阅读说明：本技术 一种吡哆醛在制备治疗卵巢癌的药物中的用途 (Application of pyridoxal in preparation of medicine for treating ovarian cancer ) 是由 令狐华 李若男 詹诗杰 刘斌 于 2020-12-28 设计创作，主要内容包括：本发明属于分子生物学和医药技术领域,公开了一种吡哆醛在制备治疗卵巢癌的药物中的用途,吡哆醛制备成药学上接受的任何一种制剂。验证吡哆醛在制备治疗卵巢癌的药物中的用途的方法包括：获取吡哆醛,并利用PBS配制为50mM工作液,利用0.22μm滤器避光抽滤灭菌；利用xCELLigence工作站及对应的培养板进行活细胞无标记计数检测。本发明通过实验证实了吡哆醛可抑制卵巢癌细胞增殖,且抑制作用十分显著,因此可用于制备治疗卵巢癌的药物,或制备研究卵巢癌发生发展机制的相关试剂。吡哆醛是维生素B6中间代谢产物之一,而维生素B6是水溶性维生素,其应用安全性较高,因此吡哆醛在治疗卵巢癌方面具有很强的临床应用可能。(The invention belongs to the technical field of molecular biology and medicine, and discloses an application of pyridoxal in preparing a medicine for treating ovarian cancer. The method for verifying the use of pyridoxal for the preparation of a medicament for the treatment of ovarian cancer comprises: obtaining pyridoxal, preparing 50mM working solution by using PBS, and performing suction filtration and sterilization by using a 0.22 mu m filter in dark place; and (3) utilizing an xCELLigence workstation and a corresponding culture plate to carry out living cell label-free counting detection. Experiments prove that pyridoxal can inhibit the cell proliferation of ovarian cancer, has obvious inhibition effect, and can be used for preparing medicaments for treating ovarian cancer or preparing related reagents for researching the occurrence and development mechanism of ovarian cancer. Pyridoxal is one of the intermediate metabolites of vitamin B6, while vitamin B6 is a water-soluble vitamin, and the application safety is high, so that the pyridoxal has strong clinical application possibility in the aspect of treating ovarian cancer.)

1. Use of pyridoxal in the preparation of a medicament for the treatment of ovarian cancer.

2. Use of pyridoxal according to claim 1 for the preparation of a medicament for the treatment of ovarian cancer, said pyridoxal being prepared in any of the pharmaceutically acceptable formulations.

3. Use of pyridoxal in the preparation of an agent for inhibiting ovarian cancer cell proliferation.

4. Use of pyridoxal according to claim 3 for the preparation of an agent that inhibits the proliferation of ovarian cancer cells, wherein said ovarian cancer cells are SKOV3, OVCAR3, 3AO, or ES-2.

5. A method for validating the use of pyridoxal according to claim 2 for the preparation of an agent for inhibiting the proliferation of ovarian cancer cells, said method for validating the use of pyridoxal for the preparation of an agent for inhibiting the proliferation of ovarian cancer cells comprising:

step one, obtaining pyridoxal, preparing the pyridoxal into a working solution by using PBS, and performing suction filtration and sterilization by using a filter in a dark place;

and secondly, performing label-free counting detection on the living cells by using an xCELLigence workstation and a corresponding culture plate.

6. The method for verifying the use of pyridoxal for the preparation of an agent for inhibiting ovarian cancer cell proliferation according to claim 5, wherein, in the first step, pyridoxal is obtained and formulated into 50mM working solution using PBS, and sterilized by suction filtration with a 0.22 μm filter in the absence of light.

7. The method according to claim 5 for use of pyridoxal in the preparation of an agent for inhibiting ovarian cancer cell proliferation, wherein in step two, complete medium is added to an E-plate 96-well plate to determine a baseline value, then well-mixed SKOV3, OVCAR3 suspension, pyridoxal, and complete medium are added to make up the well fluid, and a control group is treated with an equal amount of PBS; after standing at room temperature, an automatic scanning gap was set, and cell proliferation curves were detected.

8. The method for validating the use of pyridoxal for the preparation of an agent for inhibiting the proliferation of ovarian cancer cells according to claim 7, wherein 50 μ l of complete medium is added to the E-plate96 well plate to determine the baseline value, and then the well is added with mixed SKOV3, 100 μ l of OVCAR3 suspension, 2 μ l of pyridoxal, and the complete medium is used to make up the liquid in the well to 200 μ l.

9. The method for assaying pyridoxal for use in the preparation of an agent for inhibiting ovarian cancer cell proliferation according to claim 7, wherein the working concentration of pyridoxal is up to 0.5mM and the control group is treated with an equal amount of PBS.

10. The method for validating the use of pyridoxal for the preparation of an agent for inhibiting the proliferation of ovarian cancer cells according to claim 7, wherein after 30min at room temperature, an autosampler gap is set at 15min, a cell proliferation curve is examined, and the total scan time is 72 hours.

Technical Field

The invention belongs to the technical field of molecular biology and medicines, and particularly relates to application of pyridoxal in preparation of a medicine for treating ovarian cancer.

Background

At present, the mortality rate of ovarian cancer is stable at the first of malignant tumors of female reproductive systems, the disease progression is hidden, the prognosis is poor, and the health of women is seriously threatened. The uptake of vitamin B6 was found to be associated with a reduction in the incidence of malignancies and an improvement in prognosis, pyridoxal is one of the major metabolites of vitamin B6, both phosphorylated (PLP) and non-Phosphorylated (PL) forms. Although the phosphorylated form of pyridoxal (PLP) is currently considered to be the active form of vitamin B6 and may participate in the catalytic reaction as a coenzyme for a number of enzymes, the inhibitory effect of vitamin B6 on malignancies is also generally considered to be the effect of PLP. However, we repeatedly tested the anticancer efficacy of PLP and PL in ovarian cancer cells, and both of the results suggest that PLP at the same concentration has significantly less inhibitory effect on ovarian cancer cells than PL.

Through the above analysis, the problems and defects of the prior art are as follows: there is no study of the non-phosphorylated form of pyridoxal PL in tumors.

The difficulty in solving the above problems and defects is: the substance, i.e. the non-phosphorylated form of pyridoxal, which is the subject of the present invention, has traditionally been regarded as the inactive form of vitamin B6, and its function has not yet been recognized.

The significance of solving the problems and the defects is as follows: the non-phosphorylated pyridoxal PL is definitely the vitamin B6 component with the anticancer activity, and is favorable for fully utilizing the component to carry out anticancer treatment so as to obtain better ovarian cancer resistant efficacy and have more reasonable cost-effectiveness ratio.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an application of pyridoxal in preparing a medicament for treating ovarian cancer.

The invention is realized by the application of pyridoxal in preparing the medicine for treating ovarian cancer.

Further, the pyridoxal is prepared into any pharmaceutically acceptable preparation.

Another object of the present invention is to provide a use of pyridoxal in the preparation of an agent for inhibiting the proliferation of ovarian cancer cells; the ovarian cancer cell is SKOV3, OVCAR3, 3AO or ES-2.

Another object of the present invention is to provide a method for verifying the use of pyridoxal in the preparation of an agent for inhibiting ovarian cancer cell proliferation, the method for verifying the use of pyridoxal in the preparation of an agent for inhibiting ovarian cancer cell proliferation comprising:

step one, obtaining pyridoxal, preparing 50mM working solution by using PBS, and performing suction filtration and sterilization by using a 0.22 mu m filter in dark place;

and secondly, performing label-free counting detection on the living cells by using an xCELLigence workstation and a corresponding culture plate.

Further, in the second step, the detection of the label-free counting of the living cells by using the xcelligene workstation and the corresponding culture plate comprises:

adding 50 mu l of complete culture medium into an E-plate96 well plate to determine a baseline value, adding 100 mu l of mixed SKOV3 and OVCAR3 suspension and 2 mu l of pyridoxal into the well, and supplementing the complete culture medium to 200 mu l of liquid in the well to enable the working concentration of the pyridoxal to reach 0.5mM, and treating a control group with the same amount of PBS; after standing at room temperature for 30min, setting the automatic scanning gap for 15min, detecting the cell proliferation curve, and the total scanning time is 72 hours.

By combining all the technical schemes, the invention has the advantages and positive effects that: experiments prove that pyridoxal can inhibit the cell proliferation of ovarian cancer, has obvious inhibition effect, and can be used for preparing medicaments for treating ovarian cancer or preparing related reagents for researching the occurrence and development mechanism of ovarian cancer. PL is one of vitamin B6 intermediate metabolites, and vitamin B6 is a water-soluble vitamin, and the application safety is high, so that PL has strong clinical application possibility in the aspect of treating ovarian cancer.

The invention detects and compares the influence of pyridoxal PL and PLP in a phosphate form on the proliferation capacity of ovarian cancer cells through living cell unmarked counting, and the inhibition effect of PL on ovarian cancer cells under the same concentration is obviously higher than that of PLP. Further research and development of new uses of PL would be beneficial to fully utilize the compound and facilitate the treatment of clinically relevant diseases.

The invention proves that PL can inhibit the proliferation of ovarian cancer cells through experiments, so that PL can be used for preparing a medicament for treating ovarian cancer or preparing a related reagent for researching the occurrence and development mechanism of ovarian cancer. Meanwhile, the invention proves that the inhibition effect of pyridoxal on the proliferation of ovarian cancer cells is much more obvious than that of other substances on the proliferation of ovarian cancer cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

Fig. 1 is a flowchart of a method for verifying the use of pyridoxal in the preparation of an agent for inhibiting ovarian cancer cell proliferation, provided by an embodiment of the invention.

FIG. 2 is a diagram illustrating the results of a free-label counting experiment for viable cells according to an embodiment of the present invention.

FIG. 3 is a schematic representation of the clonal growth of cells after 6 days of treatment with PBS or 0.5mM PL of SKOV3 provided in an example of the present invention.

FIG. 4 is a schematic representation of the clonal growth of cells after treatment of OVCAR3 for 6 days with PBS or 0.5mM PL provided by an embodiment of the present invention.

FIG. 5 is a schematic diagram showing the clonal growth of ES-2 cells treated with PBS or 0.5mM PL for 6 days.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems of the prior art, the present invention provides a use of pyridoxal in preparing a medicament for treating ovarian cancer, which is described in detail below with reference to the accompanying drawings.

The pyridoxal provided by the embodiment of the invention is an intermediate metabolite of vitamin B6 and is oxidized by pyridoxine, and the chemical formula of the pyridoxal is as follows: 3-hydroxy-5-hydroxymethyl-2-methylpyridine-4-formaldehyde, the molecular structural formula is:

the application of the pyridoxal provided by the embodiment of the invention in preparing the medicine for treating ovarian cancer.

The application of the pyridoxal provided by the embodiment of the invention in preparing the agent for inhibiting the proliferation of ovarian cancer cells; the ovarian cancer cell is SKOV3, OVCAR3, 3AO or ES-2.

As shown in fig. 1, the method for verifying the use of pyridoxal in preparing an agent for inhibiting ovarian cancer cell proliferation provided by the embodiment of the invention comprises the following steps:

s101, obtaining pyridoxal, preparing 50mM working solution by using PBS, and performing suction filtration and sterilization by using a 0.22 mu m filter in dark place;

and S102, performing label-free counting detection on the living cells by using an xCELLigence workstation and a corresponding culture plate.

In step S102, the detection of the label-free counting of living cells using the xcelligene workstation and the corresponding culture plate according to the embodiment of the present invention includes:

adding 50 mu l of complete culture medium into an E-plate96 well plate to determine a baseline value, adding 100 mu l of mixed SKOV3 and OVCAR3 suspension and 2 mu l of pyridoxal into the well, and supplementing the complete culture medium to 200 mu l of liquid in the well to enable the working concentration of the pyridoxal to reach 0.5mM, and treating a control group with the same amount of PBS; after standing at room temperature for 30min, setting the automatic scanning gap for 15min, detecting the cell proliferation curve, and the total scanning time is 72 hours.

The technical effects of the present invention will be further described with reference to specific embodiments.

Example 1:

1. PL and PLP were purchased from Sigma and prepared as 50mM working solutions in PBS, and sterilized by suction filtration through 0.22 μm filters in the dark. Detection of viable cell label-free counts (RTCA) was performed using the xCELLigence workstation and corresponding plate (E-plate 96).

2. PL and PLP can inhibit the proliferation of ovarian cancer cells, and the inhibition ability of PL on the proliferation of ovarian cancer cells is obviously stronger than that of PLP with the same concentration.

Add 50. mu.l complete medium to E-plate96 well plate to determine baseline, and add well mixed SKOV3, 100. mu.l OVCAR3 suspension (1X 10)⁵Pieces/ml), 2. mu. lPL or PLP, and make up to 200. mu.l of liquid in the wells with complete medium to achieve a working PL and PLP concentration of 0.5mM, control group treated with equal amount of PBS. After standing at room temperature for 30min, setting the automatic scanning gap for 15min, detecting the cell proliferation curve, and the total scanning time is 72 hours. Referring to fig. 2, no-marker counting experiments of viable cells showed that PL significantly inhibited the growth of ovarian cancer cell lines SKOV3, OVCAR3, 3AO and ES-2, and the inhibitory effect was significantly higher than that of PLP (. about.p)<0.01)。

The results indicate that PL can be used for preparing a medicament for treating ovarian cancer or preparing a related reagent for researching the occurrence and development mechanism of ovarian cancer. In the research process, the inhibition effect of PL on the proliferation of ovarian cancer cells is obviously better than that of other compounds.

The technical effects of the present invention will be described in detail with reference to experiments.

In this experiment, 500 ovarian cancer SKOV3, OVCAR3, ES-2 cells and 500. mu.l of complete medium were added to each well of a 24-well plate, and PL or an equal volume of PBS was added to the wells to give a final PL concentration of 0.5 mM. After culturing in an incubator at 37 ℃ for 6 days, cells were fixed with 4% paraformaldehyde, and the number and size of cell colonies formed were observed after staining with crystal violet (FIG. 3-FIG. 5).

FIG. 3 clonal growth of cells after 6 days of PBS or 0.5mM PL treatment with SKOV 3; FIG. 4 clonal growth of cells after 6 days of treatment of OVCAR3 with PBS or 0.5mM PL; FIG. 5 clonal growth of ES-2 cells after 6 days of PBS or 0.5mM PL treatment.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.