Application of combination of okannin and epirubicin in prevention and / or treatment of colon cancer

Abstract

The invention discloses application of combination of okannin and epirubicin in prevention and / or treatment of colon cancer, relates to the technical field of biological medicine, and is technically characterized in that a pharmaceutical composition comprises okannin and epirubicin, the preferable molar ratio is 15: 1 or 7.5: 1, and the pharmaceutical composition aims to reduce the dosage of epirubicin, reduce toxic and side effects and enhance the curative effect through a synergistic effect. The invention also comprises a method for detecting the influence of the pharmaceutical composition on colon cancer cell viability and apoptosis, and an animal model for evaluating the influence on the tumor growth of a colon cancer tumor-bearing mouse. The pharmaceutical composition disclosed by the invention can be used for remarkably inhibiting the proliferation of colon cancer cells and enhancing the apoptosis promoting effect of epirubicin, and shows a good anti-tumor effect and relatively low visceral organ toxicity in an animal model. The invention provides a safe and effective new treatment strategy for colon cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of okanamin combined with epirubicin in preventing and / or treating colon cancer. Background Art

[0002] Colon cancer is one of the most common gastrointestinal malignancies in the world. Although the specific cause of colon cancer has not yet been fully elucidated, it is known that more than 80% of patients have no family history, and lifestyle factors such as diet, obesity, smoking and drinking may increase the risk of disease. Currently, surgery-assisted chemoradiotherapy is the main treatment for colon cancer. Although this treatment can prolong the survival of patients, the toxic side effects of chemotherapy drugs on normal tissues and the development of drug resistance in patients limit the efficacy of chemotherapy.

[0003] Epirubicin (Epi) is an antibiotic antitumor drug that interferes with the transcription process by intercalating between DNA nucleobase pairs, preventing the formation of mRNA, and thus inhibiting the synthesis of DNA and RNA. As a common drug in chemotherapy, the use of epirubicin is limited by dose-related toxicity, and patients may develop resistance to it. Therefore, the use of okanidin combined with epirubicin in the prevention and / or treatment of colon cancer mentioned in the present invention aims to reduce the dosage of epirubicin through synergistic effects, while reducing its toxic side effects, providing a new strategy for the treatment of colon cancer. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide the use of oxanin combined with epirubicin in the prevention and / or treatment of colon cancer. The combination of oxanin and epirubicin for colon cancer has obvious synergistic effect.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: the use of oxanthate combined with epirubicin in the prevention and / or treatment of colon cancer, the pharmaceutical composition comprises oxanthate and epirubicin, wherein the molar ratio of oxanthate to epirubicin is 15:1.

[0006] Furthermore, the molar ratio of oxanthate to epirubicin in the pharmaceutical composition can also be 7.5:1.

[0007] Furthermore, the pharmaceutical composition is used to prepare a drug for inhibiting the activity of colon cancer cells.

[0008] Furthermore, the pharmaceutical composition is used to prepare a drug for inhibiting tumor growth in colon cancer-bearing mice.

[0009] Furthermore, the pharmaceutical composition includes but is not limited to injections, pills, tablets, granules, and capsules.

[0010] Another object of the present invention is to provide a method for detecting the effect of oxanthate combined with epirubicin on the viability of colon cancer cells, the method comprising using the MTT method to detect the effect of different concentrations of oxanthate and epirubicin on the viability of HCT116 cells, and calculating the cell viability.

[0011] Another object of the present invention is to provide a method for detecting the effect of oxanin combined with epirubicin on apoptosis of colon cancer cells, the method comprising using the Annexin V / PI method to detect the effect of oxanin and epirubicin on apoptosis of HCT116 cells.

[0012] Furthermore, the method compares the effects of epirubicin or oxanthene alone with the combined use of the two drugs on apoptosis of colon cancer cells to evaluate the enhancing effect of oxanthene on the pro-apoptotic effect of epirubicin.

[0013] Another object of the present invention is to provide an animal model for evaluating the effects of oxanthene combined with epirubicin on tumor growth in colon cancer-bearing mice. The model is established by establishing a colon cancer CT26 transplanted tumor mouse model and administering oxanthene and epirubicin in combination to evaluate the inhibitory effect on tumor growth and the toxic reaction to important organs.

[0014] Compared with the prior art, the beneficial effects of this scheme are as follows: the present invention combines oxanin with epirubicin to achieve a synergistic effect of the two, significantly inhibit the proliferation of colon cancer cells HCT116, and reduce the dosage of epirubicin, thereby reducing the dose-related toxic and side effects. Secondly, the experimental results show that this combination therapy can significantly enhance the apoptotic effect of epirubicin on colon cancer cells, and the apoptosis rate of the combination therapy group is significantly increased compared with the single drug use. In addition, in the colon cancer-bearing mouse model, the tumor inhibition effect of oxanin combined with epirubicin is significantly better than that of single drug use, and the tumor inhibition rate reaches 75.18%, showing a strong in vivo anti-tumor activity. At the same time, the scheme can also improve the organ index of mice and reduce the toxic reaction of epirubicin to important organs. In summary, this scheme not only improves the therapeutic effect, but also effectively reduces the toxic and side effects of chemotherapy drugs, providing a safer and more effective treatment option for patients with colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram showing the effects of okanamin and epirubicin on HCT116 cell viability in Example 1 of the present invention;

[0016] Figure 2 Schematic diagram of the effect of okananin combined with epirubicin on HCT116 cell viability in Example 1 of the present invention;

[0017] Figure 3This is a schematic diagram of the calculation of the combined index of oxanthate and epirubicin on HCT116 cells in Example 1 of the present invention;

[0018] Figure 4 This is a graph showing the experimental results of detecting the effect of oxanthate combined with epirubicin on apoptosis of HCT116 cells using the Annexin V / PI method in Example 1 of the present invention;

[0019] Figure 5 This is a graph showing the effect of okananin combined with epirubicin on promoting apoptosis of colon cancer cells HCT116 in Example 1 of the present invention;

[0020] Figure 6 This is a schematic diagram of the in vivo pharmacodynamic study results of okananin combined with epirubicin on a colon cancer CT26 transplanted tumor mouse model in Example 2 of the present invention;

[0021] Figure 7 is a schematic diagram of the effect of okananin combined with epirubicin on tumor growth in tumor-bearing mice in Example 2 of the present invention;

[0022] Figure 8 This is a schematic diagram of the effects of okananin combined with epirubicin on the organ indexes of tumor-bearing mice in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] The present invention provides the use of Okanin combined with Epirubicin (Epi) in the preparation of a drug for preventing and / or treating colon cancer. The Okanin combined with Epirubicin of the present invention significantly inhibits the activity of colon cancer cells, and the Okanin combined with Epirubicin significantly inhibits the growth of tumors in colon cancer-bearing mice. The sources of Okanin and Epirubicin of the present invention are not particularly limited, and can be commercially available products well known to those skilled in the art, or can be obtained by a preparation method disclosed in the prior art.

[0026] In the present invention, the molar ratio of okananin to epirubicin is 15: 1 and 7.5: 1, preferably 15: 1. The combined index of okananin combined with epirubicin in the present invention on colon cancer cells is less than 1, and has a significant synergistic effect.

[0027] In the present invention, the drug includes oxanthate and epirubicin and pharmaceutical excipients compatible with oxanthate and epirubicin to prepare dosage forms that are conducive to administration, such as but not limited to aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder aerosol, sustained-release agent and controlled-release agent. The pharmaceutical excipients of the present invention can be those conventionally used in various preparations, such as but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; they can also be selected for use in order to be compatible with the substances, such as emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc.

[0028] Example 1: In vitro experiments of oxanin and epirubicin on colon cancer cells HCT116

[0029] 1. MTT assay to detect the effects of oxanthate and epirubicin on HCT116 cell viability

[0030] HCT116 cells in logarithmic phase were collected and 8×10 3 The cells were plated into a 96-well plate and cultured for 24 hours at 37°C and 5% CO2. The experiment was divided into a blank group and a drug-treated group. After the cells adhered to the wall, PBS solution was added to the blank group, and different concentrations of okanamin (12.5, 25, 50, and 100 μM) and epirubicin (0.5, 1, 2, 4, 8, and 16 μM) were added to the drug-treated group, with 3 parallel wells in each group. The culture was continued for 48 hours to observe the cell morphology, 10 μL of MTT solution was added to each well, and after culturing for 4 hours at 37°C and 5% CO2, the supernatant was discarded, 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals, and after shaking on a microporous oscillator for 10 minutes, the absorbance of each well was measured at a wavelength of 490 nm using an enzyme reader, and the cell viability was calculated according to the following formula. The results are as follows Figure 1 shown.

[0031] Cell viability = OD value of drug-treated group / OD value of blank group × 100%.

[0032] The results are as follows Figure 1 As shown, oxanthate and epirubicin can inhibit the proliferation of HCT116 cells, and with the increase of the administration concentration of oxanthate and epirubicin, the effect of inhibiting cell proliferation gradually increased, which was significantly different from the blank group.

[0033] 2. MTT assay to detect the effect of oxanthate combined with epirubicin on HCT116 cell viability and calculate the combined index

[0034] MTT assay was used to determine the cell viability of HCT116 cells treated with oxanthate (10, 20, and 30 μM) and epirubicin (1, 2, and 4 μM) for 48 h. Figure 2 shown.

[0035] CompuSyn software was used to calculate the combined index CI value after combined use. When CI < 1, it indicates that the two drugs have a synergistic effect, when CI = 1, it indicates that the two drugs have an additive effect, and when CI > 1, it indicates that the two drugs have an antagonistic effect. Figure 3 shown.

[0036] like Figure 3 As shown in the figure, when the concentration ratio of oxanin and epirubicin is 7.5:1 and 15:1, the combined effect of the two drugs on inhibiting the proliferation of HCT116 cells is more obvious than that of single drugs, and the corresponding CI value is less than 1, showing a synergistic effect. The combined use of oxanin and epirubicin can reduce the dosage of epirubicin and the cumulative dose of epirubicin under the condition of producing the same inhibitory effect on colon cancer cells, and reduce the probability of toxic side effects caused by the dosage of epirubicin. Based on the above, the concentration ratio of oxanin: epirubicin = 15:1 was selected for subsequent experiments.

[0037] 3. Annexin V / PI method to detect cell apoptosis

[0038] HCT116 cells in logarithmic phase were collected and plated at 100 × 10 4The cells were plated in 6-well plates and cultured for 24 hours at 37°C and 5% CO2. The experiment was divided into a blank group, an epirubicin group, an okanin group, and a combined drug group. The blank group was added with PBS solution, and the drug group was added with 2 mL of a final concentration of 2 μM Epi, 30 μM Okanin, and a combination of the two drugs (2 μM Epi + 30 μM Okanin) solution. After culturing for 24 hours, the cells to be tested were digested with EDTA-free trypsin and collected into EP tubes, and centrifuged at 1000 rpm for 3 minutes. Carefully aspirate and discard the supernatant, wash the cell pellet with 1 mL of pre-cooled PBS buffer, and discard the supernatant. One of the blank groups was divided into two parts, one of which was treated in boiling water for 1 minute, mixed with the other untreated part, centrifuged and discarded the supernatant, and pre-cooled 1×Binding Buffer was added to make up to 1.5 mL, and divided into 3 tubes, one of which was a blank control tube and two tubes were single-stained tubes. 5 μL Annexin V-FITC or 10 μL PI were added to the single-stained tubes and incubated at room temperature in the dark for 5 min. The drug-treated group was resuspended in 500 μL 1× Binding Buffer, and 5 μL Annexin V-FITC and 10 μL PI were added. The cells were incubated at room temperature in the dark for 5 min and detected and analyzed by flow cytometry. Each experimental group was repeated three times. The experimental results are shown in Figure 4 shown.

[0039] like Figure 5 As shown in the figure, compared with the blank group, the apoptosis rate of the epirubicin group and the oxanthate group increased, but compared with the epirubicin group and the oxanthate group, the apoptosis rate of the two-drug combination group increased significantly, and the difference was statistically significant (P < 0.05). This shows that compared with the use of epirubicin or oxanthate alone, the combined use of the two has a better effect on the apoptosis of colon cancer cells HCT116, indicating that oxanthate can enhance the apoptosis-promoting effect of epirubicin on colon cancer cells HCT116.

[0040] Example 2: In vivo pharmacodynamics of okananin combined with epirubicin on a mouse model of colon cancer CT26 xenografts

[0041] 1. Establishment of the CT26 colon cancer transplant mouse model

[0042] Forty 6-week-old male BALB / C mice were fed with normal drinking water and feed, and were adapted to feeding in the animal house for 1 week. The experiment was divided into a normal group, a model group, an epirubicin group, an oxadiazine group, and a combination group, with 8 mice in each group. CT26 cells were cultured, washed with sterile PBS solution, digested with trypsin, and then digested with complete medium. The cells were collected and centrifuged at 1000 rpm / min for 5 minutes, the supernatant was discarded, and the cells were washed twice with sterile PBS solution, and then resuspended with sterile PBS solution and the cell concentration was adjusted to 5×10 7Except for the normal group, each mouse was subcutaneously injected with 100 μL of CT26 cell suspension at the right armpit.

[0043] 2. Drug administration

[0044] The tumor grows to 80-100 mm 3 BALB / C mice were randomly divided into 4 groups. The model group was intraperitoneally injected with an equal volume of normal saline every day, the epirubicin group was intraperitoneally injected with 2.5 mg / kg epirubicin solution every day, the oxanthate group was intraperitoneally injected with 40 mg / kg oxanthate solution every day, and the combination group was intraperitoneally injected with 2.5 mg / kg epirubicin + 40 mg / kg oxanthate.

[0045] The mice were weighed before administration every day and detailed records were kept. After 10 days of administration, the mice were weighed and killed the next day after the last administration. The heart, liver, spleen, lung, kidney and tumor tissues were taken and weighed to explore the effects of okananin combined with epirubicin on tumor growth in tumor-bearing mice and the toxic reactions to important organs. The tumor inhibition rate and organ index were calculated. The specific results are as follows: Figure 6 shown.

[0046] Tumor inhibition rate (%) = (average tumor weight of mice in the model group - average tumor weight of mice in the drug administration group) / average tumor weight of mice in the model group × 100%

[0047] Organ index (%) = mouse organ mass (mg) / mouse body mass (g) × 100% Figure 7-8 As shown, the tumor size and mass of the epirubicin group, oxanthene group, and combination group were all smaller than those of the model group, indicating that epirubicin, oxanthene, and the combination of the two drugs can inhibit the growth of colon cancer tumors in vivo. The tumor inhibition rates of epirubicin and oxanthene alone were 54.70% and 37.65%, respectively. The tumor inhibition rate reached 75.18% after the combination of the two drugs, indicating that the combined use of the two drugs is more effective in preventing colon cancer than the use of epirubicin or oxanthene alone. In addition, compared with the epirubicin group, the combined administration of oxanthene and epirubicin can improve the organ index of mice, indicating that oxanthene can reduce the adverse reactions of epirubicin.

[0048] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for preventing and / or treating colon cancer, characterized in that: The pharmaceutical composition comprises oxanthate and epirubicin, wherein the molar ratio of oxanthate to epirubicin is 15:

1.

2. The pharmaceutical composition according to claim 1, characterized in that: The molar ratio of oxanin to epirubicin in the pharmaceutical composition can also be 7.5:

1.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that: The pharmaceutical composition is used for preparing a drug for inhibiting the activity of colon cancer cells.

4. The pharmaceutical composition according to any one of claims 1 or 2, characterized in that: The pharmaceutical composition is used for preparing a medicine for inhibiting tumor growth in mice bearing colon cancer.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The pharmaceutical composition includes but is not limited to injections, pills, tablets, granules, and capsules.

6. A method for detecting the effect of okananin combined with epirubicin on the viability of colon cancer cells, characterized by: The method includes using the MTT method to detect the effects of different concentrations of oxanthate and epirubicin on the viability of HCT116 cells, and calculating the cell viability.

7. A method for detecting the effect of okananin combined with epirubicin on apoptosis of colon cancer cells, characterized by: The method comprises using Annexin V / PI method to detect the effects of oxanthate and epirubicin on apoptosis of HCT116 cells.

8. The method according to claim 7, characterized in that: The method compares the effects of epirubicin or oxanthene alone and the combined use of the two drugs on apoptosis of colon cancer cells to evaluate the enhancing effect of oxanthene on the apoptosis-promoting effect of epirubicin.

9. An animal model for evaluating the effect of okananin combined with epirubicin on tumor growth in colon cancer-bearing mice, characterized by: The model is established by establishing a colon cancer CT26 transplanted tumor mouse model and administering oxadiazine and epirubicin in combination to evaluate the inhibitory effect on tumor growth and the toxic response to important organs.

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