阅读说明：本技术 Cy7-Cl及其在制备抗结直肠癌药物中的应用 (Cy7-Cl and application thereof in preparation of anti-colorectal cancer drugs ) 是由 张磊 汤昆 王群 邱娜 崔昆丽 史霄 滕铁山 程小霞 陈明亮 刘楠 贾爽爽 梁 于 2021-09-07 设计创作，主要内容包括：本发明属于化工医药技术领域,本发明涉及1,7-二[N-(3,5-二(2-(2-甲氧乙氧基)乙氧基)乙氧基)苄基-3,3-二甲基-3H-吲哚]-4-氯-3,5-环己烯基三碳菁溴化盐染料及其在制备抗结直肠癌药物中的应用(简称Cy7-Cl)。MTT结果显示：Cy7-Cl显著抑制结直肠癌HCT116细胞、SW480细胞活力。AnnexinV-FITC/PI双染凋亡检测结果显示：Cy7-Cl显著诱导细胞凋亡。本发明的小分子化合物Cy7-Cl作为新的抗结直肠癌药物或者其辅助成分进行开发,抑制结直肠癌效果显著,将为治疗和治愈结直肠癌提供新的途径和手段。(The invention belongs to the technical field of chemical medicines, and relates to a 1, 7-bis [ N- (3, 5-bis (2- (2-methoxyethoxy) ethoxy) benzyl-3, 3-dimethyl-3H-indole ] -4-chloro-3, 5-cyclohexenyl tricarbocyanine bromide dye and application thereof in preparation of anti-colorectal cancer drugs (Cy 7-Cl for short). MTT results show that: cy7-Cl significantly inhibited colorectal cancer HCT116 cell and SW480 cell viability. The annexin V-FITC/PI double-staining apoptosis detection result shows that: cy7-Cl significantly induced apoptosis. The small molecular compound Cy7-Cl is developed as a novel anti-colorectal cancer drug or an auxiliary component thereof, has a remarkable effect of inhibiting colorectal cancer, and provides a novel way and means for treating and curing colorectal cancer.)

1, 7-bis [ N- (3, 5-bis (2- (2-methoxyethoxy) ethoxy) benzyl-3, 3-dimethyl-3H-indole ] -4-chloro-3, 5-cyclohexenyl tricarbocyanine bromide dye, abbreviated as Cy 7-Cl; the chemical structure of the Cy7-Cl is shown below:

2. use of the 1, 7-bis [ N- (3, 5-bis (2- (2-methoxyethoxy) ethoxy) benzyl-3, 3-dimethyl-3H-indole ] -4-chloro-3, 5-cyclohexenyl tricarbocyanine bromide dye of claim 1 in the preparation of a medicament for the treatment of colorectal cancer.

3. The use of Cy7-Cl for the preparation of an anti-colorectal cancer drug according to claim 2, wherein Cy7-Cl inhibits colorectal cancer cell line HCT116 or SW480 cell viability.

4. The use of Cy7-Cl in the preparation of an anti-colorectal cancer drug according to claim 2, wherein the Cy7-Cl is effective in inhibiting the activity of colorectal cancer cells at a concentration of 0.04875-6.25 μ M.

5. Use of Cy7-Cl according to claim 1 for the preparation of an anti-colorectal cancer drug, which Cy7-Cl induces production of reactive oxygen species by colorectal cancer HCT116 cells or SW480 cells.

6. The use of Cy7-Cl according to claim 2 for the preparation of an anti-colorectal cancer drug, wherein the Cy7-Cl is localized in mitochondria of HCT116 cells or SW480 cells of colorectal cancer.

7. Use of Cy7-Cl according to claim 2 for the preparation of a medicament against colorectal cancer, which Cy7-Cl induces mitochondrial pathway apoptosis in colorectal cancer HCT116 cells or SW480 cells.

Technical Field

The invention belongs to the technical field of chemical medicines, and particularly relates to a novel tricarbocyanine dye Cy7-Cl and application thereof in preparation of anti-colorectal cancer medicines.

Background

The incidence and mortality of malignant tumors has increased year by year and has become one of the most serious diseases threatening human health. Colorectal cancer is a common digestive tract malignant tumor in clinic, the occurrence of which is usually related to dietary change and environmental factors, and the morbidity and mortality are high. Colorectal cancer is the fourth leading cause of death worldwide, the third most common cancer in men and the second most common cancer in women, with the second highest mortality rate. Colorectal cancer remains a leading cause of cancer-related death in the world. In China, according to the national cancer statistical data published by the national cancer center in 2019, the incidence rate of colorectal cancer is the third national cancer, accounting for 9.8 percent of the total population of cancer patients, which seriously harms the health of residents in China, and has become a public health problem to be solved urgently. The clinical treatment mode of colorectal cancer mainly comprises surgical treatment, radiotherapy and chemotherapy. The major clinical chemotherapeutic drugs for colorectal cancer include: oxaliplatin, irinotecan and fluorouracil drugs (such as 5-fluorouracil). However, drugs such as oxaliplatin, irinotecan and 5-fluorouracil all have predictable hepatotoxicity, so that the clinical application of the drugs is limited. Therefore, the development of a colorectal cancer treatment drug with high efficiency and low toxicity is imminent.

At present, the heptamethine cyanine dye is widely researched and applied to tumor diagnosis, chemotherapy, photodynamic therapy and photothermal therapy in the field of tumor diagnosis and treatment. Heptamethine cyanine dyes themselves have low cytotoxicity, and thus, in chemotherapy research, cyanine molecules often act as indicators in chemotherapeutic drug delivery systems or by photothermal or photodynamic therapy. The Shi topic group reports the research on tumor treatment by using a series of heptamethine cyanine compounds modified by benzene rings, multiple carbon chains, carboxyl groups and the like through a photothermal photodynamic principle, the series of compounds are different from the compound modified structure of the invention, and the compound Cy7-Cl of the invention can induce colorectal cancer cell apoptosis without the photothermal and photodynamic effects. The Ja-Hyoung Ryu subject group obtains a high molecular material for treating tumors by applying photodynamic through assembling a multi-carbon chain modified heptamethine cyanine compound and hyaluronic acid. However, the nanomaterial-based multifunctional photosensitizer is faced with many problems such as stable large-scale preparation process, mostly absorbed and retained in reticuloendothelial organs, long-term toxicity, etc., resulting in that most nanomaterial-based multifunctional photosensitizers remain in the early development stage. Therefore, the development of the chemical micromolecule heptamethine cyanine compound which does not depend on a nanometer high molecular material and does not need complex chemical connection but is based on the inherent multifunctional characteristics of the chemical structure of the compound is expected to avoid the problems, greatly improves the potential of clinical transformation application, and promotes and expands the prospect of tumor optical treatment.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel cyanine dye Cy7-Cl and a novel application thereof as a medicine, in particular to an application of Cy7-Cl in preparation of an anti-colon cancer medicine.

In order to achieve the purpose, the invention adopts the following technical scheme: the chemical structure of the Cy7-Cl is shown below:

the relevant properties are as follows:

chemical name: 1, 7-bis [ N- (3, 5-bis (2- (2-methoxyethoxy) ethoxy) benzyl-3, 3-dimethyl-3H-indole ] -4-chloro-3, 5-cyclohexenyl tricarbocyanine bromide dye, abbreviated as Cy 7-Cl.

The molecular formula is as follows: c₇₂H₁₀₀O₁₆N₂ClBr; molecular weight: 1364.9257, respectively; the detection mode is as follows:¹HNMR,¹³c NMR, HRMS; the characteristics are as follows: the product is light blue powder; the source is as follows: synthesized in the laboratory. Pharmacological properties: insoluble in water and soluble in DMSO.

Further, the concentration range of Cy7-Cl for inhibiting colorectal cancer cells is 0.04875-6.25 μ M.

The invention provides a method for inhibiting the activity of colorectal cancer cells in vitro, which comprises the steps of adding Cy7-Cl into a culture solution of tumor cells, and adding Cy7-Cl into the culture solution to obtain final concentrations of 0.04875 mu M, 0.0975 mu M, 0.195 mu M, 0.39 mu M, 0.78 mu M, 1.56 mu M, 3.12 mu M and 6.25 mu M.

Flow cytometry and fluorescence microscopy results showed that Cy7-Cl induced the production of active oxygen in colorectal cancer HCT116 cells, SW480 cells, by adding Cy7-Cl to the culture broth of tumor cells at final concentrations of Cy7-Cl of 0.195. mu.M, 0.78. mu.M, 3.12. mu.M, respectively; laser confocal results showed that Cy7-Cl was localized to mitochondria. Therefore, the invention discloses a method for locating mitochondria, which adds Cy7-Cl into a culture solution of tumor cells, and the final concentration of the added Cy7-Cl is 0.78 mu M.

The invention also provides a method for inducing colorectal cancer cell apoptosis in vitro, which comprises the steps of adding Cy7-Cl into a culture solution of tumor cells, wherein the final concentrations of the Cy7-Cl are respectively 0.195 mu M, 0.78 mu M and 3.12 mu M.

The rectal cancer cell can be colorectal cancer HCT116 cell and SW480 cell.

The invention also provides an anti-colorectal cancer medicament, the active component of the anti-colorectal cancer medicament is Cy7-Cl, and the synthetic route is as follows:

the invention has the beneficial effects.

The Cy7-Cl synthesized by the invention is a cyanine derivative, most of the compounds play an anti-tumor role through photothermal photodynamic therapy at present, and Cy7-Cl can play an anti-tumor role without photothermal photodynamic. Particularly, the invention provides application of Cy7-Cl in preparation of anti-colorectal cancer drugs. MTT results show that: cy7-Cl significantly inhibited the proliferation of colorectal cancer cells and produced reactive oxygen species and localized to the mitochondria. The annexin V-FITC/PI double-staining apoptosis detection result shows that: cy7-Cl induced apoptosis in colorectal cancer cells. The small molecular compound Cy7-Cl is developed as a new anti-colorectal cancer drug or an auxiliary component thereof, has a remarkable effect of inhibiting colorectal cancer, and provides a new treatment way and means for treating and curing tumors.

Drawings

FIG. 1.Cy7-Cl structural identification¹H NMR(400MHz,DMSO-d₆) And (4) map data.

FIG. 2 structural identification of Cy7-Cl¹³C NMR(400MHz,DMSO-d₆) And (4) map data.

FIG. 3 Cy7-Cl structural identification HRMS (ESI) profile data.

FIG. 4. assay of Cy7-Cl for inhibition of colorectal cancer HCT116 (panels A, C) and SW480 (panels B, D) cell viability; the E diagram shows the inhibitory activity of positive control CY5-PY-1 on SW480 cells. P < 0.05; p < 0.01; p < 0.001.

FIG. 5. measurement of the absence of significant effect of Cy7-Cl on the viability of colorectal cancer HCT116 (panel A) and SW480 (panel B) cells in combination with laser irradiation, and measurement of the absence of significant effect of laser irradiation on Cy7-Cl by extracellular photothermal assay (panel C).

FIG. 6 measurement of the effect of the small molecule inhibitors Ferrostatin (iron death inhibitor) (panels A, B), Nerostatin (necrosis inhibitor) (panels C, D), N-acetylcysteine (antioxidant) (panels E, F), Z-VAD-FMK (apoptosis inhibitor) (panels G, H) in combination with Cy7-Cl on the viability of colorectal cancer cells HCT116 and SW 480. Assay of the combination of N-acetylcysteine, Z-VAD-FMK and Cy7-Cl to promote proliferation of HCT116 (panel E, G) and SW480 (panel F, H) cells. P < 0.05; p < 0.01; p < 0.001.

FIG. 7. assay of Cy7-Cl to induce localization of colorectal cancer cells HCT116 (panel A) and SW480 (panel B) into mitochondria. P < 0.05; p < 0.01; p < 0.001.

FIG. 8 measurement of reactive oxygen species production by Cy7-Cl induced colorectal cancer HCT116 (panels A, C) and SW480 (panels B, D) cells. P < 0.05; p < 0.01; p < 0.001.

FIG. 9 Cy7-Cl induced a decrease in mitochondrial membrane potential in HCT116 (panel A) and SW480 (panel B) cells.

FIG. 10 measurement of the decrease of ATP levels induced by Cy7-Cl in cells of HCT116 (panel A) and SW480 (panel B) of colorectal cancer. P < 0.05; p < 0.01; p < 0.001.

FIG. 11 assay of Cy7-Cl to induce apoptosis in colorectal cancer HCT116 (panel A) and SW480 (panel B). P < 0.05; p < 0.01; p < 0.001.

FIG. 12 measurement of up-regulation of pro-and anti-apoptotic protein expression by Cy7-Cl (panels A, B).

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:

step (1) synthesis of N- (3, 5-bis (2- (2-methoxyethoxy) ethoxy) benzyl-2, 3, 3-trimethyl-3H-indole bromide salt (3).

A100 mL two-neck round-bottom flask was charged with Compound 1(1.95g, 12.27mmol, 1.25eq), Compound 2(4.86g, 9.81mmol, 1.0eq) and acetonitrile 40mL, stirred for 10min, and reacted at 60 ℃ under argon protectionAfter 72h, TLC monitoring reaction is finished, acetonitrile is removed by rotary evaporation, and red viscous liquid is obtained by vacuum drying. And (3) separating the crude product by column chromatography (200-mesh silica gel 300 meshes, mobile phase: methanol: dichloromethane: 1:20) to obtain a red solid compound 3: 3.71g, yield: 79.3 percent.¹H NMR(400MHz,DMSO-d6)δ7.20(d,J＝1.6Hz,1H),7.04(dd,J＝1.6Hz,8.4Hz,1H),6.72(dd,J＝1.6Hz,8.4Hz,1H),6.61(d,J＝8.4Hz,1H),6.37(m,3H),4.67(s,2H),4.01-3.99(m,4H),3.91-3.87(m,2H),3.69-3.67(m,4H),3.55-3.48(m,12H),3.42-3.39(m,4H),3.22(s,6H),1.32(s,6H)。

Step (2) Synthesis of 1, 7-bis [ N- (3, 5-bis (2- (2-methoxyethoxy) ethoxy) benzyl-3, 3-dimethyl-3H-indole ] -4-chloro-3, 5-cyclohexenyl tricarbocyanine bromide dye (Cy 7-Cl for short)

Placing the compound 3(781.6mg,1.36mmol,2.0eq), the compound 4(117.4mg,0.68mmol,1.0eq) and sodium acetate (167.3mg,2.04mmol,3.0eq) in a 100mL Schlenk bottle, adding 40mL of acetic anhydride, heating to 100 ℃ under the protection of argon, reacting for 4h, monitoring the reaction by TLC, cooling to room temperature, removing the solvent by rotary evaporation to obtain a crude product, and separating by column chromatography (300-400-mesh silica gel, mobile phase: methanol: dichloromethane: 1:15) to obtain a green solid compound, namely cy 7-cl: 399.1mg, yield: and 43 percent.¹H NMR(400MHz,DMSO-d₆)δ8.28-8.24(d,J＝14Hz,2H),7.68-7.66(d,J＝7.6Hz,2H),7.42-7.41(m,4H),7.33-7.28(m,2H),6.49-6.48(m,2H),6.43-6.40(m,6H),5.43(s,4H),4.04-4.01(m,8H),3.69-3.67(m,8H),3.54-3.46(m,24H),3.40-3.37(m,8H),3.20(s,12H),2.58-2.57(m,4H),1.78-1.76(m,2H),1.71(s,12H)；¹³C NMR(100MHz,DMSO-d₆)δ172.8,160.0,148.7,143.4,142.3,140.8,137.3,128.7,126.9,125.4,122.6,111.7,105.5,102.3,100.0,71.2,69.9,69.8,69.6,68.8,67.3,58.0,49.1,48.5,47.1,27.6,25.8,21.1,20.2；HRMS(ESI)：calcd for C₇₁H₁₀₀ClN₂O₁₆Br[M-Br]⁺1283.6761，found[M-Br]⁺1283.0201，[M-Br+Na]²⁺653.9965。

The Cy7-Cl structure identification map data are shown in figure 1, figure 2 and figure 3.

Cy7-Cl was dissolved in DMSO and prepared as a 50mM stock solution for use.

Growth inhibition of Cy7-Cl on colorectal cancer cells was determined using example 1, MTT method and cell cloning experiments

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% CO₂100U/mL penicillin and 100. mu.g/mL streptomycin in RPMI1640 complete medium was cultured at 37 ℃ for 12 hours, Cy7-Cl was added at different concentrations (0.04875. mu.M, 0.0975. mu.M, 0.195. mu.M, 0.39. mu.M, 0.78. mu.M, 1.56. mu.M, 3.12. mu.M, 6.25. mu.M, respectively), and at each concentration, 5-fold wells were set, and culture was continued for 48 hours, and the culture solution was discarded, and the cell viability was measured by MTT reagent.

The determination method comprises the following steps: adding a pre-prepared MTT reaction solution into 15 mu L/well, continuously culturing for 4h, sucking and discarding supernatant, adding DMSO into 100 mu L/well to dissolve the reduction product, reading absorbance value at 490nm wavelength, calculating cell viability, determining Cy7-Cl intervention well absorbance value/control well absorbance value as cell viability, and calculating IC of Cy7-Cl for colorectal cancer HCT116 cells and SW480 cells₅₀The value is obtained.

IC₅₀Refers to the concentration of inhibitor at which cell growth is inhibited by half. This is the concentration of Cy7-Cl when the number of HCT116 cells was half of that of the control group.

As a result: IC of Cy7-Cl for colorectal cancer HCT116 cells₅₀The value was 0.8178. mu.M (FIG. 4A).

The inhibitory effect of Cy7-Cl on SW480 cells was measured in the same manner, and as a result, IC of SW480 cells was measured₅₀The value was 0.941. mu.M (FIG. 4B).

500 HCT116 cells per well were inoculated into 6 wells of cells and cultured in RPMI1640 complete medium at 37 ℃ in 5% CO2 for 24-48 hours, and then subjected to gradient drug treatment at drug concentrations of 0.195. mu.M, 0.39. mu.M, 0.78. mu.M, 1.56. mu.M, and 3.12. mu.M, respectively. After 48h of drug treatment, replacing a normal complete culture medium for continuous culture, sucking out the culture medium after a blank group grows into a certain number of macroscopic bacteria, washing for 1 time by PBS, adding 1ml of 4% paraformaldehyde into each hole for fixing for 30-60min, and washing for 1 time by PBS; adding 1ml of 1% crystal violet dye solution into each hole, and dyeing for 10-20 min; PBS washed cells several times, dried, photographed by digital camera, and the results showed: cy7-Cl significantly inhibited the proliferation of HCT116 cells (fig. 4C).

The effect of Cy7-Cl on SW480 cells was determined in the same manner, and the results showed that: cy7-Cl significantly inhibited the proliferation of SW480 cells (fig. 4D).

Next, the inhibitory activity of CY5-PY-1 (structural formula shown below) having a structure similar to that of Cy7-Cl on SW480 cells was further verified, and as a result, IC on SW480 cells was confirmed₅₀The value was 21.3 μm, and it was found that the inhibitory activity of the compound of the present invention against colon cancer cells was improved by 20 times or more, specifically, see fig. 4E.

Application example 2, MTT method to determine the Effect of laser irradiation and Cy7-Cl combination on colorectal cancer cell growth

Referring to application example 1, a 96-well plate is paved at intervals, and laser irradiation is carried out after the 96-well plate is added with medicine and cultured for 24h under the irradiation conditions of 1W, 5min and 808 nm. The effect of Cy7-Cl on colorectal cancer HCT116 cells was determined using the MTT method after 24h laser irradiation, and the results show that: the combined effect of laser irradiation and Cy7-Cl had no significant effect on HCT116 cell viability (fig. 5A).

The effect of Cy7-Cl on SW480 cells was determined in the same manner, and the results showed that: the combined effect of laser irradiation and Cy7-Cl had no significant effect on SW480 cell viability (fig. 5B).

Application example 3 measurement of the Effect of laser irradiation on Cy7-Cl by the extracellular photothermal method

Cy7-Cl was added to a culture solution of tumor cells, 200. mu.L of a Cy 7-Cl-containing medium was added to a 96-well plate, followed by laser irradiation under conditions of 1W, 5min and 808nm, at a final concentration of 0.04875-6.25. mu.M of Cy 7-Cl: photothermal did not have a significant effect on Cy7-Cl (fig. 5C).

Application example 4 MTT method to determine the influence of small molecule inhibitors Ferrostatin (iron death inhibitor), Nerostatin (necrosis inhibitor), N-acetylcysteine (antioxidant), Z-VAD-FMK (apoptosis inhibitor) on Cy7-Cl inhibition of colorectal cancer cell viability

Referring to application example 1, a certain amount of inhibitor-containing medium was prepared before adding drugs, different concentrations of Cy7-Cl were added to the inhibitor-containing medium, 5 wells were set for each concentration, the culture was continued for 48 hours, the culture solution was discarded, and the cell viability was measured using MTT reagent, and the results showed: ferrostatin and Necrostatin combined with Cy7-Cl had no significant effect on the proliferation potency of HCT116 cells (FIGS. 6A, C); n-acetylcysteine, Z-VAD-FMK in combination with Cy7-Cl significantly promoted proliferation of HCT116 cells (FIG. 6E, G).

The effect of various small molecule inhibitors on SW480 cells in combination with Cy7-Cl was determined in the same manner, and the results showed that: ferrostatin and Necrostatin in combination with Cy7-Cl had no significant effect on the proliferative capacity of HCT116 cells (FIG. 6B, D). N-acetylcysteine, Z-VAD-FMK in combination with Cy7-Cl significantly promoted SW480 cell proliferation (FIG. 6F, H).

Application example 5, Cy7-Cl, induces localization of colorectal cells to mitochondria

Taking HCT116 cells 1X 10⁴Inoculating in glass-bottom culture dish, and applying 5% CO₂100U/mL penicillin and 100. mu.g/mL streptomycin in RPMI1640 complete medium at 37 ℃ for 12 hours, 0.78. mu.M Cy7-Cl was added, and the culture was continued for 48 hours. According to the following steps of 1: 10000 of Mito-Tracker Green working solution, incubating the working solution at 37 ℃ for 5-10min, discarding the culture solution, washing with NaCl once, adding the working solution, incubating at 37 ℃ for 15-45min, sucking off waste liquid after the incubation is finished, washing with NaCl once, adding serum-free RPMI1640, and observing and photographing on a computer, wherein the result shows that: cy7-Cl induced localization of HCT116 cells into mitochondria (FIG. 7A).

The effect of Cy7-Cl on SW480 cell localization was observed in the same manner, and the results showed that: cy7-Cl induced SW480 cells to localize to mitochondria (FIG. 7B).

Application of example 6, fluorescence microscopy and flow cytometry to determine the Effect of Cy7-Cl on the production of reactive oxygen species in colorectal cancer cells

HCT116 cells were 2.5X 10⁵Perwell inoculation into 24 well plates, 5% CO₂100U/mL penicillin and 100. mu.g/mL streptomycin in RPMI1640 complete medium at 37 ℃ for 12 hours, Cy7-Cl was added at different concentrations (0.195. mu.M, 0.78. mu.M, and 3.12. mu.M, respectively), the culture was continued for 48 hours, the culture solution was discarded, and the ratio of the concentration of penicillin to the concentration of streptomycin in the mixture was adjusted to 1: preparing a dyeing solution according to the proportion of 1000, adding the dyeing solution, and then placing 3Incubating in a cell incubator at 7 ℃ for about 40min, washing with PBS for 2 times after the incubation is finished, adding a little serum-free RPMI1640, and carrying out on a computer for observation and photographing, wherein the result shows that: cy7-Cl significantly induced an increase in reactive oxygen levels in HCT116 cells (FIG. 8A).

The effect of Cy7-Cl on the reactive oxygen species production by SW480 cells was also observed, and the results showed that: cy7-Cl significantly induced an increase in SW480 cellular reactive oxygen species levels (FIG. 8B).

HCT116 cells were 1X 10⁵Perwell inoculation into 6 well plates, 5% CO₂RPMI1640 complete medium of 100U/mL penicillin and 100 μ g/mL streptomycin was cultured at 37 ℃ for 12h, different concentrations (0.195 μ M, 0.78 μ M, 3.12 μ M, respectively) of Cy7-Cl were added, culture was continued for 48h, the culture solution was discarded, the cells were digested with EDTA-free trypsin and collected in a centrifuge tube, washed 3 times with PBS, and the waste solution was discarded according to 1: preparing a staining solution according to the proportion of 1000, adding the staining solution, incubating in a cell culture box at 37 ℃ for about 40min, and blowing and beating the cells upside down every 5 min. After incubation, centrifugation is carried out, PBS is washed for 2 times, and flow cytometry is used for detection, so that the result shows that: cy7-Cl significantly induced an increase in reactive oxygen levels in HCT116 cells (FIG. 8C).

The same procedure measures the effect of Cy7-Cl on ROS production by SW480 cells and shows that: cy7-Cl significantly induced an increase in SW480 cellular reactive oxygen species levels (FIG. 8D).

Application example 7, JC-1 staining method to measurement of Cy7-Cl induced mitochondrial Membrane potential drop

Referring to application example 6, paving a 24-hole plate and adding medicine, preparing 4mL JC-1 dyeing working solution, specifically: 10. mu.L JC-1(200 mL) +1.6mL ultrapure water +0.4mL JC-1 staining buffer (5 mL). The waste liquid is sucked off, washed once with NaCl, 500. mu.L JC-1 working solution is added into each hole, and the incubation is carried out for about 40min at 37 ℃. 5mL of staining buffer was prepared, specifically: 1mL JC-1 staining buffer +4mL ultrapure water. After incubation, the waste was aspirated and washed 2 times with JC-1 staining buffer. After washing, adding 500 μ L complete culture medium into each well, keeping out of the sun, operating the machine to observe and take pictures, and displaying the result: cy7-Cl significantly induced a decrease in mitochondrial membrane potential of HCT116 cells (FIG. 9A).

The JC-1 fluorescent probe can quickly and sensitively detect the mitochondrial membrane potential change. When the mitochondrial membrane potential is high, JC-1 is gathered in a matrix of mitochondria to form a polymer which can generate red fluorescence; when the mitochondrial membrane potential is low, JC-1 does not aggregate in the matrix of mitochondria, and then JC-1 is a monomer and can generate green fluorescence. This makes it very convenient to detect changes in mitochondrial membrane potential through the conversion of fluorescence color. The decrease in cell membrane potential can be readily detected by the transition of JC-1 from red fluorescence to green fluorescence.

The effect of Cy7-Cl on the mitochondrial membrane potential of SW480 cells was observed in the same manner, and the results showed that: cy7-Cl significantly induced a decrease in the mitochondrial membrane potential of SW480 cells (FIG. 9B).

Effect of application example 8, Cy7-Cl on ATP production by colorectal cancer cells

Referring to application example 6, 6-well plates were placed, the cells were added, 200. mu.L of the extract was added to each EP tube, and the cells were lysed. After lysis, centrifugation is carried out at 4 ℃ and 12000g for 5min, the supernatant is taken and dissolved in ice bath for standby application, 100 μ L of ATP detection working solution is required for each sample, and ATP detection reagent diluent and ATP detection reagent are mixed according to the proportion of 1: 9. After 100 mul of detection working solution is added into a septal hole of a 96-pore plate, standing for 5min at room temperature, adding 20 mul of sample into each hole, detecting by a multifunctional microplate reader, and displaying the result: cy7-Cl significantly induced a decrease in ATP levels in HCT116 cells (FIG. 10A).

ATP levels decrease during apoptosis, indicating impaired or decreased mitochondrial function, usually simultaneously with a decrease in mitochondrial membrane potential.

The effect of Cy7-Cl on ATP production by SW480 cells was determined in the same manner, and the results showed that: cy7-Cl significantly induced a decrease in ATP levels in SW480 cells (FIG. 10B).

Application example 9, annexin V-FITC/PI double staining assay Cy7-Cl induces apoptosis in colorectal cancer cells

Referring to application example 6, 6-well plates were plated, and the plates were incubated at 37 ℃ in 5% CO2 incubator RPMI1640 complete medium for 48 hours. Cells were digested with EDTA-free trypsin and collected in a centrifuge tube, washed 3 times with PBS, and 500. mu.L Binding Buffer, 5. mu.L (2.5. mu.g/ml) Annexin-V-FITC and 5. mu.L (50. mu.g/ml) PI (propidium iodide) were added, respectively, carefully protected from light, and stained for 15min before measurement with a flow cytometer. The staining results for Annexin-V-FITC and PI show that: cy7-Cl significantly induced apoptosis of HCT116 cells (fig. 11A).

The effect of Cy7-Cl on SW480 apoptosis was determined in the same manner, and the results showed that: cy7-Cl significantly induced SW480 apoptosis (fig. 11B).

Application example 10, Western blot method to determine the Effect of Cy7-Cl on apoptosis-related protein expression

Preparing glue; sampling; electrophoresis, membrane conversion constant current 300mA, 1.5 h; sealing the PVDF membrane shaking table by using skim milk and incubating for 1 h; immersing the PVDF membrane in a primary antibody by using a hybridization bag, and incubating overnight at 4 ℃; TBST washing for 3 times, each time for 10 min; incubating the secondary antibody for 1h in a shaking table at room temperature; TBST washing for 3 times, each time for 10 min; and finally, carrying out protein detection. The results show that: cy7-Cl regulates the expression of HCT116 apoptosis-related proteins, such as up-regulating the expression of pro-apoptotic proteins Bax, Cytc, and clear-caspase 3, and down-regulating the expression of anti-apoptotic protein BCL-2, indicating that Cy7-Cl significantly induces HCT116 apoptosis (FIG. 12).

The effect of Cy7-Cl on SW480 apoptosis was determined in the same manner, and the results showed that: cy7-Cl regulates the expression of SW480 apoptosis-related proteins, for example, up-regulating the expression of pro-apoptotic proteins Bax, Cytc and clear-caspase 3 and down-regulating the expression of anti-apoptotic protein BCL-2, indicating that Cy7-Cl significantly induces SW480 apoptosis (FIG. 12).