CN100333788C - Tumor cell apoptosis accelerating medicine - Google Patents

Abstract

The present invention discloses a tumor cell apoptosis accelerating medicament. The active ingredient of the tumor cell apoptosis accelerating medicament provided by the present invention is Jlip protein. The medicament contains a medicinal carrier which can be accepted by a human body. The medicament can accelerate the apoptosis of the breast cancer cells and the pulmonary cancer cells and can be used for clinically treating tumors. The medicament has important theoretic significances and actual significances.

Description

A kind of medicine that quickens apoptosis of tumor cells

Technical field

The present invention relates to a kind of medicine, particularly a kind of medicine that quickens apoptosis of tumor cells.

Background technology

Activator protein-1 (activator protein-1, AP-1) be certified the earliest transcription factor, belong to alkaline leucine zipper class superfamily (basic domain leucine zipper superfamily, bZIP) member is that many cellular signal transduction approach are at endonuclear joint.AP-1 can regulate and control many important cell life processes, as cell proliferation, apoptosis, survival, differentiation, canceration etc.The AP-1 activity is subjected to rigorous, dynamic regulation and control, and the adjusting albumen in multiple physiological stimulation, environmental factors and the born of the same parents can be regulated and control the activity of AP-1.The regulation and control of AP-1 are hot fields that the AP-1 researcher is paid close attention to.C-Jun is the core constituent of AP-1 family, also is the transcription activating protein that function is the most powerful among the AP-1 family member (transcriptional activator).C-Jun albumen is encoded by proto-oncogene c-jun, 331 aminoacid of total length, molecular weight 39kDa.The existing c-Jun of discovering has more than 60 interaction protein, is maximum among the AP-1 family member.A common feature of all known c-Jun interaction proteins is, with complete molecular forms performance function.

Inventor place laboratory has started the large-scale cDNA order-checking of people 22 tire liver in all pregnant ages in 1998, the original intention of this plan be overall understanding 22 tire liver in all pregnant ages gene expression characteristics, excavate new cytokine, be that clinical practice and new drug development are sought new target spot.Examining order was finished in 2000, had measured more than 15,000 expressed sequence tag (EST) altogether, contained 1660 knowns, had set up so far the gene expression profile of system, maximum-norm the most.Finish the bioinformatic analysis of the ESTs of more than 5000 new genes of representative, determined 110 emphasis that contain the cDNA of total length ORF as new functional gene research.In these 1660 knowns transcription factor, transcribe related gene and signal transducers and amount to 234, account for 14.1%, thus in the tire liver still undiscovered transcribe with transcription regulatory factor in have the factor of playing an important role in some generations, the development probably in important diseases (as tumor).

Jlip (c-Jun leucine zipper interacting protein) is a molecule that certain research clue is arranged in above-mentioned 110 genes.Its cDNA total length 1633bp, Genbank number of registration AF291105, long 409 aminoacid of encoding proteins, the N end contains a PH domain (pleckstrin homology domain), C 72 aminoacid of end (wherein comprising a leucine zipper structure) and mice JZA-20 peptides homologous very high (up to 96%), the latter is Chevray in 1992 etc. with c-Jun leucine zipper district is that bait utilizes yeast two-hybrid to seek the fragment that c-Jun finds when conjugated protein, prompting Jlip may combine with c-Jun and regulates and control the AP-1 activity, and called after Jlip (c-Jun leucine zipper interacting protein) in view of the above.But at present not to conjugated protein report the in c-Jun leucine zipper district in people source, being polypeptide fragment and Chevray obtains, is not complete albumen, the polypeptide that obtains is not done further checking and functional study yet.

Both contained the PH domain with a part, the compositing characteristic that contains leucine zipper structure again is rarer.At present, have only a few molecule that the function report is arranged, as AFAP-110, beta Pix, APPL and Vav (Baisden et al., 2001; Kim et al., 2001; Mitsuuchi et al., 1999; Romero﹠Fischer, 1996).Wherein have only proto-protein Vav and AP-1 that function association is arranged, it can activate the JNK/AP-1 path.But Vav is indirect to the regulation and control of AP-1, depends on the existence of Rac and Vav intramolecularly DH domain, and leucine zipper structure does not participate in regulation and control.

The structural analysis of Jlip molecule is shown there is not the DNA land in the N end of its leucine zipper structure, because of rather than typical bZIP molecule; Intramolecularly does not contain other DNA land yet, thereby neither DNA conjugated protein, therefore got rid of as transcription factor may.And the PH domain with phospholipids incorporate characteristic is anchored to it plasma membrane probably, therefore, should be anchored on the plasma membrane by Jlip, participate in the c-Jun in the syncaryon again, on convention, be contradiction, hint that also Jlip may participate in the function of AP-1 with undiscovered new control methods still.

A Canadian laboratory has also obtained identical gene, Bosc etc. are main direction with research CK2, with CK2 α is bait protein screening human B cell library, obtains this gene, called after CKIP-1 (casein kinase 2interacting protein-1).Bosc etc. have verified the interaction of CKIP-1 and CK2 α in vivo with in the experiment in vitro, preliminary analysis location in the tissue distribution of CKIP-1 and the cell.Whether they attempt to inquire into CKIP-1 is the substrate of CK2 to the influence or the CKIP-1 of the kinase activity of CK2, but what obtain all is negative findings.Therefore, they only infer that CKIP-1 may be the adjusting albumen of CK2.So about the Jlip molecule, existing certain research basis more has problems not have to solve: does not prove conclusively as yet though the C end of Jlip may combine with c-Jun (1); (2) whether Jlip regulates and control the activity of AP-1/c-Jun, unclear; (3) physiological function of Jlip is not reported.

The innovation and creation content

The purpose of this invention is to provide a kind of medicine that quickens apoptosis of tumor cells.

The medicine of acceleration apoptosis of tumor cells provided by the present invention, its active component are Jlip albumen.

When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.

Medicine of the present invention can be made various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.

Medicine of the present invention can quicken the apoptosis of breast cancer cell, lung adenocarcinoma cell.

The present inventor finds that Jlip albumen can strengthen the sensitivity of tumor cell to apoptosis inducing factors such as tumor necrosis factor, cycloheximides, is a new apoptosis of tumor cells potentiation molecule.Medicine of the present invention can be assisted and is used for treating clinically tumor, has great importance.

Description of drawings

Fig. 1 is Jlip/SK-BR-3 and pcDNA3.1/SK-BR-3 cell proliferation curve

Fig. 2 is subjected to the apoptosis curve of cell behind the TNF/CHX signal stimulus for Jlip/SK-BR-3 and pcDNA3.1/SK-BR-3

Fig. 3 is subjected to the activity change curve of cell caspase-3 behind the TNF/CHX signal stimulus for Jlip/SK-BR-3 and pcDNA3.1/SK-BR-3

Fig. 4 is

The specific embodiment

Material

Tumor necrosis factor (TNF) is available from Chemicon; (cycloheximide is CHX) available from Sigma for cycloheximide; Recombination human source caspase-3 is available from Pharmingen; Caspase inhibitor DEVD-CHO is available from Clontech; The pcDNA3.1-Myc plasmid is available from Invitrogen; PcDNA3.1-Myc-Jlip plasmid construction the primer is P8181 and P8183, insert sheet segment length 1.2kb, be inserted between the HindIII and KpnI site of carrier, (primer sequence is P8181:caa gct tcg aat tct cgc ccg cgc ccg ctg gga atg; P8183:cag agg tac ctt cat cag gct ctt); Hyclone (FBS) is available from Hyclone; G418 is available from Gibco; FuGene 6 transfection reagents are available from Roche.

Embodiment 1, Jlip quicken the apoptosis and the mechanism thereof of tumor cell

1, the growth rate of Jlip/SK-BR-3 and pcDNA3.1/SK-BR-3 cell

(1) Jlip/SK-BR-3 stablizes the screening of strain

PcDNA3.1-Myc and pcDNA3.1-Myc-Jlip plasmid (the coding C-terminal contains the Jlip albumen of Myc label) transfection breast cancer cell SK-BR-3, the SK-BR-3 cell is cultivated with RPMI1640 (15%FBS).Carry out in 6 orifice plates with the FuGene6 transfection method.After 3 days, change the fresh medium that contains G418 (800 μ g/ml), continue to cultivate about 2 weeks, changed liquid and remove dead cell 1 time in per 2 days.After treating that the clone grows, the picking clone also is transferred to continuation amplification culture in 24 orifice plates, then is extended to successively in 12 orifice plates, 6 orifice plates, the culture bottle.Identify positive colony by Westernblot.Through the screening in 3 weeks, obtained about 20 of the monoclonals of stably express Jlip, all be accredited as the positive through Western blot, obtained the contrast clone of transfection empty carrier simultaneously.

(2) growth rate of Jlip/SK-BR-3 and pcDNA3.1/SK-BR-3 cell

24 orifice plates (1 * 10 are spread in Jlip/SK-BR-3 and the strain of pcDNA3.1/SK-BR-3 cytotostatic ⁵Individual cells/well), cell counting every day (blood counting chamber direct count) was counted 3 holes every day, averages, and amounts to 7 days, changed 1 time culture fluid at the 4th day.Experiment repeats 3 times, gets wherein representative 1 secondary data mapping, obtains cell growth curve.

The result does not have difference although show the form of two groups of cells as shown in Figure 1, and growth rate is also all than comparatively fast, but still difference as can be seen, and the ability of cell proliferation of empty carrier group is stronger.Among Fig. 1, error bars is represented standard deviation.

2, Jlip/SK-BR-3 and the strain of pcDNA3.1/SK-BR-3 cytotostatic are subjected to the apoptosis situation of cell behind the TNF/CHX signal stimulus

When Jlip/SK-BR-3 and the strain of pcDNA3.1/SK-BR-3 cytotostatic grow to fusion rate 90% left and right sides, remove serum starvation overnight, add the TNF of 20ng/ml and the CHX of 10 μ g/ml, induce and handle 2-8hr.TNF prepares with water, and CHX prepares with ethanol ,-20 ℃ of preservations.

Through the inductive cell of TNF/CHX with trypsinization, centrifugal, add PBS (containing 15%FBS)+70% ethanol (the two volume ratio 3: 7), place more than the 4hr for-20 ℃, centrifugal, add RNaseA digestion, dye with propidium iodide (PI), two groups of SK-BR-3 of analyzing and testing on the stream type cell analyzer stablize strain to the TNF/CHX signal stimulus after the ratio of apoptotic cell, the result is as shown in Figure 2.

Cross and express the generation that Jlip itself can not be apoptosis-induced.Be subjected to the stimulation of TNF/CHX when cell after, to be higher than matched group in the apoptosis rate Jlip of the 8hr inner cell that is detected group, statistical analysis shows, significant difference during 2hr (P＜0.05), 4hr is to 8hr difference extremely significantly (P＜0.01), handle back 6hr as TNF/CHX, about 27.0 ± 2.5% Jlip group cell generation apoptosis, this moment, the empty carrier group had only 11.8 ± 1.9% apoptosis takes place.Therefore, Jlip expresses can strengthen the sensitivity of breast cancer cell to tumor necrosis factor, quickens the apoptosis of breast cancer cell.

3, Jlip quickens the mechanism of apoptosis of tumor cells

In order to illustrate the mechanism that Jlip quickens apoptosis of tumor cells, with DEVD-CHO-AFC is substrate, measure the explanation of test kit (available from Clontech) according to ApoAlertcaspase-3 and carry out the Caspase-3 determination of activity, the different absorbance value reflection caspase-3 activity of fluorogenic substrate.

The result as shown in Figure 3, after showing that TNF/CHX stimulates, the caspase-3 activity of Jlip group cell just can be seen activation at 2hr, and at this activated 4hr that appears at of matched group, can see that in 6hr phase scope when interior two groups difference is apparent in view.In later time phase, this difference is no longer obvious, shows that the activation of caspase-3 this moment may be near plateau.Research in the past found once that caspase-3 was that CHX is apoptosis-induced necessary, can the inductive apoptosis of antagonism CHX (Woo et al., 1998) such as the mouse bone marrow cells neutrophil cell that caspase-3 practices shooting.

In order to prove conclusively the importance that caspase-3 takes place for apoptosis, before TNF/CHX adds, the specific inhibitor DEVD-CHO that adds caspase-3 in advance, the result as shown in Figures 2 and 3, show that DEVD-CHO can block the inductive apoptosis of TNF/CHX and caspase-3 activates, shown the importance of caspase-3.In addition, also observe, the expression of Jlip not cell cycle produces significantly influence.

The inhibitor of Caspase-3 can be blocked the effect of inductive apoptosis of TNF/CHX and Jlip promotion apoptosis, and it is apoptosis-induced necessary to show that caspase-3 is not only TNF/CHX, also is that Jlip mediated Apoptosis enlarge-effect is necessary.Some is similar to other caspase-3 substrates that have complete different structure with Jlip this effect of Jlip, as MEKK1, RET and Bcl-2 (Widmann et al., 1998; Bordeaux et al., 2000; Chenget al., 1997).

From present embodiment as can be seen, J1ip growth capable of inhibiting cell increases the apoptosis induction sensitivity of SK-BR-3 breast cancer cell to TNF/CHX; Jlip is the potentiation molecule of an apoptosis.

Embodiment 2, Jlip reduce the colony of lung adenocarcinoma cell Glc-82 under condition of culture and form ability

1, the screening of Jlip/Glc-82 and the strain of pcDNA3.1/Glc-82 cytotostatic

PcDNA3.1-Myc and pcDNA3.1-Myc-Jlip plasmid transfection lung adenocarcinoma cell Glc-82, the Glc-82 cell is cultivated with RPMI1640 (10%FBS).Transfection, screening and authentication method are with the method for SK-BR-3 cytotostatic strain among the embodiment 1.

2, the colony formation ability of Jlip/Glc-82 and the strain of pcDNA3.1/Glc-82 cytotostatic is measured

Prepare the soft agar flat board earlier.Preparation 2 * RPMI1640 culture fluid, 1.2% and 0.7% low melting-point agarose gel.With 1.2% agarose gel and equal-volume 2 * RPMI1640 mixing, be laid in the 60mm diameter culture dish, as bottom glue.The strain cell is stablized in trypsinization then, makes single cell suspension, counting, bed board, 600 cells of each culture dish inoculation.With 0.7% agarose gel and equal-volume 2 * RPMI1640 mixing, maintain about 40 ℃, in the time of peptic cell as top layer glue.Cell is joined rapidly in the top layer glue, be laid on above the bottom glue.In incubator, cultivate about 2 weeks the number of cell clones that family planning is long.

The result show that the empty carrier group obtains 267 ± 84.01 colonies, and Jlip expression group is had to 135.25 ± 21.19 colonies, significant difference (P＜0.05) as shown in Figure 4.Colony formation ability has been represented the vicious transformation ability of tumor cell, and therefore, the expression of Jlip can suppress the vicious transformation of tumor cell significantly.

From present embodiment as can be seen, Jlip can suppress the malignant proliferation of tumor cell effectively.

Claims (6)

1, a kind of medicine that quickens apoptosis of tumor cells, its active component is a Jlip albumen.

2, medicine according to claim 1 is characterized in that: described tumor cell is breast cancer cell, lung adenocarcinoma cell.

3, medicine according to claim 1 is characterized in that: also contain the acceptable pharmaceutical carrier of human body in the described medicine.

4, the application of Jlip albumen in preparation acceleration apoptosis of tumor cells medicine.

5, medicine according to claim 4 is characterized in that: described tumor cell is a breast cancer cell.

6, medicine according to claim 4 is characterized in that: described tumor cell is a lung adenocarcinoma cell.

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