CN107213153B

CN107213153B - Application of zoledronic acid compound in preparation of medicine for treating cancer

Info

Publication number: CN107213153B
Application number: CN201710448499.6A
Authority: CN
Inventors: 彭莹; 林建国; 邱玲; 李珂; 吕高超; 徐栋; 刘清竹
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2017-06-14
Filing date: 2017-06-14
Publication date: 2020-09-08
Anticipated expiration: 2037-06-14
Also published as: CN107213153A

Abstract

The invention provides an application of a zoledronic acid compound in preparing a medicine for treating cancer, wherein the zoledronic acid compound can be used for treating cancer, can be widely used for treating breast cancer, neuroblastoma, osteosarcoma, prostatic cancer, liver cancer, gastric cancer and colon cancer, and particularly has a remarkable effect in treating colon cancer, and the IC50 of 72h of the existing zoledronic acid (ZOL) acting on HCT116 cells is 25.8 mu M measured, and the zoledronic acid compound (M) disclosed by the invention₄IDP) had an IC50 of 0.98. mu.M on HCT116 cells for 72h, compared to an IC50 of ZOL which is M₄IC50 of IDP 26.3 times, M₄IDP has a more potent cytotoxic effect on HCT116 cells than ZOL and has a lower IC50 value, M₄IDP has great potential to be exploited for the treatment of colon cancer.

Description

Application of zoledronic acid compound in preparation of medicine for treating cancer

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to an application of a zoledronic acid compound in preparation of a cancer treatment drug.

Background

Bisphosphonates (BPs) are organic analogs of inorganic pyrophosphate (PPi) which are organic compounds characterized by two P-C bonds to the same carbon atom. By varying the substituents attached to the carbon atoms, various BPs have been developed. Among them, zoledronic acid (ZOL) is a third generation bisphosphonate, and to carbon atoms are attached a hydroxyl group and an imidazole ring containing 2 nitrogen atoms (FIG. 22).

The zoledronic acid and other bisphosphonates can be specifically combined with hydroxyapatite in bone, have strong bone resorption resisting effect, inhibit actin formation by directly changing the shape of osteoclast, interfere the absorption and reabsorption of osteoclast to bone, also inhibit the release of peptide in bone matrix, break the vicious circle called 'seed and soil mechanism' between tumor and bone formation, and inhibit the growth of tumor cells. Therefore, the zoledronic acid can be widely used for treating osteoporosis, osteitis deformans, hypercalcemia, tumor-related bone diseases and the like in clinic. Recent research shows that zoledronic acid has direct antiproliferative and proapoptotic activity on various cancer cells such as brain glioma, liver cancer, prostate cancer, breast cancer and the like, so that zoledronic acid is expected to be a new weapon for treating the cancers.

Colorectal cancer (CRC) is a cancer originating in the colon or rectum, and is also the third most common cancer in men and the second most common cancer in women worldwide. Colorectal cancer is endemic, with almost 55% occurring in more developed regions. With the development of social economy and the change of life style of people, the incidence rate of colorectal cancer in China is on a rapid rising trend. Colorectal cancer ranks fourth in cancer mortality worldwide, and current therapies often fail to eliminate potential metastatic foci, and new therapies remain to be developed. Some studies have shown that zoledronic acid inhibits colorectal cancer cell proliferation and causes apoptosis, an effect that has also been demonstrated in mouse transplanted tumor models. However, because the research on the aspect is very few, the inhibition effect and the potential mechanism of the bisphosphonate such as zoledronic acid on the growth of colorectal cancer cells are not well researched, and because the molecular weight (Mr 272) of zoledronic acid is small, the influence of the structure is large, and small differences in the structure can generate large influence on the drug effect of the bisphosphonate such as zoledronic acid, the drug effect result of the bisphosphonate such as zoledronic acid on cancer cells is difficult to predict, the optimization of the structure of the bisphosphonate such as zoledronic acid and the further discovery of high-efficiency and low-toxicity drugs have great challenge and practical significance, and the systematic screening of pharmacodynamics has important value. However, the treatment of cancer with the zoledronic acid compound 1-hydroxy-2- (4-methyl-1H-imidazolyl) -ethane-1, 1-bisphosphonic acid has not been disclosed in the prior artThe related reports of the symptoms, such as synthesis of 4-methylimidazole bisphosphate and the like⁹⁹Tc^mThe zoledronic acid compound 1-hydroxy-2- (4-methyl-1H-imidazolyl) -ethane-1, 1-diphosphonic acid (M) disclosed in marker₄IDP) is used only for⁹⁹Tc^mMarking, not proposing M₄Whether IDP has an anticancer effect or not, let alone the intensive studies on its inhibitory effect on cancer cells and its potential mechanism, and therefore on bisphosphonates such as M mentioned above₄The use of IDP in the treatment of cancer, such as colon cancer, still requires further development and investigation.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide the application of the zoledronic acid compound in preparing the medicine for treating the cancer.

Therefore, the invention provides the application of the zoledronic acid compound in preparing the medicine for treating the cancer; the zoledronic acid compound has a structure shown in a formula (I):

the use of the compound, wherein the cancer comprises breast cancer, neuroblastoma, osteosarcoma, prostate cancer, liver cancer, stomach cancer and colon cancer.

The medicine for treating cancer comprises medicines for resisting human breast cancer cells MDA-MB-231, neuroblastoma cells SH-SY5Y, osteosarcoma cells U2OS, prostate cancer cells PC-3, liver cancer cells SMMC-7721, stomach cancer cells SGC-7901 and colon cancer cells HCT 116.

The invention also provides an anticancer drug which takes the compound shown in the formula (I) as an effective component.

The anticancer drug is a clinically acceptable preparation prepared by taking the compound shown in the formula (I) as an active ingredient and selectively adding conventional auxiliary materials according to a conventional process.

The preparation of the anticancer drug comprises tablets, capsules, granules, syrups, powders, pills, tinctures, vinum, soft extracts, freeze-dried powders, pastilles or mixtures.

The technical scheme of the invention has the following advantages:

1. the invention provides an application of a zoledronic acid compound in preparing a medicine for treating cancer, wherein the zoledronic acid compound can be used for treating cancer, and has an inhibiting effect on the growth of human breast cancer cells MDA-MB-231, neuroblastoma cells SH-SY5Y, osteosarcoma cells U2OS, prostate cancer cells PC-3, liver cancer cells SMMC-7721, stomach cancer cells SGC-7901 and colon cancer cells HCT116, so that the zoledronic acid compound can be widely used for treating breast cancer, neuroblastoma, osteosarcoma, prostate cancer, liver cancer, stomach cancer and colon cancer, particularly has a remarkable effect on the aspect of treating colon cancer, and the IC50 of the existing zoledronic acid (ZOL) which acts on HCT116 cells for 72h is measured to be 25.8 mu M, and the zoledronic acid compound (M39M) disclosed by the invention can be used for treating cancer₄IDP) had an IC50 of 0.98. mu.M on HCT116 cells for 72h, compared to an IC50 of ZOL which is M₄IC50 of IDP 26.3 times, M₄IDP has a more potent cytotoxic effect on HCT116 cells than ZOL and has a lower IC50 value, M₄IDP has great potential to be developed and applied to the treatment of colon cancer, and is verified to be M₄When the concentration of IDP is up to 5 mu M, the cell activity of human normal liver cells LO2 is not obviously influenced, and when M is used, the cell activity is not influenced₄When the concentration of IDP is as high as 2.5 mu M, the cell activity of human normal gastric mucosa cell GES-1 is not obviously influenced, which shows that M₄IDP has good selective inhibition effect on human colon cancer cell HCT116, M₄IDP is hopefully developed into a high-efficiency and low-toxicity medicament for treating human colon cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a scheme showing the preparation of 2- (4-methyl-1H-imidazolyl) acetic acid, a compound prepared in example 1 of the present invention¹An H-NMR spectrum;

FIG. 2 is a diagram of the preparation of 1-hydroxy-2- (4-methyl-1H-imidazolyl) -ethane-1, 1-bisphosphonic acid, compound prepared in example 1 of the present invention¹An H-NMR spectrum;

FIG. 3 shows M in Experimental example 1 of the present invention₄FIG. 2 shows the results of experiments in which IDP inhibited MDA-MB-231, SH-SY5Y, U2OS, PC-3, SMMC-7721, SGC-7901 and HCT116 cell proliferation;

FIG. 4 shows M in Experimental example 2 of the present invention₄CCK-8 analysis result chart of HCT116 cell proliferation inhibited by IDP with different action time; results are expressed as mean ± SD,. p<0.05，**p<0.01, compared to respective controls;

FIG. 5 shows different concentrations M in Experimental example 2 of the present invention₄CCK-8 assay of IDP or ZOL on HCT116 cells for 72 h; results are expressed as mean ± SD,. star.p<0.01；

FIG. 6 shows different concentrations M in Experimental example 2 of the present invention₄The result of CCK-8 analysis of the effect of IDP on HCT116, LO2 and GES-1 cells for 72 h; results are expressed as mean ± SD,. star.p<0.01, with the same concentration M₄Comparing the cell viability of HCT116 cells under IDP;^##p<0.01, compared to respective controls;

FIG. 7 is a graph showing the results of a colony formation experiment in Experimental example 2 of the present invention;

FIG. 8 is a histogram of the colony formation experiment in Experimental example 2 of the present invention; p <0.01, compared to control;

FIG. 9A shows M in Experimental example 3 of the present invention₄A flow cytometry analysis result graph of the cell cycle when IDP acts for 0 h;

FIG. 9B shows M in Experimental example 3 of the present invention₄A flow cytometry analysis result graph of the cell cycle when IDP acts for 24 h;

FIG. 9C shows M in Experimental example 3 of the present invention₄A flow cytometry analysis result graph of the cell cycle when IDP acts for 48 hours;

FIG. 9D shows M in Experimental example 3 of the present invention₄A flow cytometry analysis result graph of the cell cycle when IDP acts for 72 h;

FIG. 10 shows M in Experimental example 3 of the present invention₄The distribution ratio of the cell population in G1 and S, G2/M phases of different action time of IDP results; results are expressed as mean ± SD,. p<0.05，**p<0.01, compared to respective controls;

FIG. 11 shows M in Experimental example 3 of the present invention₄The effect of IDP on HCT116 cell cycle-related protein expression;

FIG. 12 shows the results of the Annexin V/PI double staining assay in Experimental example 3 of the present invention, wherein bar is 50 μm;

FIG. 13 shows the results of Hoechst33258 staining analysis in Experimental example 3 of the present invention;

FIG. 14 shows M in Experimental example 3 of the present invention₄The effect of IDP on the expression of apoptosis-related proteins cleared PARP and active-caspase 3;

FIG. 15 shows M in Experimental example 3 of the present invention₄Experimental results for IDP promotion of ROS accumulation in HCT116 cells; results are expressed as mean ± SD,. p<0.05，**p<0.01, compared to a control;

FIG. 16 shows M in Experimental example 3 of the present invention₄The result of the influence of IDP on the expression of the endoplasmic reticulum stress related marker protein;

FIG. 17 shows the results of MDC staining test in Experimental example 3 of the present invention;

FIG. 18 shows M in Experimental example 3 of the present invention₄The effect of IDP on the expression level of autophagy-related proteins;

FIG. 19 shows M in Experimental example 3 of the present invention₄The effect of IDP on HCT116 autophagy flow resulted in bar being 20 μm;

FIG. 20 shows M in Experimental example 3 of the present invention₄The result of the effect of IDP on the expression of PTEN protein of HCT116 cells and key effector molecules of PI3K/Akt/mTOR pathway;

FIG. 21 shows M in Experimental example 3 of the present invention₄The possible mechanistic patterns of IDP-induced G1 phase arrest, apoptosis and autophagy of HCT116 cells;

FIG. 22 is the chemical structure of ZOL.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The compound shown as the formula (I) can be synthesized according to 4-methylimidazole diphosphonic acid and the synthesis method thereof⁹⁹Tc^mThe synthesis method of 4-methylimidazole bisphosphate disclosed in marker "can also be synthesized according to the preparation method described in example 1 below.

Example 1

This example provides a process for preparing a compound of formula (I), comprising the steps of:

the synthetic route of the compound (I) is as follows:

synthesis of Compound Ethyl 2- (4-methyl-1H-imidazolyl) acetate represented by Q1 and formula (A-2)

A150 mL three-necked flask was charged with 8.2g (0.1mol) of 4-methylimidazole represented by the formula (A-1), KOH8.4g (0.15mol), and K₂CO₃13.8g (0.1mol), tetrabutylammonium bromide 0.7g (2mmol) and CH₂Cl₂75mL of the mixture was stirred at room temperature for 0.5h, 11.2mL (0.1mol) of ethyl bromoacetate was slowly added dropwise thereto, the mixture was refluxed for 8h and then filtered, and the filter cake was made of CH₂Cl₂Washing with 40mL for 2 times, washing the filtrate with 150mL of saturated NaCl solution, separating the layers, and purifying the organic phase with anhydrous Na₂SO₄After drying, the solvent was distilled off to obtain 10.6g of a tan viscous liquid, i.e., ethyl 2- (4-methyl-1H-imidazolyl) acetate, which is a compound represented by the formula (A-2).

Synthesis of Compound 2- (4-methyl-1H-imidazolyl) acetic acid represented by Q2 and formula (A-3)

Adding 8.4g (0.05mol) of ethyl 2- (4-methyl-1H-imidazolyl) acetate of the compound (A-2) obtained in the step Q1, 100mL of distilled water and 1mL of concentrated hydrochloric acid into a 250mL three-neck flask, heating and refluxing for reaction for 8H, adding 2g of activated carbon, refluxing and decoloring for 0.5H, then carrying out hot filtration to obtain a light brown aqueous solution, distilling to remove the solvent to obtain a light brown viscous liquid, adding acetone into the viscous liquid, stirring, precipitating a solid, carrying out suction filtration, drying, and then recrystallizing by using isopropanol to obtain 6.1g of a white solid, namely the compound 2- (4-methyl-1H-imidazolyl) acetic acid of the compound (A-3), wherein the yield is as follows: 54.8 percent; a white solid; mp: 153-156 ℃; IR (KBr) (. sigma./cm)^-1：3328，1644，1603，1450；ESI-MS(m/z)：139(M)；¹H NMR(500MHz,DMSO)：9.06(s,1H,CH_ring),7.45(s,1H,CH_ring),5.14(s,2H,CH ₂COOH),2.22(s,3H,CH ₃) ), [ isomers: 9.00(s,1H, CH)_ring),7.42(s,1H,CH_ring),5.10(s,2H,CH ₂COOH),2.22(s,3H,CH ₃)](see FIG. 1 for¹H-NMR spectrum); c₆H₈N₂O₂Elemental analysis (calculated)/%: c: 51.38%, H: 5.67%, O: 22.95%, N: 19.99 percent.

Q3, Compound 1-hydroxy-2- (4-methyl-1H-imidazolyl) -ethane-1, 1-bisphosphonic acid (M) of formula (I)₄IDP) Synthesis

A250 mL three-necked flask was charged with 5.6g (0.04mol) of 2- (4-methyl-1H-imidazolyl) acetic acid, which is the compound represented by (A-3) obtained in step Q2, 40mL of chlorobenzene, and 85% by mass of H₃PO₄8mL, uniformly mixing, heating the mixture to 100 ℃, stirring and reacting for 0.5H, then slowly dropwise adding 10.4mL of phosphorus trichloride (after dropwise adding is finished for about 0.5H) when the temperature is reduced to 65 ℃, continuously heating to 100 ℃, reacting for 3H, cooling, adding 50mL of 9mol/L hydrochloric acid for reflux reaction for 6H, cooling, pouring the reaction liquid into cold ethanol, separating out a solid, performing suction filtration, drying, and recrystallizing with water to obtain 3.4g of a white solid, namely the target final product, namely the compound 1-hydroxy-2- (4-methyl-1H-imidazolyl) -ethane-1, 1-diphosphonic acid (M) shown in the formula (I)₄IDP). The yield is 29.7%; white solid；mp：227-231℃；IR(KBr)，σ/cm^-1：3398，3200，2930，2875，1665，1620，1448，1245；ESI-MS(m/z)：285(M^-)；¹H NMR(500MHz,DMSO)：7.54(3,1H,CH_ring),6.86(s，1H，CH_ring)，4.30(s,2H,NCH ₂)，2.01(s,3H,CH ₃) And [ isomer: 7.78(s,1H, CH)_ring),6.57(s,1H,CH_ring),4.30(s,2H,CH ₂COOH),2.14(s,3H,CH ₃)](see FIG. 2 for¹H-NMR spectrum); c₆H₁₂N₂O₇P₂Elemental analysis (calculated)/%: c: 25.19%, H: 4.18%, O: 39.11%, N: 9.83%, P: 21.69 percent.

Example 2

This example provides an anticancer agent containing the compound represented by formula (I) prepared in example 1 as an active ingredient.

Furthermore, the anticancer drug is a clinically acceptable preparation prepared by taking the compound shown in the formula (I) as an active ingredient and selectively adding conventional auxiliary materials according to a conventional process, wherein the preparation comprises tablets, capsules, granules, syrups, powders, pills, tinctures, vinum, soft extracts, freeze-dried powders, lozenges or mixtures.

Examples of the experiments

The compound 1-hydroxy-2- (4-methyl-1H-imidazolyl) -ethane-1, 1-diphosphonic acid (M) shown as the formula (I) in the invention₄IDP) inhibitory effect on cancer cells

The following experimental examples relate to the following experimental materials:

fetal Bovine Serum (FBS), antibiotics, DMEM, purchased under Gibco brand in the united states;

hoechst33258, dansyl cadaverine (MDC), MTT, DMSO from Sigma, usa;

annexin V/PI staining kit purchased from Roche;

CCK-8 kit, crystal violet, cell cycle analysis kit, Reactive Oxygen Species (ROS) analysis kit, RIPA lysate, mChery-GFP-LC 3B expressing adenovirus (Ad-mChery-GFP-LC 3B), and antibodies such as p27, Cyclin D1, PARP, active-caspase3, CHOP, Bip, LC3B, PTEN, phospho-GSK-3 beta (Ser9), GSK-3 beta, Bad and the like, were purchased from Biyunnan corporation;

antibodies such as phospho-Rb (Ser780), ERO1L, p62, ATG5, phospho-PDK1(Ser241) were purchased from Abcam;

antibodies such as phospho-Akt (Ser473), Akt, phospho-mTOR (Ser2448), mTOR, phospho-p70S6K (Thr389) and the like were purchased from CST corporation, USA;

PERK, beta-actin, HRP labeled secondary antibody and Western Blotting Luminol reagent are products of Santa Cruz company in the United states;

human breast cancer cell line MDA-MB-231, human neuroblastoma cell line SH-SY5Y, human osteosarcoma cell line U2OS, human prostate cancer cell line PC-3, human liver cancer cell line SMMC-7721, human gastric cancer cell line SGC-7901, human colon cancer cell line HCT116, human liver cell line LO2 and human gastric mucosal cell line GES-1 were purchased from American ATCC company in culture medium of 90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin, 5% CO at 37 deg.C₂Culturing under the condition;

medicine preparation: m₄IDP is prepared in example 1 and zoledronic acid (ZOL) is a commercially available product, M₄Both IDP and ZOL were more than 98% pure, dissolved in sterile water to a concentration of 10mM, and stored at-20 ℃ for use diluted with medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) to the desired concentration.

Experimental example 1M₄Inhibitory Effect of IDP on various human cancer cells

This example used CCK-8 to assay for M₄Growth inhibitory effect of IDP on human-derived breast cancer cell MDA-MB-231, neuroblastoma cell SH-SY5Y, osteosarcoma cell U2OS, prostate cancer cell PC-3, liver cancer cell SMMC-7721, stomach cancer cell SGC-7901 and colon cancer cell HCT 116. The specific method comprises the following steps:

experimental groups: the above cancer cells were plated in 96-well plates at a density of 4000 cells/well, and 100. mu.L of the medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) was plated in each well at 37 ℃ with 5% CO₂After culturing for 24h under the condition, adding the extract into each well respectivelyInto 100. mu.L of a mixture containing M at various concentrations₄The medium of IDP, said M₄The concentration of IDP was 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M and 60. mu.M in this order, at 37 ℃ with 5% CO₂Culturing under the above conditions for 72h, adding 20 μ L CCK-8 solution into each well, and continuing to culture at 37 deg.C and 5% CO₂Culturing for 4h under the condition, and detecting the absorbance value of the culture solution under the condition of 450nm by using a SpetraMax M5 enzyme-labeling instrument to obtain an OD value of an experimental group; control group: only 200. mu.L of medium, M, was added per well₄Healthy HCT116 cells with an IDP concentration of 0. mu.M were used as a control group, and OD values of the control group were obtained according to the experiment. Cell viability was expressed as the percentage of the OD of the experimental group to the OD of the control group. The results are shown in FIG. 3, M₄IDP has inhibitory effect on all of the above-mentioned cancer cells, and the effect is enhanced with increasing drug concentration. Wherein M is₄The IDP has the strongest inhibitory effect on human colon cancer HCT116 cells, which indicates that M₄IDP has higher inhibition potential on human colon cancer HCT116 cells.

Experimental example 2M₄Inhibitory Effect of IDP on human Colon cancer HCT116 cells

2.1 cytotoxicity assays

2.1.1 detection of M Using CCK-8 assay₄Growth inhibitory effect of IDP on HCT116 cells. The specific method comprises the following steps:

experimental groups: HCT116 cells were plated at 4000/well density into 96-well plates with 100. mu.L of the medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) per well at 37 ℃ with 5% CO₂After culturing for 24h under the condition, 100 μ L of different M concentrations are added into each well₄IDP medium, said medium containing different concentrations of M₄M in IDP Medium₄The concentrations of IDP were 0.3125. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 5% CO at 37 ℃ respectively₂Culturing under the conditions for 24h, 48h or 72h, adding 20 μ L CCK-8 solution into each well, and continuing to culture at 37 deg.C and 5% CO₂Culturing for 4h under the condition, and detecting the absorbance value under the condition of 450nm by using a SpetraMax M5 enzyme-labeling instrument; control group: only 200. mu.L of medium, M, was added per well₄Health with 0 μ M concentration of IDPHCT116 cells were used as a control group, and OD values of the control group were obtained according to the experiment. Cell viability was expressed as the percentage of the OD of the experimental group to the OD of the control group, see FIG. 4, M₄IDP has inhibitory effect on HCT116 cells, and the effect is enhanced with concentration or time, and after 24 hr, the concentration is as low as 2.5 μ M₄IDP can inhibit HCT116 cell activity, and after 48 hr, the concentration of M is as low as 0.3125 μ M₄IDP can inhibit HCT116 cell activity, and after 72h, M₄The greater ability of IDP to inhibit HCT116 cell activity indicates that M is present₄IDP has a high inhibitory potential against HCT116 cells.

2.1.2 comparison of M Using CCK-8 analysis₄Growth inhibitory effects of IDP and ZOL on HCT116 cells. The specific method comprises the following steps:

experimental groups: HCT116 cells were plated at 4000/well density into 96-well plates with 100. mu.L of the medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) per well at 37 ℃ with 5% CO₂After culturing for 24h under the condition, 100 μ L of different M concentrations are added into each well₄IDP or ZOL medium containing M at different concentrations₄M in IDP or ZOL Medium₄The concentration of IDP or ZOL was 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, respectively, at 37 ℃ with 5% CO₂Culturing for 72h, adding 20 μ L CCK-8 solution into each well, culturing for 4h, and detecting absorbance value at 450nm with Speramax M5 enzyme-labeling instrument to obtain M₄OD values of IDP or ZOL experimental groups; control group: only 200. mu.L of medium, M, was added per well₄Healthy HCT116 cells with 0. mu.M concentration of IDP or ZOL were used as a control group, and OD values of the control group were obtained according to the experiment. Cell viability was expressed as the percentage of the OD of the experimental group to the OD of the control group. The results are shown in FIG. 5, from which M is known₄IDP was more cytotoxic than ZOL on HCT116 cells and M was measured₄The IC50 of the IDP on HCT116 cells for 72h was 0.98. mu.M, and the IC50 of ZOL on HCT116 cells for 72h was determined to be 25.8. mu.M, ZOL being the same as M₄IDP vs. HCT116 at 72h IC50, ZOL at IC50 was M₄26.3 times IDP, show M₄IDP has a stronger cytotoxic effect on HCT116 cells than ZOLAnd has a lower IC50 value, M₄IDP is promising for applications in the treatment of colon cancer.

2.1.3 comparison of M Using CCK-8 analysis₄Growth inhibitory effects of IDP on human colon cancer cells HCT116 and human liver cells LO2, human gastric mucosal cells GES-1. The specific method comprises the following steps:

spreading human colon cancer cell HCT116, human liver cell LO2, and human gastric mucosa cell GES-1 at 4000/well density to 96-well plate, wherein each well contains 100 μ L of the culture medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), and the culture medium is cultured at 37 deg.C and 5% CO₂After culturing for 24h under the condition, 100 μ L of different M concentrations are added into each well₄The medium of IDP, said M₄The concentration of IDP was 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, and 10. mu.M in this order, 5% CO at 37 ℃₂Culturing under the above conditions for 72h, adding 20 μ L CCK-8 solution into each well, and continuing to culture at 37 deg.C and 5% CO₂Culturing for 4h under the condition, and detecting the absorbance value of the culture solution under the condition of 450nm by using an American SpetraMaxM5 enzyme-labeling instrument to obtain an OD value of an experimental group; control group: only 200. mu.L of medium, M, was added per well₄Healthy cells with an IDP concentration of 0. mu.M were used as a control group, and the OD value of the control group was obtained according to the experiment. Cell viability was expressed as the percentage of the OD of the experimental group to the OD of the control group. The results are shown in FIG. 6, M₄The cytotoxic effect of IDP on human colon cancer HCT116 cells is stronger than that of human normal liver cells LO2 and human normal gastric mucosa cells GES-1, and the concentration of M is as high as 5 mu M₄The inhibition rate of IDP on HCT116 reaches 65%, but the IDP has no obvious influence on the cell activity of LO 2; m at a concentration of up to 2.5. mu.M₄The inhibition rate of IDP on HCT116 reaches 55%, but the IDP has no obvious influence on the cell activity of GES-1. This indicates that M is compared with the normal human cells LO2 and GES-1₄IDP has good selective inhibition effect on human colon cancer cell HCT116, M₄IDP is hopefully developed into a high-efficiency and low-toxicity medicament for treating human colon cancer.

2.2 cloning experiments

HCT116 cells were plated at a density of 300/well in 12-well plates with 1mL of the medium per well (90% DMEM, 10)% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h of incubation under conditions, the medium was replaced with various concentrations of M₄IDP medium containing various concentrations of M₄M in IDP Medium₄The IDP concentrations were 0. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, respectively, and continued at 37 ℃ with 5% CO₂After 14 days of culture under the conditions, the HCT116 cells were fixed with methanol and stained with 1% crystal violet for 10min, rinsed well with water and dried in air, and then observed under a microscope and clones containing more than 50 cells were counted. The results are shown in FIGS. 7 and 8, using M₄The number of clones of IDP-treated cells was significantly less than that of the cells without M addition₄Number of IDP-treated cell clones, this inhibitory effect being dependent on M₄The increase in IDP concentration is enhanced.

Experimental example 3M₄IDP causes human colon cancer HCT116 cell G1 phase block, apoptosis and autophagy by blocking PI3K/Akt/mTOR

3.1 cell cycle Studies

HCT116 cells were plated at 2 × 10⁵Individual/well density was plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP was treated for 0h (control), 24h, 48h, 72h, respectively. Then, HCT116 cells were collected, fixed with 75% ethanol by volume, and then, HCT116 cells were resuspended in a 50. mu.g/mL PI solution, the treated cells were analyzed by a Flow cytometer of BD company, Germany, and the data obtained were analyzed by Flow Jo software. The results are shown in FIGS. 9A-D and FIG. 10, where it can be seen that M increases with time₄IDP causes accumulation of cells in the G1 phase, and cells in the S and G2/M phases gradually decrease.

It is well known that the cell cycle is negatively regulated by CDK, p27, etc., and positively regulated by the cyclin-CDK complex. To study M₄The possible mechanism of IDP causing G1 phase block is that the expression of some proteins related to G1/S checkpoint, such as p27, cyclin D1 and phosphine-Rb, is detected by Western Blotting method, and HCT116 cell is treated with 2 × 10⁵Of one/holeDensity plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 ℃₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP (dichloro-diphenyl-trichloroethane) treatment of HCT116 cells for 0h, 24h, 48h and 72h, then cracking of HCT116 cells by using a proper amount of RIPA lysate, 12,000 × g high-speed centrifugation for 5min, taking supernatant, measuring the protein concentration of the supernatant by using a BCA method, adding a proper volume of 6 × SDS loading buffer solution into the solution containing 30 mu g of protein, boiling water bath for 5min, then carrying out short-time centrifugation, then loading all the samples into 10% SDS polyacrylamide gel for electrophoresis, setting the voltage for electrophoresis as 100V, transferring the protein on the 10% SDS polyacrylamide gel onto a PVDF membrane by using a Bio-Rad wet electric transfer membrane instrument after the electrophoresis is finished, transferring the protein onto the PVDF membrane by using 100V voltage for 1h, transferring the protein onto the PVDF membrane by using p27, CyclinD1 or pho-Rb antibody with the dilution concentration of 1:1000 after the membrane transfer, and obtaining the detection result shown in figure 11₄IDP increased the expression of CDK inhibitory molecule p27 and inhibited the expression of cyclin D1 and phospho-Rb (p-Rb), indicating that M₄The G1 phase block caused by IDP may be attributed to its effect on p27, CyclinD1 and phospho-Rb.

3.2 apoptosis assay

Experimental group (M)₄IDP) HCT116 cells were treated with 3 × 10⁴One/well density was plated in 24-well plates, 500. mu.L of the medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) per well, 5% CO at 37 ℃₂After 24h incubation under conditions, 2.5. mu.M M was used₄Treating with IDP for 72h, then absorbing the culture medium, adding Annexin V/PI staining solution prepared according to the requirements of the kit, incubating at room temperature for 10min, observing under an Olympus IX51 fluorescence microscope, and taking a picture; the Control group (Control) was performed according to the above experimental group procedure, but only 500. mu.L of the medium was added to each well, i.e., without M₄IDP-treated healthy HCT116 cells. The results are shown in FIG. 12, where the arrows indicate apoptotic cells whose cell membranes were stained green by Annexin V-FITC, the circles indicate apoptotic middle and late cells whose nuclei were stained red by PI and dead cells, and M is shown₄IDP induced significantly more apoptotic cells than the control group.

Then makeMorphological characteristics of apoptotic cells were visualized by Hoechst33258 staining analysis by staining HCT116 cells with 3 × 10⁴One/well density was plated in 24-well plates, 500. mu.L of the medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) per well, 5% CO at 37 ℃₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP was treated for 24h, 48h, 72h, then cells were fixed with 4% paraformaldehyde, then cells were stained with 2. mu.g/mL Hoechst33258 for 5min, observed under an Olympus IX51 fluorescence microscope and photographed, and the Control (Control) was performed as described above, but only 500. mu.L of medium was added per well, i.e., no M added₄IDP-treated healthy HCT116 cells. The results are shown in FIG. 13, from which M is known₄IDP causes chromatin contraction and fragmentation of HCT116 cells (indicated by arrows).

The indicator proteins of apoptosis, namely clean PARP and active-caspase3, are detected by using a Western Blotting method, namely, the method comprises the step of dissolving HCT116 cells in 3 × 10⁴One/well density was plated in 24-well plates, 500. mu.L of the medium (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin) per well, 5% CO at 37 ℃₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP (dichloro-diphenyl-trichloroethane) treatment of HCT116 cells for 0h, 24h, 48h and 72h, cell lysis by using a proper amount of RIPA lysate, high-speed centrifugation for 5min at 12,000 × g, supernatant taking, BCA (burst amplification factor) method for measuring the protein concentration of the supernatant, adding a proper volume of 6 × SDS (sodium dodecyl sulfate) sample buffer solution into the obtained solution containing 30 mu g of protein, boiling water bath for 5min, short-time centrifugation, then full sample loading into 10% SDS polyacrylamide gel for electrophoresis, setting the voltage at 100V, after the electrophoresis is finished, using a Bio-Rad wet electric transfer membrane instrument to transfer the protein on the 10% SDS polyacrylamide gel to a PVDF membrane, using 100V voltage to transfer the membrane for 1h, using PARP, active-caspase3 and β -active antibodies with the dilution concentration of 1:1000 to detect the protein on the 10% SDS-polyacrylamide gel, and the result is shown in figure 14₄Increased duration of IDP action, which further illustrates M₄IDP causes HCT116 cells to undergo apoptosis.

3.3 intracellular ROS accumulation and endoplasmic reticulum stress Effect assay

3.3.1 intracellular ROS accumulation assay

ROS accumulation may play an important role in apoptosis by altering the internal environment of the cell, and intracellular ROS accumulation is detected by contacting HCT116 cells with 2 × 10⁵Individual/well density was plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h incubation under conditions, M was added at various concentrations₄The culture medium of IDP is treated for 24h, and the culture medium contains M with different concentrations₄M in IDP Medium₄Concentrations of IDP were 0. mu.M, 0.625. mu.M, 1.25. mu.M, 2.5. mu.M, respectively, and cells were collected as required in the Reactive Oxygen Species (ROS) assay kit, washed once with PBS, and incubated in 10. mu.M DCFH-DA protected from light at 37 ℃ for 30 min. The fluorescence signal was detected by flow cytometry. The results are shown in FIG. 15, M₄Elevated ROS levels in HCT116 cells 24h after IDP treatment, suggesting M₄IDP causes the accumulation of ROS.

3.3.2 endoplasmic reticulum stress Effect detection

ROS may cause endoplasmic reticulum stress-mediated unfolded protein reaction to influence cell growth and apoptosis, and the method for detecting endoplasmic reticulum stress effect comprises the following steps of detecting the expression condition of endoplasmic reticulum stress-related protein by using Western Blotting method, and detecting HCT116 cells with 2 × 10⁵Individual/well density was plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP treatment of HCT116 cells for 0h, 24h, 48h and 72h, collecting proteins, electrophoresis, membrane transfer, antibody incubation and exposure according to a conventional method, namely using a proper amount of RIPA lysate to lyse the cells, centrifuging at a high speed of 12,000 × g for 5min, taking supernatant, measuring the protein concentration of the supernatant by using a BCA method, adding a proper volume of 6 × SDS loading buffer solution into the obtained solution containing 30 mu g of protein, carrying out boiling water bath for 5min, centrifuging for a short time, loading all the supernatant into 10% SDS polyacrylamide gel for electrophoresis, setting the voltage at 100V during electrophoresis, and after the electrophoresis is finished, using a Bio-Rad wet electric membrane converter to polymerize 10% SDS by using a Bio-Rad wet electric membrane converterThe protein on the acrylamide gel is transferred to a PVDF membrane, the membrane is transferred for 1h by adopting 100V voltage, CHOP, Bip, ERO1L and PERK and β -actin antibodies with the dilution concentration of 1:1000 are used for detection after the membrane is transferred, the result is shown in figure 16, the expressions of endoplasmic reticulum stress-related proteins CHOP, Bip, ERO1L and PERK are all increased, and the result shows that M is M₄IDP causes endoplasmic reticulum stress effects in HCT116 cells.

3.4 autophagy study

M detection by MDC staining₄Whether IDP causes autophagy of HCT116 or not, MDC is commonly used as a specific marker stain for autophagic vesicles by contacting HCT116 cells with 2 × 10⁵Individual/well density was plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP was treated for 24h, 48h, 72h, respectively, and the cells were washed once with ice-cold PBS, and then incubated in 50 μ M MDC for 30min at 37 ℃. Cells were washed once with ice-cold PBS, fixed with 4% paraformaldehyde, observed under a fluorescence microscope and photographed, and the Control (Control) was performed as described above, but only 2mL of medium was added per well, i.e., without M₄IDP-treated healthy HCT116 cells. The results are shown in FIG. 17, M₄IDP caused accumulation of MDC-stained vacuoles, suggesting M₄IDP may cause autophagy in HCT116 cells.

Then the expression of the autophagy-related marker protein is detected by using a Western Blotting method, and the HCT116 cells are treated with 2 × 10⁵Individual/well density was plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h incubation under conditions, 2.5. mu.M M was used₄IDP treating HCT116 cells for 0h, 24h, 48h and 72h, collecting protein, electrophoresis, transferring membrane, incubating antibody and exposing according to conventional method, i.e. using appropriate amount of RIPA lysate to lyse cells, centrifuging at high speed of 12,000 × g for 5min, collecting supernatant, measuring protein concentration of the supernatant by BCA method, adding appropriate volume of 6 × SDS buffer solution into 30 μ g protein-containing solution, and boiling waterBathing for 5min, then centrifuging for a short time, then loading all samples into 10% SDS polyacrylamide gel for electrophoresis, setting the voltage at 100V during electrophoresis, transferring the proteins on the 10% SDS polyacrylamide gel onto a PVDF membrane by using a Bio-Rad wet electric transfer membrane instrument after the electrophoresis is finished, transferring the membrane for 1h by using the 100V voltage, and then detecting by using LC3B, Atg5, p62 and β -actin antibodies with the dilution concentration of 1:1000₄IDP causes autophagy in HCT116 cells, but a longer duration of action (72h) may cause diminished autophagy flow.

And finally performing autophagic flow analysis. Ad-mCherry-GFP-LC3B is an adenovirus capable of expressing mCherry-GFP-LC3B fusion protein, can effectively express fusion protein of red fluorescent protein mCherry, green fluorescent protein GFP and LC3B in target cells after infection, presents bright red and green fluorescence, and can be used for detecting autophagy of cells. After infecting the cells, mCherry-GFP-LC3B was present in the cytoplasm as diffuse yellow fluorescence (combined effect of mCherry and GFP) under a fluorescent microscope in the case of non-autophagy; in the case of autophagy, mCherry-GFP-LC3B was concentrated on the autophagosome membrane under a fluorescence microscope and appeared as yellow spots; when autophagosomes fused to lysosomes, they appeared as red spots due to partial quenching of GFP fluorescence. The specific analysis method is as follows: HCT116 cells were plated onto glass-bottomed cell culture dishes, and cells were confluent to 20% -30% for infection: the cells were first washed 2 times with medium and then infected under 20MOI Ad-mCherry-GFP-LC3B for 24h, then 2.5. mu.M M₄The infected cells were treated with IDP and observed and photographed using Olympus confocal microscope at treatment times of 0h, 3h, 6h, 16h, 24h, 48h, 72 h. The results are shown in fig. 19, where the mCherry and GFP proteins were uniformly distributed in the cytoplasm at 0h treatment time, indicating that autophagy did not occur; at 3h treatment, the mCherry and GFP proteins were visible under the fluorescent microscope to aggregate and may overlap into yellow spots (bright spots in the figure), indicating autophagosome formation, which may last for up to 24h and follow thereThe increase of the physiological time and the increase of yellow spots (bright spots in the figure) show that autophagosomes are gradually increased and mature, and the autophagy degree is gradually increased; after 48 hours of treatment, red spots are increased under a fluorescence microscope, green signals are reduced, and the red spots (arrows indicate bright spots) can be seen after overlapping, which indicates that autophagosome is fused with lysosome and autophagy flow is increased; at 72h treatment, yellow spots (bright spots within circles) and red spots (arrows indicate bright spots) in the overlaid images were both less than at 48h, indicating a diminished degree of autophagy.

3.5 Signal Path Studies

The PI3K/Akt/mTOR pathway plays an important role in regulating cell cycle, apoptosis and autophagy. To determine M₄Whether the inhibitory effect of IDP on HCT116 cells is associated with this pathway, we investigated M₄Effect of IDP on representative Signal protein molecules on this Signal pathway HCT116 cells were treated with 2 × 10⁵Individual/well density was plated in 6-well plates, 2mL of the medium per well (90% DMEM, 10% FBS, 100U/mL penicillin, 0.1mg/mL streptomycin), 5% CO at 37 deg.C₂After 24h incubation under conditions, 2.5. mu.M M was used₄Treating HCT116 cells for 0h, 24h, 48h and 72h with IDP, collecting protein, performing electrophoresis, transferring membrane, incubating antibody and exposing, namely lysing the cells with an appropriate amount of RIPA lysate, centrifuging at 12,000 × g for 5min at high speed, collecting supernatant, measuring the protein concentration of the supernatant by BCA method, adding an appropriate volume of 6 × SDS loading buffer solution to the solution containing 30. mu.g of protein, boiling water bath for 5min, centrifuging for a short time, loading the whole solution to 10% SDS polyacrylamide gel for electrophoresis at a voltage of 100V, transferring the protein on the 10% SDS polyacrylamide gel to PVDF membrane by a Bio-Rad wet electric membrane transfer apparatus after electrophoresis, transferring the protein to membrane at a voltage of 100V for 1h, transferring the protein to membrane by PTEN, phospho-PDK1(p-PDK1), phospho-t (p-phot), Akt, mTOR (mTOR), mTOR-3570, GSP-3526-3670, GSP 19-11-P-3670, and detecting the protein, the protein concentration, the antibody and the antibody are detected by BCA-3645₄IDP causes downregulation of phospho-Akt, phospho-mTOR, phospho-p70S6K and upregulation of phospho-GSK-3 β on Akt, mTThe total protein content of OR and GSK-3 β has no significant influence, the expression of pro-apoptotic protein Bad which is a downstream molecule of Akt is increased, and the expression of protein of phosphorylated form PDK1 which is an upstream kinase of Akt is reduced, which indicates that M is a protein with a high activity₄IDP may act directly on the upstream molecule of PDK 1; increased expression of PTEN, a negative regulatory molecule of PI3K/Akt pathway. These results show that M₄IDP could inhibit the PI3K/Akt/mTOR signaling pathway by up-regulating PTEN expression.

In summary, the above studies of this example show that Akt can inhibit p27 expression and increase cyclin D1 expression by inactivating GSK-3 β, thereby positively regulating G1/S cell cycle progression, and therefore, M₄IDP-induced G1 phase arrest may be associated with Akt and GSK-3 β, Bad protein is associated with the initiation of apoptosis, non-phosphorylated Bad protein can promote apoptosis by combining with and inactivating anti-apoptotic protein Bcl-2 or Bcl-xL, activated Akt can inhibit apoptosis and promote cell survival by inducing Bad phosphorylation, therefore, M₄IDP-induced apoptosis of HCT116 cells may be associated with inhibition of Akt phosphorylation and increased Bad protein expression. mTOR is a downstream effector molecule of Akt, and together with its downstream target p70S6K, exerts a negative regulatory effect on autophagy, M₄Autophagy by IDP is likely to be associated with M₄IDP is involved in the inhibition of the Akt/mTOR/p70S6K pathway. PDK1, which is an upstream molecule of Akt and a downstream molecule of PIP3, can be phosphorylated by PIP3 and causes phosphorylation of Akt. The tumor suppressor PTEN can negatively regulate the PI3K pathway, dephosphorylate PIP3 into PIP2, and thereby negatively regulate Akt activity. In summary, M₄IDP-induced arrest of G1 phase, apoptosis and autophagy in HCT116 cells may be attributed to the production of PTEN, which further affects the downstream molecules PDK1, Akt, mTOR, GSK-3 β and Bad, as shown in fig. 21.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. The use of a zoledronic acid compound in the preparation of a medicament for treating cancer; the zoledronic acid compound has a structure shown in a formula (I):

（I）；

the cancer is selected from breast cancer, prostate cancer and colon cancer.

2. The use according to claim 1, wherein the cancer treatment drug is selected from the group consisting of drugs against human-derived breast cancer cells MDA-MB-231, prostate cancer cells PC-3 and colon cancer cells HCT 116.