CN111643497B

CN111643497B - Application of Talarocvolutin A in anti-cancer drugs

Info

Publication number: CN111643497B
Application number: CN202010549488.9A
Authority: CN
Inventors: 夏勇; 杨小龙
Original assignee: JINING MEDICAL UNIVERSITY
Current assignee: JINING MEDICAL UNIVERSITY
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2021-06-29
Anticipated expiration: 2040-06-16
Also published as: CN111643497A

Abstract

The invention belongs to the application field of microbial metabolites, and particularly discloses application of Talarococcorvolutin A in preparation of a medicine for treating colon and rectal cancers or lung cancers. The concentration of talarococcovolutin A for killing cancer cells in vitro is 0.5-50 mug/mL, and the lowest concentration for effectively killing the cancer cells in 48 hours is 2 mug/mL; effective in inhibiting colon cancer cell clonogenic concentration is 2 μ g/mL, at the cellular level IC₅₀Less than 1 mu g/mL, and the effective concentration of the compound in animal level is 5-20 mg/kg. The invention has the beneficial effects that: IC (integrated circuit)₅₀Low, minimal IC of Talarococcorvolutin A at the cellular level₅₀Less than 1 microgram/mL, the effective concentration of the compound in animal level can be as low as 5mg/kg, the compound is obviously superior to cisplatin chemotherapeutic drugs, and the compound has small toxic and side effects on animals.

Description

Application of Talarocvolutin A in anti-cancer drugs

Technical Field

The invention belongs to the application field of microbial metabolites, and particularly relates to application of Talarococcorvolutin A in preparation of anti-cancer drugs, especially drugs for treating cancers of digestive systems, drugs for treating cancers of respiratory systems or foods.

Background

Cancers of the digestive system, such as colon cancer and rectal cancer, are a tumor of the digestive tract that occurs in the epithelium of human colonic mucosa. With the lack of low fiber food and trace elements and vitamins in the human diet, the continuation of poor lifestyle habits, and the rising of mental stress on survival, patients are also increasingly migrating to lower ages, with higher incidence in men. The incidence of intestinal cancer is increasing worldwide.

In the current medical level, the treatment of the above-mentioned cancers of the digestive system is mainly directed to comprehensive treatment, i.e., organic combination of radiotherapy, chemotherapy, targeted therapy and immunotherapy. The chemotherapy is an important means for treating tumors, the classical chemotherapy drugs include cisplatin, adriamycin, paclitaxel, fluorouracil and the like, the action mechanisms of the drugs are mainly to kill cancer cells by inducing early apoptosis or autophagy of the tumors, but many cancer cells are insensitive to the chemotherapy drugs and are easy to generate drug resistance to the drugs. Therefore, the development of new anticancer chemotherapeutic drugs with novel anticancer mechanisms is of great significance for the effective treatment of colorectal cancer.

On the other hand, in the current research and development of drugs, it is a great difficulty in research that how to select a suitable compound, reduce drug toxicity and reduce side effects mainly through steps of searching active ingredients, drug screening and the like. However, the synthesized compounds are more harmful to human body, so the natural compounds become the main role, and the research on the anticancer drugs of the natural small molecular compounds has become the current important target.

To date, no natural compound has been found to be effective in the treatment of cancers of the digestive system, and there is a need to find an effective natural compound that can be used in the above-mentioned drugs for cancers of the digestive system to overcome the side effects and drug resistance of patients caused by conventional chemotherapeutic drugs.

Disclosure of Invention

In order to solve the technical problems, the invention provides a natural small molecular compound with a novel anti-cancer mechanism, the compound kills cancer cells by inducing cell iron death, and if the compound is applied to a medicament for inhibiting cancers, the problem that an anti-cancer chemotherapeutic medicament based on an apoptosis inducer is easy to generate drug resistance can be avoided.

The application and the application method of Talarocvolutin A in preparing the medicines for treating digestive system cancers such as intestinal cancers, particularly colorectal cancers and duodenal cancers are the important protection contents of the invention, the effect of Talarocvolutin A after being applied to the medicines for treating the colorectal cancers is also tested and evaluated, and the result shows that the Talarocvolutin A has remarkable advantages compared with the traditional chemotherapeutic medicines, and specific evaluation and analysis are shown in specific embodiments.

In particular to the application of Talarococcorvolutinin A in preparing a medicine for treating intestinal cancer; in particular to the application of Talarocvolutin A in preparing medicaments for treating ileum, caecum, colon, rectum and duodenum cancers; and the application of Talarococcorvolutin A in preparing the medicine for treating lung cancer is also within the protection scope of the invention.

The greatest innovation of the invention is that a natural small molecular compound is developed, the value of the compound in the aspects of medicine and food health care is explored, and the compound Talarococcovolutin A is found to be used for preventing and treating digestive system cancers by inducing iron death to kill cancer cells.

Talarocelelulin A has a direct effective concentration of more than or equal to 0.5 mu g/mL in the preparation of medicaments for treating colorectal cancer;

preferably, the medicine for treating colorectal cancer further comprises pharmaceutically acceptable auxiliary materials;

preferably, the above-mentioned drug is any one of an injection, a capsule, a tablet, a granule, a powder and an aerosol. It may also be a solution, suspension or emulsion.

The infusion mode of the injection is intravenous infusion, intraperitoneal infusion, subcutaneous infusion or intramuscular infusion;

the above capsule, tablet, granule, and powder are in gastrointestinal administration form.

The medicament containing talarococcovolutin A and the food containing talarococcovolutin A are also within the protection scope of the invention.

In the application, the concentration of the Talarococcovolutin A for killing cancer cells in vitro is 1-50 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-49 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-48 mu g/ml;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-47 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-46 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-45 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-44 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-43 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-42 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-41 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-40 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-39 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-38 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-37 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-36 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-35 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-34 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-33 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-32 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-31 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-30 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-49 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-48 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-47 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-46 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-45 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-44 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-43 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-42 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-41 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-40 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-39 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-38 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-37 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-36 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-35 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-34 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-33 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-32 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-31 mu g/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-30 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-29 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-28 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-27 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-26 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-25 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-24 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-23 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-22 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-21 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-20 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-10 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-8 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 0.5-6 mug/mL;

preferably, the concentration of the talarocconvolutin A for killing cancer cells in vitro is 1-6 mug/mL;

preferably, Talaroconcelvin A kills cancer cells in vitro at a concentration of 5 μ g/mL;

talarocvolutin A at the cellular level IC₅₀Less than 1 mu g/mL, and the effective concentration of the compound in the animal level is 5-20 mg/kg;

preferably, Talarococcovolutin A has a minimum concentration of 2 μ g/mL that is effective to kill cancer cells within 48 hours; talarocelelvin A was effective at a concentration of 2 μ g/mL for inhibiting colon cancer cell clonogenic.

A method of applying talarococcovolutin a in a medicament or foodstuff, comprising the steps of:

dissolving Talarocelelulin A in an organic solvent and/or vegetable oil; then the dissolved materials are used for preparing medicines or foods;

preferably, talarococcovulin A is dissolved in any one of ethanol, DMSO and vegetable oil;

preferably, under the room temperature condition, the Talaroconvolutin A is dissolved in DMSO to ensure that the concentration of the Talaroconvolutin A is 5-20 mg/mL; preferably, the concentration is 6-20 mg/mL; preferably, the concentration is 7-20 mg/mL; preferably, the concentration is 8-20 mg/mL; preferably, the concentration is 9-20 mg/mL; preferably, the concentration is 10-20 mg/mL; preferably, the concentration is 11-20 mg/mL; preferably, the concentration is 12-20 mg/mL; preferably, the concentration is 13-20 mg/mL; preferably, the concentration is 6-19 mg/mL; preferably, the concentration is 7-19 mg/mL; preferably, the concentration is 8-19 mg/mL; preferably, the concentration is 9-19 mg/mL; preferably, the concentration is 10-19 mg/mL; preferably, the concentration is 11-19 mg/mL; preferably, the concentration is 12-19 mg/mL; preferably, the concentration is 13-19 mg/mL; preferably, the concentration is 14-19 mg/mL; preferably, the concentration is 6-18 mg/mL; preferably, the concentration is 7-18 mg/mL; preferably, the concentration is 8-18 mg/mL; preferably, the concentration is 9-18 mg/mL; preferably, the concentration is 10-18 mg/mL; preferably, the concentration is 11-18 mg/mL; preferably, the concentration is 12-18 mg/mL; preferably, the concentration is 14-18 mg/mL; preferably, the concentration is 6-17 mg/mL; preferably, the concentration is 7-17 mg/mL; preferably, the concentration is 8-17 mg/mL; preferably, the concentration is 9-17 mg/mL;

preferably, the concentration is 10-17 mg/mL; preferably, the concentration is 11-17 mg/mL; preferably, the concentration is 12-17 mg/mL; preferably, the concentration is 13-17 mg/mL;

preferably 8 mg/mL; preferably 9 mg/mL; preferably 10 mg/mL; preferably 11 mg/mL; preferably 12 mg/mL;

dissolving Talarocvolutin A into DMSO, and adding vegetable oil serving as a cosolvent into DMSO to enable the final concentration of Talarocvolutin A to be 0.2-2 mg/mL;

preferably, the concentration is 0.3-2 mg/mL; preferably, the concentration is 0.4-2 mg/mL; preferably, the concentration is 0.5-2 mg/mL; preferably, the concentration is 0.6-2 mg/mL; preferably, the concentration is 0.7-2 mg/mL; preferably, the concentration is 0.8-2 mg/mL; preferably, the concentration is 0.9-2 mg/mL; preferably, the concentration is 1.0-2 mg/mL;

preferably, the concentration is 0.2-1.9 mg/mL; preferably, the concentration is 0.3-1.9 mg/mL; preferably, the concentration is 0.4-1.9 mg/mL; preferably, the concentration is 0.5-1.9 mg/mL; preferably, the concentration is 0.6-1.9 mg/mL; preferably, the concentration is 0.7-1.9 mg/mL; preferably, the concentration is 0.8-1.9 mg/mL; preferably, the concentration is 0.9-1.9 mg/mL; preferably, the concentration is 1.0-1.9 mg/mL; preferably, the concentration is 0.4-1.9 mg/mL;

preferably, the concentration is 0.5-1.8 mg/mL; preferably, the concentration is 0.6-1.8 mg/mL; preferably, the concentration is 0.7-1.8 mg/mL; preferably, the concentration is 0.8-1.8 mg/mL; preferably, the concentration is 0.9-1.8 mg/mL; preferably, the concentration is 1.0-1.8 mg/mL; preferably, the concentration is 1.1-1.8 mg/mL; preferably, the concentration is 1.2-1.8 mg/mL; preferably, the concentration is 1.3-1.8 mg/mL; preferably, the concentration is 1.4-1.8 mg/mL; preferably, the concentration is 1.5-1.8 mg/mL;

preferably, the concentration is 0.5-1.7 mg/mL; preferably, the concentration is 0.6-1.7 mg/mL; preferably, the concentration is 0.7-1.7 mg/mL; preferably, the concentration is 0.8-1.7 mg/mL; preferably, the concentration is 0.9-1.7 mg/mL; preferably, the concentration is 1.0-1.7 mg/mL; preferably, the concentration is 1.1-1.7 mg/mL; preferably, the concentration is 1.2-1.7 mg/mL; preferably, the concentration is 1.3-1.8 mg/mL; preferably, the concentration is 1.4-1.7 mg/mL; preferably, the concentration is 1.5-1.7 mg/mL;

preferably, the concentration is 0.5-1.6 mg/mL; preferably, the concentration is 0.6-1.6 mg/mL; preferably, the concentration is 0.7-1.6 mg/mL; preferably, the concentration is 0.8-1.6 mg/mL; preferably, the concentration is 0.9-1.6 mg/mL; preferably, the concentration is 1.0-1.6 mg/mL; preferably, the concentration is 1.1-1.6 mg/mL; preferably, the concentration is 1.2-1.6 mg/mL; preferably, the concentration is 1.3-1.6 mg/mL; preferably, the concentration is 1.4-1.6 mg/mL; preferably, the concentration is 1.5-1.6 mg/mL;

preferably, the concentration is 0.5-1.5 mg/mL; preferably, the concentration is 0.6-1.5 mg/mL; preferably, the concentration is 0.7-1.5 mg/mL; preferably, the concentration is 0.8-1.5 mg/mL; preferably, the concentration is 0.9-1.5 mg/mL; preferably, the concentration is 1.0-1.5 mg/mL; preferably, the concentration is 1.1-1.5 mg/mL; preferably, the concentration is 1.2-1.5 mg/mL; preferably, the concentration is 1.3-1.6 mg/mL; preferably, the concentration is 1.4-1.5 mg/mL;

preferably, the concentration is 0.3-1.9 mg/mL; preferably, the concentration is 0.4-1.8 mg/mL; preferably, the concentration is 0.5-1.7 mg/mL; preferably, the concentration is 0.6-1.6 mg/mL; preferably, the concentration is 0.7-1.5 mg/mL; preferably, the concentration is 0.8-1.4 mg/mL; preferably, the concentration is 0.9-1.3 mg/mL; preferably, the concentration is 1.0-1.2 mg/mL;

the vegetable oil is selected from any one of corn oil, sesame oil, peanut oil, soybean oil, olive oil, rapeseed oil, peony seed oil, sunflower seed oil, cottonseed oil, hemp seed oil, tea seed oil, tung seed oil, rice oil, safflower seed oil, linseed oil, palm oil, castor oil, coconut oil, walnut oil, camellia oil and rice bran oil;

preferably, the above Talarococcovolutin A has a final concentration of 1 mg/mL.

Preferably, the administration of talarococconvolutin a in model animals (mice) is intraperitoneal injection.

TalaroceleVOlutin A was injected at an amount of 5mg/kg body weight to exert an anticancer effect in model animals (mice).

The mechanism by which talarococcovolutin a kills cancer cells is to induce iron death: iron death (Ferroptosis), an iron-dependent, lipid peroxidation-based programmed cell death mode, is a novel death mode distinct from apoptosis, autophagy, and necrosis.

The invention has the beneficial effects that:

(1) talarocelelvin A of the invention is at the cellular level IC₅₀Low, less than 1 mug/mL, effective concentration less than 5mg/kg body weight at animal level, obviously superior to cisplatin chemotherapeutic drugs;

(2) talaroconvolutin a kills cancer cells by inducing iron death, not by inducing early apoptosis and autophagy of the cells; the application of Talarocvolutin A in the medicine avoids the toxic and side effects brought to the human body by the traditional anti-cancer medicine, and simultaneously lightens the drug resistance, the dependence and the like of the human body on the traditional anti-cancer medicine;

(3) talarocelelulin A has little side effects: the administration amount of 5mg/kg body weight is 21 days, the body weight of a mouse does not change obviously, the blood of the mouse does not change obviously, and organs such as liver and kidney of the mouse do not change obviously.

Drawings

FIG. 1 is a graph showing the cancer suppressing effect of Talarococcorvolutin A at different concentrations in three different colorectal cancer cell lines;

FIG. 2 shows the effect of Talarococcovolutin A in inhibiting DNA synthesis in colon cancer cell line SW 480;

FIG. 3 shows the effect of Talarococcorvolutinin A with different concentrations on inhibiting the formation of clones when it acts on colon cancer cells for a long time;

FIG. 4 is a study of the mechanism of action of Talarococcorvolutin A in killing cancer cells;

FIG. 5 is a graph of the results of transcriptome sequencing;

FIG. 6 shows that Ferrostatin-1, an iron death inhibitor, can reverse the killing effect of Talarocvolutin A on colon cancer cells;

FIG. 7 is a transmission electron micrograph of cells treated with Talaroconvolutin A;

FIG. 8 is a comparison of the inhibition effect of Talarocvoltin A and Erastin on tumor cells, FIG. 9 is a graph of tumor change, body weight change and HE staining and immunohistochemistry of tumor bodies of mice treated with Talarocvoltin A;

FIG. 10 is a photograph showing the cancer suppressing effect of Talarococcorvolumin A on lung cancer cells; in the figure, A is the killing effect of Talaroconvolutin A on lung cancer cells A549 at different concentrations; b is the inhibiting effect of Talarococcorvolutin A on the cloning formation of lung cancer cells A549; c showed that talarocconvolutin a killed lung cancer cell a549 in a concentration-dependent manner.

Detailed Description

The present invention will be further described with reference to specific examples so that those skilled in the art may better understand the present invention, but the present invention is not limited thereto. Example 1

The method for obtaining Talarocvolutin A comprises the following steps: see: the authors of antibiotic substructures againt pathetic Fungi, talarocvolutins, From Talaromyces Convolutus: shohei Suzuki, Tomoo Hosoe, Koohei Nozawa, Ken-ichi Kawai, Takashi Yaguchi, and Shun-ichi Udagawa journal: journal of Natural Product.

Under the condition of room temperature, firstly dissolving Talarococcorvolutin A into DMSO with the concentration of 10mg/mL, and then adding corn oil into DMSO with the concentration of 0.5mg/mL by using corn oil as a cosolvent; dissolving Talarocelelulin A in DMSO for use;

inoculating the cancer cells according to 30% of the co-density, and adding Talarococcus volutinin A with different concentrations after 24 hours;

after 48 hours, the cell viability was measured by the CCK8 method.

Cells in each group were used as a control without talarococcovolutin a (100% relative viability).

As can be seen in FIG. 1, the cancer suppressing effect of Talarococconvolutin A at different concentrations in three different colorectal cancer cell lines. In the experiment, three different colorectal cancer cell lines, namely HCT116, SW480 and SW620 are cultured, after the cells are attached to the wall and grow to 50% of the common density, Talaroconvolutin A with different concentrations is added into three colorectal cancer cell columns by adopting a low serum (1% FBS) culture medium, and the killing power of the Talaroconvolutin A on the colorectal cancer cells is detected by using a CCK8 kit after 24 hours. The result shows that Talarococcorvolutin A has effective killing effect on three colorectal cancer cell lines, and the half-inhibition concentration IC50 is between 0.5 and 1.0 mu g/mL. Above a concentration of 2. mu.g/mL, tumor cells are almost completely killed.

Example 2

Regarding the effect of talarococcovolutin A with different concentrations in the colon cancer cell line SW480 in inhibiting the DNA synthesis of the cancer cells, the inventors also examined the DNA synthesis rate of the cancer cells by using Edu method after the cells were inoculated at 30% co-density for 12 hours and then incubated with talarococcovolutin A with different concentrations for 24 hours. After EdU was added to the medium for 30 minutes, the medium was discarded and the cells were fixed with 4% paraformaldehyde.

The procedure was then followed in accordance with the EdU cell proliferation kit instructions from the pecan corporation. EdU is a substance that can only intercalate into newly synthesized DNA, but cannot intercalate into original DNA, and shows red color under a fluorescent microscope; while Hoechst33342 can intercalate into all DNA and appear blue under a fluorescent microscope. The rate of cancer cell DNA synthesis can be detected visually using the ratio of red to blue.

As shown in figure 2, the ratio of EdU/Ho33342 is obviously reduced along with the increase of the concentration of Talaroconcelvin A, which indicates that Talaroconcelvin A can strongly inhibit the DNA synthesis rate of colon cancer cells.

Example 3

In order to study how different concentrations of Talarocvolutin A can inhibit colon cancer cells for a long time, the inventor used 0.5-2.0 μ g/mL of Talarocvolutin A at 50cells/cm²The inhibition of clone formation was observed every 2 days in the culture system of (1), and the results in FIG. 3 show that 0.5. mu.g/mL Talarococcovilutin A can significantly inhibit clone formation after 12 days of treatment, and 2.0. mu.g/mL can almost completely inhibit the clone formation of tumor cells.

Example 4

Regarding the mechanism of action of Talaroconvolutin a in killing cancer cells, the present inventors studied by H2DCFDA staining, and it was found that Talaroconvolutin a can cause cell membrane rupture of cancer cells (indicated by a downward arrow toward the left in fig. 4) and that Talaroconvolutin a can cause ROS accumulation inside the cells (indicated by a downward arrow toward the right) by H2DCFDA staining.

Example 5

Using transcriptome sequencing, talarocconvolutin a was found to be able to strongly induce iron death in cancer cells. In FIG. 5, A represents transcripts whose expression levels were down-regulated and up-regulated in the case of Talarocvolutin A treated colon cancer cells, the dotted line in the middle represents transcripts whose expression levels were down-regulated, the dotted line in the left represents transcripts whose expression levels were up-regulated, VPC represents the control group, VPH represents the Talarocvolutin A treated group, and each group was set in triplicate.

In FIG. 5, B shows that in the KEGG enrichment analysis, the molecules related to the iron death pathway are greatly enriched, which means that Talaroconcelvin A causes significant changes in the expression level of the molecules related to iron death.

In FIG. 5, C is the heatmap of the iron death-related protein, and the results show quantitative evidence of the molecular level of iron death caused by Talarocvolutin A, wherein C represents the control group, H represents the Talarocvolutin A treatment group, and each group is in triplicate.

Fig. 5 reveals from the sequencing data point of view that the mechanism by which Talaroconvolutin a kills cancer cells is induction of iron death.

Example 6

From FIG. 6, it can be seen that the inhibitor Ferrostatin-1 of iron death can reverse the killing effect of Talarocvoltatin A on colon cancer cells, which is a side-effect of Tala A pharmacological mechanism-iron death, and from FIG. 6, it can be concluded that Talarocvoltatin A exerts a therapeutic effect on colorectal cancer by inducing iron death.

Example 7

Biological electron microscope experiment: FIG. 7 is a transmission electron micrograph of Talaroconvolutin A treated colorectal cancer cells SW 480. FIG. 7 is a photograph showing that the cells treated with Talarococcovolutin A exhibited distinct morphological characteristics of iron-dead cells: mitochondria collapse, mitochondrial cristae disappear, and cell membrane ruptures. (the organelle to which the arrow marked mt points is mitochondria, which is in the form of an oval in shape and the mitochondrial membrane is intact and the inside has a ridge; after Talarocvoltutin A treatment, the morphology of mitochondria becomes abnormal and the mitochondria shrinks and the membrane is incomplete and the internal ridge disappears), cell membrane disruption (the arrow marked mem points to the cell membrane, which is in the untreated cell, which is intact; after Talarocvoltutin A treatment, the cell membrane becomes vacuolated and ruptured). Fig. 7 reveals the anticancer action mechanism of Talaroconvolutin a from the aspect of cell morphology: exerts therapeutic effects on colorectal cancer by inducing iron death.

Example 8

Compared with the talarococcovolutin A provided by the invention and similar products, such as Erastin which achieves the aim of inhibiting cancer by inducing the death of iron by cancer cells, the inventor carries out the following experiments:

comparative experiment: to compare the present invention with the current world recognized anti-cancer capacity of Erastin, an iron death inducer. The following experiments were performed: SW480 cells were cultured in DMEM (high glucose) medium containing 10% FBS, and Talarotinomycin A or Erastin (Erastin is available from MCE) was added at a concentration of 1-30uM at a cell density of 40%, respectively, and after 24 hours of culture, the cell viability was measured by CCK-8. FIG. 8 shows that Talarococcovolutin A can inhibit SW480 cell proliferation by more than 50% at 5uM, but Erastin at the same concentration can only slightly inhibit the same tumor cells. At 10uM, Talarococcovolutin A almost completely inhibited cell proliferation, but Erastin inhibited cell proliferation by less than 50%. It can be seen from the graph and experimental data in fig. 8 that: talarocelelvin A has obviously better cancer inhibition effect than currently accepted commercial iron death inducer Erastin.

Example 9

Mouse experiments:

in vivo experiments: talarocelelvin A is used for researching the cancer inhibition effect in a mouse (Balb/c nude mouse is taken as an experimental object in the invention) model. In Balb/c nude mice, subcutaneous tumor of colon cancer cell is 5 multiplied by 10 under axilla of each mouse⁶When the tumor volume grows to 300 mm³On the left and right, the samples were randomly divided into two groups of 5. The administration dosage is 5mg/kg body weight, specifically, Talarococcorvolutin A is prepared by dissolving high concentration (10 mg/mL) in DMSO, dissolving in corn oil (concentration is 1 mg/mL), and then the administration frequency is once for 2 days, the administration mode is intraperitoneal injection, and the control group is injected with the same volume of corn oil. Measuring the tumor volume every 3 days until the average tumor volume of one group of the control group or the administration group reaches 1600mm³. Then, the mice are sacrificed in an euthanasia mode, subcutaneous tumor bodies are picked, formalin fixation is carried out on the tumor bodies, and the cell proliferation marker Ki67 is detected by HE staining and immunohistochemical staining after the next pathological section. The A, B results in FIG. 9 show that the tumor volume was significantly smaller in the group administered 21 days after administration than in the control group. Panel C shows that tumor growth was inhibited in mice from the administered group, and the tumor cell proliferation rate was decreased in mice from the administered group as seen from Ki67 immunohistochemistry results. Panel D shows that there was no significant change in body weight of the mice over the 21 day dosing treatment. In addition, the routine test result of the blood of the experimental mouse shows that the body weight of the mouse has no obvious change. Animal experiments show that: talarocelelulin A has good anti-tumor performance. FIG. 9 illustrates the anti-tumor activity of Talarococcorvolutin A, and low toxicity and side effects from the in vivo experimental point of view.

Example 10

The concentration of the talarococcovolutin A for killing cancer cells in vitro is 0.5-50 mug/mL; talarocvolutin A at the cellular level IC₅₀Less than 1 mu g/mL, and the effective concentration of the compound in the animal level is 5-20 mg/kg;

the lowest concentration of Talarocvolutin A for 48 hours to effectively kill cancer cells is 2 mug/mL; talarocelelvin A was effective at a concentration of 2 μ g/mL for inhibiting colon cancer cell clonogenic.

When the Talarocvolutin A is applied, the Talarocvolutin A is dissolved in any one of ethanol, DMSO and vegetable oil at room temperature; the concentration is 5-20 mg/mL, and the vegetable oil is selected from any one of corn oil, sesame oil, peanut oil, soybean oil, olive oil, rapeseed oil, peony seed oil, sunflower seed oil, cottonseed oil, hemp seed oil, tea seed oil, tung seed oil, rice oil, safflower seed oil, linseed oil, palm oil, castor oil, coconut oil, walnut oil, camellia oil and rice bran oil.

The experiments comprise verification of the cancer inhibition effect of Talaroconcelvin A in different colorectal cancer cell lines, detection of the effect of Talarocvolutin A in inhibiting the DNA synthesis of cancer cells in a colon cancer cell line SW480, detection of the effect of Talarocvolutin A in killing the cancer cells, sequencing results of transcriptome, and experiments that Ferrostatin-1, an iron death inhibitor, can reverse the killing effect of Tala A on colon cancer cells, electron micrographs, animal tumor changes, weight changes, HE staining of tumor bodies and immunohistochemistry, and the following conclusion can be drawn:

talarococconvolutin a kills cancer cells by inducing cellular iron death, and has a very significant inhibitory effect on cancer cells such as colon cancer cells; if the compound is applied to the medicines for inhibiting cancer cells, the treatment idea of cancer is necessarily widened, and a high-efficiency and low-toxicity treatment mode is provided;

2. through mouse experiments, the proliferation rate of tumor cells of a mouse is found to be obviously reduced, and the body weight of the mouse has no obvious change after administration treatment, so that the Talaroconvolutin A has good tumor inhibition effect on animals when being applied to a medicament; it is understood that talarococcovitin a is useful for animals such as domestic animals (e.g., pork pigs), pets (e.g., pet dogs, pet cats), poultry, birds, and zoo animals, in addition to suppressing cancer cells such as colon cancer in humans, and that excellent antitumor effects can be obtained by feeding such animals when talarococcovitin a is prepared as a feed.

Example 11

Regarding the study of the killing effect of talarococcovolutin a on lung cancer cells, the present inventors conducted the following experiments:

non-small cell lung cancer A549 is taken as a research object, and the cancer inhibition effect of Talaroconvolutin A on lung cancer cells is researched by a CCK8 kit and two means of clone formation. Culturing lung cancer cells in F-12K culture medium containing 10% FBS, adding 0-10ug/mL Talarococcorvolutin A in 5% CO when CO-density grows to 50%_2，After culturing for 48 hours in a 37-degree cell culture box, the cell culture solution is replaced, and then the activity of the cells is detected by using a CCK8 kit. As shown in the figure A, when the concentration of Talarocvolutin A exceeds 1.5ug/mL, the composition has obvious killing effect on lung cancer cells A549, and when the concentration of Talarocvolutin A reaches 5mg/mL, the composition can almost completely inhibit the proliferation of A549. In FIG. B, the inhibitory effect of Talarocvolutin A on the formation of lung cancer cell A549 clone is shown, TalWhen the concentration of aroconvoltin A reaches 2ug/mL, the inhibition ability to the A549 clone formation is shown, and when the concentration of Talaroconvoltin A reaches 3 ug/mL, the A549 can hardly form the cell clone group. In fig. C, evidence that Talaroconvolutin a kills lung cancer cells a549 in a concentration-dependent manner is given from changes in cell morphology: talarocvolutin A at 2ug/mL can cause A549 to show necrotic cells (shown by black arrows), Talarocvolutin A at 3 ug/mL can cause A549 to lose the original cell morphology, and when the concentration of Talarocvolutin A is further increased, A549 becomes slender and is accompanied by massive cell death. Thus, the above experiment fully proves the inhibition effect of talarococcovolutin A on lung cancer cells.

Example 12

Talarococcorvolutin a may be applied to food products, such as the use of talarococcorvolutin a in food/food processing; application of Talarocvolutin A in liquid food and solid food; the application of Talaroconvolutin A in granular food or solid powder food or microcapsule food or semi-liquid food or paste food or frozen food; and Talaroconvolutin A in solid beverage, liquid beverage, and granular food, are also within the scope of the invention.

For example, a jelly, pudding, yogurt, candy, chewing gum containing talarococcovlutin a, and the like, are within the scope of the present invention; talarocelelulin A can be mixed with carrier such as excipient, binder, disintegrating agent, lubricant, solvent, binder, surfactant, water-soluble polymer, etc., and supplemented with other materials, such as conventional food additives, to prepare foods in various forms. The food of the present invention may be prepared by mixing the ingredients usually used in the field of food as appropriate, and selecting an appropriate material from these ingredients according to specific needs, as long as the desired effect is not impaired.

Talarocelelulin A-containing food product, comprising other conventional food additives such as: mouthfeel improving agents, such as: a sweetener selected from the group consisting of stevia, licorice, disodium glycyrrhizinate, tripotassium and trisodium glycyrrhizinate, sucrose, lactose, sorbitol, isomaltitol, lactitol, isomaltitol, glucose, high fructose syrup, erythritol, xylitol, sorbitol, xylitol, maltitol, neohesperidin dihydrochalcone, saccharin, sodium cyclamate, aspartame, sodium cyclamate, thaumatin, oligosaccharide alcohols, panthenol, fructooligosaccharide, galactose, acesulfame potassium, sucralose, fructose, aspartame, sodium cyclamate, aspartame, mogroside, honey, glycyrrhizin, ammonium glycyrrhizinate, monopotassium glycyrrhizinate, stachyose, raffinose, oligosaccharide, polyfructose, polyglucose, xylooligosaccharide, oligomannose, isomaltitol, oligomannose and solid polyethylene glycol;

the sour agent is citric acid, malic acid, citric acid, ascorbic acid, gluconic acid, L-malic acid, fumaric acid, tartaric acid, sodium montmorillonoid, fruit vinegar, etc.;

the edible essence is natural essence or synthetic essence; mainly fruit essence or vegetable essence; the method comprises the following steps: grape essence, strawberry essence, henry steudnera tuber essence, corn essence, peanut essence, juicy peach essence, blueberry essence, watermelon essence, milk essence, orange essence, jasmine essence, banana essence, rose essence, vanilla essence, orange essence, apple essence, mango essence, litchi essence and the like;

the stabilizer comprises: CMC, sodium alginate, pectin, gelatin, chitin, chitosan, sodium caseinate, xanthan gum, guar gum, Arabic gum, carrageenan, agar, konjac gum, gellan gum, beta-cyclodextrin, starch, stearate, monoglyceride, sucrose ester, diglycerol monoglyceride stearate, tetraglycerol monoglyceride stearate, succinylglycerol, diacetylsuccinylglycerol, sodium lactoyl stearate, sorbitan triglyceride stearate, sorbitan monoglyceride, sorbitan oleate, polyoxyethylene sorbitan stearate, propylene glycol monoglyceride, and polyoxyethylene sorbitan oleate; propylene glycol alginate, propylene glycol alginate;

the following auxiliary materials can also be included, such as: antioxidants such as ascorbic acid, preservatives, coloring agents, color fixatives, leavening agents, and the like; specifically, the extract can be used for preparing resistant dextrin, milk powder, sodium bicarbonate, ammonium bicarbonate, protein powder, sodium benzoate, potassium sorbate and lactic acid; emulsifiers such as polyglycerin fatty acid esters, sucrose fatty acid esters and enzymatically decomposed lecithin; in addition, it also contains nutrition enhancer, plant extract, functional components and trace elements.

For example, in the preparation of liquid beverage, Talaroconvolutin A after dissolution is taken, and then sweetener, sour agent, edible essence, stabilizer, preservative and antioxidant are optionally added;

when preparing powdery solid beverage, the Talarococcorvolutin A after dissolving is taken, and then sweetener, sour agent, edible essence, preservative and antioxidant are optionally added;

when the microcapsule is prepared, the Talarococcorvolutin A after dissolution is taken, and then optional stabilizer, preservative, antioxidant, sweetener and the like are added.

The above materials are merely exemplary, not exhaustive, and are selected from the above materials, but are not limited to the above species. The preparation method of the food is the same as the conventional food preparation method, and only the dissolved Talaroconvolutin A is added into the food; the amount added is the same as the effective dose in example 10.

Similarly, talarococcovolutin a of the present invention is applicable to the treatment of other cancer drugs besides the preparation of digestive cancer drugs and respiratory cancer drugs, because the mechanism of talarococcovolutin a for inhibiting the growth of all tumor cells is similar, and therefore, the application of talarococcovolutin a to other cancer drug treatment is also within the scope of the present invention.

Claims

Application of Talaroconcelvin A in preparation of medicines for treating colon cancer, rectal cancer or lung cancer.
2. The use of claim 1, wherein Talaroconvolutin a kills cancer cells in vitro at a concentration of 0.5 to 50 μ g/mL.
3. The use of any one of claims 1 to 2, wherein Talaroconcelvin A kills cancer cells in vitro at a concentration of 1 to 20 μ g/mL.
4. The use of any one of claims 1 to 2, wherein Talaroconcelvin A kills cancer cells in vitro at a concentration of 2 to 10 μ g/mL.
5. The use of any one of claims 1 to 2, wherein Talaroconcelvin A kills cancer cells in vitro at a concentration of 3 to 8 μ g/mL.
6. The use of any one of claims 1 to 2, wherein Talaroconcelvin A kills cancer cells in vitro at a concentration of 4 to 5 μ g/mL.
7. Use according to any one of claims 1 to 2, wherein talarococcovolutin a is at the cellular level IC₅₀Less than 1 mu g/mL, and the effective concentration of the compound in animal level is 5-20 mg/kg.
8. The use of any one of claims 1 to 2, wherein the lowest concentration of talarocconvolutin a that is effective at killing cancer cells for 48 hours is 2 μ g/mL.
9. The use of any one of claims 1 to 2, wherein talarocconvolutin a is effective at a concentration of 2 μ g/mL for inhibiting colon cancer cell clonality.
10. The use according to claim 1, wherein Talaroconvolutin a is used for the prevention or treatment of colon or rectal cancer by killing cancer cells by inducing iron death;

the Talaroconcelvin A has direct effective concentration of 0.6 mug/mL or more when being applied to the preparation of the medicines for treating colon cancer and rectal cancer.
11. The use of claim 1, wherein the colon cancer drug, rectal cancer drug further comprises pharmaceutically acceptable excipients.
12. The use of claim 1, wherein the medicament is any one of an injection, a capsule, a tablet, a granule, a powder, and an aerosol.
13. The use of claim 12, wherein the injectable formulation is administered by intravenous infusion, intraperitoneal infusion, subcutaneous infusion or intramuscular infusion.
14. The use according to claim 12, wherein the capsule, tablet, granule or powder is administered from the gastrointestinal tract.