A pharmaceutical composition for treating liver cancer and its application

Abstract

The present invention provides a pharmaceutical composition for treating liver cancer and its application, belonging to the field of biomedicine technology. A pharmaceutical composition for treating liver cancer comprises scutellarin C and dihydroisotanshinone I in a mass ratio of (9-11): (5-15). The present invention also provides an application of the pharmaceutical composition in the preparation of an anti-liver cancer drug. Scutellarin C and dihydroisotanshinone I induce ferroptosis in liver cancer cells, thereby achieving the purpose of treating liver cancer and reducing the risk of liver cancer recurrence.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a pharmaceutical composition for treating liver cancer and application thereof. Background Art

[0002] Primary liver cancer is a type of cancer with a high morbidity and mortality rate. Primary liver cancer mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The three types differ greatly in pathogenesis, biological behavior, pathological histology, treatment methods and prognosis, of which HCC accounts for 75% to 85% and ICC accounts for 10% to 15%. Common treatments for cancer include surgery, chemotherapy, radiotherapy, etc., and when necessary, they are also combined with targeted therapy, biological therapy and other methods.

[0003] Scutellarin C is a sesquiterpenoid compound that can be isolated from Scutellaria baicalensis. It has attracted more and more attention from researchers due to its multiple pharmacological activities such as anti-tumor, anti-viral, anti-liver fibrosis, anti-parasitic and cardioprotective. Dihydroisotanshinone I is an extract from the rhizome of Salvia miltiorrhiza, which is the most commonly used Chinese medicine for promoting blood circulation and removing blood stasis in clinical practice. Related literature reports that Salvia miltiorrhiza has cardiovascular effects such as increasing coronary blood flow, dilating coronary arteries, and preventing myocardial ischemia, and is widely used in the study of cardiovascular diseases.

[0004] At present, there is no report on the application of chrysanthemum lactone C and dihydroisotanshinone I in the treatment of liver cancer. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a pharmaceutical composition for treating liver cancer, which combines thallolide C and dihydroisotanshinone I to reduce the recurrence rate of liver cancer by inducing ferroptosis in liver cancer cells.

[0006] The present invention provides a pharmaceutical composition for treating liver cancer, comprising cypermethrin C and dihydroisotanshinone I;

[0007] The mass ratio of the chrysanthemum lactone C and dihydroisotanshinone I is (9-11): (5-15).

[0008] Preferably, the mass ratio of the indusia lactone C to dihydroisotanshinone I is 2:(2-3).

[0009] Preferably, the mass ratio of the indusia lactone C to the dihydroisotanshinone I is 2:3.

[0010] The invention provides application of the pharmaceutical composition in preparing anti-liver cancer drugs.

[0011] Preferably, the anti-liver cancer effect is manifested in at least one of the following aspects: inhibiting the growth of liver tumors, reducing the activity of liver cancer cells, and inducing ferroptosis of liver cancer cells.

[0012] Preferably, the inducing liver cancer cell ferroptosis comprises increasing Fe 2+ , the content of at least one of malondialdehyde and reactive oxygen species and / or reduce the expression of ferroptosis-related proteins in liver cancer cells.

[0013] Preferably, the dosage form of the drug includes at least one of the following: injection, injection and sustained-release preparation;

[0014] The concentration of scutellaria lactone C in the injection is not less than 20 μM; the concentration of dihydroisotanshinone I is not less than 30 μM.

[0015] The present invention provides application of the pharmaceutical composition in preparing an in vitro liver cancer cell inhibitor.

[0016] Preferably, the mass ratio of the indusia lactone C and dihydroisotanshinone I is (9-11): (5-15).

[0017] The present invention provides a method for inhibiting the growth of liver cancer cells in vitro, wherein the liver cancer cells are placed in an environment containing cypermethrin C and / or dihydroisotanshinone I for growth;

[0018] The concentration of the chrysanthemum lactone C is not less than 20 μM;

[0019] The concentration of the dihydroisotanshinone I is not less than 30 μM.

[0020] The present invention provides a pharmaceutical composition for treating liver cancer, comprising scutellarin C and dihydroisotanshinone I; the mass ratio of scutellarin C and dihydroisotanshinone I is (9-11): (5-15). In the embodiment of the present invention, the CCK8 method is first used to detect the effect of scutellarin C and dihydroisotanshinone I in different mass ratios on the activity of liver cancer cells. The results show that compared with the control group and the solvent group, the scutellarin C and dihydroisotanshinone I compound groups with different concentrations can significantly reduce the activity of liver cancer cells, reducing the cell survival rate to below 60%, and as the concentration of dihydroisotanshinone I increases, the cell survival rate gradually decreases. At the same time, the liver cancer animal model administration experiment shows that compared with the control group, the scutellarin C and dihydroisotanshinone I compound administration group can significantly reduce the tumor volume, and there is no significant difference with the positive control. It can be seen that the pharmaceutical composition provided by the present invention can inhibit the growth and vitality of liver cancer cells, reduce the volume of liver cancer tumors, and achieve the purpose of effectively treating liver cancer. The pharmaceutical composition provided by the present invention not only greatly improves the pharmaceutical value of cypermethrin C and dihydroisotanshinone I, but also provides new means and new ideas for the treatment of liver cancer and the development of clinical drugs for the treatment of liver cancer.

[0021] The present invention provides the use of scutellarin C or dihydroisotanshinone I in the preparation of anti-liver cancer drugs. In the embodiment of the present invention, the CCK8 method is first used to detect the effect of administration of different concentrations of scutellarin C (5~20μM) or dihydroisotanshinone I (10~30μM) on the activity of liver cancer cells. The results show that compared with the control group and the solvent group, scutellarin C or dihydroisotanshinone I significantly reduce the survival rate of liver cancer cells, and are concentration-dependent. At the same time, the liver cancer animal model administration experiment shows that compared with the control group, scutellarin C or dihydroisotanshinone I administration groups can significantly reduce tumor volume, and there is no significant difference with the positive control. It can be seen that scutellarin C or dihydroisotanshinone I alone have the efficacy of reducing the activity of liver cancer cells and inhibiting the growth of liver cancer tumors in the body, which provides new means and new ideas for the treatment of liver cancer and clinical drugs for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The results of the cell activity test (CCK8 method) on the viability of HePG2 liver cancer cells are shown in Figure A, where 5 μM, 10 μM and 20 μM of scutellarin C were administered alone; B was the result of 10 μM, 20 μM and 30 μM of dihydroisotanshinone I administered alone; C was the result of 20 μM scutellarin C and 10 μM, 20 μM and 30 μM of dihydroisotanshinone I administered in combination; D was the result of 10 μM oxaliplatin and 5 μM, 10 μM and 20 μM scutellarin C administered in combination; E was the result of 10 μM oxaliplatin and 10 μM, 20 μM and 30 μM of dihydroisotanshinone I administered in combination;

[0023] Figure 2 The effects of different concentrations of single drug and drug combination on Fe 2+ , MDA and ROS content detection results, where A is Fe 2+ A is the result graph of MDA content; B is the result graph of MDA content; C is the result graph of ROS content; "*" indicates P <0.05; “**” indicates P <0.01; "***" means P <0.001;

[0024] Figure 3 The results of the effects of single drugs and drug combinations on the expression of ferroptosis-related proteins in HePG2 liver cancer cells are shown in Figure 1, where A is the WB result graph, B and C are bar graphs of WB results, and the vertical axis is the "relative ratio of ferroptosis protein / reference protein", and "*" indicates P <0.05; “**” indicates P <0.01; "***" means P <0.001;

[0025] Figure 4 Figure 2 is the detection result of tumor volume growth in each experimental group. DETAILED DESCRIPTION

[0026] The present invention provides a pharmaceutical composition for treating liver cancer, comprising thiamethoxam lactone C and dihydroisotanshinone I; the mass ratio of thiamethoxam lactone C to dihydroisotanshinone I is (9-11): (5-15).

[0027] In the present invention, the mass ratio of the thallolide C to dihydroisotanshinone I is preferably 2: (2-3), more preferably 2: 3. The present invention does not impose any special restrictions on the source of the thallolide C, and it can be obtained by using commodity purchase methods known in the art. In the embodiment of the present invention, the thallolide C is purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0028] Formula I.

[0029] In the present invention, the molecular formula of dihydroisotanshinone I is C 18 H 14 O3, CAS No. 20958-18-3, structural formula as shown in Formula II. The present invention does not impose any special restrictions on the source of the dihydroisotanshinone I, and it can be obtained by using commodity purchase methods known in the art. In the embodiment of the present invention, the dihydroisotanshinone I is purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0030] Formula II.

[0031] In the present invention, the dosage form of the drug is preferably an injection, an injection or a sustained-release preparation. The injection preferably also includes an excipient. The excipient is preferably water for injection. The concentration of scutellarin C in the injection is preferably not less than 5 μM, more preferably 10~20 μM. In one embodiment of the present invention, scutellarin C at concentrations of 5 μM, 10 μM and 20 μM was used to treat liver cancer cells HePG2, and the cell activity was determined by CCK8 method. The results showed that compared with the blank control group and the solvent (DMSO) group, the 5 μM scutellarin C administration group can significantly inhibit cell activity, and the degree of inhibition is concentration-dependent. In another embodiment of the present invention, a liver cancer mouse model experiment was carried out, and the results showed that compared with the blank control, the 20 mg / kg scutellarin C administration group can significantly reduce the volume of liver cancer tumors, which is concentration-dependent.

[0032] In the present invention, the concentration of dihydroisotanshinone I in the injection is preferably not less than 10 μM, more preferably 20-30 μM. In one embodiment of the present invention, hepatoma cell HePG2 was treated with dihydroisotanshinone I at concentrations of 10 μM, 20 μM, and 30 μM, respectively, and the cell activity was determined by CCK8 method. The results showed that compared with the blank control group and the solvent (DMSO) group, the 10 μM dihydroisotanshinone I administration group was able to significantly inhibit cell activity, and the degree of inhibition was concentration-dependent. In another embodiment of the present invention, a liver cancer mouse model experiment was carried out, and the results showed that compared with the blank control, the 30 mg / kg dihydroisotanshinone I administration group was able to significantly reduce the liver cancer tumor volume, which was concentration-dependent.

[0033] The invention provides application of the pharmaceutical composition in preparing anti-liver cancer drugs.

[0034] In the present invention, the anti-liver cancer agent is preferably manifested in at least one of the following aspects: inhibiting the growth of liver tumors, reducing the activity of liver cancer cells, and inducing liver cancer cell ferroptosis. The induction of liver cancer cell ferroptosis preferably includes increasing Fe 2+, malondialdehyde and reactive oxygen content and / or reduce the expression of liver cancer cell ferroptosis-related proteins. The liver cancer cell ferroptosis-related proteins preferably include GPX4 and / or SLC7A11. In an embodiment of the present invention, cell viability experiments show that the compound of scutellarin C and dihydroisotanshinone I has more significant advantages than the single administration of scutellarin C and dihydroisotanshinone I. At the same time, liver cancer animal model experiments show that the compound of scutellarin C and dihydroisotanshinone I is more conducive to inhibiting tumor volume, which indicates that the pharmaceutical composition has a more obvious inhibitory effect on liver cancer growth.

[0035] In the present invention, ferroptosis is a programmed cell death that depends on iron and ROS, which is different from apoptosis, autophagy and necrosis at the morphological, biochemical and genetic levels. Ferroptosis is a new therapeutic strategy to overcome the drug resistance of cancer cells. In addition, ferroptosis can selectively target invasive cancer cells and is also expected to improve the efficacy of immunotherapy and overcome the drug resistance of immunotherapy. In order to further explore the anti-liver cancer mechanism of the pharmaceutical composition, cypermethrin C or dihydroisotanshinone I, the present invention respectively measured Fe in liver cancer cells. 2+ , MDA and ROS content and ferroptosis-related protein expression. The results showed that the pharmaceutical composition, cypermethrin C or dihydroisotanshinone I can increase the Fe 2+ , MDA and ROS content, and reduce the content of GPX4 protein and SLC7A11. It can be seen that the pharmaceutical composition, cypermethrin C or dihydroisotanshinone I can achieve the efficacy of treating liver cancer and reducing the recurrence rate of liver cancer by inducing ferroptosis in liver cancer cells.

[0036] In the present invention, the dosage form of the drug preferably includes an injection; the concentration of scutellarin C in the injection is preferably not less than 20 μM, more preferably 22-30 μM, further preferably 23-28 μM, and most preferably 25 μM; the concentration of dihydroisotanshinone I is preferably not less than 30 μM, more preferably 32-40 μM, further preferably 33-38 μM, and most preferably 35 μM. The present invention has no special restrictions on the preparation method of the injection, and the preparation method of the injection well known in the art can be used. The usage and dosage of the injection is 1.1 mg / kg mouse body weight scutellarin C and 1.6 mg / kg mouse body weight dihydroisotanshinone.

[0037] In view of the fact that thiamethoxam lactone C and / or dihydroisotanshinone I have the property of inhibiting the activity of liver cancer cells in vitro, the present invention provides the use of thiamethoxam lactone C and dihydroisotanshinone I in the preparation of in vitro liver cancer cell inhibitors.

[0038] The invention provides a method for inhibiting the growth of liver cancer cells in vitro, wherein the liver cancer cells are placed in an environment containing thallolide C and / or dihydroisotanshinone I for growth; the concentration of the thallolide C is not less than 20 μM; the concentration of the dihydroisotanshinone I is not less than 30 μM.

[0039] In the present invention, the environment containing thiamethoxam lactone C and / or dihydroisotanshinone I is preferably a liver cancer cell culture medium containing thiamethoxam lactone C and / or dihydroisotanshinone I. The growth temperature of the liver cancer cells is preferably 36-38° C., more preferably 37° C. The growth conditions of the liver cancer cells are preferably 5% CO2 conditions.

[0040] The following is a detailed description of a pharmaceutical composition for treating liver cancer and its application provided by the present invention in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] Effects of cypermethrin C and / or dihydroisotanshinone I on the activity of hepatoma cells

[0043] HePG2 liver cancer cells were grown in DMEM medium containing 10% fetal bovine serum by mass percentage. The antibiotic concentrations in the medium were 100 U / ml penicillin and 100 μg / ml streptomycin. The cell culture was carried out in an incubator at a temperature of 37°C and a carbon dioxide concentration of 5%. The cells were digested with trypsin and prepared into a cell suspension. 5×10 3 Cells / well were seeded in 96-well plates, and 100 μl of cell suspension was added to each well. The cells were pre-cultured in a 37°C, 5% CO2 incubator for 24 h. The culture medium was replaced with 200 μl of 5 μM, 10 μM and 20 μM TC alone, 10 μM, 20 μM and 30 μM dihydroisotanshinone I (DT) alone, and 20 μM TC and 10 μM, 20 μM and 30 μM dihydroisotanshinone I in combination. The medium containing solvent (DMSO) was set as the control group, and the cell group containing only the medium was set as the blank control (con). Three replicates were set for each sample concentration. The culture plates were incubated in the incubator for 24 h, and 10 μl of CCK-8 solution was added to each well (be careful to avoid the formation of bubbles, otherwise it will affect the reading of the OD value). The plates were incubated in the incubator for 1-4 h, and the absorbance at 450 nm was measured using an enzyme reader.

[0044] The effect on the viability of HePG2 liver cancer cells was shown in the cell activity test (CCK8 method). Figure 1 Compared with the blank control and solvent groups, 5 μM, 10 μM and 20 μM of cypermethrin C alone could significantly reduce the survival rate of liver cancer cells (see Figure 1 A in the middle), while 10 μM, 20 μM and 30 μM dihydroisotanshinone I alone can also significantly reduce the survival rate of liver cancer cells (see Figure 1 B), and the degree of inhibition was dependent on the drug concentration. The combined administration of 20 μM thiamethoxam C and 10 μM, 20 μM and 30 μM dihydroisotanshinone I not only significantly reduced the cell survival rate compared with the blank control and solvent groups, but also further reduced the cell survival rate compared with thiamethoxam C or dihydroisotanshinone I. Therefore, the combined use of thiamethoxam C and dihydroisotanshinone I can further reduce the viability of liver cancer cells. 10 μM oxaliplatin was co-administered with 5 μM, 10 μM and 20 μM thiamethoxam C and 10 μM, 20 μM and 30 μM dihydroisotanshinone I (see Figure 1 D and E in the figure). The combined use of scutellarin C and dihydroisotanshinone I significantly improved the inhibitory effect on tumor cells compared with the use of either drug alone. However, when scutellarin C or dihydroisotanshinone I was used alone in combination with oxaliplatin, the inhibitory effect on tumor cells did not increase with the increase in drug concentration. This shows that the combination of the two drugs can achieve a better tumor inhibition effect.

[0045] Example 2

[0046] Fe 2+ , MDA and ROS contents were measured

[0047] Experimental steps: The HePG2 liver cancer cells used were cultured under the same conditions as above. The drugs included: 10 μM dihydroisotanshinone I (LG), 20 μM dihydroisotanshinone I (MG), 30 μM dihydroisotanshinone I (HG), 20 μM cypermethrin C + 30 μM dihydroisotanshinone I (TG). A control group (CG) was also set up;

[0048] HePG2 cells were treated with drugs for 24 h and then with Fe 2+ Kit, MDA kit and CMH2DCFDA fluorescent dye for intracellular Fe 2+ , MDA and ROS contents were measured.

[0049] Effects of different concentrations of single drugs and drug combinations on Fe 2+ The results of MDA and ROS content detection are shown in Figure 2 .Depend on Figure 2 It can be seen that compared with the control group, different concentrations of single drugs and drug combinations can increase the Fe 2+ , MDA and ROS contents, and compared with the single-drug group, the drug combination could further significantly increase the Fe2+ , MDA and ROS contents.

[0050] Example 3

[0051] Effect on the expression of ferroptosis-related proteins in HePG2 liver cancer cells.

[0052] Experimental steps: The HePG2 liver cancer cells used were cultured under the same conditions as described above. The drug groups included administration of only 20 μM cypermethrin C (TC), only 30 μM dihydroisotanshinone I (DT), and 20 μM cypermethrin C combined with 30 μM dihydroisotanshinone I (TC+DT).

[0053] HePG2 cells were treated with drugs for 24 hours, digested with trypsin and collected. Rinse three times with pre-cooled PBS, add 100μl cell lysis buffer RIPA and lyse on ice for 10 minutes. After centrifugation at 4℃, collect the supernatant and determine the protein concentration by BCA method. After calculating the appropriate loading concentration, add the protein sample to the appropriate proportion of loading buffer and denature at high temperature for 10 minutes. Prepare polyacrylamide gel with a separation gel content of 12% and a concentration gel content of 5%, load the sample and run the gel at 90V for 50 min and 160V for 160 min. Transfer the protein to PVDF membrane at 70 V for 90 min. After blocking the membrane at room temperature for 2 hours, rinse three times with TBST, add the corresponding antibody and incubate overnight, rinse three times with TBST, incubate with the corresponding secondary antibody for 2 hours, rinse three times with TBST, expose to color, and use software for processing and analysis. At the same time, β-actin was used as a control protein.

[0054] The experimental results are shown in Figure 3 Compared with the control group, different concentrations of single drugs and drug combinations can reduce the expression of GPX4 and SLC7A11 in liver cancer cells to varying degrees, and compared with the single drug group, the drug combination can further significantly reduce the expression of GPX4 and SLC7A11 in liver cancer cells.

[0055] Example 4

[0056] Drug administration experiment on liver cancer animals

[0057] Take 10 5HePG2 hepatoma cell lines were ectopically inoculated subcutaneously in the axilla of immunodeficient mice. After 2 weeks, tumor formation was perceptible by touch. At this time, the tumors were ectopically treated with a combination of 20 mg / kg cypermethrin C and 30 mg / kg dihydroisotanshinone I (TC+DT drug combination), 20 mg / kg cypermethrin C alone (TC), and 30 mg / kg dihydroisotanshinone I alone (DT) as experimental groups; there was also a PBS injection group as a control group; and an injection of oxaliplatin injection as a positive control group. There was no significant difference in the weight of the mice.

[0058] The results are as follows Figure 4 Compared with the blank control group, single cypermethrin C and dihydroisotanshinone I as well as the TC+DT drug combination can effectively inhibit tumor growth, and the TC+DT drug combination has a stronger ability to inhibit liver cancer tumor volume.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating liver cancer, characterized in that: It is composed of scutellaria lactone C and dihydroisotanshinone I; The mass ratio of the chrysanthemum lactone C and dihydroisotanshinone I is 2: (2-3).

2. The pharmaceutical composition for treating liver cancer according to claim 1, characterized in that: The mass ratio of the chrysanthemum lactone C and dihydroisotanshinone I is 2:

3.

3. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of anti-liver cancer drugs.

4. The use according to claim 3, characterized in that: The anti-liver cancer effect is manifested in at least one of the following aspects: inhibiting the growth of liver tumors, reducing the activity of liver cancer cells and inducing ferroptosis of liver cancer cells.

5. The application according to claim 4, characterized in that: The method of inducing liver cancer cell ferroptosis comprises increasing Fe 2+ , the content of at least one of malondialdehyde and reactive oxygen species and / or reduce the expression of ferroptosis-related proteins in liver cancer cells.

6. The use according to claim 3, characterized in that: The dosage form of the drug includes at least one of the following: injection, injection and sustained-release preparation; The concentration of scutellaria lactone C in the injection is not less than 20 μM; The concentration of dihydroisotanshinone I is not less than 30 μM.

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