The composition of N1-methyladenosine or N6-methyladenosine and cisplatin and its use in preparing anti-tumor drugs

Abstract

The present invention discloses a composition of N1-methyladenosine or N6-methyladenosine and cisplatin and its use for preparing an anti-tumor drug; it claims a composition composed of N1-methyladenosine or N6-methyladenosine and cisplatin; it also claims the use of this composition for preparing an anti-non-small cell lung cancer drug. The present invention discovers that the combined use of N1-methyladenosine and cisplatin can produce an obvious synergistic inhibitory effect on cisplatin-sensitive non-small cell lung cancer, cisplatin moderately resistant non-small cell lung cancer, and cisplatin highly resistant non-small cell lung cancer; the combined use of N6-methyladenosine and cisplatin can produce an obvious synergistic inhibitory effect on cisplatin moderately resistant non-small cell lung cancer and cisplatin highly resistant non-small cell lung cancer. Therefore, the composition of N1-methyladenosine or N6-methyladenosine and cisplatin has the prospect of being developed into a drug for anti-non-small cell lung cancer.

Description

Technical Field

[0001] The invention belongs to the field of chemistry and relates to a compound composition, in particular to a composition of N1-methyladenosine or N6-methyladenosine and cisplatin and the use of the composition in preparing anti-tumor drugs. Background Art

[0002] Lung cancer is one of the most common cancers in humans and has become the leading cause of cancer-related death worldwide. According to histopathological characteristics, about 85% of lung cancer patients have non-small cell lung cancer, and most patients with non-small cell lung cancer are already in the advanced stage when diagnosed, with a five-year survival rate of only 17.4%. Currently, chemotherapy, molecular targeted therapy and immunotherapy are the main treatments for advanced non-small cell lung cancer, but they all produce varying degrees of drug resistance, seriously affecting clinical efficacy.

[0003] Cisplatin (DDP) is an alkylating cytotoxic chemotherapy drug with significant anti-cancer effects and a broad anti-cancer spectrum. It is the main chemotherapy for non-small cell lung cancer. However, the efficacy of cisplatin alone in the treatment of non-small cell lung cancer is only 15%, and the efficacy of combined chemotherapy is 15-40%. Tumor resistance is one of the main reasons limiting its clinical efficacy. Therefore, how to specifically reverse cisplatin resistance in non-small cell lung cancer is a key scientific issue that needs to be solved urgently in the clinic.

[0004] Epigenetics mediated by small molecule modified metabolites is closely related to tumor resistance. 1 A), N6-methyladenosine (6-Methyladenosine, m 6 A) is a methylated adenosine. Studies have reported that RNA-bound N1-methyladenosine and N6-methyladenosine play a key role in tumor cell proliferation and metastasis, and the individualization of chemotherapy drug effects. However, there are few reports on the role of free N1-methyladenosine and N6-methyladenosine in tumors. At present, no studies at home and abroad have shown that the combination of free N1-methyladenosine or N6-methyladenosine and cisplatin can play a synergistic role in inhibiting non-small cell lung cancer and reversing its cisplatin resistance. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a composition of N1-methyladenosine or N6-methyladenosine and cisplatin and the use of the composition in preparing anti-tumor drugs.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A composition consisting of N1-methyladenosine and cisplatin.

[0008] Use of the composition composed of N1-methyladenosine and cisplatin for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-sensitive non-small cell lung cancer, and the molar ratio of N1-methyladenosine to cisplatin in the composition is 2000:(10-20) or 4000:(1.25-20).

[0009] Use of the composition composed of N1-methyladenosine and cisplatin for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-moderately resistant non-small cell lung cancer, and the molar ratio of N1-methyladenosine to cisplatin in the composition is (1000-4000):(2.5-40).

[0010] Use of the composition composed of N1-methyladenosine and cisplatin for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-highly resistant non-small cell lung cancer, and the molar ratio of N1-methyladenosine to cisplatin in the composition is (3.125-200):1.

[0011] Use of N1-methyladenosine and cisplatin in combination for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-sensitive non-small cell lung cancer, cisplatin-moderately resistant non-small cell lung cancer or cisplatin-highly resistant non-small cell lung cancer.

[0012] A composition composed of N6-methyladenosine and cisplatin.

[0013] Use of the composition composed of N6-methyladenosine and cisplatin for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-moderately resistant non-small cell lung cancer, and the molar ratio of N6-methyladenosine to cisplatin in the composition is (500-2000):(2.5-40).

[0014] Use of the composition composed of N6-methyladenosine and cisplatin for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-highly resistant non-small cell lung cancer, and the molar ratio of N6-methyladenosine to cisplatin in the composition is (500-2000):40.

[0015] Use of N6-methyladenosine and cisplatin in combination for preparing a drug for treating non-small cell lung cancer, wherein the non-small cell lung cancer is cisplatin-moderately resistant non-small cell lung cancer or cisplatin-highly resistant non-small cell lung cancer.

[0016] Beneficial effects:

[0017] The present invention discovers that the combination of N1-methyladenosine and cisplatin can produce obvious synergistic inhibitory effects on cisplatin-sensitive non-small cell lung cancer, cisplatin moderately resistant non-small cell lung cancer, and cisplatin highly resistant non-small cell lung cancer; the combination of N6-methyladenosine and cisplatin can produce obvious synergistic inhibitory effects on cisplatin moderately resistant non-small cell lung cancer and cisplatin highly resistant non-small cell lung cancer. Therefore, the composition of N1-methyladenosine or N6-methyladenosine and cisplatin has the prospect of being developed into a drug for treating non-small cell lung cancer. Detailed implementation manners

[0018] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention thereby.

[0019] I. Experimental materials and reagents

[0020] Cells: Human non-small cell lung cancer cell line A549 and cisplatin-resistant cell line A549 / DDP were purchased from the National Experimental Cell Resource Sharing Platform (Shanghai). Among them, A549 / DDP is derived from its parental A549 cell line, and A549 cells were screened and cultured by the method of increasing drug concentration combined with high-dose shock. Before the experiment, its resistance index (RI) was measured to ensure its drug resistance.

[0021] Drugs: Cisplatin (Cisplatin, DDP, CAS No.: 15663-27-1), N6-methyladenosine (6-Methyladenosine, m 6 A, CAS No.: 1867-73-8) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and N1-methyladenosine (1-Methyladenosine, m 1 A, CAS No.: 15763-06-1) was purchased from Beijing J&K Scientific Ltd.

[0022] Reagents: RPMI-1640 medium (Gibco, USA); fetal bovine serum FBS (Gibco, USA); penicillin-streptomycin (Hyclone, USA); 0.5% trypsin-EDTA solution (Boster, Wuhan Boster Biological Engineering Co., Ltd.); PBS (Boster, Wuhan Boster Biological Engineering Co., Ltd.); cell culture grade DMSO (Sigma-Aldrich, USA); Cell Counting Kit 8 (CCK-8) (Glpbio, USA).

[0023] Consumables: 10 cm cell culture dish (SORFA, China); 15, 50 mL centrifuge tubes (JET BIOFIL, China); cell cryopreservation tubes (SORFA, China); 96-well cell culture plates (SORFA, China).

[0024] II. Experimental Methods

[0025] 1. Establishment of drug-resistant cell models

[0026] Moderate drug-resistant cell model: The concentration-increasing method was adopted. According to the half-maximal inhibitory concentration (IC 50 ) of cisplatin on A549 cells, 0.1 μg / mL cisplatin was selected as the starting concentration to induce the cells. After the A549 cells were passaged and cultured until they adhered to the wall, the culture medium was replaced with the medium containing 0.1 μg / mL cisplatin. When the cell confluence reached 80%-90%, the cells were passaged. The cells were passaged at this concentration for 1-2 weeks until the cells could grow stably. Thereafter, the concentration was gradually increased in a gradient of 0.05 μg / mL until the cells could grow and be passaged stably at a concentration of 2.0 μg / mL. The total induction time was 12 months. The induced cell line was named A549 / DDP, and its RI was measured to be 5.4, belonging to moderate drug-resistant cells.

[0027] Highly drug-resistant cell model: The A549 / DDP cells induced for 12 months above were induced by a large dose of 5 μg / mL cisplatin for 72 h, and then cultured in RPMI-1640 medium without drugs for 2 weeks. The RI of the cells was measured to be 10.4, belonging to highly drug-resistant cells.

[0028] 2. Inhibitory effect of m 1 A combined with DDP on non-small cell lung cancer and its cisplatin-resistant cells in vitro

[0029] When the A549 and A549 / DDP cells reached the logarithmic growth phase, they were digested and centrifuged, resuspended in complete medium, and inoculated into 96-well plates at a density of 5×10 3 cells / well, with 100 μL of cell suspension in each well. For A549 sensitive cells, five concentrations of DDP (1.25, 2.5, 5, 10, 20 μM) and two concentrations of m 1 A (2, 4 mM) were set; for A549 / DDP moderately drug-resistant cells, five concentrations of DDP (2.5, 5, 10, 20, 40 μM) and three concentrations of m 1 A (1, 2, 4 mM) were set, and for A549 / DDP highly drug-resistant cells; five concentrations of DDP (5, 10, 20, 40, 80 μM) and five concentrations of m 1 A (0.25, 0.5, 1, 2, 4 mM) were set. In the experiment of m 1 A combined with DDP, a blank group, a control group, an m 1 A single-use group, a DDP single-use group, and groups of different concentrations of DDP combined with different concentrations of m 1 A were set, with 3 replicates in each group, to investigate m 1Inhibitory effect of the combination of A and DDP on the survival rate of A549 sensitive cells and highly cisplatin-resistant A549 / DDP cells. After culturing the cells overnight in an incubator at 37°C with 5% CO2, the blank group and the control group were replaced with fresh complete medium, and the drug administration group was replaced with fresh complete medium containing different concentrations of each drug. After the cells grew in the incubator for 48 h, 10 μL of CCK-8 reagent was added to each well. After mixing, the mixture was placed in the incubator for 2 h, and then the absorbance (OD) value of each well was measured using a multifunctional microplate reader at a detection wavelength of 450 nm. At the same time, blank wells (without cells, blank medium) and control wells (with cells, without drug administration) were set. The cell inhibition rate was calculated based on the average value of three wells in each drug administration concentration group. Cell inhibition rate formula: Cell inhibition rate (%) = [1 - (OD of drug-treated cells - OD of blank) / (OD of control cells - OD of blank)] × 100%.

[0030] 3. m 6 Inhibitory effect of the combination of A and DDP on cisplatin-resistant cells of non-small cell lung cancer in vitro

[0031] When the A549 / DDP cells reached the logarithmic growth phase, they were digested and centrifuged, resuspended in complete medium, and inoculated into 96-well plates at a density of 5×10 3 cells / well, with 100 μL of cell suspension in each well. For moderately cisplatin-resistant A549 / DDP cells, five concentrations of DDP (2.5, 5, 10, 20, 40 μM) and three concentrations of m 6 A (0.5, 1, 2 mM) were set; for highly cisplatin-resistant A549 / DDP cells, five concentrations of DDP (5, 10, 20, 40, 80 μM) and three concentrations of m 6 A (0.5, 1, 2 mM) were set. In the experiment of the combination of m 6 A and DDP, a blank group, a control group, a group with m 6 A alone, a group with DDP alone, and groups with different concentrations of DDP combined with different concentrations of m 6 A were set, with 3 replicate wells in each group, to investigate the inhibitory effect of the combination of m 6 A and DDP on the survival rate of highly cisplatin-resistant A549 / DDP cells (the subsequent experimental method was the same as that described under "2. Inhibitory effect of the combination of m 1 A and DDP on the survival rate of non-small cell lung cancer and its cisplatin-resistant cells in vitro"). Since m 6 A had no significant effect on the survival rate of A549 sensitive cells, the effect of the combination of m 6 A and DDP on the survival rate of A549 sensitive cells was not investigated.

[0032] 4. Data processing

[0033] The experimental data were processed using Graphpad Prism 7.0 software, and the calculated results of cell inhibition rate were expressed as Mean±SD.

[0034] 5. Evaluation indicators for drug synergistic effect

[0035] According to the inhibition rate Jin Zhengjun Q value to judge the effect of the combination of N1-methyladenosine or N6-methyladenosine and DDP: Q = E a+b / (E a +E b -E a ×E b ), E a and E b are the inhibition rates of the two drugs used alone respectively, and E a+b is the inhibition rate of the combined drug use; the numerator in the formula represents the "measured combined effect", and the denominator is the "expected combined effect"; Q < 0.85 is the antagonistic effect, 0.85 ≤ Q < 1.15 is the additive effect, and Q ≥ 1.15 is the synergistic effect.

[0036] III. Experimental results

[0037] 1. Inhibitory effect of the combination of m 1 A and DDP on non-small cell lung cancer and its cisplatin-resistant cells

[0038] The inhibition rates and Q value calculation results of different concentrations of DDP combined with different concentrations of m 1 A on A549 sensitive cells, A549 / DDP moderately and highly resistant cells are shown in Table 1, Table 2 and Table 3 respectively. The results show that 2 mM m 1 A combined with DDP (10 - 20 μM), 4 mM m 1 A combined with various concentrations of DDP exerted a synergistic inhibitory effect on the proliferation of A549 sensitive cells (Table 1); various concentrations of m 1 A combined with DDP could synergistically inhibit the proliferation of A549 / DDP moderately resistant cells (Table 2); after various concentrations of m 1 A combined with DDP, it exerted a synergistic or additive inhibitory effect on the proliferation of A549 / DDP highly resistant cells, and only 40 μM DDP combined with various concentrations of m 1 A could produce a synergistic inhibitory effect (Table 3).

[0039] Table 1 Inhibition rate and Jin Zhengjun Q value of different concentrations of DDP combined with m 1 A on A549 sensitive cells

[0040]

[0041]

[0042] Table 2 Inhibition rate of different concentrations of DDP combined with m 1 A on A549 / DDP moderately drug-resistant cells and Jin Zhengjun Q value

[0043]

[0044] Table 3 Inhibition rate of different concentrations of DDP combined with m 1 A on A549 / DDP highly drug-resistant cells and Jin Zhengjun Q value

[0045]

[0046] 2. Inhibitory effect of m 6 A combined with DDP on cisplatin-resistant cells of non-small cell lung cancer in vitro

[0047] Inhibitory rates and Q value calculation results of different concentrations of DDP combined with different concentrations of m 6 A on A549 / DDP moderately and highly drug-resistant cells are shown in Table 4 and Table 5 respectively. The results show that each concentration of m 6 A combined with DDP exerts a synergistic effect on inhibiting the proliferation of A549 / DDP moderately drug-resistant cells (Table 4); only 40 μM DDP combined with each concentration of m 6 A exerts a synergistic effect on inhibiting the proliferation of A549 / DDP highly drug-resistant cells. The low-concentration combination of the two (5 μM DDP + 0.5 mM m 6 A) produces an antagonistic effect, and the remaining concentration combinations are all additive effects (Table 5).

[0048] Table 4 Inhibition rate of different concentrations of DDP combined with m 6 A on A549 / DDP moderately drug-resistant cells and Jin Zhengjun Q value

[0049]

[0050] Table 5 Inhibition rate of different concentrations of DDP combined with m 6 A on A549 / DDP highly drug-resistant cells and Jin Zhengjun Q value

[0051] The above results show that m 1 A combined with DDP can produce obvious synergistic inhibitory effects on cisplatin-sensitive non-small cell lung cancer, cisplatin-moderately drug-resistant non-small cell lung cancer, and cisplatin-highly drug-resistant non-small cell lung cancer; m 6 A combined with DDP can produce obvious synergistic inhibitory effects on cisplatin-moderately drug-resistant non-small cell lung cancer and cisplatin-highly drug-resistant non-small cell lung cancer. Therefore, m 1 A or m 6The composition of A and DDP has the prospect of being developed into a drug for treating non-small cell lung cancer.

[0052] The function of the above embodiments is to specifically introduce the substantial content of the present invention. However, those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Use of a composition for the preparation of a medicament for treating non-small cell lung cancer, characterized in that: The non-small cell lung cancer is cisplatin-sensitive non-small cell lung cancer, and the composition consists of N1-methyladenosine and cisplatin, and the molar ratio of the two is 2000:10 to 20 or 4000:1.

25.

2. Use of a composition for the preparation of a drug for treating non-small cell lung cancer, characterized in that: The non-small cell lung cancer is cisplatin-moderately resistant non-small cell lung cancer, and the composition consists of N1-methyladenosine and cisplatin, and the molar ratio of the two is 1000 to 4000:2.5 to 40.

3. Use of a composition for the preparation of a drug for treating non-small cell lung cancer, characterized in that: The non-small cell lung cancer is cisplatin-highly resistant non-small cell lung cancer, and the composition consists of N1-methyladenosine and cisplatin, and the molar ratio of the two is 3.125 to 25:

1.

4. Use of a composition for the preparation of a medicament for treating non-small cell lung cancer, characterized in that: The non-small cell lung cancer is cisplatin-moderately resistant non-small cell lung cancer, and the composition consists of N6-methyladenosine and cisplatin, and the molar ratio of the two is 500 to 2000:2.5 to 40.

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