Application of pitavastatin in the preparation of drugs for reversing tumor multidrug resistance

Abstract

The present invention discloses the application of pitavastatin in the preparation of a drug for reversing tumor multidrug resistance. The present invention finds that pitavastatin can significantly enhance the sensitivity of oxaliplatin-resistant human colorectal cancer cells to multiple tumor chemotherapy drugs (including oxaliplatin, irinotecan and regorafenib), and reverse the multidrug resistance of oxaliplatin-resistant human colorectal cancer. The present invention also finds that pitavastatin can significantly enhance the sensitivity of cisplatin-resistant human non-small cell lung cancer cells to multiple tumor chemotherapy drugs (including cisplatin, oxaliplatin and paclitaxel), and reverse the multidrug resistance of cisplatin-resistant human non-small cell lung cancer. Therefore, pitavastatin has the application prospect of being developed into a drug for reversing tumor multidrug resistance.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of pitavastatin in preparing a drug for reversing tumor multidrug resistance. Background Art

[0002] Chemotherapy and targeted therapy are important means of treating tumors in clinic, but tumor cell resistance and multidrug resistance (MDR) seriously limit their efficacy, and there is no effective intervention plan. Colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) are common malignant tumors in clinic. At present, the treatment of CRC is still mainly oxaliplatin (OXP) or irinotecan (CPT-11) supplemented with other targeted drugs. During the progression and treatment of cancer, other chemotherapy drugs or targeted drugs are often used for patients with OXP resistance. However, after CRC patients develop resistance, they usually also develop resistance to other chemotherapy drugs or targeted drugs, leading to treatment failure. NSCLC currently still uses chemotherapy based on platinum drugs followed by targeted drug adjuvant therapy. However, after developing resistance to platinum drugs such as cis-platinum (DDP) and carboplatin, multidrug resistance also occurs, which seriously limits the efficacy of chemotherapy drugs. The clinical MDR phenomenon has led to the failure of chemotherapy for many tumors and reduced survival rates. Therefore, how to reverse tumor MDR has become the main difficulty in current clinical tumor treatment.

[0003] Pitavastatin is a drug suitable for treating hyperlipidemia and familial hypercholesterolemia, with high safety and good efficacy. The inventors of the present application disclosed in previous studies that pitavastatin has a relatively significant inhibitory effect on HCT116 / SW620 colorectal cancer cells (CN114886894A). Summary of the invention

[0004] The first purpose of the present invention is to provide the use of pitavastatin in the preparation of a drug for reducing the multidrug resistance of platinum chemotherapy drug-resistant tumors, the second purpose is to provide the use of pitavastatin in the preparation of a drug for enhancing the sensitivity of platinum chemotherapy drug-resistant tumors to chemotherapy drugs, the third purpose is to provide the use of a composition of pitavastatin and regorafenib, oxaliplatin or irinotecan in the preparation of a drug for treating human colorectal cancer resistant to oxaliplatin, and the fourth purpose is to provide the use of a composition of pitavastatin and paclitaxel, cisplatin or oxaliplatin in the preparation of a drug for treating human non-small cell lung cancer resistant to cisplatin.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] The use of pitavastatin in the preparation of a drug for reducing the multidrug resistance of platinum-based chemotherapy drug-resistant tumors; wherein the multidrug resistance refers to the resistance of tumor patients to two or more tumor chemotherapy drugs.

[0007] Preferably, the platinum-based chemotherapy drug-resistant tumor is human colorectal cancer that is resistant to oxaliplatin.

[0008] More preferably, the two or more tumor chemotherapy drugs include oxaliplatin, irinotecan and regorafenib.

[0009] Preferably, the platinum-based chemotherapy drug-resistant tumor is human non-small cell lung cancer resistant to cisplatin.

[0010] More preferably, the two or more tumor chemotherapy drugs include cisplatin, oxaliplatin and paclitaxel.

[0011] Application of pitavastatin in the preparation of drugs for enhancing the sensitivity of platinum-based chemotherapy-resistant tumors to chemotherapy drugs.

[0012] Preferably, the platinum-based chemotherapy drug-resistant tumor is human colorectal cancer that is resistant to oxaliplatin, and the chemotherapy drugs include oxaliplatin, irinotecan and regorafenib.

[0013] Preferably, the platinum-based chemotherapy drug-resistant tumor is human non-small cell lung cancer resistant to cisplatin, and the chemotherapy drugs include cisplatin, oxaliplatin and paclitaxel.

[0014] Use of a combination of pitavastatin, regorafenib, oxaliplatin or irinotecan in preparing a drug for treating human colorectal cancer resistant to oxaliplatin.

[0015] Application of a combination of pitavastatin, paclitaxel, cisplatin or oxaliplatin in preparing a medicine for treating human non-small cell lung cancer resistant to cisplatin.

[0016] Beneficial effects:

[0017] The present invention finds that pitavastatin can significantly enhance the sensitivity of oxaliplatin-resistant human colorectal cancer cells to multiple tumor chemotherapy drugs (including oxaliplatin, irinotecan and regorafenib), and reverse the multidrug resistance of oxaliplatin-resistant human colorectal cancer. The present invention also finds that pitavastatin can significantly enhance the sensitivity of cisplatin-resistant human non-small cell lung cancer cells to multiple tumor chemotherapy drugs (including cisplatin, oxaliplatin and paclitaxel), and reverse the multidrug resistance of cisplatin-resistant human non-small cell lung cancer. Therefore, pitavastatin has the application prospect of being developed into a drug for reversing tumor multidrug resistance. DETAILED DESCRIPTION

[0018] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0019] 1. Experimental Materials

[0020] 1. Cell lines

[0021] The cisplatin-resistant cell line A549 / DDP of human non-small cell lung cancer was purchased from the Shanghai National Laboratory Cell Resource Sharing Platform and identified by Shanghai Yihe Biotechnology Co., Ltd.; culture conditions: RPMI-1640 basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, cultured in 5% CO2, 37°C.

[0022] The oxaliplatin-resistant human colorectal cancer cell line HCT116 / L was purchased from Shanghai Meixuan Biotechnology Co., Ltd. and identified by Shanghai Yihe Biotechnology Co., Ltd.; culture conditions: RPMI-1640 basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, cultured in 5% CO2, 37°C.

[0023] 2. Reagents and consumables

[0024] Standards: Irinotecan (CPT-11, purity ≥ 99%, CAS: 97682-44-5), oxaliplatin (OXP, purity ≥ 99%, CAS: 61825-94-3), and paclitaxel (PTX, purity ≥ 99%, CAS: 33069-62-4) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Cisplatin (DDP, purity ≥ 99%, CAS: 15663-27-1) was purchased from Sigma-Aldrich, USA. Regorafenib (Reg, purity ≥ 99%, CAS: 755037-03-7) was purchased from Shanghai MCE Biotechnology Co., Ltd., pitavastatin (Ptv, purity > 99%, CAS: 147526-32-7), pravastatin (Pra, purity > 96%, CAS: 81093-37-0), simvastatin (Stv, purity ≥ 97%, CAS: 79902-63-9) and lovastatin (Ltv, purity ≥ 98%, CAS: 75330-75-5) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0025] Reagents: RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from Gibco, USA; 0.25% EDTA trypsin solution and PBS solution were purchased from Wuhan Boster Biotechnology Co., Ltd.; penicillin-streptomycin was purchased from HyClone, USA; dimethyl sulfoxide (DMSO) was purchased from Beijing Solebow Technology Co., Ltd.; Cell Counting Kit 8 (CCK-8) cell proliferation and cytotoxicity detection kit was purchased from Nanjing Shengxing Biotechnology Co., Ltd.

[0026] Consumables: 10cm cell culture dishes, cell freezing tubes, multi-well cell culture plates, 15 and 50mL centrifuge tubes; cell counting chamber; 0.22μm sterile filter membrane.

[0027] 3. Instruments and equipment

[0028] Pipette gun (Eppendorf, Germany); biological safety cabinet (Thermo, USA); cell culture incubator (Thermo, USA); biological inverted microscope (Jiangnan Yongxin Optical Co., Ltd., Nanjing, China); cell counter (Ruiyu Biotechnology Co., Ltd., Shanghai, China); 1 / 100,000 electronic analytical balance (Sartorius, Germany); low-speed desktop centrifuge (Anting Scientific Instrument Factory, Shanghai, China); electric blast drying oven (Boxun Industrial Co., Ltd. Medical Equipment Factory, Shanghai, China); electric constant temperature water bath (Yiheng Scientific Instrument Co., Ltd., Shanghai, China); microplate constant temperature oscillator (Aosheng Instrument Co., Ltd., Hangzhou, China); vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen, China); multifunctional microplate reader (Tecan Infinite M200 Pro, Switzerland); KH-250DB CNC ultrasonic cleaner (Hechuang Ultrasonic Instrument, Kunshan, China).

[0029] 2. Experimental Methods

[0030] 1. Solution preparation

[0031] Preparation of DDP mother solution: Accurately weigh an appropriate amount of DDP, add PBS buffer, shake and vortex in a 55°C water bath to dissolve, and prepare a mother solution with a concentration of 1.0 mg / mL. Filter with a 0.22μm sterile filter membrane, transfer to a sterile centrifuge tube, and store at 4°C in the dark. Before use, add complete culture medium to the DDP mother solution in proportion to dilute to the target concentration.

[0032] Preparation of OXP mother solution: Accurately weigh an appropriate amount of OXP, add PBS buffer, shake and vortex in a 55°C water bath to dissolve, and prepare a mother solution with a concentration of 1.0 mg / mL. Filter with a 0.22μm sterile filter membrane, transfer to a sterile centrifuge tube, and store at 4°C in the dark. Before use, add complete culture medium to the OXP mother solution in proportion to dilute to the target concentration.

[0033] Preparation of CPT-11 mother solution: Accurately weigh an appropriate amount of CPT-11, add DMSO buffer, and prepare a mother solution with a concentration of 20mM. Filter with a 0.22μm sterile filter membrane, transfer to a sterile centrifuge tube, and store at 4℃ in the dark. Before use, add complete culture medium to the CPT-11 mother solution in proportion to dilute to the target concentration.

[0034] Preparation of Reg mother solution: Accurately weigh an appropriate amount of Reg, add DMSO buffer to prepare a mother solution with a concentration of 10mM, filter with a 0.22μm sterile filter membrane, transfer to a sterile centrifuge tube, and store at 4℃ in the dark. Before use, add complete culture medium to the Reg mother solution in proportion to dilute to the target concentration.

[0035] Preparation of PTX mother solution: Accurately weigh an appropriate amount of PTX, add DMSO buffer, and prepare a mother solution with a concentration of 10 mg / mL. Filter with a 0.22 μm sterile filter membrane, transfer to a sterile centrifuge tube, and store at 4°C in the dark. Before use, add complete culture medium to the PTX mother solution in proportion to dilute to the target concentration.

[0036] Preparation of complete culture medium: Pipette an appropriate amount of culture medium, add 1 / 9 volume of FBS and 1% penicillin-streptomycin, shake well, and prepare a complete culture medium containing 10% FBS. Refrigerate at 4°C and use up within one week.

[0037] Preparation of cell freezing solution: Prepare the solution before use by mixing FBS:DMSO=9:1 to prepare a suitable volume of cell freezing solution. Keep the solution away from light and store at 4°C.

[0038] 2. Cell culture

[0039] (1) Cell recovery

[0040] Heat the water bath to 37°C in advance, take out the cells frozen in the liquid nitrogen tank, quickly put the cryotube into the water bath to thaw, and shake it continuously; when the liquid in the tube is completely melted, wipe the liquid outside the tube, blow it evenly with a pipette in the clean bench, aspirate the cell suspension, transfer it to a centrifuge tube containing 10 times the volume of cell suspension culture medium, centrifuge for 5 minutes at 1000 rpm, discard the supernatant, add 2 mL of complete culture medium preheated to 37°C, blow it evenly and transfer it to the culture bottle, mark it and culture it in an incubator with 5% CO2 and 37°C.

[0041] (2) Cell passaging

[0042] After HCT116 / L and A549 / DDP cells are revived, they are cultured for about 2 days. The cells are taken out of the incubator and observed under a microscope. If the degree of cell aggregation reaches 80%-90%, they are subcultured. Place the culture medium, trypsin, and PBS in a water bath and preheat to 37°C; discard the old culture medium, wash twice with 3mL PBS, add 1mL trypsin to digest and continue to observe under a microscope. When the cell gaps increase, the cells retract the protruding tentacles and become round, quickly add 2mL of culture medium to terminate digestion. Puff the cell suspension to make it uniform, and divide it into bottles at a ratio of 1 to 4. According to the cell growth status, change the medium every 48 hours and subculture 2-3 times a week.

[0043] (3) Cell cryopreservation

[0044] After the cells are digested and centrifuged, the supernatant is discarded, and the cells are resuspended in 1 mL of cell freezing solution. After being pipetted and mixed, the cells are transferred to a 2.5 mL cryotube. The cryotube is placed in a cryobox, stored in a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for long-term storage the next day.

[0045] 3. CCK-8 assay to detect Ptv reversal of drug resistance and multidrug resistance in HCT116 / L

[0046] (1) The OXP, CPT-11 and Reg stock solutions were diluted with culture medium to different concentrations, and HCT116 / L cells were given culture medium containing OXP at concentrations of 1.56, 3.13, 6.25, 12.50, 25.00, and 50.00 μg / mL, or culture medium containing CPT-11 at concentrations of 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 μM, or culture medium containing Reg at concentrations of 0.63, 1.25, 2.50, and 5.0 μM to determine the IC values ​​of various anti-tumor drugs against HCT116 / L cells. 50 value.

[0047] The above different gradient concentrations of OXP, CPT-11 and Reg were combined with 2.5 μM Ptv for IC 50 Value determination.

[0048] The inhibitory effect of Ptv alone on HCT116 / L was determined.

[0049] (2) Prepare cell suspension and count cells. Dilute the cell suspension with culture medium according to the counting results to meet the standards of CCK-8 experiment (cell density should be 5×10 3 / well), inoculate the cell suspension in a 96-well plate (100 μL / well), take out the 96-well plate after the cells adhere to the wall for 24 hours, discard the old culture medium, add 100 μL of culture medium containing the above concentration of drugs to each well, and continue to culture in the incubator for 48 hours. At the same time, set up blank wells (no cell suspension, only culture medium) and control wells (with cells but without drug culture medium).

[0050] (3) After 48 h, 10 μL of CCK-8 reagent was added to each well, mixed and incubated for another 2 h. The absorbance (OD) value of each well was measured using an enzyme-linked immunosorbent assay (ELISA) at a detection wavelength of 450 nm.

[0051] (4) The cell inhibition rate was calculated based on the average value of three wells in each dosing concentration group.

[0052] Cell survival rate (%) = (addition cell OD - blank OD) / (control OD - blank OD) × 100

[0053] GraphPad Prism 8.0 software was used to draw the survival curves of the two cell lines at different drug concentrations, and the half inhibition rate IC was calculated. 50 and reversal multiples.

[0054] In addition, as a comparison, the effects of other statins (lovastatin Ltv, pravastatin Pra, simvastatin Stv) on reversing HCT116 / L resistance were determined. The determination concentration and method were the same as Ptv. HCT116 / L cells are oxaliplatin-resistant cell lines, so OXP was selected as the anti-tumor drug.

[0055] 4. CCK-8 assay to detect Ptv reversing drug resistance and multidrug resistance of A549 / DDP

[0056] The DDP, OXP and PTX stock solutions were diluted to different concentrations with culture media, respectively. A549 / DDP cells were given culture media containing DDP at concentrations of 5.00, 10.00, 20.00, 40.00, 60.00, and 80.00 μM, or culture media containing OXP at concentrations of 3.13, 6.25, 12.50, 25.00, 50.00, and 100.00 μg / mL, or culture media containing PTX at concentrations of 0.15, 0.31, 0.63, 1.25, and 2.50 μg / mL to determine the IC values ​​of various antitumor drugs against A549 / DDP. 50 value.

[0057] The above-mentioned different gradient concentrations of DDP, OXP and PTX were combined with 2.5 μM Ptv for IC 50 Determination.

[0058] The inhibitory effect of Ptv alone on A549 / DDP was determined.

[0059] The other steps are the same as “3. CCK-8 method to detect Ptv's reversal of drug resistance and multidrug resistance of HCT116 / L”.

[0060] 3. Experimental Results

[0061] 1. Evaluation of the effect of pitavastatin in reversing drug resistance and multidrug resistance in HCT116 / L cells

[0062] We determined the inhibitory effect of Ptv alone on HCT116 / L cells and the IC values ​​of the anticancer drugs OXP, CPT-11, and Reg on HCT116 / L cells in the presence or absence of Ptv. 50 The results show that:

[0063] (1) Ptv alone had no inhibitory effect on HCT116 / L cells. Taking the cell survival rate of the control group as 100%, the cell survival rate of the 2.5 μM Ptv alone administration group was (93.48±7.44)%.

[0064] (2) IC values ​​of the inhibitory effects of antitumor drugs OXP, CPT-11, and Reg on HCT116 / L cells in the absence of Ptv 50 The values ​​were 17.46 μg / mL, 17.47 μM, and 4.13 μM, respectively. The IC values ​​of Ptv against HCT116 / L cells in the presence of 2.5 μM were 50 The values ​​were significantly reduced to 1.61μg / mL, 6.37μM, and 0.64μM, respectively, with reversal factors as high as 10.84, 2.74, and 6.45 times. A more intuitive comparison is shown in Table 1.

[0065] Table 1 Evaluation of the effect of pitavastatin in reversing drug resistance and multidrug resistance of HCT116 / L cells

[0066]

[0067] The experimental results showed that pitavastatin can significantly enhance the sensitivity of oxaliplatin-resistant human colorectal cancer cells to a variety of tumor chemotherapy drugs (including oxaliplatin, irinotecan and regorafenib), and reverse the multidrug resistance of oxaliplatin-resistant human colorectal cancer.

[0068] 2. Evaluation of the effect of pitavastatin in reversing drug resistance and multidrug resistance of A549 / DDP cells

[0069] We determined the inhibitory effect of Ptv alone on A549 / DDP cells and the IC values ​​of the inhibitory effects of antitumor drugs DDP, OXP, and PTX on A549 / DDP cells in the presence or absence of Ptv. 50 The results show that:

[0070] (1) Ptv alone had no inhibitory effect on A549 / DDP cells. Taking the cell survival rate of the control group as 100%, the cell survival rate of the group treated with 15 μM Ptv alone was still as high as (95.64±8.25)%.

[0071] (2) IC values ​​of the inhibitory effects of antitumor drugs DDP, OXP, and PTX on A549 / DDP cells in the absence of Ptv 50 The values ​​were 8.53 μM, 24.18 μg / mL, and 1.16 μg / mL, respectively. The IC values ​​of Ptv against A549 / DDP cells in the presence of 2.5 μM Ptv were 50 The values ​​were significantly reduced to 3.54μM, 6.48μg / mL, and 0.30μg / mL, respectively, with reversal factors as high as 2.41, 3.73, and 3.87 times. A more intuitive comparison is shown in Table 2.

[0072] Table 2 Evaluation of the effect of pitavastatin in reversing drug resistance and multidrug resistance of A549 / DDP cells

[0073]

[0074] The experimental results showed that pitavastatin can significantly enhance the sensitivity of cisplatin-resistant human non-small cell lung cancer cells to a variety of tumor chemotherapy drugs (including cisplatin, oxaliplatin and paclitaxel), and reverse the multidrug resistance of cisplatin-resistant human non-small cell lung cancer.

[0075] 3. Evaluation of the effect of other statins on reversing drug resistance and multidrug resistance of HCT116 / L cells

[0076] For comparison, we tested the effects of other statins (lovastatin Ltv, pravastatin Pra, simvastatin Stv) on reversing the drug resistance of HCT116 / L. The test results are shown in Table 3.

[0077] Table 3 Evaluation of the effects of various statins in reversing drug resistance in HCT116 / L cells

[0078]

[0079] The results of this experiment showed that none of the other three statins tested had the resistance-reversing activity of pitavastatin.

[0080] The above experimental results show that pitavastatin can significantly enhance the sensitivity of oxaliplatin-resistant human colorectal cancer cells to a variety of tumor chemotherapy drugs (including oxaliplatin, irinotecan and regorafenib), and reverse the multidrug resistance of oxaliplatin-resistant human colorectal cancer; pitavastatin can significantly enhance the sensitivity of cisplatin-resistant human non-small cell lung cancer cells to a variety of tumor chemotherapy drugs (including cisplatin, oxaliplatin and paclitaxel), and reverse the multidrug resistance of cisplatin-resistant human non-small cell lung cancer. Therefore, pitavastatin has the application prospect of being developed into a drug that reverses tumor multidrug resistance.

[0081] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but 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 pitavastatin in the preparation of a drug for enhancing the sensitivity of platinum-based chemotherapy-resistant tumors to chemotherapy drugs; wherein: The platinum chemotherapy drug-resistant tumor is human colorectal cancer resistant to oxaliplatin, and the chemotherapy drug is oxaliplatin, irinotecan or regorafenib; or the platinum chemotherapy drug-resistant tumor is human non-small cell lung cancer resistant to cisplatin, and the chemotherapy drug is cisplatin, oxaliplatin or paclitaxel.

2. Use of a combination of pitavastatin and regorafenib, a combination of pitavastatin and oxaliplatin, or a combination of pitavastatin and irinotecan in the preparation of a drug for treating human colorectal cancer that is resistant to oxaliplatin.

3. Use of a combination of pitavastatin and paclitaxel, a combination of pitavastatin and cisplatin, or a combination of pitavastatin and oxaliplatin in the preparation of a drug for treating human non-small cell lung cancer resistant to cisplatin.

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