Application of EZH2 inhibitor in preparation of drug for resisting temozolomide drug resistance

文档序号：293982 发布日期：2021-11-26 浏览：3次中文

阅读说明：本技术 Ezh2抑制剂在制备抵抗替莫唑胺耐药性药物中的应用 (Application of EZH2 inhibitor in preparation of drug for resisting temozolomide drug resistance ) 是由何志承平轶芳时雨姚小红曾晖刘庆郭海涛卞修武于 2021-08-11 设计创作，主要内容包括：本发明涉及EZH2抑制剂新的药物用途,涉及EZH2抑制剂在制备抵抗替莫唑胺(Temozolomide,TMZ)耐药性药物中的应用。以及EZH2抑制剂在制备提高胶质瘤细胞对TMZ敏感性药物中的应用。本发明通过大量一系列的体内外功能实验发现HOXA5表达与TMZ的IC50值呈显著正相关,研发了HOXA5在胶质瘤干细胞调控及TMZ耐药性产生过程中的作用。证明使用EZH2抑制剂GSK343可以部分回复由HOXA5过表达所引起的肿瘤细胞抵抗TMZ的杀伤作用,也可以使用EZH2抑制剂提高胶质瘤细胞对TMZ的敏感性,EZH2抑制剂可以作为TMZ耐药患者的联合用药选择。(The invention relates to a novel medicinal application of an EZH2 inhibitor, and relates to an application of an EZH2 inhibitor in preparing a medicament for resisting Temozolomide (TMZ) drug resistance. And the application of the EZH2 inhibitor in preparing the medicine for improving the sensitivity of glioma cells to TMZ. According to the invention, a large number of in vivo and in vitro functional experiments show that the expression of HOXA5 is in significant positive correlation with the IC50 value of TMZ, and the effect of HOXA5 in the process of glioma stem cell regulation and TMZ drug resistance generation is developed. It is proved that GSK343 can partially recover the killing effect of tumor cells against TMZ caused by over-expression of HOXA5 by using an EZH2 inhibitor, the sensitivity of glioma cells to TMZ can be improved by using an EZH2 inhibitor, and the EZH2 inhibitor can be used as a drug combination selection of TMZ resistant patients.)

Use of an EZH2 inhibitor for the preparation of a medicament against temozolomide resistance.

2. The use of claim 1 wherein the EZH2 inhibitor is used in combination with temozolomide.

3. The use according to claim 1 or 2, wherein the EZH2 inhibitor is GSK 343.

Application of an EZH2 inhibitor in preparation of a medicament for improving sensitivity of glioma cells to temozolomide.

5. The use of claim 4 wherein the EZH2 inhibitor is used in combination with temozolomide.

6. The use of claim 4 or 5, wherein the EZH2 inhibitor is GSK 343.

Technical Field

The invention belongs to the technical field of biological molecular medicines, and relates to an application of an EZH2 inhibitor in preparation of a drug for resisting temozolomide drug resistance.

Background

Brain gliomas are the most common intracranial primary malignancies of the adult central nervous system, including 4 types of astrocytomas, oligodendrogliomas, ependymomas, and mixed gliomas, while the highest grade of Glioblastoma (GBM) among astrocytomas is the most common adult intracranial malignancy, with the world health organization grading to grade iv.

GBM grows infiltratively, often invades surrounding normal tissues, is very difficult to completely excise by surgery, and has high recurrence rate. Currently, the treatment of GBM in the clinic generally employs a combination of maximal safe range surgical resection, Temozolomide (TMZ) chemotherapy, and adjuvant radiotherapy. The best treatment modalities for GBM are currently consistent with the concept of resecting as many tumors as possible within a certain safety margin and using STUPP protocol chemoradiotherapy, i.e. chemotherapy should be synchronized throughout the course of the radiotherapy. Unfortunately, GBM has been found and actively treated to have a median survival time of only 14.6 months, approximately only 30% of patients who survive two years, less than 10% of patients who survive 5 years, and a short survival time, which is highly undesirable, one of the most significant reasons being patient resistance to the chemotherapeutic drug TMZ.

TMZ is a standard chemotherapeutic drug for the treatment of glioblastoma and inhibits the growth of GBM by alkylating N-7 or O-6 on guanine residues, thereby improving overall survival of GBM patients. It is not effective in some patients, i.e., producing TMZ resistance. Some originally sensitive tumor cells can also gradually generate acquired drug resistance in the chemotherapy process to cause tumor progression and recurrence, and the effect of postoperative chemotherapy is greatly influenced. Thus, the use of TMZ in the treatment of GBM patients is largely limited.

It is widely accepted by the academia that the existence of glioma stem cells causes GBM cells to generate drug resistance to TMZ and other drugs, thereby causing poor treatment effect, but the mechanism of the drug resistance is still to be further clarified. Auffinger et al believe that after treatment with chemotherapeutic drugs such as TMZ, some tumor cells in glioma will transform into glioma stem cells, and these cells will exhibit stronger drug resistance to chemotherapeutic drugs, affecting the therapeutic effect. While Gong et al found that TMZ, although it caused death of glioma cells, had a minor effect on glioma stem cells, this effect was correlated with the expression of the multiple drug-resistant ATP-binding cassette (ABC) transporter G2 gene.

Therefore, how to reduce the TMZ drug resistance of GBM patients, improve the chemotherapy sensitivity of GBM patients and reduce the recurrence rate of tumors of patients is a problem to be solved by GBM clinical treatment.

Disclosure of Invention

In view of the above, the present invention aims to provide a new medical application of an EZH2 inhibitor, which reduces TMZ drug resistance of GBM patients, improves chemotherapy sensitivity of GBM patients, and reduces the recurrence rate of tumors in patients.

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

use of an EZH2 inhibitor for the preparation of a medicament against temozolomide resistance.

Further, EZH2 inhibitors were used in combination with temozolomide.

Further, the EZH2 inhibitor is GSK 343.

Application of an EZH2 inhibitor in preparation of a medicament for improving sensitivity of glioma cells to temozolomide.

Further, EZH2 inhibitors were used in combination with temozolomide.

Further, the EZH2 inhibitor is GSK 343.

The invention has the beneficial effects that: according to the invention, a large number of in vivo and in vitro functional experiments show that the expression of HOXA5 is in significant positive correlation with the IC50 value of TMZ, and the effect of HOXA5 in the regulation and control of glioma stem cells and the generation process of TMZ drug resistance is developed; further studies demonstrated that knockdown HOXA5 in combination with TMZ was effective in inhibiting tumor growth and extending survival in mice. The molecules interacting with HOXA5 and the expression relationship of the molecules are continuously found, and researches show that binding of HOXA5 and an EZH2CXC structural domain activates an innovative regulatory mechanism of a downstream classical molecule H3k27me3, so that the application of an EZH2 inhibitor GSK343 can partially recover the killing effect of tumor cells against TMZ caused by HOXA5 overexpression, the application of an EZH2 inhibitor can improve the sensitivity of glioma cells to TMZ, and the EZH2 inhibitor can be used as a medication choice for TMZ-resistant patients. The research result provides a new medicinal direction and experimental basis for targeting glioma stem cells and improving the clinical treatment effect of TMZ.

Drawings

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 results of experiments with the expression level of HOXA5 and with the knockdown and overexpression of HOXA5 in glioma cells.

FIG. 2 is a graph of HOXA5 expression correlated with prognosis of TMZ-treated GBM patients.

FIG. 3 shows the results of various experiments on mouse orthotopic transplantable tumors.

FIG. 4 is a network diagram of a HOXA5 interacting molecule.

FIG. 5 is a graph showing the expression of immunofluorescence double staining and immunohistochemical staining of HOXA5 and EZH 2.

Fig. 6 is a graph of the results of various experiments demonstrating the interaction of HOXA5 with EZH 2.

Fig. 7 is a graph of the results of various experiments demonstrating the interaction of over-expressed HOXA5 and the EZH2 inhibitor GSK 343.

Fig. 8 is a graph of the results of various experiments demonstrating that EZH2 inhibitors can increase glioma cell sensitivity to TMZ.

FIG. 9 is a HOXA5 shRNA lentiviral vector map.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

Example 1

1. Glioma cell lines and primary glioma stem cells were examined with QRT-PCR, respectively: HOXA5 mRNA expression levels in U87 (glioma cell line), GBM-1 (primary glioblastoma cells), T98G (human brain glioma cells), LN229 (brain glioma cells), GSC-2 (primary glioma stem cells), and GSC-3 (primary glioma stem cells). The detection method comprises the following steps:

1) cells in logarithmic growth phase were collected, centrifuged to collect the pellet.

2) The feijie RNA extraction kit is used for extracting RNA.

3) After the determination of the RNA concentration, a reverse transcription reaction system (20. mu.l) was set at an RNA concentration of 50. mu.g/. mu.l: RNA X. mu.l, 5 XPrime Script RT Master Mix 4. mu.l, DEPC water (supplemented to 20. mu.l, i.e. 20-X-4. mu.l). The reverse transcription reaction conditions were set as follows: 30min at 37 deg.C → 5sec at 85 deg.C → 4 deg.C forever.

4) RT-PCR: carrying out real-time fluorescent quantitative PCR detection on the cDNA obtained by reverse transcription in the step 3), wherein the specific reaction system is set as follows: cDNA 1. mu.l, 2 XSSYBR Premix Ex Taq 5. mu.l, the two are premixed and added into 8 connecting tubes according to 6. mu.l of each hole; 0.5. mu.l of forward primer, 0.5. mu.l of reverse primer, ddH₂O3. mu.l, and the three were premixed and added to a cap of an 8-tube set in an amount of 4. mu.l per well. Combining the 8-connecting pipe and the 8-connecting pipe cover, fully and uniformly mixing by using a palm centrifuge, and loading on a machine.

The PCR reaction system is set as follows:

and after the computer is operated, analyzing the result by using software.

The primer sequences required for this experiment are shown in table 1 below:

TABLE 1

2. Knockdown and overexpression of HOXA5 in glioma stem cells and differentiated glioma cells, respectively

Glioma cells are transfected on the basis of the constructed HOXA5 overexpression plasmid and shRNA sequence, and the next functional experiment is carried out after the puro screening and the flow cytometry sorting of GFP positive expression cells.

HOXA5 overexpression plasmid construction

The primer sequences are shown in table 2 below:

TABLE 2

Forward	gctctagaatgagctcttattttgtaaac	SEQ No.7
			Reverse	ataagaatgcggccgc tcagggacggaaggcccctc	SEQ No.8

The over-expression sequence is shown in SEQ No. 9.

ATGAGCTCTTATTTTGTAAACTCATTTTGCGGTCGCTATCCAAATGGCCCGGACTACCAGTTGCATAATTATGGAGATCATAGTTCCGTGAGCGAGCAATTCAGGGACTCGGCGAGCATGCACTCCGGCAGGTACGGCTACGGCTACAATGGCATGGATCTCAGCGTCGGCCGCTCGGGCTCCGGCCACTTTGGCTCCGGAGAGCGCGCCCGCAGCTACGCTGCCAGCGCCAGCGCGGCGCCCGCCGAGCCCAGGTACAGCCAGCCGGCCACGTCCACGCACTCTCCTCAGCCCGATCCGCTGCCCTGCTCCGCCGTGGCCCCCTCGCCCGGCAGCGACAGCCACCACGGCGGGAAAAACTCCCTAAGCAACTCCAGCGGCGCCTCGGCCGACGCCGGCAGCACCCACATCAGCAGCAGAGAGGGGGTTGGCACGGCGTCCGGAGCCGAGGAGGACGCCCCTGCCAGCAGCGAGCAGGCGAGTGCGCAGAGCGAGCCGAGCCCGGCGCCGCCCGCCCAACCCCAGATCTACCCCTGGATGCGCAAGCTGCACATAAGTCATGACAACATAGGCGGCCCGGAAGGCAAAAGGGCCCGGACGGCCTACACGCGCTACCAGACCCTGGAGCTGGAGAAGGAGTTCCACTTCAACCGTTACCTGACCCGCAGAAGGAGGATTGAAATAGCACATGCTCTTTGCCTCTCCGAGAGACAAATTAAAATCTGGTTCCAAAACCGGAGAATGAAGTGGAAAAAAGATAATAAGCTGAAAAGCATGAGCATGGCCGCGGCAGGAGGGGCCTTCCGTCCCTGA

The HOXA5 shRNA lentiviral vector map is shown in FIG. 9. The viral vector construction framework is shown in table 2:

TABLE 2

And (3) analyzing a sequencing result:

HOXA5 ShRNA-1 (shown as SEQ No. 10)

nntggnatnaatttgactgtaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccggccggactaccagttgcataatctcgagattatgcaactggtagtccggttttttgaattcggatccattaggcggccgcgtggataaccgtattaccgccatgcattagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcgctaccggacgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccnngagggcgagggcgatgccacctacggcaagctgacctgaagttcatctgcaccaccggcaagctgcccntgccctggacccaccctcgtgaccacccnnacnacggcgtgcagtgcnttcagcngctnncnaccacatnngcagcacganntnnnagtcggcatgcccnnnntacgnnccannnnnnnacatcttcttnaaggacgaacggnnactncanagnnnn

HOXA5 ShRNA-2 (shown as SEQ No. 11)

nnnnngataanttgactgtaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccggctgagtatctgagcgtttaaactcgagtttaaacgctcagatactcagttttttgaattcggatccattaggcggccgcgtggataaccgtattaccgccatgcattagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcgctaccggacgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccncaagttcagcgtgtccggcgaggggcgaagggcgatgccaccttacggctagctgaccctgaagttcatctgcaccannggcagctgcccgtgcccttgnnncacccctcnnngaccaccctgaactacggcgtgcagtgnnnagccgcttacccgacnacatgnncagcacgaacttnncagntcgnnntnncnnaaggctacgtcaggagcncacnatctttcttcag

FIG. 1 is the results of experiments with the expression level of HOXA5 and with the knockdown and over-expression of HOXA5 in each glioma cell line and primary glioma stem cells U87, GBM-1, T98G, LN229, GSC-2 and GSC-3 in which the expression level of HOXA5 mRNA is structurally shown in A in FIG. 1, and the IC50 values reflecting the tumor cell killing effect of Temozolomide (TMZ) are shown in B in FIG. 1, and correlation analysis is performed, and the C results in FIG. 1 show that HOXA5 expression is positively correlated with the IC50 value of TMZ, indicating that the killing effect of TMZ may be directly influenced by the high or low expression of HOXA 5. Next, we knock down and over-express HOXA5 in glioma stem cells and differentiated glioma cells respectively, and after detecting protein expression of apoptosis signal pathway related markers, the expression of BAX, clearage-caspase 3 and clearage-PARP is obviously increased after the expression of HOXA5 is knocked down, and the expression of the proteins is also reduced after the expression of HOXA5 is over-expressed (D in fig. 1), and the above results suggest that the expression change of HOXA5 can obviously influence the killing effect of TMZ on glioma cells and is directly reflected on the expression change of apoptosis signal pathway markers. In order to more intuitively reflect the condition that TMZ kills tumor cells after HOXA5 expression is changed, the proportion of apoptotic cells is detected by using flow cytometry, and as a result, the proportion of cells which are positively expressed by annexin V is obviously reduced after HOXA5 is over-expressed (E in figure 1), and the proportion of positive cells is obviously increased after HOXA5 is knocked down (F in figure 1). Finally, we detected that HOXA5 expression was significantly higher than that of non-drug resistant cells in the constructed T98G-resistant cell line (G in fig. 1), and based on this, after knocking down HOXA5 expression, IC50 values were significantly decreased again, suggesting that these cells partially restored sensitivity to TMZ killing (H in fig. 1).

Example 2

Validation of clinical pathological significance of HOXA5 expression in TMZ-treated glioblastoma specimens

The relationship between the expression of HOXA5 and TMZ IC50 value was confirmed by in vitro functional experiments in example 1, and then the relationship between the expression of HOXA5 and the curative effect of TMZ was first verified in a database, and it was found that the level of HOXA5 expression in the group treated with TMZ could effectively distinguish the prognosis of patients (A in FIG. 2), while the level of HOXA5 expression in the untreated group was not correlated with the curative effect of TMZ (B in FIG. 2), indicating that the curative effect of TMZ is closely correlated with the level of HOXA5 expression. Next, we analyzed HOXA5 expression in combination with MGMT promoter methylation, a common indicator for evaluating TMZ efficacy, and the results showed that the level of expression of HOXA5 in MGMT methylated group could effectively differentiate patient prognosis, while the level of expression of HOXA5 in MGMT unmethylated group had no correlation with patient prognosis (C in fig. 2), indicating that the development of MGMT independent drug resistance could be correlated with HOXA5 expression, suggesting the importance of evaluating HOXA5 expression and TMZ efficacy based on MGMT methylation.

Example 3 HOXA5 in combination with TMZ is effective in inhibiting tumor growth and prolonging survival of mice

The relationship between HOXA5 expression and TMZ efficacy was continued to be detected in mouse orthotopic transplants:

1) collecting knockdown HOXA 5-expressing glioma stem cells and control cells in logarithmic growth phase, adjusting cell density to 2 × 10 after centrifugal resuspension counting⁶One per ml.

2) After 4-6 weeks old female NOD/SCID mice were anesthetized by intraperitoneal injection using prepared pentobarbital sodium, 5. mu.l of cell suspension was injected perpendicularly and slowly at 0.5cm to the right and back at the intersection of the median anterior cerebral line and the lateral canthus line of both eyes using a protein microsyringe, the depth of injection was 0.5cm, and gentle and slow withdrawal was noted after completion of injection.

3) After 7 days of tumor implantation, treatment with temozolomide was started for 5 days, wherein the control group (Ctr) was injected with a solution without temozolomide and the experimental group (TMZ) was treated at 10 mg/kg; meanwhile, continuous feeding of doxycycline supplemented aqueous solution to HOXA5 knock-down group (TMZ + sh HOXA5) induced HOXA5 knock-down expression.

4) Mice were observed daily for survival and brain tissue was harvested immediately after symptoms had developed and cryosectioned and paraffin tissue samples prepared.

4) In vivo imaging experimental detection is carried out on 5 days, 12 days, 19 days and 26 days after establishment of the transplanted tumor model. The method specifically comprises the steps that 200ul luciferin substrate is injected into the abdominal cavity of each mouse, and after 10min, the anesthetized mice are detected by a living body imaging system.

5) After the mice naturally die, recording the time and analyzing the life cycle in time.

Fig. 3 shows the experimental results of in situ transplanted tumors in mice, and first in vivo animal imaging experiments show that TMZ alone can effectively inhibit the growth rate of glioma cells compared with the control group, but in the case of the tumor of the later TMZ-alone treatment group continuously growing slowly, the tumor of the HOXA5 knocked down combined with the TMZ treatment group grows more slowly (A, B in fig. 3), which suggests that HOXA5 knocking down may increase the killing effect of TMZ. In addition, statistical analysis of survival in tumor bearing mice revealed that knockdown HOXA5 in combination with TMZ treatment was also effective in prolonging survival in mice (C in fig. 3).

Example 4

The results of the above studies indicate that HOXA5 expression can affect glioma cell TMZ resistance, and next we further clarified the regulatory mechanism of HOXA5 using bioinformatics analysis of molecules that interact with HOXA 5. After screening in glioma databases and combining 4 database analyses to obtain 39 molecules associated with HOXA5 expression (a in fig. 4), we next analyzed the network that probably interacts with HOXA5 by online database analysis (B in fig. 4), and cross-combining the two sets of data we found 13 molecules including PBX1, MEIS1, EZH2 as molecules that probably interact with HOXA5, and further tested one by one.

The expression relationship of HOXA5 and its potentially interacting molecules was examined by immunofluorescence double staining of TMZ-treated glioma stem cells and immunohistochemical staining of glioblastoma samples, respectively. Results co-localization of HOXA5 with EZH2 expression was found after immunofluorescence double staining of TMZ-treated glioma stem cells (a in fig. 5). Expression of both was also detected by immunohistochemical staining in glioblastoma samples and HOXA5 expression was positively correlated with EZH2 expression (B in fig. 5).

HOXA5 acts as a transcription factor, and may be regulated by binding to the EZH2 promoter region to regulate its expression, or by a relationship of protein interaction. To clarify the regulation patterns of both, the regulation patterns were verified experimentally, respectively, and there was no significant change in EZH2 mRNA levels after over-expression of HOXA5 (a in fig. 6), suggesting that HOXA5 regulates EZH2 probably through its protein-protein interaction. We next performed Co-IP experiments in glioma stem cells and found that either HOXA5 or EZH2 antibodies captured the profilin (fig. 6B), suggesting that the mode of action of HOXA5 and EZH2 is achieved by binding of proteins to each other in vivo. To further clarify the relationship between the binding of the two and the influence of HOXA5 on TMZ resistance, we first examined the expression of EZH2 and its downstream effector molecule H3k27me3 after knocking down the expression of HOXA5 in TMZ-treated glioma stem cells, and found that both expressions were also reduced (C in fig. 6). In functional recovery experiments for detecting the proportion of apoptotic cells, we found that the expression of EZH2 was knocked down on the basis of the over-expression of HOXA5, and the results showed that the reduction of the proportion of apoptotic cells caused by the over-expression of HOXA5 (D in fig. 6) could be partially recovered, while the recovery effect was also found in the detection of the apoptotic signal pathway and protein expression of signal pathways downstream of EZH2 (E in fig. 6), suggesting that the HOXA5-EZH2 pathway affects the importance of TMZ drug resistance.

Example 5

Finally, we repeated the above experiment using the EZH2 inhibitor GSK343 (concentration 1mg/ml, dilution in medium at 1:100 ratio, effect 24 hr). We found that after cell treatment with GSK343, based on over-expression of HOXA5, expression of EZH2 and downstream signaling pathway H3k27me3 proteins both appeared to be suppressed after activation (a in fig. 7). While GSK343 treatment was also found to partially revert the protective effect on cell killing due to over-expression of HOXA5 in the detection of apoptotic cells by flow cytometric staining (B and C in fig. 7).

An EZH2 inhibitor can improve sensitivity of glioma cells to temozolomide

It has been previously shown that GSK343, an EZH2 inhibitor, partially reverts to the killing of tumor cells against TMZ caused by over-expression of HOXA 5. Further preliminary investigations were made regarding the effect of using GSK343 alone. We first compared the apoptosis of glioma cells after TMZ treatment alone and combined with GSK343 treatment and found that both the level of apoptosis signaling pathway marker protein expression (a in fig. 8) and the proportion of apoptotic cells (B in fig. 8) appeared to be elevated. Next, GSK343 was added to T98G-resistant cells, respectively, and the protein level (C in fig. 8) and IC50 values (D in fig. 8) of the apoptosis signaling pathway marker and the proportion of apoptotic cells (E in fig. 8) were found to return to the non-resistant cell level. Whereas over-expression of HOXA5 after use of GSK343 inhibitor resulted in glioma cells developing resistance to TMZ again (fig. 8F). In addition, there was also an increase in the protein level of the apoptosis signaling pathway marker after knocking down EZH2 expression alone (G in fig. 8). The above results suggest the value and the relevant mechanism of the application of the EZH2 inhibitor GSK343 in the treatment of drug resistance in TMZ. GSK343, an EZH2 inhibitor downstream of HOXA5, can be used in combination with temozolomide as a dosing option for temozolomide resistant patients.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

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