CN107099576A - A kind of applications of cell-cycle arrest agent KR in human breast cancer cell - Google Patents

Abstract

The invention provides a kind of applications of cell-cycle arrest agent KR in human breast cancer cell, belong to Biochemistry and Molecular Biology technical field.A kind of applications of cell-cycle arrest agent KR in human breast cancer cell, step is as follows：(1) cell recovery；(2) cell culture；(3) medicine is prepared；(4) cell is handled；(5) cell proliferation inhibition rate is detected；(6) cell cycle is detected.Beneficial effects of the present invention：The data foundation of the present invention can raise CDKN1A in KR and cause ATP to exhaust, cause DNA biosynthesis blocks, and cell-cycle arrest has on the basis of in-vitro multiplication inhibitory activity of highly significant in the S phases.

Description

Application of a cell cycle blocker KR in human breast cancer cells

technical field

The invention relates to the anti-tumor effect of cytokinin kinetin riboside (KR) as a cell cycle blocker on breast cancer cell lines, and belongs to the technical field of biochemistry and molecular biology.

Background technique

Breast cancer is a malignant tumor that occurs in the glandular epithelial tissue of the breast. According to statistics from the International Agency for Research on Cancer (IARC) of the World Health Organization, there were about 1.55 million new cases of breast cancer in women worldwide in 2015, accounting for all women. About 25% of the incidence of malignant tumors; death due to breast cancer accounts for about 15% of all female malignant tumor deaths, accounting for 2% of all female deaths. Like most other countries, breast cancer has become the most common cancer in Chinese women. In Shanghai and other eastern coastal cities, breast cancer has surpassed lung cancer to become the cancer with the highest incidence rate in women; currently, the number of new breast cancer cases and deaths in China every year Accounting for 12.2% and 9.6% of the world respectively. Conventional drugs clinically used to treat breast cancer have significant toxic and side effects, although they play an important role in the treatment. It has been proved by a lot of practice that using modern technology, the active ingredients of natural active substances obtained from natural products can treat breast cancer while reducing the toxic and side effects of radiotherapy and chemotherapy. Therefore, it is very necessary to find natural medicines that effectively improve the treatment efficiency and have little toxic and side effects to treat breast cancer.

Cytokinin CK (cytokinin) is a type of plant hormone discovered in the 1950s. It plays a decisive role in the growth and development of plants, such as: promoting cell division, tissue differentiation, lateral bud growth, inhibiting adventitious roots, Lateral root formation, inhibition of senescence, regulation of plant apical dominance, transmission of nutrient quotes, etc., naturally occurring cytokinins are derivatives of adenine, which are formed by replacing the H at the N6 position of purine with other groups. Activity varies widely with side chain length, degree of unsaturation, and other properties. Cytokinins exist in the following forms: free, nucleoside, nucleoside-5' phosphate, 3-, 7-, 9- and O-glycosides and amino conjugates. Common naturally occurring cytokinins are: zeatin (Zeatin, ZT), zeatin riboside (Zeatin riboside, ZR), dihydrozeatin (dHZT), isopentenyl adenosine (i6A ). At present, more and more studies have found that CK not only regulates the growth and development of plants, but also plays an important role in the proliferation and differentiation of animal cells.

KR (kinetin riboside) is a nucleoside form of cytokinin CK that exists in plants. Its common name is kinetin nucleoside, its molecular formula is C ₁₅ H ₁₇ N ₅ O ₅ , and its molecular weight is 347.3. The common structural feature of nucleoside compounds is composed of sugar groups and bases. From the chemical structure, there are different degrees of similarities between natural nucleosides and nucleoside compounds, so it is speculated that KR can also interfere or directly act on proteins, Nucleic acid biosynthesis, which interferes with tumor cell and viral replication, plays an important role in antitumor and antiviral aspects. According to experiments, KR is an anti-proliferative agent that can induce apoptosis in some cell lines. The principle is that it can up-regulate CDKN1A to cause ATP depletion, resulting in cell cycle arrest in G2/M phase. It has strong toxic and pro-apoptotic effects on cancer cells in vitro. When breast cancer cells are treated with KR, cell cycle arrest and/or apoptosis are caused in different degrees.

Contents of the invention

The present invention aims at the above-mentioned prior art, and the present invention provides the anti-tumor application of KR in human breast cancer cell lines.

Technical scheme of the present invention:

A kind of application of cell cycle blocker KR in human breast cancer cells, the steps are as follows:

(1) Cell recovery: Take out the cryopreservation tube containing the cryopreserved human breast cancer cell line MCF7 from the -80°C refrigerator, quickly put it in a 37°C water bath, shake it slightly, and wait for the liquid to melt; The human breast cancer cell line MCF7 was suspended in a centrifuge tube containing sterile RPMI 1640 culture medium containing 10% inactivated fetal calf serum, centrifuged at 1000rpm for 5min, after centrifugation, discard the supernatant, flick the cells gently, and enter Suspend the cells in sterile RPMI 1640 culture medium and a centrifuge tube, transfer them to a culture bottle and shake gently left and right to make the cells in the culture bottle evenly distributed, and obtain the revived breast cancer cell line MCF7;

(2) Cell culture: Place the revived human breast cancer cell line MCF7 in an incubator with a concentration of 5% CO ₂ , a relative humidity of 90%, and a temperature of 37°C, and observe the cells after 10 hours. If they have adhered to the wall, Aspirate all the culture medium, add the same amount of RPMI 1640 culture medium again; wait for the cells to grow to 70%-80% of the bottom area of the culture flask, and passage once;

(3) Drug preparation: dissolve the KR powder with aseptic serum-free RPMI 1640 medium, so that the mother solution concentration of the KR solution reaches 1.2 mM, and continue to dilute the mother solution of the dissolved KR solution to The concentration is 2μM-200μM, filter sterilized, and store at room temperature;

(4) Treatment of cells: Digest the cells in the logarithmic growth phase with trypsin, count the cells, dilute the cells to a density of 3×10 ⁴ -5×10 ⁴ cells/ml, mix them by pipetting, and inoculate the cells in In a 96-well plate, 100 μl of human breast cancer cell line MCF7 was taken from each well. After 12 hours, after the cells had adhered to the wall, 7 groups of experiments were set up, with 4 replicate holes in each group. Group 1: blank control group; Group 2: 120 μM KR The 3rd group: 20 μ M KR group; The 4th group: 10 μ M KR group; The 5th group: 2 μ M KR group; The 6th group: 1 μ M KR group; The 7th group: 0.2 μ M KR group; After lightly mixing, incubate for 2 days in an incubator with a concentration of 5% CO ₂ , a relative humidity of 90%, and a temperature of 37°C to detect the inhibitory activity of cell proliferation;

(5) Detection of cell proliferation inhibition rate: the cells treated in step (4) were incubated in an incubator with a concentration of 5% CO ₂ , a relative humidity of 90%, and a temperature of 37°C for 48 hours, with 4 replicate wells each ;Use MTT kit for detection, add 20 μl MTT solution to each well of cell suspension, place in 37°C, CO ₂ incubator and incubate for 3 hours, measure the absorbance of each group of cells at 490nm, and record the data;

(6) Cell cycle detection: Digest the cells in the logarithmic growth phase with trypsin, count the cells, take an appropriate amount of cells to dilute, the diluted cell density is 1×10 ⁵ to 1.5×10 ⁵ cells/ml, and mix well by pipetting , the cells were inoculated in a 6-well plate, and 2ml of human breast cancer cell line MCF7 was taken from each well. After 12 hours, after the cells were attached to the wall, a blank control group and a 10 μM KR group were set, and KR was added to the 10 μM KR group, so that the concentration of KR was 10 μM, mixed gently, placed in an incubator with a concentration of 5% CO ₂ , a relative humidity of 90%, and a temperature of 37°C for 36 hours, and divided into two samples with a 15ml centrifuge tube, according to the cell cycle and apoptosis Instructions for Death Detection Kit Fix cells and prepare propidium iodide staining buffer. After 12 hours, add PBS to each sample to wash several times, resuspend cells, add required propidium iodide staining buffer to each sample and incubate at 37°C in the dark 30min, and finally washed twice with PBS; 300-mesh sieve was used for detection on a BD FACSCalibur flow cytometer after filtration.

Beneficial effects of the present invention: The data of the present invention are based on the fact that KR can upregulate CDKN1A to cause ATP depletion, resulting in blocked DNA synthesis, cell cycle arrest in S phase, and very significant anti-proliferation activity in vitro.

Description of drawings

Figure 1 shows the inhibitory effect of different concentrations of KR on the proliferation of human breast cancer cells.

Figure 2 shows the effect of KR on the cell cycle of human breast cancer cells. 2(a) Control group. 2(b) Cell cycle of MCF7 cells after 10 μM KR treatment for 36 h.

detailed description

The specific implementation manners of the present invention will be further described below in conjunction with the accompanying drawings and technical solutions.

Cells, kits and reagents used in the present invention: human breast cancer MCF7 cell line, fetal bovine serum, double antibody (penicillin/streptomycin), RPMI 1640 medium, MTT kit, cell cycle and apoptosis staining kit, KR.

Example 1

In vitro proliferation inhibitory activity of KR on human breast cancer cells: the revived human breast cancer cells MCF7 were cultured in sterile RPMI1640 containing 10% FBS inactivated fetal bovine serum, 1% penicillin/1% streptomycin (double antibody) cultured in an incubator at 37°C, 5% CO ₂ and saturated humidity, and the cells grew to about 70%-80% and were passaged once. Continue culturing to obtain human breast cancer cells in an active growth state, and collect the cells. Inoculate 5× ¹⁰³ cells in the logarithmic growth phase in a 96-well plate containing 100 μl RPMI 1640 culture medium per well, and add 10 μl of 120 μM, 200 μM, 100 μM, 20 μM, 10 μM, 2 μM concentrations according to the experimental requirements KR, put them in the incubator and incubate for 2 days, each with 4 duplicate wells. The MTT kit was used for detection, and 20 μl of MTT solution was added to each well of cells, and placed in a CO ₂ incubator at 37° C. for 3 hours. The absorbance of each group of cells at 490nm was measured, the data was recorded, and the data of each group was analyzed by variance analysis method of repeated measures data, the results are shown in Figure 1. It can be seen from Fig. 1 that the proliferation rate of the human breast cancer cell line MCF7 was significantly lower than that of the control group by the application of different concentrations of KR, indicating that KR can inhibit the proliferation rate of the human breast cancer cell line MCF7.

Example 2

KR induces apoptosis of MCF7 cells: Digest the above-mentioned cultured cells in the logarithmic growth phase with trypsin, count the cells, take an appropriate amount of cells to dilute, the diluted cell density is 1×10 ⁵ ~1.5×10 ⁵ cells/ml, pipette Mix evenly, inoculate the cells in a 6-well plate, take 2ml of human breast cancer cell line MCF7 from each well, and after 12 hours, after the cells adhere to the wall, set up a blank control group and a 10 μM KR group, and add 200 μl of 100 μM KR to the 10 μM KR group (the working concentration is 10 μM KR), mixed gently, placed in an incubator with a concentration of 5% CO ₂ , a relative humidity of 90%, and a temperature of 37°C for 36 hours, and divided into two samples in a 15ml centrifuge tube. Apoptosis Detection Kit Instructions Fix the cells and prepare propidium iodide staining buffer. After 12 hours, add 10ml PBS to each sample to wash twice, resuspend the cells, add 500μl propidium iodide staining buffer to each sample and incubate at 37°C in the dark 30min, and finally washed twice with PBS; 300-mesh sieve was used to filter and then detected by BD FACSCalibur flow cytometer; the results are shown in Figure 2, and it can be seen from Figure 2 that compared with the control group, the application of KR can up-regulate CDKN1A and cause ATP Depletion leads to the blockage of DNA synthesis and cell cycle arrest in S phase, which has strong toxic and pro-apoptotic effects on human breast cancer cell line MCF7 in vitro. When breast cancer cell MCF7 is treated with KR, it leads to cell cycle arrest and/or apoptosis.

Claims (1)

1. applications of a kind of cell-cycle arrest agent KR in human breast cancer cell, it is characterised in that step is as follows：

(1) cell recovery：The cryopreservation tube that will be equipped with freezing Breast cancer lines MCF7 takes out from -80 DEG C of refrigerators, puts rapidly Enter in 37 DEG C of water-baths, gentle agitation, treat that liquid melts；By the Breast cancer lines MCF7 in cryopreservation tube be suspended in containing In the centrifuge tube of the sterile RPMI 1640 culture mediums of 10% inactivated fetal bovine serum, 1000rpm, 5min centrifugations, after centrifugation terminates, Supernatant is abandoned, cell of gently upspringing enters sterile RPMI 1640 culture mediums with centrifuge tube, cell being suspended, is transferred to again Left and right gently shakes in blake bottle, is uniformly distributed the cell in blake bottle, the breast carcinoma cell strain MCF7 recovered；

(2) cell culture：The Breast cancer lines MCF7 of recovery is placed in concentration for 5%CO₂, relative humidity be 90%, temperature To be cultivated in 37 DEG C of incubators, cell is observed after 10h, if adherent, nutrient solution is all suctioned out, equivalent is added again RPMI 1640 culture mediums；Treat cell length to blake bottle floor space 70%~80% pass on 1 time；

(3) medicine is prepared：KR powder is dissolved with the sterile culture mediums of RPMI 1640 without serum, makes the mother liquor of KR solution dense Degree reaches 1.2mM, continue with the sterile culture mediums of RPMI 1640 by the mother liquor of KR solution dissolve be diluted to concentration be 2 μM- 200 μM, filtration sterilization, room temperature preservation；

(4) cell is handled：Cell in exponential phase is digested with pancreatin, after cell count, dilution, diluting cells density For 3 × 10⁴~5 × 10⁴Individual/ml, piping and druming is mixed, and cell is inoculated in 96 orifice plates, and 100 μ l Breast cancer lines are taken per hole After MCF7,12h, after after cell attachment, 7 groups of experiments, every group of 4 multiple holes, the 1st group are set：Blank control group；2nd group：120μΜ KR groups；3rd group：20 μ Μ KR groups；4th group：10 μ Μ KR groups；5th group：2 μ Μ KR groups；6th group：1 μ Μ KR groups；7th group： 0.2 μ Μ KR groups；The corresponding medicines of 10 μ l are added into each group respectively, after gently mixing, concentration are placed in for 5%CO₂, it is relatively wet Degree is to be incubated 2 days in the incubator that 90%, temperature is 37 DEG C, detection cell inhibitory effect activity；

(5) cell proliferation inhibition rate is detected：By the cell after step (4) processing, concentration is placed in for 5%CO₂, relative humidity be 90%th, temperature is is incubated 48h, each 4 multiple holes in 37 DEG C of incubator；Detected using MTT kits, per hole cell suspension In plus 20 μ l MTT solution, be placed in 37 DEG C, CO₂3h is incubated in incubator, the absorbance of each group cell at 490nm, record is determined Data；

(6) cell cycle is detected：Cell in exponential phase is digested with pancreatin, after cell count, takes appropriate cell to enter Row dilution, diluting cells density is 1 × 10⁵~1.5 × 10⁵Individual/ml, piping and druming is mixed, and cell is inoculated in 6 orifice plates, is taken per hole After 2ml Breast cancer lines MCF7,12h, after after cell attachment, blank control group and 10 μ Μ KR groups are set, to 10 μ Μ KR is added in KR groups, the concentration for making KR is 10 μ Μ, after gently mixing, be placed in concentration for 5%CO₂, relative humidity be 90%, temperature Spend in the incubator for 37 DEG C and be incubated 36h, two samples are distributed into 15ml centrifuge tubes, detect and try according to cell cycle and apoptosis Agent box specification is fixed cell and prepared after the dye solution of iodate third, 12h, and PBS is added in each sample and is washed repeatedly, is resuspended thin Born of the same parents, 37 DEG C of lucifuges of the third dye solution of iodate are incubated 30min needed for each sample is added, and are finally washed twice with PBS；300 mesh sieves With machine testing on BD FACSCalibur flow cytometers after net filtration.