GB2611905A

GB2611905A - Use of pyridoxal in preparation of drugs for treating ovarian cancer

Info

Publication number: GB2611905A
Application number: GB2219507.7A
Authority: GB
Inventors: Linghu Hua; Li Ruonan; Zhan Shijie; Liu Bin
Original assignee: First Affiliated Hospital of Chongqing Medical University
Current assignee: First Affiliated Hospital of Chongqing Medical University
Priority date: 2020-12-28
Filing date: 2021-12-03
Publication date: 2023-04-19
Also published as: WO2022143010A1; CN112691103A; CN112691103B; GB202219507D0

Abstract

A use of pyridoxal in preparation of drugs for treating ovarian cancer. Pyridoxal is prepared into any pharmaceutically accepted preparation. A method for verifying the use of pyridoxal in preparation of the drugs for treating ovarian cancer. A use of pyridoxal in preparation of reagents for inhibiting proliferation of ovarian cancer cells, and a use of pyridoxal in preparation of related reagents for the study of pathogenesis and development mechanisms of ovarian cancer.

Description

USE OF PYRIDOXAL IN PREPARATION OF DRUGS FOR TREATING OVARIAN

CANCER

CROSS REFERENCE TO RELATED APPLICATION

[1] The present application claims priority to the Chinese Patent Application No. 202011577585.5, filed with the China National Intellectual Property Administration (CNIPA) on December 28, 2020, and entitled "USE OF PYRIDOXAL IN PREPARATION OF DRUG FOR TREATING OVARIAN CANCER", which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

121 The present disclosure belongs to the technical field of molecular biology and medicine, and particularly relates to a use of pyridoxal in preparation of a drug for treating ovarian cancer.

BACKGROUND ART

[3] At present, the mortality rate of ovarian cancer ranks first among malignant tumors of female Cr) reproductive system. The ovarian cancer has insidious disease progression and poor prognosis, seriously threatening women's health. Vitamin B6 intake has been found to be associated with a reduction in the occurrence of malignancies and an improved prognosis; pyridoxal is one of the main metabolites of the vitamin B6, which has two types: phosphorylated form (pyridoxal phosphate, PLP) o and non-phosphorylated form (PL). PLP is currently considered to be an active form of the vitamin B6, which may participate in catalytic reactions as a coenzyme of various enzymes, and an inhibitory effect of the vitamin B6 on malignant tumors is also generally considered to be an effect of the PLP. The problems and defects in the prior art are: no research on the pyridoxal (PL) in non-phosphorylated form in tumors has been found, but the pyridoxal in non-phosphorylated form is traditionally considered to be an inactive form of the vitamin B6, with functions not been recognized.

SUMMARY

[4] Aiming at the problems existing in the prior art, the present disclosure provides a use of pyridoxal in preparation of a drug for treating ovarian cancer. Anticancer efficacies of PLP and pyridoxal are repeatedly verified in ovarian cancer cells, and results show that an inhibitory effect of the PLP on the ovarian cancer cells is significantly inferior to that of the PL at the same concentration.

[5] The present disclosure provides a use of pyridoxal in preparation of a drug for treating ovarian cancer.

161 In one embodiment, the pyridoxal may be prepared into any pharmaceutically acceptable preparation, [7] The present disclosure further provides a use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells, where the ovarian cancer cells may be selected from SKOV3, OVCAR3, 3A0, and ES-2.

[8] The present disclosure further provides a method for verifying the use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells, including the following steps: [9] step 1, preparing a 50 mM working solution by using the pyridoxal with a PBS, and conducting suction filtration and sterilization in the dark by using a 0.22 pm filter; and 1101 step 2, conducting xCELLigence real-time cell analysis by using an xCELLigence workstation and a corresponding culture plate.

1111 In one embodiment, in the step 2, the xCELLigence real-time cell analysis is conducted by using an xCELLigence workstation and a corresponding culture plate, where includes: [12] measuring a baseline value by adding 50 pl of a complete medium to an 96-well E-plate; adding 100 pl of a fully-mixed suspension of the SKOV3/0VCAR3/3A0/ES-2 and 2 pl of the pyridoxal to a well, and supplementing liquid in the well with the complete medium to 200 pl, such that the pyridoxal has a working concentration of 0.5 mM, while treating a control group with an equal amount of the PBS; setting an automatic scanning gap at 15 min to detect a cell proliferation curve for a total scanning time of 72 h after being allowed to stand at room temperature for 30 min. [13] Combined with all the above technical solutions, the advantages and positive effects of the present disclosure are as follows: it is confirmed that the pyridoxal may inhibit the proliferation of ovarian cancer cells, with an extremely significant inhibitory effect. Therefore, the pyridoxal may be used to prepare drugs for treating ovarian cancer, or to prepare relevant reagents for studying a mechanism of occurrence and development of the ovarian cancer. Pyridoxal is one of the intermediate metabolites of vitamin B6, and the vitamin B6 is a water-soluble vitamin, with a relatively high use safety. Therefore, the pyridoxal has a strong clinical use possibility in the treatment of ovarian cancer.

[14] In the present disclosure, the effects of pyridoxal and its phosphorylated form PLP on the proliferation ability of ovarian cancer cells are detected and compared by xCELLigence real-time cell analysis, and the inhibitory effect of pyridoxal on ovarian cancer cells is significantly higher than that of the PLP at a same concentration. Further research and development of new uses of the pyridoxal may be beneficial to make full use of the compound and promote the treatment of clinically relevant diseases.

1151 In the present disclosure, it is confirmed that the pyridoxal may inhibit the proliferation of ovarian cancer cells. Therefore, the pyridoxal may be used to prepare drugs for the treatment of ovarian cancer, or to prepare relevant reagents for studying mechanism of occurrence and development of the ovarian cancer. In addition, it is proved that the inhibitory effect of pyridoxal on the proliferation of ovarian cancer cells is much more significant than that of other substances on the proliferation of ovarian cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

1161 In order to explain the technical solutions of the examples of the present disclosure more clearly, drawings that need to be used in the examples of the present disclosure are briefly introduced below. Obviously, the drawings described below are only some examples of the present disclosure, and other drawings may also be obtained from these drawings by those of ordinary skill in the art without creative work.

1171 FIG. I is a flowchart of a method for verifying use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells provided by an example of the present disclosure; 1181 FIG. 2 is a schematic diagram of results of an xCELLigence real-time cell analysis provided

in an example of the present disclosure;

1191 FIG. 3 is a schematic diagram of colony formation ability of cell after SKOV3 provided in the example of the present disclosure is treated with a PBS or 0.5 mM pyridoxal for 6 d; [20] FIG. 4 is a schematic diagram of colony formation ability of cell after OVCAR3 provided in the example of the present disclosure is treated with a PBS or 0.5 mM pyridoxal for 6 d; 1211 FIG. 5 is a schematic diagram of colony formation ability of cell after 3A0 provided in the example of the present disclosure is treated with a PBS or 0.5 mM pyridoxal for 6 d; 1221 FIG. 6 is a schematic diagram of colony formation ability of cell after ES-2 provided in the example of the present disclosure is treated with a PBS or 0.5 mM pyridoxal for 6 d.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[23] To make the objective, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described below in detail below with reference to the embodiments. Understandably, the specific embodiments described herein are merely intended to explain the present disclosure but not to limit the present disclosure.

1241 In view of the problems existing in the prior art, the present disclosure provides a use of pyridoxal in preparation of a drug for treating ovarian cancer, and the present disclosure will be further described in detail below in conjunction with drawings 1251 In the embodiment of the present disclosure, the pyridoxal is an intermediate metabolite of vitamin B6, which is obtained from the oxidation of pyridoxine, and has a chemical formula of: 3-hy droxy-5-hydroxymethy1-2 -m ethyl pyri di n e-4-carb al dehyde, and a molecular structure of: [26] 1271 The present disclosure provides a use of pyridoxal in preparation of a drug for treating ovarian cancer.

[28] The present disclosure further provides a use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells, where the ovarian cancer cells are selected from SKOV3, OVCAR3, 3A0, and ES-2.

1291 As shown in FIG. 1, the present disclosure further provides a method for verifying the use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells, including the following steps: [30] S101, a 50 mM of working solution is prepared by using the pyridoxal with a PBS, and suction filtration and sterilization are conducted in the dark by using a 0.22 pm filter; and [31] S102, xCELLigence real-time cell analysis is conducted by using an xCELLigence workstation and a corresponding culture plate.

[32] In step S102, the xCELLigence real-time cell analysis is conducted by using an xCELLigence workstation and a corresponding culture plate by: [33] a baseline value is measured by adding 50 tl of a complete medium to an 96-well E-plate; 100 pl of a fully-mixed suspension of the SKOV3, the OVCAR3, the 3A0, or the ES-2 and 2!Al of the pyridoxal are added to a well, and liquid in the well is supplemented with the complete medium to 200 il, such that the pyridoxal has a working concentration of 0.5 mM, while a control group is treated with an equal amount of the PBS; an automatic scanning gap is set at 15 min to detect a cell proliferation curve (FIG. 2) for a total scanning time of 72 h after being allowed to stand at room temperature for 30 min. [34] The technical solutions of the present disclosure will be further described below in conjunction with specific examples.

[35] Example 1

[36] L Pyridoxal and PLP each were purchased from Sigma-Aldrich, prepared into a 50 mM working solution with a PBS, and suction filtration and sterilization were conducted in the dark by using a 0.22 pm filter, xCELLigence real-time cell analysis was conducted by using an xCELLigence workstation and a corresponding culture plate (96-well E-plate).

[37] 2. Pyridoxal and PLP each could inhibit the proliferation of ovarian cancer cells, and an inhibitory ability of the pyridoxal on the proliferation of ovarian cancer cells was significantly stronger than that of the PLP at a same concentration.

[38] A baseline value was measured by adding 50 pl of a complete medium to an 96-well E-plate; pl of a fully-mixed suspension (1x105 cells/ml) of the SKOV3, the OVCAR3, the 3A0, or the ES-2 and 2 p1 of the pyridoxal or the PLP were added to a well, and liquid in the well was supplemented with the complete medium to 200 R1. such that the pyridoxal or the PLP had a working concentration of 0.5 mM, while a control group was treated with an equal amount of the PBS. An automatic scanning gap was set at 15 min to detect a cell proliferation curve for a total scanning time of 72 h after being allowed to stand at room temperature for 30 min. Referring to FIG. 2, the xCELLigence real-time cell analysis showed that the pyridoxal significantly inhibited the growth of ovarian cancer cell lines SKOV3, OVCAR3, 3A0, and ES-2, with an inhibitory effect significantly higher than that of the PLP (*, P<0.01). ;[39] The above results suggested that the pyridoxal can be used to prepare drugs for treating ovarian cancer, or to prepare relevant reagents for studying the mechanism of occurrence and development of the ovarian cancer. Moreover, during the research, it is found that the inhibitory effect of pyridoxal on the proliferation of ovarian cancer cells is significantly better than that of some other compounds. ;[40] The technical effects of the present disclosure will be described in detail below in conjunction with the experiments. ;[41] In this experiment, 500 of ovarian cancer cells SKOV3, OVCAR3, 3A0, or ES-2 and 500 pl of a complete medium were added to each well of a 24-well plate, and pyridoxal or an equal volume of a PBS was added to the well to make the pyridoxal have a final concentration of 0.5 mM. The cells were fixated with 4% paraformaldehyde, and stained with crystal violet to observe the number and size of cell clones after culturing in a 37°C incubator for 6 d (FIG. 3 to FIG. 6). ;[42] FIG. 3 shows the colony formation ability of SKOV3 treated with PBS or 0.5 mM pyridoxal for 6 d; FIG. 4 shows the colony formation ability of OVCAR3 treated with PBS or 0.5 mM pyridoxal for 6 d; FIG. 5 shows the colony formation ability of 3A0 treated with PBS or 0.5 mM pyridoxal for 6 d; and FIG. 6 shows the colony formation ability of ES-2 treated with PBS or 0.5 mM pyridoxal for 6 d. 0.5 mM pyridoxal significantly inhibited the ability of the 4 of ovarian cancer cell lines to form clones. ;[43] The foregoing are merely descriptions of the specific embodiments of the present disclosure, and the protection scope of the present disclosure is not limited thereto. Any modification, equivalent replacement, improvement, etc. made within the technical scope of the present disclosure by a person skilled in the art according to the spirit and principle of the present disclosure shall fall within the protection scope of the present disclosure *

Claims

WHAT IS CLAIMED IS: 1. Use of pyridoxal in preparation of a drug for treating ovarian cancer.
2. The use of pyridoxal in preparation of a drug for treating ovarian cancer according to claim 1, wherein the pyridoxal is prepared into any pharmaceutically acceptable preparation.
3. Use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells.
4. The use of pyridoxal in preparation of a reagent for inhibiting proliferation of ovarian cancer cells according to claim 3, wherein the ovarian cancer cells are selected from SKOV3, OVCAR3, 3A0, and ES-2.
5. A method for verifying the use according to claim 3 or 4, comprising the following steps: step 1, preparing a working solution using the pyridoxal with a PBS, and conducting suction filtration and sterilization in the dark using a filter; and step 2, conducting xCELLigence real-time cell analysis by using an xCELL gence workstation and a corresponding culture plate.
6. The method according to claim 5, wherein in the step 1, 50 mM of the working solution is prepared by using the pyridoxal with the PBS, and the suction filtration and sterilization is conducted in the dark by using a 0.22 im filter.
7. The method according to claim 5, wherein in the step 2, a baseline value is measured by adding a complete medium to an E-plate 96-well plate; a fully-mixed suspension of SKOV3, OVCAR3, 3A0, or ES-2 and the pyridoxal are added to a well, and liquid in the well is supplemented with the complete medium, while a control group is treated with an equal amount of the PBS; an automatic scanning gap is set to detect a cell proliferation curve after being allowed to stand at room temperature.
8. The method according to claim 7, wherein further comprising measuring the baseline value by adding 50 ill of the complete medium to the 96-well E-plate; adding 100 pl of the fully-mixed suspension of the SKOV3, the OVCAR3, the 3A0, or the ES-2 and 2 pl of the pyridoxal to the well, and supplementing the liquid in the well with the complete medium to 200 pl.
9. The method according to claim 7, wherein the pyridoxal has a working concentration of 0.5 m Nil, and the control group is treated with the equal amount of the PBS.
10. The method according to claim 7, wherein the automatic scanning gap is set at 15 mm to detect the cell proliferation curve for a total scanning time of 72 h after being allowed to stand at room temperature for 30 min.
11. A drug for treating ovarian cancer, wherein active ingredient of the drug is pyridoxal.
12. Use of pyridoxal in preparation of a relevant reagent for studying a mechanism of occurrence and development of ovarian cancer.