Use of terpenoids isolated from the succulent stems of krameria triandra for the preparation of a tumor multi-drug resistance reversing agent

Abstract

The application belongs to the technical field of medicine, and provides a new use of a terpenoid compound HYL100-5-2 contained in a plant Pereskia aculeata, and specifically, application in anti-tumor multidrug resistance. The application relates to a terpenoid natural product extracted from the succulent stem of the Pereskia aculeata, which has the effect of reversing the multidrug resistance of tumors, and can be used as an auxiliary drug in tumor drug treatment. The application proves through multiple experiments that the HYL100-5-2 has a high effect of reversing multidrug resistance. The HYL100-5-2 is expected to be developed into a new type of specific reversing agent drug for multidrug-resistant tumors.

Description

Technical Field

[0001] This invention relates to HYL100-5-2, a terpenoid natural product extracted from the stem of the plant *Hypericum perforatum*, which can reverse multidrug resistance in tumors. It can increase the concentration of chemotherapeutic drugs in cells, thereby enhancing the sensitivity of cancer cells to chemotherapeutic drugs and improving the efficacy of tumor chemotherapy. Therefore, it has a good adjuvant effect in anti-tumor treatment. Background Technology

[0002] Malignant tumors are a major category of diseases that seriously threaten human health. However, multidrug resistance in tumors is a significant reason for chemotherapy failure in cancer patients, as drug resistance in tumor cells greatly reduces the effectiveness of chemotherapeutic drugs. Multidrug resistance results from the interaction of multiple factors within tumor cells, with the main mechanism being the high expression of ABC transporters within tumor cells, which expel drugs from the cell and reduce their concentration within the cell. Therefore, it is essential to find highly effective and low-toxicity multidrug resistance reversal agents to improve the efficacy of cancer treatment.

[0003] The ABC transporter family is a large class of transmembrane proteins, typically composed of two transmembrane domains and two ATP-binding domains. Their main function is to bind substrate molecules, hydrolyze ATP, and alter the protein structure, thereby transporting drugs from inside the cell to outside, reducing the effective intracellular drug concentration and preventing therapeutic effects. Within the ABC family, MRPs, P-gp, and BCRP proteins play crucial roles in the development of multidrug resistance in tumor cells. P-gp protein was the first discovered and most widely studied ABC transporter. When chemotherapeutic drugs come into contact with tumor cells, the drug enters the cell and binds to p-gp, leading to ATP hydrolysis and a conformational change in p-gp, thus expelling the drug from the cell. MRP1, a member of the ABC transporter family discovered by Cole et al. in 1992 in the H69AR drug-resistant human small cell lung cancer cell line, also functions to expel drugs from the cell and shares some sequence homology with p-gp. However, MRP1 cannot independently transport unmodified chemotherapeutic drugs, but it can transport drugs bound to glutathione. BCRP is a protein in the ABC transporter family discovered in 1998 that is associated with tumor drug resistance. Unlike most ABC transporters, BCRP needs to form a dimer to function. Studies have found that BCRP is highly expressed in many tumor cells.

[0004] The application of multidrug resistance reversal agents is an important means to overcome multidrug resistance in tumors, and a large number of compounds with multidrug resistance reversal activity have been discovered. First-generation reversal agents mainly work by directly inhibiting the efflux of p-gp proteins. Examples include verapamil and cyclosporine A, which competitively bind to the substrate binding site to reverse the efflux of transport proteins. However, they require high doses to achieve good effects, and these high doses produce significant toxic side effects, making their clinical application difficult. Second-generation reversal agents include dextrorotatory verapamil and PSC833; however, PSC833, when used in combination with antitumor drugs in clinical trials, easily causes various complications and serious adverse consequences. Third-generation reversal agents overcome the limitations of second-generation reversal agents by utilizing structure-activity relationships and combinatorial chemistry, thus more effectively inhibiting p-gp function. However, due to poor clinical efficacy or serious side effects, they have not entered clinical application.

[0005] Many natural products, such as plant polyphenols and curcumin, can reverse multidrug resistance mediated by ABC transporters. Through pharmacological experiments, reversal experiments, and efflux experiments, we found that HYL100-5-2, extracted from the stem of *Euphorbia hirta*, can reverse multidrug resistance in tumors. Summary of the Invention

[0006] The first objective of this invention is that HYL100-5-2 has no toxic side effects on tumor cells.

[0007] This invention further discloses that HYL100-5-2 can reverse multidrug resistance in tumors and increase the accumulation of chemotherapeutic drugs in cells.

[0008] This invention further discloses the effect of HYL100-5-2 on p-gp protein expression. Attached image description: Figure 1 HYL100-5-2 structural formula; Figure 2 The graph shows the toxicity test results of doxorubicin hydrochloride on HLA and HLA / DDP. Figure 3 The graph shows the toxicity test results of mitoxantrone hydrochloride on HELA and HELA / DDP. Figure 4 The graph shows the toxicity test results of HYL100-5-2 against HLA and HLA / DDP. Figure 5 The graph shows the toxicity test results of 5 μM and 10 μM HYL100-5-2 and 10 μM Verapamil combined with doxorubicin hydrochloride on hela / ddp. Figure 6The graph shows the toxicity test results of 5 μM and 10 μM HYL100-5-2 and 10 μM Verapamil combined with mitoxantrone hydrochloride on hela / ddp. Figure 7 The experimental diagram shows the effect of HYL100-5-2 on the efflux function of MRPs proteins; Figure 8 The experimental graph shows the effect of HYL100-5-2 on the efflux function of p-gp protein; Figure 9 An experimental diagram showing the accumulation of doxorubicin hydrochloride in cells for HYL100-5-2; Figure 10 An experimental diagram showing the intracellular accumulation of mitoxantrone salt in HYL100-5-2; Figure 11 The experimental graph shows the effect of HYL100-5-2 concentration on ABCB1 transcription levels; Figure 12 The experimental graph shows the effect of the duration of HYL100-5-2 treatment on ABCB1 transcription levels; Detailed Implementation Pharmacological Experiment 1: Toxicity Test of HYL100-5-2, Doxorubicin Hydrochloride (ADM), and Mitoxantrone Hydrochloride (MT) on HLA and HLA / DDP principle: Cell Counting Kit-8 is a rapid and highly sensitive assay kit based on WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonyl)-2H-tetrazole monosodium salt), widely used for the detection of cell proliferation and cytotoxicity. In the presence of an electron-coupling reagent, it can be reduced by mitochondrial dehydrogenases to generate a highly water-soluble orange-yellow formazan. The intensity of the color is inversely proportional to cytotoxicity. The OD value is measured at 450 nM using a microplate reader, indirectly reflecting the number of viable cells. Experimental methods: HeLa and HeLa / DPP cells were spaced at a density of 2.5 x 10⁻⁶. 3 Inoculate each well of a 96-well plate with a volume of 100 μL, setting up three replicates of 96 wells. Add sterile distilled water around the perimeter of the 96-well plate. Incubate the 96-well plate at 37°C with 5% CO2 for 24 hours. Eight suitable drug concentrations were designed and added to the wells. The blank control was added with only 100 μL of culture medium, and the negative control was added with 100 μL of cell suspension without the test drug.

[0009] After adding the test drug, place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0010] After 24 hours, add 10 μL of cck-8 reagent to each well and incubate at 37°C in a 5% CO2 incubator for 4 hours. Detect the absorbance at 450 nm using a microplate reader.

[0011] Processing data Cell viability = (Experimental group absorbance - Blank control absorbance) / (Negative control absorbance - Blank control absorbance) x 100%.

[0012] Experimental results: HYL100-5-2 has very low toxicity to HELA and HELA / DDP, with IC50 values ​​of 70.81±2.0786 μM and 77.35±3.0880 μM, respectively.

[0013] The IC50 values ​​of ADM for HELA and HELA / DDP were 0.4177±0.0134 μM and 1.0027±0.03129 μM, respectively.

[0014] The IC50 values ​​of MT for HELA and HELA / DDP are 0.043 ± 0.0074 μM and 0.2092 ± 0.0131 μM, respectively.

[0015] Pharmacological Experiment 2: Reversal Experiment of HYL100-5-2 principle: HYL100-5-2 can enhance the concentration of doxorubicin hydrochloride and mitoxantrone hydrochloride in cells, thereby reducing the IC50 of doxorubicin and mitoxantrone hydrochloride on HLA / DDP. Methods: HeLa / DPP cells were plated using the cytotoxic method described above, and the test drug was added in combination with 5 μM and 10 μM HYL100-5-2, respectively.

[0016] Experimental results: According to CCK-8 assay results, the IC50 of human cervical cancer cell line HeLa cells against doxorubicin hydrochloride was 0.4177±0.013 μM, while the IC50 of its resistant cell line HeLa / DDP cells against doxorubicin hydrochloride was 1.0027±0.03129 μM, with a resistance fold of 2.4-fold. When the reversal agent standard 10 μM Verapamil was used in combination with doxorubicin hydrochloride, the IC50 of HeLa / DDP cells against doxorubicin hydrochloride was 0.3371±0.0835 μM, with a reversal fold of 2.97-fold. When 5 μM HYL100-5-2 was used in combination with doxorubicin hydrochloride, the IC50 of HeLa / ddp cells against doxorubicin hydrochloride was 0.7102 ± 0.1131 μM, with a reversal fold of 1.41. When 10 μM HYL100-5-2 was used in combination with doxorubicin hydrochloride, the IC50 of HeLa / ddp cells against doxorubicin hydrochloride was 0.3573 ± 0.0989 μM, with a reversal fold of 2.81. This indicates that HYL100-5-2 can increase the drug toxicity of the anticancer drug doxorubicin hydrochloride to drug-resistant HeLa / ddp cells, and the working concentration of HYL100-5-2 is positively correlated with its reversal effect.

[0017]

[0018] According to CCK-8 assay results, the IC50 of the human cervical cancer cell line HeLa cells against mitoxantrone hydrochloride was 0.043±0.0074 μM, while the IC50 of its resistant cell line HeLa / DDP cells against mitoxantrone hydrochloride was 0.2092±0.0131 μM, with a resistance fold of 4.87-fold. When the reversal agent standard 10 μM Verapamil was used in combination with mitoxantrone hydrochloride, the IC50 of HeLa / DDP cells against mitoxantrone hydrochloride was 0.0700±0.0306 μM, with a reversal fold of 2.99-fold. When 5 μM HYL100-5-2 was used in combination with mitoxantrone hydrochloride, the IC50 of mitoxantrone hydrochloride in HeLa / ddP cells was 0.0926 ± 0.0225 μM, with a reversal fold of 2.26. When 10 μM HYL100-5-2 was used in combination with mitoxantrone hydrochloride, the IC50 of mitoxantrone hydrochloride in HeLa / ddP cells was 0.0467 ± 0.0254 μM, with a reversal fold of 4.48. This indicates that HYL100-5-2 can increase the drug toxicity of the anticancer drug mitoxantrone hydrochloride to drug-resistant HeLa / ddP cells, and the working concentration of HYL100-5-2 is positively correlated with its reversal effect.

[0019]

[0020] Pharmacological Experiment 3: Effects of HYL100-5-2 on the efflux function of MRPs, p-gp, and BCRP proteins principle: The effect of HYL100-5-2 on the efflux function of MRPs, p-gp, and BCRP proteins can alter the fluorescence intensity of the corresponding fluorescent dyes in cells.

[0021] method: HeLa and HeLa / DPP cells were grouped at a density of 1 x 10⁻⁶. 4 Inoculate each well of a 96-well plate with a volume of 200 μL, setting up three replicates of 96 wells. Add sterile distilled water around the perimeter of the 96-well plate. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 96 hours.

[0022] The reversal agent standard Verapamil was prepared at 2 mM, the dye Rhodamine 123 at 50 ug / ml, the reversal agent standard MK571 at 5 mM, the dye BCECF-AM at 200 μM, the reversal agent standard Ko143 at 2 Mm, and the dye hoechst-33342 at 50 ug / ml. The test sample HYL100-5-2 was prepared at 2.5 mM.

[0023] After adding the standard and the test sample HYL100-5-2, incubate in a 37℃, 5% CO2 constant temperature incubator for 15 min.

[0024] Add fluorescent dye after 15 minutes and incubate for 4 hours; set up three duplicate wells for each sample.

[0025] Remove the 96-well plate from the incubator, wash each well twice with 200 μL of cold PBS, discard the PBS, and perform the assay on a full-wavelength multi-mode microplate reader.

[0026] The excitation wavelength of Rho123 was 480 nm, and the emission wavelength was 550 nm. The final concentration of Rho123 fluorescent dye for detecting p-gp protein was 2 ug / ml, the final concentration of the standard Verapamil was 10 μM, and the concentrations of the test sample HYL100-5-2 were 5 μM and 10 μM, respectively.

[0027] Experimental results: HYL100-5-2 increased the intracellular accumulation of dyes Rho123 and BCECF-AM. At a concentration of 10 μM, the intracellular fluorescence intensity of Rho123 was stronger than that at 5 μM. However, at the same concentration of 10 μM, the intracellular fluorescence intensity of Rho123 was stronger with HYL100-5-2 than that of Verapamil, while the intracellular fluorescence intensity of BCECF-AM was lower than that of MK571 at 10 μM. This indicates that the intracellular accumulation of fluorescent dyes by HYL100-5-2 is concentration-dependent.

[0028] Pharmacological Experiment 4: Effect of HYL100-5-2 on the intracellular accumulation of doxorubicin hydrochloride and mitoxantrone hydrochloride Principle: The effect of HYL100-5-2 on ABC transporters influences the efflux of doxorubicin hydrochloride and mitoxantrone hydrochloride. method: HeLa and HeLa / DPP cells were grouped at a density of 1 x 10⁻⁶. 4 Inoculate each well of a 96-well plate with a volume of 200 μL, setting up three replicates of 96 wells. Add sterile distilled water around the perimeter of the 96-well plate. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 96 hours.

[0029] The reversal agent standard Verapamil was prepared at 2 mM, doxorubicin hydrochloride at 0.1 mM, the reversal agent standard Ko143 at 2 mM, and mitoxantrone hydrochloride at 100 ug / ml. The test sample HYL100-5-2 was prepared at 2.5 mM.

[0030] After adding the standard and the test sample HYL100-5-2, incubate in a 37℃, 5% CO2 constant temperature incubator for 90 min.

[0031] After 90 minutes, fluorescent dye was added and incubated for 4 hours; three replicates were set for each sample.

[0032] Remove the 96-well plate from the incubator, wash each well twice with 200 μL of cold PBS, discard the PBS, and perform the assay on a full-wavelength multi-mode microplate reader.

[0033] The excitation wavelength of doxorubicin hydrochloride was 480 nm, the emission wavelength was 550 nm, and the final concentration was 1 μM. The final concentration of the standard Verapamil was 10 μM. The concentrations of the test sample HYL100-5-2 were 5 μM and 10 μM, respectively.

[0034] The excitation wavelength of mitoxantrone hydrochloride was 600 nm, and the emission wavelength was 680 nm. The final concentration used was 1 ug / ml, the final concentration of the standard Verapamil was 10 μM, and the final concentrations of HYL100-5-2 were 5 μM and 10 μM, respectively.

[0035] Experimental results: HYL100-5-2 can increase the accumulation of doxorubicin hydrochloride and mitoxantrone hydrochloride in cells, and the accumulation of doxorubicin hydrochloride and mitoxantrone hydrochloride in cells is higher with high concentration of HYL100-5-2 than with low concentration of HYL100-5-2.

[0036] Pharmacological Experiment 6: Effects of HYL100-5-2 on ABCB1 Transcription Levels method: 1. Extraction of total RNA from cells (1) Prepare a 1‰ concentration (V / V) solid-phase RNase remover and wipe pipettes, laminar flow hood surfaces, etc. with the prepared RNase remover. Irradiate the laminar flow hood with UV lamp for 30 min and pre-cool the high-speed centrifuge.

[0037] (2) Centrifuge at 1000 rpm for 3 min, discard the supernatant, and collect the cells. Add 1 mL of TRIzol reagent and pipette repeatedly with 1 mL of the solution to lyse all the cells and obtain the cell lysate.

[0038] (3) After repeated blowing and shaking, place the tube at room temperature for 5 minutes on a clean bench to allow the nucleic acid and protein to fully dissociate. Add 0.2 mL of chloroform to every 1 mL of Trizol reagent, tighten the cap, shake vigorously manually for about 15 seconds, and then let it stand at room temperature for 3 minutes in a clean bench.

[0039] (4) Centrifuge at 4 ℃ for 12000 rpm for 15 min. After centrifugation, carefully place the centrifuge tube on ice and transfer it to a clean bench.

[0040] (5) Carefully aspirate 400-500 μL of the colorless aqueous upper phase (if less, aspirate less) using a 200 μL pipette and transfer it to a new 1.5 mL enzyme-free centrifuge tube. When aspirating, the pipette tip should touch the intermediate layer of DNA and protein impurities (intermediate layer, white).

[0041] (6) Add an equal volume of the aspirated aqueous phase and pre-cooled isopropanol, tighten the cap, and gently invert to mix. Let stand on ice for 10 min.

[0042] (7) Centrifuge at 4 ℃ and 12000 rpm for 10 min to allow the RNA to precipitate to the bottom of the centrifuge tube.

[0043] (8) Discard the supernatant. When discarding the supernatant, be careful not to discard the RNA as well. Add 1 mL of 75% ethanol prepared with pre-cooled enzyme-free water to each tube and wash twice. Gently pipette to remove residual isopropanol and salt. Centrifuge at 12,000 rpm for 5 min at 4 ℃.

[0044] (9) The supernatant can be aspirated with a pipette, and the RNA precipitate can be dried (evaporation at room temperature or vacuum drying). Note that the RNA precipitate should not be completely dried, otherwise it will be difficult to see and dissolve.

[0045] (10) Dissolve the RNA precipitate in an appropriate amount (10–40 μL) of RNase-free water. This RNA solution can be used for further experiments or stored at -80 °C.

[0046] 2. RNA reverse transcription into cDNA (1) Take 2 μL of RNA solution and dilute it 50 times with RNase-free water. Use an eppendorf BioPhotometer plus instrument to determine the concentration and purity of the RNA sample.

[0047] (2) Calculate the volume required for 500 ng RNA, add 2 μL of 4×Gdna wiper Mix, and bring the volume to 8 mL with RNase-free water. Gently pipette and incubate at 42 °C for 2 min to remove genomic DNA from the template RNA.

[0048] (3) Add 2 μL of 5×HiScript ll qRT SuperMix ll directly to the reaction tube from the previous step and gently mix with a pipette.

[0049] (4) After adding all the ingredients, 25 °C for 10 min.

[0050] (5) Reverse transcription was performed at 50 ℃ for 30 min.

[0051] (6) Inactivate reverse transcriptase at 85 ℃ for 5 min.

[0052] (7) Store the first-strand cDNA at -20 ℃ for later use.

[0053] 3 Real-Time PCR Real-Time PCR Primer Table

[0054] Establish a 10 μL reaction system

[0055] Set Real-Time PCR reaction conditions

[0056] Results Analysis The purpose of this experiment was to compare and analyze the changes in the transcription levels of certain genes in Jurkat cells relative to the control group, so the 2-ΔΔCt method was used. This analytical method can directly calculate the fold change in the expression of the same gene mRNA between the control and experimental groups; the housekeeping gene GAPDH was used as an internal reference to correct the values ​​and reduce errors.

[0057] fold ratio (experimental group / control group) = 2 - [(Ct target gene - Ct internal reference) experimental group - (Ct target gene - Ct internal reference) control group].

[0058] Experimental results: Treatment with hela / ddp at concentrations of 2.5 μM and 52.5 μM had no effect on ABCB1 transcription. At a concentration of 10 μM, it had a weak downregulation effect on ABCB1 transcription; treatment with hela / ddp at 10 μM for 24 h, 48 h, and 72 h had no effect on ABCB1 transcription.

[0059] In summary, cytotoxicity and fluorescence efflux assays confirmed that HYL100-5-2 has minimal toxicity to HLA and HLA / DDP, and can increase the intracellular accumulation of doxorubicin hydrochloride and mitoxantrone hydrochloride, making it a promising adjuvant drug in the anticancer process. To better treat cancer patients, the development of highly effective, non-toxic multidrug resistance reversal agents is particularly important. Pharmacological and pharmacodynamic studies indicate that HYL100-5-2 has a good reversal effect; when used in combination with other chemotherapeutic drugs, it can effectively treat tumors, with rapid onset and long-lasting effects, inhibiting tumor cell efflux and thus improving the efficacy of cancer treatment.

Claims

1. The use of compound HYL100-5-2 of the following structural formula (Ⅰ) in the preparation of a multidrug resistance reversal agent for human cervical cancer, wherein said compound HYL100-5-2 is used in combination with doxorubicin hydrochloride or mitoxantrone hydrochloride.

Images