CN104292302B - A kind of polypeptide and its application for strengthening tumour cell to antitumor drug sensitiveness - Google Patents

Abstract

The invention discloses a polypeptide for enhancing the sensitivity of tumor cells to antitumor drugs and its application. Specifically, a polypeptide for enhancing the sensitivity of tumor cells to antitumor drugs and inhibiting tumor cell migration, and fusion proteins, complexes, nucleic acid sequences, nucleic acid vectors, host cells and pharmaceutical compositions related to the polypeptide are disclosed . The invention also provides the application of the polypeptide in the preparation of drugs for enhancing the sensitivity of tumor cells to anti-tumor drugs and inhibiting the migration of tumor cells.

Description

A polypeptide that enhances the sensitivity of tumor cells to antitumor drugs and its application

technical field

The invention relates to the technical field of biomedicine, in particular to a polypeptide for enhancing the sensitivity of tumor cells to antitumor drugs and its application.

Background technique

Chemokines are a large class of small molecular proteins with chemotaxis in the members of the cytokine superfamily. Among them, the interaction between the chemokine CXCL12 and its receptor CXCR4 located on the surface of various tumor cell membranes has important effects on tumor development, Invasion and metastasis play an important role (Fukuda S., Broxmeyer H.E. and Pelus L.M., Blood, 2005, 105, 3117-3126). By interfering with the interaction between CXCR4 and CXCL12, it can inhibit the invasion, metastasis and drug resistance of tumor cells in vivo, providing a possible way for the treatment of tumor diseases (Burger J.A. and Peled A., Leukemia, 2009, 23, 43-52) .

Leukemia is a malignant disease of hematopoietic tissue. Due to the residue of drug-resistant leukemia cells in the body after chemotherapy and the invasion and metastasis of leukemia cells in the body, it is still a huge challenge to completely cure leukemia (Gerard C. and Rollins B.J., Nat. Immunol., 2001 ,2,108-115), the development of new drugs and treatment methods is an important goal to improve the cure rate of leukemia.

Contents of the invention

The inventor obtained a group of polypeptides through a lot of experiments and creative work, and found that this group of polypeptides has the ability to enhance the sensitivity of tumor cells to anti-tumor drugs and inhibit the migration of tumor cells, and has the potential to be used as a therapeutic or adjuvant therapy for tumors (especially leukemia) and breast cancer) drug potential.

The invention provides the following technical solutions:

In the first aspect, the present invention provides a polypeptide for enhancing the sensitivity of tumor cells to antitumor drugs and inhibiting the migration of tumor cells. The polypeptide has an amino acid sequence selected from SEQ ID NO: 1-4:

(1) GGFDRRNANFNDI (SEQ ID NO: 1),

(2) GGYDLDLSVARL (SEQ ID NO: 2),

(3) GGQGCFRRNTVDDWISITRAL (SEQ ID NO: 3), and

(4) GGTRALAFFDC (SEQ ID NO: 4).

The polypeptide can be prepared by artificial chemical synthesis.

In a second aspect, the present invention provides a fusion protein comprising the polypeptide described in the first aspect.

In the third aspect, the present invention provides a complex obtained by coupling the polypeptide described in the first aspect with a macromolecule carrier. The macromolecule carrier includes, for example, protein carriers such as bovine serum albumin, human serum albumin, keyhole albumin, bovine thyroglobulin, and other gamma globulins. The coupling can be glutaraldehyde method, MBS method, carbodiimide method, halogenated nitrobenzene method and active ester method, etc.

In a fourth aspect, the present invention provides a nucleic acid sequence encoding the polypeptide described in the first aspect.

In the fifth aspect, the present invention provides a nucleic acid vector comprising the nucleic acid sequence described in the fourth aspect.

In a sixth aspect, the present invention provides a host cell comprising the nucleic acid vector of the fifth aspect. For example, it may be a host cell transformed with the nucleic acid vector, typical but non-limiting examples, such as microbial host cells such as Escherichia coli host cells and the like.

In the seventh aspect, the present invention provides a pharmaceutical composition, which comprises the polypeptide described in the first aspect or the fusion protein described in the second aspect or the complex described in the third aspect or the complex described in the fourth aspect. The nucleic acid sequence described in the fifth aspect or the nucleic acid vector described in the fifth aspect or the host cell described in the sixth aspect.

In the eighth aspect, the present invention provides the polypeptide described in the first aspect or the fusion protein described in the second aspect or the complex described in the third aspect or the nucleic acid sequence described in the fourth aspect or the nucleic acid sequence described in the fifth aspect Use of the nucleic acid vector or the host cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect in the preparation of drugs for enhancing the sensitivity of tumor cells to anti-tumor drugs and inhibiting tumor cell migration. Preferably, the tumor cells are leukemia cells and/or breast cancer cells.

The present invention also provides a method for treating or adjuvantly treating tumors (especially leukemia and breast cancer), comprising administering to humans or animals an effective amount of the polypeptide described in the first aspect or the fusion protein described in the second aspect or the fusion protein described in the third aspect. The compound described in the fourth aspect or the nucleic acid sequence described in the fourth aspect or the nucleic acid vector described in the fifth aspect or the host cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect.

The beneficial effects of the present invention are: the polypeptide described in the present invention has the ability to enhance the sensitivity of tumor cells to anti-tumor drugs and inhibit the migration of tumor cells, and can be used as a drug for treating or adjuvantly treating tumors (especially leukemia and breast cancer).

Description of drawings

Figure 1 is the results of light scattering experiments of C1-C4 polypeptide molecules in 1×PBS solution containing 1% DMSO, in which Represents the signal intensity curve of the negative control PBS; Represents the signal intensity curve of the C1 polypeptide solution; Represents the signal intensity curve of the C2 polypeptide solution; Represents the signal intensity curve of the C3 polypeptide solution; Shows the signal intensity curve of the C4 polypeptide solution.

Figure 2 is the results of staining rate of C1-C4 polypeptide molecules on K562 cells in flow cytometry, in which: (a) K562 cells labeled only with FITC-SA, (b) K562 cells labeled with C1 and FITC-SA , (c) C2 and FITC-SA labeled K562 cells, (d) C3 and FITC-SA labeled K562 cells, (e) C4 and FITC-SA labeled K562 cells. 10 ^∧ 0 means that the relative intensity of the fluorescent signal produced by the detected cells is 10 ^∧ 0 (ie 10 ⁰ ), and the rest are similar.

Figure 3 is the results of the effect of C1-C4 polypeptide molecules on the viability of K562 cells, in which: the negative control is K562 cells without polypeptide molecules, and the other groups are added with 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1μg/mL C1-C4 polypeptide.

Figure 4 shows the effect of C1-C4 polypeptide molecules on cyclophosphamide (CPA) sensitization and killing of K562 cells, in which: (a) 10ng/mL, 100ng/mL, 500ng/mL, 1μg/mL of C1 polypeptide The sensitization effect of the molecule on 0mM, 5mM, and 10mM CPA; (b) the sensitization effect of 10ng/mL, 100ng/mL, 500ng/mL, and 1μg/mL C2 polypeptide molecules on 0mM, 5mM, and 10mM CPA; ( c) Sensitization effect of 10ng/mL, 100ng/mL, 500ng/mL, 1μg/mL C3 polypeptide molecules on 0mM, 5mM, 10mM CPA; (d) 10ng/mL, 100ng/mL, 500ng/mL, 1μg The sensitization effect of C4 polypeptide molecule/mL to 0mM, 5mM, 10mM CPA.

Fig. 5 is a graph showing the results of sensitization of 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1μg/mL C2 polypeptide molecules to 0mM, 6mM CPA.

Fig. 6 is a graph showing the affinity results between C3 polypeptide molecules and HL-60 cells, wherein the control is the cell group only added with FITC-SA.

Fig. 7 is a result diagram of the affinity ELISA method of C1-C4 polypeptide molecules to HL-60 cells, wherein the blank control is a PBS control group without polypeptide molecules.

Figure 8 is the results of the effect of C1-C4 polypeptide molecules on the viability of HL-60 cells, in which: the negative control is HL-60 cells without polypeptide molecules, and the other groups are added with 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1μg/mL C1-C4 polypeptide.

Figure 9 is a graph showing the effect of C1 and C3 polypeptide molecules on HL-60 cell apoptosis, in which the control is a negative control that only adds culture medium solution without adding C1 or C3 polypeptide molecules, and the remaining groups are added with 100nM, 1μM, 10 μM of C1 or C3 polypeptide molecules.

Figure 10 is a graph showing the inhibitory effect of C1-C4 polypeptide molecules on the migration of HL-60 cells induced by CXCL12 molecules, where the control is the migration rate of HL-60 cells when no chemokine CXCL12 is added; The migration rate of HL-60 cells at CXCL12; the migration rates of HL-60 cells after adding C1-C4 polypeptide molecules in the other groups.

Fig. 11 is a graph showing the results of the inhibitory effect of C3 polypeptide molecules on the migration of HL-60 cells induced by MS-5 cells.

Fig. 12 is a graph showing the inhibitory effect of C3 polypeptide molecules on MS-5 cells inducing HL-60 cells to adhere to MS-5.

Fig. 13 is a graph showing the results of C3 polypeptide molecule's inhibitory effect on CXCL12 molecule-induced HL-60 cytoskeleton protein assembly captured by laser confocal microscope (1000 times magnification).

Figure 14 is an electrophoresis result diagram of the inhibitory effect of C3 polypeptide molecules on the signaling pathway induced by CXCL12 molecules on HL-60 cells, wherein 1, 2, 3, and 4 represent four parallel swimming lanes, p38 represents p38 protein, and P- p38 means phosphorylated p38 protein, Erk means Erk protein, P-Erk means phosphorylated Erk protein, "+" means adding corresponding substance (CXCL12 or C3), "-" means not adding corresponding substance (CXCL12 or C3).

Fig. 15 is the result diagram of the effect of C3 polypeptide molecule on 10-hydroxycamptothecin (Cam) killing HL-60 cells, wherein the control means no treatment, C3 means C3 polypeptide treatment, and 10-hydroxycamptothecin means 10-hydroxycamptothecin Tree-camptothecin treatment, 10-hydroxycamptothecin + C3 means firstly treated with C3 polypeptide and then treated with 10-hydroxycamptothecin.

Fig. 16 is a diagram showing the affinity results of C3 polypeptide molecules to tumor-related cells NB4 cells, wherein the control is the cell group only added with FITC-SA.

Figure 17 is a graph showing the inhibitory effect of C3 polypeptide molecules on the migration of NB4 cells induced by CXCL12 molecules.

Figure 18 is a graph showing the inhibitory effect of C3 polypeptide molecules on the migration of NB4 cells induced by MS-5 cells.

Fig. 19 is a graph showing the inhibitory effect of C3 polypeptide molecules on MS-5 cells inducing NB4 cells to adhere to MS-5.

Figure 20 is a graph showing the effect of C3 polypeptide molecule on 10-hydroxycamptothecin (Cam) killing NB4 cells, wherein the control means no treatment, C3 means C3 polypeptide treatment, and 10-hydroxycamptothecin means 10-hydroxycamptothecin Treatment, 10-Hydroxycamptothecin+C3 means firstly treated with C3 polypeptide and then treated with 10-Hydroxycamptothecin.

Fig. 21 is a diagram showing the affinity results of C3 polypeptide molecules to tumor-related cells THP-1 cells, wherein the control is the cell group only added with FITC-SA.

Fig. 22 is a graph showing the inhibitory effect of C3 polypeptide molecules on the migration of THP-1 cells induced by CXCL12 molecules.

Figure 23 is a graph showing the inhibitory effect of C3 polypeptide molecules on the migration of THP-1 cells induced by MS-5 cells.

Fig. 24 is a graph showing the inhibitory effect of C3 polypeptide molecules on MS-5 cells inducing THP-1 cells to adhere to MS-5.

Fig. 25 The results of the inhibitory effect of the C3 polypeptide molecule on the THP-1 cytoskeleton protein assembly induced by the CXCL12 molecule captured by the laser confocal microscope (1000 times magnification).

Fig. 26 is a graph showing the affinity of C3 polypeptide molecules to U937 cells, wherein the control is the cell group only added with FITC-SA.

Figure 27 is a graph showing the inhibitory effect of C3 polypeptide molecules on the migration of U937 cells induced by CXCL12 molecules.

Figure 28 is a graph showing the inhibitory effect of C3 polypeptide molecules on the migration of U937 cells induced by MS-5 cells.

Figure 29 is a graph showing the inhibitory effect of C3 polypeptide molecules on MS-5 cells inducing U937 cells to adhere to MS-5.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Embodiment 1: the synthesis of polypeptide

Polypeptides were synthesized according to the sequences shown in Table 1, and diluted to appropriate concentrations in experiments as needed.

Table 1 Synthetic peptides

name sequence C1 Biotin-GGFDRRNANFNDI C2 Biotin-GGYDLDLSVARL C3 Biotin-GGQGCFRRNTVDDWISITRAL C4 Biotin-GGTRALAFFDC

Wherein, the amino acid sequences of C1-C4 polypeptides respectively correspond to SEQ ID NO: 1-4 in the summary of the invention. The role of biotin (Biotin) is to combine with streptavidin (SA) in the enzyme-linked immunological reaction, which is a commonly used technical means, and has no substantial impact on the function of the polypeptide of the present invention.

Example 2: Polypeptide Molecule Aggregation State and Stability Experiment in Solution

Preparation of 1×PBS solution: Weigh 0.4g NaCl, 0.01g KCl, 182mg Na ₂ HPO ₄ 12H ₂ O, 12mgKH ₂ PO ₄ , 0.01g NaN ₃ , dissolve in 50ml secondary water, filter with 0.22μm water Membrane filtration, set aside.

The C1-C4 polypeptide molecules were made into a 30 μM 1×PBS solution containing 1% DMSO. The prepared polypeptide solution was placed in a 120r/min, 37°C constant temperature shaker and kept for 36 hours. A 1×PBS solution containing 1% DMSO was used as a negative control.

From the moment the sample preparation is completed, take out 400 μL of the sample solution from the test solution every 4 hours, and add it to a 1cm×1cm quartz cuvette. In a fluorescence spectrophotometer (SL55), with 405 nm as the excitation wavelength, the emitted light signal at 405 nm was collected. The slit width is 1.0nm, 1.0nm. Static light scattering reflects changes in the aggregation state of polypeptide molecules in solution over time. As shown in Figure 1, as time goes on, due to the influence of the mechanical shearing action of the liquid environment, the C1-C4 polypeptide molecules will change from a state with a certain aggregation size to a monodisperse state. From the 4th hour, the aggregation state of the polypeptide molecules remained stable, and the experimental results showed that the intensity (a.u.) tended to be stable.

Example 3: Affinity experiment of polypeptide molecules to leukemia-related cells K562 cells

K562 cells were used as a model for studying leukemia cells. In the ELISA experiment, 500,000 fixed K562 cells were plated in a 96-well culture plate. After drying, PBS containing 100nM polypeptide molecules was added to each well and incubated with the cells at 37°C. After 3 hours, the non-specific adsorption was removed. After the polypeptide was added, horseradish peroxidase-labeled streptavidin (HRP-SA) was added to it for incubation, and then the substrate reaction of horseradish peroxidase was added, and the multifunctional microplate reader was used to determine its Absorption at 490nm.

In the flow cytometric analysis experiment, 500,000 fixed K562 cells were plated in a 96-well culture plate, and after drying, PBS containing 100nM polypeptide was added to each well to incubate with the cells at 37°C, and removed after 3 hours After non-specifically adsorbed peptides, the proportion of cells bound to peptide molecules with fluorescent groups is detected. The excitation wavelength is 490nm, and the detection emission wavelength is 525nm. As shown in Figure 2, the C1-C4 polypeptide molecules can all label K562 cells (the fluorescent staining rate is four times greater than that of the negative control). Among them, C3 polypeptide has the strongest affinity to K562 cells, which is 65.30%.

Example 4: Experiment of the influence of polypeptide molecules on the viability of K562 cells

K562 was used as a model system for studying leukemia cells. In a Conning 96-well plate, 40,000 cells were cultured per well, using RPMI-1640 medium (containing 10% North American calf serum, 1‰ streptomycin). 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1μg/mL C1-C4 polypeptides were incubated with K562 cells for 72 hours. At the 72nd hour after incubation, the upper medium in the culture plate was sucked away, the fresh medium was mixed with the MTS (single solution cell proliferation detection kit) solution at a ratio of 5:1, and 120ul of the mixture was added to each well and reacted at 37°C for 1.5 h. In a continuous spectrum multifunctional microplate reader (Tecan infinite M200), the absorbance value at 490 nm was measured.

As shown in Figure 3, when 10 ng/mL-1 μg/mL of C1-C4 polypeptide molecules were added to K562 cells, the cell survival rate was not significantly different from that of K562 cells alone, and the cell survival rate after adding the polypeptide There is no linear relationship with the amount of polypeptide added. That is, the C1-C4 polypeptide basically does not affect the survival rate of K562 cells.

Example 5: Effect of polypeptide molecules on CPA killing K562 cells

K562 was used as a model system for studying leukemia cells. In a Conning 96-well plate, 40,000 cells were cultured per well, using RPMI-1640 medium (containing 10% North American calf serum, 1‰ streptomycin). 10ng/mL, 100ng/mL, 500ng/mL, 1μg/mL C1-C4 polypeptide molecules were incubated with K562 cells for 24 hours. CPA was added to the mixed system of the polypeptide and K562 cells so that the final concentrations of CPA were 5 mM and 10 mM respectively. After another 48 hours, the upper medium in the culture plate was aspirated, the fresh medium was mixed with the MTS solution at a ratio of 5:1, and 120 μL of the mixture was added to each well to react at 37° C. for 1.5 h. In a continuous spectrum multifunctional microplate reader (Tecan infinite M200), the absorbance value at 490 nm was measured.

As shown in Figures 4a-4d, C1-C4 polypeptides can increase the killing effect of CPA on K562 cells to varying degrees and increase the drug sensitivity of K562. Among them, the C1, C2, and C4 polypeptides can significantly enhance the killing effect of CPA on cells at a concentration of 100 ng/mL. At a concentration of 1 μg/mL, the C3 polypeptide can increase its killing effect on 10 mM CPA to a certain extent.

In a Conning 96-well plate, 40,000 cells were cultured per well, using RPMI-1640 medium (containing 10% North American calf serum, 1‰ streptomycin). 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1μg/mL of C2 polypeptide were incubated with K562 cells for 24 hours. Add CPA to the mixed system of the polypeptide and K562 cells, so that the final concentration of CPA is 6mM. After 48 hours, the upper medium in the culture plate was sucked away, the fresh medium was mixed with the MTS solution at a ratio of 5:1, and 120 μL of the mixture was added to each well to react at 37°C for 1.5 h. In a continuous spectrum multifunctional microplate reader (Tecan infinite M200), the absorbance value at 490 nm was measured.

As shown in Figure 5, when the concentration of C2 polypeptide is 500ng/mL (the concentration of CPA is 6mM), it can significantly increase the killing effect of CPA (P=0.0034).

That is, the C1-C4 polypeptide molecules can effectively increase the drug sensitivity of leukemia cells.

Example 6: Affinity of polypeptide molecules to HL-60 cells-flow cytometry analysis method experiment

HL-60 cells were used as a model for studying leukemia cells. Seed 500,000 HL-60 cells in serum-free medium containing 1% DMSO into a 24-well culture plate, add C3 polypeptide molecules at a final concentration of 10 μM and incubate for 4 h. After the cells were collected, they were washed twice with PBS, and then incubated in 5 μg/ml FITC-SA solution at 4° C. for 0.5 h. Wash twice with PBS and filter through a 100-mesh cell strainer. The cell group with only FITC-SA added was used as a negative control. In a flow cytometer, the proportion of cells that are labeled is detected.

As shown in Figure 6, the polypeptide molecule has a high binding force to HL-60 cells.

Example 7: Affinity of polypeptide molecules to tumor-related cells HL-60 cells-ELISA method experiment

HL-60 cells were used as a model for studying leukemia cells. Spread 500,000 fixed HL-60 cells into a 96-well culture plate, add PBS containing 100nM polypeptide to each well and incubate with the cells at 37°C after attachment, and remove the non-specifically adsorbed polypeptide after 3 hours , and then add horseradish peroxidase-labeled streptavidin (HRP-SA) to incubate, then add horseradish peroxidase substrate reaction, and use a multifunctional microplate reader to measure its concentration at 490nm Absorbance.

As shown in FIG. 7 , C1-C4 polypeptide molecules all bind to HL-60 cells to varying degrees.

Example 8: Effect of polypeptide molecules on the viability of tumor-related cells HL-60 cells

HL-60 was used as a model system for studying leukemia cells. In a Conning 96-well plate, 100,000 cells were cultured per well, using RPMI-1640 medium (containing 10% North American calf serum, 1‰ streppenicillin). 0ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1μg/mL C1~C4 polypeptides were incubated with HL-60 cells for 24, 48, 72 hours. At 24, 48, and 72 hours after incubation, aspirate the upper medium in the culture plate, mix the fresh medium and MTS solution at a ratio of 5:1, add 120 μL of the mixture to each well and react at 37°C for 1.5 hours. In a continuous spectrum multifunctional microplate reader (Tecaninfinite M200), the absorbance value at 490 nm was measured.

As shown in Figure 8, when 10ng/mL～1μg/mL of C1～C4 polypeptide was added to HL-60 cells, the cell survival rate was not significantly different from that of HL-60 cells alone, and after adding the polypeptide There is no linear relationship between cell viability and the amount of added polypeptide. That is, the C1-C4 polypeptides basically do not affect the viability and proliferation ability of HL-60 cells.

Example 9: Effect of polypeptide molecules on HL-60 cell apoptosis

HL-60 cells were used as a model for studying leukemia cells. HL-60 cells were inoculated in a 24-well plate, 500,000 cells per well, and 100nM-10μM C1 or C3 polypeptide solution was added to the well plate, and the same volume of medium solution was added to the control group, and incubated for 24 hours. After that, the cells were washed twice with PBS, resuspended with binding buffer (10mM Hepes/NaOH, pH7.4, 140mM NaCl, 2.5mMCaCl), stained with FITC-Annexin V solution for 3 minutes, and then added propidium iodide (PI ) solution together in the dark for 10 minutes, and then detected and analyzed by flow cytometry.

As shown in FIG. 9 , the apoptosis rate of HL-60 cells treated with 100 nM-10 μM C1 or C3 polypeptide has no significant difference compared with untreated cells. That is, the C1 and C3 polypeptides basically do not induce the apoptosis of HL-60 cells.

Example 10: Experiment of the inhibitory effect of polypeptide molecules on the migration of HL-60 cells induced by CXCL12 molecules

HL-60 cells were used as a model for studying leukemia cells. HL-60 cells were incubated with 200ng/mL C1-C4 polypeptide in serum-free medium solution for 1 hour, then added to the upper chamber, 150,000 cells per chamber, and 200ng/ml CXCL12 solution in the lower chamber. After 24 hours of migration, the cell solution in the lower chamber was collected, counted and counted after centrifugation. After further treating the HL-60 cells with a C3 solution with a concentration ranging from 0.1 to 10 μM, the mobility of the cells was detected.

As shown in Figure 10, when no chemokine CXCL12 was added (control experimental group), the migration rate of HL-60 cells was low. After adding CXCL12 to the lower chamber, the cells showed a strong ability to migrate to the lower chamber. After adding 200ng/mL C1-C4 polypeptide molecules to the upper chamber to incubate the cells, the migration ability of HL-60 cells was reduced by 40%, 38%, 36%, and 17% in turn. That is, the C1-C4 polypeptide molecules can effectively inhibit the metastasis and migration of tumor cells induced by chemokines. Moreover, the reduction of the mobility is proportional to the concentration of the polypeptide. When the concentrations of the C3 polypeptide are 0.1 μM, 1 μM, and 10 μM, the migration rates of HL-60 cells are 65±9.7%, 61±3.6%, respectively, compared with the CXCL12 group. 36±5.7%.

Example 11: Inhibitory Effect of Polypeptide Molecules on Migration of HL-60 Cells Induced by MS-5 Cells

HL-60 cells were used as a model for studying leukemia cells. After HL-60 cells were stained with calcein for 10 minutes, they were washed with medium solution, and then incubated with 10 μM C3 polypeptide in serum-free medium solution for 1 hour, and added to the small chamber, 150,000 cells per chamber, and the lower chamber was 100,000 MS-5 cells per well were treated with 8 μg/ml mitomycin for 2 hours and left overnight. After incubation for 6 hours, the cell solution in the lower chamber was collected, and after centrifugation, the fluorescence value of the cell solution was detected at an excitation light of 488 nm and an absorption light of 514 nm.

As shown in Figure 11, compared with the control, MS-5 cells induced more HL-60 cells to migrate to the lower chamber, while the response ability of HL-60 cells pretreated with C3 polypeptide to MS-5 was significantly reduced, and the cells The mobility dropped to 74±9.0%.

Example 12: Inhibitory effect of polypeptide molecules on MS-5 cells inducing HL-60 cells to adhere to MS-5

HL-60 cells were used as a model for studying leukemia cells. Pre-seed 100,000 MS-5 cells per well in a 24-well plate and let stand overnight. HL-60 cells were stained with calcein for 10 minutes, incubated with 0.1-10 μM C3 polypeptide solution in serum-free medium for 2 hours, and added to 24-well plates with MS-5 cells at the number of 500,000 cells per well. After incubation for 1 hour, the non-adherent cells in the supernatant were collected, centrifuged and concentrated, and the fluorescence value of the cell solution was detected at the excitation light of 488nm and the absorption light of 514nm, and the adhesion rate was calculated. HL-60 cells adhered to MS-5 cells were photographed under a fluorescent microscope.

The results are shown in Figure 12. Compared with the control, the adhesion ability of HL-60 cells pretreated with C3 polypeptide to stromal cells was significantly reduced, and the adhesion rate gradually decreased with the increase of polypeptide concentration. When the peptide concentration was 0.1μM, 1μM, 10μM, the adhesion rates of HL-60 cells were 0.78±0.16, 0.69±0.15, 0.64±0.21, respectively.

Example 13: Experiment of the inhibitory effect of polypeptide molecules on the assembly of HL-60 cytoskeleton proteins induced by CXCL12 molecules

HL-60 cells were used as a model for studying leukemia cells. After 500,000 HL-60 cells were treated with 10 μM C3 polypeptide for 1 hour, they were incubated with 200 ng/ml CXCL12 at 37°C for 1 hour; the HL-60 cells were collected, and the cells were fixed with 2% paraformaldehyde at 37°C for 30 minutes, PBS After washing twice, permeabilize the membrane with 0.5% Triton X-100/PBS for 5 min, wash twice with PBS, block with 1% BSA/PBS for 5 min, dry and use 2 μg/ml rhodamine-labeled phalloidin solution at 37 ℃ for 40 min, and then washed 3 times with 0.5% Tween20/PBS. Finally, the microfilament skeleton structure of cells was observed by laser confocal microscope.

As shown in Figure 13, after HL-60 cells were activated by CXCL12, the microfilaments were more concentrated on the cell membrane, and the cytoplasm was relatively aggregated under the action of microfilaments, while the cells treated with C3 polypeptide first had more microfilaments in the nucleus under the action of CXCL12. Around, the microfilaments in the cytoplasm are relatively loose and diffuse.

Example 14: Inhibitory effect experiment of polypeptide molecules on the signaling pathway induced by CXCL12 molecules on HL-60 cells

HL-60 cells were used as a model for studying leukemia cells. HL-60 cells were resuspended in serum-free medium and added to a 24-well plate, with 500,000 cells per well. After incubation for 0.5 h, a C3 polypeptide solution with a final concentration of 10 μM was added to incubate for 1 h, and the same volume of DMSO was added to the control group. Afterwards, 200ng/ml of CXCL12 was added to the wells, and after incubation for 1 h, the reaction was terminated with cold PBS and the cells were collected. Cell pellets were lysed at 4°C for 30 min with RIPA lysis buffer containing 1 mM PMSF and phosphatase inhibitor cocktail. Afterwards, centrifuge the lysed cell liquid at 4°C, 15000 rpm/min, for 10 min, take the supernatant, and use the BCA method to detect the protein concentration. Add 30 μg protein to the sample well, separate by 12% polyacrylamide electrophoresis and transfer to 0.45 μm PVDF membrane, block with 1% skimmed milk and incubate with specific primary antibody and HRP-labeled secondary antibody successively. Visually observe and analyze the results.

The results are shown in Figure 14. CXCL12 can increase the phosphorylation levels of P38 and ERK in cells, and the antagonistic polypeptide can inhibit the effect of this activation pathway of CXCL12, and the antagonistic polypeptide can reduce the phosphorylation of P38 and ERK in cells to a certain extent. The level of CXCR4/CXCL12 axis was confirmed to be blocked by the antagonistic polypeptide.

Example 15: Effect of polypeptide molecules on HL-60 cells killed by 10-hydroxycamptothecin (Cam)

HL-60 cells were used as a model for studying leukemia cells. 150,000 mouse bone marrow stromal cells MS-5 were seeded into 24-well plates and adhered overnight. 500,000 HL-60 cells resuspended in serum-free medium were seeded into a 96-well plate, and then C3 polypeptide molecules with a final concentration of 10 μM containing 1% DMSO were added and incubated for 1 h. Transfer the cells in the 96-well plate to a 24-well plate with MS-5 cells or a 24-well plate containing only the same volume of medium, and add C3 polypeptide molecules with a final concentration of 10 μM containing 1% DMSO to the plate, After 4 hours of incubation, 50nM 10-hydroxycamptothecin (Cam) was added, and after 24 hours, the HL-60 cells in the 24-well plate were collected, washed twice with PBS, and then washed with binding buffer (10mM Hepes/NaOH, pH7.4, 140mM NaOH Cl, 2.5mM CaCl ₂ ) resuspended, FITC-Annexin V solution was dark stained for 3 minutes, PI solution was added to stain together for 10 minutes, and detected and analyzed by flow cytometry.

As shown in Figure 15, MS-5 cells can protect HL-60 cells from the killing effect of 10-hydroxycamptothecin to a certain extent, and improve the survival of HL-60 cells. However, after pretreatment with C3 polypeptide, the protective effect of MS-5 cells on HL-60 cells was reduced to a certain extent, thereby increasing the sensitivity of 10-hydroxycamptothecin.

Example 16: Affinity experiment of polypeptide molecules to tumor-associated cells NB4 cells

NB4 cells were used as a model for studying leukemia cells. The serum-free medium containing 500,000 NB4 cells in 1% DMSO was planted in a 24-well culture plate, and C3 polypeptide molecules with a final concentration of 10 μM were added to incubate for 4 h. After the cells were collected, they were washed twice with PBS, and then incubated in 5 μg/ml FITC-SA solution at 4° C. for 0.5 h. Wash twice with PBS and filter through a 100-mesh cell strainer. The cell group with only FITC-SA added was used as a negative control. In a flow cytometer, the proportion of cells that are labeled is detected.

As shown in Figure 16, the polypeptide molecule has a high binding force to NB4 cells.

Example 17: Experiment of the inhibitory effect of polypeptide molecules on the migration of NB4 cells induced by CXCL12 molecules

NB4 cells were used as a model for studying leukemia cells. NB4 cells were incubated with C3 polypeptide solution at a concentration of 0.1-10 μM in serum-free medium solution for 1 hour, then added to the upper chamber, 150,000 cells per chamber, and 200 ng/ml CXCL12 solution in the lower chamber. After 24 hours of migration, the cell solution in the lower chamber was collected, counted and counted after centrifugation.

As shown in Figure 17, the migration rate of NB4 cells was low when no chemokine CXCL12 was added. After adding CXCL12 to the lower chamber, the cells showed a stronger ability to migrate to the lower chamber. When the concentrations of C3 peptides were 0.1 μM, 1 μM, and 10 μM, the migration rates of NB4 cells were 76±8.8%, 57±18.5%, and 41±11.1%, respectively, compared with the CXCL12 group.

Example 18: Inhibitory Effect of Polypeptide Molecules on Migration of NB4 Cells Induced by MS-5 Cells

NB4 cells were used as a model for studying leukemia cells. After NB4 cells were stained with calcein for 10 minutes, they were washed once with medium solution, and then incubated with 10 μM C3 polypeptide in serum-free medium solution for 1 hour, and added to the small chamber, 150,000 cells per chamber, and 8 μg/ml was used in the lower chamber. 100,000 MS-5 cells per well after being treated with Mitomycin for 2 hours. After incubation for 6 h, the cell solution in the lower chamber was collected, and after centrifugation, the fluorescence value of the cell solution was detected at the excitation light of 488 nm and the absorption light of 514 nm.

As shown in Figure 18, compared with the control, MS-5 cells induced more NB4 cells to migrate to the lower chamber, while the response ability of NB4 cells pretreated with C3 polypeptide to MS-5 was significantly reduced, and the cell migration rate decreased to 68±6.1%.

Example 19: Inhibitory effect of polypeptide molecules on MS-5 cells inducing NB4 cells to adhere to MS-5

NB4 cells were used as a model for studying leukemia cells. Pre-seed 100,000 MS-5 cells per well in a 24-well plate and let stand overnight. NB4 cells were stained with calcein for 10 minutes and incubated with 0.1-10 μM C3 polypeptide solution in serum-free medium for 2 hours, and added to 24-well plates with MS-5 cells at the number of 500,000 cells per well. After incubation for 1 h, the non-adherent cells in the supernatant were collected, centrifuged and concentrated, and the fluorescence value of the cell solution was detected at the excitation light of 488nm and the absorption light of 514nm, and the adhesion rate was calculated. While NB4 cells adhered to MS-5 cells were photographed under a fluorescent microscope.

The results are shown in Figure 19. Compared with the control, the adhesion ability of NB4 cells pretreated with C3 polypeptide to stromal cells was significantly reduced, and the adhesion rate gradually decreased with the increase of polypeptide concentration. When the peptide concentration was 0.1 μM, 1 μM and 10 μM, the adhesion rates of NB4 cells were 0.91±0.09, 0.63±0.08, and 0.41±0.27, respectively.

Example 20: Effect of polypeptide molecules on NB4 cells killed by 10-hydroxycamptothecin

NB4 cells were used as a model for studying leukemia cells. 150,000 mouse bone marrow stromal cells MS-5 were seeded into 24-well plates and adhered overnight. 500,000 N B4 cells resuspended in serum-free medium were seeded into a 96-well plate, and then C3 polypeptide molecules with a final concentration of 10 μM containing 1% DMSO were added and incubated for 1 h. Transfer the cells in the 96-well plate to a 24-well plate with MS-5 cells or a 24-well plate containing only the same volume of medium, and add C3 polypeptide molecules with a final concentration of 10 μM containing 1% DMSO to the plate, After incubation for 4 hours, 11nM 10-hydroxycamptothecin was added. After 24 hours, the NB4 cells in the 24-well plate were collected, washed twice with PBS, and then washed with binding buffer (10mM Hepes/NaOH, pH7.4, 140mM NaCl, 2.5mM CaCl ₂ ) resuspended, FITC-AnnexinV solution was darkened and stained for 3 minutes, then added PI solution and stained together for 10 minutes, and detected and analyzed by flow cytometry.

As shown in Figure 20, MS-5 cells can protect NB4 cells from the killing effect of 10-hydroxycamptothecin to a certain extent, and improve the survival of NB4 cells. However, after pretreatment with C3 polypeptide, the protective effect of MS-5 cells on NB4 cells was reduced to a certain extent, thereby increasing the sensitivity of 10-hydroxycamptothecin.

Example 21: Affinity experiment of polypeptide molecules to tumor-associated cells THP-1 cells

THP-1 cells were used as a model for studying leukemia cells. Seed 500,000 THP-1 cells in 1% DMSO serum-free medium into a 24-well culture plate, add C3 polypeptide molecules at a final concentration of 10 μM and incubate for 4 h. After the cells were collected, they were washed twice with PBS, and then incubated in 5 μg/ml FITC-SA solution at 4° C. for 0.5 h. Wash twice with PBS and filter through a 100-mesh cell strainer. The cell group with only FITC-SA added was used as a negative control. In a flow cytometer, the proportion of cells that are labeled is detected.

As shown in Figure 21, C3 has high binding ability to THP-1 cells.

Example 22: Experiment of the inhibitory effect of polypeptide molecules on the migration of THP-1 cells induced by CXCL12 molecules

THP-1 cells were used as a model for studying leukemia cells. THP-1 cells were incubated with C3 polypeptide solution at a concentration of 0.1-10 μM in serum-free medium solution for 1 hour, then added to the upper chamber, 150,000 cells per chamber, and 50 ng/ml CXCL12 solution in the lower chamber. After 24 hours of migration, the cell solution in the lower chamber was collected, counted and counted after centrifugation.

As shown in Figure 22, THP-1 cells had a lower migration rate in the absence of the addition of the chemokine CXCL12. After adding CXCL12 to the lower chamber, the cells showed a stronger ability to migrate to the lower chamber. When the concentrations of C3 polypeptide were 0.1 μM, 1 μM, and 10 μM, the migration rates of THP-1 cells were 90±5.4%, 76±5.0%, and 65±17.8%, respectively, compared with CXCL12 group.

Example 23: Experiment of the inhibitory effect of polypeptide molecules on the migration of THP-1 cells induced by MS-5 cells

THP-1 cells were used as a model for studying leukemia cells. After THP-1 cells were stained with calcein for 10 minutes, they were washed with medium solution, and then incubated with 10 μM C3 polypeptide in serum-free medium solution for 1 hour, and added to the small chamber, 150,000 cells per chamber, and 8 μg for the lower chamber. MS-5 cells left overnight after being treated with mitomycin/ml for 2 hours, 100,000 cells per well. After incubation for 6 h, the cell solution in the lower chamber was collected, and after centrifugation, the fluorescence value of the cell solution was detected at the excitation light of 488 nm and the absorption light of 514 nm.

As shown in Figure 23, compared with the control, MS-5 cells induced more THP-1 cells to migrate to the lower chamber, while the response ability of THP-1 cells pretreated with C3 polypeptide to MS-5 was significantly reduced, and the cells The mobility dropped to 77±10.5%.

Example 24: Experiment of the inhibitory effect of polypeptide molecules on MS-5 cells inducing THP-1 cells to adhere to MS-5

THP-1 cells were used as a model for studying leukemia cells. Pre-seed 100,000 MS-5 cells per well in a 24-well plate and let stand overnight. THP-1 cells were stained with calcein for 10 min and incubated with 0.1-10 μM C3 polypeptide solution in serum-free medium for 2 h, and added to a 24-well plate with MS-5 cells at the number of 500,000 cells per well. After incubation for 1 h, the non-adherent cells in the supernatant were collected, centrifuged and concentrated, and the fluorescence value of the cell solution was detected at the excitation light of 488nm and the absorption light of 514nm, and the adhesion rate was calculated. While THP-1 cells adhered to MS-5 cells were photographed under a fluorescent microscope.

The results are shown in Figure 24. Compared with the control, the adhesion ability of THP-1 cells pretreated with C3 polypeptide to stromal cells was significantly reduced, and the adhesion rate gradually decreased with the increase of polypeptide concentration. When the peptide concentration was 0.1 μM, 1 μM, and 10 μM, the adhesion rates of THP-1 cells were 0.72±0.19, 0.59±0.08, and 0.51±0.23, respectively.

Example 25: Experiment of the inhibitory effect of polypeptide molecules on THP-1 cytoskeleton protein assembly induced by CXCL12 molecules

THP-1 cells were used as a model for studying leukemia cells. After 500,000 THP-1 cells were treated with 10 μM C3 polypeptide for 1 hour, they were incubated with 50 ng/ml CXCL12 at 37°C for 1 hour; THP-1 cells were collected and fixed with 2% paraformaldehyde at 37°C for 30 minutes, PBS After washing twice, permeabilize the membrane with 0.5% Triton X-100/PBS for 5 min, wash twice with PBS, block with 1% BSA/PBS for 5 min, dry and use 2 μg/ml rhodamine-labeled phalloidin solution at 37 ℃ for 40 min, and then washed 3 times with 0.5% Tween20/PBS. Finally, the microfilament skeleton structure of cells was observed by laser confocal microscope.

As shown in Figure 25, the microfilament skeleton of THP-1 cells after CXCL12 activation is relatively tight, and the surface of the cell membrane is irregular, and a synapse-like structure is formed, which corresponds to the enhancement of cell migration and adhesion capabilities, while The cells treated with C3 polypeptide first had a more diffuse and irregular skeleton arrangement after the action of CXCL12, mostly around the nucleus, similar to the control.

Example 26: Affinity experiment of polypeptide molecules to U937 cells

U937 cells were used as a model to study leukemia cells. The serum-free medium containing 500,000 U937 cells in 1% D MSO was seeded into a 24-well culture plate, and C3 polypeptide molecules with a final concentration of 10 μM were added to incubate for 4 h. After the cells were collected, they were washed twice with PBS, and then incubated in 5 μg/ml FITC-SA solution at 4° C. for 0.5 h. Wash twice with PBS and filter through a 100-mesh cell strainer. The cell group with only FITC-SA added was used as a negative control. In a flow cytometer, the proportion of cells that are labeled is detected.

As shown in Figure 26, the polypeptide molecule has a high binding force to U937 cells.

Example 27: Experiment of the inhibitory effect of polypeptide molecules on the migration of U937 cells induced by CXCL12 molecules

U937 cells were used as a model to study leukemia cells. U937 cells were incubated with C3 polypeptide solution at a concentration of 0.1-10 μM in serum-free medium solution for 1 hour, then added to the upper chamber, 150,000 cells per chamber, and 50 ng/ml CXCL12 solution in the lower chamber. After 24 hours of migration, the cell solution in the lower chamber was collected, counted and counted after centrifugation.

As shown in Figure 27, the migration rate of U937 cells was low when no chemokine CXCL12 was added. After adding CXCL12 to the lower chamber, the cells showed a stronger ability to migrate to the lower chamber. When the concentrations of C3 polypeptide were 0.1 μM, 1 μM, and 10 μM, the migration rates of U937 cells were 85±15.3%, 90±2.2%, and 73±12.3%, respectively, compared with the CXCL12 group.

Example 28: Inhibitory Effect of Polypeptide Molecules on Migration of U937 Cells Induced by MS-5 Cells

U937 cells were used as a model to study leukemia cells. After U937 cells were stained with calcein for 10 minutes, they were washed once with medium solution, and then incubated with 10 μM C3 polypeptide in serum-free medium solution for 1 hour, and added to the small chamber, 150,000 cells per chamber, and 8 μg/ml for the lower chamber. 100,000 MS-5 cells per well after being treated with Mitomycin for 2 hours. After incubation for 6 h, the cell solution in the lower chamber was collected, and after centrifugation, the fluorescence value of the cell solution was detected at the excitation light of 488 nm and the absorption light of 514 nm.

As shown in Figure 28, compared with the control, MS-5 cells induced more U937 cells to migrate to the lower chamber, while the response ability of U937 cells pretreated with C3 polypeptide to MS-5 was significantly reduced, and the cell migration rate decreased to 79±10.9%.

Example 29: Inhibitory effect of polypeptide molecules on MS-5 cells inducing U937 cells to adhere to MS-5

U937 cells were used as a model to study leukemia cells. Pre-seed 100,000 MS-5 cells per well in a 24-well plate and let stand overnight. U937 cells were stained with calcein for 10 min and incubated with 0.1-10 μM C3 polypeptide solution in serum-free medium for 2 h, and added to a 24-well plate with MS-5 cells at the number of 500,000 cells per well. After incubation for 1 h, the non-adherent cells in the supernatant were collected, centrifuged and concentrated, and the fluorescence value of the cell solution was detected at the excitation light of 488nm and the absorption light of 514nm, and the adhesion rate was calculated. U937 cells adhered to MS-5 cells were photographed under a fluorescent microscope.

The results are shown in Figure 29. Compared with the control, the adhesion ability of U937 cells pretreated with C3 polypeptide to stromal cells was significantly reduced, and the adhesion rate gradually decreased with the increase of polypeptide concentration. When the peptide concentration was 0.1 μM, 1 μM, 10 μM, the adhesion rates of U937 cells were 0.87±0.09, 0.67±0.15 or 0.37±0.08, respectively.

The above experiments show that the polypeptide of the present invention has the ability to enhance the sensitivity of tumor cells to antitumor drugs and inhibit the migration of tumor cells, and can be used as a drug for treating or adjuvantly treating tumors (especially leukemia and breast cancer).

Those skilled in the art will understand that: although the specific implementation of the present invention has been described in detail, according to all teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (8)

1. a kind of be used to strengthen polypeptide of the tumour cell to antitumor drug sensitiveness and suppression tumor cell migration, its feature exists In the amino acid sequence such as SEQ ID NO of the polypeptide：Shown in one of 1~4.

A kind of 2. fusion protein for including polypeptide as claimed in claim 1.

3. a kind of polypeptide as claimed in claim 1 and compound obtained from macromolecular carrier coupling.

A kind of 4. nucleotide sequence for encoding polypeptide as claimed in claim 1.

A kind of 5. nucleic acid carrier for including nucleotide sequence as claimed in claim 4.

A kind of 6. host cell for including nucleic acid carrier as claimed in claim 5.

7. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition includes polypeptide as claimed in claim 1 or as weighed Profit requires fusion protein described in 2 or compound as claimed in claim 3 or nucleotide sequence as claimed in claim 4 or such as Nucleic acid carrier or host cell as claimed in claim 6 described in claim 5.

8. a kind of polypeptide as claimed in claim 1 is being prepared for strengthening leukemia tumor cells to antitumor drug sensitiveness And the application in the medicine of suppression leukemia tumor cells migration；

Wherein, the leukemia tumor cells are in K562 cells, HL-60 cells, NB4 cells, THP-1 cells or U937 cells Any one or at least two combination.