CN110787296A - Pharmaceutical composition for preventing or treating pancreatic cancer and kit for detecting pancreatic cancer - Google Patents
Pharmaceutical composition for preventing or treating pancreatic cancer and kit for detecting pancreatic cancer Download PDFInfo
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- CN110787296A CN110787296A CN201810865178.0A CN201810865178A CN110787296A CN 110787296 A CN110787296 A CN 110787296A CN 201810865178 A CN201810865178 A CN 201810865178A CN 110787296 A CN110787296 A CN 110787296A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a pharmaceutical composition for preventing or treating pancreatic cancer and a kit for detecting pancreatic cancer, the pharmaceutical composition for preventing or treating pancreatic cancer, the pharmaceutical composition being capable of reducing or inhibiting a) the biological activity of BCAT2 or b) the expression of a gene encoding BCAT 2; a kit comprising reagents for detecting BCAT 2; the invention discloses BCAT2 for the first time for promoting pancreatic cancer cell proliferation, knocking BCAT2 out in vivo for inhibiting pancreatic duct intraepithelial neoplasia (PanIN), and provides a pharmaceutical composition for preventing and treating pancreatic cancer and a pancreatic cancer evaluation kit.
Description
Technical Field
The invention relates to the field of medicine and health, in particular to a pharmaceutical composition for preventing or treating pancreatic cancer and a kit for detecting pancreatic cancer.
Background
Pancreatic ductal intraepithelial neoplasia (PanIN) is a precancerous lesion of malignant pancreatic ductal adenocarcinoma, for which there is currently a lack of sufficient knowledge about its development.
Metabolic reprogramming is one of the first proposed tumor metabolic features by Otto Warburg, which mainly refers to tumor cells that preferentially undergo glycolysis even when oxygen content is sufficient. With the continuous study of tumor metabolism, the concept of Warburg effect is also continuously expanded. In addition to glycolysis, the generalized Warburg effect is also continuously covered in other metabolic pathways such as fatty acid metabolism, amino acid metabolism and one-carbon unit cycle, etc. (Hanahan and Weinberg, 2011).
KRAS mutations are present in about 90% of pancreatic cancers (Halbrook and Lysitis, 2017). In the KRAS mutant PDAC animal model, the concentration of Branched Chain Amino Acids (BCAAs) in plasma has been significantly elevated early in PDAC, suggesting that BCAA metabolism may be associated with the development of PDAC (Mayers et al, 2014). Interestingly, studies have shown that in the KRAS mutation-induced cancer model, development of non-small cell lung cancer is BCAT dependent, while development of pancreatic cancer is BCAT independent (Mayers et al, 2016). However, recent studies have shown that overexpression of BCAT2 significantly promotes tumor cell proliferation in malic enzyme-deficient PDACs (Dey et al, 2017). Clearly, these conflicting reports suggest that intensive research is still required to elucidate the complex relationship between BCAA and PDAC development.
Branched-chain amino acids are essential amino acids, including leucine, isoleucine and valine branched-chain amino acid transaminase (BCAT) and branched-chain amino acid ketoacid dehydrogenase complex (BCKDC) are two key enzymes of BCAA catabolism (shimomura et al, 2001). BCAT is involved in catalyzing the first reaction of BCAA, mainly two subtypes, one is BCAT1 or BCATc, which is mainly located in the cytosol, and the other is BCAT2 or bcatm, which is mainly located in mitochondria, although they are distributed differently in the cell, but all catalyze the same chemical reaction, i.e., transfer of the amino group on the branched-chain amino acid to alpha-ketoglutarate (α -KG), producing the corresponding branched-chain ketoacid (BCKA) and glutamate, in which pyridoxal phosphate (PLP) is used as a coenzyme (Ichihara and Koyama, 1966; Taylor and Jenkins,1966) further acetyl-a CoA and succinyl-CoA are finally produced under the catalysis of a series of related coenzymes such as BCKDC.
However, existing studies have not elucidated which subtypes of BCAT affect pancreatic cancer, and more studies are directed to the close association of BCAT1 with cancer.
U.S. patent No. US20130072397a1, which discloses a method for diagnosing and prognostically determining tumors using BCAT1 protein, discloses a method for diagnosing tumors, particularly brain tumors, and a method for assessing the prognosis of patients with such tumors based on the determination of the expression of BCAT1 in a patient sample.
As described above, although existing studies suggest that BCAT is closely related to cancer, it has not been elucidated which subtype of BCAT affects pancreatic cancer.
Disclosure of Invention
In view of the above technical problems, the present invention provides a pharmaceutical composition for treating pancreatic cancer, which is capable of reducing or inhibiting a) the biological activity of BCAT2 or b) the expression of a gene encoding BCAT 2.
Preferably, the pharmaceutical composition comprises a BCAT2 inhibitor; the BCAT2 inhibitor comprises diterpene, triterpene, benzimidazole, sulfonyl hydrazide, and derivatives and prodrugs thereof.
Preferably, the pharmaceutical composition comprises an antibody directed against BCAT2 or a BCAT2 fragment.
Preferably, the pharmaceutical composition comprises an shRNA against the BCAT2 gene; more preferably, the base sequence of the shRNA is shown in SEQ ID NO 1-6.
Preferably, the pharmaceutical composition further comprises other pharmaceutical excipients.
The invention also discloses the use of a BCAT2 inhibitor in the manufacture of a medicament for the prevention and treatment of pancreatic cancer, the BCAT2 inhibitor being capable of reducing or inhibiting a) the biological activity of BCAT2 or b) the expression of a gene encoding BCAT 2.
Preferably, the BCAT2 inhibitor comprises diterpenes, triterpenes, benzimidazoles, sulfonylhydrazides, and derivatives and prodrugs thereof.
Preferably, the BCAT2 inhibitor comprises an antibody directed against BCAT2 or a BCAT2 fragment.
Preferably, the BCAT2 inhibitor comprises shRNA against the BCAT2 gene.
The invention also discloses a kit containing the BCAT 2.
Preferably, the BCAT2 test kit comprises qPCR primers for the amount of BCAT2 transcribed RNA when the biological sample is fresh tissue of pancreatic cancer; further preferably, the base sequences of the qPCR primers are SEQ ID NO. 7 and SEQ ID NO. 8.
Preferably, the BCAT2 detection kit comprises an immunohistochemical method for the expression level of BCAT2 protein when the biological sample is a paraffin section of pancreatic cancer tissue.
Preferably, the BCAT2 test kit comprises a biological sample that is a living organism that binds to BCAT2 metabolic enzyme activity18F or11Positron Emission Tomography (PET) imaging of N-labeled amino acid derivatives as leucine, isoleucine and valine analogs.
The invention also discloses application of the reagent for detecting BCAT2 in preparation of a product for detecting pancreatic cancer.
Preferably, the pancreatic cancer of the invention is pancreatic ductal adenocarcinoma.
Compared with the prior art, the technical scheme of the invention has the following advantages: the invention discloses BCAT2 for the first time to promote pancreatic cancer cell proliferation, knock-out BCAT2 in vivo to inhibit pancreatic ductal intraepithelial neoplasia, and provides a pharmaceutical composition for preventing and treating pancreatic cancer and a pancreatic cancer assessment kit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 shows Western Bolt (WB) results of 7 samples of example 2 of the present invention;
FIG. 2 is the BCAA content in the cell depletion medium of 7 samples according to example 2 of the present invention;
FIG. 3 is a graph of the effect of BCAT2 on the acid production rate of cells according to example 2 of the present invention;
FIG. 4 is a graph of the effect of BCAT2 on oxygen consumption rate of example 2 of the present invention;
FIG. 5 shows the intracellular pair of BCAT2 in example 2 of the present inventionNADH/NAD+And the effect of the levels of acetyl-CoA (ace-CoA) and succinyl-CoA;
FIG. 6 is BCAT1 protein levels in pancreatic ductal cells and pancreatic ductal carcinoma cells of example 2 of the present invention;
FIG. 7 shows the efficiency of the knockdown of BCAT2 by BXPC3 cells of example 2 of the invention;
FIG. 8 shows the knockdown efficiency of SW1990 cells BCAT2 of example 2 of the present invention;
FIG. 9 shows the results of KRAS activating mutation over-expression in three cell lines and KRAS knock-down in three cell lines in example 3 of the present invention;
FIG. 10 is a graph of the effect of KRAS knockdown on BCAT2 and TRIM21 interaction in SW1990, PANC1 and AsPC1 cells of example 3 of the invention;
FIG. 11 is the change in the content of BCAT2 after treatment in example 3 of the present invention;
FIG. 12 is the effect of KRAS on BCAA metabolism of example 3 of the present invention;
FIG. 13 shows the results of co-transfection of BCAT2-Flag with a plasmid of HA-UB into HEK293T cells according to example 3 of the present invention;
FIG. 14 shows the results of co-transfection of BCAT2-Flag with HA-UB plasmid in MG 132-treated HEK293T cells knocked-down in TRIM1 according to example 3 of the present invention;
fig. 15 is protein levels of intracellular BCAT2 knock-down TRIM1 of example 3 of the invention;
FIG. 16 is a graph showing the effect of overexpression of KRAS activating mutation in BxPC3 cells in example 3 on the interaction between BCAT2 and TRIM 21;
FIG. 17 is a graph of the effect of overexpressing BCAT2 and knocking down BCAT2 on the content of BCAAs in cell-depleting media according to example 1 of the present invention;
FIG. 18 is a cell growth curve of example 1 of the present invention;
FIG. 19 is a test of clonality of cells according to example 1 of the present invention;
FIG. 20 is a lower view of an immunohistochemical staining mirror of pancreatic tissue Bcat2 of mice of a control group and a KC group in example 1 of the present invention;
FIG. 21 is a photograph under immunohistochemical staining mirrors of Ck19, Bcat2 and Ki67 in serial sections of KC mouse pancreatic tissue of example 1 of the present invention;
FIG. 22 is a graph showing the protein expression level of BCAT2 after overexpression of BCAT2-Flag plasmid in H6C7 and HPNE cells by WB of example 1 of the present invention;
FIG. 23 is a graph of the effect of BCAT inhibitor 2 treatment of example 1 of the present invention on the content of BCAAs in cell-depleting media;
FIG. 24 is an in-mirror image of the results of cloning of H6C7 and HPNE cells overexpressing BCAT2, respectively, in example 1 of the present invention;
FIG. 25 is a graph showing the effect of overexpression of BCAT2 by H6C7 and HPNE cells, respectively, on cell migration in example 1 of the present invention;
FIG. 26 is an in-lens picture of the results of cell clone formation in SW1990 and BxPC3 cells knocking down BCAT2 according to example 1 of the present invention;
FIG. 27 is a test of the cell migration results of SW1990 and BxPC3 cells knocking down BCAT2 according to example 1 of the present invention.
FIG. 28 is a photograph under immunohistochemical staining mirror of pancreatic tissue Bcat2 of mouse in control group and KCB group according to example 1 of the present invention;
FIG. 29 is a statistical chart of pancreatic tissues of mice in the control group and KCB group according to different stages according to example 1 of the present invention;
FIG. 30 is a photograph under immunohistochemical staining mirrors of BCAT2 in para-cancerous and pancreatic cancer tissue of example 1 of the invention.
Detailed Description
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Example 1BCAT2 promotes proliferation of pancreatic ductal carcinoma cells
1. The BCAT2 inhibitor sulfonyl hydrazide type BCAT2 inhibitor was used at a dose of 7.5 micromolar (μ M), 30 μ M, 60 μ M.
2. Preparing a mouse model, preparing a transgenic mouse model for conditional knockout of Bcat2, and hybridizing different transgenic mice to obtain Pdx1-cre respectively; LSL-KRAS G12D (KC for short), Pdx 1-Cre; bcatflox/floxAnd LSL-KrasG 12D; bcat2flox/folx(ii) a A mouse model of Pdx1-Cre (KCB for short). The KC mouse model can well simulate the process of generation and development of pancreatic cancer premalignant panIN stage and pathological manifestations thereof (Hingorani et al, 2003), while the KCB mouse model can observe the influence of BCAT2 on the generation and development of panIN.
3. The effect of BCAT2 on BCAA uptake by pancreatic ductal epithelial cells and PDAC cells was examined. Results as shown in fig. 17 and 22, stability-over-expressing BCAT2 significantly increased the ability of H6C7 and HPNE cells to take up BCAAs; while stability knockdown of BCAT2 caused a significant decrease in the ability of SW1990 and BxPC3 cells to take up BCAAs. Among them, fig. 17 shows that BCAT2 promotes uptake of BCAAs by cells. In H6C7 and HPNE cells overexpressing BCAT2, the content of BCAAs in the cell-depleting medium was examined (fig. 17, left panel); BCAT2 knockdown in SW1990 and BxPC3 cells, the content of BCAAs in cell-depleted medium was examined (fig. 17, right panel). *: p < 0.05. FIG. 22 is an identification of stable cell lines overexpressing BCAT 2. WB identified stable expression of BCAT2 in H6C7 (fig. 22, left panel) and HPNE (fig. 22, right panel) cells. Furthermore, as BCAT2 inhibitor treatment time was extended and treatment concentration increased, the ability of cells to consume BCAAs was also gradually diminished (as shown in figure 23). Figure 23 is a graph of BCAT2 inhibitor down-regulating cellular uptake of BCAAs. BxPC3 cells were treated with different concentrations of BCAT2 inhibitor for 24 hours (h) (fig. 23, left panel), 48 hours (fig. 23, right panel), and the content of BCAAs in the cell-depleted medium was examined. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
4. Counting the number of cells at different time points growth curves were prepared to examine the effect of BCAT2 on proliferation of pancreatic ductal epithelial cells and PDAC cells. As a result, as shown in fig. 18, overexpression of BCAT2 in immortalized normal pancreatic cells significantly promoted cell proliferation of H6C7 and HPNE. Simultaneously, over-expression of BCAT2 significantly promoted the clonogenic and migratory abilities of the above cells (FIG. 19, left panel; FIGS. 24-25). In contrast, knockdown of BCAT2 in cancerous cells significantly inhibited the proliferation of SW1990 and BxPC3 cells (FIG. 18, bottom panel), and attenuated the clonogenic and migratory abilities of the cells (FIG. 19, right panel; FIGS. 26-27). *: p < 0.05; **: p < 0.01; ***: p < 0.001. As shown in fig. 19, BCAT2 promoted clonal formation of pancreatic ductal epithelial cells. Overexpression of BCAT2 in H6C7 and HPNE cells, respectively, promoted cell clonogenic (fig. 19, left panel), and stable knock-down of BCAT2 in SW1990 and BxPC3 cells, respectively, inhibited PDAC cell clonogenic (fig. 19, right panel). *: p < 0.05; **: p < 0.01. Fig. 24 is a graph of the promotion of pancreatic ductal epithelial cell clonogenic by overexpression of BCAT 2. H6C7 and HPNE cells stably over-expressed BCAT2, respectively, and two weeks later colony formation was recorded by microscopic photography. Fig. 25 shows that BCAT2 promotes cell migration. H6C7 and HPNE cells stably overexpressed BCAT2, respectively, and cell migration was detected and quantified by Transwell membrane crossing assay (fig. 25, left panel). The scale bar is 200 μm. *: p < 0.05; **: p < 0.01. Figure 26 shows that knockdown of BCAT2 inhibits PDAC cell clonogenic. Stable knock-down of BCAT2 in SW1990 and BxPC3 cells, respectively, was followed two weeks later by microscopic picture recording of colony formation (fig. 26, left) and quantification (fig. 26, right). The scale bar is 200 μm. **: p < 0.01. Figure 27 shows that knockdown of BCAT2 inhibits PDAC cell migration. BCAT2 was stably knocked down in SW1990 and BxPC3 cells, respectively, and cell migration was detected and quantified in the transwell membrane crossing assay (fig. 27, left). The scale bar is 200 μm. *: p < 0.05; **: p < 0.01.
The above data indicate that BCAT2 promotes BCAA uptake by pancreatic ductal epithelial cells and PDAC cells, and regulates proliferation and migration of pancreatic ductal epithelial cells and PDAC cells.
5. In order to investigate the function of BCAT2 under physiological and pathological conditions in vivo, normal mice and specimens of pancreas tissues of KC and KCB mice were collected, and by immunohistochemical staining (IHC), it was found that BCAT2 was expressed in pancreatic acinar cells of both normal control mice (including WT, Pdx1-Cre and LSL-KRAS G12D) and KC mice, but not in pancreatic ductal epithelial cells of normal control group (fig. 20). However, Bcat2 exhibited high expression in ductal epithelial cells in the PanIN phase of KC group, even higher than that of pancreatic cells (fig. 20). In addition, expression of Bcat2 in ductal epithelial cells could not be detected in the KCB group (fig. 28, 29). Further, serial sections of the same tissue blocks were taken and IHC staining was performed on pancreatic tissues of KC group mice with Ck19 (catheter marker molecule), Ki67 (cell proliferation marker molecule) and Bcat2 antibody, respectively. We found that expression of Ck19, Ki67 and Bcat2 co-localized within pancreatic ductal tissue where PanIN lesions occurred (fig. 21). IHC staining was performed on pancreatic tissues of KCB mice, and it was found that knocking out Bcat2 significantly inhibited the generation and development of PanIN (FIG. 28, FIG. 29). IHC identified the expression level of Bcat2 in mouse pancreas (16 weeks of age, 6). Triangles indicate normal ductal tissue, five stars indicate acinar tissue, arrows indicate PanIN stage ductal. The scale bar is 50 μm. IHC identified the expression levels of CK19, Bcat2 and Ki67 in mouse pancreas (16 weeks old, 5). Arrows indicate Ck19, Bcat2, and Ki67 staining, respectively. The scale bar is 12.5 μm. In addition, we performed IHC staining of pancreatic cancer and its paracarcinoma tissues separately and we found that BCAT2 was significantly highly expressed in pancreatic cancer tissues (FIG. 30)
The data show that Bcat2 is highly expressed in ductal epithelial cells in the PanIN stage, early in pancreatic carcinogenesis, suggesting that Bcat2 plays an important role in the development of PanIN. In addition, clinical sample data further demonstrate that BCAT2 also plays an important role in the development of pancreatic cancer.
The mechanism by which the BCAT2 inhibitor of the present example prevents and treats pancreatic cancer is as follows.
Example 2 significant upregulation of BCAT2 in pancreatic cancer, thereby promoting BACC metabolism
To investigate the relationship of branched-chain amino acid metabolism to pancreatic cancer, we focused on branched-chain amino acid transaminase (BCAT), the first key enzyme involved in branched-chain amino acid catabolism. It includes two subtypes, BCAT1 located in the cytoplasm and BCAT2 located in the mitochondria. The relationship of branched chain amino acid metabolism to pancreatic cancer was analyzed by the following numerous experiments.
1. The protein levels of BCAT1 and BCAT2 were detected in immortalized normal pancreatic ductal epithelial cells and pancreatic ductal carcinoma cells, respectively.2 immortalized normal pancreatic ductal epithelial cells hTERT-HPNE and HPDE6C7 (hereinafter referred to as HPNE and H6C7), and 5 pancreatic ductal carcinoma cells were selected as the subjects of the study.the protein levels of BCAT2 in pancreatic cells were detected using Western Blot, HPNE and H6C7 were immortalized normal pancreatic ductal epithelial cells, PANC1, BxPC3, ASPC1, Capan1, and SW1990 were PDAC cell lines, and quantified using β -actin as an internal reference. the data of Western bolt are shown in FIGS. 1 and 6, in pancreatic cancer cells, the protein level of BCAT2 was significantly up-regulated compared with immortalized normal cells (shown in FIG. 1), whereas the protein of PAAT 1 was up-regulated only in PANC1 cells, and was significantly unchanged in other pancreatic ductal carcinoma cells (shown in FIG. 6).
2. Further, to verify whether BCAT2 upregulation in pancreatic ductal cells promotes BCAA metabolism. After culturing the cells for 24h, the amount of BCAAs in the medium consumed by each cell was measured, and it was found that the amount of BCAAs consumed by pancreatic ductal carcinoma cells was 1.5-2.5 times that of HPNE, which is a normal pancreatic ductal carcinoma cell, and that the expression of BCAT2 was positively correlated, and that the BCAAs consumption ability of PDAC cells was enhanced (as shown in FIG. 2).
3. To investigate the regulation of BCAT2 on BCAA metabolic flux, BCAT2 was first examined separately for its effects on cellular glycolysis and aerobic respiration using Seahorse technology, where cellular acid production rate (ECAR) represents cellular glycolysis capacity and Oxygen Consumption Rate (OCR) represents cellular aerobic respiration capacity. Knockdown of BCAT2 caused cellular increase in ECAR and decrease in OCR. As shown in the upper panel of FIG. 3, in BxPC3 cells of control or knockdown BCAT2, glucose (glucose), oligomycin (oligomycin) and 2-deoxy-D glucose (2-DG) were added sequentially at the indicated time points. Drawing an ECAR curve; as shown in the lower panel of fig. 3, in BxPC3 cells of control or knockdown BCAT2, oligomycin (oligomycin), trifluorocyanophenylhydrazone (FCCP) and rotenone (rotenone) were added sequentially at the indicated time points. And drawing an OCR curve. *: p < 0.05; **: p < 0.01; ***: p < 0.001. BCAT2 inhibitors down-regulate cellular OCR levels. As shown in figure four, BCAT2 inhibitor treated BxPC3 cells were added sequentially with oligomycin (oligomycin), trifluorocyanophenylhydrazone (FCCP) and rotenone (rotenone) at the indicated time points. And drawing an OCR curve. *: p < 0.05; **: p < 0.01. The results show that: in BxPC3 cells, knockdown of BCAT2 was able to significantly enhance cellular ECAR levels while attenuating OCR levels (as shown in fig. 3 and 7). In agreement, the cellular OCR levels were also significantly downregulated following BCAT2 inhibitor treatment of BxPC3 cells (as shown in FIG. 4)
4. Intracellular NADH/NAD detection+And a ratio of (B)/(A), and BCAALevels of the free metabolites acetyl-CoA (ace-CoA) and succinyl-CoA (suc-CoA). The results show that after BCAT2 knockdown in SW1990 cells, the NADH/NAD + ratio was significantly up-regulated, while the intracellular ace-CoA and suc-CoA concentrations were not significantly changed (FIGS. 5 and 8). As shown in FIG. 5, knock-down of BCAT2 upregulates intracellular NADH/NAD ratios but does not affect ace-CoA and suc-CoA. In SW1990 cells with BCAT2 knocked down, the kit detected the ratio of NADH/NAD +, and LC-MS detected acetyl-CoA (ace-CoA) and succinyl-CoA (suc-CoA). **: p is a radical of<0.01; n.s. represents no significant difference.
Example 3KRAS activating mutations stabilize BCAT2 protein
KRAS activating mutations are present in about 90% of pancreatic ductal carcinomas (Kanda et al, 2012). Therefore, we further investigated whether KRAS activating mutations modulate the protein levels of BCAT 2.
1. Three KRAS wild-type cell lines, namely H6C7, HPNE pancreatic ductal epithelial cells and BxPC3 cells, were selected and the KRAS activating mutant (KRAS G12V) was overexpressed in these three cell lines, and it was found that KRAS mutations up-regulate BCAT2 protein levels without affecting the mRNA levels of BCAT 2. H6C7, HPNE and BxPC3 cells overexpress KRAS activating mutant KRAS G12V, SW1990, PANC1 and AsPC1 cells down-regulate KRAS, BCAT2 and KRAS, respectively, by WB protein levels, β -actin was quantified in-cells Q-PCR examining BCAT expression levels of BCAT2 in each cell line. n.s. represents no significant difference, KRAS G12V up-regulates BCAT2 protein levels 1.9, 1.7 and 1.2 times, while KRAS 68627 up-regulate mRNA levels by 1.9, 1.7 and 1.2 times, and by translational modifications (left, right) as depicted in the map, 9, 18. 9, 8, 18. C469, 9, 18. and 18. C469).
2. BCAT2 was found to be significantly ubiquitinated in HEK293T cells co-transfected with plasmids of BCAT2-Flag and HA-UB (see FIG. 13).
3. Reversible regulation of ubiquitination consists mainly of 4 enzymes, E1, E2, E3 ubiquitin ligase and Deubiquitinase (DUB) (leekemaker and Ovaa, 2017). Among them, E3 ubiquitin ligase is a substrate-specific enzyme of ubiquitination mediated degradation pathway (Hershko and Ciechanover, 1998). Protein molecules interacting with BCAT2 were screened by tandem affinity purification and mass spectrometry (TAP-MS), and the results of identification showed that high scores were obtained for Trimotif protein 21(TRIM 21). It was suggested that TRIM21 might be an E3 ligase that regulates BCAT2 protein degradation. After knock-down of TRIM21, the level of ubiquitination of BCAT2 was significantly down-regulated (see fig. 14). Furthermore, there was a distinct increase in the intracellular protein levels of BCAT2 after knockdown of endogenous TRIM21 in H6C7, SW1990, AsPC1 and PANC1 cells, respectively (see fig. 15). These results indicate that TRIM21 is the E3 ubiquitin ligase of BCAT 2.
4. To further elucidate the molecular mechanism of KRAS G12V in regulating BCAT2 protein levels. As shown in figure 10, knockdown KRAS enhanced the interaction of BCAT2 with TRIM21 in SW1990, PANC1 and AsPC1 cells. KRAS was knocked down in SW1990, PANC1 and AsPC1 cells, and endogenous IP and WB detected the interaction of BCAT2 and TRIM21, respectively. BCAT2 was quantified as an IP internal reference. Accordingly, as shown in fig. 16, KRAS G12V was overexpressed in BxPC3 cells, and KRAS G12V was found to significantly block the binding of TRIM21 and BCAT 2. BxPC3 cells overexpressing KRAS G12V bound MG132 treatment for 6h and endogenous IP and WB detected the interaction of BCAT2 and TRIM 21. TRIM21 was quantified as an IP internal control. As shown in fig. 11, overexpression of KRASG12V significantly extended the half-life of BCAT 2. H6C7 cells over-expressed KRAS G12V, CHX treatment at different times, WB detected BCAT2 protein amount (FIG. 11, left panel) and quantified (FIG. 11, right panel). *: p < 0.05).
These data indicate that KRAS mutations are able to block the binding of BCAT2 and E3 ubiquitin ligase TRIM21, reduce the level of ubiquitination of BCAT2, and stabilize the protein level of BCAT 2.
5. The influence of KRAS on the metabolism of BCAA is further detected by LC-MS, and the over-expression of KRAS is foundG12VCan up-regulate BCAA and α -ketoisocaproic acid (KIC) levels in H6C7 cells, promote BCAAs catabolism (figure 12, left panel), whereas, knock-down KRAS causes a decrease in BCAA and KIC levels in PANC1 cells, inhibiting BCAAs catabolism (figure 12, right panel). KRAS is overexpressed in H6C7 cellsG12VDetecting the content of BCAAs related metabolites by LC-MS; KR knockdown in PANC1 cellsAnd (3) detecting the content of the BCAAs related metabolites by using AS and LC-MS. *: p is a radical of<0.05;**:p<0.01;***:p<0.001。
The results of the above studies indicate that KRAS mutation promotes BCAA catabolism, while BCAT2 is a key enzyme in BCAA catabolism.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
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Claims (15)
1. A pharmaceutical composition for preventing or treating pancreatic cancer, wherein said pharmaceutical composition is capable of reducing or inhibiting a) the biological activity of BCAT2 or b) the expression of a gene encoding BCAT 2.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises a BCAT2 inhibitor; the BCAT2 inhibitor comprises diterpene, triterpene, benzimidazole, sulfonyl hydrazide, and derivatives and prodrugs thereof.
3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises an antibody directed against BCAT2 or a BCAT2 fragment.
4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises an shRNA against the BCAT2 gene.
5. The pharmaceutical composition of claim 4, wherein the base sequence of the shRNA is shown in SEQ ID NO 1-6.
6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutical excipient.
Use of a BCAT2 inhibitor, which is capable of reducing or inhibiting a) the biological activity of BCAT2 or b) the expression of a gene encoding BCAT2, for the preparation of a medicament for the prevention and treatment of pancreatic cancer.
8. The BCAT2 inhibitor of claim 7, comprising a diterpene, a triterpene, a benzimidazole, a sulfonyl hydrazide, and derivatives, prodrugs thereof.
9. The BCAT2 inhibitor of claim 7, comprising an antibody against BCAT2 or a BCAT2 fragment.
10. The BCAT2 inhibitor of claim 7 comprising an shRNA against BCAT2 gene; the base sequence of shRNA is shown in SEQ ID NO 1-6.
11. A kit comprising reagents for detecting BCAT 2.
12. The kit for detecting BCAT2 of claim 11, wherein said kit comprises qPCR primers to detect the amount of BCAT2 transcribed RNA.
13. The kit for detecting BCAT2 of claim 12, wherein the base sequences of the qPCR primers are SEQ ID NO. 7 and SEQ ID NO. 8.
14. Application of a reagent for detecting BCAT2 in preparation of products for detecting pancreatic cancer.
15. The pancreatic cancer of any one of claims 1-7 or 14 which is ductal pancreatic cancer.
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CN113777309A (en) * | 2021-09-07 | 2021-12-10 | 复旦大学附属肿瘤医院 | Application of autoantibody in preparation of pancreatic ductal adenocarcinoma diagnostic kit |
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CN114073697B (en) * | 2021-01-19 | 2023-01-24 | 复旦大学附属肿瘤医院 | Application of BCAT2 inhibitor in preparation of medicines for preventing and/or treating BCAT 2-mediated related metabolic diseases |
CN113777309A (en) * | 2021-09-07 | 2021-12-10 | 复旦大学附属肿瘤医院 | Application of autoantibody in preparation of pancreatic ductal adenocarcinoma diagnostic kit |
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