CN109438542B

CN109438542B - Novel cardiac glycoside monomeric compound and application thereof in preparation of antitumor drugs

Info

Publication number: CN109438542B
Application number: CN201811024872.6A
Authority: CN
Inventors: 马龙; 苏艳芳; 满淑丽
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2018-09-04
Filing date: 2018-09-04
Publication date: 2022-04-05
Anticipated expiration: 2038-09-04
Also published as: CN109438542A

Abstract

The invention relates to a novel cardiac glycoside monomeric compound and application thereof in preparing antitumor drugs, MTT experiments are utilized to verify that HTF-1 can induce apoptosis of HeLa cells of human cervical cancer, MCF-7 cells of human breast cancer and HepG2 cells of human liver cancer and has dose and time dependence, and then the influence of HTF-1 on the apoptosis, cell cycle, proliferation, migration, invasion and related protein expression level of HeLa cells is respectively detected by using HeLa cells as research objects and utilizing a cell flow analysis technology, a wound healing experiment, a Transwell cell experiment, a protein immunoblotting method and the like. The HTF-1 can inhibit the proliferation, migration and invasion of cancer cells and induce the HeLa cells to undergo apoptosis, so that the cell cycle is arrested in the S phase.

Description

Novel cardiac glycoside monomeric compound and application thereof in preparation of antitumor drugs

Technical Field

The invention belongs to the field of antitumor drug screening, and particularly relates to a novel cardiac glycoside monomer compound and application thereof in preparing antitumor drugs.

Background

The tumor is one of the most serious diseases endangering human health at present, although the traditional chemotherapy and radiotherapy successfully improve the cure rate of various malignant tumors, the pain of patients is relieved. But also has the defects of strong side effect, easy relapse after operation and the like. The traditional Chinese medicine is a material basis for treating diseases in traditional Chinese medicine, has the characteristics of multiple components and multiple target points, can act on different links of tumor attack, has the advantages of low toxic and side effects, capability of improving the immunity of the organism, difficulty in generating drug resistance and the like, and makes the research of searching novel antitumor drugs from natural products reluctant.

Ranunculaceae plant of Thezaumatococcus (Helleborus thibetanus Franch) is a unique plant in China, mainlyIt is distributed in the northwest of Sichuan, the south of Gansu and the south of Shaanxi. Chimonanthus praecox, commonly known as Xiaotao Erqin, has been used as a national medicine for treating urethritis, cystitis, traumatic injury, sore, furuncle, pyogenic infections, and the like. Extracts of several plants of the genus sneeze have immunostimulating and anti-inflammatory properties. The chemical components of the sinopodophyllum hexandrum are gradually researched, and some steroid saponins, one pregnane, spironolsulfate and several toad dihydroxyenoic acid lactone compounds are further separated and identified. However, little research has been seen on the biological activity of some monomeric compounds of this species. Cardiac glycosides are a class of drugs with selective cardiotonic action due to their action on cell surface Na⁺/K⁺Specific inhibition of ATPase, cardiac glycosides have been widely recognized for their therapeutic effects in the treatment of congestive heart failure and cardiac arrhythmias. However, little is known about the possible anticancer activity of cardiac glycosides and certain derivatives thereof. In the research, a novel cardiac glycoside monomeric compound HTF-1 separated from the traditional Chinese medicine poliomyelia has strong anticancer activity, and a potential molecular mechanism is tried to be explored.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a novel cardiac glycoside monomer compound and application thereof in preparing an anti-tumor medicament, and the HTF-1 provided by the invention can inhibit the proliferation, migration and invasion of cancer cells and induce HeLa cells to undergo apoptosis so that the cell cycle is arrested in the S phase. In addition, HTF-1 was able to activate caspase 9-dependent apoptotic pathways and double-stranded DNA breaks (DSBs), while HTF-1 was found to activate JNK but inhibit ERK and PI3K-Akt-mTOR pathways.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

a novel cardiac glycoside monomer compound has the following structural formula:

use of a novel cardiac glycoside monomer compound in the preparation of a medicament for treating tumors.

Application of novel cardiac glycoside monomeric compound in preparation of preparations for inducing apoptosis of human cervical cancer HeLa cells, human breast cancer MCF-7 cells and human liver cancer HepG2 cells.

Application of novel cardiac glycoside monomeric compound in preparation of PI3K-Akt-mTOR signal pathway/protein inhibitor.

Application of novel cardiac glycoside monomeric compound in preparation of ERK signal pathway/protein inhibitor.

Application of novel cardiac glycoside monomeric compound in preparation of Caspase 9 protein agonist.

Application of novel cardiac glycoside monomeric compounds in preparation of gamma H2AX protein agonists.

Application of novel cardiac glycoside monomeric compound in preparation of JNK signal pathway/protein agonist.

The invention has the advantages and positive effects that:

1. the part of the HTF-1 in vitro induction of cancer cell apoptosis in the invention is acted by the mechanism, so that the invention is expected to enrich the understanding of human beings on cardiac glycoside compounds, discover that the compounds have strong anticancer activity and pave the way for the research and development of anticancer drugs based on natural products.

2. The invention firstly carries out biological activity research on cardiac glycoside with a new structure, thereby finding that the cardiac glycoside has strong in-vitro anticancer activity. It can inhibit cancer cells dose-dependently with minimal toxicity to normal human cells. HTF-1 can induce apoptosis, inhibit cell proliferation, migration and invasion, and cause HeLa cell cycle arrest in S phase. Meanwhile, HTF-1 activates caspase 9, reduces the expression of some anti-apoptotic proteins such as survivin and Bcl-2, activates JNK, inhibits ERK and PI3K-Akt-mTOR pathways, and the combined action of the mechanisms leads cancer cells to undergo apoptosis.

3. The HTF-1 can be used as a medicine for controlling the development of cancers to provide reliable experimental basis. Although HTF-1 is not significantly toxic to normal human cells, its toxicity cannot be completely ruled out, thus requiring further pharmacological experiments in vivo. The natural product has rich medicinal resources, and the HTF-1 is found to be a promising anti-cancer drug through preliminary research at present. Meanwhile, the mechanism of HTF-1 in vitro antitumor is elucidated, the human knowledge of cardiac glycoside is enriched, and the application range of the cardiac glycoside compound is expanded.

Drawings

FIGS. 1A-D show the in vitro anti-cancer activity assay of HTF-1. FIG. 1A: HTF-1 chemical structural formula; FIG. 1B, FIG. 1C and FIG. 1D: respectively detecting the cell survival rates of HeLa, MCF-7 and HepG2 after HTF-1 treatment by an MTT experiment; FIG. 1E: cytotoxicity of HTF-1 on cancer cells versus normal cells selected for the experiment (48 hours of HTF-1 treatment).

FIGS. 2A-C are images of HeLa cells after HTF-1 treatment. FIG. 2A: brightfield microscopy pictures (20X) after 24 hours of treatment of HeLa cells with different concentrations of HTF-1; FIG. 2B: after HeLa cells are treated by HTF-1 with different concentrations for 24 hours, Hoechst H33342 is used for dyeing a fluorescent picture; FIG. 2C: HeLa cells were treated with different concentrations of HTF-1 for 24 hours and then subjected to scanning electron microscopy.

FIGS. 3A-E are flow charts of 24 hour apoptosis of HTF-1 treated HeLa cells. Fig. 3A-3D: apoptosis detection flow charts of a control group, a 100nM treatment group, a 200nM treatment group and a 500nM treatment group respectively; FIG. 3E: histogram of apoptosis rate statistics. Arithmetic sum of Q2 and Q3 as the apoptosis rate for each group. P < 0.05; p < 0.01; p < 0.005. Error bars are obtained from three independent replicates.

FIGS. 4A-D are cell cycle distribution flow charts of HTF-1 treated HeLa cells for 24 hours. Fig. 4A-4C: hela cells were treated with HTF-1 at 0nM, 100nM and 200nM for 24-hour period distribution flow chart; FIG. 4D: cell cycle distribution statistical histogram.

FIGS. 5A-E are graphs showing the effect of HTF-1 treatment on cancer cell proliferation. FIG. 5A: cloning to form an experimental picture; FIG. 5B: a photograph of a single clone; FIG. 5C: statistical results of colony formation rate histogram, p <0.005, error bars are obtained from independent triplicate experiments; FIG. 5D: ki-67 staining experimental picture; FIG. 5E: EdU label experimental pictures.

FIGS. 6A-B are graphs of the effect of HTF-1 treatment 24 on HeLa cell migration and invasion. FIG. 6A: in vitro wound healing experiments; FIG. 6B: transwell cell experiments. P < 0.05; p < 0.01; p < 0.005. Error bars are obtained from three independent replicates.

FIGS. 7A-C are WB experiments examining the effect of different doses of HTF-1 on the expression levels of the relevant proteins in HeLa cells treated for 24 hours. FIG. 7A: proteins associated with apoptosis and DNA double strand breaks; FIG. 7B: MAPK signaling pathway related proteins; FIG. 7C: PI3K-Akt-mTOR signal pathway related protein.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments, which are illustrative only and not limiting, and the scope of the present invention is not limited thereby.

The invention has proved that the new cardiac glycoside monomer compound extracted from Chinese medicine podophyllum hexandrum has anticancer activity through experiments, and is named as HTF-1, the method is as follows:

firstly, MTT experiments prove that HTF-1 can induce apoptosis of human cervical cancer HeLa cells, human breast cancer MCF-7 cells and human liver cancer HepG2 cells and has dose and time dependence.

Then, the influence of HTF-1 on apoptosis, cell cycle, proliferation, migration, invasion and related protein expression level of the HeLa cells is detected by taking the HeLa cells as research objects and utilizing a cell flow analysis technology, a wound healing experiment, a Transwell chamber experiment, a protein immunoblotting method (Western blotting) and the like.

Research results show that HTF-1 can inhibit proliferation, migration and invasion of cancer cells and induce HeLa cells to undergo apoptosis, so that the cell cycle is arrested in the S phase. In addition, HTF-1 was able to activate caspase 9-dependent apoptotic pathways and double-stranded DNA breaks (DSBs), while HTF-1 was found to activate JNK but inhibit ERK and PI3K-Akt-mTOR pathways.

Secondly, the specific operation of the steps is as follows:

2.1 Compounds and antibodies

Compounds were purchased from Sigma-Aldrich or Aladdin Biotech, Inc., unless otherwise noted. The basement membrane matrigel invasion cell was purchased from BD corporation, usa. Commercial antibody sources: antibodies to phosphorylated ERK1/2, phosphorylated JNK, phosphorylated mTOR, phosphorylated Akt, JNK, p38, PI3K, Ki-67, FEN1, and survivin were purchased from Santa Cruz biotechnology company. Phosphorylated PI3K, phosphorylated p70S6k, p70S6k, Bcl-2, clear caspase 3, clear caspase 7, clear caspase 9, gamma H2AX, Akt and mTOR were purchased from Cell Signaling Technology, USA. PARP and phosphorylated p38 antibodies were purchased from biyuntian biotechnology (Beyotime). ERK antibodies were purchased from bosch de biotechnology limited. Internal reference antibodies to GAPDH and β -actin were purchased from Tianjin Sanjian Biotechnology Ltd (Tianjin Sungene Biotech). HRP-labeled secondary antibodies were purchased from Invitrogen, life technologies ltd. The Annexin V-FITC apoptosis assay kit is purchased from Tianjin three-arrow biotechnology limited. The EdU assay kit was purchased from lebo biotechnology limited, guangzhou.

2.2 preparation of HTF-1

Taking about 8kg of the Chinese medicinal materials of the small podophyllum hexandrum, crushing, soaking in about 10L of 95% ethanol for 2 weeks, and collecting the extracting solution; then, the medicinal materials are extracted by hot reflux with about 10L of 95% ethanol (2h multiplied by 2), then extracted by hot reflux with about 10L of 60% ethanol (2h multiplied by 2), concentrated under reduced pressure, the concentrated solutions are combined, about 5L of distilled water is added to prepare suspension, petroleum ether, chloroform, ethyl acetate and n-butanol are sequentially used for extraction, 934g of n-butanol extract is obtained, the suspension passes through a D-101 macroporous resin column, and gradient elution is sequentially carried out by water and 30%, 50%, 70% and 95% ethanol. The 30% ethanol eluted fraction (378g) was subjected to silica gel column chromatography and eluted with ethyl acetate-methanol gradient. Washing the obtained fraction Fr.10-25 with methanol, and (1) passing the obtained insoluble substance (800mg) through a C18 chromatographic column, and performing gradient elution with methanol-water. The obtained fraction Fr.1-13 (600mg) was passed through a C18 column and eluted with methanol-water (37.5: 62.5). Washing the obtained fraction Fr.9-11 with methyl ether to obtain HTF-1(316 mg); (2) the resulting mother liquor (27.6g) was subjected to silica gel column chromatography and chloroform-methanol gradient elution, and the resulting fraction Fr.16-18 was washed with methanol to give HTF-1(57 mg).

2.3 culture of cells

Culturing human cervical cancer HeLa cell, human breast cancer MCF-7 cell, human liver cancer HepG2 cell, and human normal liver cell L02 in DMEMCulturing in the medium. DMEM medium was supplemented with 10% Fetal Bovine Serum (FBS), 1% diabody (penicillin/streptomycin P/S), 1% glutamine. Cells were incubated at 37 ℃ with 5% CO₂Culturing under the condition.

2.4MTT assay

Control, experimental and blank groups were set. Collecting logarithmic phase cells, adjusting the concentration of cell suspension, adding 200. mu.L of culture medium to each well, plating to 8000 cells/well in 5% CO₂And incubating in a cell culture box at 37 ℃, adding a fresh drug-containing culture medium containing 3% FBS after the cells are cultured for 24 hours and adhere to the walls, setting 3 multiple holes for each drug concentration, and respectively detecting the cell viability conditions for 24 hours and 48 hours. After adding 20. mu.L of MTT (ready-to-use, 5mg/mL MTT solution in DPBS buffer) to each well and continuing the culture for 4 hours, the culture medium in the wells was discarded, and 200. mu.L of dimethyl sulfoxide (DMSO) was added to each well to dissolve the precipitate, and the mixture was placed on a shaker and shaken at a low speed for 10 minutes. Detecting the absorbance of each well at 490nm wavelength with an enzyme linked immunosorbent detector (A)₄₉₀nm). Cell viability ═ average a of experimental groups₄₉₀Average of blank groups A₄₉₀) V (average A of control group)₄₉₀Average of blank groups A₄₉₀) X 100%. The experiment was repeated three times. The concentration effect curve is plotted with the horizontal axis of drug concentration and the vertical axis of cell survival rate to determine the median Inhibitory Concentration (IC)₅₀)。

2.5 clone formation experiments

The experiment was divided into control and experimental groups. In 3cm cell culture dishes, 800 HeLa cells were inoculated per dish, and after 3-5 days of culture, the control group was not treated with HTF-1, and the experimental group was treated with 50nM HTF-1. The cells are continuously cultured for 7-8 days. During the cell culture period, the cells were changed every 3-4 days. Finally, after the colonies were formed, they were stained with 0.1% crystal violet and photographed. The experiment was repeated three times. Statistical analysis was performed on the number of colony formations. The control group was defined as 100%, and the relative colony formation rate of the experimental group was calculated.

2.6 wound healing experiments

The experiment was divided into control and experimental groups. HeLa cells were seeded in 6-well plates, the amount of cells was adjusted, cultured for 24 hours and allowed to growLong until complete fusion. Subsequently, a white plastic tip was used to scratch perpendicularly to the petri dish. The scraped loose HeLa cells were washed with fresh medium. Then adding fresh drug-containing medium containing 3% FBS, and adding 5% CO at 37 deg.C₂The cells were cultured in an incubator, and pictures of scratched areas of HeLa cells were taken with an optical inverted microscope at 0, 24, and 48 hours, respectively. The experiment was repeated three times and the cell mobility was calculated. Cell mobility (%) (average width of 0 hr scratch-24/48 hr scratch average width)/0 hr scratch average width 100%.

2.7 cell invasion assay

The experiment sets up a control group and an experimental group. Matrigel was thawed overnight at 4 ℃ and diluted 1:8 with pre-cooled serum-free medium. The cell chambers were placed in a 24-well plate, 40. mu.L of diluted matrigel was added to each chamber, and the gel was completely solidified by incubation in an incubator at 37 ℃ for 4 hours. HeLa cell suspensions were prepared in medium containing 3% FBS, counted and 5X 10 cells were added per well⁴Cells (experimental group cells were previously treated with HTF-1), the final volume in each chamber was 300. mu.L, and 600. mu.L of medium containing 10% FBS was added to the lower chamber. The cell culture plate was placed at 37 ℃ with 5% CO₂And (4) culturing for 48 hours in a cell culture box, and taking out. Cells were fixed with 4% paraformaldehyde in the dark for 30 min and washed 3 times with DPBS. Cells that failed to invade the chamber were carefully removed with a cotton swab and stained with 0.1% crystal violet stain for 15 minutes. And counted under a microscope and photographed. The control group was defined as 100%, and the relative cell invasion rate was calculated.

2.8 detection of apoptosis by annexin V and PI double staining

The experiment was divided into control and experimental groups, and HeLa cells were seeded in 6-well plates, treated with HTF-1 at different concentrations for 24 hours after the cells were attached, and then digested with trypsin without EDTA. HeLa cells were washed with DPBS and resuspended in 1 × binding buffer (10mM HEPES, 140mM NaCl, 2.5mM CaCl)₂0.1% bovine serum albumin, pH 7.4). Adjusting the cell density to 1X 10⁶Per mL, 100. mu.L of cells (1X 10) were added to each tube⁵Individual cell), and 5. mu. Lannexin V-FITC and 5. mu. were added theretoL Propidium Iodide (PI) solution and incubation for 15 min at room temperature in the absence of light. After the staining was completed, detection was performed using a flow cytometer (BD FACSCalibur).

2.9PI Monostain assay cell cycle

The experiment is divided into a control group and an experimental group, HeLa cells are inoculated in a 6-well plate, and after the cells are attached to the wall, the HeLa cells are respectively treated with HTF-1 with different concentrations for 24 hours. Then trypsinized, the cells were collected, washed with DPBS, suspended in 70% ice-cold ethanol and fixed overnight at 4 ℃. Subsequently, the cells were resuspended in DPBS containing 50U/mLRNA enzyme and 50. mu.g/mL Propidium Iodide (PI), and then incubated for 15 minutes in the absence of light. After staining was completed, cell cycle detection was performed using a flow cytometer (BD FACSCalibur).

2.10 Western blotting technique (Western blot, WB)

The experiment is divided into a control group and an experimental group, HeLa cells are inoculated in a 6-well plate, after the cells are attached to the wall, HTF-1 with different concentrations is respectively added to treat the cells for 24 hours, a culture medium is aspirated, the cells are digested by pancreatin, and total protein in the cells is extracted by RIPA strong lysate (protease inhibitor and phosphatase inhibitor are added when in use). After mixing the total protein and the loading buffer solution at a ratio of 1:1, boiling the mixture at 95 ℃ for 5 minutes, performing SDS-polyacrylamide gel electrophoresis, and transferring the mixture to a PVDF membrane. The membranes were blocked in PBST buffer containing 5% skim milk powder for 1 hour at room temperature. The primary antibody was then incubated overnight at 4 ℃. The secondary antibody was incubated at room temperature for 2 hours. Using the chemiluminescence (ECL) method, and by Sage Creation MiniChemi^TMThe imaging system performs exposure imaging.

2.11Hoechst H33342 staining

The experiment was divided into control and experimental groups. The cell slide was placed in a 6-well plate, and HeLa cells (3X 10)⁵/well) were seeded on cell slides in 6-well plates and kept overnight. HeLa cells were treated with different concentrations of HTF-1 for 24 hours. The cell slides were then washed three times with cold DPBS and fixed in cold methanol at-20 ℃ for 5 minutes. Then incubated with Hoechst H33342 for 15 minutes at room temperature in the dark, dried in the dark, and photographed using a fluorescence inverted microscope.

2.12Ki-67 staining fluorescence imaging

The experiment was divided into control and experimental groups. The cell slide was placed in a 6-well plate, and HeLa cells (3X 10)⁵/well) were seeded on cell slides in 6-well plates and kept overnight. HeLa cells were treated with different concentrations of HTF-1 for 24 hours. Thereafter, the cell slides were washed three times with cold DPBS and fixed in cold methanol at-20 ℃ for 5 minutes. And stained with Ki-67 for 2 hours at room temperature. After staining, cells were washed three times with DPBS. Staining with 5 μ M Hoechst H33342 and secondary antibody for 20 min in the dark. After drying in the dark, the photographs were taken using a fluorescent inverted microscope.

2.13EdU labelling assay

The experiment was divided into control and experimental groups. Log phase cells were collected, the concentration of the cell suspension was adjusted, HeLa cells (6000/well) were seeded into 96-well plates, and then treated with HTF-1 at various concentrations in the presence of a medium containing 3% FBS. At 5% CO₂After 24 hours of incubation at 37 ℃ in an incubator, cells were incubated for 4 hours with 100. mu. LEdU solution per well. Cells were washed 2 times 5 min each with DPBS. Cell immobilization: cells were fixed with 4% paraformaldehyde in the dark for 30 min at room temperature. Then 2mg/mL glycine was added to each well and incubated for 5 minutes in order to neutralize the paraformaldehyde and ensure the staining reaction system. Cells were then washed with DPBS. mu.L of penetrant (0.5% TritonX-100 DPBS) was added to each well, incubated for 10 minutes, and washed once with DPBS for 5 minutes. Apollo staining: add 100. mu.L per well

The staining reaction was incubated for 30 min at room temperature in the dark. Adding 100 mu L of penetrant into each hole, incubating for 10 minutes, adding 100 mu L of methanol into each hole, and washing for 2 times, each time for 5 minutes; DNA staining: each well was incubated with 100. mu.L of 5. mu.M Hoechst H33342 for 30 minutes at room temperature in the absence of light, followed by 3 washes with DPBS. Finally, the cell images were taken using a fluorescence inverted microscope.

2.14 scanning Electron microscope image

The experiment was set up as a control group and an experimental group. HeLa cells (1X 10)⁵/well) were seeded on cell slides in 6-well plates and kept overnight. Different concentrations of HTF-1 were added and incubated for 24 hours. Thereafter, willSlides were washed with DPBS and fixed with 2.5% glutaraldehyde for 2 hours at room temperature. The samples were washed twice more with DPBS for 10 minutes each. The cell slides were dehydrated twice in sequence with different concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%) for 15 minutes each time. The coverslip was allowed to dry overnight and then observed under a Hitachi SU1510 scanning electron microscope.

Third, result and discussion

3.1HTF-1 is capable of inducing apoptosis and inhibiting proliferation of cancer cells

First, we investigated HTF-1 antitumor activity in vitro using a cell model. FIG. 1A shows the chemical structure of HTF-1, and FIG. 1A shows that HTF-1 has a typical cardiac glycoside structure, having a sterol core, an unsaturated lactone ring, and a glucosyl group attached at the C-3 position. HTF-1 contains a six-membered unsaturated pyrone ring and should be defined as bufadienolide (bufadienolide). In FIGS. 1B, C and D, the in vitro anti-tumor activity of HTF-1 was studied using cell models including HeLa (human cervical cancer), MCF-7 (human breast cancer) and HepG2 (human liver cancer cell). FIGS. 1B, C and D show that HTF-1 is cytotoxic to all three cell lines in a time and dose dependent manner. And HTF-1 showed anticancer effects as low as 50nM for HeLa cells. IC of HTF-1 on HeLa cells₅₀Values were 546.3. + -. 145.4nM (24 h) and 158.0. + -. 44.9nM (48 h). Furthermore, IC of HTF-1 on MCF-7 cells₅₀IC values of 1025. + -. 228.6nM (24 h) and 238.7. + -. 58.9nM (48 h) for HepG2 cells₅₀Values were 1407. + -. 184.4nM (24 hours) and 220.9. + -. 35.2nM (48 hours). In contrast to the other two cell lines, HeLa cells were more sensitive to the effect of HTF-1, and we therefore decided to use HeLa cells as a cell model for subsequent studies. The ideal anti-cancer drug should be selectively toxic to cancer cells. As shown in FIG. 1E, HTF-1 was used to treat human hepatoma cell HepG2 and human normal hepatocyte L02 at different concentrations, and HTF-1 had strong cytotoxicity to HepG2 but very weak cytotoxicity to L02, indicating that HTF-1 had good selectivity.

FIG. 2A shows that the cells in the control group have good adherence, clear cell outline, good refractivity and vigorous cell growth. After treatment with 200nM and 500nM HTF-1, the cell morphology changed greatly: the cell volume generally decreased, the morphology became round, the cell-to-cell definitive junctions disappeared and detached from the surrounding cells, indicating that the cytoskeleton was damaged and adhesion was inhibited. As shown in FIG. 2B, the control fluorescence photograph shows that the nuclei of the live cells show a uniform distribution of bright blue fluorescence, while the HTF-1 treated cells show excessively bright nuclei. Arrows indicate nuclei in cells that have been over-stained by DNA fragmentation and chromatin shrinkage, which are characteristic of apoptotic cells. As shown in FIG. 2C, the control group HeLa cells were found to have dense microvilli around them. After HTF-1 treatment, HeLa cells shrink severely, microvilli become thinner and tend to disappear, and many protrusions and vesicles appear on the surface of cell membranes, which are also remarkable characteristics of apoptosis.

Flow cytometry was used to detect apoptosis using annexin V-FITC and PI double staining. As shown in fig. 3, each flow diagram consists of four quadrants, i.e., Q1 through Q4. Q1 (upper left quadrant), Q2 (upper right quadrant), Q3 (lower right quadrant) and Q4 (lower left quadrant) represent nude, late, early and live cells, respectively. The sum of Q2 and Q3 in the four quadrants of the flow chart is commonly used to indicate the extent of apoptosis. The results show that the apoptosis rate of the HTF-1 experimental group is remarkably increased compared with that of the control group, and the HTF-1 experimental group has dose dependence.

3.2HTF-1 is able to cause cancer cell cycle arrest in S phase

FIG. 4 shows the effect of HTF-1 treatment on cell cycle distribution 24 hours after HeLa cells. The S phase is the period of DNA replication of the cells, and the low dose group (100nM HTF-1) had less S phase cell accumulation and the higher dose group (200nM HTF-1) had about 73% increase in S phase cells compared to the control. The percentage of cells in S phase after HTF-1 treatment increased, indicating that growth inhibition of HeLa cells by HTF-1 was associated with cell cycle arrest in S phase. And as shown in FIG. 4C, HTF-1 was able to induce typical Sub-G1 apoptotic peaks in HeLa cells.

3.3HTF-1 inhibits the proliferation, migration and invasion of cancer cells

To investigate the effect of HTF-1 on cancer cell proliferation, a clonogenic experiment was performed. The colony formation assay measures the ability of individual cells to form cell colonies, and the ability of cancer cells to produce colonies when plated at low density reflects their proliferative potential. We examined the effect of HTF-1 on the proliferative capacity of individual HeLa cells using a clonogenic assay. The number of cell colonies formed by the control group was much greater than the 50nMHTF-1 experimental group as shown in FIG. 5A. In addition, the size of the single cell colony, as shown in FIG. 5B, was much smaller in the experimental group than in the control group. Statistical analysis showed significant differences in colony formation rates between the control and experimental groups (fig. 5C).

Ki-67 is a nuclear protein closely associated with cell proliferation and is considered to be a marker of cell proliferation. EdU (5-ethynyl-2-deoxyuridine) is a thymidine analogue that can be incorporated into replicating DNA molecules during cell proliferation instead of thymine and is widely used to monitor DNA synthesis. Therefore, we used Ki-67 staining experiments (FIG. 5D) and EdU labeling experiments (FIG. 5E) to assess the effect of HTF-1 on cancer cell proliferation viability. The experimental results show that with increasing HTF-1 dose, Ki-67 and EdU fluorescence decrease, indicating that cell proliferation is inhibited after HTF-1 treatment.

The above results indicate that HTF-1 can effectively inhibit the proliferation of tumor cells.

An in vitro wound healing assay was used to assess the effect of HTF-1 on cancer cell migration. Wound healing experiments are a well-established and low-cost technique for cellular experiments to study cancer cell migration under different treatment conditions. As shown in fig. 6A, the cells of the control group continued to grow, and the gaps of the scratch became narrow at 24 hours and 48 hours, indicating strong migration ability of the cancer cells. While the HTF-1 treated group migrated slowly at 24 and 48 hours, indicating that HTF-1 had the ability to inhibit HeLa cell migration in vitro. The transwell experiment was used to evaluate the effect of HTF-1 on HeLa cell invasion. This experiment is a tumor cell invasion model, and tumor cell invasion capacity is evaluated by counting the number of cells that cross the matrix membrane. The cells in the control group were able to cross the matrix membrane as shown in fig. 6B, indicating that HeLa cells have a strong invasive ability. And the number of the cancer cells passing through the matrix membrane is obviously reduced in the 50nM HTF-1 experimental group, which shows that the HTF-1 can obviously inhibit the invasive capability of the cancer cells.

3.4HTF-1 is able to activate caspase 9 and JNK and inhibit ERK and PI3K/Akt/mTOR pathways

Further study on the molecular mechanism of HTF-1 acting on tumor cells was carried out. As shown in FIG. 7A, when HeLa cells were treated with HTF-1 at 50nM to 200nM, both

caspases

9 and 3 were activated and cleaved. Therefore, we determined that HTF-1 causes HeLa cell apoptosis through the classical caspase 9/3 pathway, i.e., the mitochondrial pathway. Caspase 7, like Caspase 3, also functions as an apoptosis executor. As shown in FIG. 7A, caspase 7 was also cleaved, suggesting that it also plays an important role in HTF-1 induced apoptosis of HeLa cells.

Double-stranded DNA breaks (DSBs) are widely considered to be the result of apoptosis, while γ H2AX is a marker of DNA double-stranded breaks. As shown, the expression level of γ H2AX increased with increasing dosing concentration, indicating that HTF-1 acting on cancer cells can significantly cause DNA double strand breaks.

Flap endonuclease 1(FEN1) is a structure-specific endonuclease that is important for the late-strand DNA replication, long-fragment base excision repair, and ribonucleotide excision repair pathways, making it the central link in maintaining genomic stability. Cancer cells often over-activate the DNA repair system to repair unlimited DNA replication, and overexpression of FEN1 is seen in several cancers. As shown in fig. 7A, the expression level of FEN1 decreased with the increase of the dosing concentration, which resulted in the aggravation of HeLa cell DNA damage, and thus HTF-1 showed the ability to inhibit cancer cell proliferation.

PARP (poly (ADP-ribose) polymerase) is a major cleavage target of caspase 3 and caspase 7 in vivo and plays a key role in DNA damage repair and apoptosis. Cleaved PARP has its DNA repair capacity inactivated, leading to apoptosis, and also plays a role in cancer treatment as a drug target for chemotherapeutic drugs. As shown, the PARP splice is increased in the cells with increasing HTF-1 concentration. Survivin is a member of the Inhibitor of Apoptosis Protein (IAP) family, and functions as an inhibitor of caspase 3. As shown, Survivin expression was down-regulated with increasing dosing concentration. Bcl-2 is considered to be an important anti-apoptotic protein that functions in mitochondria, inhibiting the release and transfer of mitochondrial cytochrome c. Bcl-2 expression was down-regulated with increasing concentrations of HTF-1 treated groups. The above results indicate that the down-regulation of PARP, Bcl-2 and Survivin in cells all contribute to the anti-cancer activity of HTF-1.

Further examine the effect of the relevant protein pathways in HeLa cells after HTF-1 treatment. The phosphorylation levels of JNK, ERK and p38 were first examined. As shown in fig. 7B, phosphorylated JNK was upregulated, whereas ERK activation was inhibited and phosphorylated p38 was barely affected. The mitogen-activated protein kinase (MAPK) superfamily contains extracellular signal-regulated protein kinase 1/2(ERK1/2), C-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK). These are key proteins in cancer development that regulate cancer cell differentiation, proliferation, migration, metastasis, apoptosis, and inflammation. Notably, the role of the MAPK superfamily in cancer treatment is complex, as they are linked to cell type, drug concentration, drug mechanism, and even apoptosis and cell survival time.

The phosphatidylinositol 3-kinase (PI 3K)/protein kinase b (akt)/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of the balance between cellular function, survival and apoptosis. It plays a crucial role in tumorigenesis and in the course of therapy. Activation of this pathway results in cell proliferation, stimulation of mitosis, metastatic capacity, angiogenesis, and, at the same time, inhibition of apoptosis and reduced therapeutic efficacy. As shown in FIG. 7C, HTF-1 inhibited the phosphorylation of PI3K, Akt, mTOR, and p70S6 kinase. It is shown that HTF-1 can inhibit this pathway to induce apoptosis in cancer cells.

Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Claims

1. The application of a novel cardiac glycoside monomeric compound in the preparation of preparations for inducing apoptosis of HeLa cells of human cervical carcinoma, MCF-7 cells of human breast cancer and HepG2 cells of human liver cancer is disclosed, wherein the structural formula of the novel cardiac glycoside monomeric compound is as follows:

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