CN109793726B - Application of benzethonium chloride in preparation of medicine for preventing and treating lung cancer - Google Patents
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Abstract
The invention discloses application of benzethonium chloride in preparation of a medicine for preventing and treating lung cancer. According to the invention, in-vitro and in-vivo experiments show that benzethonium chloride can inhibit the growth and proliferation of lung cancer, and the benzethonium chloride has the advantages of small toxic and side effects, high safety and low price, so that the benzethonium chloride can be used as a medicine for treating lung cancer. In addition, the invention also finds that the combined use of the benzethonium chloride and the gefitinib obviously inhibits the growth of the lung cancer cells compared with the use of the gefitinib alone, which indicates that the addition of the benzethonium chloride can increase the sensitivity of the lung cancer cells to the gefitinib, so that the benzethonium chloride can be used as a sensitizer of the gefitinib or used for treating the lung cancer by combining the benzethonium chloride and the gefitinib.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of benzethonium chloride in preparation of a medicine for preventing and treating lung cancer.
Background
Lung cancer is one of the most common malignant tumors and one of the most refractory diseases in the world, and the incidence rate is on the rise in recent years. Because the differentiation between the lung cancer and the benign lung disease is difficult, and the early symptoms of the lung cancer are not obvious, the disease condition of most patients is developed to the middle and late stage when the patients visit the clinic, so that the optimal treatment period is missed, and the treatment effect is seriously influenced. At present, lung cancer treatment means comprise operation treatment, radiotherapy, chemotherapy, immunotherapy, traditional Chinese medicine treatment and the like, however, the postoperative recurrence and poor prognosis of patients are caused by the tolerance of cancer to radiotherapy and chemotherapy, the treatment difficulty is increased, and anticancer drugs commonly used in clinic often have certain toxic and side effects and are expensive. Therefore, the search for safe and efficient anti-tumor drugs for inhibiting the growth of lung cancer is inevitable.
Benzethonium chloride (Benzenthonium chloride) is approved by the Food and Drug Administration (FDA) of the United states for use as a first-aid product, is a sterilizing disinfectant, is also a novel preservative, has good surface activity, and is widely used as a preservative and bactericide in the fields of daily chemicals, medicines and the like. However, for lung cancer, no relevant literature and patent reports that benzethonium chloride has an inhibitory effect on the growth of lung cancer cells.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the application of benzethonium chloride in preparing the medicine for preventing and treating lung cancer.
The invention also aims to provide the application of benzethonium chloride and gefitinib in preparing the anti-tumor combined medicament.
The purpose of the invention is realized by the following technical scheme: application of benzethonium chloride in preparing medicine for preventing and treating lung cancer is disclosed.
The benzethonium chloride can achieve the purpose of treating the lung cancer by inhibiting the growth and/or proliferation of the lung cancer.
The effective concentration of the benzethonium chloride is 5-20 mu M; preferably 10 to 20 μ M.
Use of benzethonium chloride in the manufacture of a medicament for inhibiting the growth and/or proliferation of lung cancer cells.
The effective concentration of the benzethonium chloride is 5-20 mu M; preferably 10 to 20 μ M.
The lung cancer cell is preferably A549 cell or H1299 cell.
Application of benzethonium chloride and gefitinib in preparing anti-tumor combined medicine.
The tumor is preferably lung cancer.
An anti-tumor combination drug, comprising benzethonium chloride and gefitinib.
The effective concentration of benzethonium chloride in the anti-tumor combined medicine is 5-20 mu M (preferably 5 mu M), and the effective concentration of gefitinib is 5-20 mu M (preferably 5 mu M).
Use of benzethonium chloride for the manufacture of a medicament for increasing the sensitivity of lung cancer cells to gefitinib.
The medicine for preventing and treating lung cancer and the anti-tumor combined medicine can also contain one or more pharmaceutically acceptable carriers or auxiliary materials; the auxiliary material is at least one of a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant or a lubricant.
The medicine can be prepared into a medicinal preparation by adopting a conventional method in the field; comprises oral administration pharmaceutical preparations, such as capsules, pills, tablets, oral liquid, granules, tinctures and the like.
Application of benzethonium chloride in preparing sensitizer for gefitinib.
Compared with the prior art, the invention has the following advantages and effects:
1. benzethonium chloride is mainly used for sterilizing and disinfecting agents, but no relevant report and patent exists at present for the inhibition effect of benzethonium chloride on lung cancer. The invention combines in vitro and in vivo experiments, and the result shows that benzethonium chloride can inhibit the growth and proliferation of lung cancer, is an FDA approved drug, has small toxic and side effect, high safety and low price, can be dissolved in water and orally taken, assists in lung cancer treatment, can provide a new drug choice for clinical treatment of lung cancer, and has good development and utilization prospects.
2. Gefitinib is used as a first-line drug for lung cancer, which has serious influence on the treatment effect due to drug resistance, and the invention finds that benzethonium chloride can increase the sensitivity of lung cancer cells to gefitinib, so that the combination of benzethonium chloride and gefitinib can be used for treating lung cancer.
Drawings
FIG. 1 is a graph of the effect of benzethonium chloride on lung cancer cell proliferation; wherein, the graph A shows the influence of benzethonium chloride with different concentrations on the growth capacity of A549 cells; panel B shows the effect of different concentrations of benzethonium chloride on the growth capacity of H1299 cells.
FIG. 2 is a graph of the effect of benzethonium chloride on apoptosis of lung cancer cells; wherein, the graph A shows the influence of different concentrations of benzethonium chloride on apoptosis of A549 and H1299 cells; FIG. B is a statistical result of apoptosis of A549 cells induced by benzethonium chloride with different concentrations; and the graph C shows the statistical result of the H1299 cell apoptosis induced by different concentrations of benzethonium chloride.
FIG. 3 is a graph of the effect of benzethonium chloride on the tumorigenic capacity of lung cancer cells in nude mice; wherein, panel A is subcutaneous tumor in nude mice; panel B is the change in tumor volume size; panel C shows the body weight change of nude mice.
FIG. 4 is a graph of the results of benzethonium chloride increasing the sensitivity of lung cancer cells to gefitinib; wherein, the graph A shows the influence of different drugs on the growth capacity of A549 cells; panel B is the effect of different drugs on the growth capacity of H1299 cells.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Example 1 assay of WST-1 cell Activity
1. Cell plating: a549 and H1299 cells (purchased from American type Culture Collection, ATCC) cell bank were collected, counted, plated in 96-well plates in an amount of 3000 cells per well, and cultured overnight.
2. And (3) drug treatment: benzethonium chloride was diluted with DMEM complete medium to treatment concentrations (0, 5, 10, 15, 20 μ M), and then the original medium in the 96-well plate was replaced for 24, 48 and 72h, respectively.
WST-1 detection: according to the following steps of 1: 10(w/v), diluting WST-1(WST-1 cell proliferation assay kit) with DMEM complete medium to obtain a culture medium containing WST-1; then, the medium originally in the 96-well plate was replaced with the medium containing WST-1, and the medium containing WST-1 was additionally added to the wells without cultured cells as a blank at 100. mu.L per well. After incubating the 96-well plate in a constant temperature incubator at 37 ℃ for 1h, the plate is read by a microplate reader (450 nm). The whole process is carried out in dark.
4. As a result: the results are shown in fig. 1A and 1B, which show that with the drug treatment of concentration gradient and time gradient, the growth of lung cancer cells is significantly inhibited, i.e. benzethonium chloride can be used to inhibit the proliferation of lung cancer cells.
Example 2 apoptosis
1. And (3) drug treatment: a549 and H1299 cells were collected and plated in 6-well plates to a cell density of about 40%, and the cells were treated by adding DMEM medium containing 0, 10, 15 and 20. mu.M benzethonium chloride, respectively.
2. Collecting cells: after 48h of drug treatment, apoptotic cells in the supernatant were collected, centrifuged at 1100rpm for 3min and then discarded, adherent cells were digested with 0.25% trypsin, collected in the same centrifuge tube, centrifuged at 1100rpm for 3min and then discarded. Staining was performed after two washes with PBS buffer.
3. Dyeing: the cells were resuspended in 500. mu.L Binding Buffer (apoptosis detection kit), 5. mu.L Annexin V-FITC was added to each tube and mixed well, 5. mu.L Propidium Iodide was added and mixed well, and then the reaction was carried out for 15min at room temperature in the dark.
4. Flow detection: after the staining was finished, the staining was checked by a C6 flow cytometer, and before the machine was operated, the sample was filtered through a 400 mesh sieve to remove particles. Wherein Annexin V-FITC is green fluorescence, an FL1 channel is selected for detection, and a Propidium Iodide is red fluorescence, and an FL2 or FL3 channel is selected for detection.
5. As a result: the results are shown in fig. 2, which shows that benzethonium chloride treatment induced apoptosis of cells significantly.
EXAMPLE 3 nude mice subcutaneous tumor formation experiment
1. The animal experiment of the subject strictly follows the guiding principles of animal nursing institutions and is approved by the ethical committee of animal experiments of river-south university.
2. The experimental animals used in the subject are female nude mice (Balb/c-nu, purchased from southern medical university laboratory animal center) of 6-8 weeks in total 15, wherein 5 control groups and 10 experimental groups construct nude mice subcutaneous tumorigenesis models:
(1) cell collection: cells were collected and counted at 1X 10 per nude mouse6The total number of cells was calculated from the amount of A549 cells, and the cells were resuspended in PBS buffer and mixed with matrigel (purchased from spring Biotech, Guangzhou) at a volume ratio of 1: 1.
(2) Inoculation: after the mice were anesthetized (the degree of anesthesia was evaluated by painless and painful stimulation to confirm that the nude mice were under anesthesia), the above-mentioned resuspended cells mixed with matrigel were taken out with a 1mL syringe (25G needle) and injected subcutaneously into the nude mice.
3. One week after cell injection, nude mice were observed for tumor formation and measured, and when tumors reached a size of 5 × 5, administration by intraperitoneal injection and intragastric administration was started. In the experiment, 3 benzethonium chloride concentration gradients (50 mg/mL of benzethonium chloride mother liquor prepared by DMSO is firstly diluted into the use concentration by sterile water, and each mouse is administrated with 100 mu L of the benzethonium chloride mother liquor each time) are arranged, and two administration modes are adopted. 0mg/kg, 5mg/kg (intraperitoneal injection) and 10mg/kg (intragastric administration), respectively, of 5 of the above-mentioned drugs were administered every two days. 6. During the administration, body weight of nude mice and size of transplanted tumor were measured every two days. Two weeks later, the nude mice were euthanized and the tumors were removed.
4. As a result: the results are shown in FIG. 3. FIGS. 3A and 3B show the results of subcutaneous tumor formation in nude mice, and the tumor growth curves show that subcutaneous tumor formation in nude mice is significantly inhibited by benzethonium chloride. The benzethonium chloride can inhibit the growth of lung cancer tumor. And fig. 3C is a statistical result of the body weights of the three groups of mice after administration, showing that there is no significant difference in body weight between the three groups of mice, demonstrating the safety of the use of benzethonium chloride as an antitumor agent.
Example 4 Benzochloraz increases sensitivity of Lung cancer cells to Gefitinib
1. Cell plating: a549 and H1299 cells were collected, counted, plated in 96-well plates at 3000 cells per well, and cultured overnight.
2. And (3) drug treatment: benzethonium chloride and gefitinib were diluted to 5 μ M with DMEM complete medium, respectively, and then the original medium in the 96-well plate was replaced for 72 hours.
WST-1 detection: according to the following steps of 1: 10(w/v), diluting WST-1(WST-1 cell proliferation assay kit) with DMEM complete medium to obtain a culture medium containing WST-1; then, the medium originally in the 96-well plate was replaced with the medium containing WST-1, and the medium containing WST-1 was additionally added to the wells without cultured cells as a blank at 100. mu.L per well. After incubating the 96-well plate in a constant temperature incubator at 37 ℃ for 1h, the plate is read by a microplate reader (450 nm). The whole process is carried out in dark.
4. As a result: as shown in fig. 4, the gefitinib alone significantly inhibited the growth of lung cancer cells compared to the combination group, and there was a significant difference, indicating that the addition of benzethonium chloride can increase the sensitivity of lung cancer cells to gefitinib.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. Application of benzethonium chloride in preparing medicine for preventing and treating lung cancer is disclosed.
2. Use of benzethonium chloride according to claim 1 for the preparation of a medicament for the prevention and treatment of lung cancer, characterized in that: the benzethonium chloride can achieve the purpose of treating the lung cancer by inhibiting the growth and/or proliferation of the lung cancer.
3. Use of benzethonium chloride according to claim 1 for the preparation of a medicament for the prevention and treatment of lung cancer, characterized in that: the effective concentration of the benzethonium chloride is 5-20 mu M.
4. The application of benzethonium chloride in preparing medicine for inhibiting growth and/or proliferation of lung cancer cell is characterized by that:
the lung cancer cell is A549 or H1299 cell.
5. The application of benzethonium chloride and gefitinib in preparing the anti-tumor combined medicament is characterized in that: the tumor is lung cancer.
6. An anti-tumor combined medicament is characterized in that: including benzethonium chloride and gefitinib.
7. The anti-tumor combination drug according to claim 6, characterized in that: the effective concentration of benzethonium chloride in the anti-tumor combined medicine is 5-20 mu M, and the effective concentration of gefitinib is 5-20 mu M.
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