CN106176700B - Application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament - Google Patents

Abstract

The invention discloses application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament, niclosamidum is found through experiments that treated that the expression of intracellular KSHV or EBV related genes is substantially reduced in inventor, and the release of extracellular virion also decreased significantly.These inhibiting effect are in concentration dependent, and just have good antivirus action in the concentration of not overt toxicity, provide strong theoretical foundation and experimental basis for the research and development of further antiviral drugs, have important research and development value and development significance.Based on this, niclosamidum can be developed as safely and effectively anti-KSHV or EBV drugs.

Description

Application of niclosamide in the preparation of anti-tumor herpes virus drugs

technical field

The invention relates to a new application of a compound, in particular to the application of niclosamide in the preparation of anti-tumor herpes virus drugs.

Background technique

Kaposi's sarcoma-associated herpesvirus KSHV (Kaposi's sarcoma-associated herpesvirus) is a member of the herpesvirus family. Kaposi's sarcoma induced by KSHV infection is a common malignant tumor in AIDS patients, about 20% of AIDS patients will be accompanied by Kaposi's sarcoma, and the mortality rate of AIDS-KS patients is extremely high, and its 5-year survival rate is only about 8%. In the normal population, more than 95% of individuals infected with KSHV do not show clinical symptoms and become ill. However, in immunosuppressed patients, such as AIDS patients, organ transplant patients, and chemotherapy and radiotherapy patients, KSHV has a high infection rate and great harm, which can lead to Kaposi's sarcoma (KS), primary exudate Lymphoma (primary effusionlymphoma, PEL) and multicentric castleman disease (MCD) and other diseases. In recent years, high KSHV infection rates and malignant tumors have also been found in other diseases (such as HBV chronic hepatitis, drug abuse and senile diseases), which are gradually attracting attention.

Epstein-barr virus (EBV) and KSHV belong to the same γ-herpes virus. EBV can be transmitted through saliva, and some studies in recent years have shown that this virus can also be transmitted through sexual behavior. At present, EBV infects more than 95% of individuals over the age of 5 in the world. After infection, EBV exists in the body of the carrier for life, showing a latent infection state, which can cause a variety of human diseases, including infectious mononucleosis (Infection Mononucleosis) , IM), oral hairy leukoplakia, neurological diseases multiple sclerosis (Multiple Sclerosis, MS), Gianotti-Crosti syndrome, erythema multiforme (Erythema Multiforme, EM), acute vulvar ulcer and multiple sclerosis after solid organ transplantation Lymphoproliferative disorders. Currently, commonly used drugs that inhibit viral DNA synthesis to treat herpes virus infection (such as acyclovir and its related compounds) are less effective against these two virus infections, and vaccines are still in the research and development stage.

Niclosamide, as an anthelmintic, has been recognized by the World Health Organization for its safety. In addition, studies have found that niclosamide may have a certain effect on the treatment of bacterial infections. There are no clear experimental data showing that niclosamide has pharmaceutical activity reports on KSHV and EBV.

Contents of the invention

The object of the present invention is to provide the application of niclosamide in the preparation of anti-tumor herpes virus drugs.

The inventor used techniques such as Western Bolt and Real-time quantitative PCR to detect the expression of KSHV-related genes in KSHV-positive cells BCBL1 and the content of extracellular virus particles; detect EBV-positive cells, including lymphocytes P3HR-1 and epithelial cells HNE1 The expression of EBV-related genes and the content of extracellular virus particles in -2089. Experiments found that the expression of KSHV or EBV-related genes in cells treated with niclosamide was significantly reduced, and the release of extracellular virus particles was also significantly reduced. These inhibitory effects are concentration-dependent, and have good antiviral effects at concentrations without obvious toxicity, which provides a strong theoretical basis and experimental basis for further research and development of antiviral drugs, and has important research and development value and development significance. Based on this, niclosamide can be developed as a safe and effective anti-KSHV or EBV drug.

Description of drawings

Figure 1: The effect of niclosamide on the expression of KSHV latency protein LANA, lytic phase protein RTA, K8, and ORF64 in BCBL1 cells;

Figure 2: Effects of niclosamide on the expression of EBV-related proteins EA-D and ZTA in P3HR-1 cells;

Figure 3: The effect of niclosamide on the expression of EBV-related proteins EA-D and ZTA in HNE1-2089 cells;

Figure 4: Effect of niclosamide on virus production in BCBL1 cells;

Figure 5: Effect of niclosamide on the virus production outside P3HR-1 cells;

Figure 6: Effect of niclosamide on HNE-2089 extracellular virus production;

Figure 7: Effect of niclosamide on the survival of PBMC cells.

Detailed ways

The technical solution of the present invention will be further described below in combination with experiments.

In the following experiments, unless otherwise specified, the reagents, equipment and methods used in the present invention are conventionally purchased reagents, equipment and methods commonly used in this technical field.

Experimental principle: EBV and KSHV are gamma-herpes viruses, and their life cycle is divided into incubation period and lysis period. After the cells are infected, they enter the incubation period after a short acute reaction, and after special stimulation, the cells enter the lysis period for virus amplification. reproduce. Therefore, detecting the expression of related proteins in the incubation period and lysis period can be used as a means to evaluate the effect of drugs on the two viruses.

In the present invention, it is abbreviated as: TPA, euphorbia diterpene ester; NaB, sodium butyrate

Experiment 1: Effects on the expression of KSHV latency protein LANA, lytic phase protein RTA, K8, ORF64 in BCBL1 cells

1) Take well-grown BCBL1 cells and inoculate them in a 6-well transparent flat-bottom plate, with 2×10 ⁶ cells per well. The medium used is a complete medium: RPMI1640, 10% fetal bovine serum and 1% double antibody, culture conditions are 5% carbon dioxide, 37°C;

2) After 12 hours, add the inducer TPA, the final concentration is 20ng/mL;

3) After 3 hours, gradient concentrations of niclosamide were added, and the final concentrations were 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, and 1 μM;

4) After culturing for 48 hours, cells were harvested at 1000 rpm for 10 minutes, and Western blot experiments were performed.

The results are shown in Figure 1: the expression of KSHV-associated proteins RTA, K8, and ORF64 in BCBL1 cells was affected by the concentration of niclosamide in a certain concentration-dependent manner.

Experiment 2: The effect of niclosamide on the expression of EBV-related proteins EA-D and ZTA in P3HR-1 cells

1) Take well-grown P3HR-1 cells and inoculate them in a 6-well transparent flat-bottomed plate, with 2×10 ⁶ cells per well. The medium used is a complete medium: RPMI1640, 10% fetal bovine serum and 1% double antibody, culture conditions are 5% carbon dioxide, 37°C;

2) Add inducers TPA (final concentration: 20 μg/ml) and NaB (final concentration: 3 mM) after passage for 12 hours;

3) Niclosamide was added after 3 hours of induction, and the final concentrations were 0 μM, 0.01 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM;

4) After 48 hours after adding the inducer, the cells were harvested for western blot experiments.

The results are shown in Figure 2: In P3HR-1 cells, the expression of EBV-related proteins ZTA and EA-D was affected by the concentration of niclosamide in a certain concentration-dependent manner.

Experiment 3: The effect of niclosamide on the expression of EBV-related proteins EA-D and ZTA in HNE1-2089 cells

1) Take well-grown HNE1-2089 cells and inoculate them in 6-well transparent flat-bottomed plates at a ratio of 1:4;

2) After 12h, the cells adhered to the wall, and the inducers TPA (final concentration: 20μg/ml) and NaB (final concentration: 3mM) were added

3) After 3 hours, add niclosamide at gradient concentrations, the final concentrations are 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM;

4) After culturing for 48 hours, the cells were scraped out from the plate with a cell scraper, rinsed with cold PBS, centrifuged at 1000 rpm, 10 min, and 4°C to collect the cells for Western blotting.

The experimental results are shown in Figure 3: the expression of EBV-related proteins ZTA and EA-D in HNE1-2089 cells was affected by the concentration of niclosamide, showing a certain concentration dependence.

Experiment 4: Effect of niclosamide on virus production in BCBL1 cells

1) Take the well-growing cell line BCBL1 and plant it in a 48-well plate with a cell dosage of 1×10 ⁵ /well. Divide the cells into a non-induced group (3 wells) and an induced group (21 wells);

2) After 3 hours, the induction group was added with inducer TPA, the final concentration was 20ng/mL;

3) Add corresponding concentrations of niclosamide after 3 hours, the concentrations are 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, respectively, and 3 wells are prepared for each concentration;

4) Harvest the cells after 120 hours, take 10000g, and take the supernatant at 4°C;

5) Extracting extracellular viral DNA, the method is as follows:

a) Take 200 μL supernatant, add 2 μL DNase I, 20 μL 10×DNase I buffer, and incubate in a 37°C incubator for 1 hour;

b) Add 10 μL of 0.5M EDTA to the above liquid, mix well, and terminate the reaction;

c) Inactivate in a water bath at 80°C for 10 minutes;

d) Add 20 μL proteinase K solution, followed by 200 μL AL buffer, vortex and mix, and bathe in 56°C water bath for 10 minutes;

e) Add 440 μL of phenol chloroform to extract the DNA in the virus particles, and vortex to mix;

f) 12000rpm, centrifuge at 4°C for 10min;

g) Separate the layers after centrifugation, take about 300 μL of the supernatant, and transfer it to another ep tube. Avoid pumping phenol chloroform into the lower layer;

h) Add 6 μL of 5M NaCl solution, add 750 μL of absolute ethanol, and finally add 1 μL of glycogen;

i) Mix well and place in -80°C refrigerator for 1 hour;

j) Take the sample out of the refrigerator, centrifuge at 12000rpm, 4°C for 30min, and remove the supernatant;

k) DNA precipitate appears at the bottom of the Ep tube, wash with 1mL of 70% ice ethanol, cover it, and turn it upside down several times;

l) 12000rpm, centrifuge at 4°C for 10min, discard the supernatant;

m) 12000rpm, centrifuge at 4°C for 10min to remove residual alcohol;

n) Open the lid to allow excess alcohol to evaporate;

o) After 3 minutes, add 40 μL ddH ₂ O to dissolve the DNA;

6) Real-time quantitative PCR to detect virus content.

The result is shown in Figure 4. The results showed that niclosamide inhibited the production of KSHV in BCBL1 cells in a concentration-dependent manner.

Experiment 5: The effect of niclosamide on the virus production outside P3HR-1 cells;

1) The well-growing cell line P3HR-1 was planted in a 48-well plate with a cell dosage of 1×10 ⁵ /well, and the cells were divided into a non-induced group and an induced group;

2) After 3 hours, the induction group was added with inducers TPA and NaB, and the final concentrations were 20ng/mL and 3mM, respectively;

3) After induction for 3 hours, add corresponding concentrations of niclosamide, the concentrations are 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM, respectively, and each concentration is repeated for 3 wells;

4) Harvest the cells after 120 hours, centrifuge at 10,000g for 10 minutes, and take the supernatant at 4°C;

5) Extracting viral DNA, the method is as follows:

a) A． Take 200 μL supernatant, add 2 μL DNase I, 20 μL 10×DNase I buffer, and incubate in a 37°C incubator for 1 hour;

b) Add 10 μL of 0.5M EDTA to the above liquid, mix well, and terminate the reaction;

c) Inactivate in a water bath at 80°C for 10 minutes;

d) Add 20 μL proteinase K solution, followed by 200 μL AL buffer, vortex and mix, and bathe in 56°C water bath for 10 minutes;

e) Add 440 μL of phenol chloroform to extract the DNA in the virus particles, and vortex to mix;

f) 12000rpm, centrifuge at 4°C for 10min;

g) Separate the layers after centrifugation, take about 300 μL of the supernatant, and transfer it to another ep tube. Avoid pumping phenol chloroform into the lower layer;

h) Add 6 μL of 5M NaCl solution, add 750 μL of absolute ethanol, and finally add 1 μL of glycogen;

i) Mix well and place in -80°C refrigerator for 1 hour;

j) Take the sample out of the refrigerator, centrifuge at 12000rpm, 4°C for 30min, and remove the supernatant;

k) DNA precipitate appears at the bottom of the Ep tube, wash with 1mL of 70% ice ethanol, cover it, and turn it upside down several times;

l) 12000rpm, centrifuge at 4°C for 10min, discard the supernatant;

m) 12000rpm, centrifuge at 4°C for 10min to remove residual alcohol;

n) Open the lid to allow excess alcohol to evaporate;

o) After 3 minutes, add 40 μL ddH ₂ O to dissolve the DNA;

6) Real-time quantitative PCR to detect virus content.

The result is shown in Figure 5. The results showed that niclosamide can inhibit EBV extracellular virus release in a concentration-dependent manner.

Experiment 6: The effect of niclosamide on the virus production outside HNE-2089 cells;

1) The well-growing cell line HNE1-2089 was planted in a 24-well plate with a cell dosage of 2×10 ⁵ /well, and the cells were divided into a non-induced group and an induced group;

2) After 12 hours, the cells adhered to the wall, and the inducers TPA and NaB were added, and the final concentrations were 20ng/mL and 3mM, respectively;

3) After induction for 3 hours, add corresponding concentrations of niclosamide, the concentrations are 0 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM, respectively, and each concentration is repeated for 3 wells;

4) Harvest the cells after 120 hours, centrifuge at 10,000g for 10 minutes, and take the supernatant at 4°C;

5) Extracting viral DNA, the method is as follows:

a) Take 200 μL supernatant, add 2 μL DNase I, 20 μL 10×DNase I buffer, and incubate in a 37°C incubator for 1 hour;

b) Add 10 μL of 0.5M EDTA to the above liquid, mix well, and terminate the reaction;

c) Inactivate in a water bath at 80°C for 10 minutes;

d) Add 20 μL proteinase K solution, followed by 200 μL AL buffer, vortex and mix, and bathe in 56°C water bath for 10 minutes;

e) Add 440 μL of phenol chloroform to extract the DNA in the virus particles, and vortex to mix;

f) 12000rpm, centrifuge at 4°C for 10min;

g) Separate the layers after centrifugation, take about 300 μL of the supernatant, and transfer it to another ep tube. Avoid pumping phenol chloroform into the lower layer;

h) Add 6 μL of 5M NaCl solution, add 750 μL of absolute ethanol, and finally add 1 μL of glycogen;

i) Mix well and place in -80°C refrigerator for 1 hour;

j) Take the sample out of the refrigerator, centrifuge at 12000rpm, 4°C for 30min, and remove the supernatant;

k) DNA precipitate appears at the bottom of the Ep tube, wash with 1mL of 70% ice ethanol, cover it, and turn it upside down several times;

l) 12000rpm, centrifuge at 4°C for 10min, discard the supernatant;

m) 12000rpm, centrifuge at 4°C for 10min to remove residual alcohol;

n) Open the lid to allow excess alcohol to evaporate;

o) After 3 minutes, add 40 μL ddH ₂ O to dissolve the DNA

6) Real-time quantitative PCR to detect virus content.

The result is shown in Figure 6. The results showed that niclosamide can inhibit the extracellular virus production of EBV in EBV-infected epithelial cells in a concentration-dependent manner.

Experiment 7: The effect of niclosamide on the survival of PBMC cells

MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboymethoyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt) is a newly synthesized tetrazole compound, which can It is reduced to colored formazan products by various dehydrogenases in the mitochondria of living cells, and its color depth is highly correlated with the number of living cells of some sensitive cell lines within a certain range. According to the measured absorbance value (OD value) of 490n, the number of viable cells is judged. The larger the OD value, the stronger the cell activity, and the less toxic the drug. The specific experimental operation is as follows:

1) inoculate cells, with the RPMI medium that contains 10% fetal calf serum, human peripheral blood mononuclear cell PBMC is inoculated to 96 well plates with 10000 cells per well, and volume per well is 100ul;

2) Niclosamide was added after 12 hours, and the final concentrations were 0 μM, 0.01 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM;

3) After culturing for 48 hours, add 20ul of MTS solution to each well, and continue to incubate in the incubator for 2~4 hours;

4) Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, and observe the cytotoxicity of the compound to PBMC cells.

The experimental results are shown in Figure 7, and the experimental results show that when CC50>80 μM, the toxicity of the antiviral compounds is low, and there is no cytotoxicity in PBMC cells.

The experimental results showed that the expression of KSHV or EBV-related genes in the cells treated with niclosamide was significantly reduced, and the release of extracellular virus particles was also significantly reduced. These inhibitory effects are concentration-dependent, and have good antiviral effects at concentrations without obvious toxicity, which provides a strong theoretical basis and experimental basis for further research and development of antiviral drugs, and has important research and development value and development significance.

Therefore, niclosamide can play a better therapeutic effect on diseases caused by KSHV and EBV by inhibiting the replication and expression of KSHV and EBV, especially Kaposi's sarcoma and primary effusion lymphoma caused by KSHV. , Multi-center castor chronic disease has good preventive and therapeutic effects.

Niclosamide can be used in combination with other active ingredients against oncogenic herpesviruses to inhibit the replication and expression of KSHV and EBV through various pathways, and to treat diseases caused by KSHV and EBV, especially Kaposi’s sarcoma and primary tumors caused by KSHV. It plays a better role in the treatment of exudative lymphoma and multi-center castor chronic disease.

Claims (1)

1. application of the niclosamidum in preparing anti-tumorigenesis herpesvirus medicament, wherein the tumorigenesis herpesviral is EBV.