Effect of MTX on central stem cells

Methotrexate:

Methotrexate is an anti-folate antitumor drug, which inhibits the growth and reproduction of tumor cells by inhibiting the synthesis of dihydrofolate reductase.

Main uses:

Methotrexate can be used as folic acid antagonist, effective inhibitor of dihydrofolate reductase, immunosuppressive agent, and tissue culture reagent. This product is a good anti-tumor drug for the treatment of acute lymphatic leukemia, lymphoma, malignant mole, choriocarcinoma, squamous cell carcinoma, lung cancer, cervical cancer, breast cancer and osteosarcoma. After oral administration of methotrexate for 12 hours, 90% of the original drug was excreted from the urine, and the main metabolite was 7-hydroxy methotrexate.

Introduction to the treatment of methotrexate: Chemotherapy is one of the important means of treating malignant tumors. Since the 1960s, high-dose methotrexate treatment methods have been used in clinical practice, and some conventional chemotherapy of malignant tumors has achieved good results. However, methotrexate (MTX) kills tumor cells and also causes a number of adverse reactions to normal tissue cells, especially some renewed tissues (such as bone marrow) in the body. In recent years, the neurotoxic effects of methotrexate have attracted the attention of clinicians. Studies have confirmed that high-dose intravenous and intrathecal administration of MTX is associated with demyelinated white matter necrosis, oligodendrocyte loss, axonal swelling, brain softening and white matter atrophy. Neural stem cells are a group of cells with self-renewal ability and multi-directional differentiation potential in the central nervous system, which play a key role in the development and maintenance of the nervous system.

The toxic effects of methotrexate on neural stem cells (NSC) cultured in vitro can be found in historical literature. Its toxic mechanism has been explored in vitro. The NSCs in fetal rat cerebral cortex were cultured in vitro and identified by Nestin immunofluorescence. Morphological changes of NSC after MTX intervention were observed under microscope. The effects of MTX on the proliferation of NSCs were detected by 5-bromo deoxyuridine (BrdU) incorporation and Nestin/BrdU immunohistochemical double labeling. Cell cycle analysis was performed by flow cytometry.

According to the historical literature research, the growth, survival and apoptosis of neural stem cells (methotrexate cell culture) cultured in vitro by MTX were observed, and the methotrexate mechanism was explored.

The effects of methotrexate:

Effect of methotrexate on the morphology of neural stem cells

After observation with an inverted microscope, after 24 hours of normal cell culture, some cells were deposited on the bottom wall of the culture flask, and some cells were suspended in the culture medium. The cells appeared in pairs. After 2 to 3 days of culture, cell clusters of different sizes were observed. The larger cell mass is composed of dozens of cells with bright cell edge, strong refractive index, and it is in a suspended state. That is, the neurospheres gradually increase with the prolongation of the culture time, and each neurosphere increases on the 6th day. Neurosphere is formed by the aggregation of tens or even hundreds of cells, and the cells are arranged in a compact manner. After being cultured for 5 days in MTX, the growth state of the 40g/l MTX group was basically the same as that of the control group, while the cells of the 400g/LMTX intervention group were in a nutritionally deficient state, the cell spheres were relatively small and the number of spheres was relatively small.

Effect of methotrexate on the proliferation of neural stem cells:

The primary cultured 6d-proliferated cells were subjected to NestinBrdU immunofluorescence single labeling and Nestin/BrdU immunofluorescence double-labeled staining under fluorescence microscope. 10 non-overlapping field and double-labeled positive cells were randomly selected from each group for counting. The proportion of cells in the 400g/lMTX group was lower than that in the control group, and the percentage of double-labeled positive cells in the total cells was also lower than that in the control group.

Effect of MTX on the concentration of Hcy in neural stem cells:

Homocysteine is an important metabolic intermediate of sulfur-containing amino acids in humans. Homocysteine, also known as homocysteine, is produced by the conversion of methionine to methyl groups in the body. There are two ways to go. One is that Hcy can be catalyzed by cystathionine condensation enzyme and cystathionine. The formation of cysteine requires the participation of vitamin B6, or the combination of thiol oxidation to form homocysteine. In addition, Hcy can re-synthesize methionine under the auxiliary action of folic acid and vitamin B12. Catalyzed by methionine synthetase, and must have N5-methyltetrahydrofolate as a donor to the methyl group, the latter being produced by tetrahydrofolate catalyzed by 5,10-methylenetetrahydrofolate reductase.

Methotrexate is a class of anti-metabolic chemicals that interfere with the synthesis of thymidine and purines and inhibit DNA by competitively inhibiting dihydrofolate reductase and blocking it from catalyzing the conversion of folic acid to tetrahydrofolate. It is more sensitive to cells with faster cell proliferation, such as neural stem cells, so it affects the synthesis of protein and RNA to some extent in cell proliferation. The results of experiment show that MTX has toxic effects on neural stem cells cultured in vitro, and has a certain dose-dependent relationship. In addition, cell cycle analysis confirmed that MTX can interfere with NSC cell cycle progression, inhibiting cells from G1 phase into S phase, resulting in an increase in G1 phase ratio, suggesting that MTX can specifically act on S phase, allowing cells to arrest in G1 phase, so MTX has a certain nhibitory effect on DNA synthesis. Due to MTX intervention, the methyl cycle process is blocked, the synthesis of 5-methyltetrahydrofolate is reduced, and it is impossible to provide sufficient methyl group to Hcy.and cannot be converted into methionine (Met), resulting in Hcy accumulation. This study confirmed that MTX can cause a relative excess of Hcy in neural stem cells, further demonstrating that MTX plays an important role in the generation and metabolism of Hcy.

The experiments have shown the concentration of Hcy in the 400g/L MTX group and the control group increased to the maximum on the 5th day and then decreased with time. The Hcy concentration in the MTX group was significantly higher than that in the control group at the 135th and 7th day.

In addition, a large number of experiments have shown that accumulation of Hcy in the body can lead to hyperhomocysteinemia (HHcy). HHcy is very common in neurological diseases such as Alzheimer's disease and Parkinson's disease, and it is currently believed that HHcy is the cause of Alzheimer's disease. Hcy is an independent risk factor for neurodegenerative diseases and has certain damage to the central nervous system. Kruman et al. have shown that Hcy has a damaging effect on rat hippocampal neurons cultured in vitro and low concentration of Hcy can promote neuronal apoptosis. It has been found that in experimental models of AD animals, the incubation of hippocampal neurons with Hcy can lead to apoptosis or increased cell sensitivity to A-amyloid-induced cell death. It was first reported in 2003 that Hcy can promote astrocyte apoptosis in rat cerebral cortex. Therefore, the authors speculate that the toxic effects of MTX on NSCs may be related to the increase of Hcy concentration.

Methotrexate is an antagonist of folic acid, and excessive intake of methotrexate can cause a deficiency of folic acid in the body. Experiments have shown that the lack of folic acid in the diet can weaken the repair function of DNA, increase the damage of DNA, and induce apoptosis of nerve cells. In addition, Craciuneseu et al. found that folic acid deficiency in late pregnancy of mice can reduce the proliferation of embryonic brain NSCs and increase the apoptosis of embryonic brain cells. The authors used MTX to intervene in vitro culture of NSCs, which is consistent with the above-mentioned reports of neuronal apoptosis induced by folate deficiency. 

Other drugs:

Aza-serine is similar to glutamine, which inhibits the formation of UTP→CTP. 2. Methotrexate is an analog of folic acid, which can competitively inhibit dihydrofolate reductase, so that folic acid cannot be reduced to dihydrofolate and tetrahydrofolate, thereby inhibiting the synthesis of purine nucleotides for the treatment of tumors. 3.6-Mercaptopurine (6MP) has a chemical structure similar to that of hypoxanthine, which competitively inhibits hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and prevents the reductive synthesis pathway of purine nucleotides. 4. It is not similar to hypoxanthine except for molecular exchange, so it can inhibit xanthine oxidase, thereby inhibiting the production of uric acid and treating gout. 5. The structure of 5-fluorouracil (5-FU) is similar to that of thymine and can be converted into FdUMP and FUTP in vivo. Similar to the structure of dUMP, FdUMP is an inhibitor of thymidylate synthase, which hinders the synthesis of dTMP and interferes with the synthesis of RNA molecules, thereby achieving anti-tumor effects.