CN108354939B - Application of acetyl bufalin in the preparation of antitumor drugs - Google Patents

Abstract

The invention discloses an application of acetyl bufalin in preparing antitumor drugs, wherein the structure of the acetyl bufalin is shown as a formula (I), the acetyl bufalin can improve antitumor curative effects, and particularly has a good inhibition effect on lung cancer cells.

Description

Application of acetyl bufalin in the preparation of antitumor drugs

technical field

The invention belongs to the field of biomedicine, and in particular relates to the application of acetylbufalin in the preparation of antitumor drugs.

Background technique

Tumor is one of the main causes of human death, and its morbidity and mortality are on the rise every year. Statistics show that since 2010, malignant tumor has become the leading cause of death for urban and rural residents. Visible tumor prevention and treatment is very urgent. Drug therapy is one of the main means of tumor treatment. Although many anti-tumor drugs have been developed, which can effectively prolong the life of patients or improve the quality of life of patients, the research and development of tumor drugs still faces great challenges. For example, anti-tumor drugs are mostly cytotoxic drugs, which have obvious side effects. The clinical application of these drugs is limited. In the past two decades, targeted therapy for tumors has developed rapidly. Drugs targeting tumor signaling proteins such as imatinib and trastuzumab have shown promising therapeutic effects and low toxic side effects. However, the emergence of acquired drug resistance and the variability of tumor genomes make targeted therapy also face great challenges.

Bufalin, a cardiac glycoside substance, with the molecular formula C ₂₄ H ₃₄ O ₄ , is derived from the serous fluid secreted by the posterior glands and skin of Chinese giant toads and black-orbited toads. The most toxic ligand. Studies have shown that bufalin mainly has the functions of strengthening the heart, anesthesia and analgesia, and also has a certain effect on the nervous system.

At present, some studies have reported that bufalin has a certain anti-cancer effect, but its anti-cancer effect is not good, and its anti-cancer mechanism and target are not clear.

SUMMARY OF THE INVENTION

The invention provides the application of acetyl bufalin in the preparation of antitumor drugs, and the acetyl bufalin has higher curative effect.

A kind of application of acetyl bufalin in the preparation of antitumor medicine, the structure of described acetyl bufalin is shown in formula (I):

In the present invention, the acetyl bufalin is a derivative of bufalin, and the test results show that the antitumor efficacy can be improved by the derivative.

Preferably, the antitumor drug is used for the treatment of lung cancer.

The present invention selects three human non-small cell lung cancer (NSCLC) cell lines PC-9, A549, H460, and observes the effect of acetylbufalin on the proliferation of NSCLC cells through MTT experiment. The results showed that the proliferation of NSCLC cell lines could be inhibited in a dose-dependent manner after 48 hours of treatment, among which H460 had the lowest IC50 and was the most sensitive to the drug. Preferably, the antitumor drug is used to inhibit the proliferation of human non-small cell lung cancer cell lines PC-9, A549 or H460.

The experimental results showed that acetylbufalin can concentration-dependently up-regulate the tumor suppressor protein Bax and down-regulate the expression of the tumor-promoting protein Bcl-2, thereby inducing the apoptosis of NSCLC. Preferably, the acetylbufalin is used to up-regulate the tumor suppressor protein Bax and down-regulate the expression of the tumor-promoting protein Bcl-2.

The test results showed that acetylbufalin could inhibit the expression of G2/M-related proteins in a concentration-dependent manner. Preferably, the acetyl bufalin is used to induce cell G2/M phase arrest.

The test results showed that acetylbufalin can inhibit the phosphorylation of STAT3 in a concentration-dependent manner, and 400nM can significantly inhibit the phosphorylation of STAT3. Preferably, the acetylbufalin is used to inhibit STAT3 phosphorylation.

Compared with the prior art, the beneficial effects of the present invention are embodied in:

In the present invention, by further derivatizing bufalin, the obtained acetylbufalin has better inhibitory effect on tumor cells.

Description of drawings

Figure 1 shows the inhibitory effect of acetyl bufalin on the proliferation of NSCLC cells in Example 2, wherein, the effects of A. bufalin (Bu) and acetyl bufalin (Ac-Bu) on the proliferation of PC-9 cells. B. Inhibitory effect of acetyl bufalin on the proliferation of PC-9, A549 and H460 cells.

FIG. 2 is a graph showing the results of detecting PC-9 apoptosis by Hochest kit staining of cell nuclei in Example 3. FIG.

FIG. 3 is a graph showing the effect of flow cytometry in the detection of apoptosis of PC-9 and A549 cells induced by acetylbufalin in Example 3. FIG.

FIG. 4 is a schematic diagram of the results of WB analysis of apoptosis-related proteins Bcl-2 and Bax in Example 3. FIG.

FIG. 5 is a schematic diagram showing the results of inhibition of NSCLC cell colony formation by acetyl bufalin in Example 4. FIG.

FIG. 6 is a schematic diagram showing the result of cell cycle arrest after acetyl bufalin acted on PC-9 cells for 16 h in Example 5. FIG.

FIG. 7 is a schematic diagram showing the results of inhibition of NSCLC cell cycle-related protein expression by acetyl bufalin in Example 5. FIG.

FIG. 8 is a schematic diagram showing the results of inhibition of STAT3 phosphorylation by acetyl bufalin in PC-9, H460 and A549 cells in Example 6. FIG.

Detailed ways

The synthesis of embodiment 1 acetylbufalin

Bufalin (15 mg, 0.0388 mmol) and triethylamine (0.39 mmol) were dissolved in dry dichloroethane (1 mL), then acetyl chloride (0.39 mmol) was added and the reaction mixture was stirred at room temperature for 19 hours. The solvent was spun dry, followed by column chromatography on silica gel (DCM/MeOH=50:1 to 20:1) to give the product (17 mg, 100% yield) as a pale yellow solid.

The reaction formula is as follows:

The product characterization data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.82 (dd, J=9.6, 4.0 Hz, 1H), 7.22 (s, 1H), 6.26 (d, J=9.6 Hz, 1H), 5.07 (s, 1H) ,2.49-2.40(m,1H),2.26-2.15(m,1H),2.09-1.99(m,4H),1.94-1.81(m,2H),1.80-1.35(m,18H),1.27(s, 3H), 0.69(s, 3H); ¹³ C NMR (100MHz, CDCl3) δ 170.7, 162.3, 148.6, 146.7, 122.6, 115.3, 85.4, 70.4, 51.3, 48.4, 42.4, 40.9, 36.8, 35.9, 35.2, 32.8, 30.5, 30.5, 28.7, 26.4, 25.1, 23.7, 21.5, 21.4, 21.3, 16.5; ESI-HRMS: calcd.forC ₂₆ H ₃₇ O ₅ ⁺ (M+H) ⁺ 429.2636, found 429.2637.

Example 2 Acetobufalin inhibits the proliferation of NSCLC cells

Three human non-small cell lung cancer cell lines PC-9, A549, H460 were selected, and the effects of bufalin (Bu) and acetylbufalin (Ac-Bu) on the proliferation of these cells were observed by MTT assay. NSCLC cells were seeded in 96-well plates (the cell concentration was 5000/well), 100ul RPMI1640 medium was added to each well and cultured overnight. After the cells adhered, the medium was changed, and different concentration gradients of bufalin and acetylbufalin were added. Control Groups were added an equal amount of DMSO. After culturing for 48 hours, MTT solution was added, and the culture was continued for 4 hours. The supernatant was discarded, DMSO was added to dissolve, the absorbance value was detected, and the IC50 was determined. The results are shown in Figure 1: the inhibitory effect of acetyl bufalin on PC-9 cell proliferation was about 5 times higher than that of bufalin (Figure 1A), and acetyl bufalin could dose-dependently inhibit the proliferation of NSCLC cell lines (Figure 1A). 1B).

Example 3 Acetobufalin induces apoptosis of NSCLC cells.

We stained the nucleus with Hoechst 33258 apoptosis staining kit, and judged the apoptosis by observing the color of the nucleus. The results are shown in Figure 2. It was found that different concentrations of acetylbufalin (100nM, 200nM, 400nM) affected PC-9 cells After 24 hours, the color of the nucleus changed from normal blue to bright blue, indicating that the cells were undergoing apoptosis, and the apoptosis became more obvious with the increase of the concentration.

We used Annexin V-FITC/PI double-staining flow cytometry to detect the effect of acetylbufalin-induced apoptosis in NSCLC cells. The NSCLC cells were inoculated in 6-well plates, and after culturing for 18 hours, the experimental group was added with different concentrations of acetyl bufalin (final concentrations were 100nM, 200nM, 400nM), and the control group was added with the same amount of DMSO. The results of dose-dependent induction of apoptosis in NSCLC cells are shown in Figure 3 .

We treated PC-9, A549, and H460 with different concentrations of acetylbufalin (100nM, 200nM, 400nM), and the control group was added with the same amount of DMSO, and the protein was extracted after 24h of treatment, and the total cell protein extract (70mg) was detected by western blotting. Apoptosis-related proteins Bcl-2 and Bax, GAPDH was used as an internal reference, and the results are shown in Figure 4. It was found that acetylbufalin can concentration-dependently up-regulate the tumor suppressor protein Bax and down-regulate the expression of the tumor-promoting protein Bcl-2, thereby inducing apoptosis in NSCLC .

Example 4 Acetyl bufalin inhibits NSCLC cell colony formation

Tumor cells can proliferate indefinitely to form cell colonies. Through colony formation experiments, we can understand the effect of compounds on cell proliferation. The NSCLC cells were seeded in a 6-well plate (the cell concentration was 1×10 ³ /well), and the cell suspension was repeatedly pipetted to make the cells fully diffuse. After pre-incubation for 18 hours, the experimental group was added with different concentrations of bufalin (final concentrations were 100nM, 200nM, 400nM, and the solvent was DMSO), and the control group was added with the same amount of DMSO. The culture was continued for 24 hours, and the culture medium was replaced with fresh RPMI1640 for one week to form a single cell colony. The results are shown in FIG. 5 , and it can be seen from FIG. 5 that acetylbufalin can inhibit the colony formation of these three NSCLC cells in a dose-dependent manner.

Example 5 Acetobufalin induces cell cycle arrest in NSCLC

We used the nucleic acid dye PI to label DNA and detect cell cycle arrest by flow cytometry. The PC-9 cells were inoculated in 6-well plates, and after culturing for 18 hours, the experimental group was added with different concentrations of acetyl-bufalin (final concentrations were 100nM, 200nM, 400nM), and the control group was added with the same amount of DMSO, and it was found that the effect of acetyl-bufalin for 16h Afterwards, it was able to block the cell cycle in a dose-dependent manner and blocked in the G2/M phase. The results are shown in Figure 6.

At the same time, the levels of various target proteins in the total cell protein extract (70 mg) were detected by western blotting, and the cycle-related proteins MDM2, CyclinB1, CDC2 and P53 were analyzed, and GAPDH was used as an internal reference. The results are shown in Figure 7. It was found that acetylbufalin can Concentration-dependent inhibition of cycle-related protein expression.

Example 6 Acetyl bufalin exerts anti-tumor effect by inhibiting STAT3

We further explored the mechanism and direct target of acetylbufalin's antitumor effect. We treated human lung cancer cells PC-9, A549, and H460 with different concentrations of acetylbufalin (final concentrations: 100 nM, 200 nM, 400 nM), and detected STAT3 and p-STAT3 in total cell protein extracts (70 mg) by western blotting For the protein level, GAPDH was used as an internal reference, and the results are shown in Figure 8. It was found that acetylbufalin can inhibit the phosphorylation of STAT3 in a concentration-dependent manner, and 400nM can significantly inhibit the phosphorylation of STAT3. Therefore, acetylbufalin may exert its anti-tumor effect by inhibiting STAT3, and STAT3 may be its direct target.

Claims (5)

1. the application of a kind of acetyl bufalin in the preparation of antitumor medicine, it is characterized in that, the structure of described acetyl bufalin is as shown in formula (I):

(I) The antitumor drug is used for treating non-small cell lung cancer. 2. the application of acetyl bufalin according to claim 1 in the preparation of antitumor drug, it is characterized in that, described antitumor drug is used for inhibiting human non-small cell lung cancer cell line PC-9, A549 or H460 proliferation. 3. the application of acetyl bufalin according to claim 1 in the preparation of antitumor drug, it is characterized in that, described acetyl bufalin is used for up-regulating tumor suppressor protein Bax, down-regulating the expression of tumor-promoting protein Bcl-2 . 4. The application of acetyl bufalin according to claim 1 in the preparation of antitumor drugs, wherein the acetyl bufalin is used for inducing cell G2/M phase arrest. 5 . The application of acetyl bufalin according to claim 1 in the preparation of antitumor drugs, wherein the acetyl bufalin is used for inhibiting STAT3 phosphorylation. 6 .

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