CN107951892A - Zinc protoporphyrin is preparing enhancing Human colorectal cancer cells to the application in 5 FU 5 fluorouracil chemosensitivity medicine - Google Patents

Abstract

The invention discloses zinc protoporphyrin to prepare enhancing Human colorectal cancer cells to the application in 5 fluorouracil chemotherapy sensitive drugs, belongs to chemotherapeutics technical field.The present invention provides basis for new drug development and innovative treatments, suitable for carrying out chemotherapy to Human colorectal carcinoma patient, improves the Chemotherapy of 5 fluorouracil medicines.

Description

Preparation of zinc protoporphyrin enhances chemosensitivity of human colorectal cancer cells to 5-fluorouracil use in sex medicine

technical field

The invention relates to the application of zinc protoporphyrin in the preparation of drugs for enhancing the chemosensitivity of human colorectal cancer cells to 5-fluorouracil, and belongs to the technical field of chemotherapeutic drugs.

Background technique

Colorectal cancer is one of the most common gastrointestinal tumors. According to the International Agency for Research on Cancer (IARC) of the World Health Organization, there were approximately 1.36 million new cases of colorectal cancer in the world in 2012, ranking among the most malignant. Tumor ranks third, after lung cancer and breast cancer; about 690,000 deaths, after lung cancer, liver cancer and gastric cancer, ranks fourth among malignant tumors. my country is a low-incidence area of colorectal cancer. In recent years, with the development of my country's economy, changes in diet structure and lifestyle, the incidence of colorectal cancer is on the rise. According to statistics, as of 2016, the number of new cases of colorectal cancer in my country has reached 331,000, including 159,000 deaths.

At present, the main treatment is surgical resection of the tumor at the primary site, followed by chemotherapy, radiotherapy, immunotherapy and other means of adjuvant treatment. Effective chemotherapy can not only kill residual cancer cells, but also prevent the further development of cancer cells. Diffusion and transfer. The usual chemotherapy regimen is combined chemotherapy based on 5-fluorouracil (5-FU).

Contents of the invention

The invention provides the application of zinc protoporphyrin in the preparation of drugs for enhancing the chemosensitivity of human colorectal cancer cells to 5-fluorouracil, and provides a basis for new drug research and development and innovative therapy. The technical scheme adopted is as follows:

The invention provides an application of zinc protoporphyrin (Zinc protoporphyrin, ZnPP) in preparing a drug for enhancing the chemosensitivity of human colorectal cancer cells to 5-fluorouracil.

The present invention studies the effect of heme oxygenase-1 (hemeoxygenase-1, HO-1) on 5-fluorouracil (5-FU) chemosensitivity and its As a possible mechanism, the inventors applied different concentrations of zinc protoporphyrin (ZnPP) and cobalt protoporphyrin (CoPP) alone or in combination with 5-FU to act on human colorectal cancer cells, and observed the inhibitory effect of drugs on cell growth by Alaram Blue method; Quantitative PCR was used to analyze the expression changes of HO-1 gene; the apoptosis rate was detected by flow cytometry; the possible molecular mechanism was analyzed by detecting Caspase-3 and reactive oxygen species (ROS). The results showed that all kinds of cells expressed HO-1, and the expression of HO-1 could be enhanced by using 5-FU alone or 5-FU combined with CoPP, but CoPP could induce it, and 5-FU combined with ZnPP could reduce the expression of HO-1. -1 expression, and can significantly inhibit the growth of human colorectal cancer cells in a dose-dependent manner; 5-FU combined with ZnPP significantly increased the cell growth inhibition rate and apoptosis rate compared with 5-FU alone (P< 0.05), 5-FU combined with CoPP could weakly reduce the cell growth inhibition rate and apoptosis rate (P>0.05); compared with the blank control group, the intracellular ROS activity of 5-FU alone increased significantly, and 5-FU combined ZnPP can further increase the activity of intracellular ROS (P<0.05), while the combination of CoPP can reduce the level of intracellular ROS activity (P<0.05).

In summary: the inventors found for the first time that ZnPP can enhance the chemosensitivity of human colorectal cancer cells to 5-FU, which may be related to the increase in intracellular ROS activity and intracellular Caspase-3 activity.

The invention provides a basis for new drug research and development and innovative therapy. The invention is suitable for performing chemotherapy on human colorectal cancer patients and improving the chemotherapy effect of 5-fluorouracil.

Description of drawings

Figure 1 is the RT-PCR analysis of HO-1 gene expression in each treatment group of RKO cells;

(A, HO-1; B, β-Actin; A and B: M is Marker DL2000; 1 is the blank control group; 2 is the group added with DMSO; 3 is the group added with 5-FU; 4 is the group added with 0.5 μmol CoPP+ 5-FU group; 5 is the group adding 1 μmol CoPP+5-FU; 6 is the group adding 5 μmol CoPP+5-FU; 7 is the group adding 10 μmol CoPP+5-FU; 8 is the group adding 0.5 μmol ZnPP+5-FU; 9 is the group added with 1 μmol ZnPP+5-FU; 10 is the group added with 5 μmol ZnPP+5-FU; 11 is the group added with 10 μmol ZnPP+5-FU).

Figure 2 is the RT-PCR analysis of HO-1 gene expression in each treatment group of HCT8 cells;

(A, HO-1; B, β-Actin; A and B: M is Marker DL2000; 1 is the blank control group; 2 is the group added with DMSO; 3 is the group added with 5-FU; 4 is the group added with 0.5 μmol CoPP+ 5-FU group; 5 is the group adding 1 μmol CoPP+5-FU; 6 is the group adding 5 μmol CoPP+5-FU; 7 is the group adding 10 μmol CoPP+5-FU; 8 is the group adding 0.5 μmol ZnPP+5-FU; 9 is the group added with 1 μmol ZnPP+5-FU; 10 is the group added with 5 μmol ZnPP+5-FU; 11 is the group added with 10 μmol ZnPP+5-FU).

Figure 3 is the RT-PCR analysis of HO-1 gene expression in each treatment group of HT29 cells;

(A, HO-1; B, β-Actin; A and B: M is Marker DL2000; 1 is the blank control group; 2 is the group added with DMSO; 3 is the group added with 5-FU; 4 is the group added with 0.5 μmol CoPP+ 5-FU group; 5 is the group added with 1 μmol CoPP+5-FU; 6 is the group added with 5 μmol CoPP+5-FU; 7 is the group added with 0.5 μmol ZnPP+5-FU; 8 is the group added with 1 μmol ZnPP+5-FU; 9 is the group added with 5 μmol ZnPP+5-FU;).

Figure 4 shows the relative expression of HO-1 gene in each treatment group of RKO cells analyzed by quantitative PCR.

Figure 5 shows the relative expression of HO-1 gene in each treatment group of HCT8 cells analyzed by quantitative PCR.

Figure 6 shows the relative expression of HO-1 gene in each treatment group of HT29 cells analyzed by quantitative PCR.

Figure 7 shows the cell inhibition rate detected by the Alaram Blue method.

Figure 8 is the detection of cell apoptosis by flow cytometry in each treatment group of HCT8 cells;

(a, 1 μmol ZnPP combined with 200 μmol 5-FU group; b, 5 μmol ZnPP combined with 200 μmol 5-FU group; c, 10 μmol ZnPP combined with 200 μmol 5-FU group; d, 1 μmol CoPP combined with 200 μmol 5-FU group; e, 5 μmol CoPP combined with 200 μmol 5-FU group; FU group; f, 10 μmol CoPP combined with 200 μmol 5-FU group; g, blank control group; h, DMSO group; I, 200 μmol 5-FU group.

Figure 9 Flow cytometric detection of apoptosis ratios in each treatment group.

Figure 10 is the effect of each treatment group on the ROS activity of cells.

Figure 11 is the effect of each treatment group on the activity of Caspase-3 in HCT8 cells.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the examples.

Example 1:

1 Materials and methods

1.1 Materials

1.1.1 Reagents

RKO cells; HT29 cells; HCT8 cells and other cells were preserved in the laboratory; RPMI-1640 medium, DMEM medium, double antibodies, fetal bovine serum, and Alaram Blue kit were purchased from Life Technology Company; reactive oxygen species, Caspase3, and apoptosis Detection kits and Bradford protein concentration detection kits were purchased from Biyuntian Biotechnology Co., Ltd.; HO-1 and internal reference β-actin primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.; RT-PCR kits were purchased from ThermoScientific.

1.1.2 Instruments

Autoclave (HVE-50, Japan Hariyama Company); two-person single-sided purification workbench (SW-CJ-2FD, Suzhou Purification Equipment Co., Ltd.); full-featured microplate detector (SYNERGY/H1, American Biotek Company) ; CO ₂ incubator (BB150, American Thermo Company); inverted microscope (DMi8, German LEICA Company); centrifuge (5810R, German Eppendorf Company).

1.2 Method

1.2.1 Cell recovery and culture

Take the cryopreservation tubes containing HCT8, RKO, and HT29 cells out of the -80°C refrigerator, put on film gloves, thaw them quickly in a 37°C water bath, transfer the thawed suspension to a centrifuge tube, centrifuge at 1000rpm for 5min, discard For the supernatant, the cell suspension was transferred to a culture dish containing complete culture solution, and placed in a CO ₂ incubator for cultivation. Observe the cells every day. If the cell adherent confluence reaches 80%, it can be passaged by trypsinization.

1.2.2 Determination of the optimal drug concentration

Take a 50ml centrifuge tube on the analytical balance, adjust to zero, take the drug (ZnPP, ZnPP, 5-FU) into the centrifuge tube with a medicine spoon, take it out and fully dissolve it with DMSO, so that the concentration is 0.01mol/L, and divide it into 1.5ml Centrifuge tubes, labeled. Seal with parafilm and store in a -20°C refrigerator. According to the experimental results of Alaram Blue, it is determined that the drug concentration of 5-FU is 200 μmol, the concentration of CoPP drugs is 0.5 μmol to 10 μmol, and the concentration of ZnPP drugs is 0.5 μmol to 10 μmol. The DMSO group was set up separately, 200 μmol 5-FU drug group was used alone, 0.5 μmol CoPP, 1 μmol CoPP, 5 μmol CoPP, and 10 μmol CoPP were added after 200 μmol 5-FU was added for 24 hours, and 0.5 μmol ZnPP was added respectively in the ZnPP group after 200 μmol 5-FU was added for 24 hours , 1 μmol ZnPP, 5 μmol ZnPP, 10 μmol ZnPP, after 24 hours of treatment, collect and detect various indicators.

1.2.3 Cell preparation

Take well-grown cells in the logarithmic growth phase, digest with trypsin, dilute the culture medium to a cell suspension with a concentration of 5×10 ⁴ /mL, inoculate 2 mL per well into a 6-well plate, and transfer to CO ₂ incubator culture. Wash and discard the original culture medium of adherent cells after overnight culture, and add 1 mL of new complete culture medium. Except for the DMSO group, add DMSO corresponding to the volume of 5-FU, and add 5-FU with a final concentration of 200 μmol to each well. , so that the volume of the culture medium was 2 mL. After culturing for 24 hours, discard the culture medium, wash with PBS for 3 times, add 2 mL of culture medium containing medicine to the DMSO group set in 1.2. Into the well plate, each concentration of 3 in parallel, after 24 hours of culture to detect the indicators.

1.2.4 Quantitative PCR detection of HO-1 mRNA expression in human colorectal cancer cells

Referring to the reverse transcription manual of Thermo Scientific, cDNA was synthesized in a 20 μL system using 1 μg of total RNA as a template. HO-1 upstream primer 5'-CTTCAAGCTGGTGATGGC-3'(SEQ ID NO.1), downstream primer 5'-TGGAGCCGCTTCACATAG-3'(SEQ ID NO.2), product 219bp; β-Actin upstream primer 5'-TCCCTGGAGAAGAGCTACGA- 3' (SEQ ID NO.3), the downstream primer 5'-AGCACTGTGTTGGCGTACAG-3' (SEQ ID NO.4), the product is 194bp. Gene expression was detected with Takara's fluorescent quantitative reagents. The reaction program of fluorescent quantitative PCR is 95°C, 30s; 95°C, 5s; 60°C, 30s; 72°C, 34s; 40cycles.

1.2.5 Detection of intracellular oxidative stress levels ROS, Caspase3 and apoptosis

The cells treated with the combination of drugs in the 96-well plate were tested for the level of oxidative stress in the cells by referring to the method of the Biyuntian Active Oxygen Detection Kit (S0033). In the 6-well plate, the cells treated with the drug combination were used to detect the expression of Caspase 3 activity in cells according to the method of Beyontian Caspase 3 Activity Detection Kit (C1115). In the 6-well plate, the cells treated with the drug combination were used to detect the apoptosis of the cells by flow cytometry according to the method of Beyontian Annexin V-FITC Cell Apoptosis Detection Kit (C1063).

2 results

2.1 Expression of HO-1 gene in different drug treatment groups

RT-PCR and quantitative PCR were used to analyze the expression of HO-1 gene in HCT8, RKO, and HT29 cells in each drug treatment group, and the results are shown in Figures 1 to 6. The results showed that a small amount of HO-1 expression could be seen in the control group in each treatment group, and the application of 5-FU drugs could induce a significant increase in the expression of HO-1 mRNA, and the expression of HO-1 mRNA further increased after 5-FU drugs combined with CoPP, and The gene expression of HO-1 also increased with the increase of the concentration of CoPP. The combination of 5-FU drug with ZnPP can reduce the expression of HO-1mRNA, and with the increase of the concentration of ZnPP, the expression of HO-1mRNA is gradually reduced. It indicated that exogenous ZnPP could reduce the expression of HO-1 gene, while CoPP could increase the expression of HO-1 gene. However, in HT29 cells, the expression of the gene was not detected because the cells in the 0.5 μmol CoPP combined with 5-FU group and the 0.5 μmol ZnPP combined with 5-FU group shed more cells.

2.2 The effect of each drug concentration on cell proliferation

Alaram Blue detection showed that different concentrations of 5-FU and ZnPP could significantly inhibit the proliferation of human colorectal cancer cells in a concentration-dependent manner, and the inhibition rate was enhanced as the concentration increased (Figure 7). CoPP had little effect on the proliferation of human colorectal cancer cells, and there was no statistically significant difference compared with the control group. When 5-FU was combined with different concentrations of ZnPP, the cell proliferation rate of each group was significantly lower than that of the 5-FU group alone (P<0.05), and it was concentration-gradient dependent; while 5-FU combined with different concentrations of CoPP, the cell proliferation rate of each group The rate was slightly higher than that of the 5-FU group alone.

2.3 Comparison of cell apoptosis rate in each treatment group

The results of flow cytometry (FCM) analysis of the proportion of apoptosis cells showed (Figure 8 and Figure 9): 5-FU alone had little effect on colorectal cancer cells, and the number of advanced apoptotic cells was significantly higher than that of the no-drug group increased; when combined with ZnPP, the apoptosis rate of colorectal cancer cells increased in a concentration-dependent manner, and the difference was extremely significant compared with the single 5-FU group (p<0.01); while when combined with CoPP, the cell The apoptotic rate decreased, and the difference was not significant compared with the control group (p>0.05).

As shown in Figure 10, the ROS activity of cells treated with 5-FU alone was significantly higher than that of the blank control group (P<0.05), especially in RKO cells and HT29 cells, and the ROS activity of cells increased again after 5-FU combined with different concentrations of ZnPP The increase in HCT8 cells was more obvious, and it was in a concentration gradient, and the difference was statistically significant compared with the single drug group (P<0.05); while the ROS activity of 5-FU combined with different concentrations of CoPP was significantly reduced, and the ROS activity in RKO cells And HT29 cells are particularly obvious, and the concentration gradient (P <0.05). 2.5 Analysis of relative activity of Caspase-3 in each group

As shown in Figure 11, the Caspase-3 activity of cells treated with 5-FU alone was significantly higher than that of the normal control group (P<0.05). Not significant, but presenting a concentration gradient, the difference was statistically significant compared with the single drug group (P<0.05); while 5-FU combined with different concentrations of CoPP decreased the activity of Caspase-3 significantly, and it was not significant in HCT8 cells at low concentrations of CoPP. Obviously, and in a concentration gradient (P<0.05), the decrease of Caspase-3 activity in HT29 cells was not obvious.

3 Discussion

HO-1 has cytoprotective functions, namely anti-oxidation, anti-inflammation and anti-apoptosis, and plays an increasingly important role in the occurrence and development of tumors. In recent years, studies have found that many solid tumors have high expression of HO-1, and the expression is more obvious during hypoxia and radiotherapy and chemotherapy. In tumor cells, the expression of HO-1 is closely related to tumor diffusion, metastasis and anti-apoptosis. Fang et al pointed out that HO-1 can inhibit tumor cell apoptosis, thereby promoting tumor growth. HO-1 can inhibit apoptosis by reducing intracellular prooxidative substances and increasing the level of bilirubin. Zhang Junkai et al. reported that inhibiting HO-1 in drug-resistant liver cancer Bel/Fu cell lines can reduce the concentration of chemotherapeutic drugs and mRNA expression, and inhibiting HO-1 affects the expression of MCR-1.

In this experiment, the level of HO-1 gene was detected in the cells treated with different drugs. The results showed that after 5-FU combined with ZnPP, the expression of HO-1 gene in the cells was down-regulated (Figure 1 to Figure 6), and the cell activity decreased, indicating that Reducing HO-1 can inhibit the activity of colorectal cancer cells, and after applying CoPP to colorectal cancer cells, the expression of HO-1 gene in the cells is up-regulated, and the activity of colorectal cancer cells does not change significantly (Figure 7). After the chemotherapy drug 5-FU was applied, the expression of HO-1 in the cells increased; the combined application of CoPP could further induce the expression of HO-1, and the cell inhibition rate and apoptosis rate were significantly lower than those of 5-FU alone, while the combination of ZnPP inhibited the expression of HO-1. After the expression of HO-1 in cells, the rate of cell inhibition and apoptosis increased significantly. This indicates that CoPP induces the expression of HO-1 to reduce the sensitivity of colorectal cancer cells to 5-FU chemotherapy injury, and the application of ZnPP can enhance the sensitivity of colorectal cancer cells to 5-FU chemotherapy by blocking the expression of HO-1. It shows that the HO-1 inducer CoPP can indeed induce the expression of HO-1 and protect the cells, while the HO-1 inhibitor ZnPP can inhibit the expression of HO-1 and promote its apoptosis.

In addition, the application of 5-FU to inhibit the growth of colorectal cancer cells. After combined with ZnPP, the cells became round and even apoptosis was more obvious, indicating that the combination of ZnPP and chemotherapy drug 5-FU has a sensitizing effect. After being used in combination with CoPP, the cell growth state was relatively better than that of the 5-FU group alone, indicating that CoPP can weaken the effect of 5-FU on cell growth state.

After detecting cell apoptosis by flow cytometry, it was found that after 5-FU combined with ZnPP, the number of apoptotic cells increased; and after the use of CoPP, the number of apoptotic cells decreased ( FIG. 9 ). The experimental results indicated that ZnPP increased cell apoptosis by inhibiting HO-1, and CoPP inhibited cell apoptosis by inducing the expression of HO-1 to protect cells.

Active oxygen is very important to promote cell mitosis and induce cell proliferation. Within a certain concentration range, this ability to promote cell proliferation is positively correlated with the level of active oxygen. If it is too low in cells, it will inhibit cell mitosis and affect cell proliferation. proliferation. In Bai Hua's study on the effect of NAC on the proliferation activity of embryonic liver cells, it was confirmed that there is a positive correlation between the level of intracellular ROS and cell proliferation. It is now generally believed that cell apoptosis is the result of a series of highly regulated cysteine protease caspase cascade reaction (cascade) events, and caspase-3 (also known as CPP32, apopain) has been confirmed to be in the downstream of the cascade reaction. Degradation of the corresponding substrate in the cell leads to cell death. It shows that ZnPP can increase the inhibitory effect of 5-FU drugs on cell growth, and ZnPP affects cell growth by inhibiting the expression of HO-1 gene.

Because HO-1 is closely related to cell protection and anti-oxidative stress. RKO cells, HCT8 cells, and HT29 cells treated with different drugs were tested for intracellular ROS levels, Caspase 3 activity, and cell apoptosis. The results showed that: compared with the 5-FU group alone, after the combination of ZnPP and 5-FU, the level of intracellular reactive oxygen species increased and the activity of Caspase3 increased, which corresponded to a significant increase in the rate of cell apoptosis; while CoPP combined with 5-FU Compared with the 5-FU group alone, the level of active oxygen in the cells decreased, the activity of Caspase 3 and the apoptosis rate of the FU combined treatment group decreased.

In summary, this experiment explored the effect of heme oxygenase-1 on the chemosensitivity of human colorectal cancer cells to 5-fluorouracil. In the concentration range of 0.5 to 10 μmol in the experiment, the HO-1 inhibitor ZnPP can increase the sensitivity of human colorectal cancer cells to 5-fluorouracil. Colorectal cancer cells are sensitive to 5-FU drugs, and HO-1 agonist CoPP can slow down the chemotherapeutic effect of 5-FU drugs. Its mechanism of action may be to affect the level of reactive oxygen species produced in cells by changing the expression of HO-1 gene, and indirectly inhibit the activity of Caspase 3, thereby reducing the rate of cell apoptosis. As a classic chemotherapeutic drug for tumor treatment, 5-FU can reduce the expression level of HO-1 gene and increase the sensitivity of 5-FU and other chemotherapeutic drugs through ZnPP and other drugs that inhibit HO-1 gene expression in vitro. It provides a new idea for the medicine or clinical practice of the treatment of rectal tumors or other types of cancer.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore Therefore, the protection scope of the present invention should be defined by the claims.

Claims (1)

1. The application of zinc protoporphyrin in the preparation of drugs for enhancing the chemosensitivity of human colorectal cancer cells to 5-fluorouracil.