CN113197890A

CN113197890A - Application of tricin in preparation of antitumor drugs

Info

Publication number: CN113197890A
Application number: CN202110471949.XA
Authority: CN
Inventors: 颜培宇; 梁丽娴; 周华; 李润泽; 李佳馨; 谢莹; 刘良
Original assignee: Macau University of Science and Technology
Current assignee: Macau University of Science and Technology
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-03

Abstract

The invention belongs to the field of medicines, and discloses application of tricin in preparation of antitumor drugs, wherein the tumors comprise lung cancer, glioma and pancreatic cancer. The tricin provided by the invention has the effects of inhibiting a PRKCA/SPHK/S1P signal pathway and an anti-apoptosis signal pathway, and can effectively play an obvious role in inhibiting the growth of various tumors, especially KRAS lung cancer mutant tumor cells. It is also proposed that tricin may also act as a KRAS mutation inhibitor, PRKCA inhibitor or S1P modulator, modulating cell growth and apoptosis progression.

Description

Application of tricin in preparation of antitumor drugs

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of tricin in preparation of antitumor drugs.

Background

Lung cancer is a serious health-threatening cancer with high morbidity and mortality, of which non-small cell lung cancer (NSCLC) is a more common type of lung cancer. The existing research shows that the occurrence of the non-small cell lung cancer is closely related to the KRAS mutation of the cancer gene. Scientists have long been dedicated to develop drugs targeting KRAS mutations, but no cure for KRAS mutations is available, and some serious side effects may occur in the reported novel small molecule inhibitors.

PRKCA is one of 10 members of the protein kinase C family, and plays an important role in regulating cell invasion, proliferation and apoptosis in a variety of tumor types, including NSCLC. PRKCA is also highly expressed in NSCLC patients, and thus inhibition of PRKCA is considered as a method for treating NSCLC.

Sphingosine-1-phosphate (S1P) is regulated by sphingosine kinases 1 and 2(SPHK1 and SPHK2) in sphingolipid metabolism, is considered to be a bioactive lipid molecule involved in various cellular processes such as cell proliferation and immunoregulation. S1P is involved in the progression of many diseases, such as sepsis, rheumatoid arthritis and cancer, and reduction in SPHK1, SPHK2 and S1P can result in alleviation of symptoms.

Therefore, it is desired to provide a drug having a better antitumor effect, particularly a significant therapeutic effect on lung cancer.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the tricin provided by the invention has the effects of inhibiting a PRKCA/SPHK/S1P signal path and an anti-apoptosis signal path, and can effectively play an obvious role in inhibiting the growth of various tumors, especially KRAS lung cancer mutant tumor cells.

The invention provides an application of tricin in preparing anti-tumor drugs, wherein the tumors comprise lung cancer, glioma and pancreatic cancer.

The tricin is a flavonoid natural component, can be extracted from rice, wheat or reed rhizome, and has antiinflammatory, diabetes preventing, anti-angiogenesis and antivirus effects. The above-mentionedThe molecular formula of the tricin is C₁₇H₁₄O₇Molecular weight is 330.29, and the structural formula is shown as follows:

the test shows that the tricin has obvious selective cytotoxicity on tumor cells of various tissues, wherein the tricin has obvious inhibiting effect on proliferation, anti-apoptosis, migration and clone formation of lung cancer cells.

Preferably, the lung cancer is non-small cell lung cancer. Experiments in the invention show that the tricin has good inhibition effect on the growth of three lung cancer cell lines such as H358 cells, H2122 cells, LLC cells and the like, and the toxicity of the tricin on the three lung cancer cell lines is named as follows: h358> H2122> LLC, wherein H358 cells and H2122 cells are human non-small cell lung cancer cells, which shows that the inhibition and killing effects of the tricin on the non-small cell lung cancer cells are stronger.

The invention also provides an anti-tumor medicament for treating lung cancer, glioma and pancreatic cancer, wherein the anti-tumor medicament contains tricin.

Preferably, the anti-tumor medicine further comprises pharmaceutically acceptable auxiliary materials.

More preferably, the pharmaceutically acceptable adjuvant comprises at least one of a solvent, a filler, a lubricant, a disintegrating agent, a buffering agent, a cosolvent, an antioxidant, a bacteriostatic agent, an emulsifier, a binder or a suspending agent.

Preferably, the dosage form of the antitumor drug comprises injection, powder injection, granules, capsules, tablets, powder, dripping pills, oral liquid or suppositories.

The invention also provides a KRAS mutation inhibitor, which comprises tricin.

The invention also provides a PRKCA inhibitor, which comprises tricin.

The invention also provides an S1P regulator, wherein the S1P regulator comprises tricin.

Compared with the prior art, the invention has the following beneficial effects:

the invention indicates that the natural product tricin has obvious inhibition effect on proliferation, anti-apoptosis, migration and clone formation of tumor cells, in particular to KRAS^G12CThe lung cancer mutant tumor cells have obvious cytotoxicity, and have no obvious cytotoxicity to lung fibroblast of normal people. Moreover, the invention indicates that the tricin can inhibit PRKCA/SPHK/S1P signal path and anti-apoptosis signal path, thus can be used as KRAS mutation inhibitor, PRKCA inhibitor or S1P regulator to regulate cell growth and apoptosis process.

Drawings

FIG. 1 shows the results of growth inhibition of Lewis lung carcinoma cell xenograft tumors by tricin;

FIG. 2 shows the results of MTT assay of tricin on different cells;

FIG. 3 shows the results of experiments on cloning formation of LLC cells by tricin;

FIG. 4 shows the results of an apoptosis assay of LLC cells with tricin;

FIG. 5 shows the results of expression of Prkca, Sphk1 and Sphk2 genes of tricin in LLC cells;

FIG. 6 shows the effect of PRKCA on the induction of apoptosis by tricin.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Reagents and cell culture

Pubchem CID: 5281702, CAS #: 520-32-1) and cisplatin (CAS #: 15663-27-1) were both purchased from MedChemexpress. In vivo experiments: dissolving tricin in a solution consisting of 10% DMSO, 10% PEG400, 10% Tween-80 and 70% PBS; cisplatin is dissolved in deionized water. In vitro experiments: the tricin was dissolved in DMSO.

LLC (murine Lewis lung carcinoma cells) and H2122 (human KRAS)^G12CMutant lung cancer cells), H358 (human KRAS)^G12CMutant lung cancer cells), HepG2 (human liver cancer cells), HCT-8 (human colon cancer cells), U-87MG (human glioma cells), miapac-2 (human pancreatic cancer cells), and CCD-19Lu (normal human lung fibroblasts) were used as test materials, and all of these cells were purchased from american type culture collection bank (ATCC). Subjecting the cells to 5% CO at 37 deg.C₂Cultured in an incubator in which U-87MG, Miapaca-2 and LLC cells were cultured in DMEM medium. CCD-19Lu cells were cultured in MEM medium. HepG2, HCT-8, H2122 and H358 cells were cultured in RPMI1640 medium.

Data in the following examples are presented as mean ± standard deviation, processed using Graph prism7.0 software, and data differences between groups were compared using one-way analysis of variance (ANOVA) or t-test, with P <0.05 indicating significant differences and P <0.01 and P <0.001 indicating very significant differences.

Example 1: establishment and intervention of tumor-bearing mouse model

Preparing a lung cancer subcutaneous transplantation tumor model: culturing lung cancer cell strain LLC, and subculturing. Cells were collected at the log phase of the cells and formulated at a concentration of 5X 10⁵One/100. mu.L cell suspension was injected subcutaneously into the right forelimb of C57BL/6J mice, and the tumor was allowed to grow to volume after about 7 days>50mm³I.e. the tumor grafting is successful.

After the tumor inoculation is successful, the mice are divided into 4 groups at random, each group comprises 6-7 mice, and each group comprises: 1) control group (PBS group); 2) positive control cisplatin group (2 mg/kg); 3) high dose group of tricin (50 mg/kg); 4) low dose group of tricin (25 mg/kg). PBS control group and the medical group of high dose and low dose of tricin are respectively injected into abdominal cavity once a day, 0.2 ml is injected each time, tumor volume is measured once every 3 days, and the weight is weighed. The cisplatin group was intraperitoneally injected every three days, 0.2 ml each time. Wherein tumor volume is ((length × width)/2). When body weight loss was > 20% or tumors were >20 mm in diameter either, mice were sacrificed and tumor bodies weighed. After 15 days of dosing, all mice were sacrificed and tumor mass weighed. Tumor inhibition rate calculation formula: the inhibition ratio (%) - (control tumor weight-experimental tumor weight)/control tumor weight × 100%.

The test result is shown in figure 1, and the tricin can obviously inhibit the growth of Lewis lung cancer cell xenograft tumor. The tricin group delayed LLC tumor growth compared to the control group (shown as A in FIG. 1); during the treatment period, the mice in the tricin group did not lose weight significantly, the positive control cisplatin group inhibited tumor growth, but the mice lost weight significantly (shown as B in fig. 1); FIG. 1C is a photograph showing representative tumors in each group, the size of the tumor volume being consistent with the results shown in A in FIG. 1; as shown in FIG. 4, after 15 days of experiment, the tumor inhibition rates of the cisplatin group, the low-dose tricin group and the high-dose tricin group were 54.49% (P <0.001), 43.55% (P <0.01) and 65.03% (P <0.001), respectively.

Example 2: MTT assay

HepG2, HCT-8, U-87MG, Miapaca-2, LLC, H2122, H358 and CCD-19Lu cells were seeded in 96-well microplates (3000 wells 5000/well). After the cells were adhered, 100 μ l of tricin at various concentrations (150, 75, 37.5, 18.75 and 9.375 μmol/l) was added as a test group and DMSO was used as a control group; after 72 hours of incubation, 10. mu.l of 0.5 mg/ml MTT solution was added and incubated at 37 ℃ for 4 hours in an incubator. After 4 hours, the supernatant was aspirated, 100. mu.l of dimethylsulfoxide (SDS) was added, and the absorbance was measured at a wavelength of 570nm and 650nm using an absorbance meter. IC50 was calculated using GraphPad Prism7.0 software, and each of the above experiments was performed independently in triplicate.

The test results are shown in FIG. 2, in which A-H represent LLC (murine Lewis lung carcinoma cell) and H2122 (human KRAS), respectively^G12CMutant lung cancer cells), H358 (human KRAS)^G12CMutant lung cancer cells), HepG2 (human liver cancer cells), HCT-8 (human colon cancer cells), U-87MG (human glioma cells), Miapaca-2 (human pancreatic cancer cells), and CCDMTT test results of 19Lu (normal human lung fibroblast) cells, which result in the IC50 values of tricin on different cells: LLC: 77.98 + -1.21 micromoles/liter; h2122: 38.46 ± 1.12 micromoles per liter; h358: 30.78 + -1.21 micromoles/liter; HepG 2: 88.69 + -1.17 micromoles/liter; HCT-8: 149.90 + -1.10 micromoles/liter; u-87 MG: 58.91 + -1.08 micromoles/liter; miapaca-2: 82.20 + -1.10 micromoles/liter; CCD-19 Lu: > 300 micromoles/liter. The results show that the tricin has growth inhibitory effect on various tumor cells, wherein the growth inhibitory effect on human KRAS^G12CThe mutant lung cancer cells (H2122 and H358) were best inhibited.

Example 3: clone formation experiments

LLC cells were seeded in 6-well plates, 500 cells per well. After cell adhesion, the cells were incubated in medium containing different concentrations (0, 12.5, 25, 50, 100. mu. mol/l) of tricin for about 10 days, with the medium being changed every three days. When colony formation was evident, LLC cells were fixed with 4% Paraformaldehyde (PFA) for 30 minutes at 4 ℃. And then stained with a crystal violet solution for at least 30 minutes. After washing away the crystal violet solution, the colonies were photographed.

The results are shown in FIG. 3, where A shows that tricin shows dose-dependence on clonal formation of LLC cells; in FIG. 3B shows that tricin shows dose dependence on LLC cell migration; in FIG. 3, C shows the migration distance statistics of LLC cells, all experiments were performed in triplicate, and the data are presented as mean. + -. standard deviation (. SP <0.05,. SP <0.01), compared to the migration distance of 0. mu. mol/l of isoflavones. The results show that the tricin inhibits the clonogenic capacity of LLC cells in a dose-dependent manner and has obvious inhibition effect on LLC cell migration in a dose-dependent manner.

Example 4: apoptosis assay

LLC cells (2X 10)⁵Cells/well) were seeded in 6-well plates for 24 hours, and LLC cells were incubated with the indicated concentrations (0, 50, 100, and 150 μmol/l) of tricin for 24 hours. After subsequent resuspension with 100. mu.l of 1 XBinding buffer, LLC cells were stained with 2. mu.l of Annexin-V FITC and 4. mu.l of PI reagent for 15 minutes in the dark. Apoptosis details were calculated using a BD Aria III flow cytometerCell number, data were analyzed using FlowJo v10 software.

The results are shown in FIG. 4, wherein A in FIG. 4 represents the percentage of apoptosis of LLC cells by different concentrations of tricin calculated by flow cytometry; in FIG. 4, B represents the statistics of apoptosis. All experiments were performed in triplicate and data expressed as mean ± standard deviation (. about.p <0.01,. about.p <0.001) compared to the percentage of apoptosis of 0 μmol/l of tricin; FIG. 4C shows the Western blot analysis of p-AKT, T-AKT, Bcl-XL, full-length PARP and cleared PARP protein expression levels 24 hours after treatment with tricin; flow cytometry detection results show that the malt flavone obviously increases the LLC apoptosis rate. Western blot results show that in LLC cells, tricin increased the expression level of cleared PARP, decreased the expression level of full-length PARP, Bcl-XL and AKT phosphorylation, and was dose-dependent, but that tricin had little effect on total AKT.

Example 5: western blot experiment and quantitative real-time PCR experiment

Western blot experiment

LLC cells were treated with 0, 50, 100, 150. mu. mol/l of tricin (2X 10 per well), respectively⁵Individual cells). After 24 hours, 1 × RIPA buffer was added. After 15 minutes, the cells were scraped from the surface of the dish, and the lysed suspension was centrifuged (12000rpm, 4 ℃,10 minutes) to collect the supernatant. Total protein concentration was determined using Pierce BCA protein assay kit. The protein was cooked at 100 ℃ for 10 minutes. 2 microliters of molecular weight marker and an average amount (about 40 micrograms) of protein were added to a 10% SDS-acrylamide gel. After completion of the electrophoresis, the protein was transferred to a nitrocellulose membrane (NC) at 300mA for 2 hours. The membranes were incubated in 5% skim milk for 1 hour. The membrane was then incubated with the relevant primary antibody (1:1000 dilution) overnight in a shaker at 4 ℃. The next day, the membranes were incubated for 2 hours in luciferin-bound rabbit antibody diluted 1:10000, protected from light. Finally, the gray value of each band was detected using a LI-COR Odessy imaging scanner.

Quantitative real-time PCR experiment

LLC cells were treated with different concentrations (0, 50, 100, 150. mu.M) of tricin for 24hThen (c) is performed. The total cellular RNA was then extracted by Trizol reagent. After reverse transcription of RNA into cDNA, high-efficiency real-time quantitative PCR technology of ViiATM7 was used. By using 2^-△△CtThe method calculates the gene expression level. Primers were purchased from GenePharma and the primer sequences are shown below:

Prkca-F：5’-TGGTCTCTCAGTCTCTCGCA-3’(SEQ ID NO.1)；

Prkca-R：5’-ACAAAGTGGGCTGGATAGGC-3’(SEQ ID NO.2)；

Sphk1-F：5’-CTTGATAGTGTTCCCTGGGGG-3’(SEQ ID NO.3)；

Sphk1-R：5’-GGGGACGAAGTCGAGATGAG-3’(SEQ ID NO.4)；

Sphk2-F：5’-ATGATCGGAGCTTGCTGGAC-3’(SEQ ID NO.5)；

Sphk2-R：5’-GCCAGGCCAAGTGTTGAAAG-3’(SEQ ID NO.6)；

Gapdh-F：5’-TGACCTCAACTACATGGTCTACA-3’(SEQ ID NO.7)；

Gapdh-R：5’-CTTCCCATTCTCGGCCTTG-3’(SEQ ID NO.8)。

in order to verify the action mechanism of the tricin for inhibiting LLC cell proliferation, the invention carries out a series of molecular researches. The results are shown in FIG. 5, in which A-C in FIG. 5 represent RNA expression levels (. times.P <0.01,. times.P <0.001) of Prkca, Sphk1 and Sphk2, respectively, after 24 hours LLC treatment with isoflavones by quantitative PCR, compared to RNA expression levels of isoflavones at 0. mu. mol/l; d in FIG. 5 shows the results of Western blotting of the expression levels of p-PRKCA, T-PRKCA, SPHK1 and SPHK2 proteins after 24 hours of treatment with tricin.

Quantitative PCR results show that the expressions of Prkca, Sphk1 and Sphk2 genes are all obviously reduced after the treatment of the tricin with different concentrations. Meanwhile, the western blot detection result shows that the protein level result is consistent with the gene level result. The tricin significantly reduced the protein levels of phosphorylated PRKCA, SPHK1 and SPHK2, but the effect of tricin on total PRKCA was small.

Example 6: stable transfection assay

LLC cells were seeded in 6-well plates (approximately 1.8X 10)⁵-2.0×10⁵One/hole). After 24 hours, 1. mu.g of plasmid (sh-Control or sh-Prkca) and 2. mu.gLiposome 2000 was mixed with 800. mu.l of opti-mem medium to incubate the cells. After 4-6 hours, change to DMEM medium. After the LLC cells are cultured for 48 hours, a 96-well plate and 1 microgram/ml puromycin are used for screening and constructing a stable sh-Prkca knockout LLC cell line, and a western blot method is adopted to verify whether the gene is successfully knocked out. After successful knockout, transfected LLC cells were then treated with 100. mu.M/l of tricin for 24 hours and apoptosis was detected by flow cytometry.

The results are shown in FIG. 6, in which A in FIG. 6 is the result of deletion of Prkca gene; in FIG. 6, B is the detection result of the flow cytometer after the transfected LLC cells are treated with the tricin; in FIG. 6, C is the statistic result of apoptosis. All experiments were performed in triplicate and data expressed as mean ± standard deviation (. P <0.01,. P < 0.001). The results show that after the LLC cells with Prkca knocked out are treated by the tricin, apoptotic cells are obviously reduced, and the PRKCA is proved to be an important medium for inducing apoptosis by the tricin.

Example 7: capsule preparation

Taking 400g of the tricin, adding 1000g of lactose and 1200g of starch, uniformly mixing, using starch slurry as an adhesive, carrying out wet granulation, drying, adding 20g of magnesium stearate, uniformly mixing, filling into capsules, and preparing the capsules.

SEQUENCE LISTING

<110> Australian university of science and technology

Application of <120> tricin in preparation of antitumor drugs

<130> 1

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

tggtctctca gtctctcgca 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

acaaagtggg ctggataggc 20

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<400> 3

cttgatagtg ttccctgggg g 21

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

ggggacgaag tcgagatgag 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

atgatcggag cttgctggac 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

gccaggccaa gtgttgaaag 20

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

<400> 7

tgacctcaac tacatggtct aca 23

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence

<400> 8

cttcccattc tcggccttg 19

Claims

1. The application of the tricin in the preparation of antitumor drugs is characterized in that the tumors are lung cancer, glioma and pancreatic cancer.

2. The use of claim 1, wherein the lung cancer is non-small cell lung cancer.

3. An antitumor drug for treating lung cancer, glioma and pancreatic cancer, which is characterized by comprising tricin.

4. The antitumor drug as claimed in claim 3, wherein the antitumor drug further comprises a pharmaceutically acceptable excipient.

5. The antitumor drug as claimed in claim 4, wherein the pharmaceutically acceptable adjuvant comprises at least one of a solvent, a filler, a lubricant, a disintegrant, a buffer, a cosolvent, an antioxidant, a bacteriostatic agent, an emulsifier, a binder or a suspending agent.

6. The antitumor drug as claimed in claim 4, wherein the dosage form of the antitumor drug comprises injection, powder injection, granules, capsules, tablets, powder, dripping pills, oral liquid or suppositories.

7. A KRAS mutation inhibitor comprising tricin.

8. A PRKCA inhibitor comprising tricin.

9. An S1P modulator, wherein the S1P modulator comprises tricin.