阅读说明：本技术 MII-tt-DTT及其在制备抗结直肠癌药物中的应用 (MII-tt-DTT and application thereof in preparing anti-colorectal cancer drugs ) 是由 张磊 程小霞 王群 汤昆 贾爽爽 崔昆丽 陈明亮 刘楠 梁雅喻 李慧珍 田志立 于 2021-09-23 设计创作，主要内容包括：本发明属于化工医药技术领域,具体涉及MII-tt-DTT在制备抗癌药物中的应用,所述MII-tt-DTT的化学结构如下所示：MTT结果显示：MII-tt-DTT能显著抑制结直肠肿瘤细胞的增殖,并定位到线粒体。Annexin V and PI双染凋亡检测结果显示：MII-tt-DTT能诱导细胞凋亡；Western Blot结果显示：MII-tt-DTT能诱导细胞铁死亡。本发明的小分子化合物MII-tt-DTT作为新的抗结直肠肿瘤药物或者其辅助成分进行开发,抑制肿瘤效果显著,将为治疗和治愈结直肠肿瘤提供新的途径和手段。(The invention belongs to the technical field of chemical medicines, and particularly relates to an application of MII-tt-DTT in preparing an anticancer drug, wherein the chemical structure of the MII-tt-DTT is as follows: MTT results show that: MII-tt-DTT significantly inhibited proliferation of colorectal tumor cells and was localized to the mitochondria. The Annexin V and PI double-staining apoptosis detection result shows that: MII-tt-DTT can induce apoptosis; the Western Blot results show: MII-tt-DTT induces cellular iron death. The small molecular compound MII-tt-DTT is developed as a new anti-colorectal tumor medicament or an auxiliary component thereof, has obvious tumor inhibition effect, and provides a new way and a new hand for treating and curing colorectal tumorsAnd (4) section.)

3- (2- (N-methyl-3, 3-dimethyl-3H-indole iodate) -1-vinyl) dithieno [3,2-b:2',3' -d ] thiophene, abbreviated as MII-tt-DTT, the chemical structure of which is shown below

2. Use of MII-tt-DTT according to claim 1 for the preparation of a medicament against colorectal cancer.

3. The use of MII-tt-DTT according to claim 2, for the preparation of an anti-colorectal cancer medicament, wherein the MII-tt-DTT inhibits the cell viability of the colorectal cancer cell lines HCT116, SW480 or DLD-1.

4. The use of claim 2, wherein the MII-tt-DTT is used at a concentration of 0.0975-25 μ M.

5. The use of MII-tt-DTT for the preparation of an anti-tumor medicament according to claim 2, wherein the MII-tt-DTT inhibits the growth of subcutaneous tumor bearing of the colorectal cancer cell line HCT116 BALB/C nude mouse at a dose of 1 mg/kg.3d.

6. Use of the MII-tt-DTT according to claim 2, for the preparation of an anti-colorectal cancer medicament, to induce apoptosis in colorectal cancer cell lines HCT116, SW480 or DLD-1.

7. Use of the MII-tt-DTT according to claim 2 for the preparation of an anti-colorectal cancer medicament, which localizes in and causes mitochondrial damage to the colorectal cancer cell lines HCT116, SW480 or DLD-1 mitochondria.

8. Use of the MII-tt-DTT according to claim 2, for the preparation of an anti-colorectal cancer medicament, for inducing the production of reactive oxygen species by the colorectal cancer cell lines HCT116, SW480 or DLD-1.

9. Use of MII-tt-DTT according to claim 2 for the preparation of a medicament against colorectal cancer, which induces a decrease in the antioxidant system activity of the colorectal cancer cells xCT-GPX 4.

10. The use of MII-tt-DTT according to claim 2 for the manufacture of a medicament for the treatment of colorectal cancer, which induces an increase in total iron content in colorectal cancer cells, which in turn induces the onset of colorectal cancer cell iron death.

Technical Field

The invention belongs to the technical field of chemical medicines, and particularly relates to application of MII-tt-DTT in preparation of an anti-colorectal cancer medicine.

Background

Colorectal cancer is one of the most common malignancies of the digestive tract, and is the third most prevalent in the incidence of malignancies, with mortality being the second most prevalent. The incidence of colorectal cancer is increasing, and the treatment and prevention of colorectal cancer have attracted wide social attention. So far, the clinical treatment modes for colorectal cancer mainly include surgery, radiotherapy and chemotherapy. The surgery treatment has great damage to the body of the patient, and the development stage, the position and the like of the tumor determine the feasibility of the surgery. The existing chemotherapeutic drugs such as 5-fluorouracil, oxaliplatin, irinotecan and the like have side effects of different degrees. Therefore, the development of new therapeutic drugs for colorectal cancer remains a primary task for many researchers.

Thiophene (thiolene), a heterocyclic compound, is widely found in nature, and particularly, Thiophene-fused ring structures are contained in the effective molecular structures of some antitumor drugs, and show good antitumor activity, so that Thiophene (thiolene) is widely applied to the field of medical treatment. Thiophene has been reported to have cytotoxic activity when exposed to long wavelength ultraviolet light. It is excited by ultraviolet light (wavelength 300-400 nm) and visible light to initiate high-reactivity singlet oxygen¹O₂) The latter produces oxidation reactions on biological molecules (including lipids, proteins and nucleic acids) which are toxic to the organism.

Indoles are compounds of pyrrole in parallel with benzene, also known as benzopyrrole. Indole derivatives are widely distributed in nature, the structures of a plurality of natural compounds contain indole rings, and some indole derivatives are closely related to life activities. The indole compounds are heterocyclic derivative alkaloids and have remarkable physiological activity. A large number of studies at home and abroad show that the indole or indole structure has an anticancer effect. The research shows that melatonin secreted by pineal body is an indole hormone with obvious antitumor activity. The research shows that the pine cone body of the tumor-bearing animal is removed, and the tumor development is promoted as a result. And the growth stimulation to the tumor can be eliminated by administering a certain amount of melatonin.

Indole compounds widely exist in natural products and bioactive drug molecules, and are widely applied to agricultural chemistry and pharmaceutical industry. Recent studies have found that thienoindole derivatives can also be widely used in the design of light emitting devices and light emitting materials, but have relatively few applications in pharmaceutical research, which may be related to less research on the synthesis of the thienoindole derivatives selectively mono-substituted or di-substituted, and further enhancement and breakthrough are needed. The MII-tt-DTT is a trithiophene indole derivative, emits red fluorescence by itself, and is expected to be applied to treatment of tumors. There is currently no study on the activity associated with this compound.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a compound MII-tt-DTT with a new structure and a new application thereof as a medicine, namely the application of the MII-tt-DTT in preparing a medicine for resisting colorectal cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the MII-tt-DTT in preparing the antitumor drugs is as follows:

the relevant properties are as follows:

chemical name: 3- (2- (N-methyl-3, 3-dimethyl-3H-indole Iodide) -1-vinyl) dithieno [3,2-b:2',3' -d ] thiophene, MII-tt-DTT for short (note: indole Iodide indole index II for short, indole monoiodide MII for short);

the molecular formula is as follows: c₂₁H₁₈NS₃I; molecular weight: 507.47, respectively; the detection mode is as follows:¹HNMR，¹³CNMR, HRMS; the characteristics are as follows: the product is light red powder; the source is as follows: the subject is composed. Pharmacological properties: insoluble in water and soluble in DMSO.

In particular to application of MII-tt-DTT in preparing anti-colorectal cancer drugs. The compounds are derivatives of indolyltrithiophene.

Further, the concentration of the anti-colorectal cancer effect of the MII-tt-DTT is 0.0975-25 mu M.

The invention provides a method for inhibiting in vitro tumor cell proliferation, which comprises the steps of adding MII-tt-DTT into a culture solution of tumor cells, wherein the final concentration of the added MII-tt-DTT is 0.0975-25 mu M.

The invention also provides a method for inducing in vitro tumor cell apoptosis and iron death, which comprises the steps of adding MII-tt-DTT into a culture solution of tumor cells, wherein the final concentration of the added MII-tt-DTT is 0.0975-25 mu M.

The tumor cells can be colorectal cancer HCT116 cells, colorectal cancer SW480 cells and colorectal cancer DLD-1 cells.

The invention also provides an anti-colorectal cancer medicament, and the active component of the anti-colorectal cancer medicament is MII-tt-DTT.

The MII-tt-DTT synthesis route is as follows:

compared with the prior art, the invention has the beneficial effects.

The invention provides application of MII-tt-DTT in preparation of antitumor drugs. MTT results show that: MII-tt-DTT significantly inhibited proliferation of colorectal tumor cells and was able to co-localize with mitochondria. The Annexin V and PI double-staining apoptosis detection result shows that: MII-tt-DTT induces apoptosis. ROS and Fe²⁺The content detection result and the result of Western Blot detection TFR1 show that: MII-tt-DTT induces cellular iron death. The small molecular compound MII-tt-DTT is developed as a new anti-colorectal tumor medicament or an auxiliary component thereof, has obvious tumor inhibition effect, and provides a new way and means for treating and curing colorectal tumors.

Drawings

FIG. 1: identification of MII-tt-DTT structure¹H NMR (400MHz, DMSO-d6) profile data;

FIG. 2: identification of MII-tt-DTT structure¹³C NMR (100MHz, DMSO-d 6));

FIG. 3: MII-tt-DTT structure identification HRMS (MALDI-DHB) map data;

FIG. 4: HCT116, SW480, DLD-1 cell morphology results after MII-tt-DTT administration; B-D: MTT results; e: plate cloning results;

FIG. 5: a: tumor-bearing model test-tumor volume; b: tumor-bearing model test-tumor-bearing weight; c: toxicity test-NS group, 5-Fu group, MII-tt-DTT group nude mouse pictures; d: toxicity test-visceral HE staining results; e: tumor-bearing model test-tumor-bearing HE staining result; F-G: toxicity test-liver injury assay; H-I: toxicity test-renal injury assay; j: toxicity test-nude mouse body weight; K-O: toxicity test-organ coefficient;

FIG. 6: the effect of MII-tt-DTT combined with NAC, VE, Z-VAD-FMK, Necrostatin-1, Apoptosis on cell proliferation;

FIG. 7: a: flow results of MII-tt-DTT induced apoptosis; B-D: streaming quantization results; e: western Blot is used for detecting the effect of MII-tt-DTT on the expression of BaX, Bcl-2, Cytc and cleared-Cas 3; F-Q: western Blot quantification results;

FIG. 8: a: (iii) mitochondrial co-localization results; B-D: an ATP detection result;

FIG. 9: JC-1 staining result;

FIG. 10: a: detecting the result of ROS fluorescence; B-D: detecting results by an ROS flow cytometer; E-G: detecting the result of ROS flow cytometry after the combined action of MII-tt-DTT and NAC;

FIG. 11: A-C: GSH detection results; D-F: GSSG detection result; G-I: cys detection results; j: western Blot is used for detecting the influence of MII-tt-DTT on XCT and GPX4 expression; K-P: western Blot quantification results;

FIG. 12: A-C: fe²⁺/Fe³⁺Detecting the result; D-F: detecting the result of ROS flow cytometry after the combined action of MII-tt-DTT and DFO; G-I: fe after combined action of MII-tt-DTT and DFO²⁺/Fe³⁺Detecting results by a flow cytometer; J-L: MTT detection result after the combined action of MII-tt-DTT and DFO; m: western Blot to detect the effect of MII-tt-DTT on the expression of NCOA4 and TRF1 of cells; N-S: western Blot quantification results; in the figure, P<0.05；**，P<0.015；***，P<0.001；

Detailed Description

In order to make the technical purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is further described with reference to specific examples, but the implementation is intended to explain the present invention and should not be construed as a limitation of the present invention, and those who do not specify specific techniques or conditions in the examples follow the techniques or conditions described in the literature in the field or follow the product specification.

The experimental method comprises the following steps:

preparation of Compounds

Specifically, compound 1(91.3mg, 0.41mmol, 1.0eq), compound 2(122.4mg, 0.41mmol, 1.0eq), potassium acetate (7.7mg, 0.59mmol, 1.4eq) are taken out and put into a 50mL Schlenk bottle, 15mL of ethanol is added, 100 ℃ is reacted for 12 hours under the protection of argon, the reaction is stopped, the solution is cooled to room temperature, a crude product is obtained by rotary evaporation and solvent removal, and the compound MII-tt-DTT is obtained by column chromatography (ethyl acetate: petroleum ether ═ 60: 1): 85.1mg, yield: 41 percent.

Identification of MII-tt-DTT structure¹HNMR、¹³CNMR and HRMS are shown in FIGS. 1-3.

MII-tt-DTT was dissolved in DMSO and prepared as 100mM stock solution for use.

The effect of MII-tt-DTT on colorectal cancer cell proliferation was determined using example 1, MTT, plate cloning.

HCT116 cells (purchased from the cell bank of the culture Collection of the national academy of sciences) at 3X 10³Perwell inoculation into 96 well plates, 5% CO₂After culturing in DMEM complete medium containing 100U/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ for 12 hours, MII-tt-DTT was added at different concentrations (25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M, 0.78. mu.M, 0.39. mu.M, 0.195. mu.M and 0.0975. mu.M, respectively), and the culture was continued for 48 hours with 5-fold-setting of each concentration, and the culture solution was discarded, and the cell survival rate was determined using MTT reagent.

The determination method comprises the following steps: washing cells once with serum-free medium, adding a pre-prepared MTT reaction solution into 15 mu L/hole, continuously culturing for 4h, sucking and removing supernatant, adding DMSO into 100 mu L/hole to dissolve reduction products, shaking table for 10min, reading absorbance value at 490nm wavelength, calculating cell survival rate, determining MII-tt-DTT intervention hole absorbance value/control hole absorbance value as cell survival rate value, and calculating IC of MII-tt-DTT on HCT116 cells₅₀The value is obtained.

IC₅₀Refers to the concentration of inhibitor at which cell growth is inhibited by half. This is the concentration of MII-tt-DTT at which HCT116 cells were half as large as the control.

As a result: IC of MII-tt-DTT on HCT116 cells₅₀The value was 1.957. mu.M (see FIG. 4B).

MII-tt-DTT pairs were determined in the same mannerInhibition of colorectal cancer SW480 cells, colorectal cancer DLD-1 cells, and IC of the same on SW480 cells, DLD-1 cells₅₀The values were 3.249. mu.M, 3.274. mu.M. (see FIGS. 4C-D).

HCT116 cells were seeded at 500 cells/well in 6-well cell culture plates. After the cells are attached to the wall, gradient drug treatment is carried out at 37 ℃ and CO₂After the incubator is used for 48 hours, fresh DMEM complete culture medium is replaced to continue the culture. And after the blank group of cells grow into a certain number of macroscopic clone spots, carrying out crystal violet staining and taking a picture.

As a result: MII-tt-DTT showed significant inhibition of HCT116 cell proliferation (see FIG. 4B).

The inhibition effect of MII-tt-DTT on the proliferation of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells was determined by the same method, and as a result, the MII-tt-DTT significantly inhibited the proliferation of SW480 cells and DLD-1 cells (see FIGS. 4C-D).

Application example 2, Effect of MII-tt-DTT on colorectal cancer subcutaneous tumor-bearing in vivo and toxicity test of tumor-bearing model nude mice

Collecting HCT116 cells in logarithmic growth phase, resuspending in physiological saline to density of 1 × 10⁷Cell suspension per mL. After the skin of the injection site of the nude mouse is disinfected, the cell suspension is injected subcutaneously into the subcutaneous tissue of the right axilla of the nude mouse. 7 days after inoculation, the blank control group, the 5-fluorouracil group and the experimental group were administered with physiological saline, 3.48mg/mL of 5-fluorouracil and 1mg/kg of MII-tt-DTT drug treatment, respectively. Treatment was performed every 3 days for 30 days.

After 30 days later, three groups of nude mice were anesthetized, blood was taken from the eyeballs, and the heart, liver, spleen, lung, kidney and tumor were excised, and the tumor and organs were weighed and recorded. After being treated, the eyeball blood is detected by a special animal analyzer according to the following indexes: lymphocyte (LYM), leukocyte (WBC), Hemoglobin (HGB) and Red Blood Cell (RBC). Liver and kidney index detection according to kit instructions: alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), urea nitrogen (BUN), and Creatinine (CR). The organs were examined for histomorphology.

As a result: MII-tt-DTT did not cause substantial damage to the heart, liver, spleen, lung, and kidney of the mice and significantly inhibited tumor growth, and the mice did not lose weight significantly compared to the control group (see FIG. 5).

Application example 3 Effect of Small molecule inhibitors in combination with MII-tt-DTT treatment on colorectal cancer cell proliferation

HCT116 cells were 3X 10³Perwell inoculation into 96 well plates, 5% CO₂DMEM complete medium with 100U/mL penicillin and 100. mu.g/mL streptomycin was incubated at 37 ℃ for 12 h. A culture medium containing NAC (1mM) was prepared, MII-tt-DTT was diluted with the medium to various concentrations (25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.12. mu.M, 1.56. mu.M, 0.78. mu.M, 0.39. mu.M, 0.195. mu.M, 0.0975. mu.M, respectively), and then the cells were subjected to the administration of 5-fold wells at each concentration, followed by culturing for another 48 hours, discarding the culture solution, and the MTT reagent was used to determine the cell survival rate.

The same method measures the combined action of MII-tt-DTT and VE, Z-VAD-FMK, Necrostatin-1 and Apoptosis on the inhibition of the proliferation of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells, and as a result, the MII-tt-DTT has obvious inhibition effect on the proliferation of SW480 cells and DLD-1 cells (see figure 6).

Application example 4, MII-tt-DTT induces apoptosis in colorectal cancer cells

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. Cells were digested with EDTA-free trypsin and collected in a centrifuge tube, washed 3 times with PBS, and 5. mu.L (2.5. mu.g/ml) of Annexin-V-FITC and 5. mu.L (50. mu.g/ml) of PI (propidium iodide) were added, stained for 15min and then assayed by flow cytometry for levels of apoptosis using FlowJo 7.6.1.

The staining results for Annexin-V-FITC and PI show that: MII-tt-DTT induced apoptosis of HCT116 cells (see FIGS. 7A-D).

The effect of MII-tt-DTT on the apoptosis of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells was determined in the same manner, and the results showed that: MII-tt-DTT induced apoptosis in SW480 cells, DLD-1 cells (see FIGS. 7A-D).

Taking HCT116 cells 1X 10⁶The cells were plated on 60mm cell culture dishes. After gradient drug treatment, CO at 37 deg.C₂Complete culture of incubator DMEMAnd culturing in the medium for 48h, and extracting the protein. Western Blot was used to detect the expression of BaX, Bcl-2, Cytc, and cleared-Cas 3.

The Western Blot detection result shows that: MII-tt-DTT induced the expression of BaX, Cytc, cleared-Cas 3 in HCT116 cells to increase, and Bcl-2 expression to decrease (see FIG. 7E-Q).

The same method measures the effect of MII-tt-DTT on the expression of colorectal cancer SW480 cells, colorectal cancer DLD-1 cells BaX, Bcl-2, Cytc, cleared-Cas 3, and the results show that: MII-tt-DTT induced apoptosis in SW480 cells, DLD-1 cells (see FIGS. 7E-Q).

Application example 5, MII-tt-DTT localizes to mitochondria in colorectal cancer cells and induces mitochondrial damage

Taking HCT116 cells 1X 10³Inoculating the cells in a glass-bottom cell culture dish, administering the cells after the cells adhere to the wall, continuously culturing for 48h, removing the culture medium, adding a Mito-Green staining solution (Mito-Green: complete culture medium is 1:3000) which is incubated in advance into the culture dish, incubating for 40min at 37 ℃, removing the staining solution, washing with NaCl twice, adding a certain amount of fresh culture medium, observing fluorescence by using a laser confocal microscope, and taking pictures. And (5) performing light-shielding treatment in the whole process.

As a result: MII-tt-DTT was able to localize in mitochondria of HCT116 cells (see FIG. 8A).

The same method was used to determine the effect of MII-tt-DTT on mitochondrial co-localization in SW480 and DLD-1 cells from colorectal cancer, and the results showed that MII-tt-DTT was localized in mitochondria in SW480 and DLD-1 cells (see FIG. 8A).

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. Cells were digested with EDTA-free trypsin and collected in a centrifuge tube and lysed on ice by adding 200 μ L of lysis buffer. After cracking, centrifuging at 4 ℃ and 12000g for 5min, and taking the supernatant to be tested. Preparing equivalent ATP detection working solution according to the proportion of the ATP detection reagent and ATP detection reagent diluent being 1:9, adding 100 mu L of ATP detection working solution into detection holes of a 96-well plate, and standing for 5min at room temperature to consume background ATP. Adding 20 μ L of sample into each hole after 5min, mixing with gun, and separating for 2sThe RLU value was determined. And (5) performing light-shielding treatment in the whole process.

As a result: MII-tt-DTT induced an increase in ATP levels in mitochondria in HCT116 cells (see FIG. 8B).

The effect of MII-tt-DTT on mitochondrial ATP levels in SW480 and DLD-1 cells of colorectal cancer was determined in the same manner, and it was shown that MII-tt-DTT induced increased ATP levels in mitochondria in SW480 and DLD-1 cells (see FIGS. 8C-D).

Taking HCT116 cells 1X 10³Inoculating the cells in a 24-hole cell culture dish, performing gradient administration on the cells after the cells are attached to the wall, continuously culturing for 48h, removing the culture medium, washing with PBS once, adding 500 mu L of fresh complete culture medium into each hole, adding 500 mu L of JC-1 dyeing working solution prepared in advance (JC-1 is diluted according to the proportion of adding 8mL of ultrapure water into each 50 mu L of JC-1(200X), then adding 2mL of JC-1 dyeing buffer solution (5X), and uniformly mixing to obtain the JC-1 dyeing working solution) and fully and uniformly mixing. 37 ℃ and CO₂Incubate for 20 min. During the incubation, an appropriate amount of JC-1 staining buffer (1X) was prepared at a rate of 4mL of ultrapure water per 1mL of JC-1 staining buffer (5X), and placed in an ice bath. After the incubation was completed, the supernatant was aspirated, washed 2 times with JC-1 staining buffer (1X), and then 500. mu.L of fresh complete medium was added and observed under a fluorescent microscope.

The observation result of the fluorescence microscope shows that: MII-tt-DTT induced a decrease in mitochondrial membrane potential of HCT116 cells (see FIG. 9).

The effect of MII-tt-DTT on mitochondrial membrane potential of SW480 and DLD-1 cells of colorectal cancer was determined in the same manner, and it was shown that MII-tt-DTT induced a decrease in mitochondrial membrane potential of SW480 and DLD-1 cells (see FIG. 9).

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. (1) Flow cytometry detection: EDTA-free trypsinization and collection of cells in centrifuge tubes, dilution of DCFH-DA to a final concentration of 10. mu. mol/L in serum-free medium, resuspension of cells, CO at 37 ℃₂Incubating for 20min in the incubator, reversing and uniformly mixing once every 3-5 min, and then measuring by using a flow cytometer. (2) And (3) fluorescent microscope detection: removal of cellsThe culture medium was diluted with 1mL of DCFH-DA at 37 ℃ and CO₂After incubation for 20min in the incubator, the fluorescence intensity is detected by using 488nm excitation wavelength and 525nm emission wavelength.

Flow detection and fluorescence detection results show that: MII-tt-DTT induced an increase in ROS levels in HCT116 cells (see FIGS. 10A-B).

The effect of MII-tt-DTT on ROS production levels in SW480 and DLD-1 cells of colorectal cancer was also determined by the same method, and the results showed that MII-tt-DTT induced increased ROS levels in SW480 and DLD-1 cells (see FIGS. 10A-D).

Application example 6, MII-tt-DTT induces iron death in colorectal cancer cells

1. GPX4 pathway

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. Digesting by using EDTA-free trypsin, collecting cells in a centrifuge tube, washing by using PBS for 2 times, adding a reagent with 3 times of cell precipitation volume for resuspending the cells, repeatedly freezing and thawing for 2-3 times, centrifuging for 10min at the temperature of 4 ℃ and 8000g, and collecting supernatant to be tested at the temperature of 4 ℃. 20 mu L of sample, 140 mu L of reagent II and 40 mu L of reagent III are sequentially added into a sample tube, mixed uniformly and stood for 2min to detect the absorbance at 412nm (a blank hole A1 and a sample hole A2), and delta A is A2-A1. The kit is a Solibao GSH content detection kit.

The OD measurement results showed that: MII-tt-DTT induced an increase in GSH levels in HCT116 cells (see FIG. 11A).

The effect of MII-tt-DTT on GSH production levels in SW480 and DLD-1 cells of colorectal cancer was determined in the same manner, and it was shown that MII-tt-DTT induced an increase in GSH levels in SW480 and DLD-1 cells (see FIGS. 11B-C).

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. Digesting by trypsin without EDTA, collecting cells in a centrifuge tube, washing by PBS for 2 times, adding a reagent with 3 times of cell precipitation volume for resuspending cells, repeatedly freezing and thawing for 2-3 times, centrifuging at 4 ℃ and 8000g for 10min, and collecting supernatant to be tested at 4 ℃. Add 10. mu.L of sample to the sample tubeMixing the product with 0.2. mu.L reagent II, and incubating at 37 deg.C for 30 min. After the incubation is finished, 70 mu L of reagent three, 10 mu L of reagent four, 10 mu L of reagent five and 1 mu L of reagent six are sequentially added (the time is started when the reagent six is added), the mixture is rapidly mixed, 30s and 150s absorbances A1 and A2 at 412nm are detected, and delta A is calculated to be A2-A1. The kit is a Solibao GSSG content detection kit.

The OD measurement results showed that: MII-tt-DTT induced an increase in GSSG levels in HCT116 cells (see FIG. 11D).

The effect of MII-tt-DTT on the GSH production levels in SW480 and DLD-1 cells of colorectal cancer was determined in the same manner, and it was shown that MII-tt-DTT induced an increase in GSSG levels in SW480 and DLD-1 cells (see FIGS. 11E-F).

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. EDTA free Trypsin digestion and cell Collection in centrifuge tubes according to cell number (10)⁴Adding a first reagent into the extract at a ratio of 500-1000: 1 (mL), ultrasonically breaking cells (ice bath, 200W, ultrasonic for 3s, interval of 10s, repeated for 30 times), centrifuging at 4 ℃ for 10min at 8000g, and collecting supernatant to be tested at 4 ℃. And sequentially adding 10 mu L of sample, 80 mu L of reagent II and 10 mu L of reagent III into the sample tube, uniformly mixing, standing at room temperature for 15min, and detecting the absorbance value at 600 nm. The kit is a kit for detecting the built cysteine (Cys) in Nanjing.

The OD measurement results showed that: MII-tt-DTT induced a decrease in Cys content in HCT116 cells (see FIG. 11G).

The effect of MII-tt-DTT on Cys production levels in SW480 and DLD-1 cells from colorectal cancer was determined in the same manner, and it was shown that MII-tt-DTT induced a decrease in Cys levels in SW480 and DLD-1 cells (see FIGS. 11H-I).

Taking HCT116 cells 1X 10⁶The cells were plated on 60mm cell culture dishes. After gradient drug treatment, CO at 37 deg.C₂And extracting protein after culturing in a DMEM complete medium in an incubator for 48 hours. Western Blot detects the expression of xCT and GPX 4.

The Western Blot detection result shows that: MII-tt-DTT induced a decrease in the intracellular expression of xCT, GPX4 in HCT116 cells (see FIG. 11J).

The same method measures the effect of MII-tt-DTT on the expression of colorectal cancer SW480 cells, colorectal cancer DLD-1 cells xCT, GPX4, and the results show that: MII-tt-DTT induced a decrease in the expression of xCT, GPX4 in SW480 cells and DLD-1 cells (see FIG. 11J).

To summarize: MII-tt-DTT further induces cell iron death by GPX 4-mediated increases in GSH, GSSG levels and decreases in Cys levels in HCT116 cells.

The effect of MII-tt-DTT on iron death of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells was determined in the same manner, and the results showed that: MII-tt-DTT induced iron death in SW480 cells, DLD-1 cells (see FIGS. 11A-P).

2. TFR1 pathway

Taking HCT116 cells 1X 10⁴Seeded in 6-well cell culture plates. After gradient drug treatment, CO at 37 deg.C₂Incubator DMEM complete medium was cultured for 48 h. Digesting by trypsin without EDTA, collecting cells in a centrifuge tube, washing by PBS for 1 time, centrifuging at 4 ℃ and 3500rpm for 10min, and taking supernatant to be tested. Adding 150 μ L of iron color developing agent into each sample, mixing, boiling in water bath for 5min, cooling with flowing water, 3500rpm/min, centrifuging for 10min, collecting supernatant, and measuring OD value at 520 nm.

The OD measurement results showed that: MII-tt-DTT Induction of Fe in HCT116 cells²⁺/Fe³⁺The content increased (see fig. 12A).

The same method is used for determining MII-tt-DTT Fe in SW480 cells and DLD-1 cells of colorectal cancer²⁺/Fe³⁺The content influence shows that the MII-tt-DTT induces SW480 and DLD-1 cells to be Fe²⁺/Fe³⁺The content was increased (see FIGS. 12B-C).

Taking HCT116 cells 1X 10⁶The cells were plated on 60mm cell culture dishes. After gradient drug treatment, CO at 37 deg.C₂And extracting protein after culturing in a DMEM complete medium in an incubator for 48 hours. Western Blot detects the expression of transferrin receptors TFR1, NCOA 4.

The Western Blot detection result shows that: MII-tt-DTT induced an increase in expression of TFR1 and NCOA4 in HCT116 cells (see FIG. 12M).

The same method was used to determine the effect of MII-tt-DTT on the expression of TFR1 and NCOA4 in colorectal cancer SW480 cells and colorectal cancer DLD-1 cells, showing that: MII-tt-DTT induced an increase in expression of TFR1 and NCOA4 in SW480 cells and DLD-1 cells (see FIG. 12M).

After addition of the iron chelator DFO, intracellular Fe of HCT116, SW480, DLD-1 was again measured according to the above method²⁺/Fe^{3 +}Content, ROS production content and cell viability. The OD measurement results showed that: combined effect of MII-tt-DTT and DFO compared to MII-tt-DTT alone, Fe in HCT116, SW480, DLD-1 cells²⁺/Fe³⁺The content and ROS content are reduced, and the cell activity is increased. (see FIGS. 12D-F)

To summarize: MII-tt-DTT mediates intracellular Fe in HCT116 via TFR1²⁺/Fe³⁺The content and ROS content are increased, and the cell iron death is further induced.

The effect of MII-tt-DTT on iron death of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells was determined in the same manner, and the results showed that: MII-tt-DTT induced pig death in SW480 cells, DLD-1 cells (see FIGS. 12A-M).