Application of 3 beta-Hydroxylup-12-en-28 in medicine for treating paclitaxel drug-resistant lung cancer

Abstract

The invention belongs to the field of medicines, and provides an application of 3 beta-Hydroxylup-12-en-28 in a medicine for treating lung cancer with taxol drug resistance. The structural formula of the 3 beta-Hydroxylup-12-en-28 is as follows: # imgabs0 # 3 beta-Hydroxylup-12-en-28 can be used for reducing the cell activity of highly drug-resistant paclitaxel-resistant cells of the human non-small cell lung cancer and reducing the expression of drug-resistant related proteins P-gp, MRP1 and ABCG2 which are highly expressed by the paclitaxel-resistant cells of the human non-small cell lung cancer, so that the effect of resisting the drug resistance of the paclitaxel is exerted.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the application of 3β-Hydroxylup-12-en-28 as an anti-resistance agent in lung cancer. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] 3β-Hydroxylup-12-en-28 is a pentacyclic triterpene compound containing TPP+. It is a new derivative that introduces TPP+ into oleanolic acid. Through cell activity detection, it was found that 3β-Hydroxylup-12-en-28 has a high toxicity to tumor cells, especially paclitaxel-resistant human non-small cell lung cancer cells. At present, the research on 3β-Hydroxylup-12-en-28 is still in the initial stage, and there is no clinical trial and application of 3β-Hydroxylup-12-en-28.

[0004] Lung cancer remains the cancer with the highest cancer mortality rate worldwide, with non-small cell lung cancer (NSCLC) accounting for 90% of all cases. Because approximately 70-80% of these patients are not eligible for radical surgery due to advanced disease at the time of diagnosis, chemotherapy is currently the main treatment for NSCLC. However, the prognosis of patients with advanced NSCLC remains poor due to the low response rate and toxic side effects of chemotherapy. Paclitaxel is widely used as a standard first-line chemotherapy drug in clinical chemotherapy for NSCLC. Paclitaxel can be used as an early adjuvant drug for NSCLC and can improve the quality of life and life expectancy of patients with advanced NSCLC. However, paclitaxel resistance is a common problem during chemotherapy for NSCLC patients, which ultimately leads to tumor recurrence. Therefore, paclitaxel resistance is one of the biggest obstacles to successful treatment, which urgently requires the discovery and development of drugs targeting resistance.

[0005] In cancer chemotherapy, the increased efflux of chemotherapeutic drugs from target cells can impair the efficacy of the drugs, leading to a decrease in the intracellular drug concentration. ABC transporters are the largest family of transmembrane transporters in organisms. The typical structural feature of ABC transporters is a four-domain structure, consisting of two cytoplasmic nucleotide binding domains (NBDs) that bind and hydrolyze ATP and two transmembrane domains (TMDs) that recognize and transport substrates. Among them, P-glycoprotein (MDR1, P-gp), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) are the most important drug transporters and are associated with therapeutic resistance in human lung cancer. These proteins pump various endogenous or exogenous substrates out of cells to produce a detoxification effect. At the same time, this process also leads to a decrease in intracellular drug concentration and resistance. Overexpression of ABC transporters is one of the key biomarkers of drug resistance in human lung cancer. P-glycoprotein (MDR1, P-gp), encoded by subfamily B member 1 (ABCB1), is the most studied member of the ABC transporters and has been shown to confer drug resistance to various cancer cells. These transporters have broad substrate specificity, and they mediate resistance to a wide range of anticancer drugs. P-gp transports hydrophobic compounds, while ABCC1 and ABCG2 transport hydrophobic drugs and large anionic compounds. Overexpression of P-gp has been well established to confer resistance to paclitaxel, with levels significantly higher in human lung cancer A549 paclitaxel-resistant cells (A549 / Taxol) than in their parental cells A549. ABCC1 confers resistance to chemotherapeutic drugs in various cancer types, including acute myeloid and lymphocytic leukemia, non-small cell lung cancer, prostate cancer, breast cancer, and neuroblastoma. ABCG2 is a major survival factor for cancer stem cells, which exist in tumors as a unique chemoresistant population and contribute to tumor recurrence and metastasis. ABCG2 promotes the efflux of multiple anticancer drugs to protect cancer stem cells. High expression of ABCG2 has been detected in multiple cancer types, and three drug-resistant proteins are closely associated with paclitaxel-resistant lung cancer cells, which encourages extensive research on discovering drugs that inhibit the expression of ABC transporters.

[0006] The paper "Progress in Research on the Anti-tumor Mechanism of Oleanolic Acid and Its Derivatives" records that this type of substance has the effect of reversing multidrug resistance. However, due to the large number of oleanolic acid and its derivatives, the industry has not yet found suitable oleanolic acid derivatives as highly effective anti-resistance agents for paclitaxel-resistant lung cancer. Summary of the invention

[0007] In order to solve the above problems, the present invention provides the use of 3β-Hydroxylup-12-en-28 as an anti-resistance agent in lung cancer.

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

[0009] The first aspect of the present invention provides the use of 3β-Hydroxylup-12-en-28 in the preparation of a drug for treating paclitaxel-resistant lung cancer. The structural formula of the 3β-Hydroxylup-12-en-28 is as follows:

[0010]

[0011] First, the present invention uses 3β-Hydroxylup-12-en-28 to perform cell activity and proliferation detection on human non-small cell lung cancer cells (A549) and paclitaxel-resistant cells (A549 / Taxol) and human normal lung epithelial cells BEAS-2B, i.e., CCK8 experiment. The results show that 3β-Hydroxylup-12-en-28 has the strongest sensitivity to human non-small cell lung cancer paclitaxel-resistant cells A549 / Taxol, and the weakest cytotoxicity to human normal lung epithelial cells BEAS-2B. The results of CCK8 experiments and data analysis showed that after 3β-Hydroxylup-12-en-28 (0.5μmol / L, 1μmol / L, 1.5μmol / L, 2μmol / L, 2.5μmol / L) treated A549, A549 / Taxol, BEAS-2B cells for 48h, the half inhibitory concentration (IC50) of A549, A549 / Taxol, BEAS-2B cells was 1.14±0.23μmol / L, 0.98±0.12μmol / L, Further experiments showed that after 3β-Hydroxylup-12-en-28 (0.5μmol / L, 1μmol / L, 1.5μmol / L, 2μmol / L, 2.5μmol / L) was used to treat A549, A549 / Taxol, and BEAS-2B cells for 24h, 48h, and 72h, the proliferation inhibitory effect of 3β-Hydroxylup-12-en-28 on A549 and A549 / Taxol cells increased with time.

[0012] In some embodiments, 3β-Hydroxylup-12-en-28 can inhibit the proliferation of non-small cell lung cancer cells and non-small cell lung cancer paclitaxel-resistant cells.

[0013] In some embodiments, the inhibitory effect of 3β-Hydroxylup-12-en-28 on the proliferation of non-small cell lung cancer cells and paclitaxel-resistant non-small cell lung cancer cells increases with time, that is, 3β-Hydroxylup-12-en-28 inhibits the proliferation of human non-small cell lung cancer in a time-dose-dependent manner.

[0014] In some embodiments, the non-small cell lung cancer paclitaxel-resistant cells include: A549 / Taxol.

[0015] In some embodiments, the concentration of 3β-Hydroxylup-12-en-28 is 0.5 to 2.5 μM.

[0016] In some embodiments, the cells are treated with 3β-Hydroxylup-12-en-28 for 24 to 72 hours.

[0017] The second aspect of the present invention provides the use of 3β-Hydroxylup-12-en-28 in the preparation of a drug for inhibiting the expression of ABC transporters. The structural formula of 3β-Hydroxylup-12-en-28 is as follows:

[0018]

[0019] The expression of P-gp, MRP1 and ABCG2 proteins is closely related to the drug resistance state of tumor cells, and the expression of the three proteins is positively correlated with the drug resistance of cells. First, the present invention uses western blot experiment to test whether the three drug resistance proteins P-gp, MRP1 and ABCG2 are highly expressed in human non-small cell lung cancer paclitaxel-resistant cells. A549 cells and A549 / Taxol cells are plated in different six-well plate wells at the same density, and proteins are extracted after full growth. Protein immunoblotting is used for testing. The results show that the three drug resistance proteins are highly expressed in human non-small cell paclitaxel-resistant cells. Human non-small cell lung cancer paclitaxel-resistant cells are treated with different concentrations of 3β-Hydroxylup-12-en-28 (0.5μM, 1μM, 2μM) for 48h, and the expression of p-gp is detected by Western Blot method and cell immunofluorescence color method. The results show that 3β-Hydroxylup-12-en-28 treatment significantly reduces the expression of P-gp in a concentration-dependent manner.

[0020] In some embodiments, the ABC transporters include: P-gp, MRP1, ABCG2.

[0021] In some embodiments, the concentration of 3β-Hydroxylup-12-en-28 is 0.5 to 2.5 μM.

[0022] The third aspect of the present invention provides the use of 3β-Hydroxylup-12-en-28 in the preparation of a drug for resisting paclitaxel-resistant cells of human non-small cell lung cancer.

[0023] The present invention firstly uses CCK8 method to test whether the paclitaxel-resistant cells of human non-small cell lung cancer have reached a certain drug resistance multiple to carry out the experiment, and paclitaxel (0.03 μmol / L, 0.06 μmol / L, 0.12 After treating human non-small cell lung cancer A549 cells with paclitaxel (5μmol / L, 0.025μmol / L, 0.05μmol / L, 0.1μmol / L) for 48h and treating human non-small cell lung cancer paclitaxel-resistant cells with paclitaxel (1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L) for 48h, CCK8 was used to verify the paclitaxel resistance multiple. The results showed that the half inhibitory concentration (IC50) of paclitaxel on human non-small cell lung cancer cells was 0.03±0.01μmol / L, and the half inhibitory concentration (IC50) of paclitaxel on human non-small cell lung cancer paclitaxel-resistant cells was 3.16±0.65μmol / L, and the resistance multiple reached 1 05 times, indicating that human non-small cell lung cancer paclitaxel-resistant cells are highly resistant. Secondly, human non-small cell lung cancer and paclitaxel-resistant cells were treated with common clinical lung cancer chemotherapy drugs, that is, doxorubicin (ADR) (0.0625μmol / L, 0.125μmol / L, 0.25μmol / L, 0.512μmol / L) was used to treat human non-small cell lung cancer and paclitaxel-resistant cells for 48 hours, and CCK8 detection was used after 48 hours. The results showed that the half inhibitory concentration (IC50) of doxorubicin for human non-small cell lung cancer cells was 0.17±0.14μmol / L, and the half inhibitory concentration (IC50) of doxorubicin for human non-small cell lung cancer paclitaxel-resistant cells was 1.10±0.24μmol / L, and the resistance multiple was 6.5 times. Human non-small cell lung cancer and paclitaxel-resistant cells were treated with vincristine (VCR) (0.3125μmol / L, 0.0625μmol / L1.25μmol / L2.5μmol / L) for 48 hours and then CCK8 was used for detection. The results showed that the half inhibitory concentration (IC50) of vincristine on human non-small cell lung cancer cells was 0.29±0.04μmol / L, and the half inhibitory concentration (IC50) of vincristine on human non-small cell lung cancer paclitaxel-resistant cells was 2.41±0.07μmol / L, and the resistance multiple was 9.7 times. These results showed that human non-small cell lung cancer paclitaxel-resistant cells were highly resistant and also showed a certain resistance to other chemotherapy drugs, indicating the ability of 3β-Hydroxylup-12-en-28 to selectively resist the resistance of human non-small cell lung cancer paclitaxel-resistant cells.

[0024] In a fourth aspect, the present invention provides a drug for resisting paclitaxel-resistant cells of human non-small cell lung cancer, the active ingredients of which include: 3β-Hydroxylup-12-en-28;

[0025] The structural formula of 3β-Hydroxylup-12-en-28 is as follows:

[0026]

[0027] The effective amount of 3β-Hydroxylup-12-en-28 is 0.5 to 2.5 μM.

[0028] Beneficial effects of the present invention

[0029] (1) 3β-Hydroxylup-12-en-28 can reduce the cell viability of highly resistant human non-small cell lung cancer paclitaxel-resistant cells and reduce the expression of resistance-related proteins P-gp, MRP1, and ABCG2 that are highly expressed in human non-small cell lung cancer paclitaxel-resistant cells, thereby exerting an anti-paclitaxel resistance effect.

[0030] (2) Human non-small cell lung cancer cells resistant to paclitaxel are highly resistant and also resistant to common clinical lung cancer chemotherapy drugs. 3β-Hydroxylup-12-en-28 has high cytotoxicity to human non-small cell lung cancer cells resistant to paclitaxel, and can inhibit the proliferation of human non-small cell lung cancer cells and paclitaxel-resistant cells in a time-dose-dependent manner. 3β-Hydroxylup-12-en-28 has low cytotoxicity to human normal lung epithelial cells BEAS-2B. 3β-Hydroxylup-12-en-28 can inhibit the expression of resistance-related proteins P-gp, MRP1, and ABCG2 that are highly expressed in human non-small cell lung cancer cells resistant to paclitaxel, achieving the effect of resisting paclitaxel resistance in lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 It is the chemical structure of 3β-Hydroxylup-12-en-28 of Example 1 of the present invention.

[0033] Figure 2The effect of 3β-Hydroxylup-12-en-28 on the activity of A549, A549 / Taxol and BEAS-2B cells, (A) 3β-Hydroxylup-12-en-28 of Example 1 of the present invention inhibited the proliferation of A549, A549 / Taxol and BEAS-2B cells in a dose-dependent manner, and inhibited the proliferation of A549 and A549 / Taxol cells in a time-dependent manner, (B) CCK8 detected the cell viability of A549 and A549 / Taxol cells after 24 hours, 48 ​​hours and 72 hours of treatment with 3β-Hydroxylup-12-en-28 at different concentrations. Data are provided as the mean ± SD of three or more different experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001vs.control group.

[0034] Figure 3 The effect of 3β-Hydroxylup-12-en-28 on the expression of P-gp, MRP1, and ABCG2 in A549 / Taxol cells in Example 2 of the present invention, (A) Western blot analysis of P-gp, MRP1, and ABCG2 protein expression in A549 / Taxol cells and A549 cells, data are provided as the mean ± SD of three or more different experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. control group. (B) Western blot analysis of 3β-Hydroxylup-12-en-28 inhibiting the expression of P-gp, MRP1, and ABCG2 proteins in A549 / Taxol cells, data are provided as the mean ± SD of three or more different experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001vs.control group (C) Immunofluorescence assay to detect the effect of 3β-Hydroxylup-12-en-28 on the expression of P-gp, MRP1, and ABCG2 proteins. Green fluorescence shows P-gp, MRP1, and ABCG2, and blue shows DAPI (bar, 50 μm).

[0035] Figure 4The paclitaxel-resistant human non-small cell lung cancer cells of Example 3 of the present invention are highly resistant and resistant to common clinical lung cancer chemotherapy drugs. (A) Paclitaxel treatment of A549 and A549 / Taxol cells for 48 hours was used for detection by CCK8 experiment. (B) Doxorubicin treatment of A549 and A549 / Taxol cells for 48 hours was used for detection by CCK8 experiment. (C) Vincristine treatment of A549 and A549 / Taxol cells for 48 hours was used for detection by CCK8 experiment. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. control group. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0037] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0038] Example 1 Effect of 3β-Hydroxylup-12-en-28 on the activity of paclitaxel-resistant non-small cell lung cancer cells and normal lung epithelial cells

[0039] The experiment was divided into a blank group (control), a 3β-Hydroxylup-12-en-28 drug group (0.5μM, 1μM, 1.5μM, 2μM, 2.5μM), and human non-small cell lung cancer (A549) and paclitaxel-resistant cells (A549 / Taxol) and normal lung epithelial cells BEAS-2B were treated with drugs for 48 hours before cell activity and proliferation detection, namely CCK8 experiment. Figure 2 As shown in (A), the results showed that 3β-Hydroxylup-12-en-28 had the strongest sensitivity to paclitaxel-resistant human non-small cell lung cancer cells and the weakest cytotoxicity to normal lung epithelial cells BEAS-2B. The CCK8 experimental results and data analysis showed that after 48 hours of action of 3β-Hydroxylup-12-en-28, the half-inhibitory concentration (IC50) for A549, A549 / Taxol, and BEAS-2B cells was 1.14±0.23μmol / L, 0.98±0.12μmol / L, and 2.32±0.01μmol / L.

[0040] CCK8 was used to detect the cell viability of human non-small cell lung cancer cells and paclitaxel-resistant cells treated with 3β-Hydroxylup-12-en-2 at different concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 2.5) for 24 hours, 48 ​​hours and 72 hours, as shown in Figure 3. Figure 2 As shown in (B).

[0041] The results showed that the proliferation inhibitory effect of 3β-Hydroxylup-12-en-28 on A549 cells and A549 / Taxol cells increased in a time- and concentration-dependent manner.

[0042] Example 2 Effect of 3β-Hydroxylup-12-en-28 on the Expression of P-gp, MRP1 and ABCG2 Proteins in Paclitaxel-Resistant Human Non-Small Cell Lung Cancer Cells

[0043] The present invention studies the effect of 3β-Hydroxylup-12-en-28 on the expression of P-gp, MRP1 and ABCG2 in paclitaxel-resistant A549 / Taxol cells of human non-small cell lung cancer. The expression of P-gp, MRP1 and ABCG2 in A549 / Taxol cells and A549 cells was analyzed by Western blot. Figure 3 As shown in (A), the experimental group was divided into a blank group and a drug-treated group (0.5 μmol / L, 1 μmol / L, and 2 μmol / L). A549 / Taxol cells were treated with the drug for 48 h, and Western blot was used to analyze the effect of 3β-Hydroxylup-12-en-28 on the expression of P-gp, MRP1, and ABCG2 proteins in the cells. Figure 3 As shown in (B), the experimental group was divided into a blank group and a drug-treated group (0.5 μmol / L, 1 μmol / L, and 2 μmol / L). A549 / Taxol cells were treated with drugs for 48 h, and the effect of 3β-Hydroxylup-12-en-28 on P-gp expression was detected by immunofluorescence assay. Figure 3 As shown in (C).

[0044] It can be seen that 3β-Hydroxylup-12-en-28 can inhibit the expression of P-gp, MRP1, and ABCG2 proteins in a concentration-dependent manner.

[0045] Example 3 3β-Hydroxylup-12-en-28 against paclitaxel-resistant human non-small cell lung cancer cells

[0046] After human non-small cell lung cancer A549 cells were treated with paclitaxel (0.03μmol / L, 0.06μmol / L, 0.125μmol / L, 0.025μmol / L, 0.05μmol / L, 0.1μmol / L) for 48h and human non-small cell lung cancer paclitaxel-resistant cells were treated with paclitaxel (1μmol / L, 2μmol / L, 3μmol / L, 4μmol / L, 5μmol / L, 6μmol / L) for 48h, CCK8 was used to verify the paclitaxel resistance multiple. The results showed that the half inhibitory concentration (IC50) of paclitaxel on human non-small cell lung cancer cells was 0.03±0.01μmol / L, and the half inhibitory concentration (IC50) of paclitaxel on human non-small cell lung cancer paclitaxel-resistant cells was 3.16±0.65μmol / L, and the resistance multiple reached 105 times. Figure 4 As shown in (A), secondly, human non-small cell lung cancer and paclitaxel-resistant cells were treated with common clinical lung cancer chemotherapy drugs, that is, human non-small cell lung cancer and paclitaxel-resistant cells were treated with doxorubicin (ADR) (0.0625μmol / L, 0.125μmol / L, 0.25μmol / L, 0.512μmol / L) for 48 hours, and CCK8 was used for detection. The results showed that the half inhibitory concentration (IC50) of doxorubicin for human non-small cell lung cancer cells was 0.17±0.14μmol / L, and the half inhibitory concentration (IC50) of doxorubicin for human non-small cell lung cancer paclitaxel-resistant cells was 1.10±0.24μmol / L, and the drug resistance multiple was 6.5 times, as shown in Figure 2. Figure 4 As shown in (B). Human non-small cell lung cancer and paclitaxel-resistant cells were treated with vincristine (VCR) (0.3125μmol / L, 0.0625μmol / L1.25μmol / L2.5μmol / L) for 48 hours and then tested with CCK8. The results showed that the half inhibitory concentration (IC50) of vincristine on human non-small cell lung cancer cells was 0.29±0.04μmol / L, and the half inhibitory concentration (IC50) of vincristine on human non-small cell lung cancer paclitaxel-resistant cells was 2.41±0.07μmol / L, and the drug resistance was 9.7 times. Figure 4 As shown in (C), these results indicate that human non-small cell lung cancer cells resistant to paclitaxel are highly resistant and also show certain resistance to other chemotherapy drugs, indicating the ability of 3β-Hydroxylup-12-en-28 to resist the resistance of human non-small cell lung cancer cells resistant to paclitaxel.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. 3 The use of β-Hydroxylup-12-en-28 in the preparation of a drug for treating paclitaxel-resistant lung cancer, characterized in that: The structural formula of 3β-Hydroxylup-12-en-28 is as follows:

2. The use according to claim 1, characterized in that 3β-Hydroxylup-12-en-28 can inhibit the proliferation of non-small cell lung cancer cells and paclitaxel-resistant non-small cell lung cancer cells.

3. The use according to claim 1, characterized in that The inhibitory effect of 3β-Hydroxylup-12-en-28 on the proliferation of non-small cell lung cancer cells and paclitaxel-resistant non-small cell lung cancer cells increased with time.

4. The use according to claim 2, characterized in that The non-small cell lung cancer paclitaxel-resistant cells include: A549 / Taxol.

5. The use according to claim 2, characterized in that The concentration of 3β-Hydroxylup-12-en-28 is 0.5 to 2.5 μM. 6.3 Use of β-Hydroxylup-12-en-28 in the preparation of a drug for inhibiting the expression of ABC transporters, characterized in that: The structural formula of 3β-Hydroxylup-12-en-28 is as follows:

7. The use according to claim 6, characterized in that The ABC transporters include: P-gp, MRP1, and ABCG2.

8. The use according to claim 6, characterized in that The concentration of 3β-Hydroxylup-12-en-28 is 0.5 to 2.5 μM. 9.3 Application of β-Hydroxylup-12-en-28 in the preparation of drugs to combat paclitaxel-resistant human non-small cell lung cancer cells.

10. A drug for resisting paclitaxel-resistant cells of human non-small cell lung cancer, characterized in that: Active ingredients include: 3β-Hydroxylup-12-en-28; The structural formula of 3β-Hydroxylup-12-en-28 is as follows: The effective amount of 3β-Hydroxylup-12-en-28 is 0.5 to 2.5 μM.

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