CN1981872B - Use of PUMA in tumor chemoradiotherapy sensibilization - Google Patents
Use of PUMA in tumor chemoradiotherapy sensibilization Download PDFInfo
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- CN1981872B CN1981872B CN 200510130277 CN200510130277A CN1981872B CN 1981872 B CN1981872 B CN 1981872B CN 200510130277 CN200510130277 CN 200510130277 CN 200510130277 A CN200510130277 A CN 200510130277A CN 1981872 B CN1981872 B CN 1981872B
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Abstract
本发明涉及PUMA基因的新用途，特别是外源PUMA基因或包含有PUMABH3结构域的cDNA及蛋白的导入在制备增加肿瘤细胞对化疗药物和放射治疗的敏感性药物中的用途。The present invention relates to a new use of PUMA gene, especially the use of introducing exogenous PUMA gene or cDNA and protein containing PUMABH3 domain in the preparation of drugs for increasing the sensitivity of tumor cells to chemotherapeutic drugs and radiotherapy.
Description
Technical field
The cDNA and the proteic importing that the present invention relates to the new purposes of PUMA gene, particularly external source PUMA gene or include PUMABH3 domain (LRRMADDLN) increase tumor to the purposes in chemotherapeutics and the radiocurable sensitive drug in preparation.
Background technology
Chemotherapy of tumors is an indispensable important means in the malignant tumor Comprehensive Treatment; Become the main method of some tumor radical cure; (new adjuvant chemotherapy) chemotherapy can make the cure rate of part cancer increase before operation back (adjuvant chemotherapy) and the operation, can increase the excision chance of multiple solid tumor in local late period.But the various chemotherapeutics (alkylating agent, antimetabolitas, AGPM class, antineoplastic plant medicine thing, platinum class etc.) of different effects mechanism all have toxic and side effects in various degree, as: bone marrow depression, gastrointestinal reaction, the heart, lung, liver, kidney, bladder, neurotoxicity etc.Radiotherapy is the method except that operation and the topmost treatment tumor the chemotherapy, but it has bigger toxic and side effects to propagation tissue slower or that lose multiplication capacity, like the radium necrosis of radiation fibrosis of lung, brain and spinal cord.These toxic and side effects have had a strong impact on and have put, the application of chemotherapy in clinical, seek a kind of can the enhancing and put, chemotherapeutic efficacy, lower its therapeutic dose, thereby the material that alleviates its toxic and side effects is the target that the medical personnel seek assiduously.
Now, the known radiotherapy of people and most of chemotherapeutics are mainly all removed tumor cell through cell death inducing.The importing of the short apoptosis-related genes of external source might play increase put, the effect of chemosensitivity, this is confirmed in people's research to p53 in the past.
PUMA (p53 up-regulated modulator of apoptosis) is newfound Bcl-2 protein family a member of calendar year 2001 report, gains the name because of it can and have powerful apoptosis-promoting effect by the p53 rapid induction.It interacts through BH3 domain and other Bcl-2 family protein, and inducing cell pigment c discharges, and causes apoptosis.PUMA albumen is positioned on the mitochondrial membrane, and (the 141-149 amino acids be that apoptosis-induced institute is requisite LRRMADDLN), and C 43 amino acid residues of end (151-193 position) is that it is in mitochondrion location and apoptosis-induced necessary to the BH3 domain.It is proteic approaching only to contain the PUMA expression structure of 93 amino acid residues of C end apoptosis-induced level and total length PUMA.The apoptosis-induced ability of PUMA is powerful fast, and the human large intestine cancer DLD1 cell of expressing p53 occurs late 9 hours (like chromatin agregation, the nuclear segmentization) than the DLD1 apoptosis morphological change of expressing PUMA.
Summary of the invention
CDNA and the albumen that the object of the present invention is to provide a kind of PUMA gene or include PUMA BH3 domain increase in preparation put, the new purposes in the chemosensitivity medicine.In other words, the invention reside in and provide that a kind of tumor is put, chemotherapeutic sensitizer.
Chemotherapeutics of the present invention includes, but are not limited to:
Platinum class: cDDP (cisplatin cisplatin), CBP (carboplatin carboplatin), L-OHP (Oxaliplatin oxaliplatin), NDP (Nedaplatin nedaplatin) etc.
Microtubule inhibitor: PTX (Taxol paclitaxel), TXT (Taxotere taxotere), VLB (Vinblastine vinblastine), VCR (Vincristione vincristine), VNR (Vinorelbine vinorelbine) etc.
Anti-metabolism: 5-Fu (5-Fluorouracil five fluorouracil), 6-TG (Thioguanine thioguanine), AG337 (Nolatrexed is nolatrexed), FT-207 (Tegafur ftorafur), HCFU (Carmofur carmofur), 5`-DFUR (Doxifluridine FTL), UFT (UFT), Ara-C (Cytarabine cytosine arabinoside) etc.
Topoisomerase II inhibitor: VP16-213 (etoposide etoposide), VM-26 (teniposide VM-26) etc.
Anthracene nucleus antineoplastic antibiotic: ADR (adriamycin amycin), DAM (daunomycin daunorubicin), aklavine (aclacinomycin), epirubicin (epirubicin), Carubicin (idarubiein), mitoxantrone (mitoxatrone) etc.
Preferably cisplatin cDDP, paclitaxel taxol, five fluorouracil 5-Fu, oxaliplatin Oxaliplatin, etoposide etoposide, amycin adriamycin.
Radiotherapy of the present invention includes but not limited to gamma-rays.
Medicine of the present invention is that the PUMA gene imports carrier or includes the cDNA of PUMA BH3 domain and the medicine that albumen forms.
The present invention can adopt and well known to a person skilled in the art that range gene imports carrier; Can adopt the adenovirus mediated system that the PUMA gene (Ad-Δ BH3) of external source PUMA gene (Ad-PUMA) and disappearance BH3 domain is imported; Through people's cancer nude mice tumor model in body outer cell line, the body; The effect of the collaborative chemotherapeutics of confirmation Ad-PUMA and radiotherapy, enhancing antitumor curative effect does not then possess above-mentioned functions by the gene constructed Ad-Δ BH3 of PUMA that lacks the BH3 domain.Described carrier is not limited to replication-defective adenoviral vector; Can also comprise the method that other various exogenous genes import; Like particle gun, liposome method, receptor-mediated transduction etc.; And other virus carrier system, described other viral vector comprises, replication competent adenovirus, herpes simplex virus, retrovirus, adeno-associated virus, vaccinia virus etc.Success import the cDNA include PUMA BH3 domain and albumen also can play increase put, the effect of chemosensitivity.
The chemotherapeutics of PUMA or its BH3 domain ability enhanced sensitivity also is not limited to several kinds cited in the following experiment.
Usually the use of Ad-PUMA comprises:
Treatment experiment for mice can be adopted and not wait dosage according to different situations (tumor size, treatment number of times, blanking time etc.): general 5 * 10
8PFU～5 * 10
9PFU/ time, successive administration is 5 times once a day, also can be administered once in per two days or three days totally three times.
Those skilled in the art know the medicine for treatment dosage of Ad-p53 for human body, and treatment group dosage, blanking time are not waited, and are described as by its product description: weekly, and each 10
12VP (is equivalent to 3.3 * 10
10PFU), the local multi-point injection of tumor tissue.
Ad-PUMA of the present invention can be with reference to using dosage and the application method of Ad-p53.
The present invention is through experiment confirms in a series of external, bodies: import external source PUMA gene; In comprising in the entity tumors such as breast carcinoma, cervical cancer, pulmonary carcinoma, hepatocarcinoma, cancer of pancreas and the esophageal carcinoma of multiple histological types; Can strengthen the chemotherapeutics and the radiocurable antitumor action of multiple different effects mechanism; Then do not possess this function behind the external source PUMA gene delection BH3 domain, and the importing of PUMA is more remarkable than the importing of p53 gene with the Synergistic of chemotherapeutics.The importing of external source PUMA does not change the influence of various chemotherapeutics to the cell cycle of tumor cell itself, and this synergism is because apoptosis-induced collaborative causing.
Description of drawings
Figure 1A shows that Ad-PUMA associating chemicotherapy significantly suppresses the growth of A549 cell.
The chemotherapeutic that Figure 1B shows various dose separately or with Ad-PUMA Combined Treatment A549 cell after cell growth rate.
Fig. 2 A shows that Ad-PUMA significantly strengthens the inductive A549 apoptosis of amycin.
Fig. 2 B shows that Ad-PUMA significantly strengthens the inductive A549 apoptosis of gamma-rays.
The different virus that shows Fig. 3 infects cell survival rate behind the esophageal cancer cell 72h.
Fig. 4 shows that the dull and stereotyped colony of KYSE150 cell forms situation.
Fig. 5 shows that different factors handle the variation of KYSE-150 cell cycle behind the 48h.
Fig. 6 A shows the karyomorphism after the DAPI dyeing.
Fig. 6 B shows that 50MOIAd-PUMA infects KYSE150 different time points apoptotic cell percentage ratio.
Fig. 6 C shows that 200MOIAd-p53 infects KYSE150 different time points apoptotic cell percentage ratio.
Fig. 6 D shows that Ad-PUMA and chemotherapeutic synergy 36hr respectively organize apoptotic cell percentage ratio.
Fig. 7 representes to adopt the Auele Specific Primer (upper reaches 5 '＞GTCCTCAGCCCTCGCTCT＜3 ' downstream 5 '＞CTGCTGCTCCTCTTGTCTCC＜3 ') of PUMA to detect the change electrophoretogram of PUMA in the mRNA level.
Fig. 8 shows KYSE150 transplanted tumor in nude mice growth curve chart.
Fig. 9 shows the KYSE150 transplanted tumor in nude mice tissue of peeling off.
Figure 10 A shows that Ad-PUMA significantly suppresses the lung carcinoma cell growth.
The form of cell changes behind Figure 10 B demonstration PI staining detection Ad-PUMA infection H1299 lung carcinoma cell.
Apoptotic cell percentage ratio behind Figure 10 C demonstration flow cytometer detection Ad-PUMA infection H1299 lung carcinoma cell.
Figure 11 shows that Ad-PUMA is more remarkable than Ad-p53 cell growth inhibiting.
Figure 12 A shows A549 transplanted tumor in nude mice growth curve chart.
Figure 12 B shows that Ad-PUMA suppresses the growth of A549 transplanted tumor in nude mice.
Figure 12 C shows H1299 transplanted tumor in nude mice growth curve chart.
Figure 12 D shows that Ad-PUMA significantly induces the H1299 transplanted tumor in nude mice to organize apoptosis.
The specific embodiment
The research of Ad-PUMA and chemotherapy drugs in combination effect in the embodiment one various tumor cell lines
Method:
Adopt means as well known to those skilled in the art to obtain the PUMA gene, and it is imported adenovirus, obtain Ad-PUMA (adenovirus-PUMA).Then various tumors below the exponential phase (breast carcinoma, cervical cancer, hepatocarcinoma, pulmonary carcinoma, cancer of pancreas) cell line cell is inoculated in 96 orifice plates, according to the cell number that the discrepancy adjustment of cell volume between different cell lines is inoculated, overnight incubation.Described Ad-PUMA is (before the infection cell of adenovirus-PUMA); Discard former culture medium; Wash gently 3 times with PBS; Add 100 μ l and contain the serum-free RPMI1640 culture medium of a certain amount of Ad-PUMA, make it reach certain infection multiplicity (multiplicity of infection), the Ad-PUMA virion that infection ability is arranged of some is arranged in promptly average each cell.Owing to the sensitivity of every kind of cell line to Ad-PUMA there are differences, the infection multiplicity between each cell line is different.96 orifice plates are positioned over 37 ℃, 5%CO
2Incubator is cultivated 90min, during whenever rock culture dish gently once at a distance from 15min is cross, viral liquid is evenly distributed.Add the RPMI1640 complete medium (containing 10%FBS, 100U/ml penicillin, 0.1mg/ml streptomycin) that contains the finite concentration chemotherapeutics in the different then holes respectively.Add the concentration of chemotherapeutics definite principle be growth inhibition ratio＜60% that this chemotherapeutics is made the time spent pair cell separately.Wherein the concentration of cDDP, taxol, 5-Fu is respectively 5 μ M, 5nM, 10 μ M in HeLa, SiHa, MGC803, A549, the P3 cell line; The concentration of 5-Fu is 20 μ M in the BEL7402 cell line, and is surplus the same; The concentration of cDDP, taxol, 5-Fu adopts the absorbance of mtt assay with each porocyte of ELIASA detection after being respectively 2 μ M, 2nM, 10 μ M. drug treating 72hr in the MCF-7 cell line, calculates the suppression ratio of each processed group cell growth.Each processed group is established 3 parallel holes, repeated experiments 2-3 time.
Result: see table 1.
In the tumor cell line of histological types (breast carcinoma, cervical cancer, hepatocarcinoma, pulmonary carcinoma and cancer of pancreas), external source imports the cytotoxicity that PUMA gene (Ad-PUMA) can strengthen the chemotherapeutics (as: cisplatin cDDP, paclitaxel taxol, five fluorouracil 5-Fu etc.) of multiple different effects mechanism.After the Ad-PUMA coupling of low dosage; The growth inhibition ratio of chemotherapeutics pair cell has increased by 10.39%～54.51% respectively; Wherein cervical cancer tumer line SiHa is the most responsive to Ad-PUMA; As long as external source imports the Ad-PUMA of 2MOI, in promptly average each SiHa cell 2 reorganization PUMA adenovirus particles that infection ability is arranged are arranged, just can significantly strengthen the cytotoxicity of various chemotherapeutics.
Table 1
Different disposal group inhibitory rate of cell growth (%)
Ad-PUMA and chemotherapeutics synergism and Study on Mechanism thereof in embodiment two lung cancer cell lines
1.Ad-PUMA significantly reduce the IC50 of different chemotherapeutic in the A549 lung carcinoma cell
Ad-PUMA10MOI infects the A549 cell; Taxol, Cisplatin, 5-FU, Etoposide and Adriamycin with variable concentrations behind the 24hr handle cell; Adopt the MTS method to detect the absorbance of each porocyte after continue cultivating 48hr, detect each chemotherapeutic absorbance of effect separately simultaneously, with untreated cell as contrast; Calculate each processed group cell growth rate, calculate the IC50 that each chemotherapeutic reaches associating Ad-PUMA separately.Each processed group is established 3 parallel holes, repeated experiments 3 times.
Result Ad-PUMA in the A549 lung cancer cell line can significantly reduce the IC50 of various chemotherapeutic; Minimumly can it be reduced to 1/3 original (Adriamycin0.76 μ g/ml reduces to 0.22 μ g/ml), the highest IC50 of making is lower than 1/10 original (Taxol and 5-FU) (table 2).This explanation Ad-PUMA all has significant chemotherapy sensitizing effect in each lung cancer cell line.
Table 2
Chemotherapeutic reaches and the symphyogenetic IC50 of Ad-PUMA separately in the A549 cell
2.Ad-PUMA strengthen the research of lung cancer cell line A549 to chemosensitivity
Adopt with embodiment one identical method and obtain Ad-PUMA, the infection intensity infection A549 cell with 10MOI carries out different disposal Taxol:5nM behind the 24hr; 5-FU:5-Fluorouracil, 25 μ g/ml; Oxa:Oxaliplatin, 25 μ M; Cis:Cisplatin, 25 μ M; Etp:Etoposide, 2 μ M; Adr:Adriamycin, 0.2 μ g/ml.Adopt the MTS method to detect the absorbance of each porocyte after continuing to cultivate 48hr, the suppression ratio of making the time spent cell growth with each chemotherapeutic separately is reference, relative cell growth inhibited multiple after calculating Ad-PUMA and each the chemotherapeutic coupling.And each chemotherapeutic carried out significance test separately and with the symphyogenetic dosage effect trend of Ad-PUMA.Each processed group is established 3 parallel holes, repeated experiments 3 times.The result sees accompanying drawing 1A, B, and inhibitory rate of cell growth is that the 2-8 that gives chemotherapeutic does not separately doubly wait after Ad-PUMA and each the chemotherapeutic coupling.Adopt GraphPadPrism IV statistical software that the dosage effect trendgram is tested, the growth inhibited that visible Ad-PUMA and each chemotherapeutic are share pair cell significantly is better than each chemotherapeutic and acts on (P＜0.05) separately.This shows that Ad-PUMA has the effect of enhancing lung cancer cell line A549 to chemotherapy drug susceptibility.
3. Ad-PUMA strengthens the Study on Mechanism of its chemosensitivity in the lung cancer cell line
The A549 cell is after 10MOI Ad-PUMA or
infect 24 hours; At the appointed time collect suspension and attached cell after adding Adriamycin 0.2 μ g/ml, detect nuclear fragmentation counting apoptotic cell percentage ratio through PI dyeing.At least 300 cells of each counting, repeated trials 3 times is got average.Shown in Fig. 2 A, use Ad-PUMA (10 MOI) or Adriamycin (0.2 μ g/ml) to handle the A549 cell separately and do not induce significant apoptosis in 72 hours, 72 hours then nearly 90% cell generation apoptosis of the two Combined Treatment A549 cell.The Ad-Δ BH3 combined chemotherapy medicine Adriamycin of same dose does not then exert an influence to the apoptosis-induced ability of the latter.This shows that the BH3 domain is that its chemotherapy sensitizing effect of PUMA performance is necessary, successful expression BH3 domain just can significantly strengthen the apoptosis-induced ability of chemotherapy in the cell.
Ad-PUMA and chemotherapeutics synergism and Study on Mechanism thereof in the embodiment three esophageal cancer cells system
1.Ad-PUMA suppress the research that different esophageal cancer cells are the growing multiplication effect
Adopt with embodiment one identical method and obtain Ad-PUMA; Respectively with different infection intensities (500MOI～Ad-PUMA 5MOI), Ad-p53 (trade name: " Gendicine "; Be that reorganization human P 53 adenovirus injection---the Shenzhen City SaiBaiNuo Gene Technology Co., Ltd produces, positive control), Ad-GFP (adenovirus of band green fluorescence protein gene GFP, negative control) infects various esophageal cancer cells; Adopt MTT to detect the absorbance of each porocyte behind the 72hr, calculate each processed group cell growth rate.Each processed group is established 3 parallel holes, repeated experiments 3～4 times.
The result sees accompanying drawing 3.It is thus clear that; At esophageal cancer cell is that infection intensity is identical among KYSE150, KYSE410, YES2, the KYSE510; Action time, the growth inhibition ratio of Ad-PUMA pair cell was significantly higher than Ad-p53 when identical, and wherein the KYSE150 cell is the most responsive to Ad-PUMA; The Ad-PUMA infection cell of 50MOI just can suppress the growth of 63.73% cell in 72 hours, and had only 51.37% at the growth inhibition ratio of the Ad-p53 pair cell of identical infection time 200MOI.
2. the synergism that Ad-PUMA and chemotherapeutics inhibition are bred in the esophageal cancer cell system
Adopt foregoing mtt assay to detect various chemotherapeutic and act on the 50% inhibition dosage IC50 (inhibitory concentration of 50%) that each esophageal cancer cell is 72hr separately.Use Ad-PUMA and each chemotherapeutic synergy of fixed dosage then, the drug level when in each esophageal cancer cell system, reaching 50% growth inhibited behind the calculating effect 72hr.Fixed Ad-PUMA dosage selection principle acts on 72hr inhibitory rate of cell growth＜10% separately for this dosage Ad-PUMA.According to formula (combination index=A
c/ A
e+ B
c/ B
e, A
eAnd B
eTwo factors that are respectively act on the concentration when reaching 50% growth inhibited, A separately separately
cAnd B
cTwo factors that then are respectively are share the concentration separately when reaching 50% growth inhibited) to calculate and share index, this index＜1 is for collaborative, and=1 is addition, and＞1 is antagonism.The result sees the following form 3:
Table 3
Ad-PUMA and chemotherapeutic suppress the synergism (IC50) of propagation in the esophageal cancer cell system
This shows that the Ad-PUMA of low infection multiplicity can significantly strengthen the sensitivity of various chemotherapeutics, after chemotherapeutics and Ad-PUMA share in each cell line, IC50 all had reduction (except the taxol among the KYSE-510) in various degree.Share index and can find out that all there are certain synergism in Ad-PUMA and various chemotherapeutic according to what calculate, and wherein 50% share index＜0.5, show that synergism is very significantly.
3.Ad-PUMA suppressing the dull and stereotyped colony of KYSE-150 cell with chemotherapy drugs in combination forms
Adopt dull and stereotyped colony to form the synergism of experimental verification Ad-PUMA and various chemotherapeutic, the result sees accompanying drawing 4.It is thus clear that 2MOI Ad-PUMA and low dose of chemotherapeutic (1 μ M cDDP, 0.5nM taxol, 2 μ M 5-Fu) synergy just can significantly reduce the colony of KYSE-150 cell and form.
4. external source imports the influence of PUMA cell cycle
KYSE150 cell behind the collection different disposal factor effect 48hr; The FACSCalibur streaming machine of Application of B D company; Adopt Cell Quest Mod Fit LT for Mac V3.0 analysis software to detect cell cycle; The result sees accompanying drawing 5, and the distribution in 48 hour cell cycles of 5MOIAd-PUMA infection cell is identical with contrast, and separately the 48 hour cell cycles of effect specificly respectively are arrested in G2/M phase and S phase for 2nM taxol and 10 μ M 5-Fu; And the Ad-PUMA of 5MOI and above-mentioned chemotherapy drugs in combination are used 48 hours, and to do the time spent separately consistent with each chemotherapeutic basically in the distribution of cell cycle.It is thus clear that the importing of external source PUMA does not change the influence of various chemotherapeutic to the tumor cell cell cycle itself.
5.DAPI staining detects Ad-PUMA and chemotherapeutics reaches symphyogenetic apoptotic cell percentage ratio separately
Collect the cell of each corresponding time point of different disposal factor, employing DAPI (4 '-6-Diamidino-2-phenylindole) staining detects apoptotic cell percentage ratio.Shown in accompanying drawing 6A, the fluorescence microscope normal nucleus nuclear membrane in observation DAPI dyeing back down is smooth, and chromatin is evenly distributed; And the apoptotic cells chromatin concentrates, marginalisation, and nuclear membrane cracking, chromatin are divided into bulk, apoptotic body occurs.Infecting the KYSE150 cell according to the apoptotic cell rate curve 50MOIAd-PUMA of DAPI dyeing back counting promptly had 50.44% cell the change of apoptotic cells nuclear to occur in 18 hours, and the nuclear change (seeing accompanying drawing 6B, C) that 200MOI Ad-p53 infection cell only had 47.98% cell to present obvious apoptosis in 48 hours.5MOI Ad-PUMA, 4 μ McDDP, 2nM taxol, the independent function cells of 10 μ M 5-Fu had 19.46%, 9.62%, 9.94%, 13.71% cell apoptosis to occur in 36 hours respectively; And when above-mentioned three kinds of chemotherapeutic still with identical concentration and 5MOI Ad-PUMA synergy 36 hours, apoptotic cells percentage ratio then reaches 32.99%, 34.72%, 39.30% (seeing accompanying drawing 6D) respectively.This Ad-PUMA and chemotherapeutic Combined application that shows low infection intensity just can significantly strengthen the apoptotic effect of induced by chemotherapeutic agents.
6.RT-PCR the expression of PUMA after detection Ad-PUMA and the low dose of chemotherapy drugs in combination effect
Collect 5MOI Ad-PUMA separately, and and low dose of chemotherapeutic (4 μ M cDDP, 2nM taxol, 10 μ M5-Fu) synergy 36hr after the KYSE-150 cell; Extract total RNA; Behind reverse transcription; Adopt the Auele Specific Primer (upper reaches 5 '＞GTCCTCAGCCCTCGCTCT＜3 ' downstream 5 '＞CTGCTGCTCCTCTTGTCTCC＜3 ') of PUMA to detect the change of PUMA in the mRNA level, the result sees accompanying drawing 7.Can find out that Ad-PUMA and low dose of various chemotherapeutic all can make cell PUMA mRNA express, and PUMA increases more obviously at the mRNA horizontal expression after the synergy.
Embodiment four KYSE-150 transplanted tumor in nude mice model validation Ad-PUMA and chemotherapeutics synergism
1.KYSE-150 the foundation of transplanted tumor in nude mice model
Centrifugal removal Digestive system behind the KYSE-150 cell dissociation with exponential phase is washed 2 times with containing two anti-Hank ' s liquid, centrifugal, counting, and the adjustment cell concentration is 1 * 10
7/ ml gets 200 μ l cell suspension inoculations in the right oxter of nude mice.Totally 35 of nude mices, 4～5 all Mus ages, body weight 14g～16g, female.
2. growth of xenografted curve
When treating that the tumor major diameter reaches 5mm～7mm, at random nude mice is divided into 7 groups.Adjustment Ad-PUMA, Ad-p53 titre make it reach 2 * 10
10PFU/ml injects once each 50 μ l, totally three times in the per 3 days tumor bodies.CDDP adopts lumbar injection, presses the administration of 3mg/kg body weight, the same adenovirus of administration time.From treatment the previous day, per 3 days major diameters with vernier caliper measurement subcutaneous transplantation tumor (a) and minor axis (b) by formula calculate tumor volume V=a * b
2/ 2, draw the growth curve of transplanted tumor, see accompanying drawing 8.It is thus clear that, the individually dosed growth that can significantly suppress transplanted tumor of Ad-PUMA, tumor growth is more slow behind the associating cDDP, and it is all obvious not as Ad-PUMA that the Ad-p53 of same dose reaches associating cDDP administration tumor killing effect separately.
3. the mensuration of transplanted tumor weight
The 18th day execution nude mice in medication strips tumor tissues (seeing accompanying drawing 9), takes by weighing tumor and weighs, and calculates tumour inhibiting rate.Particulars sees the following form:
Table 4
The tumor of different disposal group heavily reaches tumour inhibiting rate
Can find out still that from the weight of each group transplanted tumor Ad-PUMA is individually dosed can to reach 48.24% tumour inhibiting rate, tumour inhibiting rate can reach 56.24% behind the associating cDDP, compares difference with matched group very significantly, and the P value is respectively 0.001 and＜0.001.Although the Ad-p53 of same dose and cDDP administering drug combinations tumor killing effect be remarkable (P=0.014) also, its tumour inhibiting rate has only 32.68%.Ad-PUMA or Ad-p53 compare with independent injection adenovirus group with cDDP administering drug combinations group, the tumor method of double differences different all not significantly (P value is respectively 0.223,0.606), and this possibly be that the cDDP dosage of this research employing is low, causes the effect after the synergy very unobvious.
Ad-PUMA and radiotherapy synergism and Study on Mechanism thereof in embodiment five lung cancer cell lines
1. the research of its radiation sensitivity of Ad-PUMA enhancing in the lung cancer cell line
Adopt with embodiment one identical method and obtain Ad-PUMA,, accept the radiation gamma of 8Gy behind the 24hr with the infection intensity infection A549 cell of 10MOI.Continue cultivating the absorbance that adopts behind the 48hr MTS method to detect each porocyte, is reference with the suppression ratio of independent radiation gamma cell growth, relative cell growth inhibited multiple after calculating Ad-PUMA and share with radiotherapy.Each processed group is established 3 parallel holes, repeated experiments 3 times.The result sees accompanying drawing 1A, and inhibitory rate of cell growth is that independent radiotherapy is handled more than 8 times after Ad-PUMA and the radiotherapy coupling.This shows that Ad-PUMA has the effect of enhancing lung cancer cell line A549 to radiation sensitivity.
2. Ad-PUMA strengthens the Study on Mechanism of its radiation sensitivity in the lung cancer cell line
The A549 cell is after 10MOI Ad-PUMA or
infect 24 hours; Behind the 8Gy radiation gamma, at the appointed time collect suspension and attached cell, detect nuclear fragmentation counting apoptotic cell percentage ratio through PI dyeing.At least 300 cells of each counting, repeated trials 3 times is got average.Shown in Fig. 2 B, use Ad-PUMA (10 MOI) or gamma-rays (8Gy) to handle the A549 cell separately and do not induce significant apoptosis in 48 hours, 48 hours then nearly 70% cell generation apoptosis of the two Combined Treatment A549 cell.But the Ad-Δ BH3 of same dose associating gamma-rays does not then exert an influence to the apoptosis-induced ability of the latter.This shows that the BH3 domain is that its radiotherapy sensitization effect of PUMA performance is necessary, successful expression BH3 domain just can significantly strengthen the ability of radiotherapy-induced apoptosis in the cell.
The inside and outside experiment confirm BH3 of embodiment hexasomic domain is that its function of PUMA performance is necessary
1.Ad-PUMA through the various lung carcinoma cell growths of apoptosis-induced remarkable inhibition, and Ad-Δ BH3 is invalid
Adopt with embodiment one identical method and obtain Ad-PUMA; (lack aminoacid sequence is the BH3 domain of LRRMADDLN to Ad-Δ BH3; Surplus identical with PUMA), the infection intensity with 100MOI infects A549, Calu 1,128.88T, H1299, H1752, DMS53 lung carcinoma cell respectively.Infected back 48 hours, and adopted the MTS method to detect the cell growth.With untreated cell is contrast, and looking its rate of growth is 100%.For illustrating the mechanism that Ad-PUMA, Ad-Δ BH3 cell growth suppress the difference of effect, we examined it with PI (iodate third ingot) behind viral infection H1299 cell and dye in 48 hours, under phase contrast microscope and fluorescence microscope, observed.And infect the H1299 cell after 24 hours in Ad-PUMA, Ad-Δ BH3 (100MOI), carry out PI and Annexin V/DAPI dyeing, utilize flow cytometer to detect inferior G1 phase cell percentage ratio and viable apoptotic cell percentage ratio (R4, bottom right district).
The result is in 6 kinds of lung cancer cell lines that detected; Compare Ad-PUMA with untreated cell and all can cause significant growth inhibited (60%--100%), and
cell growth does not all make significant difference (accompanying drawing 10A).The cell that Ad-PUMA infects demonstrates tangible cell membrane foaming; Chromatic agglutination; Apoptosis characteristics (accompanying drawing 10B) such as nuclear segmentization, above-mentioned characteristic does not then appear in the cell that
infects.Cell cycle analysis and Annexin V dyeing confirm that all the inductive apoptosis of Ad-PUMA is remarkable; Be respectively 43% and 56%, the cell that
infects under the same case then has only 2.4% and 8% cell apoptosis (accompanying drawing 10C) to occur respectively.This explanation external source imports PUMA through the apoptosis-induced growth that can significantly suppress lung carcinoma cell; The BH3 domain that is positioned at 141 to 149 of PUMA (193 aminoacid of total length) aminoacid sequences has powerful apoptosis-promoting effect, is that its apoptosis-promoting effect of PUMA performance is necessary.
2.Ad-PUMA, Ad-p53 is to H1299 and the cytostatic comparison of A549
The Ad-PUMA of H1299 and the designated infection multiplicity of A549 cell, Ad-p53 or infection ( H1299 cell 0,2,5,10MOI; A549 cell 0,20,50,100MOI) 48hrs, as contrast, adopt the MTS method to detect with untreated cell, calculate Ad-PUMA, Ad-p53, the relative growth rate of Ad-Δ BH3 under different infection multiplicities.Visible by accompanying drawing 11,10MOI Ad-PUMA infected behind the H1299 48 hours, about 15% cell survival; 100MOIAd-PUMA infected behind the A549 cell 48 hours, about 40% cell survival.The Ad-p53 cell growth of each corresponding dosage does not all make significant difference, and Ad-Δ BH3 then cell growth does not have influence basically.This shows the more remarkable treatment effect of the Ad-PUMA of same dose than Ad-p53, and disappearance BH3 domain then makes its afunction.
3. experiment confirm BH3 domain is that its function of PUMA performance is necessary in the body
Whether PUMA (Ad-PUMA) that imports for the check external source and the PUMA (Ad-Δ BH3) of disappearance BH3 have anti-tumor activity in vivo, and we are with 4 * 10
6It is subcutaneous that individual A549 or H1299 lung carcinoma cell are injected into the female nude mice both sides of 5-6 week athymism in age, when tumor is grown to 50-100mm
3The time begin treatment experiment, each intratumor injection contains 5 * 10
8The 100 μ l PBS solution of pfu Ad-PUMA or Ad-Δ BH3 are every other day treated once totally three times.For avoiding between different animals potential individual variation to viral curative effects, Ad-PUMA and Ad-Δ BH3 are injected to the tumor of same the different sidepieces of nude mice respectively.Be designated as 0 day in the time of will treating for the first time, per 2 days major diameters with vernier caliper measurement subcutaneous transplantation tumor (a) and minor axis (b) by formula calculate tumor volume V=a * b
2/ 2, draw the growth curve of transplanted tumor, see accompanying drawing 12A.Compare Ad-Δ BH3 with independent injection PBS tumor growth is not made significant difference, 22 days gross tumor volumes after treatment reach 8 times of initial volume.The tumor growth that gives the Ad-PUMA treatment is slow, compares Ad-PUMA inhibition tumor growth with Ad-Δ BH3 and reaches 80% (P＜0.01) at least.The 22nd day execution nude mice in medication strips tumor tissues (seeing accompanying drawing 12B), and Figure 12 B the first half shows before A549 tumor-bearing mice Ad-PUMA and the Ad-Δ BH3 treatment and treats back 22 days situation.
We also treat experiment to the transplanted tumor in nude mice that the H1299 cell is set up, and whenever once contain the 100 μ l PBS solution of 5 * 108pfu Ad-PUMA or Ad-Δ BH3 at a distance from injection in 2 days, totally three times.Measured the tumor size in per 3 days, draw the growth of xenografted curve, visible Ad-PUMA suppresses tumor growth and reaches (accompanying drawing 12C) more than 80%.Cutd open in 48 hours after injection for the second time and get tumor, the frozen section of under fluorescence microscope, observing tumor is with definite adenovirus infection efficient (GFP, 200 *).It is thus clear that GFP high expressed in the tumor tissues after the treatment, the genes of interest of prompting adenovirus transduction efficiently expresses (accompanying drawing 12D).But the cell that efficiently expresses PUMA can quick apoptosis, causes the tumor tissues after the Ad-PUMA treatment to show a large amount of cell disappearances through H&E dyeing (400 *), cell intact (accompanying drawing 14D) then in the tissue that Ad-Δ BH3 or PBS handle.TUNEL dyeing (TUNEL, redness, 200 *; DAPI redyes, blueness) show a large amount of cell generation of the tumor apoptosis that gives Ad-PUMA treatment, and have apoptotic cell (accompanying drawing 12D) hardly in the tumor tissues with Ad-Δ BH3 or PBS processing.This shows that PUMA can be through apoptosis-induced effective inhibition growth of tumor, and the tumor-inhibiting action of PUMA has not existed behind the disappearance BH3.
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