CDK4 / 6 inhibitor drug-resistant invasive ductal carcinoma MCF-7 cell strain, kit and application thereof

Abstract

The invention discloses a CDK4 / 6 inhibitor drug-resistant invasive ductal carcinoma MCF-7 cell strain, a kit and application of the CDK4 / 6 inhibitor drug-resistant invasive ductal carcinoma MCF-7 cell strain. The cell strain is a human lung cancer cell with the preservation number of CCTCC (China Center For Type Culture Collection) NO: C2024219, and is named as Abe-R-MCF-7-Pa. The Abe-R-MCF-7-Pa and the kit thereof have relatively high application values in screening drugs for treating the breast cancer drug resistance and in evaluating the drugs for treating the breast cancer drug resistance.

Description

Technical Field

[0001] The present invention relates to a cell line of a CDK4 / 6 inhibitor, a kit and applications thereof, and in particular to an invasive ductal carcinoma MCF-7 cell line resistant to a CDK4 / 6 inhibitor, a kit and applications thereof. Background Art

[0002] Breast cancer is the number one malignant tumor that threatens women's health. According to statistics, the incidence and mortality of women in my country have been increasing year by year since 2000. Breast cancer includes different histological types, among which the most common is invasive ductal carcinoma (IDC), accounting for about 82%. At this time, cancer cells infiltrate and widely invade surrounding tissues. In addition to chemotherapy and endocrine therapy, new targeted therapies are becoming more and more important for the treatment of breast cancer. At the molecular and cellular level, the corresponding therapeutic drugs are targeted at cancer sites, mainly around HER2-positive carcinogenic sites, among which CDK4 / 6 inhibitors play an important role. Cyclin-dependent kinase (CDK) 4 / 6 is a key regulator of the cell cycle. It forms a complex with cyclin D, phosphorylates Rb, releases E2F, and triggers the cell cycle from the pre-DNA synthesis phase (G1 phase) to the DNA replication phase (S1 phase), thereby promoting tumor growth. However, these tumors inevitably develop drug resistance. Therefore, understanding the drug resistance mechanism of invasive ductal carcinoma and developing combined therapies are crucial to improving treatment outcomes.

[0003] Palbociclib is the world's first approved CDK4 / 6 kinase inhibitor. It is an experimental, oral, targeted CDK4 / 6 inhibitor that can selectively inhibit cyclin-dependent kinases 4 and 6 (CDK4 / 6), restore cell cycle control, and block tumor cell proliferation. A phase III trial combining Palbociclib with letrozole showed that the risk of disease progression in the subjects was reduced by 44%, and the median progression-free survival of patients treated with the combination was increased by more than one year. Despite the good efficacy, most patients still develop drug resistance. Resistance to Palbociclib involves multiple targets upstream and downstream of the CDK4 / 6 to Rb pathway, overexpression of the upstream transcription factor E2F22 and loss of downstream Rb, and cross-treatment resistance will occur during hormone therapy.

[0004] Ribociclib (KISQALI, Novartis Pharmaceuticals Corp) is an oral, highly specific CDK4 / 6 inhibitor that can target and inhibit the D1 / CDK4 and D3 / CDK6 cell cycles. CDK4 / 6 inhibitors have become one of the most popular treatments for hormone receptor-positive (HR+) / human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer. Phase III clinical trial data of the CDK4 / 6 inhibitor Ribociclib showed that the drug can significantly benefit breast cancer patients. Clinical trials (MONALEESA-7) showed that in premenopausal ER+HER2- breast cancer patients, Ribociclib combined with endocrine therapy can significantly prolong the patient's progression-free survival compared with endocrine therapy alone.

[0005] Abemaciclib is a CDK4 / 6 inhibitor and the third CDK4 / 6 inhibitor approved worldwide.

[0006] Cyclin-dependent kinases are key enzymes in cell cycle regulation. Their function requires cyclins to provide domains necessary for enzyme activity. Inhibiting CDK activity is considered an important approach to tumor treatment. Selective CDK4 / 6 inhibitors have been used to treat HR+ / HER2- advanced breast cancer. These drugs block the growth of breast cancer cells by targeting enzymes that are important in cell division. Although these therapies often shrink tumors initially, approximately 90% of patients with metastatic cancer cells and 50% of patients with stage II and III breast cancer develop drug resistance. Studies have shown that even after endocrine and cell cycle (such as CDK4 / 6) inhibition therapy, resistant cells that persist will use estrogen signals to switch to alternative growth factor receptors and reconnect cell cycle pathways. This rewiring allows cancer cells to continue to grow in the presence of estrogen and cell cycle drug inhibitors. The mechanism of tumor resistance is not single, and researchers are urgently needed to fully master the relevant knowledge and use scientific and rigorous methods to explore the mysteries. Summary of the invention

[0007] The present invention aims to establish a cell line resistant to Abemaciclib, and the establishment of a cell line can provide a necessary research model for breast cancer resistance-related research, and plays an important role in screening chemotherapy drugs targeting tumor heterogeneity. The present invention provides an invasive ductal carcinoma MCF-7 cell line resistant to CDK4 / 6 inhibitors, which is a human breast cancer cell with a deposit number of CCTCC NO: C2024219, and is named Abe-R-MCF-7-Pa.

[0008] In addition, the present invention also provides the use of the CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line described above in evaluating drugs for treating drug resistance in breast cancer.

[0009] In some embodiments, the breast cancer resistance is invasive ductal carcinoma resistance.

[0010] The present invention also provides the use of the CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line in screening drugs for treating drug-resistant breast cancer.

[0011] In some embodiments, the breast cancer resistance is invasive ductal carcinoma resistance.

[0012] The present invention also provides a kit, comprising the CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line as described above.

[0013] The present invention also provides the use of the above-mentioned kit in evaluating drugs for treating drug resistance of breast cancer.

[0014] In some embodiments, the breast cancer resistance is invasive ductal carcinoma resistance.

[0015] The present invention also provides the use of the above-mentioned kit in screening drugs for treating drug resistance of breast cancer.

[0016] In some embodiments, the breast cancer resistance is invasive ductal carcinoma resistance.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the in vitro resistance index (RI) of the cell line Abe-R-MCF-7-Pa to Abemaciclib is 7.80. The calculation formula of RI is: RI = Relative IC50 筛选获得细胞株 / Relative IC50 亲本 . Relative IC50 亲本 Relative IC50 represents the relative half-maximal effect concentration of the parental cell line used for screening; 筛选获得细胞株 Relative IC50 represents the half-maximal effect concentration of the screened cell lines. When calculating the in vitro resistance index of Abe-R-MCF-7-Pa to Abemaciclib, Relative IC50 亲本 The relative IC50 of 0.05 μmol / L obtained by experiment (see Example 2) was taken into account. 筛选获得细胞株The 0.39 μmol / L obtained by the experiment (see Example 2) was introduced to obtain an RI of 7.80 for Abemaciclib. Abe-R-MCF-7-Pa and its kit have high application value in screening drugs for treating breast cancer resistance and in evaluating drugs for treating breast cancer resistance.

[0018] The present invention provides a drug-resistant cell model for the following related studies: better study of the drug resistance problem of CDK4 / 6 inhibitors, revealing the drug resistance mechanism of invasive ductal carcinoma cells, screening chemotherapy drugs that can reverse the drug resistance of invasive ductal carcinoma, and developing more effective treatment methods for invasive ductal carcinoma cells.

[0019] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the cell growth morphology diagram of MCF-7-Parental cell line and Abe-R-MCF-7-Pa cell line.

[0021] Figure 2 This is the cell proliferation inhibition curve of Abemaciclib in MCF-7-Parental cell line and Abe-R-MCF-7-Pa cell line. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0023] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0024] The human invasive ductal carcinoma MCF-7 cell line (ECACC-86012803) used in the examples of the present invention was purchased from the ECACC cell bank, and Abemaciclib was purchased from Aladdin.

[0025] Example 1: Construction of human infiltrating ductal carcinoma cell lines resistant to Abemaciclib

[0026] The screening method is as follows:

[0027] (1) Routine thawing of frozen human invasive ductal carcinoma MCF-7 cell line was performed by routine in vitro culture and passage in an incubator at 37° C. and 5% CO 2 to expand the cells using EMEM medium (i.e., the cell culture medium used in Examples 1 and 2) containing 10% fetal bovine serum, 1% non-essential amino acids, and 2 mmol / L glutamine.

[0028] (2) Take cells in the logarithmic growth phase, count them and prepare a cell suspension with a concentration of 1×10 4 Cells / mL were plated in 2 mL per well of a six-well plate. Cell attachment was observed on the second day, and the cell culture medium containing abemaciclib at a final concentration of 28 nmol / L was replaced. Every four days, the cells were passaged once to a six-well plate, and the cell culture medium containing the same concentration of abemaciclib was added on the second day.

[0029] (3) Repeat this process for five weeks, which is a drug screening process. The drug screening process is performed six times, and the screening lasts for 6 months. The concentrations are 28nmol / L, 56nmol / L, 112nmol / L, 224nmol / L, 448nmol / L, and 900nmol / L, respectively. Finally, a cell line resistant to Abemaciclib is obtained, named Abe-R-MCF-7-Pa, with a deposit number of CCTCC NO: C2024219, a deposit date of July 31, 2024, a deposit institution of China Center for Type Culture Collection (CCTCC), and a deposit address of Wuhan University, Wuhan, China.

[0030] from Figure 1 It can be seen that the drug-resistant strain Abe-R-MCF-7-Pa is morphologically similar to the parental cell line MCF-7-Parental.

[0031] Example 2: Determination of the growth inhibition rate of Abemaciclib on Abe-R-MCF-7-Pa resistant strains

[0032] (1) The cell line Abe-R-MCF-7-Pa obtained in Example 1 and its parent cell line MCF-7-Parental were respectively subjected to in vitro expansion culture, and cells in the logarithmic growth phase were used for plating, and the cells were stained with trypan blue and the living cells were counted, and the cell concentration was adjusted to 1000 cells per well. 90 μL / well of the cell suspension was added to the 96-well culture plate as shown in Table 1, but a cell culture medium without cells was added to the blank control; the 96-well culture plate was cultured overnight in a 37° C., 5% CO2 incubator.

[0033] (2) 10 μL of drug working solution of different concentrations was added to the above-mentioned cell culture plate to make the final concentration as shown in Table 1. There were 3 replicate wells in each group. 10 μL of DMSO-cell culture medium mixture was added to the solvent control and blank control. The final DMSO concentration was 0.25% by volume.

[0034] Table 1 Schematic diagram of cell plating and drug treatment in in vitro cell activity experiment

[0035]

[0036] Note: The unit of drug concentration is μmol / L.

[0037] (3) The 96-well culture plate was returned to the incubator for 144 h. Then, 50 μL (equal to half the volume of the cell culture medium in each well) of the Cell Titer-Glo working solution of the Promega CellTiter-Glo Luminescent Cell Activity Assay Kit (Promega-G7573) was added to each well, and the cell plate was wrapped with aluminum foil to avoid light.

[0038] (4) The 96-well culture plate was shaken on an orbital shaker for 2 min to induce cell lysis. The 96-well culture plate was placed at room temperature for 10 min to stabilize the luminescent signal, and the luminescent signal was detected on a 2104 EnVision plate reader.

[0039] (5) Calculate the inhibition rate (IR) of the test drug: IR (%) = (1 – (RLU 药物 –RLU 空白对照 ) / (RLU 溶媒对照 –RLU 空白对照 ))*100%. RLU represents luminescence value. The inhibition rate of different drug concentrations was calculated in Excel, and then the inhibition curve was plotted and related parameters were calculated using GraphPad Prism software, including the minimum inhibition rate, maximum inhibition rate and relative IC 50 The results are shown in Table 2 and Figure 2 .

[0040] Table 2 Abemaciclib inhibition of MCF-7 drug-resistant cell growth

[0041]

[0042] The cell line Abe-R-MCF-7-Pa screened in vitro using Abemaciclib was determined to be resistant to Abemaciclib in vitro, and its resistance index (RI) was 7.80. Therefore, the CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line Abe-R-MCF-7-Pa screened in vitro was resistant to Abemaciclib, and the resistance performance was stable.

[0043] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line, which is a human breast cancer cell with a deposit number of CCTCC NO: C2024219 and is named Abe-R-MCF-7-Pa.

2. Use of the CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line according to claim 1 in evaluating drugs for treating breast cancer resistance.

3. The use according to claim 2, characterized in that: The breast cancer drug resistance is invasive ductal carcinoma drug resistance.

4. Use of the CDK4 / 6 inhibitor-resistant invasive ductal carcinoma MCF-7 cell line according to claim 1 in screening drugs for treating drug resistance in breast cancer.

5. The use according to claim 4, characterized in that: The breast cancer drug resistance is invasive ductal carcinoma drug resistance.

6. A kit, characterized in that: The invention relates to an invasive ductal carcinoma MCF-7 cell line resistant to the CDK4 / 6 inhibitor according to claim 1.

7. Use of the kit according to claim 6 in evaluating drugs for treating drug resistance in breast cancer.

8. The use according to claim 7, characterized in that: The breast cancer drug resistance is invasive ductal carcinoma drug resistance.

9. Use of the kit according to claim 6 in screening drugs for treating drug resistance in breast cancer.

10. The use according to claim 9, characterized in that: The breast cancer drug resistance is invasive ductal carcinoma drug resistance.

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