Adjuvant therapeutic drugs and applications for liver cancer drugs and therapeutic liver cancer drug mixtures

Abstract

Currently, the treatment regimens for most tumors are surgical resection assisted by chemotherapy, and chemotherapy resistance has now become an important factor affecting the prognosis of cancer patients. The present invention provides a new method for treating liver cancer by inhibiting FADS2 to enhance the drug sensitivity of liver cancer cells. The present invention finds that the expression level of FADS2 is up-regulated in liver cancer drug-resistant cells, and survival analysis shows that the high expression of FADS2 is related to the poor prognosis of liver cancer. Inhibiting FADS2 can significantly enhance the drug sensitivity of liver cancer cells, thereby achieving the purpose of treating liver cancer. The research results of the present invention not only provide a theoretical basis for the clinical treatment of liver cancer, but also can provide new drug targets for the development of liver cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and tumor prevention and treatment, and more specifically, to the field of treating drug resistance in liver cancer. The present invention provides a new tumor resistance marker. Fatty Acid Desatyrase 2 (FADS2), which is highly expressed in tumor tissue and contributes to tumor resistance, is used as a target for screening drugs targeting this protein and its related molecules for treating tumor resistance. Background Art

[0002] Liver cancer, one of the most common malignant tumors in my country, is characterized by high morbidity and mortality. Currently, chemotherapy remains one of the main treatments for liver cancer. However, the development of chemotherapy drug resistance during treatment is a major obstacle to treatment effectiveness. Therefore, to improve the efficacy of chemotherapy for liver cancer, studying the mechanisms of liver cancer resistance, identifying key targets for tumor resistance, and designing new drugs that enhance liver cancer treatment sensitivity are key research areas in this field.

[0003] FADS2 is a membrane protein composed of 444 amino acids with a molecular weight of 52.2 kDa; it contains a cytochrome b5-like domain, two transmembrane domains, and three histidine-rich domains (regions I, II, and III). FADS2 is abnormally expressed in malignant tumors such as breast cancer, lung cancer, liver cancer, esophageal cancer, and melanoma. Furthermore, high FADS2 expression is significantly correlated with tumor proliferation, cell migration and invasion, angiogenesis, radiotherapy resistance, clinical stage, and prognosis. FADS2 can also influence tumor cell behavior by regulating the production of metabolites in the tumor microenvironment or altering the phospholipid composition of tumor cell membranes. However, the function and mechanism of action of FADS2 in sorafenib-resistant liver cancer cells have not been reported. To investigate the role of FADS2 in sorafenib-resistant liver cancer cells, the present invention employed mass spectrometry-based quantitative proteomics screening and biological function studies, using Western blotting, plate cloning, and other methods to examine the effects of FADS2 on the proliferation and apoptosis of sorafenib-resistant liver cancer cells. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a method for treating liver cancer by inhibiting FADS2 to enhance the drug sensitivity of liver cancer cells. By inhibiting FADS2, the sensitivity of drug-resistant liver cancer cells to drugs is restored, thereby achieving the purpose of treating liver cancer.

[0005] The second purpose of the present invention is to provide a molecular target for detecting drug resistance of liver cancer, thereby providing an effective tool for detecting drug resistance of liver cancer.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for inhibiting FADS2, which is achieved by an inhibitor.

[0008] Furthermore, the inhibitors include: anti-FADS2 antibodies, small molecule inhibitors, RNA interference molecules or antisense oligonucleotides targeting FADS2 coding sequences for FADS2 protein inhibition.

[0009] Furthermore, the anti-FADS2 antibodies include: monoclonal antibodies, polyclonal antibodies and antibody fragments with immunological activity that have immunological activity against FADS2 or its active fragments and can specifically recognize and bind to the amino acid sequence or spatial structure of FADS2.

[0010] The antibodies of the present invention can be detected by immunoprecipitation, immunohistochemistry or immunofluorescence.

[0011] The small molecule inhibitors of the present invention can be prepared into various dosage forms as needed and can be administered alone or in various combinations.

[0012] The RNA interference molecule targeting the FADS2 coding sequence of the present invention is selected from shRNA, siRNA, miRNA, and dsRNA.

[0013] Furthermore, the preferred sequence of the RNA interference molecule siRNA is siRNA1, sense strand: 5'-ACGGCAAGAACUCAAAGAUTT-3' and antisense strand: 5'-AUCUUUGAGUUCUUGCCGUGG-3';

[0014] siRNA2, sense strand: 5'-ACCUGUCUGUCUACAGAAATT-3' and antisense strand:

[0015] 5'-UUUCUGUAGACAGACAGGUGG-3';

[0016] siRNA3, sense strand: 5′-ACGGCAAGAACUCAAAGAUTT-3′ and antisense strand: 5′-AUCUUUGAGUUCUUGCCGUGG-3′.

[0017] The RNA interference molecule siRNA of the present invention uses a transient transfection method.

[0018] The antisense oligonucleotides of the present invention specifically bind to FADS2 gene DNA or mRNA to inhibit FADS2 gene expression, and are molecular drugs that regulate at the gene level, including antisense DNA and antisense RNA that are artificially synthesized or expressed in vivo.

[0019] The present invention provides a method for detecting FADS2, including: immunoblotting detection based on anti-FADS2 antibodies, immunofluorescence detection based on anti-FADS2 antibodies, immunohistochemistry detection based on anti-FADS2 antibodies, targeted mass spectrometry detection and non-targeted mass spectrometry detection.

[0020] The liver cancer described in the present invention includes: primary liver cancer, secondary liver cancer, and in vitro cultured cells of primary liver cancer or secondary liver cancer.

[0021] Furthermore, primary liver cancer includes: hepatocellular carcinoma, intrahepatic bile duct carcinoma, and hepatocellular carcinoma mixed with intrahepatic bile duct carcinoma; secondary liver cancer includes: intestinal cancer, pancreatic cancer metastasis, gallbladder cancer, and bile duct carcinoma.

[0022] The drugs described in the present invention include: cisplatin, gefitinib, doxorubicin, paclitaxel, 5-fluorouracil, sorafenib, cediranib, pazopanib, axitinib, vatalanib, semasanib, sunitinib, ramucirumab and aflibercept.

[0023] The present invention provides a new method for treating liver cancer by inhibiting FADS2, thereby enhancing the drug sensitivity of liver cancer cells. The present invention found that FADS2 expression is upregulated in drug-resistant liver cancer cells, and survival analysis showed that high FADS2 expression is associated with poor prognosis of liver cancer. Inhibiting FADS2 can significantly enhance the drug sensitivity of liver cancer cells, thereby achieving the purpose of treating liver cancer. The research results of the present invention not only provide a theoretical basis for the clinical treatment of liver cancer, but also provide new drug targets for the development of liver cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 FADS2 protein is highly expressed in HepG2-R liver cancer cells resistant to sorafenib;

[0025] Figure 2 .siRNA1 and siRNA2 transfection knocked down FADS2 protein expression levels;

[0026] Figure 3 .Plate cloning experiments showed that inhibiting FADS2 expression increased the sensitivity of drug-resistant liver cancer cells to sorafenib. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to specific examples. In the following examples, the sorafenib-resistant strain HepG2-R was constructed according to the literature method (Yeh, CC; Hsu, CH; Shao, YY; Ho, WC Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer. Mol Cell Proteomics. 14(6)(2015)1527-45.); experimental methods without specific conditions are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The percentages in the solutions used are all volume ratios unless otherwise specified.

[0028] Example 1. FADS2 protein is highly expressed in HepG2-R liver cancer cells resistant to sorafenib

[0029] To investigate the expression of FADS2 in normal liver cancer cells and drug-resistant liver cancer cells, label-free quantitative proteomic analysis of HepG2 and sorafenib-resistant HepG2-R cells revealed significantly higher FADS2 abundance in HepG2-R compared to HepG2. Western blotting (see Molecular Cloning) was used to further examine FADS2 protein expression in both cell lines.

[0030] HepG2-R cells and HepG2 cells were cultured in 10 cm culture dishes respectively. When the cell density reached 80%-90% (5×10 6Cells were first washed three times with 6 ml of PBS (pH 7.4) (purchased from Gibco, USA). 300 μl of RIPA lysis buffer (purchased from Shanghai Biotech Co., Ltd.; the main components of the lysis buffer are 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and various inhibitors such as sodium orthovanadate, sodium fluoride, EDTA, and leupeptin) were added. The cells were lysed on ice for 15 minutes and centrifuged at 12,000 g for 15 minutes. The precipitate was removed to obtain the protein supernatant. Protein concentration was determined using a BCA kit (purchased from Shanghai Biotech Co., Ltd.). FADS2 protein expression levels were detected by Western blotting, with 50 μg of protein sample loaded per lane. Bio-rad electrophoresis apparatus and Bio-rad membrane transfer apparatus were purchased from Bio-rad, USA. FADS2 antibody was purchased from Gene Tex, USA, catalog number GTX64748. HSP90 antibody was used as an internal control and was purchased from Santa Cruz, catalog number SC-7947.

[0031] The results showed that the protein expression level of FADS2 in sorafenib-resistant liver cancer cells HepG2-R was significantly higher than that in the control cells HepG2, indicating that the expression of FADS2 is associated with liver cancer resistance.

[0032] Example 2. Inhibition of FADS2 expression increases the sensitivity of drug-resistant liver cancer cells to sorafenib

[0033] 1. siRNA design and synthesis

[0034] siRNA1 was designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. based on the FADS2 gene sequence, and a negative control siRNA (siRNA NC) with no sequence homology to the FADS2 gene was also provided.

[0035] siRNA NC

[0036] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3'

[0037] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'

[0038] siRNA1FADS2 sense strand: 5′-ACGGCAAGAACUCAAAGAUTT-3′;

[0039] Antisense strand: 5′-AUCUUUGAGUUCUUGCCGUGG-3′;

[0040] siRNA2FADS2, sense strand: 5′-ACCUGUCUGUCUACAGAAATT-3′;

[0041] Antisense strand: 5′-UUUCUGUAGACAGACAGGUGG-3′;

[0042] 2. Cell Transfection

[0043] HepG2-R cells (10 5 ) were plated into six-well plates. The control group was transfected with siRNA NC, and the experimental group was transfected with siRNA1 FADS2 and siRNA2 FADS2. When the cell confluence in each well reached 60%, the culture medium was discarded, and 1 mL of Opti-MEM culture medium was added for starvation treatment. 10 μl of RNAiMAX (purchased from Invitrogen) was added to each 1 mL of Opti-MEM culture medium, mixed evenly, and allowed to stand in a clean bench for 5 minutes. 2 μl of siRNA was added to each 300 μl of Opti-MEM culture medium, mixed evenly, and allowed to stand in a clean bench for 5 minutes. 300 μl of siRNA dilution was transferred to 300 μl of transfection reagent dilution, pipetted to mix evenly, and allowed to stand in a clean bench for 20 minutes. Discard the starved Opti-MEM, take the mixture of transfection reagent and siRNA and plate it into a 6-well plate, and then add 1.4 ml of Opti-MEM culture medium to make the final siRNA concentration 0.02 μM. Then place the 6-well plate in a cell culture incubator. After 6 hours, the cells were replaced with ordinary DMEM medium and cultured for 48 hours. The protein expression level was detected by immunoblotting. Protein expression level was detected by Western blotting (refer to "Molecular Cloning"). FADS2 antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA), catalog number HPA006741. HSP90 antibody was used as an internal control and was purchased from Santacruz Biotech, USA, catalog number SC-13119. The results of Western blotting showed that after transfection, the expression level of FADS2 in the experimental group was lower than that in the control group ( Figure 2 ), indicating that the transfection was successful and siRNA1 and siRNA2 could knock down the expression of FADS2.

[0044] 3. Plate cloning

[0045] HepG2-R hepatocellular carcinoma cells resistant to sorafenib were cultured in 10 cm culture dishes. 0.5 ml of trypsin was added for 2 min during the logarithmic growth phase. 6 ml of DMEM medium was added to the cells, and the cells were counted and diluted. 2 ml of cell solution (containing 10 4 cells) into each well of a six-well plate. After 48 hours, when the cells adhered to the wall and grew well, cell transfection was performed. The control group was transfected with siRNA NC, and the experimental group was transfected with siRNA1 FADS2 and siRNA2FADS2. When the cell confluence in each well reached 60%, the culture medium was discarded, the DMEM culture medium was discarded, and 1 mL of Opti-MEM culture medium was added to each well for starvation treatment. 10 μl of RNAiMAX (purchased from Invitrogen) was added to each 1 mL of Opti-MEM culture medium, mixed evenly, and allowed to stand in a clean bench for 5 minutes. 2 μl of siRNA was added to each 300 μl of Opti-MEM culture medium, mixed evenly, and allowed to stand in a clean bench for 5 minutes. Transfer 300 μl of siRNA dilution to 300 μl of transfection reagent dilution, pipet to mix evenly, and let stand in a clean bench for 20 minutes. Discard the starvation-treated Opti-MEM, take the transfection reagent and siRNA mixture and place it into a 6-well plate, and then add 1.4ml of Opti-MEM medium to make the final siRNA concentration 0.02μM. Place the 6-well plate in a cell culture incubator. After 6 hours, replace it with ordinary DMEM medium. After continuing to culture for 24 hours, add sorafenib to the final concentration of 0μM and 8μM respectively, continue to culture the cells, and observe the cell growth status. After 6 days, perform crystal violet staining (0.01% crystal violet staining solution: 270ml pure water + 30ml methanol + 0.3g crystal violet). Use 500μl of 20% acetic acid to dissolve crystal violet for 20 minutes, and detect the absorbance at 590nm.

[0046] The results showed that as the concentration of sorafenib increased, the cell survival rates in the siRNA1 FADS2 and siRNA2 FADS2 groups were significantly lower than those in the siRNA NC group, and the differences in each group were statistically significant (*p<0.05) ( Figure 3 ), the results showed that inhibiting FADS2 expression increased the sensitivity of liver cancer resistant cells to sorafenib and improved the therapeutic effect of sorafenib on liver cancer.

[0047] Example 3. FADS2 small molecule inhibitor SC 26196 increases the sensitivity of liver cancer resistant cells to sorafenib

[0048] HepG2-R and Huh7-R cells were cultured in 10 cm culture dishes respectively. 0.5 ml of trypsin was added for 2 min during the logarithmic growth phase. 6 ml of DMEM medium was added to the cells and the cells were diluted after counting. 2 ml of cell solution (containing 10 4Cells were plated in each well of a six-well plate. After 48 hours, when cells were well attached and growing, Huh7-R cells were treated with equal volumes of DMEM medium without or with the small molecule inhibitor SC26196 (purchased from Santa Cruz) to a final concentration of 0 μM (added) or 10 μM. HepG2-R cells were treated with equal volumes of DMEM medium without or with the small molecule inhibitor SC26196 to a final concentration of 0 μM or 30 μM. Cell growth was monitored and cultured for approximately 6 days. Crystal violet staining was performed (0.01% crystal violet staining solution: 270 ml of pure water + 30 ml of methanol + 0.3 g of crystal violet). Crystal violet was dissolved in 500 μl of 20% acetic acid for 20 minutes, and absorbance was measured at 590 nm.

[0049] The results showed that as the concentration of FADS2 small molecule inhibitor increased, the cell survival rate in the 10μM and 30μM groups was significantly lower than that in the 0μM group. The results indicated that the FADS2 small molecule inhibitor SC 26196 increased the sensitivity of liver cancer resistant cells to sorafenib and improved the therapeutic effect of sorafenib on liver cancer.

[0050] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention. Sequence Listing <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> Adjuvant therapy drug for liver cancer and its application and drug mixture for treating liver cancer <140> 2021113935163 <141> 2021-11-23 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 1 acggcaagaa cucaaagaut t 21 <210> 2 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 2 aucuuugagu ucuugccgug g 21 <210> 3 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 3 accugucugu cuacagaaat t 21 <210> 4 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 4 uuucuguaga cagacaggug g 21 <210> 5 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 5 acggcaagaa cucaaagaut t 21 <210> 6 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 6 aucuuugagu ucuugccgug g 21

Claims

1. A drug for the adjuvant treatment of liver cancer, which contains as its active ingredient a FADS2 protein inhibitor of liver cancer cells; The FADS2 protein inhibitor is an RNA interference molecule siRNA targeting the FADS2 coding sequence; The sequence of RNA interference molecule siRNA is one or both of the following: siRNA1, sense strand: 5′- ACGGCAAGAACUCAAAGAUTT -3′ and antisense strand: 5′-AUCUUUGAGUUCUUGCCGUGG -3′; siRNA2, sense strand: 5′-ACCUGUCUGUCUACAGAAATT-3′ and antisense strand: 5′-UUUCUGUAGACAGACAGGUGG-3′; The liver cancer drug used in the auxiliary therapeutic drug for auxiliary liver cancer treatment is sorafenib, which can restore the sensitivity of liver cancer resistant cells to the drug and improve its efficacy.

2. Use of a FADS2 protein inhibitor in the preparation of an adjuvant therapeutic drug for restoring the sensitivity of drug-resistant liver cancer cells to drugs, wherein the FADS2 protein inhibitor is one or more of siRNA1, siRNA2, or the FADS2 small molecule inhibitor SC 26196 described in claim 1, and the liver cancer drug used in the adjuvant therapeutic drug for restoring the sensitivity of drug-resistant liver cancer cells to drugs is sorafenib.

3. A drug mixture for treating liver cancer, comprising a FADS2 protein inhibitor and the liver cancer drug sorafenib; the FADS2 protein inhibitor is one or more of siRNA1, siRNA2 or the FADS2 small molecule inhibitor SC 26196 described in claim 1.

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