CN101769917A - Molecular diagnostic agent and therapeutic agent of nasopharyngeal carcinoma, reagent kit and application thereof - Google Patents

Abstract

The invention discloses a molecular diagnostic agent using epidermal growth factor receptors (EGFR) and nuclear factors kappa B (NF-kB) as an effective and specific labelled molecule for diagnosing nasopharyngeal carcinoma, and a therapeutic agent using inhibitors containing the EGFR and/or the NF-kB for treating the nasopharyngeal carcinoma. The invention also discloses a reagent kit for detecting the nasopharyngeal carcinoma, which contains reagent for determining the expressions or the activities of the EGFR or the NF-kB in an organism sample. The invention also provides a method for identifying compounds for treating the nasopharyngeal carcinoma.

Description

The molecular diagnosis agent of nasopharyngeal carcinoma and therapeutic agent, kit and application thereof

Technical field

The present invention relates to the molecular diagnosis and/or the methods of treatment of nasopharyngeal carcinoma, more specifically, relate to method, reagent, pharmaceutical composition, kit and their application of diagnosing and/or treating nasopharyngeal carcinoma via novel drug target (EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B)).This novel drug target can be used to diagnose and/or treat the validity of nasopharyngeal carcinoma, the progress degree of estimating nasopharyngeal carcinoma and/or treatment and the compound of identifying the treatment nasopharyngeal carcinoma.

Background technology

(Epstein-Barr virus EBV) is a member in the herpes virus hominis family to Epstein-Barr virus, can infect most people.This virus is relevant with many malignant tumours, and nasopharyngeal carcinoma is the most serious relevant epithelioma of Epstein-Barr virus.The Epstein-Barr virus genome is found in all nasopharyngeal carcinoma cases, yet the expression of Epstein-Barr virus encoding proteins but has only on Epstein-Barr virus nucleoprotein 1 (EBNA 1) and the latent membrane protein 1 (LMP-1) in most of nasopharyngeal carcinoma cases.The expression of LMP-1 can be induced the expression of EGF-R ELISA (EGFR) in the epidermal cell.

The disease that Epstein-Barr virus causes

Epstein-Barr virus belongs to herpesviral family, is a kind of very common virus that causes human diseases.Infection rate can reach 90% (Niederman in global crowd's scope, J., Evans, A., Subrahmanyan, L., and McCollum, R. (1970) .Prevalence, incidence andpersistence of EB virus antibody in young adults.N Engl J Med 282,361-365.).Ebv infection is more general in the children of less developed country.In developed country, ebv infection is mainly in the teenager, infection tends to the disease such as the infectious mononucleosis (infectious mononucleosis) that cause that some have manifest symptom, this disease The main concomitant symptoms has: heating, sore-throat, lymph enlargement, hepatosplenomegaly and fatiguability (Evans, A., Niederman, J., and McCollum, R. (1968) .Seroepidemiologic studies of infectiousmononucleosis with EB virus.N Engl J Med 279,1121-1127.).In case infect, Epstein-Barr virus will be present in the infected individuality throughout one's life with latent form.Latent form virus is considered to play an important role in the relevant human malignancies development of EV virus, these malignant tumours comprise: non-Hodgkin ' the s lymph cancer that nasopharyngeal carcinoma (nasopharyngeal carcinoma), Burkitt ' s lymph cancer, transplanting back lymphadenia lymthoma, Hodgkin ' s disease, acquired immune deficiency syndrome (AIDS) are correlated with, thymic carcinoma, lung cancer, cancer of the stomach and breast cancer.(Okano，M.(1998).Epstein-Barr?virus?infection?and?its?role?in?the?expanding?spectrumof?human?diseases.Acta?Paediatr.87，11-18.)

The epithelioma that nasopharyngeal carcinoma-a kind of Epstein-Barr virus is relevant

Nasopharyngeal carcinoma is the most serious relevant epithelioma of Epstein-Barr virus.In the Caucasoid of US and European, nasopharyngeal carcinoma is about 0.001% at the global average incidence of disease, in Southeast Asia and SOUTHERN CHINA area be 0.02% (this area's nasopharyngeal carcinoma incidence of disease account for all cancers 20%), in Hong Kong is 0.025%, in north African is 0.008% (Raab-Traub, N. (1992) .Epstein-Barr virus andnasopharyngeal carcinoma.Semin Cancer Biol 3,297-307.).Regardless of its geographic origin, nasopharyngeal carcinoma is always relevant with Epstein-Barr virus.The Epstein-Barr virus genome is found in all nasopharyngeal carcinoma cases, yet, the expression of Epstein-Barr virus encoding proteins but has only Epstein-Barr virus nucleoprotein 1 (EBNA 1) and latent membrane protein 1 (LMP-1) to go up [Chen in most of nasopharyngeal carcinoma cases, M., Tsai, C., Liang, C., Shu, C., Huang, C., Sulitzeanu, D., Liu, S., and Chang, Y. (1992) .Cloning and characterization of the latent membrane protein (LMP) of aspecific Epstein-Barr virus variant derived from the nasopharyngeal carcinomain the Taiwanese population.Oncogene 7,2131-2140.; Fahraeus, R., Fu, H., Ernberg, I., Finke, J., Rowe, M., Klein, G., Falk, K., Nilsson, E., Yadav, M., and Busson, P. (1988) .Expression of Epstein-Barr virus-encoded proteins innasopharyngeal carcinoma.Int J Cancer 42,329-338.].Though the correlativity of Epstein-Barr virus and nasopharyngeal carcinoma is very clear and definite, the formation mechanism of the nasopharyngeal carcinoma of Epstein-Barr virus mediation still remains to be illustrated.

The classification of nasopharyngeal carcinoma

The World Health Organization (WHO) is divided into three main hypotypes (Pizzo P.A.andPoplock A.G. (1997) Principles and Practice of Pediatric Oncology.Lippincott-Raven Publishers with nasopharyngeal carcinoma, Philadephia, PA, 3rd Edition.).The I class is a squamous cell carcinoma, and this cancer mainly influences the adult, and does not have tangible correlativity with Epstein-Barr virus.The II class is always relevant with Epstein-Barr virus with the III class, the nasopharyngeal carcinoma that is respectively nonkeratinizing (non-keratinizing) and does not break up (undifferentiated).Tumour generally is a local diffusion, often shifts to the neck lymph node.The metastases site comprises heart, lungs, liver and bone at a distance.The development of nasopharyngeal carcinoma can be divided into five stages of T0-T4, can be used for estimating prognosis [Pizzo P.A.andPoplock A.G. (1997) the Principles and Practice of Pediatric Oncology.Lippincott-Raven Publishers of pathology, Philadephia, PA, 3rd Edition.].The T0 stage shows the evidence that does not have tumour, T1s is expressed as carcinoma in situ, T1 represents that cancer exists only in a certain position of nasopharynx or has only by biopsy and could find, T2 includes the tumour in two sites, be respectively rear wall and lateral wall, T3 comprises that those have been diffused into the nasal cavity or the cancer of ccavum oropharygeum, T4 comprise those invaded encephalic or (with) cancer of cranial nerve.The T1/T2 stage can be predicted the survival rate of 54%-87%, yet is diagnosed as the tumour in T3/T4 stage, and its survival rate is quite low, only is 8%-37%.

The existing methods of treatment of nasopharyngeal carcinoma

Nasopharyngeal carcinoma is mainly treated [Pizzo P.A.and Poplock A.G. (1997) Principles and Practice of Pediatric Oncology.Lippincott-RavenPublishers by radiotherapy at present, Philadephia, PA, 3rd Edition.].Operation method is worth in the treatment of nasopharyngeal carcinoma not quite, and this is the operation because nasopharynx position difficulty undergos surgery, and when diagnosing out, tumour is often with nearly diffusion.Chemotherapy is generally as the supplementary means of radiotherapy, and the normally used factor has neoplatin (cisplatin), vincristine (vincristine), methotrexate (methotrexate), 5 FU 5 fluorouracil (5-fluorouracil) and bleomycin (bleomycin) etc.Treatment has certain effect though radiotherapy is for nasopharyngeal carcinoma, and a lot of spinoffs are also arranged simultaneously.Xerostomia is an important spinoff, considers the position of chemotherapy, this spinoff no wonder occurs.Radiotherapy also can cause multiple catarrh, and this just forces and must settle stomach tube to be avoided from the oral cavity feed to patient, and this situation can become serious further in the chemotherapy process after radiotherapy.Other ill effects also have neck fiberization (neck fibrosis), lockjaw (trismus), parmlysis of cranial nerve (cranial nerve palsies), wall neuropile weak (brachialplexus weakness), amyotrophia (muscular atrophy), hypothyroidism (hypothyroidism) and cranium face damage (craniofacial disfigurement).These spinoffs are very serious for children, because in case by radiating irradiation, facial skeleton will can significantly not grown again.The spinoff of chemotherapy is also very important, wherein mainly comprises chronic renal insufficiency (chronicrenal insufficiency), infertility (sterility), pulmonary fibrosis (pulmonary fibrosis) and peripheral nerve pathology (peripheral neuropathy).And can cause neutrophilic granulocytopenia (neutropenia), severe infections (severe infections), sometimes or even dead.Also fail to obtain very ten-strike with non-specific factor treatment such as interferon (interferon) and aciclovir (acyclovir).Therefore, we need new, effective nasopharyngeal carcinoma methods of treatment, promptly utilize Epstein-Barr virus and specific albumen thereof, target spot and their end-product to carry out site-specific inhibition treatment and the monitoring of disease progression subsequently.

Summary of the invention

What the inventor was nearest studies show that, the generation of nasopharyngeal carcinoma may and activate NF-κ B by the up-regulated expression of EGFR and cause.The signal that the activation of the up-regulated expression of EGFR and NF-κ B provides a very strong cell proliferation and survived, therefore can use EGFR and NF-κ B as the nasopharyngeal carcinoma diagnosis labeled molecule, and can use EGFR and (or) antagonist of NF-κ B treats the nasopharyngeal carcinoma that EBV causes.

Particularly, the invention provides the following:

1. the application of reagent in the diagnosticum of preparation detection nasopharyngeal carcinoma, described reagent is used for the expression or the activity of EGF-R ELISA of biosome sample (EGFR) or nuclear transcription factor-kappa B (NF-κ B) are measured.

In one embodiment, the present invention also provides the application of a kind of composite reagent in the diagnosticum of preparation detection nasopharyngeal carcinoma, and described composite reagent comprises and being used for the EGF-R ELISA (EGFR) of biosome sample or expression or the active reagent of measuring and the another kind of reagent (as the antibody at LMP-1 or EBER) that detects Epstein-Barr virus (EBV) of nuclear transcription factor-kappa B (NF-κ B).

2. reagent is used for estimating the application of diagnosticum of the validity of the progress degree of nasopharyngeal carcinoma and/or treatment in preparation, and described reagent is used for the expression or the activity of EGF-R ELISA of biosome sample (EGFR) or nuclear transcription factor-kappa B (NF-κ B) are measured.In one embodiment, described biosome sample is from making a definite diagnosis the patient who suffers from nasopharyngeal carcinoma.

3. kit that is used to detect nasopharyngeal carcinoma, it contains:

Be used for the EGF-R ELISA (EGFR) of biosome sample or the expression or the active reagent of measuring of nuclear transcription factor-kappa B (NF-κ B).In one embodiment, described kit also contains the another kind of reagent (as the antibody at LMP-1 or EBER) that detects Epstein-Barr virus (EBV).

4. the kit of the validity of progress degree that is used to estimate nasopharyngeal carcinoma and/or treatment, wherein, contain:

Be used for the EGF-R ELISA (EGFR) of biosome sample or the expression or the active reagent of measuring of nuclear transcription factor-kappa B (NF-κ B).In one embodiment, described biosome sample is from making a definite diagnosis the patient who suffers from nasopharyngeal carcinoma.In one embodiment, described kit also contains the another kind of reagent that detects Epstein-Barr virus (EBV) (as the antibody at LMP-1 or EBER, or being used to detect their other any reagent).

5. pharmaceutical composition for the treatment of nasopharyngeal carcinoma, it contains: suppress the expression of EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) or active material as effective constituent; And pharmaceutical carrier, preferred described pharmaceutical composition contain the expression that suppresses EGF-R ELISA (EGFR) or active material simultaneously and suppress the expression of nuclear transcription factor-kappa B (NF-κ B) or active material as effective constituent.

6. according to above 5 pharmaceutical composition, it also contains the active component that another kind is used for the treatment of nasopharyngeal carcinoma, be preferably chemotherapeutant, as TNF (TNF), adriamycin (ADR), Etoposide (VP-16), neoplatin (CDDP), vincristine, methotrexate, 5 FU 5 fluorouracil and bleomycin.

7. suppress the expression of EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) or active material and be used for the treatment of application in the medicine of nasopharyngeal carcinoma in preparation.

8. according to above 7 application, wherein said medicine also contains the active component that another kind is used for the treatment of nasopharyngeal carcinoma, be preferably chemotherapeutant, as TNF (TNF), adriamycin (ADR), Etoposide (VP-16), neoplatin (CDDP), vincristine, methotrexate, 5 FU 5 fluorouracil and bleomycin.

9. kit that is used for the treatment of nasopharyngeal carcinoma, it contains: the expression or the active material that suppress EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B), preferred this kit also contains the active component that another kind is used for the treatment of nasopharyngeal carcinoma, be preferably chemotherapeutant, as TNF (TNF), adriamycin (ADR), Etoposide (VP-16), neoplatin (CDDP), vincristine, methotrexate, 5 FU 5 fluorouracil and bleomycin.

10. method of compound of identifying the treatment nasopharyngeal carcinoma, described method comprises:

A., the cell of expressing EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) is provided;

B. with described compound and described cell incubation; With

Whether the expression or the activity that c. detect cell mesocuticle growth factor receptors (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) are suppressed by described compound.

Should be pointed out that each embodiment and technical scheme that the present invention is cited can carry out combination in any as required.

Detailed Description Of The Invention

EGFR is as the target spot of nasopharyngeal carcinoma treatment

EGF-R ELISA (Epidermal growth factor receptor, EGFR) and family member and cell Growth Control important relationship [Khazaie is arranged unusually, K., Schirrmacher, V., andLichtner, R. (1993) .EGF receptor in neoplasia and metastasis.CancerMetastasis Rev 12,255-274.].This family belongs to receptor tyrosine kinase, and four family members are arranged, and is respectively EGFR, Neu, ErbB-3 and ErbB-4.EGFR can cause it to form receptor dimer and autophosphorylation with combining of its respective ligand, phosphorylation will cause a signal and connection incident, this incident can activate again the mitogen-activated protein kinase activity (mitogen activatingprotein kinases, MAPK).Though the EGFR family member is essential in normal cell growth, this protein family member's overexpression also is found in epithelioma.For example, one or more EGFR family member [Shackney, S., Pollice of meeting overexpression in surpassing the human breast cancer of 30-35%, A., Smith, C., Janocko, L., Sweeney, L., Brown, K., Singh, S., Gu, L., Yakulis, R., and Lucke, J. (1998) .Intracellular coexpression of epidermalgrowth factor receptor, Her-2/neu, and p21ras in human breast cancers:evidence for the existence of distinctive patterns of genetic evolution that arecommon to tumors from different patients.Clin Cancer Res4,913-928.].EGFR also is used as treatment for cancer target spot [Baselga, J., Tripathy recently, D., Mendelsohn, J., Baughman, S., Benz, C., Dantis, L., Sklarin, N., Seidman, A., Hudis, C., Moore, J., Rosen, P., Twaddell, T., Henderson, I., and Norton, L. (1996) .PhaseII study of weekly intravenous recombinant humanized anti-p 185HER2monoclonal antibody in patients with HER2/neu-overexpressing metastaticbreast cancer.J Clin Oncol 14,737-744.; Pegram, M., Lipton, A., Hayes, D., Weber, B., Baselga, J., Tripathy, D., Baly, D., Baughman, S., Twaddell, T., Glaspy, J., and Slamon, D. (1998) .Phase II study of receptor-enhancedchemosensitivity using recombinant humanized anti-p 185HER2/neumonoclonal antibody plus cisplatin in patients with HER2/neu-overexpressingmetastatic breast cancer refractory to chemotherapy treatment.J Clin Oncol 16,2659-2671.].Herceptin, one can specific inhibition Her2/neu the peopleization monoclonal antibody, develop out in the laboratory of UCLA Dennis Slamon, and success be applied to treat special breast cancer patient's subgroup, the breast cancer of this subgroup is expressed the Her2/neu gene.Because EGFR is by the LMP-1 induced strong, and expresses in nasopharyngeal carcinoma cell, and be considered to play extremely important effect in tumour takes place, we propose to treat as us with EGFR a target spot of nasopharyngeal carcinoma.

Another target spot of NF-κ B-nasopharyngeal carcinoma treatment

LMP-1 can activate NF-κ B signal transduction pathway strongly, and this approach may play an important role in the nasopharyngeal carcinoma generating process.NF-κ B is the different poly-or together poly-dimer that is formed by Rel protein family member, and this protein family mainly comprises: p50, p52, rel-A (p65), rel-B and c-Rel.It can many heterogeneic expression as a transcription factor functionating.These genes relate to acute phase response, inflammation, lymphocyte activation, cell growth and differentiation [Grilli, M., Chiu, J.J., andLenardo, M. (1993) .NF-κ B and Rel:Participants in a MultiformTranscriptional Regulatory System.International Review of Cytology 143,1-60].The activity of NF-κ B is regulated by its mortifier I κ B usually.In many cell types, NF-κ B is positioned tenuigenin, exists with the NF-κ B/I κ B complex form of non-activity.The stimulus signal of numerous species can activate NF-κ B, comprising: virus, bacteria lipopolysaccharide, antigen receptor linker, stressors, cell factor, phorbol ester, oxygenant and UV radiation.Need the phosphorylation of I κ B and degradation process subsequently by these factor activators NF-κ B, NF-κ B just can transposition enter nucleus like this.Studies show that recently NF-κ B has strong anti-apoptotic activity.Under the usual condition, the Apoptosis that cell resistance TNF induces, and become responsive for the existence of the NF-κ B mortifier as NF-κ B.When studies show that the activity inhibited as NF-κ B in the 3T3 cell, the Ras of composition activity can kill these 3T3 cell rather than transformants, and this anti-apoptosis function that just hints NF-κ B plays an important role in cell transformation.Shown in our PRELIMINARY RESULTS, we find that NF-κ B protection HaCaT cell avoids apoptosis and raises the A1 expression of gene, and the A1 gene is a member of Bcl-2 gene family, and is relevant with anti-apoptosis and cell transformation.

Description of drawings

Fig. 1 .EBV induces the action model of nasopharyngeal carcinoma and possible therapeutic strategy;

Fig. 2. Bcl-x in the nasopharyngeal carcinoma, the expression of EGFR and EBER (using Bcl-x, EGFR or the adjacent tumor tissue section of EBER-1 specific antibody dyeing nasopharyngeal carcinoma);

Fig. 3. suppress the Apoptosis that NF-κ B can promote TNF to induce and (handle parent HeCaT and A549 cell with the TNF-α of 50ng/ml and the clone cell of the energy overexpression DA-I κ B that derives in the time that indicates.Use gel to shift and find the activity (A and B) of nucleus extract in conjunction with NF-κ B specific probe.(C) small amounts of cells is moved into 96 hole double dish, the TNF-α that adds 50ng/ml carried out the MTT monitoring analysis after 24 hours, determined the survival rate of cell);

Fig. 4. suppress NF-κ B and can promote the chemotherapeutics cell killing (to use three kinds of chemotherapeutics (Adriamycin respectively, CDDP, VP-16) handle parent's A549 cell and derive can overexpression DA-I κ B clone cell after 24 hours, carry out the MTT check and analysis, determine the survival rate of cell after the medicine thing of variable concentrations is handled);

Fig. 5. chemotherapeutics can raise the expression of Bcl-x and Bfl-1 by NF-κ B signal transduction pathway, and (A549 and A549/I κ B cell are used 50ng/ml TNF α respectively, 200 μ M VP-16,10 μ MAdriamycin and 8 μ g/ml cisplatin handled 6 hours.Collect RNA and use Northernanalysis to detect Bcl-x, the expression of Bfl-1 and GAPDH).

Embodiment

Embodiment

The cell survival gene that EGFR and NF-κ B induce is as the diagnostic method of nasopharyngeal carcinoma

We obtain and have identified to surpass 50 human nasopharyngeal carcinomas and 30 breast cancer tissue's samples in development.At first we judge by checking as the expression of LMP-1 and EBNA1 Epstein-Barr virus labels such as (nuclear antigen of an Epstein-Barr virus coding) whether these nasopharyngeal carcinoma are Epstein-Barr virus related neoplasms.Anti-LMP-1 (ab7502 is purchased from Abcam) and anti-EBER antibody (ab68991 is purchased from Abcam) immunohistochemical staining result show that nearly all nasopharyngeal carcinoma sample that we detect all is the Epstein-Barr virus positive.Then, we use corresponding monoclonal antibody (ab62, ab16901 and ab32370 are purchased from Abcam), and the method with immunohistochemical staining has detected the expression of EGFR, Her2/Neu and Bcl-x respectively.In contrast, we have also detected the expression above EGFR, Her2/Neu and Bcl-x in 30 breast cancer samples.Simultaneously we have also compared nasopharyngeal carcinoma cell and the expression of single marking in the normal cell around it, and sample is divided into four groups.So far, we have finished the assessment to EGFR, Her2/Neu and Bcl-x expression, and this uses EGFR and Bcl-x to provide first hand basis as the target of the target spot of potential diagnosis and treatment nasopharyngeal carcinoma to us.The results are shown in Table 1.

Table 1: the expression of EGFR, her2/Neu and Bcl-x in nasopharyngeal carcinoma and the breast cancer

(annotate: numeral number percent in the table)

As shown in table 1, surpass the expression of 70% nasopharyngeal carcinoma sample EGFR extreme high (++ ++), 25% sample very high (+++), it is obviously higher than adjacent Normocellular expression to be less than 5% sample, and 0% is similar to adjacent normal cell expression.In tumour cell, the overexpression of EGFR and EBER-1 can show by dyeing, and does not show (as Fig. 2) in the normal cell on every side.What is interesting is that up to the present neither one is examined overexpression Her2/Neu in all test sample, another member of EGFR family is overexpression in surpassing 30% breast cancer cell.These studies show that, EGFR specificity overexpression and not having in adjacent normal cell in nasopharyngeal carcinoma.EGFR and Her-2/Neu overexpression in 30% and 23% human breast carcinoma is respectively also found in our research.But breast cancer is not expressed LMP-1 and EBER.By the expression of one of LMP-1 in the test sample and EBER and the expression of one of EGFR and Her-2/Neu, can diagnose nasopharyngeal carcinoma thus.What is interesting is that in most breast cancer tumour samples, the overexpression of EGFR and the overexpression of Her-2/Neu do not appear in the same sample simultaneously.This result's hint, different breast cancer patients need accept different treatments according to its allelic expression.For example, the breast cancer patient of overexpression Her-2/Neu gene should treat with Her-2/Neu antagonist such as Herceptin, should treat with the EGFR antagonist when patient's tumor is overexpression EGFR.And our result shows Bcl-x overexpression in most nasopharyngeal carcinoma and breast cancer, and does not have in flanking cell.In a word, our result shows cell survival gene such as Bcl-x and the A1[Lee that EGFR and NF-κ B induce, H., Dadgostar, H., Shu, J., Cheng, Q., and Cheng, G.The NF-κ B signal transduction pathway is essential for CD40-mediatedup-regulation of the Bcl-x and Bfl-1 genes and B-cell survival.Proc.Natl.Acad.Sci.USA.96:9136-9141,1999.].Experimental result has proved in nasopharyngeal carcinoma the Bcl-x overexpression.Can be used as the diagnostic flag of nasopharyngeal carcinoma.Nasopharyngeal carcinoma diagnosis mainly relies on eb early antigen (EA) and viral capsid antigen (VCA) at present, and this diagnosis can only explain getting in touch of Epstein-Barr virus activity and the potential generation of nasopharyngeal carcinoma.The cell survival gene that we utilize EGFR and NF-κ B to induce is diagnosed the method for nasopharyngeal carcinoma, direct directed cancer cell.This method even can distinguish out the nasopharyngeal carcinoma tumour cell and adjacent normal cell.

EGFR and NF-κ B are as the medicine of nasopharyngeal carcinoma treatment

As everyone knows, the EGFR family member is relevant with the generation of solid tumor, and their mortifier such as herceptin (Genentech Inc.) have been used to the breast cancer of clinical treatment Her-2/Neu overexpression type.The micromolecule such as the Tarceva (erlotinb) (Genentech Inc) that suppress the EGFR tyrosine kinase activity are used for some treatment of cancer by drugs approved by FDA, comprising non-small cell lung cancer (NSCLC) and cancer of pancreas.The anti-egfr antibodies that many EGFR of making lose function is also grown up.Yet the EGFR inhibitor also is not used to treat nasopharyngeal carcinoma.Because the expression of EGFR is all very high in most nasopharyngeal carcinoma tumour cells, we believe that the mortifier of EGFR comprises the neutralizing antibody of micromolecular inhibitor and anti-EGFR, should be the effective nasopharyngeal carcinoma treatment factors.

In order to determine further whether the EGFR inhibitor has strong lethal effect to the NPC tumour cell really, we use EGFR inhibitor Tarceva to handle nasopharynx cancer clone C666-1[CheungST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS, Wong N, Whitney BM, Lee JC.Nasopharyngeal carcinoma cell line (C666-1) consistently harbouringEpstein-Barr virus.Int J Cancer.1999Sep 24; 83 (1): 121-6].Nasopharynx cancer clone C666-1 is hatched on 24 hole culture plates to every milliliter has 5 * 10 ⁶Begin to carry out the validity and the susceptibility experiment of EGFR inhibitor during the concentration of individual cell.The EGFR inhibitor Tarceva that in cell, adds various dose, final concentration is respectively: 0,0.1,0.3,1,3,10,30,100,300 and 1000 μ M, kept somewhere 24 hours, use then the mtt assay analysis of cells of standard survival rate (MosmannT.Rapid colorimetric assay for cellular growth and survival:application toproliferation and cytotoxicity assays.J.Immunol.Meth.1983,65,55-63), determine that whether the NPC cell is responsive to the EGFR inhibitor for treating, can also measure simultaneously and kill 50% NPC cell and whole concentration of the EGFR inhibitor of required use during the NPC cell.Experimental result shows that Tarceva kills the best IC of nasopharyngeal carcinoma cell C666-1 ₅₀Value is 1 μ M.

Then our emphasis has been assessed the effect of NF-κ B in the cancer cell survival.Because also do not have the sort of compound specificity to suppress the NF-kB pathway at present, we determine to use the I κ B (Dominant active form of I κ B, or DA-I κ B) that forms activity form to suppress NF-κ B.But we have successfully turned out the stable HeCaT cell clone of overexpression DA-I κ B.The former HeCaT cell that studies show that overexpression LMP-1 why use the HeCaT cell to be because can develop into phymatoid nasopharyngeal carcinoma in mouse.Remove the HeCaT extracellular, the A549 clone (lung cancer cell line) that we have also cultivated stable overexpression DA-I κ B (ATCC#:CCl-185) and Daudi clone (the Epstein-Barr virus transformed B lymphocytes, ATCC#:CCl-213).Utilize these clone, we have determined the effect of NF-κ B mortifier in the Apoptosis sensitization, be applicable to number of chemical therapeutic agent such as TNF (TNF-α), adriamycin (adriamycin, ADR), Etoposide (etoposide, VP-16) and neoplatin (Cisplatin, CDDP).We have compared the allelic expression of parent's normal cell and DA-I κ B express cell to utilize the Affymetrix genetic chip, identify the target gene of many NF-κ B.More than research shows that not only NF-κ B is a valuable treatment of cancer target spot, has also increased us for NF-κ B and cancer generation and the machine-processed understanding of chemical drug resistance relation.

In order to study the effect of NF-κ B in cell survival, we turn out HeCaT and the A549 cell of stablizing overexpression DA-I κ B.As shown in Figure 3, the overexpression of DA-I κ B is the strong inhibition TNF NF-kB activity of inducing not only, the Apoptosis that these cells of also strong sensitization TNF induces.For further research suppresses the Apoptosis that NF-κ B whether can sensitization response chemotherapeutant, after the clinical chemotherapeutant that generally use different with tens of kinds handled, we had compared father and mother for the cell survival situation of normal cell with the A549 cell of expressing DA-I κ B.As shown in Figure 4, the A549 cell all has strong resistant function to different chemotherapeutants such as ADR, VP-16 and CDDP.Yet behind I κ B inhibition NF-kB activity, the A549 cell has become responsive many for these chemotherapeutants.These data a kind of effective methods of treatment that use may become the resistant function cancer cell of uniting that shows NF-κ B mortifier and traditional chemical therapeutic agent clearly.

In order further to study the anti-medicine mechanism that cancer cell NF-κ B relies on, we use the Affymetrix genetic chip to compare the A549 cellular gene expression feature of father and mother for A549 and overexpression DA-I κ B.By chip analysis, we identify many potential relevant NF-κ B of anti-medicine mechanism and rely on gene.These NF-κ B rely on gene and comprise many Bcl-2 family genes such as Bcl-x and Bfl-1, and these genes all play a significant role in cell survival.As shown in Figure 5, many clinically generally use, be considered to can the kill tumor cell chemotherapeutant, in fact, can active cell survival genes up-regulated expression in the situation that NF-κ B relies on, this perhaps can explain the generation of the anti-medicine mechanism of cancer cell.Except that the Bcl-2 family member, we have identified that also a subgroup of redox related gene is as coding aldose reductase sample peptide (ARL-1), glutathione peroxidase 2 (GPX2), glutamyl transpeptidase (GluTrans) etc.Though these genes are not by chemotherapeutic-induced, but these genes the father and mother that chemotherapeutant had opposing for the A549 cell in high level expression, and (these cells are to the chemotherapeutant sensitivity) do not have high expressed in the A549 cell of expressing I κ B.Further research needs to understand the effect of these genes in anti-medicine mechanism.

Our The above results shows in nasopharyngeal carcinoma that just tumour cell rather than normal cell on every side give expression to for example Bcl-X of high-level cells survival gene.Our research has also proved and has suppressed the expression that NF-κ B path just may stop Bcl-X, caused the apoptosis of cancer cell.The strategy of another novelty of this data suggest NPC therapy is promptly used the combination of EGFR inhibitor and NF-kB inhibitor.More effective than using wherein any one separately in order to determine this combination treatment, we will carry out three groups of experiments, promptly use the combined treatment NPC cell of EGFR inhibitor Tarceva, NF-kB inhibitor PS1145 and two inhibitor respectively.With nasopharyngeal carcinoma cell is that C666-1 is hatched on 24 hole culture plates to every milliliter and had 5 * 10 ⁶Begin to experimentize during the concentration of individual cell.Add the NF-kB inhibitor PS 1145 of various dose in cell, final concentration is respectively: 0,0.1,0.3,1,3,10,30,100, and 300 and 1000 μ M kept somewhere 24 hours, used the survival rate of the mtt assay analysis of cells of standard then.It is 10 μ M that the optium concentration of using PS1145 to kill 50% NPC cell is measured in interpretation of result.

Use the employed polypharmaceutic composition of experiment of killing nasopharyngeal carcinoma cell to contain Tarceva and PS1145 EGFR and NF-kB inhibitor associating compound.Compound medicine is added the C666-1 cell, and it will be more effective that 50% NPC cell is killed the combined therapy nasopharyngeal carcinoma that found that EGFR inhibitor and NF-kB inhibitor.

Conclusion

Our invention has proved that EGFR and Bcl-X can be used as the biological target of nasopharyngeal carcinoma diagnosis and treatment.The NPC that our data suggest EBV causes grows up by pungency regulation and control EGFR, and EGFR provides a strong cell division signal, activates NF-κ B path, makes cell breed and the generation canceration rapidly.

Our invention has two kinds of main application:

1) we can use EGFR and Bcl-X as the biological target of diagnosing nasopharyngeal carcinoma.

The diagnostic method of nasopharyngeal carcinoma all is only limited to and detects the antibody that the EBV virus protein causes at present.The limitation of these methods be to be used for the nasopharyngeal carcinoma people at highest risk examination and can not be as making a definite diagnosis foundation.And our result is as shown in Fig. 2, and EGFR and Bcl-X only highly express in nasopharyngeal carcinoma cell.Therefore can be used from the target one of EBV and make a definite diagnosis nasopharyngeal carcinoma.

2) use EGFR inhibitor Tarceva or NF-kB inhibitor PS1145 can both kill nasopharyngeal carcinoma cell separately, use Tarceva and PS1145 treatment nasopharyngeal carcinoma more effective yet unite.

Claims (10)

1. the application of reagent in the agent of preparation nasopharyngeal carcinoma diagnosis, described reagent is used for the expression or the activity of EGF-R ELISA of biosome sample (EGFR) or nuclear transcription factor-kappa B (NF-κ B) are measured.

2. reagent is used for estimating the application of diagnosticum of the validity of the progress degree of nasopharyngeal carcinoma and/or treatment in preparation, and described reagent is used for the expression or the activity of EGF-R ELISA of biosome sample (EGFR) or nuclear transcription factor-kappa B (NF-κ B) are measured.

3. kit that is used to detect nasopharyngeal carcinoma, it contains:

Be used for the EGF-R ELISA (EGFR) of biosome sample or the expression or the active reagent of measuring of nuclear transcription factor-kappa B (NF-κ B).

4. the kit of the validity of progress degree that is used to estimate nasopharyngeal carcinoma and/or treatment, wherein, contain:

Be used for the EGF-R ELISA (EGFR) of biosome sample or the expression or the active reagent of measuring of nuclear transcription factor-kappa B (NF-κ B).

5. pharmaceutical composition for the treatment of nasopharyngeal carcinoma, it contains: suppress the expression of EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) or active material as effective constituent; And pharmaceutical carrier, preferred described pharmaceutical composition contain the expression that suppresses EGF-R ELISA (EGFR) or active material simultaneously and suppress the expression of nuclear transcription factor-kappa B (NF-κ B) or active material as effective constituent.

6. according to the pharmaceutical composition of claim 5, it also contains the active component that another kind is used for the treatment of nasopharyngeal carcinoma, be preferably chemotherapeutant, as TNF (TNF), adriamycin (ADR), Etoposide (VP-16), neoplatin (CDDP), vincristine, methotrexate, 5 FU 5 fluorouracil and bleomycin.

7. suppress the expression of EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) or active material and be used for the treatment of application in the medicine of nasopharyngeal carcinoma in preparation.

8. according to the application of claim 7, wherein said medicine also contains the active component that another kind is used for the treatment of nasopharyngeal carcinoma, be preferably chemotherapeutant, as TNF (TNF), adriamycin (ADR), Etoposide (VP-16), neoplatin (CDDP), vincristine, methotrexate, 5 FU 5 fluorouracil and bleomycin.

9. kit that is used for the treatment of nasopharyngeal carcinoma, it contains: the expression or the active material that suppress EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B), preferred this kit also contains the active component that another kind is used for the treatment of nasopharyngeal carcinoma, be preferably chemotherapeutant, as TNF (TNF), adriamycin (ADR), Etoposide (VP-16), neoplatin (CDDP), vincristine, methotrexate, 5 FU 5 fluorouracil and bleomycin.

10. method of compound of identifying the treatment nasopharyngeal carcinoma, described method comprises:

A., the cell of expressing EGF-R ELISA (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) is provided;

B. with described compound and described cell incubation; With

Whether the expression or the activity that c. detect cell mesocuticle growth factor receptors (EGFR) and/or nuclear transcription factor-kappa B (NF-κ B) are suppressed by described compound.

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