Multi-target anti-breast cancer drug composition and its application

Abstract

The present invention belongs to the field of biomedicine technology, and relates to an anti-breast cancer drug composition based on multiple targets and applications thereof; specifically provides an anti-breast cancer drug composition with BIRC5, NAMPT and TOP1 as combined therapeutic targets and applications thereof; the drug composition comprises a BIRC5 inhibitor, a NAMPT inhibitor and a TOP1 inhibitor; the present invention uses the BIRC5 inhibitor, the NAMPT inhibitor and the TOP1 inhibitor in combination, which can produce a synergistic anti-tumor effect on different breast cancer cell lines, and is stronger than the anti-tumor effect of a single inhibitor or a combination of any two inhibitors, and provides a new treatment scheme for the clinical treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to an anti-breast cancer drug composition based on multiple targets and its application, and specifically to an anti-breast cancer drug composition with baculovirus inhibitor of apoptosis protein repeat sequence 5 (BIRC5), nicotinamide phosphoribosyltransferase (NAMPT) and topoisomerase I (TOP1) as combined therapeutic targets and its application. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women, and its incidence rate currently ranks first in the world, seriously threatening human life and health. At present, commonly used treatment methods include the use of chemotherapy drugs, targeted therapy, radiotherapy, etc. Among them, targeted therapy mainly uses a single targeted drug. However, as a highly heterogeneous malignant tumor, the pathogenesis of breast cancer is complex and diverse, involving multiple gene mutations and dysregulation of multiple signal pathways. The therapeutic effect of a single targeted drug is limited and there are problems such as tumor resistance.

[0003] Baculovirus inhibitor of apoptosis protein repeat 5 (BIRC5) is an important member of the apoptosis inhibitor protein family. It is almost not expressed in normal adult differentiated tissues, but is significantly expressed during embryonic development and most tumor tissues. It has the dual effects of promoting cell proliferation and inhibiting cell apoptosis. BIRC5 is highly expressed in a variety of breast cancers and is significantly correlated with patient survival and prognosis.

[0004] Nicotinamide phosphoribosyltransferase (NAMPT) is a mammalian nicotinamide adenine dinucleotide (NAD + ) is a key rate-limiting enzyme in the salvage synthesis pathway. It is highly expressed in a variety of malignant tumor tissues and is positively correlated with tumor proliferation, metastasis and poor prognosis. Since rapid proliferation, metastasis and angiogenesis require more NAD+, tumor cells are highly dependent on NAD+ levels, making them sensitive to NAMPT activity regulation.

[0005] Topoisomerase I (TOP1) plays an important role in DNA replication, transcription, recombination and repair by catalyzing single-strand breaks of DNA and releasing supercoiling and torsional tension of DNA molecules. However, the clinical application of TOP1 inhibitors such as topotecan and irinotecan is limited due to problems such as myelosuppression toxicity and drug resistance.

[0006] Therefore, drug combination therapy targeting key targets of breast cancer is an important strategy. Compared with single targeted therapy, multi-target drug combination therapy has many advantages, such as improving efficacy, reducing host toxicity and adverse reactions, and reducing the occurrence of tumor resistance. It has become one of the future development trends of tumor treatment. In view of the problems of poor efficacy, large toxic and side effects, and tumor resistance of existing treatment options, it is urgent to find synergistic drugs for combination therapy to improve treatment efficacy, reduce toxic and side effects, reverse tolerance, and prolong patient survival. Summary of the invention

[0007] In view of this, the present invention provides an anti-breast cancer drug composition based on multiple targets and its application, in particular, an anti-breast cancer drug composition with BIRC5, NAMPT and TOP1 as combined therapeutic targets and its application, wherein the drug composition can produce a synergistic anti-tumor effect on different breast cancer cell lines.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention first provides an anti-breast cancer drug composition based on multiple targets, wherein the targets include BIRC5, NAMPT and TOP1 as combined treatment targets.

[0010] Preferably, the pharmaceutical composition comprises a BIRC5 inhibitor, a NAMPT inhibitor and a TOP1 inhibitor.

[0011] Preferably, the BIRC5 inhibitor comprises siRNA of the BIRC5 gene or LQZ-7I; the sense strand sequence of the siRNA is shown in SEQ ID NO:1, and the antisense strand sequence is shown in SEQ ID NO:2.

[0012] Preferably, the NAMPT inhibitor comprises siRNA of the NAMPT gene or FK866; the sense strand sequence of the siRNA is shown in SEQ ID NO:3, and the antisense strand sequence is shown in SEQ ID NO:4.

[0013] Preferably, the TOP1 inhibitor comprises siRNA or Topotecan of the TOP1 gene; the sense strand sequence of the siRNA is shown in SEQ ID NO:5, and the antisense strand sequence is shown in SEQ ID NO:6.

[0014] The present invention also provides application of the pharmaceutical composition in preparing anti-breast cancer drugs.

[0015] The present invention also provides an anti-breast cancer drug, which comprises the anti-breast cancer drug composition.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides an anti-breast cancer drug composition with BIRC5, NAMPT and TOP1 as combined therapeutic targets. By using a BIRC5 inhibitor, a NAMPT inhibitor and a TOP1 inhibitor in combination, the composition can inhibit the proliferation of breast cancer cells and has a synergistic anti-tumor effect, which is stronger than the anti-tumor effect of a single inhibitor or a combination of any two inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of using BIRC5, NAMPT and TOP1 siRNAs alone or in combination on the expression of BIRC5, NAMPT and TOP1 in MCF7 cells in Example 1 are shown.

[0019] Figure 2 The effects of using BIRC5, NAMPT and TOP1 siRNAs alone or in combination on the expression of BIRC5, NAMPT and TOP1 in MDA-MB-231 cells in Example 1 are shown.

[0020] Figure 3 The figure shows the effect of siRNA of BIRC5, NAMPT and TOP1 used alone or in combination on the proliferation of MCF7 cells in Example 2.

[0021] Figure 4 The figure shows the effects of siRNA of BIRC5, NAMPT and TOP1 used alone or in combination on the proliferation of MDA-MB-231 cells in Example 2.

[0022] Figure 5 The effects of LQZ-7I, FK866 and Topotecan alone or in combination on tumor growth in 4T1 tumor-bearing mice in Example 4 are shown.

[0023] Figure 6 The following is the effect of LQZ-7I, FK866 and Topotecan used alone or in combination on the tumor weight of 4T1 tumor-bearing mice in Example 4.

[0024] Figure 7 The effects of LQZ-7I, FK866 and Topotecan alone or in combination on the body weight of 4T1 tumor-bearing mice in Example 4 are shown. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, which are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the reagents, equipment and methods used in the present invention are conventional commercially available reagents, equipment and methods used in the art.

[0026] Example 1: Inhibitory effects of siRNAs of BIRC5, NAMPT and TOP1 alone or in combination on the expression of BIRC5, NAMPT and TOP1 in MCF7 and MDA-MB-231 cells

[0027] (1) Cell culture: MCF7 and MDA-MB-231 cells (both purchased from the cell bank of the Chinese Academy of Sciences in Shanghai) were inoculated into T25 culture flasks with complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured at 37°C and 5% CO2. The cells were subcultured after the cell growth density reached 80%-90%. The culture medium in the culture flask was discarded, 1 mL of trypsin cell digestion solution was added, and the cells were placed in an incubator for 2-3 minutes. After the cells were observed to be rounded and the gaps increased under a microscope, two times the complete culture medium was added to terminate the digestion, and the cell suspension was collected and centrifuged at 1000 rpm / min for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium. The cells were divided into culture flasks at a subculture ratio of 1:2 and cultured in an incubator.

[0028] (2) Cell inoculation: When the cells grow to the logarithmic growth phase, trypsin is added for digestion. After termination of digestion, the supernatant is discarded by centrifugation and diluted with complete culture medium. The cells are inoculated into a 6-well plate at a density of 15,000 cells / well and a seeding volume of 2 mL / well. The plates are placed in an incubator and cultured for 24 h before use in subsequent experiments.

[0029] (3) siRNA transfection: BIRC5, NAMPT and TOP1 siRNA single transfection groups, three target gene siRNA co-transfection groups, negative control group (NC group, transfected with siRNA with no homology to the target gene sequence) and blank control group (BC group, no siRNA transfection treatment) were set up. When the cell density reached 60%-80%, transfection was performed. 200 μL of DMEM high-glucose medium without serum was added to each sterile centrifuge tube, and then 6 μL of 20 μM target gene siRNA and 10 μL of GP-transfect-Mate transfection reagent (all purchased from Suzhou Jima Gene) were added. Among them, the three target gene siRNA co-transfection groups needed to add 6 μL of siRNA of the corresponding target gene, and let it stand at room temperature for 5 minutes to prepare the siRNA culture medium mixture and the transfection reagent culture medium mixture.

[0030] Add the transfection reagent culture medium mixture dropwise to the siRNA culture medium mixture, mix gently, and let stand at room temperature for 15-20 minutes to prepare the transfection complex. During the preparation of the transfection complex, replace 1.6mL of complete culture medium in the 6-well plate. Add 400μL of transfection complex to the well, shake gently to evenly distribute it, and place it in the incubator for culture. After 5 hours, replace the complete culture medium, place it in the incubator for 48 hours, and use qRT-PCR to detect the mRNA expression of the target gene.

[0031] Table 1. siRNA sequences of BIRC5, NAMPT and TOP1

[0032]

[0033] Note: According to the sequence listing submission requirements, the "u" in the above sequence is represented by "t" in the sequence listing.

[0034] qRT-PCR detection of mRNA expression:

[0035] (1) RNA extraction:

[0036] In the above siRNA transfection step, the culture medium was discarded 48 hours after transfection, PBS was washed, 500 μL LysisBuffer was added, pipetted vigorously 10 times and transferred to an EP tube, vortexed for 10 seconds, an equal volume of anhydrous ethanol was added to mix thoroughly, and then the liquid was added to the centrifuge column and centrifuged at 4000 rpm / min for 1 minute. The waste liquid was discarded, 500 μL Wash Buffer was added to the RNA column, and centrifuged at 12,000 rpm / min for 1 minute. The column was placed on a clean 1.5 mL centrifuge tube without RNase, opened and dried for 2 minutes. 20 μL Elution Buffer was added to the center of the membrane of the RNA column, left to stand at room temperature for 2 minutes, and centrifuged at 12,000 rpm / min for 1 minute to obtain an RNA solution. The concentration and purity of the RNA solution were determined using a Nanodrop instrument.

[0037] (2) Reverse transcription:

[0038] Take 1μg of total RNA and add it to an EP tube. Add 2μL of DNase and RNase water to 16μL. Mix by pipetting and place at room temperature for 5 minutes. Then add 4μg of 5×RT Mix and mix by pipetting. React at 42℃ for 15 minutes and dilute 10 times as a cDNA template.

[0039] (3) Primer sequence design:

[0040] The human BIRC5, NAMPT and TOP1 gene sequences were downloaded from the NCBI database (the NCBI reference sequences were: NM_001168.3, NM_005746.3 and NM_003286.4, respectively). The primer sequences of the target genes were designed using the Shanghai Bioengineering online primer design tool (as shown in Table 2). The specificity of the primer sequences was then verified using the BLAST tool of the NCBI database and commissioned to be synthesized by Shanghai Bioengineering.

[0041] Table 2. Primer sequences of target genes

[0042]

[0043] (4) qPCR reaction:

[0044] Prepare the qPCR reaction system according to Table 3, centrifuge briefly to mix, and set the reaction program according to Table 4. Make three parallel wells for each sample, and use GADPH primers as internal reference.

[0045] Table 3. qPCR reaction system

[0046] Element Volume (20μL system) 2×UltraSYBR Mixture 10μL Forward Primer (10μM) 0.4μL Reverse Primer (10μM) 0.4μL Template DNA 0.4μL <![CDATA[ddH2O]]> 8.8μL

[0047] Table 4. qPCR reaction program

[0048]

[0049] The experimental results are as follows Figure 1 and Figure 2 As shown, compared with the blank control group (BC group) and the negative control group (NC group), single siRNA transfection of BIRC5, NAMPT, and TOP1 and co-transfection of the three significantly reduced the expression of target gene mRNA in MCF7 and MDA-MB-231 cells, indicating that single siRNA transfection of BIRC5, NAMPT, and TOP1 was effective, and co-transfection of siRNA of the three genes could simultaneously and efficiently silence the three target genes in MCF7 and MDA-MB-231 cells.

[0050] Example 2: Inhibitory effects of siRNA of BIRC5, NAMPT and TOP1 alone or in combination on proliferation of MCF7 and MDA-MB-231 cells

[0051] Cell inoculation: When MCF7 and MDA-MB-231 cells grow to the logarithmic growth phase, add trypsin for digestion, centrifuge and discard the supernatant after termination of digestion, dilute with complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin), and inoculate the cells into 96-well plates at a density of 5,000 cells / well and an inoculation volume of 100 μL / well. Place in an incubator and continue to culture for 24 hours before use in subsequent experiments.

[0052] siRNA transfection: BIRC5, NAMPT and TOP1 siRNA single transfection groups, any two target gene siRNA co-transfection groups, three target gene siRNA co-transfection groups, negative control group (NC group, transfected with siRNA with no homology to the target gene sequence) and blank control group (BC group, no siRNA transfection treatment) were set up. When the cell density reached 60%-80%, transfection was performed. 10 μL of DMEM high-glucose medium without serum was added to each sterile centrifuge tube, and then 0.8 μL of 20 μM target gene siRNA and 0.8 μL of GP-transfect-Mate transfection reagent (both purchased from Suzhou Jima Gene) were added. Among them, 0.8 μL of siRNA of the corresponding target gene was added to the co-transfection group of multiple target gene siRNA, and the mixture was left to stand at room temperature for 5 minutes to prepare the siRNA culture medium mixture and the transfection reagent culture medium mixture.

[0053] Add the transfection reagent culture medium mixture dropwise to the siRNA culture medium mixture, mix gently, and let stand at room temperature for 15-20 minutes to prepare the transfection complex. During the preparation of the transfection complex, replace 80μL of complete culture medium in the 96-well plate. Add 20μL of transfection complex to the well, shake gently to evenly distribute it, and place it in the incubator for culture. After 5 hours, replace the complete culture medium, place it in the incubator for 48 hours, and use CCK-8 to detect cell proliferation activity.

[0054] Cell proliferation activity detection: 48 hours after transfection, aspirate the original culture medium in the well, add the detection solution containing 10% CCK-8 reagent, incubate in the incubator for 1-2 hours, and use an enzyme marker to measure the OD value at 450nm to calculate the cell survival rate. The calculation formula is:

[0055]

[0056] The experimental results are as follows Figure 3 and Figure 4 As shown, compared with the negative control group (NC group), co-transfection of siRNA of three genes had the strongest inhibitory activity on the proliferation of MCF7 and MDA-MB-231 cells, and could more effectively inhibit the proliferation of the two breast cancer cells than single gene siRNA transfection or co-transfection of siRNA of any two genes, indicating that the simultaneous use of siRNA of BIRC5, NAMPT, and TOP1 has a synergistic effect in inhibiting the proliferation of the two breast cancer cells.

[0057] Example 3: Inhibitory effects of LQZ-7I, FK866 and Topotecan alone or in combination on proliferation of MCF7 and MDA-MB-231 cells

[0058] Cell inoculation: When the cells grow to the logarithmic growth phase, add trypsin for digestion, terminate the digestion, centrifuge and discard the supernatant, dilute with complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin), and inoculate the cells into a 96-well plate with an inoculation density of 5,000 cells / well and an inoculation volume of 100 μL / well. Place the plate in an incubator and continue to culture for 24 hours before use in subsequent experiments.

[0059] Cell administration: a blank control group, a LQZ-7I single-drug group, a FK866 single-drug group, a Topotecan single-drug group, and a LQZ-7I, FK866 and Topotecan combination group were set. The molar concentrations of the single-drug groups were set to 300μM, 200μM, 100μM, 10μM, 5μM, 2.5μM, 1μM, 0.5μM, and 0.1μM, and the molar concentration of the combination group was the sum of the molar concentrations of LQZ-7I, FK866 and topotecan in the combination; among them, the combination ratios of the combination groups were set to molar ratios of 2.5:10:1, 2.5:0.1:1, 10:10:1, and 10:0.1:1.

[0060] The original culture medium in the wells was aspirated, and different concentrations of drugs and drug combinations were added respectively. Four replicate wells were set for each drug concentration, and 100 μL of drug was added to each well. 100 μL of DMEM culture medium was added to the blank control group, and the cells were transferred to a cell culture incubator and cultured for 48 h.

[0061] Cell proliferation activity detection: 48 hours after administration, the original culture medium in the well was aspirated, and the detection solution containing 10% CCK-8 reagent was added. The cells were incubated in an incubator for 1-2 hours, and the OD value at 450nm was measured using an ELISA reader to calculate the cell survival rate. The calculation formula is:

[0062]

[0063] CompuSyn software was used to calculate the drug combination index (CI) under the condition of 50% growth inhibition rate, which was used to determine the strength of the synergistic anti-tumor effect of the drug combination. A CI value <1 indicated that the drugs had a synergistic effect, a CI value = 1 indicated that the drugs had an additive effect, and a CI value > 1 indicated that the drugs had an antagonistic effect.

[0064] The experimental results are shown in Tables 5 and 6. Different proportions of LQZ-7I, FK866 and Topotecan compositions have synergistic anti-tumor effects on both MCF7 and MDA-MB-231 cells.

[0065] Table 5. IC values ​​of drugs and drug combinations in different ratios on MCF7 cells 50 and CI value

[0066]

[0067] Table 6. IC values ​​of drugs and drug combinations in different ratios on MDA-MB-231 cells 50 and CI value

[0068]

[0069] Example 4: Effects of LQZ-7I, FK866 and Topotecan alone or in combination on tumor growth and body weight in 4T1 tumor-bearing mice

[0070] Cell culture: 4T1 cells (purchased from the cell bank of the Chinese Academy of Sciences in Shanghai) were inoculated into T75 culture flasks with complete medium (1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin), cultured at 37°C and 5% CO2, and subcultured after the cell growth density reached 80%-90%. The medium in the culture flask was discarded, 3 mL of trypsin cell digestion solution was added, and the cells were placed in an incubator for 8-10 minutes. After the cell morphology became round and the gap increased under a microscope, two times the complete medium was added to terminate the digestion, and the cell suspension was collected and centrifuged at 1000rpm / min for 5 minutes. The supernatant was discarded, and the complete medium was added to resuspend, and the cells were divided into culture flasks at a subculture ratio of 1:4, and placed in an incubator for culture.

[0071] Construction of tumor-bearing mouse model: Female SPF-grade Bal b / c mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) weighing 18-22 g were used. When the cells grew to the logarithmic growth phase, trypsin was added for digestion. After digestion was terminated, the supernatant was discarded by centrifugation and the concentration of 2×10 7 0.15 mL of the cell suspension was inoculated subcutaneously in the armpit of mice.

[0072] Experimental grouping: When the tumor volume reaches 50-100mm 3 The mice were randomly divided into 8 groups, 5 in each group. LQZ-7I (3.8 mg / kg), FK866 (17 mg / kg), Topotecan (2 mg / kg), LQZ-7I (3.8 mg / kg) + FK866 (17 mg / kg), LQZ-7I (3.8 mg / kg) + Topotecan (2 mg / kg), FK866 (17 mg / kg) + Topotecan (2 mg / kg), LQZ-7I (3.8 mg / kg) + FK866 (17 mg / kg) + Topotecan (2 mg / kg) and a blank control group (blank solvent) were set up respectively. The drug solution or drug stock solution was prepared for drug administration. The drug solution was prepared according to the following method:

[0073] (1) Accurately weigh 1.9 mg of LQZ-7I, add 50 μL of DMSO, 500 μL of castor oil, and 4.45 mL of saline containing 20% ​​β-cyclodextrin in sequence, and mix well to obtain a LQZ-7I stock solution with a concentration of 0.38 mg / mL.

[0074] (2) Accurately weigh 8.5 mg of FK866, add 2.25 mL of propylene glycol, 0.25 mL of Tween 80, and 2.50 mL of ddH2O in sequence, and mix well to obtain a FK866 stock solution with a concentration of 1.7 mg / mL.

[0075] (3) Accurately weigh 1.0 mg of Topotecan and dissolve it in 5 mL of normal saline to obtain a Topotecan stock solution with a concentration of 0.2 mg / mL.

[0076] The blank control group was given blank solvents of three drug solutions, LQZ-7I, FK866 and Topotecan. Drug stock solutions and blank solvents were prepared and used immediately.

[0077] Drug administration: When the tumor volume reaches 50-100mm 3 The drugs were all administered by intraperitoneal injection every two days. The weight and growth of the mice were recorded every day from the time of tumor inoculation, and the length and width of the tumor were measured with a digital vernier caliper.

[0078] Dissection: Wait until the tumor volume reaches 1000-1500mm 3 After blood was collected from the eyeball, the mice were killed by carbon dioxide asphyxiation, and the heart, liver, spleen, lung, kidney, and tumor were collected by dissection, and the tumor weight was measured.

[0079] The experimental results are as follows Figure 5-7 As shown, compared with the blank control group, the combined drug group of LQZ-7I, FK866, and Topotecan can significantly inhibit tumor growth, and the inhibitory effect is better than that of a single drug and any two drug combinations, and has no significant effect on the weight of mice, and is relatively safe.

[0080] The above results indicate that the anti-breast cancer drug composition with BIRC5, NAMPT and TOP1 as combined therapeutic targets, the combined use of BIRC5 inhibitors, NAMPT inhibitors and TOP1 inhibitors, has a synergistic inhibitory effect on breast cancer, and is stronger than the anti-tumor effect of a single inhibitor or a combination of any two inhibitors.

Claims

1. A multi-target anti-breast cancer drug composition, characterized in that: The targets include BIRC5, NAMPT and TOP1 as combined therapeutic targets; The pharmaceutical composition comprises a BIRC5 inhibitor, a NAMPT inhibitor and a TOP1 inhibitor; The BIRC5 inhibitor includes siRNA of the BIRC5 gene or LQZ-7I, wherein the sense strand sequence of the siRNA of the BIRC5 gene is shown in SEQ ID NO: 1, and the antisense strand sequence is shown in SEQ ID NO: 2; The NAMPT inhibitor includes siRNA of the NAMPT gene or FK866, wherein the sense strand sequence of the siRNA of the NAMPT gene is shown in SEQ ID NO:3, and the antisense strand sequence is shown in SEQ ID NO:4; The TOP1 inhibitor includes siRNA or Topotecan of the TOP1 gene, the sense strand sequence of the siRNA of the TOP1 gene is shown in SEQ ID NO:5, and the antisense strand sequence is shown in SEQ ID NO:

6.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of anti-breast cancer drugs.

3. An anti-breast cancer drug, characterized in that: The medicine comprises the anti-breast cancer pharmaceutical composition according to claim 1.

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