CN110652589A - Application of GASC1 inhibitor in preparation of medicine for treating liver cancer - Google Patents
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Abstract
The invention discloses an application of a GASC1 inhibitor in preparing a medicine for treating liver cancer, which can inhibit proliferation and cloning of liver cancer cells and liver cancer cells with the 'dryness' characteristic and formation of tumors, promote the sensitivity of the liver cancer cells and liver cancer stem cells to sorafenib, reduce the drug resistance of the liver cancer stem cells to sorafenib, simultaneously enhance the apoptosis of the sorafenib on the liver cancer cells and the liver cancer stem cells, kill the liver cancer cells with the 'dryness' characteristic from the source and bring a new hope for thoroughly curing liver cancer.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of a GASC1 inhibitor in preparation of a medicine for treating liver cancer.
Background
Liver cancer (HCC) is used as a malignant tumor in the global scope, the morbidity and the mortality of Asia, particularly China are extremely high, and the traditional treatment methods such as surgery, radiotherapy, chemotherapy and the like are difficult to completely cure; at present, effective medicines are lacked for treating liver cancer, and the reason is that the liver cancer is easy to generate medicine resistance to the treatment medicines.
Liver cancer is characterized by high heterogeneity, poor prognosis and poor drug response; the occurrence, development and metastasis of liver cancer are closely related to abnormal activation of multiple signal pathways in a tumor microenvironment and cells, such as signal pathways of Notch, Ras/Raf/MAPK, WNT/beta-catenin and the like. Among them, Notch signaling pathway plays an important role in regulating liver cancer development and metastasis; researches show that the Notch signal pathway can promote the symmetric division of the liver cancer stem cells, inhibit the asymmetric division and promote the self-renewal of liver cancer and the formation of tumors. Ras/Raf/MAPK is a complex highly-conserved cell signal transduction pathway widely existing in eukaryotic cells and is very important in the processes of liver cancer occurrence and development, malignant transformation and drug resistance. The small molecule targeted drug, domega, also known as sorafenib (sorafenib), is the first drug approved by food and drug administration for the treatment of advanced HCC, is an oral multi-target kinase inhibitor, and can target serine/threonine kinase and receptor tyrosine kinase of tumor cells and growth factors on tumor blood vessels. Domestic and foreign research reports that sorafenib can delay the progress of liver cancer and prolong the life of patients in later period. At present, sorafenib is used for treating liver cancer patients, and is also used for patients with breast cancer, kidney cancer and the like, but the cancer patients are easy to generate drug resistance when the sorafenib is used. The theory of tumor Stem Cells suggests that tumor Stem Cells (CSCs) are the "seeds" for tumorigenesis and are the root cause of tumorigenesis, progression, resistance to therapy, recurrence, and metastasis. Therefore, research and development of a targeted drug capable of targeting tumor stem cells are urgently needed, the tumor stem cells are killed from the source, and the targeted drug has important theoretical value and practical significance for the pathogenesis of liver cancer and clinical development of targeted treatment strategies.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of a GASC1 inhibitor in the preparation of a medicament for treating liver cancer; the other object of the present invention is to provide a composition for treating liver cancer; the invention also aims to provide the application of the compound in preparing the medicine for treating liver cancer; the fourth purpose of the invention is to provide the application of the GASC1 inhibitor in preparing the drug for overcoming the drug resistance of liver cancer to sorafenib; the fifth purpose of the invention is to provide application of the GASC1 inhibitor in preparing a medicament for enhancing the sensitivity of sorafenib to liver cancer.
In order to achieve the purpose, the invention provides the following technical scheme:
1. application of GASC1 inhibitor in preparing medicine for treating liver cancer is provided.
Preferably, the GASC1 inhibitor is SD70 or a lentivirus or adeno-associated virus that interferes with the expression of GASC 1.
Preferably, the lentivirus interfering with GASC1 expression comprises Lv-sh-GASC1 plasmid.
Preferably, the GASC1 inhibitor is applied to preparation of drugs for inhibiting clonogenic, balling-up or tumorigenic capacity of liver cancer cells or liver cancer stem cells.
2. The complex contains a GASC1 inhibitor and sorafenib.
3. The application of the compound in preparing a medicament for treating liver cancer.
Preferably, the compound is applied to the preparation of medicines for inhibiting the clonogenic, balling-up and tumorigenic capacity of liver cancer cells or liver cancer stem cells.
Preferably, the compound is applied to preparation of a medicament for promoting apoptosis of liver cancer cells or liver cancer stem cells.
4. Application of a GASC1 inhibitor in preparing a medicament for overcoming the drug resistance of liver cancer to sorafenib.
5. Application of a GASC1 inhibitor in preparation of drugs for enhancing sensitivity of sorafenib on liver cancer.
The invention has the beneficial effects that: the invention discloses an application of a GASC1 inhibitor and sorafenib in preparation of a medicine for treating liver cancer, wherein the GASC1 inhibitor is used for interfering or inhibiting GASC1 expression to inhibit proliferation, cloning and tumor formation of liver cancer stem cells, meanwhile, the sensitivity of liver cancer stem cells to sorafenib is improved, and the apoptosis of sorafenib on the liver cancer stem cells is enhanced.
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In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the analysis of the expression of GASC1 in clinical liver cancer tissue and the clinical prognosis by databases (A: cBioPortal database analysis of the expression of GASC1 in different tumor tissues; B: three GSE databases (GSE14323, GSE25097 and GSE6764) analysis of the expression of GASC1 in clinical liver cancer tissue samples; C: Kaplan-Meier method analysis of the relationship between the expression of GASC1 in TCGA database and the prognosis of liver cancer patients).
FIG. 2 shows the effect of the GASC1 inhibitor SD70 on the clonality, tumor formation and apoptosis of hepatoma cells and hepatoma stem cells (A: the effect of the GASC1 inhibitor SD70 on the clonality is detected after treatment; B: the effect of the GASC1 inhibitor SD70 on the tumor formation is detected after treatment; and C: the effect of the GASC1 inhibitor SD70 on the apoptosis is detected after treatment).
FIG. 3 shows that inhibition of GASC1 expression can significantly reduce self-renewal of liver cancer stem cells and tumor formation (A: interference with GASC1 can significantly reduce the cloning and balling capacity of liver cancer stem cells; B: interference with GASC1 can significantly reduce the tumorigenicity capacity of liver cancer stem cells).
FIG. 4 shows the effect of GASC1 inhibitor SD70 treatment or inhibition of GASC1 expression combined with sorafenib treatment on apoptosis (A: the effect of SD70 combined with sorafenib on apoptosis of hepatoma cells and hepatoma stem cells; B: the effect of knocking out GASC1 combined with sorafenib on apoptosis of hepatoma cells).
FIG. 5 is a graph showing the effect of the GASC1 inhibitor SD70 on cell cloning and the volume of transplanted tumors (A: colony formation rate; B: tumorigenicity capacity) in combination with Sorafenib treatment of transplanted tumors in liver cancer stem cells.
FIG. 6 shows the effect of the GASC1 inhibitor SD70 in combination with sorafenib treatment on Ki-67 expression and apoptosis in transplanted tumors in liver cancer stem cells (A: Hep 3B; B: PLC/PRF/5).
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.
The hepatoma cell lines PLC/PRF/5, Huh7, HepG2, Hep3B and HEK293T used in the invention are preserved by the laboratory; the Lv-NT-shRNA plasmid and the Lv-shGASC1 plasmid are constructed by the laboratory, and are concretely seen in Liu et al cancer Research 2018,78(4): 938-949); the cell transfection kit was purchased from QIAGEN, usa; CRISPR/Cas9 control plasmid and GASC1CRISPR/Cas9 knock-out plasmid were purchased from Santa Cruz Biotechnology, Inc., USA.
Envision for use with the present inventionTMAnd DAB color developing solution from DAKO, denmark; EDTA (pH 8.0) antigen retrieval solution was purchased from Fuzhou Mi New Biotechnology development. Sorafenib (sorafenib) tablets were purchased from Bayer Scheringpharma AG, germany; SD70 was obtained from Xcessbio Biosciences Inc., USA; apoptosis detection antibody kits were purchased from Cell Signaling Technology, usa;
3/7 assay kits were purchased from Promega, USA.
Example 1 database analysis of GASC1 expression in clinical liver cancer tissue and clinical prognosis
The existing research shows that GASC1 (lysine demethylase 4C) plays an important role in regulating the processes of tumor proliferation, invasion and metastasis, tumor formation, apoptosis inhibition and the like, especially in prostate cancer and leukemia. GASC1 can regulate amino acid metabolism of tumor cell to promote tumor development. However, the role of GASC1 in liver cancer remains to be elucidated further. Based on this, the invention analyzes the GASC1 expression in different tumor tissues through the cBioPortal database, and finds that the GASC1 has different degrees of gene amplification in different tumors. Further analysis of three liver cancer GSE databases (GSE14323, GSE25097 and GSE6764) analysis of the expression of GASC1 in clinical liver cancer tissue samples, the results are shown in fig. 1. As a result, GASC1 was found to be abnormally highly expressed in the liver cancer tissue, compared with the paracarcinoma tissue. The Kaplan-Meier method analyzes the relationship between the expression of GASC1 in the TCGA database and the prognosis of the liver cancer patient, and finds that the prognosis of the liver cancer patient with high expression of GASC1 is poor, and the prognosis of the liver cancer patient with low expression of GASC1 is good. The analysis results indicate that the GASC1 can play an important role in oncogene in the process of liver cancer occurrence and development, and provides data support for targeted GASC1 to treat liver cancer.
Example 2 effects of GASC1 inhibitor SD70 on clonogenic, tumorigenicity and apoptosis of hepatoma cells and hepatoma stem cells
Preparation of GASC1 inhibitor SD70 solution: a stock solution of 100mg/mL was prepared with a volume fraction of 1% DMSO, and the required concentration was prepared in the complete medium at the time of use.
Diluting an antibody of the apoptosis detection kit: diluting according to the volume ratio of 1: 1000.
In order to clarify the biological function of GASC1 in the development of liver cancer, a liver cancer cell line Hep3B highly expressed by GASC1 and a liver cancer cell line SMMC-97H are treated by using a small molecule inhibitor SD70 of GASC1, and cell subsets with the characteristic of being dry are detected by flow-sorting the liver cancer cell line PLC/PRF/5 and the liver cancer cell line Huh7 and are respectively marked as PLC/PRF/5CSC and Huh7 CSC. The results show that the treatment of hepatoma cells and hepatoma stem cells with different concentrations of the GASC1 inhibitor SD70 (0-2.5. mu.M) can significantly inhibit the cell clonogenic capacity and has dose dependence (FIG. 2, A). Further, the experiment of NOD/SCID mouse subcutaneous transplantation tumor is carried out by using the liver cancer cells pretreated by SD70, the subcutaneous tumor forming capability of the liver cancer cells pretreated by SD70 is further proved to be remarkably reduced (figure 2, B), the liver cancer cells are treated in vitro at different concentrations or at different times of the same concentration, the expression condition of apoptosis factors is detected, and the time and dosage dependence is found to be realized (figure 2, C) when the liver cancer cells are treated by SD 70. The research proves that the GASC1 enzyme activity plays an important role in the proliferation and the tumorigenesis of the liver cancer and the inhibition of the apoptosis.
Example 3 inhibition of GASC1 expression can significantly inhibit self-renewal of liver cancer stem cells and tumor formation ability
1. Packaging, collecting and purifying lentivirus Lv-sh-GASC1 and Lv-NT-shRNA
(1) Packaging of lentiviruses Lv-sh-GASC1 and Lv-NT-shRNA
Spreading in a 10cm culture dish at a density of 5 × 106293T cells (preferably before 20 generations, the state of the cells must be good, the growth rate is high, otherwise the virus yield is greatly influenced), DMEM (v/v) containing 10% FBS in a culture solution, when the cells are confluent to 80%, DMEM (v/v) containing 10% FBS is replaced with fresh DMEM (v/v) containing 10% FBS, and the cells are used for transfection after 3-4 h.
Adding pMDLg/pRRE, pRSV-Rev and pMD2.G into Lv-sh-GASC1 and Lv-NT-shRNA lentiviral expression plasmid 15-20 μ g, respectively, adding sterile water to total volume of 450 μ L, mixing, and adding CaCl 50 μ L2.5M dropwise into the mixture2After mixing, adding 500 mu L of 2 xHBS buffer solution (the 2 xHBS buffer solution is vibrated on a vortex oscillator before being used), then placing on the vortex oscillator to vibrate for more than 10s, after the dropwise adding is finished, continuing to vibrate, placing at room temperature for 5 minutes, adding the liquid into 293T to be cultured, shaking uniformly back and forth and left and right after the adding is finished, and changing fresh 10% FBS DMEM (v/v) culture solution 6-12 hours after the adding.
(2) Collection and purification of lentivirus Lv-sh-GASC1 and Lv-NT-shRNA
Cell culture was collected once at 48 hours and 72 hours from the start of transfection, and after 48 hours, 15mL of culture was supplemented, the collected virus was centrifuged at 1,000rpm for 10min to remove cell debris, the supernatant was collected and filtered through a 0.45 μm filter, and the filtrate was transferred to a Millopore concentration column and centrifuged at 5,000rpm to concentrate the volume of liquid in the upper well to 1mL (which was added in multiple portions). Subpackaging the concentrated solution, and storing in a refrigerator at a low temperature of-80 ℃ for subsequent experiments.
2. After inhibiting the expression of GASC1, the influence of GASC1 on the self-renewal and tumor-forming ability of liver cancer stem cells is detected
Enriching cells with dry characteristics in a liver cancer cell line PLC/PRF/5 and a liver cancer cell line Huh7, inhibiting the expression of GASC1 by adopting a lentivirus infection mode, screening for 72 hours by puromycin, and then carrying out sorting and plating by a flow cytometer to detect the cell cloning, balling and tumor forming capabilities. Research results show that cell cloning and balling are performed for 14 days, and statistical analysis is performed, and the inhibition of the expression of GASC1 is found to significantly reduce the cloning and balling capacity of the liver cancer stem cells, and compared with a control group (figure 3, A); NOD/SCID mice were injected subcutaneously with interfering GASC1 expression cells and corresponding cells, tumor size was observed daily, it was observed that subcutaneous tumorigenicity was significantly enhanced in control mice compared to interfering GASC1 expression, mice were sacrificed 28 days after continuous treatment, tumors were stripped, and tumor volume and tumor weight were measured (FIG. 3, B). The results further show that the GASC1 has an important role in the self-renewal of liver cancer stem cells and the formation of tumors, and is possibly an ideal target for targeted therapy of liver cancer.
Example 4 GASC1 Effect of inhibitor SD70 treatment or inhibition of expression of GASC1 in combination with Sorafenib treatment on apoptosis
Preparation of sorafenib (sorafenib) solution: sorafenib tablets were first ground to a powder and then made up into 100mg/mL stock with a volume fraction of 1% DMSO, at the time of use, in complete medium to the desired concentration.
The CRISPR/Cas9 technology is adopted to construct a GASC1 knocked-out liver cancer cell line Hep3B and a liver cancer stem cell PLC/PRF/5 CSC: CRISPR/Cas9 control plasmid and GASC1CRISPR/Cas9 knock-out plasmid were purchased from Santa Cruz Biotechnology, Inc., USA; the cell transfection kit was purchased from QIAGEN, USA.
After the hepatoma cell line Hep3B cells of high expression GASC1 are transfected with GASC1CRISPR/Cas9(sgGASC1) plasmid for 72 hours, the hepatoma cells of GFP + are sorted out by a flow cytometer and put into a 96-well plate for monoclonal screening, after 14-21 days, the monoclones are respectively put into a 6-well plate for amplification culture, then partial cells are removed, proteins are collected for western blotting to verify the knockout efficiency, and the cells with high knockout efficiency are selected for subsequent experiments.
Apoptosis was detected using GASC1 knockout cells or after treatment with the GASC1 inhibitor SD70 in combination with sorafenib: the constructed GASC 1-knocked-out cells and corresponding control group cells are respectively set as control group cells, GASC 1-knocked-out groups and GASC 1-knocked-out groups, sorafenib groups are added, after 48 hours of treatment, through a caspase3/7 activity detection kit, through enzyme labeling instrument detection, the activity of caspase3/7 of GASC 1-knocked-out cells is obviously enhanced, and the cells are compared with the control group; compared with the GASC1 knockout group, the GASC1 group treated by combination with sorafenib group has significantly enhanced activity of the cell caspase3/7 (FIG. 4, A). The results suggest that high expression of GASC1 may be the source of liver cancer insensitivity to Sorafenib treatment.
In order to further verify the effect of inhibiting the expression of GASC1 and combining sorafenib on the apoptosis of the liver cancer cells, the GASC1 inhibitor SD70 is adopted to inhibit the activity of GASC 1. Respectively setting as a control group cell, an SD70 group and an SD70 group to be combined with a Sorafenib group, after 48 hours of treatment, detecting by a caspase3/7 activity detection kit through an enzyme labeling instrument to find that the activity of cell caspase3/7 treated by the GASC1 inhibitor SD70 is obviously enhanced, and comparing with the control group; whereas the SD70 group treated with Sorafenib in combination exhibited a significant increase in the activity of the cellular caspase3/7 compared to the SD70 group (FIG. 4, B). The results show that the inhibition of GASC1 expression or enzyme activity can significantly promote the apoptosis of sorafenib on liver cancer cells and liver cancer stem cells and inhibit the occurrence and development of liver cancer.
Example 5 effect of the GASC1 inhibitor SD70 in combination with sorafenib treatment of transplanted tumors in liver cancer stem cells on cell cloning and transplanted tumor volume.
Hepatoma cells were plated in 24-well plates with 200 cells per well, 3 replicates per sample, and 0 μ M and 2.5 μ M sorafenib, respectively, and combined with the GASC1 inhibitor SD70, were added the next day. The culture solution was aspirated every 3 days, the same concentration was added, and the treatment was continued for 4 times, i.e., 14 days later, fixation was performed with 4% by mass of paraformaldehyde, crystal violet staining was performed, and the colony formation rate of the cells in each well was observed and counted, as shown in a in fig. 5. As can be seen from A in FIG. 5, the use of Sorafenib with a concentration of 2.5. mu.M has no influence on the clonality of liver cancer cells and liver cancer stem cells, the difference is not significant, it is probable that the liver cancer cells and liver cancer stem cells have drug resistance to Sorafenib after the Sorafenib is used for a long time, and the Sorafenib with a concentration of 0.5. mu.M can significantly inhibit the formation of cell clones after being combined with SD 70. Suggesting that the reduction of the expression level of GASC1 can reduce the tolerance of the liver cancer patient to sorafenib.
According to the experiment of in vitro cell cloning, the interference of GASC1 expression or the combination of the GASC1 inhibitor and sorafenib can obviously reduce the cloning forming capability of the liver cancer cell or the liver cancer cell with the dry characteristic. To further verify the above conclusion, in NOD/SCID mice, it was verified that the combination of the GASC1 inhibitor SD70 and sorafenib could significantly reduce the clonogenic capacity of liver cancer stem cells, and the specific method was: the number of injected cells was 1X 106Or 5X 106When the subcutaneous tumor grows to 50mm3Experimental NOD/SCID mice were divided into 4 groups: (1) blank control group, no treatment; (2) sorafenib group, sorafenib gavage at 100 mg/Kg; (3) GASC1 inhibitor SD70 by intraperitoneal injection; (4) in the group of sorafenib combined with the GASC1 inhibitor SD70, sorafenib was administered at 100mg/Kg while injecting the GASC1 inhibitor SD70 intraperitoneally, and after 10-16 days of continuous treatment, mice were sacrificed, tumors were stripped off, and tumor volumes were measured (results are shown in B in FIG. 5). As can be seen from B in fig. 5, the SD70 group was able to reduce the volume of the transplanted tumor compared to the control group; the sorafenib group can inhibit the tumorigenic capacity of the liver cancer cells, but has no obvious inhibiting effect on the tumorigenic capacity of the tumor stem cells; the SD70 group in combination with the sorafenib group significantly inhibited the growth of subcutaneous transplantable tumors in mice. The results indicate that the drug resistance of the liver cancer stem cells to sorafenib is reduced after the GASC1 expression is inhibited, and finally the effect of killing tumor cells and inhibiting tumor progression is achieved.
Example 6 Effect of GASC1 inhibitor SD70 in combination with Sorafenib treatment on Ki-67 expression and apoptosis in transplanted tumors in liver cancer Stem cells
Immunohistochemistry and statistical analysis of transplantable tumors: the mass fraction of the denuded tumor in example 5 isFixing with 4% paraformaldehyde, embedding in paraffin, continuously slicing at 4-5 μm, and processing with H&E staining, observing pathological form of the transplanted tumor under a microscope, and then using EnvisionTMImmunohistochemical techniques detected Ki-67 and clear-caspase 3 expression, observed under the mirror and photographed, and the results are shown in FIG. 6. As can be seen from FIG. 6, H&E staining results show that the tumor cell morphology of the transplanted tumor of the GASC1 inhibitor SD70 combined with sorafenib treatment group is unchanged compared with the control group; the immunohistochemical detection shows that the Ki-67 in the transplanted tumor inhibiting GASC1 combined with sorafenib treatment group is remarkably reduced compared with the SD70 group and the sorafenib group; while the expression of clear-caspase 3 was significantly increased. The above research results further confirm that GASC1 is associated with the mechanism of resistance to sorafenib in liver cancer stem cells.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
- Application of a GASC1 inhibitor in preparing a medicament for treating liver cancer.
- 2. Use according to claim 1, characterized in that: the GASC1 inhibitor is SD70 or lentivirus or adeno-associated virus interfering with the expression of GASC 1.
- 3. Use according to claim 1, characterized in that: the lentivirus interfering with GASC1 expression contains Lv-sh-GASC1 plasmid.
- 4. Use according to claim 1, characterized in that: the GASC1 inhibitor is applied to the preparation of drugs for inhibiting the clonogenic, balling-forming or tumorigenic capacity of liver cancer cells or liver cancer stem cells.
- 5. A composition for treating liver cancer, comprising: the complex contains a GASC1 inhibitor and sorafenib.
- 6. Use of the complex of claim 5 in the preparation of a medicament for treating liver cancer.
- 7. Use according to claim 6, characterized in that: the compound is applied to the preparation of medicines for inhibiting the clone formation, the balling-up capability and the tumor forming capability of liver cancer cells or liver cancer stem cells.
- 8. Use according to claim 6, characterized in that: the compound is applied to the preparation of medicines for promoting the apoptosis of liver cancer cells or liver cancer stem cells.
- Application of the GASC1 inhibitor in preparing a medicament for overcoming the drug resistance of liver cancer to sorafenib.
- Application of the GASC1 inhibitor in preparing a medicament for enhancing the sensitivity of sorafenib to liver cancer.
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| AU2018200876B2 (en) * | 2012-10-02 | 2019-09-19 | Gilead Sciences, Inc. | Inhibitors of histone demethylases |
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| CN1436236A (en) * | 2000-06-12 | 2003-08-13 | 大塚制药株式会社 | GASCI gene |
| AU2018200876B2 (en) * | 2012-10-02 | 2019-09-19 | Gilead Sciences, Inc. | Inhibitors of histone demethylases |
| CN107429246A (en) * | 2014-10-31 | 2017-12-01 | 麻省理工学院 | Large-scale parallel for CRISPR combines science of heredity |
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