CN107998398A - Application of the GRP78 genes in tumor stem cell chemosensitivity medicine is improved - Google Patents

Application of the GRP78 genes in tumor stem cell chemosensitivity medicine is improved Download PDF

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CN107998398A
CN107998398A CN201711427531.9A CN201711427531A CN107998398A CN 107998398 A CN107998398 A CN 107998398A CN 201711427531 A CN201711427531 A CN 201711427531A CN 107998398 A CN107998398 A CN 107998398A
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stem cell
grp78
inhibitor
tumor stem
genes
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王志宇
王能
郑轶枫
王胜奇
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Guangdong Hospital of Traditional Chinese Medicine
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Guangdong Hospital of Traditional Chinese Medicine
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Abstract

The present invention relates to tumor stem cell chemotherapeutic area, more particularly to application of the GRP78 genes in tumor stem cell chemosensitivity medicine is improved, and the screening technique using GRP78 genes as action target spot inhibitor.The action target spot of inhibitor is GRP78 genes, the GRP78 genes are associated with tumor stem cell resistant characterization, the expression of GRP78 genes in inhibitors to inhibitor tumor stem cell, so that chemotherapeutics content raises in tumor stem cell, improves tumor stem cell chemosensitivity.The present invention helps to improve sensitiveness of the tumor stem cell to chemotherapeutics, strengthens chemotherapeutical medicine curative effect, improves life cycle and the chemotherapy response rate of tumor patient, have potential, good application prospect in tumor stem cell chemosensitivity medicine.

Description

Application of the GRP78 genes in tumor stem cell chemosensitivity medicine is improved
Technical field
The present invention relates to tumor stem cell chemotherapeutic area, more particularly to GRP78 genes are quick in raising tumor stem cell chemotherapy Application in sensitive drug.
Background technology
Malignant tumour is to threaten one of human health and the principal disease of life.According to the system of international cancer research institution Count, the whole world increases about 14,100,000 cases of cancer newly within 2012, and number of cancer deaths is up to 8,200,000, in contrast, 2008 Data are respectively 12,700,000 and 7,600,000, it is seen that the incidence and the death rate of malignant tumour are constantly raising.Statistics is shown at the same time Show that annual knurl 80% of newly swelling occurs in developing countries such as China, India, Brazil in the world at present, malignant tumour has been in China Cardiovascular and cerebrovascular disease is exceeded, has become the first big cause of the death of China resident, it accounts for the percentage of total cause of the death from the 13% of the seventies Left and right rises to more than 24% in 2009.To the year two thousand twenty, new cases are up to 3,490,000, the dead people because of malignant tumour Number will be up to 2,630,000, and the year two thousand twenty morbidity and death toll will rise 60% than 2002.Although from the nineties in last century, due to The joint of the development of early screening technology, the application of chemotherapeutics of new generation and scheme, targeted drug and biological response modifiers Intervene, and the progress of complex treatment system, tumor patient life cycle progressively extend, but Endodontic failure caused by drug resistance and then Relapse and metastasis be tumor patient main causes of death.Explore tumor drug resistance mechanism and development therapeutic targets be improve curative effect, Reduce drug resistance risk and extend the key link of survival rate.
With the development of genomics, protein science and Cell. Mol, tumor multi-medicine drug-resistant mechanism is demonstrate,proved in succession Bright and ABC (ATP-binding cassette) family protein member ABCB1, ABCC1 and ABCG2, chemotherapeutics metabolic pathway Change, drug target mutant inactive and tumor microenvironment factor (such as anoxic, vessel density) are related.The master of reversing drug resistance at present It is tactful still to suppress drug efflux, that is, develop ABC protein inhibitors and attach most importance to.A small amount of guide's chemical combination is filtered out although having developed Thing, but since specificity, security and internal the problems such as being metabolized, only a small number of medicines are in clinical experimental stage.From new angle Degree, which inquires into tumor drug resistance mechanism and development therapeutic targets, becomes the task of top priority.Tumor stem cell (cancer stem nearly ten years Cell, CSC) found in succession in the tumours such as breast cancer, lymthoma, liver cancer, cancer of the esophagus, this kind of cell has self-renewing, more To differentiation the characteristics of and powerful internal oncogenicity, it is considered to be tumour occurrence and development transfer root.Lot of documents at the same time Prove that tumor stem cell has height drug resistance, and largely enrichment expression ABC family protein members, such as ABCG2.It is but swollen at present Knurl stem cell drug resistance signaling molecule path not yet illustrates, and finds and parses the drug resistant key molecule of tumor stem cell and regulate and control logical Road, it will for effective prevention chemoresistance provides special and efficiently targets preventive means, it is thin also to possess brand-new Tumor Stem Born of the same parents' enhanced sensitivity measure.
During the occurrence and development of tumour, tumour cell is in anoxic and amino acid, glucose supplies deficiency state, draws Play unfolded protein matter and misfolded protein matter is assembled in endoplasmic reticulum, causing stress protection mechanism Non-adhesion inhibition index The activation of (UPR, unfolded protein response), can be by suspending protein translation, degraded misfolded protein etc. Mechanism eliminates or mitigates er stress, so that helper cell escape procedureization is dead.Glucose regulatory protein 78 (Glucose Regulated protein 78, GRP78) be endoplasmic reticulum system important molecule companion, can by with endoplasmic reticulum should Inductor dissociation is swashed with combining to start or close UPR reactions.In fact, Recent study is the result shows that GRP78 is that tumour is thin Born of the same parents' malignancy and drug-fast important mechanisms.But key effects of the GRP78 to tumor stem cell chemoresistance is unclear, It there is no the targeted drug for GRP78 designs at present.Therefore, it is necessary to prepared by tumor stem cell chemotherapy sensitizing medicine to GRP78 Deficiency existing for application and its inhibitor screening in thing is improved.
The content of the invention
It is an object of the invention to provide expression of the GRP78 genes in tumor stem cell is suppressed by inhibitor, to increase Application in the chemosensitivity medicine of strong tumor stem cell, and the method for screening the inhibitor.Technical scheme For：Application of the GRP78 genes in tumor stem cell chemosensitivity medicine is improved, including inhibitor, the work of the inhibitor It is GRP78 genes with target spot, using the expression of GRP78 genes in the inhibitors to inhibitor tumor stem cell, to improve Tumor Stem Sensitiveness of the cell to chemotherapeutics.
Preferably, the inhibitor include Decitabine, cefoselis sulfate, rocuronium, AT13387, Linezolid, In metolazone, madribon, Mirtazapine, Didanosine, diosmetin, rhodioloside, and trospium chloride One or more.
Preferably, the tumor stem cell is using breast carcinoma stem cell as representative.
Preferably, the breast carcinoma stem cell derives from MCF-7 cells and/or MDA-MB-231 cells.
Preferably, the inhibitor is determined by plasma resonant imaging screening analysis.
Preferably, the inhibitor is by its GRP78 protein sample binding constant with various concentrations gradient, dissociation constant and Binding affinity assays determine.
A kind of method screened using GRP78 genes as the inhibitor of action target spot, which is above-mentioned inhibitor, bag Include following steps：
Printed sample and reference substance on 3D photo-crosslinking chips；
Mixed solution to contain GRP78 albumen is used as mobile phase；
By above-mentioned printed chip and the mobile phase through plasma resonant imaging, and record the sample with it is described The affinity parameters of GRP78 albumen；And
Screened to obtain the inhibitor according to the size of above-mentioned affinity parameters.
Preferably, liquid of living again, the glycine hydrochloride salt buffer that liquid is pH=2.0 of living again are further included.
Preferably, the mobile phase includes three kinds, in three kinds of mobile phases, and the GRP78 protein concentrations are followed successively by 4 × 10-7mol/L、2×10-7Mol/L and 1 × 10-7mol/L。
Preferably, the flow velocity of the mobile phase is 2 μ l/s, binding time 300s, Dissociation time 300s.
The GRP78 alias of the present invention includes：HSPA5；78kDa glucose regulated protein；BIP； Heat shock 70kDa protein 5；Hsce70；mBiP；MIF2；Sez7.
Selection experiment tumor stem cell：
The tumor stem cell is present in the entity tumors such as breast cancer, prostate cancer, liver cancer, and molecular biosciences behaviouristics is ground Study carefully and show that tumor stem cell has the stem cell universal features such as self-renewing and Asymmetric division, and with powerful into knurl energy Power, plays a significant role during mediate tumor generation, vascularization and relapse and metastasis etc..Since breast carcinoma stem cell is in reality Find that earliest, therapy system and means are the most ripe in many tumours at present, to its resistance mechanism and gene target in body tumour The research carried out to treatment can be as the experimental basis and theoretical reference of other tumours.
Investigate the relation and molecular regulation based on GRP78 genes Yu breast carcinoma stem cell resistant characterization.It turns out that：
Chemotherapeutics can significantly raise the GRP78 of breast cancer cell MCF-7 and MDA-MB-231 and its vivo tumor model The expression of gene, while with beta-catenin and the increase of ABCG2 expressions.
For breast carcinoma resistance cell MCF-7/ADR compared with MCF-7, GRP78, beta-catenin and ABCG2 expressions are equal Significantly rise.Airflow classification experiment also proves the high expression GRP78 of breast cancer side population cell and stem cell.
Suppress GRP78 gene expressions to be remarkably improved breast carcinoma stem cell chemotherapy medicament contg and reduce beta-catenin Expression, while reduce stem cell balling-up ability.
Clinical detection prove GRP78 gene expression doses dramatically increased after chemotherapy, and with beta-catenin and ABCG2 tables It is proportionate up to content, GRP78 gene high expression patient with breast cancer's Overall survivals significantly reduce.
By High Throughput Screening Assay, the small molecule chemical combination of screening suppression GRP78 genes from BCL (1836) library of molecules Thing：
SPR (plasma resonance) technology has been used, BCL (1836) library of molecules small molecule sample is printed upon 3D photo-crosslinkings On chip, and the GRP78 protein samples of various concentrations gradient are loaded, measure binding constant Ka, dissociation constant Kd and combination are affine Power KD.Finally successfully screen 12 kinds of small molecule samples and GRP78 to interact, wherein 11 kinds close with the combination of GRP78 albumen With power (KD) 10-7~10-8In the range of, and the KD of cefoselis sulfate and GRP78 are 6.38e-9, binding ability is most strong, can make For GRP78 potential inhibitors.
Beneficial effects of the present invention are：GRP78 gene inhibitors are disclosed to can be used for preparing the increasing of tumor stem cell chemotherapy Sensitizing drug, suppresses the GRP78 gene expressions and is remarkably improved breast carcinoma stem cell chemotherapy medicament contg and reduces β-chain of rings Protein expression level, while reduce stem cell balling-up ability.Sensitiveness of the tumor stem cell to chemotherapeutics is helped to improve, is increased The effect of intense prior chemotherapy medicine, improve the quality of life of tumor patient, is improving tumor stem cell chemosensitivity drug field tool There is potential, good application prospect.
The invention discloses the potential micromolecular inhibitors of GRP78, are screened from 1836 kinds of micromolecular compounds and obtain 12 kinds There is the compound of association reaction with GRP78, wherein cefoselis sulfate has optimal binding affinity KD, based on it Compound development tumor stem cell hypersitization medicine has substantial worth, will provide potential medicine for the change of clinical cancer therapy strategy Thing, effectively avoids Endodontic failure caused by drug resistance, improves tumor patient survival rate.
Brief description of the drawings
Fig. 1 shows that chemotherapy can induce breast cancer cell GRP78 and be raised with beta-catenin/ABCG2 Expression in Vivo and in Vitro.
Fig. 2 shows GRP78 genes in enrichment expression on breast carcinoma stem cell.
Fig. 3 shows that GRP78 genes suppress to reduce breast carcinoma stem cell drug resistance and promote its differentiation.
Fig. 4 shows GRP78 in the expression on breast cancer clinical samples and its related to beta-catenin/ABCG2 signals Property.
Embodiment
In order to make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to the excellent of the present invention Embodiment is selected to be described in detail.But present disclosure is not restricted to listed illustrative embodiments.
Embodiment 1：Chemotherapy can induce breast cancer cell GRP78 and be raised with beta-catenin and ABCG2 Expression in Vivo and in Vitro
For influence of the detection chemotherapy to GRP78 expressions, the present invention have detected MCF-7 cell strainHJ2mm and MDA- Reactions of the MB-231 to three kind of one line mammary cancer chemotherapy medicine (Epi-ADM, taxol and 5 FU 5 fluorouracil).Western After the processing of blotting the results shows chemotherapeutic, two kinds of breast cancer cell GRP78 expressions significantly rise, while adjoint β- The rise of catenin and ABCG2 levels, and be in dose-dependence (A and B in Fig. 1), prompt GRP78 genes may be with change Treating response and ABCG2 expression has certain relation.In next step, it is the variation tendency after verification GRP78 expressions in vivo chemotherapy, The present embodiment distinguishes inoculating two kinds breast cancer cell using nude mice model under armpit in mammary fat pad.Treat tumour length to 5mm During × 5mm sizes, Epi-ADM (2.5mg/kg/W) intraperitoneal injection treatment 4 weeks is given.The results show Epi-ADM can significantly press down Tumour growth (C in Fig. 1) processed.Showed by immune group result at the same time, GRP78 gene expression dose liters in tumor tissues after chemotherapy Height, with the rising (D in Fig. 1) of beta-catenin and ABCG2 signal levels.This result is consistent with vitro results, it was demonstrated that GRP78 gene expression doses and chemotherapy are significantly correlated, while have synchronism with the regulation and control of beta-catenin and ABCG2 signals.
Embodiment 2GRP78 genes are in enrichment expression on breast carcinoma stem cell
Based on the discovery in embodiment 1, to study the relation between GRP78 genes and chemotherapy side effect, first to human milk Adenocarcinoma cell MCF-7 and its mdr cell MCF-7/ADR carries out chemosensitivity detection.It turns out that in MCF-7/ADR cells In, side population cell ratio significantly raises (A in Fig. 2).Flow cytometer result prompting mdr cell pumps chemotherapeutics at the same time Output capacity also dramatically increases (B in Fig. 2).To inquire into expression status of the GRP78 genes on mdr cell, to MCF-7 and its Mdr cell has carried out western blotting detections, as a result prompts GRP78 gene levels in mdr cell significantly to raise.Companion With ABCG2 and beta-catenin up-regulated expression, the close pass between GRP7 genes 8 and tumor stem cell drug resistance is further illustrated It is (C in Fig. 2).Since lot of documents research reports that drug resistance is one of characteristic of tumor stem cell.To study GRP78 bases Because whether regulate and control tumor stem cell drug resistance between exist and associate, the present invention using flow cytometer by side population cell and it is dry carefully Born of the same parents are sorted (D and F in Fig. 2) with Hochest 33342 and CD44-FITC/CD24-PI dyeing respectively, and to sub-electing Cell carry out western blotting detections, observe GRP78 genes expression.The result is shown in breast cancer side population cell and GRP78 gene expression amounts significantly raise on stem cell, while with beta-catenin and the ABCG2 signals up-regulation (E in Fig. 2 With G).The drug resistance of result of study prompting GRP78 gene mediateds may be closely related with breast carcinoma stem cell, and beta-catenin/ ABCG2 signal pathways are probably its main molecules mechanism.
Embodiment 3GRP78 genes suppress to reduce breast carcinoma stem cell drug resistance and promote its differentiation
For the effect of further research GRP78 gene regulation breast cancer cell resistant characterizations, the present invention uses The expression of GRP78 genes in GRP78siRNA silence MCF-7/ADR cells, and then sub-elect dry thin before and after siRNA is disturbed Born of the same parents carry out side population cell ratio detection.The results show GRP78siRNA apply after, in breast carcinoma stem cell side population cell ratio by 22.5% drops to 7.5% (A in Fig. 3), while intracellular Epi-ADM content significantly raises (B in Fig. 3), and with β- Catenin and ABCG2 expressions reduce (C in Fig. 3).Result of study prompting GRP78 genes are regulation and control breast carcinoma stem cells The efficiency factor of drug resistance, its mechanism are closely related with beta-catenin and ABCG2 signal pathways.In order to whether verify GRP78 It is same to influence breast carcinoma stem cell balling-up ability, the present invention added in breast carcinoma stem cell cultivating system GRP78siRNA or GRP78 polypeptides, observe its influence to breast carcinoma stem cell ball quantity of formation and size.The results show GRP78siRNA applies Afterwards, breast carcinoma stem cell ball forms number and diameter is remarkably decreased.Opposite GRP78 polypeptides can dramatically increase breast carcinoma stem cell The diameter and quantity of ball, illustrate the self-renewal capacity (D in Fig. 3) of the controllable breast carcinoma stem cell of GRP78 genes.
Embodiment 4GRP78 genes in the expression on breast cancer clinical samples and its with beta-catenin and ABCG2 signals Correlation
To further determine that the relation between GRP78 genes and breast carcinoma resistance adjusting, carrying out clinical samples detection is Steps necessary, not only can determine that results of in vitro studies, can also disclose the clinical meaning of GRP78 detections.For this reason, the present invention is to 46 Breast cancer clinical samples have carried out GRP78, the Immunohistochemical detection of beta-catenin bletilla ABCG2.The results show GRP78 bases Because expression is in significantly up-regulation (A in Fig. 4) on triple negative breast cancer sample.Since lot of documents reports triple negative breast cancer Chemotherapy side effect is poor, and postoperative recurrence shifts risk height, while studies have reported that triple negative breast cancer to contain higher Tumor Stem thin Born of the same parents group, the research of the comprehensive present invention are found, prompt high expression of the GRP78 genes on triple negative breast cancer patient's sample indirectly May be related to its breast carcinoma stem cell with high level.In next step, the present invention has extracted 10 chemotherapy for carcinoma of breast Front and rear blood carries out GRP78mRNA assays.Internal GRP78mRNA contents significantly rise after result of study shows Chemotherapy in Patients Height, and show that GRP78 genes and chemotherapy side effect have close relation (B in Fig. 4).In addition, the present invention is to GRP78 mammary gland Cancer clinical samples carry out ImmunohistochemistryResults Results analysis, the results show GRP78 gene expression doses respectively with beta-catenin and ABCG2 has positive correlation (C and D in Fig. 4), this result is consistent with Vitro Experimental Results, further illustrates that GRP78 genes can The drug resistance of breast cancer cell can be adjusted by beta-catenin.
Embodiment 5GRP78 gene inhibitor SPR the selection results
BCL (1836) library of molecules sample is printed on 3D photo-crosslinking chips, it is stringent to be beaten with reference to U.S.'s Plexera company standards Bleeding off journey performs.The inside yin and yang attribute control that company is printed on chip is (negative:DMSO and the positive:Rapamycin), control sample For measuring chip quality.Plexera using the chip handled well by SPR V3 versionsHT biologies point Sub- transactional analysis instrument carries out Real Time Monitoring to sample.Different amounts of 20mM is added in GRP78 protein liquids Tris, 50mM KCl, 5mM MgCl2 and 1mM DTT mixed solvents are diluted to 3 concentration gradients：400nM, 200nM and 100nM, Namely 4 × 10-7mol/L、2×10-7Mol/L and 1 × 10-7mol/L.Mixed solvent is used as mobile phase in whole experiment.In core The surface of piece, analyte flow velocity are 2 μ Ls-1, binding time 300s, Dissociation time 300s.Use glycine hydrochloride For Glycine-HCl (pH=2.0) solution as liquid of living again, flow velocity is 2 μ L/s, flowing time 300s.Pass through high throughput SPRi (plasma resonant imaging), the present invention have successfully screened 12 kinds of BCL small molecules samples and GRP78 albumen phase interactions respectively With, and obtained corresponding kinetic parameter and affinity parameters.Wherein there are 11 kinds of BCL small molecules samples and GRP78 albumen Binding affinity (KD) is 10-7~10-8In the range of, with reference to medium；The knot of BCL small molecules cefoselis sulfate and GRP78 albumen It is 6.38e to close affinity (KD)-9, binding ability is strong.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although passing through ginseng According to the preferred embodiment of the present invention, invention has been described, it should be appreciated by those of ordinary skill in the art that can Various changes are made to it in the form and details, the present invention that is limited without departing from the appended claims Spirit and scope.

Claims (10)

  1. Application of the 1.GRP78 genes in tumor stem cell chemosensitivity medicine is improved, it is characterised in that including inhibitor, The action target spot of the inhibitor is GRP78 genes, using the table of GRP78 genes in the inhibitors to inhibitor tumor stem cell Reach, to improve sensitiveness of the tumor stem cell to chemotherapeutics.
  2. 2. application of the GRP78 genes according to claim 1 in tumor stem cell chemosensitivity medicine is improved, it is special Sign is that the inhibitor includes Decitabine, cefoselis sulfate, rocuronium, AT13387, Linezolid, toluene quinoline azoles One kind in sulfanilamide (SN), madribon, Mirtazapine, Didanosine, diosmetin, rhodioloside, and trospium chloride or It is several.
  3. 3. application of the GRP78 genes according to claim 1 in tumor stem cell chemosensitivity medicine is improved, it is special Sign is that the tumor stem cell is using breast carcinoma stem cell as representative.
  4. 4. application of the GRP78 genes according to claim 3 in tumor stem cell chemosensitivity medicine is improved, it is special Sign is that the breast carcinoma stem cell derives from MCF-7 cells and/or MDA-MB-231 cells.
  5. 5. application of the GRP78 genes according to claim 1 in tumor stem cell chemosensitivity medicine is improved, it is special Sign is that the inhibitor is determined by plasma resonant imaging screening analysis.
  6. 6. application of the GRP78 genes according to claim 1 in tumor stem cell chemosensitivity medicine is improved, it is special Sign is：For the inhibitor by its GRP78 protein sample binding constant with various concentrations gradient, dissociation constant and combination are affine Power analysis determines.
  7. A kind of 7. method screened using GRP78 genes as the inhibitor of action target spot, it is characterised in that the inhibitor will for right The inhibitor described in 1 is sought, is comprised the following steps：
    Printed sample and reference substance on 3D photo-crosslinking chips；
    Mixed solution to contain GRP78 albumen is used as mobile phase；
    By above-mentioned printed chip and the mobile phase through plasma resonant imaging, and record the sample with it is described The affinity parameters of GRP78 albumen；And
    Screened to obtain the inhibitor according to the size of above-mentioned affinity parameters.
  8. 8. method of the screening according to claim 7 using GRP78 genes as the inhibitor of action target spot, it is characterised in that Further include liquid of living again, the glycine hydrochloride salt buffer that liquid is pH=2.0 of living again.
  9. 9. method of the screening according to claim 7 using GRP78 genes as the inhibitor of action target spot, it is characterised in that The mobile phase includes three kinds, in three kinds of mobile phases, and the GRP78 protein concentrations are followed successively by 4 × 10-7mol/L、2×10- 7Mol/L and 1 × 10-7mol/L。
  10. 10. method of the screening according to claim 7 using GRP78 genes as the inhibitor of action target spot, it is characterised in that The flow velocity of the mobile phase is 2 μ l/s, binding time 300s, Dissociation time 300s.
CN201711427531.9A 2017-12-26 2017-12-26 Application of the GRP78 genes in tumor stem cell chemosensitivity medicine is improved Pending CN107998398A (en)

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Application publication date: 20180508