3beta-Hydroxylup-12-en-28 in the treatment of paclitaxel-resistant lung cancer drugs

Abstract

The application belongs to the field of medicine, and provides application of 3beta-Hydroxylup-12-en-28 in a drug for treating lung cancer resistant to paclitaxel.The structural formula of 3beta-Hydroxylup-12-en-28 is as follows:3beta-Hydroxylup-12-en-28 can reduce the cell viability of human non-small cell lung cancer cells resistant to paclitaxel, and reduce the expression of drug resistance related proteins P-gp, MRP1 and ABCG2 highly expressed by the human non-small cell lung cancer cells resistant to paclitaxel, and therefore plays an anti-paclitaxel resistance role.

Description

TECHNICAL FIELD

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

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission by the patentee that this information constitutes prior art nor that this information is entirely relevant and pertinent to the patentee's claimed application.

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

[0004] Lung cancer is still the cancer with the highest mortality rate in the world, and non-small cell lung cancer (NSCLC) accounts for 90% of all cases. Because about 70-80% of these patients do not meet the conditions for radical surgery due to advanced disease at the time of diagnosis, chemotherapy is currently the main treatment method for NSCLC. However, due to the low response rate and toxic side effects of chemotherapy, the prognosis of patients with advanced NSCLC is still poor. 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 span 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 needs to discover and develop drugs against drug resistance.

[0005] In cancer chemotherapy, the increase of efflux of chemotherapeutic drugs from target cells will impair the efficacy of the drugs, resulting in a decrease in intracellular drug chemotherapy concentration. ABC transporters are the largest family of transmembrane transporters in organisms, and the typical structural feature of ABC transporters is a four-domain structure, consisting of two cytoplasmic nucleotide-binding domains (NBD) that bind and hydrolyze ATP and two transmembrane domains (TMD) that recognize and transport substrates, among which P-glycoprotein (MDR1, P-gp), multiple drug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2) are the most important drug transporters, which are related to the treatment resistance of human lung cancer. These proteins pump various endogenous or exogenous substrates out of cells, producing detoxification, at the same time, this process also leads to a decrease in intracellular drug concentration and drug 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 ABC transporters, which has been shown to confer resistance to various cancer cells. These transporters have a wide substrate specificity, and they mediate resistance to a variety of anticancer drugs. P-gp can transport hydrophobic compounds, while ABCC1 and ABCG2 can transport hydrophobic drugs and large anionic compounds. Overexpression of P-gp has been recognized to confer resistance to paclitaxel, and its level is significantly higher in human lung cancer A549 paclitaxel-resistant cells (A549 / Taxol) than in its parental cells A549. ABCC1 can confer resistance to chemotherapeutic drugs in various cancer types, including acute myeloid and lymphoblastic leukemia, non-small cell lung cancer, prostate cancer, breast cancer, and neuroblastoma. ABCG2 is a major survival factor for cancer stem cells, which exist as a unique chemotherapeutic-resistant population in tumors and lead to tumor recurrence and metastasis. ABCG2 promotes the efflux of various anticancer drugs to protect cancer stem cells, and high expression of ABCG2 is detected in various cancer types, and the three drug resistance proteins are closely related to the resistance of lung cancer cells to paclitaxel, which encourages extensive research on finding drugs that inhibit the expression of ABC transporters.

[0006] The paper "Research Progress on Anti-tumor Mechanism of Oleanolic Acid and Its Derivatives" records that this kind of substance has the effect of reversing multi-drug resistance, but due to the large number of oleanolic acid and its derivatives, the industry has not yet found a suitable oleanolic acid derivative as an efficient anti-drug agent for paclitaxel-resistant lung cancer. SUMMARY

[0007] To solve the above problems, the application provides the application of 3β-Hydroxylup-12-en-28 as an anti-drug agent in lung cancer.

[0008] To achieve the above purpose, the application adopts the following technical solutions:

[0009] In a first aspect, the present application provides the use of 3β-Hydroxylup-12-en-28 in the preparation of a drug for treating lung cancer resistant to paclitaxel, wherein the structure of 3β-Hydroxylup-12-en-28 is as follows:

[0010]

[0011] Firstly, the present application uses 3β-Hydroxylup-12-en-28 to detect the cell activity and proliferation of human non-small cell lung cancer cells (A549) and its paclitaxel-resistant cells (A549 / Taxol) and human normal lung epithelial cells BEAS-2B, that is, 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. Through the results of CCK8 experiment and data analysis, it is shown that 3β-Hydroxylup-12-en-28 (0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 2.5 μmol / L) treated A549, A549 / Taxol, and BEAS-2B cells for 48 h, the half-inhibitory concentration (IC50) of 3β-Hydroxylup-12-en-28 to A549, A549 / Taxol, and BEAS-2B cells is 1.14 ± 0.23 μmol / L, 0.98 ± 0.12 μmol / L, and 2.32 ± 0.01 μmol / L, respectively. Further experiments prove that 3β-Hydroxylup-12-en-28 (0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 2.5 μmol / L) treated A549, A549 / Taxol, and BEAS-2B cells for 24 h, 48 h, and 72 h, the proliferation inhibition effect of 3β-Hydroxylup-12-en-28 on A549 and A549 / Taxol cells increases with time.

[0012] In some embodiments, 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.

[0013] In some embodiments, the proliferation inhibition effect of 3β-Hydroxylup-12-en-28 on 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 taxol-resistant cells comprise A549 / Taxol.

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

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

[0017] In a second aspect of the present application, there is provided a use of 3β-Hydroxylup-12-en-28 in the preparation of a medicament for inhibiting the expression of ABC transporters, and the structural formula of 3β-Hydroxylup-12-en-28 is as follows:

[0018]

[0019] P-gp, MRP1 and ABCG2 protein expression is closely related to the drug resistance state of tumor cells, and the expression of the three is positively correlated with the drug resistance ability of cells. Firstly, the present application uses western blot experiment to test that the three drug resistance proteins P-gp, MRP1 and ABCG2 are highly expressed in human non-small cell lung cancer taxol-resistant cells. A549 cells and A549 / Taxol cells are used to test the expression of the three drug resistance proteins by western blotting. The results show that the three drug resistance proteins are highly expressed in human non-small cell taxol-resistant cells. Human non-small cell lung cancer taxol-resistant cells are treated with different concentrations of 3β-Hydroxylup-12-en-28 (0.5 μM, 1 μM, 2 μM) for 48 h, and the expression of p-gp is detected by Western Blot method and cell immunofluorescence color method. The results show that the treatment of 3β-Hydroxylup-12-en-28 significantly reduces the expression of P-gp, and is in a concentration-dependent manner.

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

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

[0022] In a third aspect of the present application, there is provided a use of 3β-Hydroxylup-12-en-28 in the preparation of a medicament for resisting drug resistance of human non-small cell lung cancer taxol-resistant cells.

[0023] The present application firstly uses CCK8 method to test whether human non-small cell lung cancer paclitaxel-resistant cells reach a certain drug resistance fold to carry out the experiment, uses paclitaxel (0.03 μmol / L, 0.06 μmol / L, 0.125 μmol / L, 0.025 μmol / L, 0.05 μmol / L, 0.1 μmol / L) to treat human non-small cell lung cancer A549 cells for 48 h, and then uses CCK8 to verify the paclitaxel-resistant drug resistance fold after paclitaxel (1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L) is used to treat human non-small cell lung cancer paclitaxel-resistant cells for 48 h, and the results show that the half inhibitory concentration (IC50) of paclitaxel to human non-small cell lung cancer cells is 0.03±0.01 μmol / L, the half inhibitory concentration (IC50) of paclitaxel to human non-small cell lung cancer paclitaxel-resistant cells is 3.16±0.65 μmol / L, and the drug resistance fold reaches 105 times, which indicates that human non-small cell lung cancer paclitaxel-resistant cells have high drug resistance, secondly, common lung cancer chemotherapy drugs are used to treat human non-small cell lung cancer and paclitaxel-resistant cells, that is, after using adriamycin (ADR) (0.0625 μmol / L, 0.125 μmol / L, 0.25 μmol / L, 0.512 μmol / L) to treat human non-small cell lung cancer and paclitaxel-resistant cells for 48 h, CCK8 is used for detection, and the results show that the half inhibitory concentration (IC50) of adriamycin to human non-small cell lung cancer cells is 0.17±0.14 μmol / L, the half inhibitory concentration (IC50) of adriamycin to human non-small cell lung cancer paclitaxel-resistant cells is 1.10±0.24 μmol / L, and the drug resistance fold is 6.5 times. After using vincristine (VCR) (0.3125 μmol / L, 0.0625 μmol / L, 1.25 μmol / L, 2.5 μmol / L) to treat human non-small cell lung cancer and paclitaxel-resistant cells for 48 h, CCK8 is used for detection, and the results show that the half inhibitory concentration (IC50) of vincristine to human non-small cell lung cancer cells is 0.29±0.04 μmol / L, the half inhibitory concentration (IC50) of vincristine to human non-small cell lung cancer paclitaxel-resistant cells is 2.41±0.07 μmol / L, and the drug resistance fold is 9.7 times. These results show that human non-small cell lung cancer paclitaxel-resistant cells have high drug resistance, and also show certain drug resistance to other chemotherapy drugs, thereby indicating that 3β-Hydroxylup-12-en-28 has the ability of selectively resisting human non-small cell lung cancer paclitaxel-resistant cell drug resistance.

[0024] In a fourth aspect of the present application, a drug for resisting human non-small cell lung cancer paclitaxel-resistant cell drug resistance is provided, and the active ingredient comprises 3β-Hydroxylup-12-en-28.

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

[0026]

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

[0028] Advantages of the present application

[0029] (1) The 3β-Hydroxylup-12-en-28 can reduce the cell viability of human non-small cell lung cancer cells resistant to paclitaxel, and reduce the expression of drug resistance related proteins P-gp, MRP1 and ABCG2 highly expressed in human non-small cell lung cancer cells resistant to paclitaxel, thus playing an anti-paclitaxel resistance role.

[0030] (2) Human non-small cell lung cancer cells resistant to paclitaxel have high drug resistance and are resistant to common lung cancer chemotherapy drugs. The 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. The 3β-Hydroxylup-12-en-28 has small cytotoxicity to human normal lung epithelial cells BEAS-2B. The 3β-Hydroxylup-12-en-28 can inhibit the expression of drug resistance related proteins P-gp, MRP1 and ABCG2 highly expressed in human non-small cell lung cancer cells resistant to paclitaxel, thereby achieving an anti-paclitaxel resistance effect in lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0031] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of these drawings are set forth to explain the present application and do not limit the present application in any manner.

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

[0033] Figure 2Figure 2 is 3β-Hydroxylup-12-en-28 effects on cell viability of A549, A549 / Taxol and BEAS-2B cells, (A) 3β-Hydroxylup-12-en-28 dose-dependently inhibited the proliferation of A549, A549 / Taxol and BEAS-2B cells, and time-dependently inhibited the proliferation of A549 and A549 / Taxol cells, (B) CCK8 assay of cell viability of A549 and A549 / Taxol cells treated with 3β-Hydroxylup-12-en-28 at different concentrations for 24 h, 48 h and 72 h. Data are presented as mean ± SD of three or more different experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. control group.

[0034] Figure 3 Figure 3 is 3β-Hydroxylup-12-en-28 effects on P-gp, MRP1, ABCG2 expression in A549 / Taxol cells, (A) Western blot analysis of P-gp, MRP1, ABCG2 protein expression in A549 / Taxol cells compared with A549 cells, data are presented as 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 inhibition of P-gp, MRP1, ABCG2 protein expression in A549 / Taxol cells, data are presented as mean ± SD of three or more different experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. control group, (C) Immunofluorescence experiment to detect the effects of 3β-Hydroxylup-12-en-28 on P-gp, MRP1, ABCG2 protein expression. Green fluorescence shows P-gp, MRP1, ABCG2, and blue shows DAPI (scale bar, 50 μm).

[0035] Figure 4Example 3 of this invention describes human non-small cell lung cancer cells resistant to paclitaxel that exhibit high drug resistance and resistance to common clinical lung cancer chemotherapy drugs. (A) A549 and A549 / Taxol cells were treated with paclitaxel for 48 hours, and the results were detected using the CCK8 assay. (B) A549 and A549 / Taxol cells were treated with doxorubicin for 48 hours, and the results were detected using the CCK8 assay. (C) A549 and A549 / Taxol cells were treated with vincristine for 48 hours, and the results were detected using the CCK8 assay. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. control group. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0038] Example 1: Effects 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 control group and two 3β-Hydroxylup-12-en-28 drug groups (0.5μM, 1μM, 1.5μM, 2μM, 2.5μM). Human non-small cell lung cancer (A549) cells, paclitaxel-resistant cells (A549 / Taxol), and normal lung epithelial cells were treated with BEAS-2B for 48 hours before cell viability and proliferation were detected using the CCK8 assay. Figure 2 As shown in Figure (A), the results revealed that 3β-Hydroxylup-12-en-28 exhibited the strongest sensitivity against paclitaxel-resistant human non-small cell lung cancer cells and the weakest cytotoxicity against normal lung epithelial cells (BEAS-2B). CCK8 assay results and data analysis showed that after 48 hours of treatment, the half-maximal inhibitory concentrations (IC50) of 3β-Hydroxylup-12-en-28 against A549, A549 / Taxol, and BEAS-2B cells were 1.14±0.23 μmol / L, 0.98±0.12 μmol / L, and 2.32±0.01 μmol / L, respectively.

[0040] The cell viability after 24 hours, 48 hours and 72 hours of treatment of human non-small cell lung cancer and its taxol-resistant cells with 3β-Hydroxylup-12-en-2 at different concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 2.5) was detected by CCK8, as shown in Figure 2 (B).

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

[0042] Example 2 Effect of 3β-Hydroxylup-12-en-28 on P-gp, MRP1 and ABCG2 protein expression in human non-small cell lung cancer taxol-resistant cells

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

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

[0045] Example 3 Anti-drug resistance of 3β-Hydroxylup-12-en-28 against human non-small cell lung cancer taxol-resistant cells

[0046] After human non-small cell lung cancer A549 cells were treated with paclitaxel (0.03 μmol / L, 0.06 μmol / L, 0.125 μmol / L, 0.025 μmol / L, 0.05 μmol / L, 0.1 μmol / L) for 48 h and human non-small cell lung cancer paclitaxel-resistant cells were treated with paclitaxel (1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L) for 48 h, the CCK8 was used to verify the paclitaxel-resistant fold, and the results showed that the half inhibitory concentration (IC50) of paclitaxel on human non-small cell lung cancer cells was 0.03 ± 0.01 μmol / L, the half inhibitory concentration (IC50) of paclitaxel on human non-small cell lung cancer paclitaxel-resistant cells was 3.16 ± 0.65 μmol / L, and the drug resistance fold was 105, as shown in FIG. 1(A). Figure 4 Next, human non-small cell lung cancer and paclitaxel-resistant cells were treated with a common clinical lung cancer chemotherapy drug, i.e., human non-small cell lung cancer and paclitaxel-resistant cells were treated with adriamycin (ADR) (0.0625 μmol / L, 0.125 μmol / L, 0.25 μmol / L, 0.512 μmol / L) for 48 h, and then the CCK8 was used for detection, and the results showed that the half inhibitory concentration (IC50) of adriamycin on human non-small cell lung cancer cells was 0.17 ± 0.14 μmol / L, the half inhibitory concentration (IC50) of adriamycin on human non-small cell lung cancer paclitaxel-resistant cells was 1.10 ± 0.24 μmol / L, and the drug resistance fold was 6.5, as shown in FIG. 1(B). Figure 4 Human non-small cell lung cancer and paclitaxel-resistant cells were treated with vincristine (VCR) (0.3125 μmol / L, 0.0625 μmol / L, 1.25 μmol / L, 2.5 μmol / L) for 48 h, and then the CCK8 was used for detection, and the results showed that the half inhibitory concentration (IC50) of vincristine on human non-small cell lung cancer cells was 0.29 ± 0.04 μmol / L, the half inhibitory concentration (IC50) of vincristine on human non-small cell lung cancer paclitaxel-resistant cells was 2.41 ± 0.07 μmol / L, and the drug resistance fold was 9.7, as shown in FIG. 1(C). Figure 4 These results showed that human non-small cell lung cancer paclitaxel-resistant cells had high drug resistance, and also showed certain drug resistance to other chemotherapy drugs, thereby indicating that 3β-Hydroxylup-12-en-28 had the ability to resist the drug resistance of human non-small cell lung cancer paclitaxel-resistant cells.

[0047] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall fall within the scope of protection of the present application.

Claims

1. Use of 3β-hydroxylup-12-en-28 in the preparation of a medicament for the treatment of non-small cell lung cancer resistant to paclitaxel, characterized in that, The structural formula of the 3β-Hydroxylup-12-en-28 is as follows: Formula I.

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

3. The use according to claim 1, wherein the compound is ###0002### The inhibition of the proliferation of non-small cell lung cancer paclitaxel-resistant cells by the 3β-Hydroxylup-12-en-28 increases with time.

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

5. The use according to claim 2, wherein the compound is ###0002### The concentration of the 3β-Hydroxylup-12-en-28 is 0.5-2.5 μM.

6. The use according to claim 1, wherein The 3β-Hydroxylup-12-en-28 is used for preparing a drug for inhibiting the expression of ABC transporter proteins.

7. Use according to claim 6, wherein The ABC transporter proteins include P-gp, MRP1, ABCG2.

8. The use according to claim 6, wherein The concentration of the 3β-Hydroxylup-12-en-28 is 0.5-2.5 μM.

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