Application of siRNA and paclitaxel combination in the preparation of drugs for treating hepatocellular carcinoma

Abstract

The present invention provides the use of an siRNA and paclitaxel composition in the preparation of a drug for treating hepatocellular carcinoma, wherein the composition is composed of siCYP1B1, siP-gp and paclitaxel in a ratio of 1:1:1. Based on research on increased expression of CYP1B1 and P-gp proteins in hepatocellular carcinoma under hypoxia and reduced drug sensitivity of hepatocellular carcinoma to paclitaxel under hypoxia, the present invention seeks siRNA that can target and knock down CYP1B1 and P-gp. The present invention uses siRNA to target and knock down CYP1B1 and P-gp, thereby reducing the expression of CYP1B1 and P-gp, reversing the drug resistance of hepatocellular carcinoma to paclitaxel, and enhancing the cell cycle arrest mediated by paclitaxel.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceuticals and relates to the application of a siRNA and paclitaxel composition in preparing a drug for treating hepatocellular carcinoma. Background Art

[0002] Liver cancer ranks sixth in incidence and third in mortality among all malignant tumors worldwide. my country, a high-incidence region for liver cancer, accounts for over half of all new cases and deaths worldwide each year. Primary liver cancer primarily includes hepatocellular carcinoma, intrahepatic bile duct carcinoma, and other rare subtypes, with hepatocellular carcinoma accounting for approximately 70%-85% of primary liver cancer cases. Due to the high mortality rate of liver cancer, chemotherapy resistance has been a hot topic in medical research.

[0003] Furthermore, the rapid proliferation of cancer cells requires significant energy and oxygen consumption. Hypoxia is a key characteristic of the tumor microenvironment (TME) of solid tumors. Studies have shown that approximately 60% of advanced solid tumors experience localized hypoxia in the TME. As a highly metabolic solid tumor, liver cancer consumes significantly more oxygen than surrounding normal tissue, with an average intratumoral oxygen content of only 0.8%.

[0004] CYP1B1 is a key member of the cytochrome P450 enzyme family (CYPs). Its expression is very low in normal human liver, but it is highly expressed in various tumor tissues, including liver cancer. Because CYP1B1 is also involved in the metabolism of anticancer drugs such as paclitaxel (PTX), its expression level is associated with paclitaxel resistance. ABC transporters primarily include multidrug resistance protein, multidrug resistance-associated protein, breast cancer resistance protein, and bile salt efflux transporters, among which P-gp (MDR1) is a key member of the multidrug resistance-associated protein family. P-gp is widely expressed in various human tissues, such as the liver, kidney, and blood-brain barrier. P-gp typically maintains intracellular homeostasis by transporting substrates from the cell to the extracellular space using the energy released by ATP hydrolysis. Because its expression can affect drug concentrations within tumor cells, it is closely associated with drug resistance. Paclitaxel is one of the efflux substrates of P-gp. Abnormal expression of P-gp can affect intracellular paclitaxel-mediated resistance of hepatocellular carcinoma to paclitaxel.

[0005] Currently, there are no experimental reports on the effects of a hypoxic microenvironment on the expression of CYP1B1 and P-gp in hepatocellular carcinoma, nor on the therapeutic efficacy of combining siCYP1B1 and siP-gp with paclitaxel for solid tumors such as liver cancer. Paclitaxel, an antimicrotubule agent, primarily binds to tubulin, affecting spindle formation during mitosis, arresting tumor cells in the G2 / M phase. However, its clinical efficacy in hepatocellular carcinoma is suboptimal. Therefore, targeting paclitaxel resistance in hepatocellular carcinoma, by leveraging siRNA to knock down CYP1B1 and P-gp, could optimize therapeutic efficacy. Summary of the Invention

[0006] The present invention aims to provide an application of an siRNA and paclitaxel composition in the preparation of a drug for treating hepatocellular carcinoma, wherein the composition is composed of siCYP1B1 (siRNA targeting CYP1B1) with a final concentration of 20 nM, siP-gp (siRNA targeting P-gp) with a final concentration of 20 nM, and paclitaxel with a final concentration of 100 nM or 10 nM in a ratio of 1:1:1.

[0007] The siRNA and paclitaxel composition provided by the present invention can enhance the therapeutic effect of paclitaxel, and through siRNA targeted knockdown of CYP1B1 and P-gp, thereby reducing the expression of CYP1B1 and P-gp, reversing the resistance of hepatocellular carcinoma to paclitaxel, and enhancing the cell cycle arrest effect mediated by paclitaxel.

[0008] The present invention's research shows that under hypoxia, the expression of the drug-metabolizing enzyme CYP1B1 and the drug transporter P-gp increases in liver cancer cells Li7 and Huh7, and that the sensitivity of liver cancer cells to paclitaxel decreases under hypoxia. Based on this experiment, the present invention designed siRNAs that can target and knock down the expression levels of the drug-metabolizing enzyme CYP1B1 and the drug transporter P-gp at both the mRNA and protein levels. Using classic P-gp inhibitors (cyclosporine A or elacridar) to inhibit P-gp efflux can increase the efficacy of paclitaxel. Using siRNA to knock down CYP1B1 or P-gp in addition to administering paclitaxel can further increase the efficacy of paclitaxel in liver cancer cells. Co-transfection of two siRNAs (siCYP1B1 and siP-gp) can further increase the sensitivity of paclitaxel and enhance the cell cycle arrest mediated by paclitaxel. This siRNA and paclitaxel combination not only more effectively knocks down CYP1B1 and P-gp, but also reverses paclitaxel resistance mediated by increased CYP1B1 and P-gp expression, enhancing paclitaxel's cell-killing and cell cycle-arresting effects, providing a novel therapeutic strategy for hepatocellular carcinoma. The experimental results provide a reference for subsequent research designing siRNAs co-targeting CYP1B1 and P-gp. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The changes in protein expression levels of CYP1B1 and P-gp in two hepatocellular carcinoma cell lines Li7 and Huh7 after hypoxia treatment.

[0010] Figure 2 This study evaluated the efficacy of paclitaxel on two hepatocellular carcinoma cell lines, Li7 and Huh7, after hypoxia treatment.

[0011] Figure 3 The P-gp inhibitor can inhibit the efflux function of P-gp in two hepatocellular carcinoma cell lines Li7 and Huh7.

[0012] Figure 4 The results are as follows: The effect of P-gp inhibitor combined with PTX in two hepatocellular carcinoma cell lines Li7 and Huh7.

[0013] Figure 5 The knockdown effect of siRNA targeting CYP1B1 or P-gp in two hepatocellular carcinoma cell lines, Li7 and Huh7.

[0014] Figure 6 This is the administration effect of the combination of siRNA and PTX in liver cancer cells Huh7.

[0015] Figure 7 The effect of the combination of siRNA and PTX on cell cycle arrest in liver cancer cells Huh7. DETAILED DESCRIPTION

[0016] The present invention is further described with reference to the accompanying drawings and embodiments.

[0017] Example 1 Increased expression of CYP1B1 and P-gp proteins in hepatocellular carcinoma cell lines Li7 and Huh7 under hypoxic conditions

[0018] Reagents and materials:

[0019] Hepatocellular carcinoma cell lines Li7 and Huh7 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. Li7 was cultured in RPMI-1640 medium (CORNING, Catalog No. 10-013-CV, supplemented with 0.11 g / L sodium pyruvate) with 10% fetal bovine serum, while Huh7 was cultured in DMEM medium (CORNING, Catalog No. 10-013-CV) with 10% GBICO fetal bovine serum. All cultures were maintained at 37°C in 5% CO2. Anhydrous cobalt chloride was purchased from Aladdin (Catalog No. 7646-79-9); anaerobic gas generation bags and anaerobic jars were purchased from Mitsubishi (Catalog No. GEN-C-1).

[0020] Experimental methods:

[0021] Li7 and Huh7 cells were treated with physical hypoxia and chemical hypoxia as hypoxic conditions. Physical hypoxia was to culture the cells in an anaerobic jar with 1% O2 for 24 hours, while chemical hypoxia was to add CoCl2 with a final concentration of 100 and 200 μM to the culture medium for 24 hours. The normoxia group (negative control group) did not receive any treatment and was simply cultured in an incubator for the same period of time. After a certain period of culture, the cell sample protein was extracted, and RIPA lysis buffer was pre-cooled on ice. Before use, the protease inhibitor PMSF with a final concentration of 1 mmol / L, Leupeptin with a final concentration of 0.5 μg / mL, and Pepstatin with a final concentration of 0.7 μg / mL were added. An appropriate amount of lysis buffer was added to each cell sample, placed in a chromatography cabinet at 4°C and gently rotated for 2 hours to fully lyse, and then centrifuged at 13,000 rpm for 10 minutes. The supernatant was stored at -80°C or used for subsequent protein quantification. The OD value was measured using a UV spectrophotometer. 280 Protein concentrations were quantified by relative readings. The concentrations of the cell protein solutions were adjusted to be the same, and protein abundance was determined by Western blot. β-actin was used as an internal control for the cell sample protein.

[0022] Experimental results:

[0023] like Figure 1 As shown in the data, the protein expression of HIF-1α (hypoxia-inducible factor-1α) increased in the two hepatocellular carcinoma cell lines after hypoxia treatment, indicating successful hypoxia induction, while the protein expression levels of CYP1B1 and P-gp increased significantly in a concentration-dependent and time-dependent manner.

[0024] Example 2

[0025] The effect of paclitaxel on hepatocellular carcinoma cell lines under hypoxic conditions is not as good as that under normoxic conditions. The sensitivity of hepatocellular carcinoma cells to paclitaxel under hypoxic conditions is not as good as that under normoxic conditions, and the drug resistance is increased.

[0026] Reagents and materials:

[0027] Paclitaxel was purchased from Nantong Feiyu (item number FY35382), CCK-8 kit was purchased from Xinsaimei (item number C6005), and other cell culture and hypoxia treatment reagents were the same as in Example 1.

[0028] Experimental methods:

[0029] 1. Preparation of paclitaxel stock solution: Prepare paclitaxel into a 1 mM stock solution using DMSO as solvent and store at -20°C;

[0030] 2. Cell Culture and Drug Addition: Li7 (3000 cells / well) and Huh7 (5000 cells / well) cells were seeded at a specific density in 96-well plates and cultured overnight. The next day, after 24 hours of adherence, the PTX-containing culture medium was serially diluted at a ratio of 1:2 or 1:4. Multiple concentration points were set, with 6 replicates per well. 100 μL of culture medium containing 10 μL of the predetermined PTX concentration was added to the replicate wells corresponding to each concentration point. The final DMSO concentration in each culture system was 0.1%. The negative control group was the experimental group that received only 0.1% DMSO. Fresh drug-containing culture medium was replaced every 24 hours. Normoxia / physical hypoxia treatment was terminated after 48 hours.

[0031] Table 1 Final concentration of paclitaxel in the two cell lines

[0032] Li7 Huh7 PTX (nmol / L) PTX (nmol / L) 15.625 3.90625 62.5 15.625 250.0 62.5 500.0 250.0 1000.0 500.0 1500.0 1000.0 3000.0 /

[0033] 3. Cell activity assay:

[0034] After drug treatment, add 100 μL of fresh culture medium containing 10 μL of CCK-8 solution to each well of the 96-well plate and incubate at 37°C in the dark for 1 to 2 hours. Read the absorbance at 450 nm and 650 nm (650 nm is the reference wavelength) using a microplate reader to calculate the cell viability at each concentration point using the following formula:

[0035] Cell survival rate = experimental group (A 450nm -A 650nm ) / control group (A 450nm -A 650nm )×100%.

[0036] 4. Experimental results:

[0037] The dose-effect curves of paclitaxel on Li7 ​​and Huh7 cells under normoxia and hypoxia are shown in Figure 2. Figure 2 As shown in the results, HCC is less sensitive to paclitaxel under hypoxic conditions than under normoxic conditions. This suggests that paclitaxel is less effective in HCC cell lines under hypoxic conditions than under normoxic conditions. HCC cells become more drug-resistant under hypoxic conditions, so finding new dosing regimens is of practical significance for solving this problem.

[0038] Example 3 Inhibition of P-gp efflux function can enhance the toxic effect of paclitaxel on liver cancer cell lines

[0039] Reagents and materials:

[0040] Elacridar (EcD) was ordered from MCE (Cat. No. 143664-11-3), cyclosporin A (CsA) was ordered from Maclean (Cat. No. 59865-13-3), and rhodamine 123 (Rho123) was ordered from Sigma-Aldrich (Cat. No. 62669-70-9). The remaining experimental reagents were the same as in Examples 1 and 2.

[0041] Experimental methods:

[0042] Preparation of Elacrida and Cyclosporine A stock solutions:

[0043] Using DMSO as solvent, elclair and cyclosporine A were prepared into 10 mM stock solutions and stored at -20°C.

[0044] P-gp activity verification experiment:

[0045] Prepare HBSS buffer, sterilize at high temperature, cool to room temperature before use, and store at 4°C. Rhodamine 123, a well-known classic substrate, was selected as a positive fluorescent probe substrate for P-gp. Cell seeding, drug addition, and fluorescence detection: Each experimental group was seeded in triplicate in 24-well plates with 1×10^5 hepatocellular carcinoma cells, Li7 or Huh7, per well and cultured for 48 h. The culture medium was discarded, and cells were washed with 37°C preheated HBSS. The HBSS was removed, and HBSS containing 10 μM Rho123 (or Rho123 and a P-gp inhibitor) was added, incubating for 2 h. The incubation medium was discarded, and the cells were washed three times with ice-cold PBS. 200 μL of cell lysis buffer (containing 0.5 M NaOH and 0.1% Triton) was added to each well. After repeated pipetting, the cell lysate was collected, and the supernatant was measured for fluorescence using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0046] Study on the cytotoxicity of combined use of P-gp inhibitors and PTX:

[0047] Li7 (3000 cells / well) and Huh7 (5000 cells / well) cells were seeded into 96-well plates at a certain density and cultured overnight. Since 2μM cyclosporine A and 1μM elacrida treatment are non-cytotoxic and can inhibit the efflux function of P-gp, the cyclosporine A / elacrida and PTX combined group was prepared by diluting PTX in a concentration gradient with complete medium containing 2μM cyclosporine A / 1μM elacrida and 0.1% DMSO; the PTX alone group was prepared by diluting PTX in a concentration gradient with complete medium containing 0.2% DMSO; the final DMSO concentration in each group of culture systems was 0.2%, 6 replicates were set for each concentration point, and new drug-containing medium was replaced every 24 hours. The subsequent cell activity detection method is the same as in Example 2.

[0048] Experimental results:

[0049] like Figure 3 As shown in Figure 2, 2 μM cyclosporine A and 1 μM elacridar can inhibit the efflux function of P-gp and have no toxicity to liver cancer cells. Figure 4 As shown in the results, the combination of P-gp inhibitors and PTX can sensitize the toxic effects of paclitaxel on liver cancer cell lines.

[0050] Example 4

[0051] siRNAs (SEQ ID NO.1 to SEQ ID NO.4) ordered for two different targets can effectively knock down the mRNA and protein expression levels of CYP1B1 and P-gp in two hepatocellular carcinoma cell lines Li7 and Huh7.

[0052] Reagents and materials: siRNA was ordered from Genemab, and the sequences are shown in Table 2 below.

[0053] Table 2

[0054]

[0055]

[0056] The remaining experimental reagents are the same as those in Examples 1 and 2.

[0057] Experimental methods:

[0058] 1. Preparation of siRNA stock solution:

[0059] Chemically synthesized siRNA should be stored as a lyophilized powder at -20°C. Before use, centrifuge at 12,000 g for 1 minute at 4°C. Dissolve and mix thoroughly with the volume indicated in the instructions for use in DEPC-treated water to obtain a 20 μM stock solution. Aliquot and store frozen at -20°C. Use within one month.

[0060] 2. Cell transfection with siRNA:

[0061] Li7 and Huh7 cells were seeded into 6-well plates at a density of about 50% and placed in a cell culture incubator overnight. Transfection of siRNA was carried out according to the instructions of the transfection reagent as follows: 200 μL per well Buffer was diluted to siRNA to be transfected (final concentration 40nM) and pipetted to mix, then 4μL was added to the system Reagent and vortex for 10 seconds to mix the system. After incubation at room temperature for 10-15 minutes, add the transfection system to a 6-well plate. After 24 hours of transfection, replace with fresh complete medium according to cell growth conditions. After 48 hours of transfection, collect cells for subsequent gene expression analysis.

[0062] Detect expression changes at the mRNA and protein levels:

[0063] Cellular RNA was extracted according to the instructions of the RNA miniprep kit. The extracted RNA was reverse transcribed into cDNA, and then quantitative reverse transcription PCR was performed to analyze the mRNA expression of the target gene. The method for extracting cell sample protein was the same as in Example 1. β-actin was used as an internal reference for cell sample mRNA and protein.

[0064] 3. Experimental results:

[0065] like Figure 5 As shown in the figure, the two siRNAs ordered for the two targets can reduce the gene expression of drug metabolizing enzyme CYP1B1 and drug transporter P-gp in liver cancer cells. Among them, siCYP1B1#1 and siP-gp#1 have better knockdown effects, so these two siRNAs were selected for subsequent experimental studies.

[0066] Example 5

[0067] The combined use of siCYP1B1 and siP-gp can sensitize the toxic effect of paclitaxel on liver cancer cell Huh7 more than the use of siCYP1B1 or siP-gp alone.

[0068] Reagents and materials:

[0069] siCYP1B1#1 and siP-gp#1 were the same as those in Example 4, paclitaxel was the same as that in Example 2, the cck-8 kit was the same as that in Example 2, and the other experimental reagents were the same as those in Example 1.

[0070] Experimental methods:

[0071] 1. Cell seeding, transfection, and drug administration:

[0072] Approximately 1×10^4 Huh7 cells were seeded in each well of a 24-well plate, with three replicate wells set up for each experimental group. After the cells adhered to the wall and cultured for 24 hours, they were transfected with siRNA. 24 hours after transfection, the drug was administered for 48 hours, and fresh drug-containing culture medium was replaced every 24 hours.

[0073] 2. Cell activity assay:

[0074] After drug treatment was terminated, 300 μL of fresh culture medium containing 30 μL of CCK-8 solution was added to each well of the 24-well plate, and the subsequent cell activity detection method was the same as in Example 2.

[0075] 3. Experimental results:

[0076] like Figure 6As shown in the results, the combined use of siCYP1B1 and siP-gp can enhance the toxic effect of paclitaxel on liver cancer cell Huh7 more than the use of siCYP1B1 or siP-gp alone.

[0077] Example 6

[0078] Compared with the administration of PTX alone, the use of siCYP1B1 or siP-gp to knock down CYP1B1 or P-gp can further enhance the cell cycle arrest effect of paclitaxel on liver cancer cells Huh7. The combined use of siCYP1B1 and siP-gp to simultaneously knock down CYP1B1 and P-gp can also further enhance the cell cycle arrest effect of paclitaxel on liver cancer cells Huh7.

[0079] Reagents and materials:

[0080] siCYP1B1#1 and siP-gp#1 were the same as those in Example 4, paclitaxel was the same as in Example 2, the cell cycle and apoptosis detection kit was purchased from Beyotime (C1052), and the other experimental reagents were the same as those in Example 1.

[0081] Experimental methods:

[0082] 1. Cell seeding, transfection, and drug administration:

[0083] Huh7 cells were seeded in 6-well plates at a density of 50%, with three replicate wells for each experimental group. After the cells adhered to the plate for 24 hours, they were transfected with siRNA. 24 hours after transfection, the medium was changed and the drug was administered for 24 hours.

[0084] 2. Cell cycle assay:

[0085] Following the instructions for the Biyuntian Cell Cycle Assay Kit, cells were digested after drug treatment and centrifuged at 1000 g for 4 minutes. The supernatant was discarded and the pellet was washed once with ice-cold PBS. 1 mL of ice-cold 70% ethanol was slowly added dropwise to the cell pellet, mixed thoroughly by pipetting, and fixed at 4°C for at least 24 hours. Following fixation, cells were centrifuged at 1000 g for 4 minutes, washed twice with ice-cold PBS, and then 500 μL of PI staining solution was added. After incubation in a 37°C water bath in the dark for 30 minutes, cell cycle analysis was performed using an ACEA NovoCyte™ series flow cytometer. Data were processed using ModFit FL 5.0 software.

[0086] 3. Experimental results:

[0087] like Figure 7As shown in the results, PTX mainly arrests liver cancer cells Huh7 in the G2 / M phase. Knockdown of CYP1B1 or P-gp alone can further enhance the cell cycle arrest effect of PTX on liver cancer cells Huh7, and the proportion of G2 / M phase increases; knockdown of both CYP1B1 and P-gp can also further enhance the cell cycle arrest effect of paclitaxel on liver cancer cells Huh7, and the proportion of G2 / M phase increases.

Claims

1. Use of a siRNA and paclitaxel composition in the preparation of a drug for treating hepatocellular carcinoma, characterized in that: The composition is composed of siCYP1B1 with a final concentration of 20 nM, siP-gp with a final concentration of 20 nM, and paclitaxel with a final concentration of 100 nM or 10 nM in a ratio of 1:1:

1. The sequence of the siCYP1B1 is shown in SEQ ID NO.1, and the sequence of the siP-gp is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The siCYP1B1 is an siRNA targeting CYP1B1, and the siP-gp is an siRNA targeting P-gp.

3. The use according to claim 1, characterized in that The application is to knock down CYP1B1 and P-gp through siRNA targeting, thereby reducing the expression of CYP1B1 and P-gp, reversing the resistance of hepatocellular carcinoma to paclitaxel, and enhancing the cell cycle arrest effect mediated by paclitaxel.

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