CN109381470B - C21 steroid saponification compound in marsdenia tenacissima and application thereof - Google Patents
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
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- A—HUMAN NECESSITIES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention relates to a C21 steroid saponin compound and application thereof in preparing anti-lung cancer drugs. The biological experiment result shows that the compound can inhibit the migration of tumor cells and make the tumor cells in the G0/G1 stage. In addition, the apoptosis of tumor cells can be induced by up-regulating Bax and down-regulating Bcl-2 expression, thereby releasing Cytochrome c and finally activating Caspase 3 and Caspase 9. Caspase 8 may also be activated and apoptosis induced by the death receptor pathway. The compound can inhibit the migration and invasion of tumor cells by down-regulating the expression of MMP-2 and MMP-9 proteins, has obvious effect and achieves the effect of resisting lung cancer.
Description
Technical Field
The invention belongs to the field of natural product compound pharmacy, and relates to a C21 steroid saponin compound and application thereof.
Background
Lung cancer poses a high threat to human health and quality of life and has become a leading cause of cancer death in recent years. In the last decade approximately 160 million people worldwide have been diagnosed with lung cancer, with over 130 deaths, and in addition, lung cancer is at the head of the lethal cancer list in men, second only to breast cancer in women. Cancer reports published in 2017 in the united states indicate that while lung cancer is not the first in the united states, it still accounts for the first of all tumor deaths, more than one-fourth of which are caused by cancer, and of which about 85% -90% are non-small cell lung cancers (NSCLC). NSCLC cells have a longer growth cycle, slower division rate, and relatively late diffuse metastasis compared to Small Cell Lung Cancer (SCLC). Currently, surgical treatment remains the mainstream of early-stage NSCLC, and for NSCLC of advanced stage or which cannot be treated by surgery, a conservative treatment scheme is mainly adopted. However, even so, the overall 5-year survival rate for lung cancer patients undergoing radical surgery is about 35-40%, with a 5-year survival rate decreasing from 77% in stage IA to 23% in stage iiia, with about 63.9% of patients dying from postoperative recurrence and metastasis. The urgent problems in medical research are to overcome lung cancer, to explore etiology, disease progression and metabolic mechanism, and to find targeted drugs for lung cancer.
The traditional Chinese medicine is a treasure inherited by China for thousands of years. Practice proves that compared with chemical drugs, the traditional Chinese medicine has unique advantages and characteristics, such as rich sources, various varieties and relatively few adverse reactions, plays a role through multiple targets and multiple channels, improves the treatment effect through the combination of multiple medicines, reduces toxic and side effects, has relatively low price and the like. Therefore, the focus of attention of various researchers is to define the effective components and the separation thereof of the traditional Chinese medicine, further research and explore the pharmacological action mechanism of the traditional Chinese medicine and develop a new anti-tumor medicine. The plant marsdenia tenacissima is widely distributed in Yunnan, Guizhou provinces and other provinces in China and is a traditional anti-tumor drug for Dai medicine in China. Recently, various preparations of cancer-eliminating drug on the market, which use marsdenia tenacissima as a raw material, have been proved to have anticancer effects. C21 steroid saponin contained in caulis Marsdeniae Tenacissimae is their main active ingredient.
Disclosure of Invention
The present inventors first evaluated the activity of Marsdenoside C (TGT-7), 11 α -O-acetyl-12 β -O-benzoyl auxin B (TGT-9), 11- α -O-2-methylbutyryl-12 β -O-benzoyl auxin B (TGT-13) and 11 α -O-benzoyl-12 β -O-acetyl succinate B (TGT-15) using the A549, Caco-2, EC-109, PC-3 and HepG2 cell lines and found that the above compounds isolated from marsdenia tenacissima were all able to effectively inhibit A549 cells and were more sensitive than other cell lines. Furthermore, we investigated the cytotoxic effects of four C21 steroid saponins at different doses on BEAS-2B normal human lung epithelial cells using trypan blue dye exclusion assay, showing that the four C21 steroid saponins are non-toxic to BEAS-2B cells in the concentration range of 0 to 100 μ M.
The migration and invasion of tumor cells are closely related to the metastasis and recurrence of cancer, and many patients die after tumor resection due to cancer metastasis. MMP-2 and MMP-9 are closely related to angiogenesis, invasion and metastasis of tumor cells. The invention discovers that four C21 steroid saponins (TGT-7, TGT-9, TGT-13 and TGT-15) can reduce the expression of MMP-2 and MMP-9 in A549 cells, thereby having effective treatment effect on cancer caused by cancer cell migration.
The outbreak of cancer at the site of fixation is mainly from tumor cell proliferation. At present, a plurality of chemotherapy drugs can activate different signal paths in cells, thereby blocking the G0/G1 phase, the S phase or the G2/M phase of tumor cells and achieving the purpose of inhibiting the proliferation of the tumor cells. Cell proliferation is closely related to the cell cycle, and cell cycle errors can lead to cell proliferation and thus the development of cancer. The invention finds that the four C21 steroid saponins (TGT-7, TGT-9, TGT-13 and TGT-15) can block the A549 cells in the G0/G1 phase, prevent the cells from developing to the S phase (DNA replication phase) and the M phase (cell division phase) and slow down the growth and proliferation of the A549 cells.
Apoptosis, which is programmed death regulated by cellular genes and belongs to the normal physiological phenomenon of cells, can maintain the stability of the internal environment of cells. The invention detects the change of the fluorescence rate of the A549 cell after dyeing by Annexin V-FITC/PI, and proves that the four C21 steroid saponins can effectively promote the apoptosis of the A549 cell. Mitochondria serve as the central regulatory pathways of endogenous apoptosis, and their status can be understood by measuring mitochondrial membrane potential. Based on this point, the invention finds that four C21 steroid saponins can reduce mitochondrial membrane potential and indicate that the C21 steroid saponins can induce apoptosis of A549 cells through a mitochondrial endogenous pathway. Reactive Oxygen Species (ROS) are important signal transduction factors that can participate in and regulate apoptosis and play an important role in the apoptotic process. ROS are mainly generated in mitochondria, and when the ROS are excessively generated, mitochondrial membrane lipid peroxidation can be caused, mitochondrial membrane potential is further influenced, cytochrome C release is promoted, and endogenous cell apoptosis is finally induced. Meanwhile, some studies show that ROS can also cause exogenous apoptosis, mainly by starting the sensitivity of tumor cells to sFasl and then activating Caspase-8 to mediate exogenous apoptosis through a death receptor Fas/Fasl pathway. The invention discovers that TGT-7, TGT-9, TGT-13 and TGT-15 can improve the ROS level in A549 cells and promote apoptosis. Cytochrome C-mediated mitochondrial apoptotic pathways are regulated by the Bcl-2 protein family. The research of the invention shows that the expression level of Bax is increased along with the increase of the concentrations of TGT-7, TGT-9, TGT-13 and TGT-15, the expression level of Bcl-2 is in negative correlation with the dosage, and the change of the two results in the increase of the Bax/Bcl-2 level, promotes the release of cytochrome C and finally induces the apoptosis. Casepase-8 is a key factor of apoptosis in a death receptor pathway, and results show that high concentration of 4C 21 steroid saponins (TGT-7, TGT-9, TGT-13 and TGT-15) can increase the expression of the cleared-caspase 8 protein, and suggest that the four C21 steroid saponins can induce apoptosis through the death receptor pathway besides the mitochondrial pathway.
The invention also relates to a pharmaceutical composition, which comprises one or more of TGT-7, TGT-9, TGT-13 and TGT-15, wherein the content of TGT-7, TGT-9, TGT-13 and/or TGT-15 compounds is 0.1-99%, and the rest is pharmaceutically acceptable carriers and/or excipients. The dosage form of the composition can be prepared into any pharmaceutically acceptable dosage form. Comprises granules, tablets, granules, soft capsules, dripping pills, ointments, injections and the like.
The invention proves that TGT-7, TGT-9, TGT-13 and TGT-15, 4C 21 steroid saponins can reduce the expression of MMP-2 and MMP-9 to inhibit the migration of A549 cells, and can be used as a medicament for treating cancers caused by the migration of non-small cell lung cancer cells. In addition, TGT-7, TGT-9, TGT-13 and TGT-15 can up-regulate Bax and down-regulate Bcl-2 expression, release cytochrome C, and finally activate Caspase-3 and Caspase-9 to induce apoptosis of A549 cells, and can be used as a medicine for treating cancers caused by proliferation of non-small cell lung cancer cells.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
FIG. 1 depicts the inhibition of cell migration and invasion in A549 cells by TGT-7, TGT-9, TGT-13, TGT-15.
FIG. 2 depicts TGT-7, TGT-9, TGT-13, TGT-15 inhibits the A549 cell cycle.
FIG. 3 depicts the effect of TGT-7, TGT-9, TGT-13, TGT-15 on A549 cell apoptosis.
FIG. 4 depicts the effect of TGT-7, TGT-9, TGT-13, TGT-15 on mitochondrial membrane potential of A549 cells.
FIG. 5 depicts the effect of TGT-7, TGT-9, TGT-13, TGT-15 on ROS levels in A549 cells.
FIG. 6 depicts the effect of TGT-7, TGT-9, TGT-13, TGT-15 on migration and apoptosis related key proteins.
FIG. 7 depicts the chemical structures of TGT-7, TGT-9, TGT-13, TGT-15.
Detailed Description
Example 1 Effect on cell migration and invasion
Experimental methods
Migration test: a549 cells at 1X 10 per well6The individual cells were seeded in 6-well plates, and when A549 cells were adhered to a density of 90%, a monolayer of A549 cells was scraped off in the middle of each well using a 20. mu.l pipette tip, washed three times with PBS, cultured for 36h with the addition of medium containing TGT-7, TGT-9, TGT-13, TGT-15 at 28. mu.M, 44. mu.M, 29. mu.M, 47. mu.M, respectively, and the control group was supplemented with DMEM medium. Three points per wound were then selected and the adjacent wound spacing was calculated using Image j.
Invasion test: the Matrigel gel was allowed to stand at 4 ℃ overnight, thawed, and then added to a serum-free medium to a final concentration of 1mg/mL to prepare a Matrigel gel. 100 μ L of the prepared matrigel was added vertically to the bottom of the upper chamber, and 600 μ L of each well containing 10% FBS was added to the lower chamber. A549 cells were resuspended in 100. mu.L of medium containing 0.1% BSA and TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M), TGT-15 (47. mu.M), and then cultured for 24 hours to allow the cells to migrate through the filter, fixed in methanol for 30 minutes, and stained with 1% crystal violet for 25 minutes. The crystal violet was washed from the surface, 5 images were taken at random under an inverted microscope, and the number of transmembrane cells in the photographs was counted.
The migration experiment results show that the cells in the control group migrated remarkably. The relative widths of the cell scratch were (0.852 + -0.087), (0.549 + -0.033), (0.909 + -0.045) and (0.538 + -0.056) after treatment with TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M) and TGT-15 (47. mu.M), respectively. The results were statistically significant compared to the control (0.443 ± 0.075) (fig. 1A). The results of the invasion experiments showed that many a549 cells of the control group were filtered from the upper to the lower part of the transwell chamber. After TGT-7(28 mu M), TGT-9(44 mu M) and TGT-13(29 mu M) treatment, A549 cells are obviously reduced after passing through the filter membrane; however, after TGT-15(47 μ M) treatment, a549 cells passing through the filter were reduced but not significantly changed compared to the control group; the invasion numbers of A549 cells are (29.87 + -0.70), (26.33 + -0.50), (58.8 + -0.92) and (66.00 + -3.74), respectively. TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M) groups were statistically significant compared to the control group (68.40. + -. 2.09) (FIG. 1B). These results indicate that TGT-7, TGT-9, TGT-13 and TGT-15 can inhibit migration and invasion of A549 cells.
Example 2 cell cycle analysis
Experimental methods
Cell cycle analysis was performed using flow cytometry. A549 cells were treated with TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M) and TGT-15 (47. mu.M) for 24 hours, collected and washed twice with ice-cold PBS. The deposited cells were shaken with 70% ethanol and suspended in a-20 ℃ refrigerator for more than 24 hours.
After the cells were fixed, they were placed on a centrifuge and centrifuged at 1000rpm for 5 minutes. The ethanol was aspirated and washed three times with ice-cold PBS. The supernatant was discarded. 0.5mL of PI/RNase was added to the cells and left to stand for about 15 minutes in the dark. Cell cycle analysis was performed immediately using flow cytometry.
A549 cells were treated with TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M), TGT-15 (47. mu.M) for 24h, then A549 cells were fixed and stained with propidium iodide, and cells were examined for A549 cell cycle phase transition using a cell treatment flow cytometer. After the A549 cells are treated by TGT-7, TGT-9, TGT-13 and TGT-15, the proportion of the cells in the G0/G1 phase is increased from (65.31 +/-3.79)% to (75.58 +/-0.44)%, (71.63 +/-2.02)%, (80.27 +/-2.13)%, and (69.17 +/-1.05)%. There was a different degree of increase in the number of cells at stage G0/G1 compared to the control group (FIG. 2A, B). The results show that TGT-7, TGT-9, TGT-13, TGT-15 can make A549 cells stagnate at G0/G1.
Example 3 apoptosis assay
Experimental methods
Apoptosis was analyzed using flow cytometry. A549 cells were treated with TGT-7 (28. mu.M, 56. mu.M), TGT-9 (44. mu.M, 88. mu.M), TGT-13 (29. mu.M, 58. mu.M), TGT-15 (47. mu.M, 94. mu.M) for 24h, then the supernatant was collected in a 15mL tip centrifuge tube, adherent cells were treated with trypsin and detached, collected into the corresponding centrifuge tube, centrifuged at 1000rpm for 5 minutes, the supernatant aspirated and washed twice with 1 × binding buffer, then stained with Annexin V and PI in sequence according to the manufacturer's instructions. Finally, a549 cells were analyzed by flow cytometry under observation with a fluorescence microscope.
Apoptotic cells were detected by Annexin V-FITC/PI double staining, and A549 cells showed more green fluorescent staining compared to the control group after 24 hours of TGT-7 (28. mu.M, 56. mu.M), TGT-9 (44. mu.M, 88. mu.M), TGT-13 (29. mu.M, 58. mu.M), TGT-15 (47. mu.M, 94. mu.M) (FIG. 3A). In addition, when apoptotic cells were quantitatively measured using a flow cytometer, the number of apoptotic cells (the sum of early and late blight cells) in the drug-treated group was significantly increased compared to the control group, and the number of apoptosis increased with the increase in concentration. These data indicate that TGT-7, TGT-9, TGT-13, TGT-15 all induced apoptosis of A549 cells in a concentration-dependent manner.
Example 4 mitochondrial membrane potential measurement
Experimental methods
JC-1 is a cationic dye that changes fluorescence by mitochondrial membrane potential (. DELTA.. psi.m). A549 cells were treated with TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M), TGT-15 (47. mu.M) for 24h and then stained with JC-1 at 37 ℃ for 20 min. Images were taken with a fluorescence microscope. When the mitochondrial membrane potential drops, the fluorescence changes from red to green. Analysis of red and green fluorescence intensities by using Image J software was used to reflect potential changes in the mitochondrial membrane.
The influence of TGT-7, TGT-9, TGT-13 and TGT-15 on the mitochondrial membrane potential of A549 cells is detected by detecting the change of red-green fluorescence ratio after JC-1 staining. The results show that the A549 cells treated by TGT-7, TGT-9, TGT-13 and TGT-15 have enhanced green fluorescence and reduced red and green fluorescence, and the red and green fluorescence ratio is respectively reduced to (3.10 +/-0.43), (2.74 +/-0.55), (9.54 +/-0.58) and (9.64 +/-1.10) from (14.24 +/-1.14) of the control group, and the statistical difference is shown (FIG. 4A). The results were then confirmed by flow cytometry experiments to be substantially identical to the above results (fig. 4B). These data demonstrate that TGT-7, TGT-9, TGT-13, TGT-15 all cause depolarization of mitochondrial membrane potentials.
Example 5 intracellular ROS detection
Experimental methods
The amount of intracellular ROS produced is measured using a ROS detection kit. After treating A549 cells with TGT-7 (28. mu.M), TGT-9 (44. mu.M), TGT-13 (29. mu.M), TGT-15 (47. mu.M) for 24h, A549 cells were cultured with 10. mu.M CDFH-DA in an incubator at 37 ℃ for 30 minutes, and then washed twice with PBS. Finally, a549 cells were observed by fluorescence microscopy and treated by flow cytometry to measure DCFH-DA fluorescence.
The effect of TGT-7, TGT-9, TGT-13, TGT-15 on ROS production in A549 cells was assessed by measuring changes in reactive oxygen species in A549 cells after DCFH-DA staining. The results of the experiments showed that the green fluorescence of A549 cells was significantly increased after treatment with TGT-7, TGT-9, TGT-13, TGT-15 compared to the control group (FIGS. 5A, B, C). These data indicate that TGT-7, TGT-9, TGT-13, TGT-15 all promote the production of reactive oxygen species in A549 cells, resulting in an increase in the concentration of reactive oxygen species.
Example 6 Western blot
Experimental methods
A549 cells treated with different concentrations of TGT-7 (28. mu.M, 56. mu.M), TGT-9 (44. mu.M, 88. mu.M), TGT-13 (29. mu.M, 58. mu.M), TGT-1 (47. mu.M, 94. mu.M) were lysed with protease inhibitors in RIPA buffer. Protein concentration was then determined by performing the BCA protein assay kit. Proteins were separated by SDS-PAGE and transferred to PVDF membrane. The corresponding proteins were analyzed using MMP-2, MMP-9, cleared-caspase 3, cleared-caspase 8, Bax, Bcl-2, and Cytochrome C primary antibodies, followed by incubation with secondary antibodies. Finally, immunoblots were quantified using ImageJ, and the images shown represent three independent experiments.
The results show that TGT-7, TGT-9, TGT-13 and TGT-15 can reduce the expression of MMP-2 and MMP-9 in A549 cells. The expression of MMP-2 and MMP-9 in A549 cells gradually decreases with the increase of the drug concentration, and is dose-dependent. In addition, TGT-7, TGT-9, TGT-13, TGT-15 at both high and low concentrations increased the expression of cytochrome C, leaved-caspase 9 and free-caspase 3, indicating that they may promote release of mitochondrial cytochrome c, activate caspase 9 and caspase 3 to induce apoptosis. High concentrations of TGT-7, TGT-9, TGT-13, TGT-15 increased the expression of clear-caspase 8, suggesting that TGT-7, TGT-9, TGT-13, TGT-15 may induce apoptosis through the mitochondrial pathway and possibly through the death receptor pathway.
The experimental data of the above examples were processed with SPSS 20.0 statistical software. Unless otherwise indicated, all data in the present invention are arithmetic means of data from independent experiments performed in triplicate. Results are expressed as mean ± Standard Deviation (SD). When the variance is uniform, the variance is analyzed using one-way variance (ANOVA), with minimal significant difference (LSD). In contrast, the Dunnett T3 test was used when the variance was not uniform. Statistically significant differences were marked as P <0.05, P <0.01, P <0.001 in the analysis of TGT-7, TGT-9, TGT-13, TGT-1 treated groups versus untreated control cells.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (5)
- Use of a C21 steroid saponin compound in the manufacture of a medicament for the treatment of cancer, wherein the cancer is caused by migration of non-small cell lung cancer cells, the C21 steroid saponin compound being selected from the group consisting of compounds of formula 2 and formula 4:
- 2. the use according to claim 1, wherein the medicament is prepared as a pharmaceutical composition.
- 3. Use according to claim 2, characterized in that the compound is present in an amount of 0.1-99% with the remainder being pharmaceutically acceptable carriers and/or excipients.
- 4. Use according to claim 3, characterized in that the composition is in the form of any pharmaceutically acceptable formulation.
- 5. The use as claimed in claim 4, wherein the composition is in the form of granules, tablets, granules, soft capsules, drop pills, ointments or injections.
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