CN105640955B - A new application of Aminoquinol - Google Patents

Abstract

The invention discloses a new application of Aminoquinol, and a series of experiments are used for testing the influence of the Aminoquinol on liver cancer cell lines HepG2 and Huh7, so that the Aminoquinol is a target CDK4/6 inhibitor, the number of cells of HepG2 and Huh7 which are stagnated in a G1 stage can be increased, and the number of cells in an S stage is reduced; can improve the apoptosis rate of HepG2 and Huh7 cells; CDK4,6, cyclinD, pho-CDK4/6 protein expression capable of inhibiting cell cycle G1 phase signal passage has anti-liver cancer effect, so that the Aminoquinol can be used for preparing anti-cancer drugs for treating liver cancer; since the Aminoquinol is a registered drug for resisting parasitic diseases by FDA, the cost of later clinical experiments and the time for drug development are shortened, and the cost and the time for developing a new anti-cancer drug are greatly saved.

Description

A new application of Aminoquinol

technical field

The invention relates to the field of medicine, in particular to a new application of Aminoquinol.

Background technique

Primary liver cancer is one of the important diseases that endanger human health, and has been listed as the focus of research and prevention by the Chinese government for many times. The treatment of liver cancer has long been a worldwide problem. The onset of liver cancer is hidden, and most of the patients are in the middle or late stage when they go to the doctor. Moreover, liver cancer is a malignant tumor with high malignancy, strong infiltration and metastasis, and easy drug resistance. The current clinical treatment of liver cancer with chemotherapy drugs is not very effective. , and there is a recurrence rate of 70-90% after surgical treatment, and the relapsed patients are resistant to chemotherapy and radiotherapy, so it is imminent to find new anti-liver cancer drugs.

Cell cycle disorder is a sign of malignant tumor formation. The activation and inactivation of cyclin-dependent kinases (CDKs) maintain the orderly progress of each phase of the cell cycle. Cyclin-dependent kinase CDK4,6 is a key regulator of the early G1 phase of the cell cycle. Inhibiting cyclin-dependent kinase CDK4,6 can inhibit the proliferation of tumor cells and is also a good tumor drug target, so Screening out drugs that can inhibit the activity of cyclin-dependent kinase CDK4, 6 can inhibit the proliferation of tumor cells and exert the effect of anti-liver cancer.

Contents of the invention

The purpose of the present invention is to provide a new application of Aminoquinol for the preparation of drugs for treating liver cancer, so as to solve the problems raised in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions:

A new use of Aminoquinol, said Aminoquinol is a target CDK4/6 inhibitor, has anti-liver cancer effect, and Aminoquinol is used to prepare anticancer drugs for treating liver cancer.

As a further solution of the present invention: the Aminoquinol has an anticancer effect on the liver cancer cell lines HepG2 and Huh7, and Aminoquinol with a concentration of 3 μM/L, 10 μM/L, and 30 μM/L interferes with the liver cancer cell lines HepG2 and Huh7 for MTT test detection, showing The time was 24h, 48h, and 72h, showing that there was a time- and dose-dependent inhibition of the increase in HepG2 and Huh7; the Aminoquinol had anticancer effects on bladder cancer cell lines RT112 and SW780, and 3 μM/L, 10 μM/L, 30 μM/L L concentration of Aminoquinol interfered with the bladder cancer cell lines RT112 and SW780 for MTT assay, and the presentation time was 24h, 48h, and 72h, showing that the time- and dose-dependent inhibition of RT112 and SW780 increased.

As a further solution of the present invention: Aminoquinol interferes with liver cancer cells HepG2 and Huh7 under the conditions of 3 μM/L, 10 μM/L, and 30 μM/L concentration by flow cytometry. After 6h, 12h, and 24h of interference, Aminoquinol increases HepG2 and Huh7 Cells stagnated in G1 phase showed a time- and dose-dependent increase trend, P<0.05. With the increase of G1 phase cells, the S phase cells decreased.

As a further solution of the present invention: detect the number of apoptosis cells after Aminoquinol interferes with liver cancer cells Huh7 and HepG2 under the concentration conditions of 3 μM/L, 10 μM/L, and 30 μM/L by flow cytometry, and the presentation time is 24h, 48h, and 72h, respectively , Aminoquinol increased the number of apoptotic cells, showing an increasing trend in a time- and dose-dependent manner, P<0.05.

As a further solution of the present invention: use Aminoquinol to interfere with liver cancer cells Huh7 and HepG2, and detect the protein expression of the signal pathway in the G1 phase of the cell cycle by Western experiments; The changes of CDK2, 4, 6, cyclinD, and pho-CDK4/6 protein expression in the pathway were detected; the results showed that the protein expression of CDK4, 6, cyclinD, pho-CDK4, and pho-CDK6 after Aminoquinol interfered with liver cancer cells Huh7 and HepG2 There was a dose-dependent downward trend, CDK2 protein expression did not change, and Aminoquinol had an inhibitory effect on CDK4/6.

Compared with the prior art, the beneficial effect of the present invention is: the present invention tests the influence of Aminoquinol on the liver cancer cell lines HepG2 and Huh7 through a series of experiments, and shows that Aminoquinol is a target CDK4/6 inhibitor, which can increase the stagnation of HepG2 and Huh7 in The number of cells in the G1 phase can reduce the number of cells in the S phase; it can increase the apoptosis rate of HepG2 and Huh7 cells; it can inhibit the expression of CDK4, 6, cyclinD, and pho-CDK4/6 protein in the G1 phase signaling pathway of the cell cycle, and has anti-liver cancer effects. Therefore, Aminoquinol can be used to prepare anticancer drugs for the treatment of liver cancer; since Aminoquinol is already registered by the FDA as an anti-parasitic drug, it shortens the cost of clinical trials in the later stage and the time for drug development, greatly saving the development time. The cost and timing of a new cancer drug.

Description of drawings

Fig. 1 is a graph showing the effect of different concentrations of Aminoquinol on the proliferation of Huh7 cells in the present invention.

Fig. 2 is a graph showing the influence of different concentrations of Aminoquinol on the proliferation of HepG2 cells in the present invention.

Fig. 3 is a graph showing the effect of different concentrations of Aminoquinol and different presentation times on the proliferation of Huh7 cells in the present invention.

Fig. 4 is a graph showing the effect of different concentrations of Aminoquinol and different presentation times on the proliferation of HepG2 cells in the present invention.

Fig. 5 is a graph showing the effect of different concentrations of Aminoquinol and different presentation times on the proliferation of RT112 cells in the present invention.

Fig. 6 is a graph showing the effect of different concentrations of Aminoquinol and different presentation times on the proliferation of SW780 cells in the present invention.

Fig. 7 is a graph showing the change of the cell number of Huh7 in the G1 phase with different concentrations of Aminoquinol and different presentation times in the present invention.

Fig. 8 is a graph showing the change of the cell number of HepG2 in the G1 phase with different concentrations of Aminoquinol and different presentation times in the present invention.

Fig. 9 is a histogram showing the effect of different concentrations of Aminoquinol on the cell cycle distribution of Huh7 in the present invention.

Figure 10 is a histogram showing the effect of different concentrations of Aminoquinol on the cell cycle distribution of HepG2 in the present invention.

Fig. 11 is a graph showing the changes in the number of Huh7 apoptotic cells at different concentrations of Aminoquinol and at different presentation times in the present invention.

Fig. 12 is a graph showing the changes in the number of apoptosis cells in HepG2 with different concentrations of Aminoquinol and different presentation times in the present invention.

Fig. 13 is a graph showing the effect of different concentrations of Aminoquinol in the present invention on inhibiting the expression of CDK2, 4, 6, cyclinD, pho-CDK4 and pho-CDK6 proteins in Huh7 and HepG2.

Fig. 14 is a bar graph showing the inhibition of CDK2 protein expression in Huh7 and HepG2 by different concentrations of Aminoquinol in the present invention.

Fig. 15 is a bar graph showing the inhibition of cyclinD protein expression in Huh7 and HepG2 by different concentrations of Aminoquinol in the present invention.

Fig. 16 is a bar graph showing the inhibition of CDK6 protein expression in Huh7 and HepG2 by different concentrations of Aminoquinol in the present invention.

Fig. 17 is a bar graph showing the inhibition of pro-CDK6 protein expression in Huh7 and HepG2 by different concentrations of Aminoquinol in the present invention.

Fig. 18 is a bar graph showing the inhibition of pro-CDK4 protein expression in Huh7 and HepG2 by different concentrations of Aminoquinol in the present invention.

Fig. 19 is a bar graph showing the inhibition of CDK4 protein expression in Huh7 and HepG2 by different concentrations of Aminoquinol in the present invention.

Detailed ways

The technical solution of this patent will be further described in detail below in conjunction with specific embodiments.

A new use of Aminoquinol, said Aminoquinol is a target CDK4/6 inhibitor, has anti-liver cancer effect, and Aminoquinol is used to prepare anticancer drugs for treating liver cancer.

The definitive experiment for this new use included the following steps:

1) Use computer idock software to screen out candidate CDK4/6 inhibitors against liver cancer against target CDK4/6:

Analyze 4,914 drugs from the FDA-certified ZINC drug database included in the dbap and fda catalogs and sort them according to the binding affinity value, find out and purchase 9 compounds with the highest scores for the next step of the experiment, They are Lifibrate, Nizofenone, fumarate, Chloracyzine, adafenoxate, Cloricromene, Sulconazole, nitrate, Aminoquinol.

2) Verify the candidate inhibitors screened for the first step through in vivo and in vitro experiments in the following laboratories:

2.1) The MTT test screened out the compound Aminoquinol with the lowest IC50 and has not been reported. The anticancer effects of 9 compounds were first checked through the MTT test. These nine compounds have anticancer effects on the liver cancer cell lines HepG2 and Huh7 respectively; Graphpad prime5 software calculates its IC50 value, compares the corresponding IC50 values of each compound, and then screens out the most effective compound Aminoquinol, whose Huh7 IC50 is 2.05 μM/L, and HepG2 IC50 is 2.35 μM/L. Aminoquinol interferes with liver cancer cell lines HepG2 and Huh7. MTT assay showed time (24h, 48h, 72h) and dose (3μM/L, 10μM/L, 30μM/L) dependent inhibitory increase effects; the Aminoquinol had anticancer effects on bladder cancer cell lines RT112 and SW780, The IC50 of the bladder cancer cell line RT112 was 4.34 μM/L, and the IC50 of SW780 was 5.06 μM/L. Aminoquinol was used to interfere with the bladder cancer cell lines RT112 and SW780 at concentrations of 3 μM/L, 10 μM/L, and 30 μM/L. 24h, 48h, 72h, showing the time and dose-dependent inhibition of RT112 and SW780 increase; as shown in Figure 1-6.

2.2) Cell cycle detection of Aminoquinol

In order to observe the inhibitory effect of Aminoquinol on liver cancer cells, the cell cycle distribution after 6h, 12h, and 24h interference of Aminoquinol under different concentrations (3, 10, 30μM/L) of liver cancer cells HepG2 and Huh7 was detected by flow cytometry , Compared with the control group, Aminoquinol greatly increased the number of cells arrested in G1 phase, and showed a time- and dose-dependent increase trend (P<0.05). At the same time, the results showed that the increase in the number of cells in G1 phase was accompanied by the increase in S phase Decrease in the number of cells; as shown in Figures 7-10.

2.3) Apoptosis detection on Aminoquinol

The number of apoptosis cells after Aminoquinol interfered with liver cancer cells Huh7 and HepG2 was detected by flow cytometry under the concentration conditions of 3μM/L, 10μM/L, and 30μM/L, and the presentation time was 24h, 48h, and 72h, respectively. increased the number of apoptotic cells, and showed a time (24h, 48h, 72h) and dose (3μM/L, 10μM/L, 30μM/L) dependent increase trend (P<0.05); as shown in Figure 11-12 shown.

2.4) Use Aminoquinol to interfere with liver cancer cells Huh7 and HepG2, and detect the protein expression of cell cycle G1 phase signaling pathways by Western experiments; after Aminoquinol interferes with liver cancer cells HepG2 and Huh7 cells, observe CDK2, 4, 6 that regulates cell cycle G1 phase signaling pathways , cyclinD, and pho-CDK4/6 protein expression changes; Western blotting experiments were used to detect the G1 phase signaling pathway proteins CDK2, 4, 6, cyclinD, and pho-CDK4/6 after Aminoquinol interfered with liver cancer cells, and the results It was shown that after Aminoquinol interfered with the liver cancer cells Huh7 and HepG2, the protein expression of CDK4, 6, cyclinD, pho-CDK4 and pho-CDK6 showed a dose-dependent decrease trend, and the expression of CDK2 protein did not change; as shown in Figure 13-19.

The above experiments show that Aminoquinol has an inhibitory effect on CDK4/6, has an inhibitory effect on liver cancer cell lines HepG2 and Huh7, and has a strong anti-liver cancer effect, so Aminoquinol can be used to prepare anticancer drugs for the treatment of liver cancer; because Aminoquinol has been approved by the FDA The registered drug for anti-parasitic diseases greatly shortens the cost of clinical trials and drug development time in the later stage, and saves the cost and time of developing a new anti-cancer drug; its application significance is significant.

The preferred implementation of this patent has been described in detail above, but this patent is not limited to the above-mentioned implementation, and within the knowledge of those of ordinary skill in the art, it can also be made without departing from the purpose of this patent. Variations.

Claims (4)

1. The application of the Aminoquinol in preparing the medicine for treating the liver cancer is characterized in that the Aminoquinol is a target CDK4/6 inhibitor and has an anti-liver cancer effect, and the Aminoquinol is used for preparing the anti-cancer medicine for treating the liver cancer.

2. The use of claim 1, wherein the cell cycle distribution of liver cancer cells HepG2 and Huh7 is detected by flow cytometry at concentrations of 3 μ M/L, 10 μ M/L and 30 μ M/L, and after 6h, 12h and 24h, the cells arrested in the G1 phase by the Aminoquinol are increased by HepG2 and Huh7, showing a trend of increasing time and dose dependence, with P <0.05 and the number of cells in the S phase is decreased with the increase in the number of cells in the G1 phase.

3. The use of claim 1, wherein the number of apoptosis of the cells after the Aminoquinol interferes with the liver cancer cells Huh7 and HepG2 under the conditions of 3 μ M/L, 10 μ M/L and 30 μ M/L concentration is detected by flow cytometry, the presentation time is 12h, 24h and 36h respectively, the Aminoquinol increases the number of apoptosis of the cells, and the presentation time and the dose dependence have the trend of increasing, and P is less than 0.05.

4. The use of claim 1, wherein the use of Aminoquinol for interfering with liver cancer cells Huh7 and HepG2 detects the expression of signal path proteins in G1 phase of cell cycle by Western experiment; after the Aminoquinone interferes with hepatoma cells HepG2 and Huh7, the change conditions of protein expression levels of CDK2,4,6, cyclinD and pho-CDK4/6 which regulate signal channels in the G1 phase of the cell cycle are detected; the results show that after the amino quinone l interferes with the hepatoma cells Huh7 and HepG2, the protein expression levels of CDK4,6, cyclinD, pho-CDK4 and pho-CDK6 show a dose-dependent descending trend, the protein expression level of CDK2 is unchanged, and the amino quinone l has an inhibiting effect on CDK 4/6.