CN108815512B

CN108815512B - Anticancer medicine and its application

Info

Publication number: CN108815512B
Application number: CN201810968867.4A
Authority: CN
Inventors: 杨刚刚; 王泽�; 常翠芳; 王改平; 齐剑英; 张全义; 张全海; 吕中原; 郗敏仰; 徐存拴
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2021-06-11
Anticipated expiration: 2038-08-23
Also published as: CN108815512A

Abstract

The invention relates to the field of anti-cancer drugs, and particularly relates to an anti-cancer drug and application thereof. An anticancer medicine comprises Rana pipiens antitumor enzyme and vitamin C. The invention takes the rat liver cancer cell strain RH-35 as a research object, studies the independent and combined action of the two drugs, evaluates the effect of the combined action of the two drugs, proves that the two drugs synergistically promote the apoptosis of the rat liver cancer RH-35 cells on the in vitro cell level, and lays a foundation for the later-stage tumor drug combined development.

Description

Anticancer medicine and its application

Technical Field

The invention relates to the field of anti-cancer drugs, and particularly relates to an anti-cancer drug and application thereof.

Background

Rana pipiens antitumor enzyme (onconase, ONC) is a novel anticancer drug, and the nuclease consisting of 104 amino acids was extracted from the oocytes and early embryonic cells of rana pipiens at the earliest. ONC is currently the first rnase to enter clinical trials, and it specifically degrades RNA to inhibit protein synthesis, thereby inducing apoptosis in tumor cells. In various tumor cell assays, ONCs selectively kill tumor cells, but have essentially no effect on normal cells. In addition, ONC also has the advantages of less adverse reaction, low immunogenicity, difficult generation of drug resistance and the like, and is an antitumor drug with great potential.

Primary liver cancer (mainly hepatocellular carcinoma, HCC)) is one of the most common malignant tumors in global morbidity and mortality, and is the leading cause of cancer-related death in the world. China is the major liver cancer country, and more than half of the liver cancer occurs in China all over the world. Clinically adopted treatment methods comprise surgical excision, liver transplantation, local ablation, chemotherapy, catheter hepatic artery intubation chemoembolization (TACE), targeted drug therapy, immunotherapy and the like, but the treatment effect of the existing method is far from satisfactory, and the survival time is prolonged to be less than 3 months. The existing antitumor drugs have more adverse reactions and high price, and the clinical large-dose use not only increases the side effects of the drugs, but also increases the treatment cost of patients. Therefore, the search for new drugs and combined administration of new drugs can improve the anti-tumor effect, reduce the side effect and cost of the drugs and have important and urgent significance for tumor treatment.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a rana pipiens anti-tumor enzyme and vitamin C which have a synergistic enhanced effect of promoting cancer cell apoptosis, particularly liver cancer, and provide a good foundation for treatment of the liver cancer.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

an anticancer medicine comprises Rana pipiens antitumor enzyme and vitamin C.

Rana parviflora antitumor enzyme (onconase, ONC) is a protein with broad spectrum of anti-solid tumor, is the first ribonuclease entering antitumor clinical tests, and is favored by the advantages of good antitumor effect, low immunogenicity, low drug resistance and the like. The existing research considers that the high dosage of the tumor drug has more side effects, so the combined drug treatment is more favored under the condition of ensuring the drug effect in clinic.

Vitamin C (VC), a water-soluble vitamin, has obvious effects on cancer treatment and alleviation of adverse reactions caused by chemotherapy, and has the advantages of low price and the like. However, the combined curative effect of the novel anticancer drug ONC and VC is not reported.

In view of the above, the invention takes the rat liver cancer cell strain RH-35 as a research object, studies the independent and combined action of the two drugs, evaluates the effect of the combined action of the two drugs and lays a foundation for the later-stage tumor drug combined development.

Specifically, the ONC and VC are used for treating the RH-35 cells by using the ONC and VC single medicine, the concentration dependence of the ONC and the VC is found to obviously inhibit cell proliferation, the cell density can be reduced, the cell nucleus is broken and the like, typical apoptosis characteristics such as small apoptotic bodies appear, and the ONC and the VC can be further verified to inhibit the cell proliferation and promote the cell apoptosis.

In addition, the ONC and VC have a synergistic inhibition effect by analyzing a graph through a CI value and an equivalent line. The action mechanisms of the two medicines are quite different from those of the existing other tumor chemotherapy medicines, and the main research on the multiple action mechanisms of ONC focuses on the specific action on RNA (tRNA), which is different from the traditional method of blocking the synthesis of DNA double strands or acting on specific protein, degrading RNA and inhibiting the synthesis of protein, and further inducing the apoptosis of tumor cells. The anti-cancer mechanism of VC may be to kill oxygen free radicals, protect normal cells from loss, induce apoptosis, and selectively kill cancer cells by inducing oxidative stress, inhibiting glycolysis and energy crisis. The apoptosis detection result of the invention indicates that the apoptosis rate of the induced cells can be greatly improved by combining the two medicines, and further proves that the combined use of the two medicines has a synergistic enhancement effect.

In conclusion, the invention preliminarily obtains the synergistic promotion of the ONC and the VC on the in vitro cell level of the two to the rat liver cancer RH-35 cell apoptosis by detecting the combined action of the ONC and the VC, which lays a foundation for further in vivo experimental research.

Further, the mass ratio of the rana parviflora antitumor enzyme to the vitamin C is 1:50-800, and preferably 1: 50.

As in the different examples, the mass ratio of rana pipiens anti-neoplastic enzyme to vitamin C is 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, and so on.

Further, the cancer is liver cancer.

Further, the medicine also comprises pharmaceutically acceptable auxiliary materials, wherein the pharmaceutically acceptable auxiliary materials comprise any one or more of diluents, excipients, disintegrants, fillers, binders, lubricants, flavoring agents, surface active agents and stabilizers.

Further, the excipient comprises any one or more of mannitol, lactose, starch, dextran and microcrystalline cellulose.

Further, the disintegrating agent comprises any one or more of polyvinylpyrrolidone, carboxymethyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl methyl cellulose.

Further, the lubricant comprises any one or a combination of talc and magnesium stearate.

Furthermore, the dosage form of the medicine comprises any one of injection, tablets, granules, pills and capsules.

The invention also provides application of the anti-cancer drug in promoting cancer cell apoptosis.

Further, the cancer cell is liver cancer.

The anticancer medicine provided by the invention is applied to treating the symptoms of liver cancer.

A method for treating liver cancer, comprising administering to a subject an effective amount of the above anti-cancer agent.

The administration may be oral, intravenous or transdermal, and is administered to a patient in need of treatment.

A pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio achievable with treatment with a drug. The level of effective dose can be determined by several factors, including the type of disease, severity of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration, rate of excretion, treatment period, concurrent use of the drug, and other factors well known in the medical arts. The agents of the invention may be administered as the sole therapeutic agent or in combination with other therapeutic agents. Also, the compositions of the present invention may be added sequentially or simultaneously with typical therapeutic agents, and the compositions may be administered in single or multiple administrations. It is important to take all of the above factors into account to administer the minimum dose that can produce the maximum effect without side effects, which can be determined by the physician according to the patient's condition, age, etc.

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

(1) the rana pipiens anti-tumor enzyme and the vitamin C are found to have the effect of synergistically enhancing and promoting the RH-35 cell apoptosis for the first time.

(2) The anti-cancer drug provided by the invention provides a good theoretical basis for treating liver cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a diagram showing the separation and purification of ONC in example 1 of the present invention;

FIG. 2 is a microscopic image showing the change in cell morphology after 24 hours of administration in example 1 of the present invention;

FIG. 3 is a view showing the change in nuclear morphology of the cell after 24 hours of the Chinese medicine of example 1 of the present invention, which is observed by Hoechst staining under an inverted fluorescence microscope;

FIG. 4 is a bar graph of the inhibition rate of ONC and VC on RH-35 cell proliferation in example 1 of the present invention;

FIG. 5 is an analysis diagram of the equivalent line of the joint action of ONC and VC in example 1 of the present invention;

FIG. 6 is a graph of the apoptosis fraction of different groups and the corresponding bar graph in example 2 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

1 materials and methods

1.1 materials

ONC solid powder (which is obtained by expression and separation and purification by a pichia pastoris system in the laboratory, the purity is higher than 95%, the yield is higher than 90%, as shown in figure 1, 1: supernatant, 2: aqueous two-phase upper phase, 3: aqueous two-phase middle phase, 4: aqueous two-phase lower phase, 5: ultrafiltration concentrated solution, 6-7: equilibrium solution elution, M: protein ladder, VC powder (provided by New country pharmaceutical products of Henan, China), rat liver cancer RH-35 cells (cell bank of Shanghai bioscience institute of Chinese academy), DMEM culture medium and fetal bovine serum FBS (Gibico company), Sulforhodamine B (Sulforhodamine B, SRB, Sigm company), Apoptosis Detection Kit PE Annexin V Apoptosis Detection Kit I (BD Pharmingent), and other reagents are all of domestic analytical purity.

1.2 Experimental methods

1.2.1 cell culture of rat hepatoma cell RH-35 in DMEM high-sugar Medium containing 10% FBS, 100U/mL penicillin and streptomycin (Invitrogen, USA) at saturated humidity, 37 deg.C, 5% CO₂Culturing in an incubator, digesting with 0.25% trypsin (containing 0.02% EDTA), carrying out conventional passage, and replacing the culture solution for 1 time within 2-3 days.

1.1.2ONC and VC Single drug treatment 0.25% cells in the logarithmic phase of tryptic digestion were seeded in 96-well plates (5X 10)³Each cell/cellWell/90. mu.L medium), 5% CO at 37 ℃₂After incubation for 24h under saturated humidity, 10. mu.L of VC (final concentration of 800, 1200, 1600, 2000, 2400. mu. mol/L) and ONC (final concentration of 2, 4, 8, 12, 16. mu. mol/L) were added at different concentrations, 10. mu.L of PBS was added to the control group, and 5 replicates of each concentration were performed for 3 replicates. After 24h of drug treatment (timely observing the growth condition of the cells under an inverted microscope and taking pictures), the SRB method detects the cell proliferation inhibition rate. Inhibition Rate (RI) — (control a value-experimental a value)/control a value × 100%. Calculating half inhibitory concentration IC of drug by linear regression₅₀(inhibitory concentration 50,IC₅₀) The value is obtained.

1.1.3 treatment of both drugs with ONC and VC in combination (final concentration IC)₅₀) Mix in different volume ratios, i.e. 4:1, 3:2, 2:3, 1:4, prepare 4 solutions, each solution was serially diluted with PBS, each dilution was the same fold, set 6 gradients. Inoculating to 96-well plate at 37 deg.C and 5% CO according to 1.1.2₂Culturing under saturated humidity for 24h, adding 10 μ L of combined drugs with different proportions and different concentrations, adding 10 μ L of PBS into control group, repeating treatment for 3 times with 5 wells for each concentration, after drug treatment for 24h (observing cell growth condition under inverted microscope and taking picture), SRB method detecting drug cell proliferation inhibition rate, each solution obtaining 2 IC₅₀Values (corresponding to ONC and VC, respectively).

1.1.4Hoechst staining after adding medicine to treat cells for 24h according to the methods of 1.1.2 and 1.1.3, adding 4% paraformaldehyde fixing solution for fixing for 15min, washing with PBS for 3 times, 5 min/time, adding Hoechst33258 for staining for 10min, washing with PBS for 3 times, 5 min/time, observing the number and morphological changes of cells under an inverted fluorescence microscope, and taking pictures.

1.1.5 Combination Index (CI), the sum of the partial forms of inhibitory concentration of two drugs, is expressed as CI ═ C_50A/IC_50A+C_50B/IC_50B，C_50AAnd C_50BRefers to the drug concentration, IC, at which the combination of A, B two drugs reaches 50% inhibition_50AAnd IC_50BRefers to the concentration of a drug that achieves 50% inhibition with a single drug. When CI is less than 1, the combination of the two medicines hasA synergistic effect; when CI is more than 1, the combination of the two medicines has antagonistic effect; when CI is 1, then a synergistic effect is considered.

1.1.6 isobologram when two drugs are used in combination, the interaction of the two drugs can be observed by calculating Fractional Inhibition Concentration (FIC) of the drugs, in which case FIC_ONCCombination therapy inhibits ONC concentration (C) at 50% cell_50ONC) Concentration of single drug ONC inhibiting 50% of cells (IC)_50ONC) And FIC_VCVC concentration (C) at 50% cell inhibition by combination_50VC) VC alone inhibited 50% cell concentration (IC)_50VC). With FIC_ONCThe value of (1) is the abscissa, FIC_VCThe value of (a) is the ordinate, the FIC value of the independent action of the two drugs is set to 1, the coordinates of the independent action of the two drugs are connected, and an analysis graph of the equivalent line (i.e., the addition line) is drawn. When the two drugs are combined, the FIC value of the ONC corresponds to the abscissa value of the combined action on the equivalent analysis chart, and the FIC value of the VC corresponds to the ordinate value of the combined action, so that the point of the combined action can be drawn. The points representing the combined action are located below, on or above the addition line, and respectively represent the synergistic, addition or antagonistic effect when the two drugs are combined.

2 results

2.1 cell morphology changes after 24h of single and combined administration are shown in FIG. 2. In FIG. 2, NC normal cells; a PBS control group; ONC single drug group (8 μmol/L); VC single drug group (1600 mu mol/L); the drug combination of ONC and VC (abbreviated as O: V-1:4, O: V-2:3, O: V-3:2 and O: V-4:1), wherein the numerical values of the drug combination of ONC and VC respectively represent different dosage ratios.

As can be seen from FIG. 2, after the drug treatment for 24h, the shapes and outlines of normal cells and control cells can be observed under an inverted microscope, the structures are compact, the adherence is firm, and the growth state is good. The cells of the administration group (single medicine group and combined group) all appear shrinkage and deformation, are in brilliant small round points, and are unstable in adherence and even fall off to die.

2.2 Observation of the change in cell nuclei using Hoechst staining under an inverted fluorescence microscope is shown in FIG. 3. In FIG. 3, NC blank groups; a PBS control group; and (2) ONC: 8 mu mol/L; VC: 1600 mu mol/L; ONC/VC: the combination medicine group (O: V-1:4, O: V-2:3, O: V-3:2 and O: V-4:1) in different proportions. The influence on the cells after the drug treatment is more visually observed, and as can be seen from fig. 3, the cell nucleus volume of the experimental group is increased, and a typical apoptosis phenomenon that the nucleus is crescent and an apoptotic body formed by sprouting and bubbling (shown by arrows of fig. 3) can be seen at a plurality of positions. Therefore, the typical apoptosis phenomena of small cell volume, chromatin condensation, margination and moon shape appear after the intervention of the medicine. The combined group has more obvious effect than the single medicine group, and the combined effect of the last two groups is more obvious.

2.3 Individual Effect of ONC and VC on RH-35

The cell survival rate of the cells subjected to the intervention of ONC (2-16 mu mol/L) and VC (800-2400 mu mol/L) with different concentrations in RH-35 cells for 24 hours is detected by adopting an SRB method, and the result shows that the survival number of the cells is gradually reduced (P is less than 0.01) along with the increase of the concentration of the medicament, and the IC of the ONC and VC acting on the cells is₅₀The values were 8. mu. mol/L and 1600. mu. mol/L (FIG. 4), respectively.

2.4 Combined index analysis

When ONC and VC are combined, the experiment is divided into four groups (O: V-1:4, O: V-2:3, O: V-3:2 and O: V-4:1) of a control group, an ONC single drug group, a VC single drug group and an ONC and VC combined group. In the control group, adherent cells have good growth state and complete cell nucleus; in the administered group, the proliferation of cells was inhibited and apoptosis was exhibited to various degrees. SRB method for determining IC of ONC and VC on liver cancer cell strain₅₀The values are shown in Table 1.

TABLE 1 IC of ONC and VC on hepatoma cell lines₅₀Value of

IC using 4 ratios in Table 1 according to the formula₅₀The CI values of the four ratios are calculated, the CI values are respectively 0.78, 0.83, 0.85 and 0.97 and are all less than 1, and the specific table is shown in table 2.

TABLE 2 combination index of ONC in combination with VC in RH-35 cells

The points representing the combined effect on the obtained isobologram were all located below the addition line (FIG. 5). Thus, the combination of ONC and VC can synergistically inhibit the proliferation of RH-35, and the optimal volume ratio of the combination of ONC and VC is 4:1, IC_50ONCAnd IC_50VC3.92 mu mol/L and 763 mu mol/L respectively.

Example 2

Flow cytometry

Taking cells in exponential growth phase, making into single cell suspension, and making into single cell suspension according to 2 × 10 per well⁵After 24h of culture in 6-well plates/mL, cells were divided into 4 groups: (1) a PBS control group; (2) single drug group ONC: 8 mu mol/L; (3) VC single drug group: 1600 mu mol/L; (4) combination group: ONC 8 mu mol/L + VC1600 mu mol/L, adding medicine for 24h, collecting cells in each well, washing twice with PBS, operating according to an apoptosis kit, and detecting the apoptosis condition by a flow cytometer (BD, USA).

Statistical analysis all data are expressed as mean ± standard deviation, differences between groups were analyzed by one-way anova with SPSS 13.0 statistical software, P <0.01 indicates significant differences, and P <0.001 indicates very significant differences.

The apoptosis detection by flow cytometry finds that as shown in fig. 6, the apoptosis rates of cells of a PBS control group and an experimental group (ONC, VC and O + V) are respectively 4.24 +/-0.36%, 15.73 +/-3.08%, 61.53 +/-2.98% and 73.65 +/-3.38%, the apoptosis rate of a single drug group of ONC and VC is remarkably reduced (P is less than 0.01), the combined apoptosis promoting effect of the two drugs is superior to that of the single drug group, and the extremely remarkable difference (P is less than 0.001) is achieved.

In conclusion, after the RH-35 cells are treated by ONC and VC with different concentrations (single medicine group) and different proportions (combined group) for 24 hours, the SRB method is used for measuring the cell activity, the change of the cell number and the cell shape after the medicine treatment is observed through microscopic examination and Hoechst staining, the combined effect of the ONC and the VC after the medicine treatment is evaluated through a Combination Index (CI) and an equivalent graphical method, and the apoptosis condition of the cells is detected through a Flow Cytometry (FCM). The results are as follows: when single drug acts on RH-35, the inhibition effect of ONC and VC on cell proliferation is strengthened along with the increase of drug concentration, and the proliferation (P is less than 0.01) and IC are obviously inhibited_50ONCAnd IC_50VCRespectively 8 mu mol/L and 1600 mu mol/L; when the combination is used, the CI values of the combination indexes are all less than 1, and an analysis diagram of an equivalent line shows that points representing the combination action of the two are all positioned below an addition line; as can be seen from microscopic examination, the cell nucleuses of the drug-treated group all showed significant apoptosis characteristics. FCM results show that the apoptosis ratio of the single medicine group is remarkably increased (P is less than 0.01), and the combined apoptosis-promoting effect of the two medicines is superior to that of the single medicine group, so that the remarkable difference (P is less than 0.001) is achieved. The conclusion is that VC in cooperation with ONC promotes the apoptosis of RH-35 cells.

The method takes the rat liver cancer cell strain RH-35 as a research object, measures the independent or combined action of ONC and VC on RH-35 by an SRB method and an FCM method, analyzes and evaluates the combined effect by combining indexes and an equivalent line, obtains the synergistic effect of the ONC and the VC on the in vitro cell level to promote the rat liver cancer RH-35 apoptosis, and lays a foundation for further in vivo experimental research.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. An anticancer drug is characterized in that the main components are rana pipiens antitumor enzyme and vitamin C;

the mass ratio of the rana pipiens anti-tumor enzyme to the vitamin C is 1: 50-800.

2. The anticancer agent according to claim 1, wherein the ratio of the amounts of said rana pipiens antitumor enzyme and vitamin C is 1: 50.

3. The anticancer agent according to claim 1, wherein said cancer is liver cancer.

4. The anticancer agent as claimed in any one of claims 1 to 3, wherein the agent further comprises pharmaceutically acceptable adjuvants selected from any one or more of diluent, excipient, disintegrant, filler, binder, lubricant, flavoring agent, surfactant and stabilizer.

5. The anticancer agent as claimed in claim 4, wherein said excipient is selected from mannitol, lactose, starch, dextran, and microcrystalline cellulose.

6. The anticancer agent as claimed in claim 4, wherein the disintegrating agent is selected from one or more of polyvinylpyrrolidone, carboxymethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose.

7. The anticancer agent as claimed in claim 4, wherein said lubricant is selected from any one or two of talc and magnesium stearate.

8. The anticancer agent of claim 4, wherein the dosage form of the agent is selected from any one of injection, tablet, granule, pill and capsule.