CN104706697A - Application of radix ginseng in inhibition of ras proto-oncogene overexpression - Google Patents

Abstract

The invention relates to the application of ginseng in inhibiting the overexpression of ras proto-oncogene, in particular to the application of ginseng in the preparation of antitumor drugs for inhibiting the overexpression of ras proto-oncogene. Studies have shown that about 90% of pancreatic cancer, 50% of colon cancer, 30%-40% of lung adenocarcinoma, and 5%-40% of leukemia are caused by the overexpression of the ras proto-oncogene. Targets of cancer drugs. Animal experiments have shown that ginseng can specifically down-regulate the overexpression of the ras proto-oncogene after mutation, but does not act on the wild-type ras proto-oncogene, so the toxicity is low. The invention provides a new application of ginseng, which can be used in the preparation of anti-tumor (tumor caused by overexpression of ras proto-oncogene) drugs.

Description

The application of ginseng in inhibiting the overexpression of ras proto-oncogene

technical field

The invention specifically relates to the application of ginseng in inhibiting the overexpression of ras proto-oncogene, and belongs to the field of traditional Chinese medicine.

Background technique

Traditional Chinese medicine ginseng is the dried root of Araliaceae ginseng Panax ginseng C. A, Mey. The main active ingredients are ginsenoside and ginseng polysaccharide. . The clinical verification of traditional Chinese medicine shows that the main effects of ginseng include nourishing vitality, nourishing the spleen and lungs, promoting body fluid and calming the nerves, improving intelligence, anti-fatigue, anti-shock, and lowering blood lipids.

About 30% of human tumors are caused by the overactivation of Ras proto-oncogene after mutation, resulting in increased expression of Ras protein. If the Ras protein is continuously activated, it can bind to the downstream effector protein and transmit the signal to the downstream signaling element, causing abnormal cell proliferation and tumorigenesis. Studies have shown that about 90% of pancreatic cancer, 50% of colon cancer, 30%-40% of lung adenocarcinoma and 5%-40% of leukemia are caused by the overactivation of ras gene.

Therefore, tumor therapy targeting the Ras protein and the signaling pathway regulated by the Ras protein is of great value in the treatment of tumors caused by ras mutations, and Ras has become a recognized target for screening anti-related malignant tumor drugs. For example, on December 20, 2005, Nexavar, a drug for advanced renal cell carcinoma approved by the US FDA, directly targets the Ras signaling pathway, which can inhibit the Raf/MEK/ERK signaling pathway and directly inhibit tumor growth.

So far, no literature has reported that ginseng can inhibit the overexpression of the ras proto-oncogene, and there is no drug on the market that uses ginseng as a raw material, which can specifically inhibit the tumor caused by the overexpression of the ras proto-oncogene.

Contents of the invention

The purpose of the present invention is to provide a new application of ginseng, especially the application of ginseng in inhibiting the overexpression of ras proto-oncogene.

Another purpose is to provide a new application of ginseng in the preparation of antitumor drugs, the said tumor is caused by the overexpression of ras proto-oncogene.

Because the ras signaling pathway is highly conserved from nematodes to humans, let-60 gene encodes a conserved Ras protein in Caenorhabditis elegans, which has 83% homology with human Ras protein. If the ras mutation, in Caenorhabditis elegans, it will cause the nematode to produce polyvagina, and in humans, it will cause the malignant proliferation of cells, leading to the occurrence of tumors. Therefore, the model organism Caenorhabditis elegans ( Caenorhabditis elegans ) overactivated mutants of the Ras/MAPK signaling pathway is widely used as a test group to screen anti-tumor drugs that inhibit the overactivation of the ras proto-oncogene pathway. Therefore, in this experiment, the Caenorhabditis elegans mutant strain was used as a model to evaluate the drugs that inhibit the overactivation of the ras proto-oncogene pathway, and it was concluded that ginseng had a significant down-regulation effect on the overexpression of the ras proto-oncogene, and had the ability to inhibit the ras proto-oncogene pathway. overactivation conclusion.

The beneficial effects of the present invention are as follows: the present invention provides a new use of ginseng, which broadens the scope of ginseng medication; ginseng has an obvious down-regulation effect on the overexpression of the ras proto-oncogene, and can specifically inhibit the overexpression of the ras proto-oncogene, Instead of directly inhibiting the proliferation and differentiation of cells, that is, the effect of ginseng is dependent on the ras pathway mechanism, rather than extensive cytotoxicity; and ginseng has no cytotoxicity, and only acts on the overactivated Ras protein, not on the wild-type Ras protein. In the future, we are expected to apply ginseng in the preparation of antitumor drugs, wherein the tumor is caused by the overexpression of ras proto-oncogene, so as to provide better treatment for patients with such tumors, and provide a basis for the treatment of some difficult cancers. Provide new avenues.

Specific examples are provided below to realize the application of ginseng according to the present invention in the preparation of antitumor drugs caused by overexpression of ras proto-oncogene, but are not limited to these examples.

Detailed ways

The preparation of embodiment one ginseng medicinal liquid

Ginseng source: purchased from Lanzhou Yellow River Medicine Market.

Weigh 9 g of ginseng; first soak in distilled water for 20 minutes, then boil on low heat, decoct for 30 minutes, constant volume, centrifuge, discard the precipitate, filter the supernatant aseptically, put it in a 4°C refrigerator for later use, and dilute to different concentrations when used.

Example The effect of Ershen liquid on Caenorhabditis elegans MT2124 (let-60 hyperactivation mutation)

Ras is a small molecular protein that participates in the regulation of many important links in animal development. The ras signaling pathway is highly conserved from nematodes to humans. The let-60 gene encodes a conserved Ras protein in Caenorhabditis elegans, which has 83% homology with human Ras proteins. If the ras mutation, in Caenorhabditis elegans, it will cause the nematode to produce polyvagina, and in humans, it will cause the malignant proliferation of cells, leading to the occurrence of tumors. Therefore, the model organism Caenorhabditis elegans ( Caenorhabditis elegans ) Ras/MAPK signaling pathway overactivation mutant is widely used as a tumor model to screen anti-tumor drugs that inhibit the overactivation of the ras proto-oncogene pathway.

MT2124 is a let-60/ras overactivated C. elegans that produces a polyvaginal phenotype. Dilute the drug solution to a certain concentration and act on Caenorhabditis elegans MT2124. If the proportion of individuals with multiple vulva in the tested nematode population can be suppressed, while the proportion of wild-type nematode individuals with only one true vulva increases, it means that the drug acts on the ras pathway , which down-regulates the overactivation of this pathway and has antitumor activity. The higher the proportion of wild-type nematodes in the population, the more pronounced the effect of the drug. It is the opposite if expressed in the proportion of mutants, where the proportion of wild type (%)=100%-ratio of mutants (%)

Experimental materials: Caenorhabditis elegans MT2124, genotype: let-60(n1046) IV, purchased from CGC (Caenorhabditis Genetics Center); Escherichia coli OP50 (uracil leakage mutant strain), as food for Caenorhabditis elegans, From CGC (Caenorhabditis Genetics Center).

Reagent preparation:

1. Preparation of NGM Plates

Element content NaCl 3.00g K ₂ HPO ₄ 2.34g KH ₂ PO ₄ 17.23g Peptone 2.50g agar 17.00g H ₂ O 1000ml

After the solid NGM medium is prepared, sterilize under high pressure at 121°C for 20 minutes, add 1ml of 5mg/ml cholesterol, 1ml of 1M MgSO ₄ , 1ml of 1M CaCl ₂ under the aseptic operating table, shake well, and pour it into the sterilized medium while it is hot. 9cm culture plate, about 20ml/plate. Stand still and wait for the medium to solidify, and set aside.

2. Preparation of M9 buffer:

Element content Na ₂ HPO ₄ 6.00g KH ₂ PO ₄ 3.00g NaCl 5.00g 1M MgSO ₄ 1.00ml H ₂ O 1000ml

3. Preparation of S solution:

name Element content S liquid formula: NaCl 5.85g the K ₂ HPO ₄ 1.00g the KH ₂ PO ₄ 6.00g the H ₂ O 1000ml

Specific experimental steps:

Cultivation of nematodes: connect nematodes to solid NGM plates coated with Escherichia coli OP50, and then culture them in an incubator at 20°C, and perform synchronization when the nematodes grow into adults.

Synchronization of nematodes: wash down the nematodes on the plate with M9 buffer, suck the washing solution into a centrifuge tube; centrifuge at 4000rpm/min for 3min, remove the supernatant; add lysate (final concentration: 3.2% NaClO and 1M NaOH); Shake on the spinner for 7 minutes to rupture the nematodes and release eggs; centrifuge at 4000rpm/min for 3 minutes, remove the supernatant, collect the precipitate to obtain a large number of nematode eggs; wash twice with M9 buffer, add 1ml of M9 buffer, and place at 20 Incubate in an incubator for 48 hours.

Drug action: Dilute the traditional Chinese medicine compound stock solution to a certain concentration, and dilute the synchronized nematodes to about 80/20μl. In a 96-well plate, add 120μl of S solution, 20μl of worm solution, and 10mg/ml Escherichia coli OP50 20μl, 5mg/ml cholesterol 20μl, appropriate diluted drug solution 20μl, each concentration set five parallel. In the blank control group, no medicinal solution was added, and the medicinal solution was replaced with S solution. Cultivate at a constant temperature of 20°C until the nematodes mature, and detect the wild-type ratio of the nematodes under a microscope. The results are shown in the table below:

Table 1 The effect of different concentrations of ginseng liquid on the wild type ratio of Caenorhabditis elegans MT2124

Note: * Compared with the blank group, P<0.05

It can be seen from the above table that ginseng can significantly increase the wild-type ratio of Caenorhabditis elegans MT2124 in a concentration-dependent manner, indicating that ginseng can inhibit the overexpression of the ras proto-oncogene and make the mutant become wild-type. At the same time, the number of Caenorhabditis elegans did not decrease during the experiment, indicating that ginseng has low toxicity and will not cause the death of Caenorhabditis elegans.

Example 3 The effect of ginseng on Caenorhabditis elegans MT8189 (lin-15 mutation inactivation)

Experimental materials: Caenorhabditis elegans MT8189, polyvulva phenotype, genotype: lin-15b(n765)X, purchased from CGC (Caenorhabditis Genetics Center); Escherichia coli OP50 (uracil leaky mutant), used as Caenorhabditis Nematode food was purchased from CGC (Caenorhabditis Genetics Center).

Concrete experimental procedure: with embodiment two.

Table 2 The effect of ginseng on Caenorhabditis elegans MT8189 (lin-15 mutation inactivation)

In Caenorhabditis elegans, lin-15(n765)X is located upstream of ras and normally inhibits the expression of ras gene. The inactivation of this gene in the MT8189 nematode strain leads to the phenotype of multiple ovipositors in the nematode.

As shown in the table, ginseng cannot inhibit the polyvulva phenotype of lin-15(n765)X. According to the principle of genetic epistasis, since ginseng can reverse the polyvulva phenotype caused by the overactivation of its downstream let60/ras proto-oncogene, then It also inhibited the polyvulva phenotype of MT8189, but the expected result was not observed in this experiment, that is, ginseng could not inhibit the polyvulva phenotype of let60/ras upstream gene lin-15(n765)X. This may be due to the fact that lin-15(n765) X. elegans contains a wild-type let60/ras copy, and ginseng only acts on overexpressed let60/ras, but is insensitive to wild-type let60/ras, that is, ginseng only acts on overexpressed let60/ras. The activated Ras protein is not sensitive to the wild-type Ras protein, which shows that ginseng only acts on the tumor cells caused by ras overactivation, but not on normal cells.

Example 4 The effect of ginseng on Caenorhabditis elegans CB1275 (lin-1 mutation inactivation)

Experimental material: Caenorhabditis elegans CB1275 Genotype: lin-1(e1275) IV was purchased from CGC (Caenorhabditis Genetics Center); Escherichia coli OP50 (uracil leakage mutant strain), as food for Caenorhabditis elegans, was purchased from CGC .

Concrete experimental procedure: with embodiment two.

Table 3 Effect of ginseng on Caenorhabditis elegans CB1275 (lin-1 mutation inactivation)

lin-1 is a gene downstream of ras, a nuclear transcription factor that can be phosphorylated by MAPK, a negative regulator of vulva development, and a signaling pathway that regulates vulva development downstream of let-60. After lin-1 mutation inactivation, nematodes have polyvaginal phenotype. Since lin-1 is a nuclear transcription factor, it regulates the process of vulva cell development. If the drug has a significant inhibitory effect on the polyvulva phenotype of the gene inactivation mutation, it means that the drug can directly inhibit the development of vulva precursor cells, prevent them from forming polyvulva and exhibit non-specific cytotoxicity.

The result of this example is that the polyvulva phenotype of the CB1275 strain nematode is not affected by ginseng, which indicates that the drug acting on the ras signaling pathway can inhibit the pseudo-vulva formed by excessive activation of ras, but cannot inhibit the pseudo-vulva formed by the inactivation of lin-1 mutation. Vaginal phenotype, indicating that ginseng acts on ras but not on lin-1, proving that the drug down-regulates the excessive activation of ras rather than directly inhibiting cell proliferation, that is, the effect of ginseng is dependent on the ras pathway mechanism, rather than extensive cytotoxic effect.

The animal experiments of the above examples show that the ginseng with anti-tumor activity involved in the present invention has an obvious down-regulation effect on the excessive activation caused by the mutation of the ras proto-oncogene, but does not act on the wild-type ras proto-oncogene, so the toxicity is low , indicating that ginseng is suitable for use in the preparation of anti-related malignant tumor drugs, and is expected to be developed into a new generation of drug preparations for the treatment of related malignant tumors caused by ras proto-oncogene mutations.

Claims (2)

1. The application of ginseng in inhibiting the overexpression of ras proto-oncogene. 2. The application of ginseng in the preparation of antitumor drugs, said tumor is caused by the overexpression of ras proto-oncogene.