CN111617251B - Use of MDM4 inhibitors as medicaments for the treatment of nasopharyngeal carcinoma - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to application of an MDM4 inhibitor as a medicine for treating nasopharyngeal carcinoma. MDM4 inhibitors can solve the problem of low sensitivity of nasopharyngeal carcinoma cells to radiation therapy by inhibiting MDM4, a key factor that is sensitive to radiation. Radiotherapy is the main therapy for treating nasopharyngeal carcinoma, but if nasopharyngeal carcinoma cells generate radiotherapy resistance, namely, under the original dosage and frequency of radiotherapy, tumor cells continue to grow and are not inhibited, radiotherapy can be disabled, and thus the disease of a patient is rapidly progressed and even died. The scheme discovers and utilizes that the activation of MDM4 is a key mechanism for causing the resistance of nasopharyngeal carcinoma to radiotherapy, and restores the sensitivity of the nasopharyngeal carcinoma to the radiotherapy by using the means of MDM4 inhibition. The scheme can be applied to the preparation of medicaments for treating nasopharyngeal carcinoma and the practical operation of research.

Description

Use of MDM4 inhibitors as medicaments for the treatment of nasopharyngeal carcinoma

Technical Field

The invention relates to the field of biomedicine, in particular to application of an MDM4 inhibitor as a medicine for treating nasopharyngeal carcinoma.

Background

Nasopharyngeal carcinoma (NPC) refers to a malignant tumor that occurs in the top and side walls of the nasopharyngeal cavity. Is one of high-incidence malignant tumors in China, and the incidence rate of the malignant tumors is the first of the malignant tumors of ear, nose and throat. Radiotherapy (i.e. radiation therapy, in this case specifically ionizing radiation) is the first choice and the main treatment for nasopharyngeal carcinoma (no local metastasis), and simple radiotherapy has good control effect on early nasopharyngeal carcinoma. However, solid tumors such as nasopharyngeal carcinoma develop some tolerance to radiation after initial radiation therapy. The reason is that under the effect of early-stage radiation, a large amount of single-strand breaks or double-strand break damages occur in DNA, DNA repair channels of cells are activated, the self-repair function of cancer cells is enhanced, the DNA escapes from the processes of apoptosis, necrosis or autophagy and the like caused by radiation, and the radiation treatment effect is poor. Even if a large dose of radiation therapy is used in the later period, the development of solid tumors still cannot be prevented, and the normal tissues are damaged greatly due to the radiation therapy, so that the patient is burdened greatly, and the life quality and the survival rate of the patient after the therapy are influenced. Therefore, there is a need to find a method or a drug for targeting nasopharyngeal carcinoma tumor tissues to enhance the radiation sensitivity of cancer cells, so as to increase the effect of radiation therapy, reduce the burden of patients and prolong the survival time of patients.

Disclosure of Invention

The invention aims to provide application of an MDM4 inhibitor as a medicine for treating nasopharyngeal carcinoma, and solves the problem of low sensitivity of nasopharyngeal carcinoma cells to radiation therapy through inhibition of MDM4 which is a key factor of radiation sensitivity.

In order to achieve the purpose, the invention adopts the following technical scheme:

use of an MDM4 inhibitor as a medicament for the treatment of nasopharyngeal carcinoma.

The principle and the advantages of the scheme are as follows: MDM4 is collectively called murine double minute 4 (murine double minute 4), also known as MDMX or HDMX. The increase of the expression level of the MDM4 protein can increase the radiotherapeutic resistance of nasopharyngeal carcinoma cells, and the MDM4 inhibitor can inhibit the expression or translation of MDM4 protein, thereby greatly reducing the generation of the MDM4 protein-mediated radiosensitivity of the nasopharyngeal carcinoma cells. Nasopharyngeal carcinoma grows in an infiltrating manner and is not clearly demarcated with surrounding tissues, and the nasopharyngeal carcinoma is difficult to completely cut off by an operation, so that the life treatment of a patient is seriously influenced. Nasopharyngeal carcinoma is mostly sensitive to radiotherapy, so radiotherapy is the main therapy for treating nasopharyngeal carcinoma. However, if the nasopharyngeal carcinoma is metastasized to a distant site in the early stage or is resistant to radiotherapy (tumor cells continue to grow but are not inhibited at the original dose and frequency of radiotherapy), the radiotherapy will fail, resulting in rapid disease progression and even death of the patient. The inventors have found that activation of MDM4 is a key mechanism for causing resistance to radiotherapy for nasopharyngeal carcinoma, and inhibition of MDM4 with an inhibitor restores the sensitivity of nasopharyngeal carcinoma to radiotherapy. The MDM4 inhibitor can be used in nasopharyngeal carcinoma with resistance to radiotherapy, and can also be used in nasopharyngeal carcinoma with sensitivity to radiation, so as to further enhance the sensitivity to radiation and reduce the dosage of radiotherapy.

It is reported in the literature that MDM4 has the function of inhibiting DNA repair (DNA damage of DNA chain breakage type), and the enhancement of the cellular DNA repair function is in positive correlation with the enhancement of the radiation resistance of the cell (ionizing radiation can cause DNA damage, thereby causing apoptosis, autophagy or necrosis of cancer cells; the stronger the DNA repair function is, the more DNA damage is repaired, the cancer cells can escape from the apoptosis, autophagy or necrosis process, namely the cancer cells can survive in a large amount), so the up-regulation of the MDM4 can increase the radiation sensitivity of the cell (the radiation resistance is reduced). However, the inventor finds that the fact is not the case through long-term research, and in nasopharyngeal carcinoma, the increase of the expression level of MDM4 protein can shield the function of P53, inhibit the activation of P53 related apoptosis pathway, increase the radiotherapeutic resistance (radiation resistance) of nasopharyngeal carcinoma cells, and cause the failure of radiotherapeutic treatment.

The inventor researches the characteristics of nasopharyngeal darcinoma gene and protein expression by means of high-throughput screening, and finds that the expression of hundreds of proteins is obviously up-regulated in nasopharyngeal darcinoma cells with radiotherapy resistance. The inventors further studied these up-regulated proteins, and found that a large number of up-regulated proteins are not directly related to the radiosensitivity (radiotherapeutic resistance) of nasopharyngeal carcinoma. The inventors have found that the MDM4 protein has a strong correlation with the radiation sensitivity of nasopharyngeal carcinoma cells through a large number of tests. In particular, in example 1, the MDM4 protein has a greater effect on the radiosensitivity of nasopharyngeal carcinoma cells: the MDM4 protein expression quantity is increased, and the radiation resistance of nasopharyngeal carcinoma cells is enhanced; the MDM4 protein expression quantity is reduced, the radiation resistance of nasopharyngeal carcinoma cells is weakened, therefore, the MDM4 protein is a key factor for the resistance of nasopharyngeal carcinoma to radiotherapy, and can be used as a target of drug action to inhibit the generation of the resistance of nasopharyngeal carcinoma to radiotherapy, thereby enhancing the sensitivity of nasopharyngeal carcinoma to ionizing radiation and enhancing the treatment effect of radiotherapy.

Further, the MDM4 inhibitor is an agent that inhibits the expression of MDM4 protein, or the MDM4 inhibitor is an agent that inhibits the activation of MDM4 protein.

In this context, an MDM4 inhibitor is understood to mean a substance which inhibits the transcription of the MDM4 gene, a substance which inhibits the translation of the mRNA of MDM4, a substance which inhibits the activation (or activation) of the MDM4 protein (the first two may also be referred to collectively as a substance which inhibits the expression of the MDM4 protein). Activation (activation) of the MDM4 protein involves a variety of means, including phosphorylation of the protein. MDM4 inhibitors are useful as promoters of the radiosensitivity of nasopharyngeal carcinoma cells in the treatment of nasopharyngeal carcinoma.

By adopting the scheme, the promoter for the radiation sensitivity degree of the nasopharyngeal carcinoma cells can increase the sensitivity degree of the nasopharyngeal carcinoma cells to radiation by directly or indirectly inhibiting the transcription of MDM4 gene, or inhibiting the translation of mRNA of MDM4, or inhibiting the expression of MDM4 protein, or inhibiting the activation of MDM4 protein, and can reduce the chemotherapeutic dose by promoting the radiation sensitivity degree, namely, the cancer cells can be killed by small-dose chemotherapy so as to resist the radiotherapeutic resistance phenomenon of the nasopharyngeal carcinoma cells. Radiotherapy resistance (radiation therapy resistance) in this scheme specifically means: under the original dose and frequency of radiotherapy, tumor cells continue to grow and are not inhibited, so that the radiotherapy is ineffective, and the phenomenon can lead the disease of a patient to rapidly progress and even die.

Further, the MDM4 inhibitor is one or a mixture of AZD1390, CHIR-124, Milciclib or NSC 207895.

Using the above protocol, AZD1390(CAS No.2089288-03-7) is a highly selective, orally bioavailable, brain-permeable inhibitor of ATM; CHIR-124(CAS No.405168-58-3) is a novel potent Chk1 inhibitor; PHA-848125(Milciclib) (CAS No.802539-81-7) is a potent, ATP-competitive CDK inhibitor; NSC207895(CAS No.58131-57-0) directly inhibits MDM4 activity. The inventor researches and discovers that the substances can inhibit the activity of MDM protein, thereby increasing the radiation sensitivity of cells and increasing the effect of radiotherapy.

Furthermore, AZD1390 had an in vitro effect concentration of 10nm and an in vitro effect time of 24 h.

With the above protocol, treatment at AZD 139010 nm concentration for 24h restored the sensitivity of CNEs to irradiation (example 4).

Further, mice with AZD1390 were administered at a daily dose of 20mg/kg for 6 days.

Using the above protocol, after a full inhibition of the MDM4 pathway in nasopharyngeal carcinoma tissues, irradiation was performed after 6 days of induction treatment with 20mg/kg AZD1390 per day to restore the sensitivity of mouse tumors to irradiation (example 5).

Furthermore, the concentration of NSC207895 was 10 μ M in vitro and the duration of in vitro action was 24 h.

Using the protocol described above, CNE cells were treated for 24h with 10. mu.M NSC207895 and inhibited the expression of MDM4 protein (example 2).

Further, the concentration of miciclib was 5 μ M in vitro and the duration of action in vitro was 24 h.

By using the above protocol, CNE cells treated with 5 μ M micciclib for 24h inhibited the expression of MDM4 protein, suggesting that micciclib may act as an MDM4 inhibitor (example 3).

Furthermore, the concentration of CHIR-124 was 5. mu.M in vitro and the duration of action in vitro was 24 h.

Using the above protocol, treatment of CNE cells with 5. mu.M CHIR-124 for 24h inhibited the expression of MDM4 protein, suggesting that CHIR-124 may act as an MDM4 inhibitor (example 3).

Further, the MDM4 inhibitor is a shRNA combination for degrading the mRNA of the MDM4 protein.

By adopting the scheme, the shRNA can degrade the mRNA of the MDM4, block the translation process of the MDM4 protein and reduce the expression quantity of the MDM4 protein. shRNA (short hairpin RNA) is cloned into an expression vector and sent into a cell body, and then can form double-stranded RNA (dsRNA) which is processed by an RNAi channel to mediate the degradation of a target mRNA sequence.

Further, the shRNA combination comprises three or more of a first shRNA with a sequence of SEQ ID NO. 1, a second shRNA with a sequence of SEQ ID NO.2, a third shRNA with a sequence of SEQ ID NO. 3, a fourth shRNA with a sequence of SEQ ID NO.4, a fifth shRNA with a sequence of SEQ ID NO.5 and a sixth shRNA with a sequence of SEQ ID NO. 6.

By adopting the scheme, three or more shRNAs are simultaneously transferred into a target cell, and the sufficient degradation of the mRNA of the MDM4 can be ensured (see example 1 and Table 1 for details).

Drawings

FIG. 1 shows the expression of MDM4 protein in CNE2 and CNE according to example 1 of the invention.

FIG. 2 shows the results of the flow-type fluorescence screening experiment (GFP fluorescent protein, CNE-Vector cell line) of example 1 of the present invention.

FIG. 3 shows the results of the flow-type fluorescence screening experiment of example 1 of the present invention (GFP fluorescent protein, CNE-shMDM4 cell line).

FIG. 4 shows the detection of the expression level of MDM4 protein by WB of example 1 of the present invention (CNE-Vector cell line and CNE-shMDM4 cell line).

FIG. 5 shows the MTT assay results of example 1 of the present invention (CNE-Vector cell line and CNE-shMDM4 cell line).

FIG. 6 shows the results of plate culture of example 1 of the present invention (CNE-Vector cell line and CNE-shMDM4 cell line).

FIG. 7 shows the statistical results of plate culture SF according to example 1 of the present invention (CNE-Vector cell line and CNE-shMDM4 cell line).

FIG. 8 shows the statistical results of the flow cytometry experiments of example 1 of the present invention (CNE-Vector cell line and CNE-shMDM4 cell line).

FIG. 9 shows WB results of example 2 of the present invention.

Figure 10 shows the qPCR results of example 2 of the invention.

FIG. 11 shows the WB results (using AZD1390) for example 3 of the present invention.

FIG. 12 shows the WB results (using CHIR-124) of example 3 of the present invention.

FIG. 13 shows WB results (using Milciclib) for example 3 of the invention.

FIG. 14 shows the results of DNA comet assay of example 4 of the present invention.

Figure 15 shows the survival curve of mice according to example 5 of the present invention (pre-experiment).

FIG. 16 shows the survival curve of the mice of example 5 of the present invention (official experiment).

Detailed Description

In examples 1-5, cell lines used in this protocol include CNE2 and CNE: nasopharyngeal carcinoma radiotherapy-sensitive CNE2 cell line (EXPASY, CVCL _6889), nasopharyngeal carcinoma radiotherapy-resistant CNE cell line (constructed in the laboratory). Antibodies used by WB in this protocol: CHECK1 (cat # 2360), p-CHECK1 (cat # 2348), CDK2 (cat # 18048) were all purchased from CST corporation; MDM4 (cat # ab49993) was purchased from Abcam corporation. Milciclib (cat # S2751), NSC207895 (cat # S2678), AZD1390 (cat # S8680), and CHIR-124 (cat # S2683) used in this protocol were purchased from Selleck, Inc.

The construction method of the CNE cell line comprises the following steps:

inoculating CNE2 cell line in RPMI 1640 culture solution containing 10% calf serum and 3% glutamine ammonia, and culturing at 37 deg.C and 5% C0₂Culturing in an incubator with saturated humidity. The basic conditions for the following irradiation were: x-ray irradiation with linear accelerator 6MVThe distance between the radioactive source and the cell is 100cm, and the tissue compensation glue with the thickness of 1cm is coated on the surface of the cell. Collecting CNE2 cells in exponential growth phase, digesting into single cell suspension, and planting in 25cm²The density of the planted cells in the culture bottle is 1.0 multiplied by 10⁴/cm²And irradiating 2Gy after the wall is attached, and continuing to carry out conventional culture for 2-3 generations. After the cells are stably grown after irradiation, the above process is repeated for 4 times, 6 times, 8Gy irradiation times and 10Gy irradiation times, 20Gy irradiation times and 3 times, 50Gy irradiation times and 100Gy irradiation times, and the whole irradiation and culture process lasts for 24 months. The surviving cells were monocloned to obtain the radiation therapy resistant cell line CNE. CNE cells were routinely passaged for more than 5 passages and used in this experimental study. And (3) the constructed CNE cell line is subjected to detection (cell genetic quality identification detection), the DNA typing of the strain is matched with the CNE cell line in the CRC database, and the cell line obtained by induction is proved to be the CNE cell line.

Example 1: research on key factors for nasopharyngeal carcinoma to resist radiotherapy

The inventor discovers that the expression level of MDM4 protein in a CNE cell line is obviously up-regulated by screening and comparing a large number of protein factors in the CNE2 cell line sensitive to nasopharyngeal carcinoma radiotherapy and a CNE cell line resistant to radiotherapy. Specifically, the inventor uses a western blot method to detect the expression of MDM4 in a nasopharyngeal carcinoma radiotherapy-sensitive CNE2 cell line and a nasopharyngeal carcinoma radiotherapy-resistant CNE cell line, and the experimental result is shown in figure 1, and the expression level of MDM4 protein in the CNE cell line is obviously higher than that of MDM4 protein in the CNE2 cell line.

An MDM4 shRNA sequence (table 1) is designed to inhibit MDM4 in CNE by taking MSnm-IRES-GFP as a Vector, cells with fluorescence expression are screened by flow fluorescence, a control cell line CNE-Vector (figure 2) with MSnm-IRES-GFP transferred into is screened, and a cell line CNE-shMDM4 (figure 3) with MSnm-shMDM4-IRES-GFP transferred into and stably interfering with the expression of MDM4 is screened. The WB detection results of the expression levels of MDM4 protein of the two cell lines are shown in FIG. 4, and it can be seen that the expression level of MDM4 protein in CNE-shMDM4 is reduced, and the cell line CNE-shMDM4 which stably interferes with the expression of MDM4 is successfully constructed.

CNE-Vector and CNE-shMDM4 cells were irradiated at different doses, and the inhibition rate of the cells after irradiation was measured by MTT, and the inhibition rate of CNE-shMDM4 cells by irradiation was found to be higher than that of CNE-Vector cells (FIG. 5).

After irradiation of 50Gy, 5000 cells of CNE-Vector, CNE-shMDM4 were added to the culture medium to prepare a single cell suspension, the suspension was cultured in a petri dish, the experiment was stopped after the cells were cloned, the number of clones was calculated after staining, the clone formation rate was calculated as the clone formation rate (PE)%, formed clone number/seeded cell number × 100%, and the survival fraction was calculated as the clone formation number of SF ═ experimental group/(cell inoculation number × PE) to find that SF of CNE-shMDM4 cells was smaller than that of CNE-Vector cells (fig. 6 and fig. 7). Apoptosis rate was calculated by Annexin V plus PI staining and was found to be greater than CNE-Vector for CNE-shMDM4 cells (FIG. 8). Indicating that interference with MDM4 expression could shift CNE to radiotherapeuticauy sensitive nasopharyngeal carcinoma cells. In this experiment, three shRNAs in Table 1 were required to be transferred into cells to achieve MDM4 inhibition (degradation of the mRNA of MDM 4). The specific process is as follows: respectively constructing plasmids (the empty plasmid is MSnm-IRES-GFP) by using the sequences of any three shRNAs shown in the table 1 to form three plasmids containing double-stranded DNA for coding the shRNAs; the constructed plasmid is transferred into a target cell, and the mRNA inhibition of MDM4 can be formed. The above specific process is a conventional means for molecular cloning in the prior art, and is not described herein. In this example, SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 were specifically used.

TABLE 1 MDM4 shRNA interference sequences (DNA sequences of shRNAs are shown)

Through the study of the present example, the inventors found that the MDM4 protein has a great influence on the radiation sensitivity of nasopharyngeal carcinoma cells: the MDM4 protein expression quantity is increased, and the radiation resistance of nasopharyngeal carcinoma cells is enhanced; the MDM4 protein expression quantity is reduced, the radiation resistance of nasopharyngeal carcinoma cells is weakened, therefore, the MDM4 protein is a key factor for the resistance of nasopharyngeal carcinoma to radiotherapy, and can be used as a target of drug action to inhibit the generation of the resistance of nasopharyngeal carcinoma to radiotherapy, thereby enhancing the sensitivity of nasopharyngeal carcinoma to ionizing radiation and enhancing the treatment effect of radiotherapy.

Example 2: mechanism research of MDM4 protein on influence on radiation sensitivity of nasopharyngeal carcinoma cells

Using immunofluorescence confocal, MDM4 was found to co-localize with P53 both in the CNE cytoplasm and nucleus, and to distribute P53 more in the cytoplasm (results images not shown). After inhibiting MDM4 with the specific inhibitor of MDM4, NSC 20789510 μ M for 24h, P53 expression was phosphorylated although slightly decreased (WB assay results are shown in fig. 9, DMSO was used as a negative control, and protein expression was observed without irradiation and with 50Gy irradiation). Indicating that NSC207895 can inhibit MDM4 and cause changes in the phosphorylation level of P53.

The expression of P21 and P53AIP1 downstream factors of P53 (primer sequences are shown in a table 2) is detected by qPCR, and the experiments are divided into a DMSO-only treatment group, a DMSO and 50Gy irradiation treatment group, an NSC 207895-only treatment group and an NSC207895 and 50Gy irradiation treatment group. NSC207895 was treated for 24h at 10. mu.M. As a result, inhibition of MDM4 was found to activate P53 downstream key factor P21, and expression of P53AIP1 (FIG. 10). It is suggested that MDM4 may affect P53 phosphorylation and its activation of downstream factors by binding to P53, thereby affecting the sensitivity of nasopharyngeal carcinoma to irradiation.

TABLE 2 downstream factor qPCR primer sequences of P53

Example 3: experimental validation of AZD1390, CHIR-124 and Milciclib to inhibit MDM4 protein expression

CNE cell lines were treated with AZD1390, CHIR-124 and Milciclib, respectively, and the effect of the three drugs on MDM4 protein expression was studied using the western blot method. The specific experimental process is as follows, digesting and mixing the cells uniformly to prepare a uniform cell suspension, inoculating the cell suspension into a 10cm culture dish, performing a test when the cells grow to 80% after plating, dissolving the drug in 5 mu M DMSO, and adding a culture medium to enable the final concentration of the drug to reach respective concentration: 10 μ M AZD1390 ( treatments 24h and 48h, respectively), 5 μ M CHIR-124 ( treatments 24h and 48h, respectively), 5 μ M Milciclib ( treatments 24h and 48h, respectively). Negative controls used 5 μ M DMSO (24 h and 48h treatment, respectively). The experimental results show that AZD1390 (FIG. 11), CHIR-124 (FIG. 12) and Milciclib (FIG. 13) all inhibit the expression of MDM4 protein in CNE, suggesting that AZD1390, CHIR-124 and Milciclib can be used as MDM4 inhibitors.

Example 4: AZD1390 cell growth inhibition assay

The effect of AZD1390 on CNE2, the growth of CNE cells, was observed. In this example, the inhibition rate of cell growth at 24h after administration was observed in MTT assay, and the inhibition rate of cell growth at 1.5X 10 after administration⁴One/well was seeded in 96-well plates and the 10nm AZD1390 was observed to have only a slight effect on CNE2, CNE cell growth, as compared to non-dosed blank wells (table 3). Experiments using DMSO as a negative control, the survival rate of CNE2 and CNE was 97.25% and 97.25% under the effect of 10nm DMSO.

Table 3: survival after treatment of nasopharyngeal carcinoma cells with different concentrations of AZD1390 (100% survival with placebo)

Using a flow cytometric apoptosis assay, the effect of AZD1390 on CNE2, CNE apoptosis was observed: we observed that treatment with AZD1390 at AZD 139010 nm concentration for 24h did not induce CNE2(DMSO 9.41 ± 1.59%, VS AZD 139010.27 ± 2.89%, p ═ 0.675) and CNE apoptosis (DMSO 9.56 ± 2.03% VS AZD 139012.23 ± 2.05%, p ═ 0.1844).

To understand the effect of AZD1390 on the sensitivity of CNE2 and CNE irradiation, we treated at different concentrations of AZD1390 for 24h and then irradiated with X-rays at different doses. The inhibition of CNE2 by irradiation, CNE growth, is shown in table 4. After the AZD 139010 nm is treated for 24 hours at the concentration, the inhibition rate of 50Gy irradiation on the growth of CNE2 is 43.46%, the inhibition rate of 100Gy irradiation on the growth of cells is 58.04%, and the inhibition on the growth of cells after 200Gy irradiation reaches 77.90%; after the treatment of AZD 139010 nm concentration for 24h, the inhibition rate of 50Gy irradiation on the growth of CNE is increased from 19.48% to 43.49%, the inhibition rate of 100Gy irradiation on the growth of cells is increased from 21.71% to 53.46%, and the inhibition rate of 200Gy irradiation on the growth of cells is increased from 27.81% to 70.54%, which is similar to the inhibition rate of irradiation on CNE 2. It can be seen that AZD 139010 nm concentration treatment for 24h can restore the sensitivity of CNE to irradiation.

TABLE 4 cell growth inhibition after AZD1390 and X-ray treatment (relative to blank control)

By using flow cytometry apoptosis detection, the apoptosis rate of CNE2 is 24.06 +/-2.75% under the irradiation dose of 50 Gy; 32.97 +/-3.11% under the irradiation dose of 100 Gy; after the CNE is treated for 24 hours by AZD 139010 nm concentration and then irradiated, the apoptosis rate of the CNE under 50Gy irradiation is increased from 9.60 +/-0.87% to 24.25 +/-2.04%; the apoptosis rate of CNE increased from 10.97 + -0.29% to 39.39 + -3.02% at a radiation dose of 100Gy, further demonstrating that treatment with AZD 139010 nm concentration for 24h restored CNE sensitivity to radiation.

The method comprises the steps of detecting the relationship between a drug and DNA damage by utilizing a comet-star electrophoresis experiment, setting a 10nM DMSO control group, a 10nM AZD1390 treatment group, a 10nM DMSO +100Gy irradiation treatment group and a 10nM AZD1390+100Gy treatment group, and applying 100Gy irradiation treatment to the 10nM DMSO +100Gy irradiation treatment group and the 10nM AZD1390+100Gy treatment group after CNE cells are treated by DMSO or AZD1390 for 24 h. The statistical result of the comet tail distance (effective moment) is shown in fig. 14, the effect of AZD1390 on DNA damage repair in CEN is observed, and it is found that AZD1390 can obviously aggravate DNA damage in CNE cells, inhibit DNA repair, and finally promote CNE cell apoptosis (P < 0.001).

Example 5: in vivo experiments

The tumor tissues of patients with nasopharyngeal carcinoma who relapse in situ in the radiotherapy process are cut into small pieces, and the small pieces are inoculated under the skin of the right hind leg of an NCG mouse (mouse immunodeficiency model) to establish a nasopharyngeal carcinoma PDX model resistant to radiotherapy. Median survival time in mice was 8 days after tumor tissue inoculation. All mice tumors grow in an infiltration manner, destroy the skin upwards, invade thigh muscles inwards, and retain the characteristic of the recurrent nasopharyngeal carcinoma in situ of the patient. Fifth day after inoculationTumor tissue growing to 100mm³Volume, NCG mice were divided into 4 groups and 6 experiments were performed per group.

The mice right hind leg tumor was irradiated with X Ray (5Gy, 10Gy, 20Gy, pre-experiment) at doses, respectively, and the effect of different irradiation doses on the mice tumor was observed. Median survival times for control (no radiation) mice were 8 days, 5Gy 7.5 days, 10Gy 9 days, 20Gy 7 days. The 20Gy irradiated mice had swollen hind legs after irradiation and failed to walk, and 4 mice had tumors liquefied and ulcerated (66.7%), and the irradiation was considered to damage the hind legs tissue of the mice, resulting in aseptic inflammation and inducing mice to die due to failure. According to death time and dissection results, the 10Gy and 5Gy irradiation dose does not affect the death time of mice (p is 0.0722) and does not cause serious tissue damage caused by irradiation, and the 10Gy is selected for subsequent experiments (figure 15). In fig. 15, a black line indicates control (no irradiation treatment), a yellow line indicates 5Gy irradiation, a green line indicates 10Gy irradiation, and an orange line indicates 20Gy irradiation.

The tumor tissues of patients with nasopharyngeal carcinoma who relapse in situ in the radiotherapy process are cut into small pieces and inoculated under the skin of the right hind leg of an NCG mouse, and a nasopharyngeal carcinoma PDX model resistant to radiotherapy is established. After successful inoculation, the tumor tissue grows to 100mm³After the volume, NCG mice were divided into 5 groups and 6 experiments were performed per group (official experiment). Compared with single irradiation (10Gy), median survival time of the single-dose group (AZD 139020 mg/Kg) is prolonged to 12 days, which is longer than that of the control group (8 days), the single-dose 10Gy irradiation group (9 days) and the simultaneous-dose irradiation group (11 days). The anatomical result shows that although the ipsilateral or contralateral lymph nodes are swollen when the mice die, pathological section HE staining shows that the cortex and pith of the lymph node structure are respectively clear, no tumor cells growing in nests are seen, and CK5/6 staining is negative, which indicates that the mice have no lymph node metastasis. The lung and liver of the mice are also normal in shape, and CK5/6 is negative, which indicates that no lung and liver metastasis exist.

The single administration group has in-situ tumor infiltration growth without reduction of the range; in-situ tumor infiltration growth in the control group, single 10Gy irradiated group and irradiated group (11 days) with concurrent administration, the range was expanded (fig. 16). In FIG. 16, the black line represents control (no irradiation and drug treatment), the blue line represents AZD 139020 mg/kg po qd (treated with drug only), the green line represents X Ray 10Gy (treated with radiation only), the yellow line represents AZD 139020 mg/kg po qd + X Ray 10Gy (irradiated immediately after dosing), and the red line represents AZD 139020 mg/kg po qd Add X Ray 10Gy (irradiated after dosing for a period of time). The experimental results suggest that: the death cause of the mice is the infiltration and damage of the primary tumor focus after inoculation; the single administration group can delay tumor infiltration but can not kill the tumor; irradiation with the administration of the drug (administration on the fourth day, irradiation starting on the fifth day, AZD 139020 mg/kg po qd + X Ray 10Gy in FIG. 16) did not increase the sensitivity of the tumor cells to irradiation.

The pre-irradiation administration time was prolonged, the administration was carried out 4-9 days after inoculation (6 days of single administration) and then the administration was continued after irradiation (10 days) (AZD 139020 mg/kg po qd Add X Ray 10Gy in FIG. 16), and the median survival time of the mice was significantly prolonged (15 days) to approximately 2 times that of the control group. And the dead mice are dissected to find that the tumor inoculated in situ at the right hind leg is obviously reduced or even disappears, and the ipsilateral and contralateral lymph nodes are not swollen. It follows from this that:

AZD1390 can restore the sensitivity of tumor-bearing tissues of a PDX model of nasopharyngeal carcinoma to radiation;

2. concurrent oral administration of AZD1390 and irradiation is not effective in restoring tumor sensitivity and the patient may not benefit;

3. after AZD1390 is used for induction treatment for 6 days, after the MDM4 pathway in nasopharyngeal carcinoma tissues is fully inhibited, irradiation is carried out, the sensitivity of the tumor to irradiation can be restored, and the obvious progress of the disease condition caused by single medicine feeding is avoided, and the side effect of the medicine is also avoided.

After 6 days of administration and irradiation with 10Gy, the regimen was continued with the best possible results to benefit the patients (results were consistent with the cellular experiments).

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Sichuan university

<120> use of MDM4 inhibitor as a medicament for the treatment of nasopharyngeal carcinoma

<130> 2020.05.22

<160> 10

<170> PatentIn version 3.5

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<400> 8

aaagtcgaag ttccatcgct c 21

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<400> 9

ggtgcccaag ttcacggag 19

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

ctggagagac ctagaccaag g 21

Claims (6)

1. Use of an MDM4 inhibitor for the manufacture of a medicament for the treatment of nasopharyngeal carcinoma, wherein the MDM4 inhibitor is one or a mixture of AZD1390, CHIR-124, miciclib and NSC 207895;

   or the MDM4 inhibitor is shRNA combination, and the shRNA combination comprises three or more of a first shRNA with a sequence of SEQ ID NO. 1, a second shRNA with a sequence of SEQ ID NO.2, a third shRNA with a sequence of SEQ ID NO. 3, a fourth shRNA with a sequence of SEQ ID NO.4, a fifth shRNA with a sequence of SEQ ID NO.5 and a sixth shRNA with a sequence of SEQ ID NO. 6.
2. 2. Use according to claim 1, characterized in that: the in vitro action concentration of AZD1390 was 10nm and the in vitro action time was 24 h.
3. 3. Use according to claim 1, characterized in that: mice with AZD1390 were administered at a daily dose of 20mg/kg and mice with AZD1390 were administered for a period of 6 days.
4. 4. Use according to claim 1, characterized in that: the concentration of NSC207895 was 10. mu.M in vitro, and the time of action was 24h in vitro.
5. 5. Use according to claim 1, characterized in that: the concentration of miciclib was 5 μ M in vitro and the duration of action in vitro was 24 h.
6. 6. Use according to claim 1, characterized in that: the concentration of CHIR-124 was 5. mu.M in vitro and the duration of action in vitro was 24 h.

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