CN105670998A - Method for calcification of cancer cells - Google Patents

Abstract

The invention discloses a method for calcifying cancer cells, which comprises the following steps: (1) inoculating the cancer cells in a medium containing folic acid and incubating them to obtain folic acid-enriched cancer cells on the surface; (2) inoculating the folic acid-enriched cancer cells on the surface The cancer cells were incubated in calcification solution to obtain calcified cancer cells. The invention also discloses the application of folic acid in the preparation of cancer cell calcification inducer. The present invention is based on the characteristics that cancer cells highly express folic acid receptors and folic acid carboxyl provides nucleation sites for calcification, forming a calcium phosphate calcification layer on the surface of cancer cells, which can inhibit cell activity, destroy the structure of cell membranes and eventually lead to the death of cancer cells; Experiments show that the calcification treatment of the present invention has no effect on blood cells, no hepatotoxicity, almost no damage to organs, and has good biological safety. Therefore, the technical solution of the present invention provides a feasible solution for tumor treatment.

Description

A method for calcifying cancer cells

technical field

The invention relates to the field of biotechnology, in particular to a method for calcifying cancer cells.

Background technique

Cancer is one of the diseases with the highest mortality rate in the world, and maintains a high incidence rate. At present, the methods of cancer treatment recognized by the international medical community are still traditional chemotherapy and radiotherapy. However, while radiotherapy and chemotherapy drugs kill cancer cells, they will cause serious damage to the body, such as kidney failure, hair loss, weight loss, etc. In addition, tumor metastasis is an important cause of death in cancer patients, and a high proportion of patients die from organ failure caused by tumor metastasis, while chemoradiotherapy drugs cannot effectively inhibit tumor metastasis. According to statistics, the annual cost of cancer treatment is very high, and it is increasing year by year, causing serious economic losses and psychological burdens to patients' families.

Although there are a variety of emerging technologies for cancer treatment, such as the use of drug carriers, by controlling the size of the carrier and modifying the tumor-specific recognition molecules on the surface, the drug can be enriched in tumor tissue, but the carrier is immunogenic. When the carrier enters the blood, it is quickly recognized and cleared by the body's immune system, thus losing its therapeutic effect. In recent years, with the deepening of scientific research, new drugs have continuously entered the market. The anticancer drug (Nivolumab) launched in 2015 is a small molecule drug designed based on the programmed cell death protein PD-1, and has achieved remarkable results in clinical trials. There are no obvious side effects, but the high cost of these drugs in the research and development stage makes the price of the drugs relatively expensive. According to statistics from the National Institutes of Health of the United States, by 2005, the cost of cancer treatment in the United States was about 209.4 billion yuan. In 2014, the cancer expenditure accounted for about 20% of the gross national product of medical care. It can be seen that patients When using this drug, you will bear high medical expenses. Tumor metastasis is an important reason why it is difficult to cure. Among many anticancer drugs, there is a serious shortage of drugs that inhibit tumor metastasis, which makes many patients helpless when metastases appear again after treatment. Therefore, it is urgent to find a safe, effective and economical method to inhibit tumor growth and metastasis.

In the human body, calcification plays an important role in the growth of bones and teeth, but pathological calcification of blood vessels can lead to cell dysfunction, eventually leading to a series of diseases, such as chronic kidney disease and cardiovascular disease. If calcification is introduced to the surface of cancer cells, it may inhibit the activity of cancer cells or eventually lead to the death of cancer cells, thus providing a new idea for cancer treatment that does not rely on drugs. In our previous research, we used chemical modification to achieve calcification of yeast cell walls. Calcification would cause yeast cells to enter a dormant period and stop dividing. This is due to the good stress resistance of yeast cells. Compared with mammalian cells, it was found that The cell membrane of mammalian cells is relatively fragile and has no cell wall protection, and some studies have shown that after the siliconization of the cell surface forms a layer of SiO2, the time of the cell does not exceed 12h, indicating that the formation of a layer of minerals on the outer layer of the cell membrane will seriously affect the activity of mammalian cells. Different from normal tissue cells, there are many specifically expressed molecular receptors on the surface of cancer cells, such as folate receptor (FR), and FR is almost not expressed in normal tissue cells, and its ligand folic acid (FA), just contains Carboxyl group that can promote calcification. Therefore, it will be an effective means to inhibit tumor growth and metastasis by taking advantage of the high expression of folic acid receptors in cancer cells and finding ways to cause calcification of cancer cells.

Contents of the invention

The invention provides a method for calcifying cancer cells, using the method to form a mineral layer on the surface of cancer cells to achieve the purpose of inhibiting the activity of cancer cells.

A method for calcifying cancer cells, comprising the steps of:

(1) Inoculating cancer cells in a medium containing folic acid and incubating them to obtain cancer cells whose surfaces are enriched in folic acid;

(2) Incubating the cancer cells enriched in folic acid on the surface in the calcification solution to prepare calcified cancer cells.

Based on the characteristic of highly expressing folic acid receptors on the surface of cancer cells, the invention inoculates the cancer cells into the culture medium containing folic acid, enriches the folic acid on the surface of the cancer cells, and increases the carboxyl content on the surface of the cancer cells. The carboxyl group provides active sites for calcification of nucleation and crystal growth, leading to calcification of cancer cells.

According to the technical method of the present invention, the calcified cancer cells obtained after the treatment are characterized by scanning electron microscopy and energy spectrum elemental analysis. cell membrane surface. Calcium phosphate covers the surface of cancer cells, restricts the physiological activities of cancer cells, significantly inhibits the activity of cancer cells, and eventually leads to the rupture of cell membranes.

Preferably, the operation step (2) is repeated several times to obtain calcified cancer cells. Add fresh calcification solution repeatedly, so that calcium ions and phosphate ions react and precipitate continuously on the carboxyl sites, and deposit more calcium phosphate on the surface of cancer cells. More preferably, repeat the operation step (2) 2 to 5 times.

Preferably, the cancer cells are human cervical cancer cells (HeLa), human breast cancer cells (MCF7) or human ovarian cells (SKVO-3). Studies have shown that the folate receptors of these three types of cells are overexpressed, which is conducive to the deposition of calcified layers. Cervical cancer, breast cancer and ovarian cancer are common malignant tumors in women. Calcification of these three types of cancer cells has guiding significance for the treatment of tumors.

In order to enrich folic acid to the greatest extent, cancer cells need to be fully contacted with the culture medium, so the inoculation density of cancer cells should not be too large. Preferably, the inoculation density of cancer cells is 40-80% of the bottom surface area of the culture well plate. More preferably, the inoculation density accounts for 60-70% of the area of the bottom surface of the culture well plate. This not only ensures effective contact between the cancer cells and the culture medium, but also makes full use of the culture well plate.

Preferably, cancer cells are washed 2-3 times with phosphate buffer before inoculation. Wash cancer cells before calcification treatment to avoid other substances from affecting the calcification effect.

Folic acid provides active sites for nucleation and crystal growth for calcification. Therefore, it is necessary to ensure that the medium contains a sufficient amount of folic acid. Preferably, in step (1), the content of folic acid in the medium is 0.2-1.2 mg/mL. The culture medium is a conventional cancer cell culture medium. Such as DMEM medium. More preferably, the content of folic acid in the medium is 0.5-1 mg/mL.

Preferably, in step (1), the incubation time is 5-30min. More preferably, the incubation time is 10-25min.

Preferably, in step (2), the calcification solution is a DMEM medium with a calcium ion concentration of 5-20 mM.

DMEM medium is used as a phosphate source, and calcium phosphate (CaP) calcification layer is formed on the cell membrane surface by adding calcium ions. Calcium ion concentration is too low, it is difficult to form a precipitate, but higher than 20mM will have toxic side effects on cells. More preferably, the calcium ion concentration in the DMEM medium is 10-15mM.

Preferably, in step (2), the incubation time is 0.25-2h. The incubation time should not be too long, as the time prolongs, the toxicity of calcium ions to the cells will also increase. More preferably 0.5-1h.

Carbon dioxide accelerates the formation of calcium phosphate. Therefore, when carbon dioxide is introduced during incubation, a small amount of carbon dioxide will dissolve in the liquid and combine with folic acid-rich calcium ions to promote the formation of precipitates. Preferably, the incubation conditions include a carbon dioxide concentration of 4-10%.

The invention also provides the application of folic acid in the preparation of cancer cell calcification inducer.

Beneficial effects of the present invention: (1) The present invention is based on the characteristics that cancer cells highly express folic acid receptors and folic acid carboxyl provides nucleation sites for calcification, and forms a calcium phosphate calcification layer on the surface of cancer cells, which can inhibit cell activity and destroy cell membranes and eventually lead to the death of cancer cells; (2) animal experiments show that the calcification treatment of the present invention has no effect on blood cells, has no hepatotoxicity, and has almost no damage to organs, and has better biological safety. Therefore, the calcification of the present invention The technical solution provides a feasible solution for tumor treatment.

Description of drawings

Figure 1 shows the results of optical microscope observation after calcification of human normal HEK293 cells and human cervical cancer cell HeLa.

Figure 2 is the SEM observation (A) and the imaging results of calcium and phosphorus in HeLa calcified cells after calcification of human normal HEK293 cells and human cervical cancer cell HeLa (B).

Figure 3 is the SEM magnified observation of CaP in the calcified layer of HeLa cells, where A is magnified 5000 times, and B is magnified 50000 times.

Fig. 4 is a graph showing the characterization results of the calcified cell sample, wherein A is X-ray diffraction analysis (XRD), and B is Fourier transform infrared spectroscopy analysis (FTIR).

Figure 5 shows the confocal observation results of HeLa cells at different times after calcification, in which the green fluorescence is the calcium phosphate in the calcified layer (stained with calcein), the red is the cell membrane (stained with PKH26), and the blue is the nucleus (stained with Hoechest33342).

Figure 6 is the results of life-and-death staining of HeLa cells 24 hours after calcification (A) and the MTT results of the survival rate of HEK293 and HeLa cells 24 hours after calcification (B). Living cells, the figure below shows the staining results of HeLa cells after 24 hours of calcification, and the results show that they are dead cells.

Figure 7 shows the results of targeted calcification of tumor tissue in mice, where picture A shows the optical photographing results of tumor tissue in different treatment groups; picture B shows the micro-CT results of calcified tumor tissue; picture C shows the TEM of tumor tissue slices after calcification Observation results, the red double-headed arrow indicates the intercellular space, and there is a large amount of calcium phosphate between the cells; D is the laser confocal result of the tumor tissue section after fluorescent staining, and the blue is the nucleus stained by Hoechst33342.

Figure 8 shows the safety evaluation of targeted calcification on mice, where A is the results of blood routine analysis of mice; B is the results of biochemical analysis of liver function in mice; C is the results of HE staining of major organs of mice.

Figure 9 shows the results of targeted calcification for anti-tumor experiments in mice, where A is the volume change of tumor growth in mice in different treatment groups; B is the weight change of mice; C is the HE staining results of tumor tissues in mice.

detailed description

The present invention will be further described below in conjunction with specific examples. These examples are for illustrative purposes only and are not intended to limit the scope of use of the present invention. In the experiment, the DMEM medium was a commercial product, and the folic acid (FA), inorganic salts, small organic molecules and proteins used were imported reagents of chemical purity or above.

Example 1

1. Preparation of targeting calcified cancer cells—A

Spread the HeLa cells in a 24-well plate, the number of cells per well is about 2×10 ⁵ , and the calcification process starts when the cells grow to about 70% of the bottom area of the well plate. First pour off the culture medium of the cells, wash with PBS 1-2 times, then add DMEM medium containing 300 μg/mLFA to the well plate, place the cells in an incubator, adjust the _CO2 concentration to 5%, and the air to 95%. After incubation at 37°C for 15 minutes, add the calcification solution of DMEM medium containing 11.8mMCa ²⁺ (including newly added 10mMCaCl ₂ ) to the well plate, place the well plate in a cell culture incubator for calcification treatment for 30 minutes, and suck off the calcification The solution was added with fresh calcification solution to continue the treatment for 60 minutes. The FA and calcification solution were treated alone as a control, and the solution was sucked off after the treatment to obtain calcified cancer cells.

3. Preparation of Targeted Calcification Cancer Cells-B

Spread the HeLa cells in a 24-well plate, the number of cells per well is about 2×10 ⁵ , and the calcification process starts when the cells grow to about 70% of the bottom area of the well plate. First, replace the medium of the cells, wash them with pH 7.2 phosphate buffer for 1-2 times, then add additional DMEM medium with 200 μg/mLFA to the well plate, and then place the cells in the incubator to adjust CO ₂ The concentration is 5%, the air is 95%, the temperature is 37°C, incubate in the incubator for 20 minutes, suck off the medium, and add 21.8mMCa ²⁺ (including newly added 20mMCaCl ₂ ) to the well plate for calcification of DMEM medium solution, and place the plate in a cell culture incubator for calcification for 30 min. The FA and calcification solution were treated alone as a control, and the solution was sucked off after the treatment to obtain calcified cancer cells.

3. Preparation of targeting calcified cancer cells—C

Spread the HeLa cells in a 24-well plate, the number of cells per well is about 2×10 ⁵ , and the calcification process starts when the cells grow to about 70% of the bottom area of the well plate. First, replace the medium of the cells, wash them with pH 7.2 phosphate buffer for 1-2 times, then add additional DMEM medium with 200 μg/mLFA to the well plate, and then place the cells in the incubator to adjust CO ₂ The concentration is 5%, the air is 95%, the temperature is 37°C, incubate in the incubator for 25 minutes, suck off the medium, and add 16.8mMCa ²⁺ (including newly added 15mMCaCl ₂ ) to the well plate for calcification of DMEM medium Place the orifice plate in a cell culture incubator for calcification treatment for 40 minutes, repeat the treatment once, and treat FA and calcification solution alone as a control, suck up the solution after the treatment, and obtain calcified cancer cells.

4. Preparation of targeting calcification cancer cells-D

Spread the HeLa cells in a 24-well plate, the number of cells per well is about 2×10 ⁵ , and the calcification process starts when the cells grow to about 70% of the bottom area of the well plate. First, replace the medium of the cells, wash them with pH 7.2 phosphate buffer for 1-2 times, then add additional DMEM medium with 200 μg/mLFA to the well plate, and then place the cells in the incubator to adjust CO ₂ The concentration is 5%, the air is 95%, the temperature is 37°C, incubate in the incubator for 25 minutes, suck off the medium, and add 16.8mMCa ²⁺ (including newly added 15mMCaCl ₂ ) to the well plate for calcification of DMEM medium Put the orifice plate in a cell incubator for calcification treatment for 30 minutes, repeat the treatment twice, and treat FA and calcification solution alone as a control, suck up the solution after the treatment, and then obtain calcified cancer cells.

5. Characterization of targeted calcified cancer cells

After the calcification of the cells is completed, fix with 4% paraformaldehyde at room temperature for 30 minutes, wash with PBS 2-3 times, and then wash and dehydrate with gradient ethanol (70%, 80%, 90%, 95% and 100%) respectively, and wait for the ethanol to volatilize Afterwards, it was observed under a scanning electron microscope, and elemental analysis was performed on the calcified layer on the surface of the calcified cells, and XRD and FTIR were used to analyze the calcified sample at the same time.

Human normal cells (HEK293) were used as a control and treated as above.

There was no significant difference in the morphology of calcified cancer cells prepared by the four methods. Taking the calcified cancer cells prepared by A as an example, the results are shown in Figures 1, 2, 3, and 4. After the HeLa cells were calcified, a calcified layer was formed on the cell surface. Figure 2 shows the composition of the calcified layer by scanning electron microscopy and energy spectrum elemental analysis. Contains calcium and phosphorus; the magnified observation of the calcified layer shows that it is formed by irregular clusters, see Figure 3; the results of the infrared spectrum in Figure 4 show that there is no crystal phase transition at wavenumbers of 1000cm ^-1 and 500cm ^-1 , combined with X- The results of ray diffraction showed that the formed calcified layer was amorphous calcium phosphate. Normal human cells (HEK293) undergo calcification treatment, and there is no calcified calcium phosphate formed on the cell surface. The above results indicate that the targeted calcification of cancer cells has been achieved by the method of the present invention.

Example 2

Laser confocal microscopy, life-and-death staining and MTT assay were used to detect the killing effect of calcification treatment on cancer cells. The specific operation is as follows:

1. Changes after cell calcification

Before calcification treatment, the cell membrane and nucleus of cancer cells were fluorescently stained with living cell dyes PKH26 and Hoechst33342, respectively. After the calcification treatment was completed, the mineral layer was fluorescently stained with the calcium-specific fluorescent dye calcein. After the staining was completed, the changes in cell morphology over time were observed with a laser confocal microscope. The results are shown in Figure 5.

2. Cell death staining

(1) HEK293, MCF7 and HeLa cells were seeded in 96-well plates at a density of 1×10 ⁴ cells/well, and after 24 hours of culture, the cells were calcified. The calcification method was the same as in Example 1.

(2) After the calcification treatment is completed, add DMEM medium and continue culturing for 24 hours.

(3) The cells were washed three times with PBS, then added with a dead-life staining reagent (Invitrogen), incubated in a cell incubator for 30 min, and observed under a fluorescent microscope.

3. MTT

(1) HEK293, MCF7 and HeLa cells were seeded in 96-well plates at a density of 1×10 ⁴ cells/well, and after 24 hours of culture, the cells were treated with calcification, and FA and calcification solution alone were used as controls.

(2) After the treatment is completed, add the culture medium and continue to cultivate for 24 hours, then add 5mg/mL MTT (20μL) to the reaction system, incubate for 4 hours, absorb the solution in the well plate, add 150μL DMSO and shake for 5-10min, and use a microplate to spectrometer A photometer (Bio-Tek) measures the absorbance at 570nm, which is proportional to the concentration of formazan (reaction product of cellular succinate dehydrogenase and MTT), which represents the activity of succinate dehydrogenase and thus indicates the active.

The results show that the calcium phosphate formed by calcification covers the surface of cancer cells, which will limit the physiological activities of cells, significantly inhibit cell activity, and eventually lead to the rupture of cell membranes, see Figure 5. The results of cell life and death staining show that targeting calcification can be used to kill cancer cells, see Figure 6A, and the survival rate of HeLa cells is inhibited by more than 60% after calcification treatment, while the survival rate of HEK29 cells can still maintain more than 80%, see Figure 6B , which is due to the low calcification efficiency caused by the low expression of folate receptors on the surface of HEK293 cells, further indicating that calcification can specifically occur on the surface of cancer cells with high expression of folate receptors.

Example 3

A HeLa tumor animal model was constructed to test the effect of targeted calcification in vivo. The operation and detection process is as follows:

(1) The HeLa cell line was inoculated subcutaneously into nude mice (20 nude mice, female) at a rate of 5×10 ⁶ per mouse as a mouse drug resistance model. After 10 days, the test was started when the tumor volume grew to 80 mm ³ .

(2) Inject 200 μL, 1 mg/mL folic acid intraperitoneally, wait for about 15 minutes, wait for the folic acid to accumulate in the tumor site, inject high-calcium DMEM medium (50 mMCa ²⁺ ) into the tumor tissue, inject once a day, and continue to treat for 15 days , to observe the effect of tumor calcification. Injection of calcification solution and folic acid alone and blank group were used as controls.

(3) Use micro-CT technology to scan the tumor, and perform three-dimensional reconstruction after scanning to determine the result of tumor calcification; slice the calcified tumor, perform fluorescent staining on the tumor slice with calcein, and use laser confocal observation after staining; The morphology of the calcified layer and the shape of the cancer cells were observed under a transmission electron microscope.

(4) In the experiment, the blood routine and biochemistry of the mice were analyzed, and the main organs of the mice were stained with HE to evaluate the safety of the method.

The results show that calcification can effectively wrap tumor cells, and the wrapped tumor tissue tumor necrosis is shown in Figure 7A; Micro-CT scanning results of the tumor, the arrows point out the tumor tissue and calcium phosphate formed by calcification, see Figure 7B; The morphology of calcium phosphate in tumor slices and the morphology of tumor cells observed by TEM indicate that calcification can be formed on the surface of cancer cells in tumor tissue, see Figure 7C; after the tumor tissue slices were stained with Hoechst33342 and the nuclei were stained by laser confocal photography, the observation results showed that the tumor cell nuclei appeared pyknotic , indicating that calcification inhibits the activity of tumor tissue, see Figure 7D. In addition, the calcification treatment has no effect on the blood cells of the mice, and has no liver toxicity, and the results of organ HE staining show that the calcification treatment has almost no damage to the organs, see Figure 8.

Example 4

A HeLa tumor mouse model was constructed to test the tumor suppressor effect of targeting calcification. The operation and detection process is as follows:

(1) The HeLa cell line was inoculated subcutaneously into nude mice (40 nude mice, female) at a rate of 5×10 ⁶ per mouse as a nude mouse model, and the test was started when the tumor volume grew to 80 mm ³ 10 days later.

(2) Inject 200 μL of 1 mg/mL folic acid intraperitoneally, and then wait for about 10 minutes. After the folic acid is enriched in the tumor site, inject 100 μL of high-calcium DMEM medium (50 mMCa ²⁺ ) into the tumor tissue once a day for 15 consecutive days. , to observe the effect of tumor calcification. Injection of calcification solution and folic acid alone and blank group were used as controls.

(3) The signs of the mice were observed daily, the body weight of the mice was recorded every 2 days, the longest diameter L and the shortest diameter B of the tumor were measured, and the mouse volume was calculated using the formula V=0.5×L×B ² .

(4) After 30 days, the mice were sacrificed, the tumors were taken out, weighed, photographed and recorded, and the tumors were stained with HE.

(5) Select the mouse cancer cell line 4T1/luc, inoculate 5×10 ⁶ cells into the mammary glands of mice (40 nude mice, female), and use it as a model after 10 days when the tumor volume grows to 80 mm ³ Start calcification treatment, and the treatment method is consistent with step (2). The small animal in vivo imaging technology was used to detect tumor metastasis in mice, and the death rate of mice was recorded at the same time.

The results showed that calcification can effectively inhibit the growth of tumor volume, see Figure 9A; in addition, the body weight of the mice did not change compared with the control group, further indicating that this method has no obvious side effects on the mice; the HE staining results showed that the HE staining of the calcified tumor would deepen , refer to Figure 9CFA/Ca treatment group, a large area of cell death has appeared around the calcified tissue. It further illustrates that the method can effectively inhibit tumor growth. Combining with FIG. 7 , it can be concluded that this method can achieve a certain targeted calcification of cancer cells in the tumor, thereby inhibiting the growth of the tumor, thereby achieving an effective effect. The mouse body weight data ( FIG. 9B ) combined with FIG. 8 shows that this method has better biological safety.

Claims (10)

1. a method for calcification cancer cell, is characterized in that, comprises the following steps:

(1) cancer cell is inoculated in the culture medium that contains folic acid, hatches, obtain the cancer cell of surface enrichment folic acid;

(2) cancer cell of surface enrichment folic acid is placed in to calcifying solution and hatches, make the cancer cell of calcification.

2. the method for claim 1, is characterized in that, repeats step (2) for several times, obtains the cancer cell of calcification.

3. the method for claim 1, is characterized in that, described cancer cell is human cervical carcinoma cell (HeLa), people's mammary glandCancer cell (MCF7) or people's gonad cell (SKVO-3).

4. the method for claim 1, is characterized in that, the inoculum density of cancer cell is cultivated orifice plate base area for growing to40-80%.

5. the method for claim 1, is characterized in that, in step (1), the content of culture medium Folic Acid is 0.2-1.2mg/mL。

6. the method for claim 1, is characterized in that, in step (1), the time of hatching is 5-30min.

7. the method for claim 1, is characterized in that, in step (2), described calcifying solution is that calcium ion concentration is 5-20The DMEM culture medium of mM.

8. the method for claim 1, is characterized in that, in step (2), incubation time is 0.25-2h.

9. the method for claim 1, is characterized in that, incubation conditions comprises that the concentration of carbon dioxide is 4-10%.

10. folic acid is in the application of preparing in cancer cell calcification derivant.