the same volume as the supernatant, turning upside down, mixing, standing at room temperature for 10min, centrifuging at 4 deg.C at 12000rpm for 10min, removing supernatant, and precipitating RNA at the bottom of the tube;
(4) adding 75% ethanol (precipitating RNA) into 1mL of 75% ethanol/mL of Trizol, gently oscillating the centrifuge tube, suspending, washing and precipitating, centrifuging at 4 ℃ and 12000rpm for 5min, and removing supernatant;
(5) drying in the air or in vacuum for 5-10 min at room temperature, dissolving the RNA sample by using 15 mu L of DEPC water, TE buffer or non-enzyme water, and quantifying the RNA concentration at 55-60 ℃ for 5-10 min according to the O.D value;
reverse transcription of RNA was performed using Evo M-MLV RT Kit with gDNA Clean for qPCR II from Ecori organisms, and reverse transcription of RNA was performed according to the instructions.
The detection of QPCR was performed using a kit comprising 0.4. mu.L of forward primer (10. mu.M), 0.4. mu.L of reverse primer (10. mu.M), dye: 10 μ L, template: 2 μ L (100 ng/. mu.L), H2O: the proportion of 7.2 mu L is used for preparing a real-time fluorescent quantitative detection system.
The sequences of the primers used for detection were as follows:
under the condition that the pore diameter of the Transwell chamber is less than 4.0 μm, the cells can not migrate and pass through. A0.3-micron Transwell cell co-culture system is selected, tumor cells are inoculated in an upper chamber, macrophages are inoculated in a lower chamber, and the influence of drug stimulation on the induction of macrophage by the tumor cells is explored through separate culture. First, THP-1 monocytes (1X 10)6Individual cells/mL) were seeded into the lower chamber of a Transwell device and treated with 100ng/mL PMA for 48h to stimulate differentiation. Next, the cells were washed 3 times with Phosphate Buffered Saline (PBS) and incubated for 24h to exclude PMA interference. Renal clear cell carcinoma cell 786-O (1X 10)5cells/mL) were seeded in the upper chamber and incubated for 24h to allow for attachment and co-culture induced macrophages. The chamber containing 786-O cells was placed directly on top of a six-well plate containing THP-1-derived macrophages, and the resulting co-culture system cells were cultured with the medium for 48h and then treated with cucurbitacin I for 24h, while the cucurbitacin I-untreated group served as a control.
To determine the effect of cucurbitacin I on macrophages in vitro, tumor-induced macrophages were cultured in RPMI-1640 medium in the absence or presence of cucurbitacin I for 24h, the expression of selected genes associated with macrophage polarization was detected by QPCR, using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a reference gene for data analysis. The results show that stimulation with PMA successfully differentiated monocyte THP-1, CD68 was significantly upregulated (fig. 2), cucurbitacin I inhibited the expression of TAM/M2-related genes CD163, CD206, FIZZ1, TGF- β, Ym1, ARG1 (fig. 3), but upregulated the expression of M1-related genes CD86, CD80, CXCL2, IL1B, iNOS, TNF (fig. 2). mRNA expression of CD163, CD206, FIZZ1, TGF-. beta.Ym 1, ARG1 was significantly reduced in inhibitor-treated co-culture systems compared to co-culture systems, with a reduction in CD163 mRNA expression level of 39.4%, 31.1% of CD206, 85.7% of FIZZ1, 19.2% of TGF-. beta.Ym 1, 82.1% of ARG1 (P <0.05) (FIG. 3). mRNA expression of CD86, CD80, CXCL2, IL1B, iNOS, TNF was significantly increased in inhibitor-treated co-culture systems compared to co-culture systems, with CD86 mRNA expression levels increased 1.666-fold, CD80 increased 2.622-fold, CXCL2 increased 5.955-fold, IL1B increased 3.249-fold, iNOS increased 3.792-fold, TNF increased 5.163-fold (P <0.01) (fig. 2).
Example 2 polarization of M1 type by cucurbitacin I to promote PMA-induced macrophage differentiation
1. Immunofluorescence assay: the multichannel high-resolution detection of a fluorescence microscope is applied, and a fluorescent antibody is used as a probe to detect corresponding antigens on the surface of a cell, so that the positioning and the expression quantity of different antigens in the cell can be determined, and the polarization condition of macrophage can be determined. The co-culture system was carried out using cell culture inserts (0.3 μm), first the THP-1 monocytes (1X 10)6Individual cells/mL) were seeded into the upper chamber of a transwell device and treated with 100ng/mL PMA for 72 hours to stimulate differentiation. Cells were washed 3 times with Phosphate Buffered Saline (PBS) and incubated for 24h to exclude PMA interference. Meanwhile, the co-culture system was divided into two groups, one group was Mega without any treatment, and the other group was Mega with 24h treatment with cucurbitacin I, 786-O cells (1X 10)5cells/mL) were seeded in the lower chamber and incubated for 24h to allow attachment. Subsequently, two sets of the after-differentiation macrophage-containing chambers were placed directly on top of a six-well plate containing 786-O cells, and the resulting co-culture system cells were cultured with RPMI-1640 mediumAnd (5) 24 h. The macrophage after common differentiation and the macrophage after cucurbitacin I treatment for 24h are respectively incubated with RPMI-1640 culture medium in a six-well plate for 24h as corresponding controls.
2. The detection result shows that the cucurbitacin I can reduce the inhibition effect of tumor cells on macrophagy M1 type polarization to a certain extent (figure 4). Compared with a co-culture system without cucurbitacin I treatment, after the tumor cells are co-cultured with the macro-phagocytes treated by cucurbitacin I, the inhibition effect of the tumor cells on M1 Marker is relieved, and the fluorescence is obviously enhanced.
Example 3 the killing effect of cucurbitacin I on renal clear cell carcinoma cell 786-O has significant time and dose dependence
1. Renal clear cell carcinoma cells 786-O were seeded into 96-well plates overnight at a density of 8000 cells per well, each group consisting of three replicates. The cells were treated with different concentrations (0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 μ M) of drug (cucurbitacin I) and then incubated at 37 ℃ for 24-48 h under the cultured cells. A drug-free Dimethylsulfoxide (DMSO) treatment was used as a control, while a blank was set. 1h before the end of incubation, 10. mu.L of CCK-8 reagent was added to each well, and the absorbance was measured at 450nm after 1h of incubation.
2. The results of the assay showed that cucurbitacin I significantly inhibited the viability of the 786-O cell line in a dose and time dependent manner (FIG. 5). The anti-proliferation effect of the compound is visible within 24 hours and is continuously increased within 48 hours, and the cucurbitacin I has a lasting and effective cytotoxic effect on renal clear cell carcinoma cells.
Example 4 inhibition of migration and colony formation of renal clear cell carcinoma cells by cucurbitacin I
1. Cell scratch test
(1) Dividing the renal clear cell carcinoma cells 786-O into four groups, incubating for 48h with drugs (cucurbitacin I) with different concentrations, and evaluating the effect of the drugs on cancer cell migration; inoculation 2 x 10 separately5The cancer cells of (a) were put in a 6-well plate, allowed to adhere overnight without serum starvation, scratched at 80-90% confluence, washed 3 times with PBS, and then photographed at scratch for 0 h; incubating the cells with drugs (0, 1/10IC50, 1/4IC50, 1/2IC50), and observing the cells at 0h, 12h, 24h, and 48hThe migration of the cells, the rate of movement was quantified by the migration distance of the cells from the reference line to the center, and the distance was quantified using imagej software.
(2) As shown in FIG. 6, the results show that cucurbitacin I has obvious inhibition effect on the migration capacity of renal clear cell carcinoma cells 786-O, and the inhibition effect has drug concentration dependence, and the higher the drug concentration is, the more obvious the migration inhibition effect is.
2. Colony formation assay
(1) Dividing the renal clear cell carcinoma cells 786-O into four groups, and after incubating with different concentrations of drug (cucurbitacin I) for 48h, evaluating the inhibition effect of the drug on the formation of cancer cell colonies to know the long-term influence of the compound on the renal clear cell carcinoma cell line; renal clear cell carcinoma cells 786-O (0, 1/10IC50, 1/4IC50, 1/2IC50) were treated with cucurbitacin I at different concentrations for 24h, washed with PBS, and 500 numbers of cancer cells were inoculated into 6-well plates, cultured for 7-14 days to observe the growth of cell colonies, fixed with 4% (v/v) methanol at room temperature for 15 minutes, washed twice with PBS, stained with 1% (w/v) crystal violet at room temperature for 10 minutes, observed by microscope and photographed, and the blue-violet color was quantified using imagej software.
(2) As shown in FIG. 7, the results indicate that cucurbitacin I has a significant inhibitory effect on colony formation of renal clear cell carcinoma cells 786-O, and that the inhibitory effect has a drug concentration dependence, and the higher the drug concentration is, the more significant the colony formation inhibition is.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
<120> application of cucurbitacin I
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ARG1-F
<400> 1
gtggaaactt gcatggacaa c 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> ARG1-R
<400> 2
aatcctggca catcgggaat c 21
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> FIZZ1-F
<400> 3
gggagtactc agtgttcct 19
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> FIZZ1-R
<400> 4
gagagggact gtactctaga c 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Ym1-F
<400> 5
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Ym1-R
<400> 6
gttccatcct cccgacagac a 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TGF-β-F
<400> 7
agctgtacca gaaatacagc a 21
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TGF-β-R
<400> 8
ataaccactc tggcgagtc 19
<210> 9
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TNF-F
<400> 9
ctctaatcag ccctctggc 19
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> TNF-R
<400> 10
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IL1B-F
<400> 11
atgatggctt attacagtgg caa 23
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> IL1B-R
<400> 12
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CXCL2-F
<400> 13
ctcaagaaca tccaaagtgt g 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CXCL2-R
<400> 14
attcttgagt gtggctatga c 21
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> iNOS-F
<400> 15
ttcagtatca caacctcagc aag 23
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> iNOS-R
<400> 16
tggacctgca agttaaaatc cc 22
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> GAPDH-F
<400> 17
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> GAPDH-R
<400> 18
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD86-F
<400> 19
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD86-R
<400> 20
ttctccgcat ctccattctc 20
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD80-F
<400> 21
cacttctgtt caggtgttat cc 22
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD80-R
<400> 22
aacagaaaca ttgtgaccac ag 22
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD206-F
<400> 23
gatgaaaggc aaggatggg 19
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD206-R
<400> 24
cttcgtgatt tcatcttgca g 21
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD163-F
<400> 25
ttgcacagat atttcagtgc ag 22
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> CD163-R
<400> 26
actgcaataa aggatgactg ac 22
Claims (10)
1. Application of cucurbitacin I in preparation of M1 type macrophage agonist and/or M2 type macrophage inhibitor.
2. Use according to claim 1, characterized in that:
the M1 type macrophage excitant is an excitant for stimulating macrophage M1 polarization, and is a preparation for up-regulating M1 related molecules CD86, CD80, CXCL2, IL1B, iNOS and TNF gene expression;
the M2 type macrophage inhibitor, namely the inhibitor for inhibiting macrophage M2 polarization, is an agent for down-regulating M2 related molecules such as CD163, CD206, FIZZ1, TGF-beta, Ym1 and ARG1 gene expression.
3. Application of cucurbitacin I in preparing medicine for treating renal cancer is provided.
4. Use according to claim 3, characterized in that:
the kidney cancer is renal clear cell carcinoma.
5. Use according to claim 3 or 4, characterized in that:
the application is one of the following applications:
the application of cucurbitacin I in preparing a growth proliferation inhibitor of renal cancer cells;
the cucurbitacin I is applied to the preparation of an invasion and transfer inhibitor of renal cancer cells;
application of cucurbitacin I in preparation of clone formation inhibitor of renal cancer cells is provided.
6. Use according to claim 5, characterized in that:
the renal cancer cell is a renal clear cell carcinoma cell 786-O.
7. Use according to claim 3 or 4, characterized in that:
cucurbitacin I is any pharmaceutically acceptable salt of cucurbitacin I.
8. Use according to claim 3 or 4, characterized in that:
cucurbitacin I is in any pharmaceutically acceptable dosage form.
9. Use according to claim 3 or 4, characterized in that:
cucurbitacin I is in any pharmaceutically acceptable dosage.
10. Use according to claim 3 or 4, characterized in that:
the cucurbitacin I and a pharmaceutically acceptable carrier are prepared into tablets, powder, granules, capsules, oral liquid, injection or corrosion inhibitor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111508173.0A CN114099519A (en) | 2021-12-10 | 2021-12-10 | Application of cucurbitacin I |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111508173.0A CN114099519A (en) | 2021-12-10 | 2021-12-10 | Application of cucurbitacin I |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114099519A true CN114099519A (en) | 2022-03-01 |
Family
ID=80364978
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111508173.0A Pending CN114099519A (en) | 2021-12-10 | 2021-12-10 | Application of cucurbitacin I |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114099519A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170106003A1 (en) * | 2014-05-28 | 2017-04-20 | Institute For Myeloma & Bone Cancer Research | Anti-cancer effects of jak2 inhibitors in combination with thalidomide derivatives and glucocorticoids |
-
2021
- 2021-12-10 CN CN202111508173.0A patent/CN114099519A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170106003A1 (en) * | 2014-05-28 | 2017-04-20 | Institute For Myeloma & Bone Cancer Research | Anti-cancer effects of jak2 inhibitors in combination with thalidomide derivatives and glucocorticoids |
Non-Patent Citations (1)
| Title |
|---|
| CURTIS J. HENRICH等: "Effects of cucurbitacins on cell morphology are associated with sensitization of renal carcinoma cells to TRAIL-induced apoptosis", APOPTOSIS, pages 79 - 89 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Park et al. | Lipopolysaccharide directly stimulates Th17 differentiation in vitro modulating phosphorylation of RelB and NF-κB1 | |
| US9333194B2 (en) | Use of ansamycin antibiotics and method of screening novel angiogenesis inhibitor | |
| CN115725446A (en) | Enterococcus faecium for resisting rectal tumors and application thereof | |
| Yang et al. | VPS9D1-AS1 overexpression amplifies intratumoral TGF-β signaling and promotes tumor cell escape from CD8+ T cell killing in colorectal cancer | |
| Wang et al. | Anti-tumor and phenotypic regulation effect of matrine on dendritic cells through regulating TLRs pathway | |
| CN114099519A (en) | Application of cucurbitacin I | |
| CN111956795A (en) | Application of chimeric antigen receptor combined anti-tumor drug taking CD99 as target | |
| WO2023280001A1 (en) | Application of mir-31-5p in acute myeloid leukemia | |
| CN1184316C (en) | Antisense oligonucleotide structure and use of cell propagation receptor IGFIK | |
| CN110664821A (en) | Application of panaxadiol in preparing medicine for inhibiting expression of PD-L1 and tumor cell proliferation protein | |
| CN116139250A (en) | Polypeptide with immunity improving, anti-tumor and life prolonging functions and application thereof | |
| CN111575232A (en) | Application of JQ1 in inhibiting expression of T lymphocyte PD-1 and/or Tim-3 | |
| CN107354159A (en) | Applications of the long-chain non-coding RNA SMAD5 AS1 siRNA in treatment of ovarian cancer | |
| CN111568891B (en) | Application of dimethyl fumarate DMF in regulating tumor metabolism and inhibiting tumor growth | |
| CN113133994B (en) | Application of tricyclic diterpene natural product Vinigrol or pharmaceutical composition thereof in preparation of anti-cancer drugs | |
| CN101502508B (en) | Application of 5-oxo-4-alkenylene-pyrazole derivative in preparing anti-tumor medicament | |
| Maziveyi et al. | Altered Tumor Necrosis Factor-Alpha Signaling Pathway Between High and Low Metastatic Carcinoma Clones Derived from Single Tumor | |
| CN113925852A (en) | New medical application of propranolol | |
| CN114990061A (en) | Culture method for induced amplification of central memory T cells | |
| CN1283802C (en) | Recombinant adenovirus and its application in malignant tumor treatment | |
| CN116473937A (en) | Bionic nano-particle with anti-malignant melanoma effect and preparation method and application thereof | |
| CN116904400A (en) | Application of calicheamicin in optimization of in-vitro CAR/TCR-T cell product preparation process | |
| CN117919395A (en) | Use of entecavir, ASO drug, mRNA therapeutic vaccine and/or interferon combination for the manufacture of a medicament for the treatment of chronic hepatitis b | |
| CN118236361A (en) | Application of taurine combined chemotherapy medicine in preparing anti-tumor medicine | |
| CN117942331A (en) | PD-1/PD-L1 small molecule inhibitor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |