Application of coniferyl aldehyde in preparation of anti-cancer drugs

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of coniferyl aldehyde in preparation of anti-cancer drugs. The invention discloses application of coniferyl aldehyde in preparation of anti-cancer drugs, coniferyl aldehyde can significantly inhibit invasion and migration ability of hepatocellular carcinoma cells, cancer cells cannot break through primary parts through key links such as interference of movement and adhesion of the cancer cells and degradation of extracellular matrixes, invasion of tumors to surrounding tissues is reduced, and the anti-cancer effect is improved. The occurrence rate of remote metastasis is reduced, the non-metastasis lifetime of the patient is prolonged, and more treatment time and survival opportunities are won for the patient; a large number of cell experiments and animal experiments prove that the medicine has no obvious liver and kidney toxicity under the treatment dosage, the tolerance of a patient to treatment is improved, the smooth proceeding of the treatment process is guaranteed, and the living quality of the patient is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of coniferyl aldehyde in the preparation of anti-cancer drugs. Background Art

[0002] Liver cancer is the third most common cause of cancer death globally, and the number of deaths continues to rise, posing a major challenge to global public health. Primary liver cancer can be divided into hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), angiosarcoma, hepatoblastoma, etc. Hepatocellular carcinoma originates from the main parenchymal cells of the liver - hepatocytes, and is the most common form of liver cancer clinically, accounting for about 75%-80% of the total cases. Most patients with hepatocellular carcinoma have a background of chronic liver diseases, such as chronic hepatitis B virus or hepatitis C virus infection, alcoholic hepatitis, non-alcoholic fatty liver disease or cirrhosis, and aflatoxin, etc.

[0003] Traditional treatment methods for hepatocellular carcinoma include surgical resection, liver transplantation, interventional therapy, radiofrequency ablation, radiotherapy and drug therapy. Currently, surgical resection remains the main and most effective way for the early treatment of hepatocellular carcinoma, but its five-year survival rate is only 50%-70%. For patients with middle and advanced liver cancer, systemic treatment mediated by drug therapy is the main treatment method, which can effectively improve the survival period. Traditional chemotherapy drugs, such as doxorubicin, etc., act on the DNA synthesis, mitosis, etc. of cancer cells by using cytotoxic drugs, and inhibit the growth and proliferation of cancer cells; conventional targeted therapy drugs, taking sorafenib as an example, mainly act on multiple signal transduction pathways of tumor cells, such as the RAF / MEK / ERK signal pathway and vascular endothelial growth factor receptor (VEGFR), etc., and inhibit the proliferation of tumor cells and tumor angiogenesis. However, whether it is traditional chemotherapy drugs or conventional targeted therapy drugs, after being used for a period of time, cancer cells are prone to develop drug resistance, resulting in a gradual decrease in the drug efficacy and even the loss of therapeutic effect, limiting the long-term therapeutic effect; moreover, while traditional chemotherapy drugs kill cancer cells, they also cause great damage to normal cells, leading to serious adverse reactions in patients such as hair loss, nausea, vomiting, bone marrow suppression, etc., reducing the quality of life and the tolerance to treatment of patients. In addition, for patients with middle and advanced hepatocellular carcinoma, the improvement of the overall survival rate and progression-free survival period by using traditional chemotherapy or conventional targeted therapy drugs alone is not significant enough to meet the clinical needs.

[0004] Current treatment methods for hepatocellular carcinoma are difficult to effectively contain the invasion and migration of cancer cells, leading to tumor spread and disease deterioration. During the treatment process, traditional anti-cancer drugs cause serious damage to the liver and kidneys, affecting the liver and kidney functions of patients. This not only reduces the quality of life of patients but may also lead to the interruption of treatment. Therefore, there is an urgent need to develop a drug that can precisely act on the molecular mechanisms and signaling pathways related to cancer cell invasion and migration, cut off the pathways of cancer cell metastasis, and prevent it from infiltrating into surrounding tissues and metastasizing to distant organs. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of coniferyl aldehyde in the preparation of anti-cancer drugs, aiming to solve the problems of large side effects and serious adverse reactions of existing treatment methods.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides the application of coniferyl aldehyde in the preparation of anti-cancer drugs.

[0008] Preferably, the anti-cancer drug has no hepatotoxicity and nephrotoxicity

[0009] The present invention provides the application of coniferyl aldehyde in the preparation of drugs for inhibiting the proliferation of hepatocellular carcinoma cells.

[0010] The present invention provides the application of coniferyl aldehyde in the preparation of drugs for inhibiting the invasion or migration of hepatocellular carcinoma cells.

[0011] The present invention provides the application of coniferyl aldehyde in the preparation of drugs for inducing apoptosis of hepatocellular carcinoma cells.

[0012] The present invention provides the application of coniferyl aldehyde in regulating the expression of DNAJBI or inhibiting the expression of RhoA.

[0013] On the other hand, the present invention provides an anti-cancer drug, and the active ingredient of the drug includes coniferyl aldehyde and / or coniferyl aldehyde derivatives.

[0014] The coniferyl aldehyde derivative is a compound obtained by derivative changes while the parent nucleus remains unchanged.

[0015] Preferably, the cancer is hepatocellular carcinoma.

[0016] Preferably, the dosage form of the drug includes one or more of injection, powder for injection, tablet, oral liquid, capsule, granule or infusion.

[0017] Preferably, the anti-cancer drug further includes pharmaceutically acceptable excipients.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) Highly efficient inhibition of invasion and migration: Coniferaldehyde can significantly inhibit the invasion and migration ability of hepatocellular carcinoma cells. By interfering with the key links such as cancer cell movement, adhesion and degradation of extracellular matrix, it prevents cancer cells from breaking through the primary site, reduces tumor invasion into surrounding tissues, reduces the incidence of distant metastasis, prolongs the patient's metastasis-free survival period, and gains more treatment time and survival opportunities for patients.

[0020] 2) Low toxicity ensures liver and kidney health: After a large number of cell experiments and animal experiments, the drug has no obvious liver and kidney toxicity at the therapeutic dose. This means that during the drug treatment, the liver and kidney functions can be maintained normal, avoiding liver and kidney damage caused by drug toxicity, such as elevated liver enzymes and renal failure.

[0021] 3) The application of coniferyl aldehyde provided in the present application has been proved through in vivo and in vitro experiments that coniferyl aldehyde can inhibit the growth and proliferation of liver cancer cells, increase the apoptosis of liver cancer cells, achieve a general inhibitory effect on liver cancer cells, and ultimately effectively exert an anti-liver cancer effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 Schematic diagram of the MTT method analysis of the cytotoxicity of 24 compounds on hepatocellular carcinoma cells, the differences were statistically significant by t-test: *p<0.05; **p<0.01; ***p<0.001.

[0024] Figure 2 This is a diagram showing the analysis of coniferaldehyde inhibiting the proliferation of hepatocellular carcinoma cells.

[0025] Figure 3 Schematic diagram of the inhibition of migration and invasion of HCC cells by coniferyl aldehyde.

[0026] Figure 4 The figure is a schematic diagram of coniferaldehyde-induced apoptosis of hepatocellular carcinoma cells. The right bar graph shows the statistical analysis of the apoptosis ratio. The t-test was used and the differences were statistically significant: *p<0.05; ***p<0.001.

[0027] Figure 5 Schematic diagram of the inhibitory effect of coniferyl aldehyde on tumor formation in vivo. (B) represents the hematoxylin and eosin (HE) staining of transplanted tumor tissue. The difference was statistically significant using t-test: *p<0.05.

[0028] Figure 6 Analysis chart of the non-significant hepatotoxic and nephrotoxic effects of coniferyl aldehyde. Among them, (A) represents hematoxylin and eosin (HE) staining of liver tissue. (B) represents hematoxylin and eosin (HE) staining of kidney tissue. (C, D) represent the analysis results of liver and kidney related biochemical indicators; liver function: ALT, TP, AST; kidney function: BUN, CRE, and UA. The t-test was used, and ns. indicates no significant difference.

[0029] Figure 7 Analysis chart of single-cell atlas of tumor microenvironment and cell characteristics of drug response from different tissue sources in HCC. Among them, (A) Tumor microenvironment atlas of 82,615 cells from peripheral blood samples (PB, n = 2), non-tumor liver (NTL, n = 8), primary tumor (PT, n = 10), portal vein tumor thrombus (PVTT, n = 2), and metastatic lymph node (MLN, n = 1) in HCC. (B) Bubble chart showing the average expression levels of characteristic genes of each cell population. The darker the color, the higher the average expression level, and the size of the dot represents the gene expression percentage. (C) UMAP visualization of the selected cells by Scissor. Red and blue dots are cells related to coniferyl aldehyde treatment and normal phenotypes. (D) Proportion of Scissor_pos and Scissor_neg cells in each cell type. (E) Average proportion of each cell type in Scissor_pos and Scissor_neg groups.

[0030] Figure 8 Analysis chart of identification of malignant cells and cell characteristics of drug response in hepatocyte subsets. Among them, (A) UMAP shows the grouping and naming of hepatocyte subsets. (B) Bubble chart shows the average expression levels of characteristic genes of each hepatocyte subset. The darker the color, the higher the average expression level, and the size of the dot represents the gene expression percentage. (C) Heatmap of inferCNV analysis. Rows in the heatmap represent cells, columns represent genes, red indicates CNV amplification, blue indicates CNV deletion, and the darker the color, the more obvious the CNV. (D) Box plot of CNV signals of T / NK cells and hepatocyte subsets. The malignant cell population was determined by copy number variation. (E) UMAP shows malignant and non-malignant cell populations. (F) UMAP visualization of the selected cells by Scissor in hepatocyte subsets. Red and blue dots are cells related to coniferyl aldehyde treatment and normal phenotypes. (G) Number of each hepatocyte subset in the Scissor_pos group and the proportion of Scissor_pos and Scissor_neg cells in each hepatocyte subset.

[0031] Figure 9This is a figure for the identification of immune cell subsets and the analysis of the characteristics of drug-responsive cells. Among them, (A) UMAP shows the grouping and naming of T&NK cell subsets. (B) In the T&NK cell subsets, UMAP visualization of the selected cells by Scissor. Red dots and blue dots are cells related to coniferyl aldehyde treatment and normal phenotypes. (C) The bubble chart shows the average expression levels of characteristic genes in each T&NK cell subset. The darker the color, the higher the average expression level, and the size of the dot represents the gene expression percentage. (E) UMAP shows the grouping and naming of Myeloid cell subsets. (F) In the Myeloid cell subsets, UMAP visualization of the selected cells by Scissor. (G) The bubble chart shows the average expression levels of characteristic genes in each Myeloid cell subset. (I) UMAP shows the grouping and naming of Plasma B cell subsets. (J) In the Plasma B cell subsets, UMAP visualization of the selected cells by Scissor. (K) The bubble chart shows the average expression levels of characteristic genes in each Plasma B cell subset. (D, H, L) The number of each cell subset in the Scissor_pos group and the proportions of Scissor_pos and Scissor_neg cells in each cell subset.

[0032] Figure 10 This is a figure for the screening and analysis of target genes related to coniferyl aldehyde treatment. Among them, (A) The volcano plot visualizes the differentially expressed genes (DEGs) between coniferyl aldehyde treatment and the control group (avg_logFC > ±1 & adjust.p < 0.05). Red indicates upregulation, blue indicates downregulation, and the gray part does not meet the screening criteria. (B) The Venn diagram visualizes the intersection genes of the SCISSOR gene set and DEGs, which are DNAJB1, HSPA1B, HSPA1A, and MS4A7. (C) The circle plot visualizes the interaction weights between hepatocyte subsets. The thicker the line, the higher the cell-to-cell communication weight. (D) The hierarchical plot visualizes the cell-to-cell interactions in the PVR, CD96, and TIGIT signaling pathways. In the left and right figures, Source is the cell subset that emits the signal. In the left figure, Target is the Hep_Sub2 and Hep_Sub4 subsets, and in the right figure, Target is the subsets other than those in the left figure. The thicker the line, the stronger the signal. (E) The violin plot visualizes the expression of ligands / receptors in hepatocyte subsets, with different subsets distinguished by different colors.

[0033] Figure 11Figure for the analysis of the expression levels of invasion and migration genes in cell populations. Among them, (A) The UMAP plot shows the expression levels of the invasion and migration genes in 5 developmental processes in each cell type. (B) The UMAP plot shows the expression levels of genes RAC1, RAC2, RHOA, RHOB, RHOC, CDC42, CCDC88A, ARPC2, WAS, TNFAIP8L2 involved in filopodia formation in each cell type. (C) The UMAP plot shows the expression levels of genes RAC1, RAC2, RHOA, RHOB, RHOC, CDC42, CCDC88A, ARPC2, WAS, TNFAIP8L2 involved in filopodia formation in the hepatocyte subset.

[0034] Figure 12 Figure for the visualization of the molecular docking results.

[0035] Figure 13 Figure for the screening and verification analysis of the pathway by which coniferyl aldehyde affects HCC. Among them, (A) Immunohistochemistry was used to examine the protein expression of RhoA, CDC42, DNAJB1, TIGIT, and PVR. (B) Western blot was used to detect the protein expression of DNAJB1, PVR, and RhoA in Hep3B and Huh-7 cells in the CA treatment group and the control group. The t-test was used, *p < 0.05, **p < 0.01, ns. no significant difference, compared with the control group.

[0036] Figure 14 Figure for the knockdown and overexpression analysis of DNAJB1 in HCC cells. Among them, (A, B) Western blot was used to detect the protein expression of DNAJB1 and RhoA after knocking down and overexpressing DNAJB1 in Hep3B and Huh-7 cells. (C) The MTT method was used to evaluate the viability of hepatocellular carcinoma cells after knocking down and overexpressing DNAJB1. The t-test was used, ns. no significant difference.

[0037] Figure 15 Figure for the inhibition of the migration and invasion of HCC cells by coniferyl aldehyde through regulating the expression of DNAJB1. Among them, (A) Western Blot shows the protein levels in Hep3B and Huh-7 with knockdown and overexpression of DNAJB1 after treatment with coniferyl aldehyde. (B, C) Coniferyl aldehyde affects the migration of HCC cells with knockdown and overexpression of DNAJB1. (D, E) Coniferyl aldehyde affects the invasion of HCC cells with knockdown and overexpression of DNAJB1. The t-test was used, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, p < 0.001, compared with the overexpression and knockdown groups.

[0038] Figure 16Analysis diagram of coniferaldehyde affecting the migration of HCC cells by regulating the expression of RhoA. Among them, (A) The migration ability of HCC cells after treatment with Rhosin hydrochloride and CA was measured by the cell scratch assay. (B) Western blot showed the protein levels of RhoA and FAK-Src signaling pathways after treatment with CA and Rhosin hydrochloride. The t-test was used, *p<0.05, **p<0.01, compared with the control group; #p<0.05, ##p<0.01, compared with the RhoA(-) group. Detailed implementation manners

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0040] The production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art without special instructions, and their names and / or abbreviations are all conventional names in the art, and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the names and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0041] There are no special restrictions on the sources of various instruments, equipment, raw materials or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0042] Example 1

[0043] Screening of effective ingredients against hepatocellular carcinoma

[0044] The present invention analyzed the cytotoxicity of 24 compounds against hepatocellular carcinoma (HCC) by the MTT method. Finally, it was detected that coniferaldehyde (CA) had a significant inhibitory effect on the proliferation of hepatocellular carcinoma cells and low toxicity to normal hepatocytes ( Figure 1 ).

[0045] Example 2

[0046] Experiment on coniferaldehyde inhibiting the growth and proliferation of hepatocellular carcinoma cells

[0047] The present invention treated HCC cells Hep3B, Huh-7 and normal liver cells MIHA with coniferaldehyde at concentrations of 40 μM, 80 μM, 120 μM, 160 μM, and 200 μM respectively, and evaluated the cell viability by the MTT method. The results are asFigure 2 As shown in A.

[0048] Result analysis: As can be seen from Figure 2 A: There is a significant positive correlation between the concentration of CA and its anti-tumor effect, that is, the higher the concentration of CA, the stronger its inhibitory effect on HCC cell proliferation. At the same time, with the prolongation of the action time, the inhibitory effect of CA on HCC cell proliferation gradually increases.

[0049] In this invention, the monoclonal formation experiment was used to verify the effect of CA on the colony formation of HCC cells. The effects of coniferyl aldehyde at concentrations of 50 μM and 100 μM on the colony formation of normal liver cells MIHA and HCC cells Huh-7 and Hep3B were measured. Sorafenib (10 μM) was used as a positive control. The t-test showed that the differences were statistically significant: **p < 0.01; ***p < 0.001. The results are as Figure 2 shown in B.

[0050] As can be seen from Figure 2 B: Using sorafenib as a positive control, the inhibitory effect of the increase in CA concentration on HCC cell colony formation is higher.

[0051] Example 3

[0052] Experiment on the inhibition of invasion or migration of hepatocellular carcinoma cells by coniferyl aldehyde

[0053] In this invention, the Transwell migration experiment (Figures A, B) and invasion experiment (Figures C, D) were used to detect the migration ability and invasion ability of HCC cells after treatment with 80 μM, 100 μM, and 120 μM coniferyl aldehyde respectively. 10 μM sorafenib was used as a positive control. The t-test showed that the differences were statistically significant: ***p < 0.001. See Figure 3 as shown.

[0054] Result analysis: As can be seen from Figure 3 it that CA can inhibit the migration and invasion abilities of HCC cell lines, and shows a concentration-dependent manner, that is, the higher the concentration, the stronger the inhibitory effect on migration and invasion abilities.

[0055] Example 4

[0056] Experiment on the induction of apoptosis of hepatocellular carcinoma cells by coniferyl aldehyde

[0057] In this invention, HCC cells Hep3B and Huh-7 were treated with 60 μM, 100 μM coniferyl aldehyde and 10 μM sorafenib for 48 h. The apoptosis of Hep3B and Huh-7 cells was analyzed by flow cytometry. The results are as Figure 4 shown,

[0058] Result analysis: As can be seen from Figure 4It can be seen that the number of apoptotic cancer cells treated with CA increased significantly: Figure 4 A It can be seen that after treatment with CA (0 μM, 60 μM, 100 μM) and sorafenib, the apoptosis rates of Hep3B were 2.53%, 3.13%, 15.94%, and 39.65% respectively; Figure 4 B It can be seen that the apoptosis rates of Huh-7 cells were 4.13%, 6.11%, 29.44%, and 48.27% respectively.

[0059] Example 5

[0060] Effect of coniferyl aldehyde on hepatocellular carcinoma at the animal level

[0061] 1. Experiment on the ability of coniferyl aldehyde to inhibit the tumorigenicity of hepatocellular carcinoma cells

[0062] The experimental animals were nude mice with tumor transplantation models. The models were established by subcutaneous injection of the Huh-7 cell line. They were divided into a control group and a treatment group. The treatment group was treated with 35 mg / kg CA to the nude mouse model. After 25 days, the in vivo effect of CA during the growth process of hepatocellular carcinoma tumors was observed.

[0063] Result analysis: Compared with the control group, after treatment with 35 mg / kg CA, both the tumor volume and weight showed significant reduction ( Figure 5 A), and the hematoxylin-eosin (HE) staining method was used to verify whether it had the morphological characteristics of glioma. The results showed that the samples had the morphological characteristics of glioma ( Figure 5 B).

[0064] 2. Experiment on the histopathological changes of coniferyl aldehyde-induced liver and kidney

[0065] In this experiment, paraffin embedding, sectioning, and HE staining were performed on the organs (liver and kidney) of mice to observe the pathological sections of the mouse liver tissue. The schematic diagrams of the results are as shown in Figure 6 A and B.

[0066] The results showed that: From Figure 6 A, it can be seen that the pathological sections of the liver tissue showed that the morphological structures of hepatocytes, hepatic sinusoids, and bile ducts were normal, without abnormal substance deposition and inflammatory reactions, and the hepatic tissue structure and cell arrangement were regular. From Figure 6 B, it can be seen that the pathological sections of the kidney tissue showed that the kidney structures, the morphologies and functions of various tissues in the CA group and the control group were normal, without abnormal substances and immune damage.

[0067] This invention verified the effects of CA on the liver and kidney functions of mice. Using professional detection kits, the liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP) in the mouse serum, and the kidney function indicators uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN) were detected, as shown in Figure 6 Analysis diagrams of liver and kidney related biochemical indicators in groups C and D.

[0068] Result analysis: As can be seen from Figure 6 C and D, compared with the control group, there were no significant differences in the levels of ALT, AST, TP, UA, CRE, and BUN in the serum of mice in the CA treatment group after statistical analysis.

[0069] Example 6

[0070] Mechanism of action of coniferyl aldehyde against hepatocellular carcinoma

[0071] This invention selected the tumor tissues of mice in the control group and the CA treatment group, performed transcriptome sequencing by Shanghai Kangcheng Biotech Co., Ltd., and then combined with the single-cell sequencing data of clinical tissue samples of hepatocellular carcinoma to explore the target cells and target genes of CA action.

[0072] 1. Single-cell landscape of hepatocellular carcinoma

[0073] First, this invention identified 12 types of cells from the single-cell atlas of the HCC tumor microenvironment from different tissue sources through marker genes, as shown in Figure 7 A and B, and then used Scissor analysis to map the results of the mouse control group and the drug treatment group to the single-cell population, where Scissor_pos was related to the drug treatment group and Scissor_neg was related to the control group.

[0074] The results showed that Scissor_pos was mainly distributed in T / NK cells, myeloid cells, hepatocytes, fibroblasts, and plasma cells, as shown in Figure 7 C and E;

[0075] Then, hepatocytes were extracted for sub-clustering to obtain 8 types of cell sub-populations, as shown in Figure 8 A and B, and T / NK cells were used as the control group;

[0076] Finally, as shown in Figure 8 C - E, Hep_Sub4 and Hep_Sub6 were defined as non-malignant cells, and Hep_Sub1, Hep_Sub2, Hep_Sub3, Hep_Sub5, Hep_Sub7, and Hep_Sub8 were defined as malignant cells.

[0077] Mapping the Scissor results to the hepatocyte sub-populations, the results are as shown in Figure 8As shown in F-G, Hep_Sub2 and Hep_Sub4 contain a relatively high proportion of Scissor_pos cells.

[0078] 2. Identification of immune cell subsets and analysis of drug-responsive cell characteristics

[0079] The present invention performs subset analysis on the characteristics of Scissor_pos immune cells, including T cells, myeloid cells, and plasma cells.

[0080] When analyzing T&NK cell subsets, the number of principal components is selected as 35 (PC = 35), and the resolution is set to 0.8 (resolution = 0.8). Through marker gene identification, 14 cell subsets as shown in Figure 9 A-D are obtained.

[0081] When analyzing myeloid cell subsets, PC = 40 and resolution = 0.5 are selected, and 11 cell subsets as shown in Figure 9 E-H are obtained;

[0082] For the Plasma B subset, PC = 26 and resolution = 0.1 are selected, and 4 cell subsets as shown in Figure 9 I-L are obtained.

[0083] 3. Potential mechanism of coniferyl aldehyde in inhibiting the invasion and migration of hepatocellular carcinoma

[0084] The present invention obtains 120 differential genes through differential analysis between the CA control group and the drug-treated group, as shown in Figure 10 A;

[0085] Taking the intersection between the differential gene set and the Scissor gene set, four intersection genes are obtained: DNAJB1, HSPA1B, HSPA1A, and MS4A7, as shown in Figure 10 B. The present invention performs CellChat analysis on the interaction patterns between hepatocyte subsets to visualize the interaction weights between hepatocyte subsets.

[0086] The results show that in the PVR, CD96, and TIGIT signaling pathways, there are specific interacting receptor-ligand pairs between the Hep_Sub2 and Hep_Sub4 subsets, as shown in Figure 10 C-E.

[0087] The results of cell experiments show that coniferyl aldehyde has a very significant inhibitory effect on the invasion and migration of hepatocellular carcinoma cells. By collecting gene sets related to the processes of tumor metastasis (Cell_adhesion, EMT_vasculogenesis, Extracellula_matrix_decomposition, Microenvironment, Pseudopods) and mapping these gene sets to single-cell populations respectively, it is found that the Pseudopods gene set has relatively high scores in each cell population, as shown in Figure 11 Figure A; then mapping the Pseudopods gene set to the large cell population and the hepatocyte subset, it is found that the observation results of RAC1, RhoA, CDC42, and ARPC2 in the large cell population and the hepatocyte subset are consistent, and they all have relatively high expression, as shown in Figure 11 Figures B and C.

[0088] Molecular docking was used to explore the interaction and binding mode of coniferyl aldehyde with ①scissor&diffgene ②cellchat ligand / receptor ③Pseudopods gene set. A series of genes with the best docking binding energy performance were screened out, including RhoA, RHOB, RHOC, CDC42, RAC1, RAC2, DNAJB1, and PVR ( Figure 12 ).

[0089] Combined with the above analysis results, five genes, namely DNAJB1, PVR, TIGIT, CDC42, and RhoA, were confirmed as the target genes for subsequent in-depth research. Through immunohistochemistry experiments and Western blot experimental results, it was finally speculated that coniferyl aldehyde may inhibit the occurrence and development of HCC by regulating the expression of DNAJB1 and RhoA ( Figure 13 A, B), and they are two independent parallel action mechanisms ( Figure 14 B).

[0090] Example 7

[0091] Verification of the functional mechanisms of the key target genes DNAJBI and RhoA 1. In in vitro experiments, it was verified that coniferyl aldehyde inhibits the migration and invasion of HCC cells by regulating the expression of DNAJB1

[0092] In this invention, Western blot was used to detect the knockdown and overexpression of DNAJB1 in Hep3B and Huh-7 cells. The results of Transwell migration and invasion experiments showed that in the HCC cell line with overexpressed DNAJB1 ( Figure 14 A), CA can further inhibit its migration and invasion ability; at the same time, in the HCC cell line with knockdown of DNAJB1, CA can reverse the change in its migration and invasion ability ( Figure 15)。Similarly, the MTT assay was used to detect whether DNAJB1 affects the proliferation of HCC cells, and the results showed no significant effect ( Figure 14 C).

[0093] 2. Coniferyl aldehyde inhibits the migration of hepatocellular carcinoma by inhibiting the expression of RhoA

[0094] In this invention, the scratch assay was used for analysis. HCC cells were treated with a RhoA inhibitor, and their migration ability was significantly inhibited. After adding CA, the migration ability was further inhibited ( Figure 16 A), and CA itself can significantly inhibit the migration of HCC cells. These series of experimental results provide an important basis for analyzing the mechanism of CA inhibiting the migration of HCC cells.

[0095] CA also has a significant inhibitory effect on the FAK-Src signaling pathway, and this effect is highly consistent with the results obtained when using a RhoA inhibitor alone ( Figure 16 B).

[0096] Based on this experimental phenomenon, it is reasonable to infer that CA is very likely to down-regulate the expression level of RhoA, thereby inhibiting the FAK-Src signaling pathway and finally effectively curbing the migration ability of HCC cells.

[0097] In summary, in vivo and in vitro experiments have proved that coniferyl aldehyde exerts a general inhibitory effect on hepatocellular carcinoma cells by inhibiting the growth and proliferation of hepatocellular carcinoma cells and increasing the apoptosis of hepatocellular carcinoma cells, and finally effectively plays the anti-hepatocellular carcinoma effect.

[0098] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Application of coniferaldehyde in the preparation of anticancer drugs.

2. The use according to claim 1, characterized in that: The anticancer drug has no liver and kidney toxicity.

3. Application of coniferaldehyde in the preparation of drugs for inhibiting the proliferation of liver cancer cells.

4. Application of coniferaldehyde in the preparation of drugs for inhibiting the invasion or migration of liver cancer cells.

5. Application of coniferaldehyde in the preparation of drugs for inducing apoptosis of liver cancer cells.

6. Application of coniferaldehyde in regulating the expression of DNAJBI or inhibiting the expression of RhoA.

7. An anticancer drug, characterized in that: The active ingredients of the drug include coniferyl aldehyde and / or coniferyl aldehyde derivatives.

8. The drug according to claim 7, characterized in that The cancer is hepatocellular carcinoma.

9. The drug according to claim 7, characterized in that The dosage form of the drug includes one or more of injection, powder injection, tablet, oral solution, capsule, granule or granule.

10. The anticancer drug according to claims 7-9, characterized in that: The anticancer drug also includes pharmaceutically acceptable excipients.

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