METHOD FOR EVALUATING CYSTINE UPTAKE ABILITY OF CELL, KIT FOR EVALUATING CYSTINE UPTAKE ABILITY OF CELL, METHOD FOR DETERMINING SELENOCYSTEINE, AND KIT FOR DETERMINING SELENOCYSTEINE

Abstract

Disclosed are a method for evaluating the cystine uptake ability of cells having the steps of contacting cells with selenocystine to allow the cells to take up selenocystine, washing away selenocystine not taken up by the cells, and crushing the cells and determining selenocystine contained in the cytoplasm and a method for determining selenocysteine having a step of contacting selenocysteine with a fluorescent dye that specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and fluorescence intensity, or reacting selenocysteine with a fluorescent dye under a condition that the fluorescent dye specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and fluorescence intensity, followed by measuring the fluorescence intensity for determining selenocystine. A kit for evaluating the cystine uptake ability of cells, containing a reducing agent that reduces selenocystine to selenocysteine, a fluorescent dye represented by the following general formula (I), and a buffer solution at a pH of 5.5 to 6.5 is disclosed.

Claims

1. A method for evaluating cystine uptake ability of cells, comprising the steps of: contacting cells with selenocystine to allow the cells to take up selenocystine; washing away selenocystine not taken up by the cells; and crushing the cells and determining selenocystine contained in cytoplasm.

2. The method for evaluating cystine uptake ability of cells according to claim 1, wherein, in the step of determining selenocystine contained in cytoplasm, selenocystine is reacted with a reducing agent to generate selenocysteine; selenocysteine is contacted with a fluorescent dye that specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and fluorescence intensity, or selenocysteine is reacted with a fluorescent dye under the condition that the fluorescent dye specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and fluorescence intensity; and the fluorescence intensity is measured to determine selenocystine.

3. The method for evaluating cystine uptake ability of cells according to claim 2, wherein, in the step of determining selenocystine contained in the cytoplasm, a fluorescent dye represented by the following general formula (I) is specifically reacted with selenocysteine under a condition of a pH of 5.5 to 6.5, ##STR00024## wherein A represents an acryloyl group or a methacryloyl group, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently an atom or an atomic group selected from the group consisting of a hydrogen atom, a hydroxyl group, a thiol group, a halogen atom, an amino group, a sulfonamide group, an azido group, and a cyano group; and a linear alkyl group, a branched alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group in which one or more hydrogen atoms are optionally replaced with other atoms or functional groups and that optionally comprises one or more of an amino group, a carbonyl group, an oxygen atom, and a sulfur atom in carbon skeletons, and among R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6, any two adjacent ones share atoms, forming a cyclic fluorescent moiety where the fluorescence wavelength and fluorescence intensity change one or both, due to the conversion of the O-A group to an OH group.

4. The method for evaluating cystine uptake ability of cells according to claim 3, wherein, in the step of determining selenocystine contained in the cytoplasm, a fluorescent dye represented by the following formula (1) is specifically reacted with selenocysteine under the condition of a pH of 5.5 to 6.5, ##STR00025## wherein R.sup.11 represents a hydrogen atom or a functional group represented by following formula (2), and R.sup.12 represents a hydrogen atom or a methyl group, ##STR00026## wherein R.sup.13 represents a hydrogen atom or a methyl group.

5. The method for evaluating cystine uptake ability of cells according to claim 4, wherein the fluorescent dye is represented by the following formula: ##STR00027##

6. The method for evaluating cystine uptake ability of cells according to claim 2, wherein the reducing agent is tris(carboxyethyl)phosphine.

7. A kit for evaluating cystine uptake ability of cells, comprising: a reducing agent that reduces selenocystine to selenocysteine, a fluorescent dye represented by the following general formula (I), and a buffer solution at a pH of 5.5 to 6.5, ##STR00028## wherein A represents an acryloyl group or a methacryloyl group, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently an atom or an atomic group selected from the group consisting of a hydrogen atom, a hydroxyl group, a thiol group, a halogen atom, an amino group, a sulfonamide group, an azido group, and a cyano group; and a linear alkyl group, a branched alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group in which one or more hydrogen atoms are optionally replaced with other atoms or functional groups and that optionally contain one or more of an amino group, a carbonyl group, an oxygen atom, and a sulfur atom in carbon skeletons, and among R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6, any two adjacent ones share atoms, forming a cyclic fluorescent moiety where the fluorescence wavelength and fluorescence intensity change one or both, due to the conversion of the 0-A group to an OH group.

8. The kit for evaluating cystine uptake ability of cells according to claim 7, wherein the fluorescent dye is represented by the following formula (1): ##STR00029## wherein R.sup.11 represents a hydrogen atom or a functional group represented by the following formula (2), and R.sup.12 represents a hydrogen atom or a methyl group, ##STR00030## wherein R.sup.13 represents a hydrogen atom or a methyl group.

9. The kit for evaluating cystine uptake ability of cells according to claim 8, wherein the fluorescent dye is represented by the following formula: ##STR00031##

10. The kit for evaluating cystine uptake ability of cells according to claim 7, wherein the reducing agent is tris(carboxyethyl)phosphine.

11. The kit for evaluating cystine uptake ability of cells according to claim 7, wherein the buffer solution is any of acetate buffer solution, phosphate buffer solution, citrate buffer solution, MES buffer solution, and Bis-Tris buffer solution.

12. A method for determining selenocysteine, comprising a step of contacting selenocysteine with a fluorescent dye that specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and fluorescence intensity, or reacting selenocysteine with a fluorescent dye under a condition that the fluorescent dye specifically reacts with selenocysteine to change in one or both of fluorescence wavelength and fluorescence intensity, followed by measuring the fluorescence intensity for determining selenocystine.

13. The method for determining selenocysteine according to claim 12, wherein, in the step of determining selenocystine, the fluorescent dye represented by the following general formula (I) is specifically reacted with selenocysteine under a condition of a pH of 5.5 to 6.5, ##STR00032## wherein A represents an acryloyl group or a methacryloyl group, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently an atom or an atomic group selected from the group consisting of a hydrogen atom, a hydroxyl group, a thiol group, a halogen atom, an amino group, a sulfonamide group, an azido group, and a cyano group; and a linear alkyl group, a branched alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group in which one or more hydrogen atoms are optionally replaced with other atoms or functional groups and that optionally contain one or more of an amino group, a carbonyl group, an oxygen atom, and a sulfur atom in the carbon skeletons, and among R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6, any two adjacent ones share atoms, forming a cyclic fluorescent moiety where the fluorescence wavelength and fluorescence intensity change one or both, due to the conversion of the O-A group to an OH group.

14. The method for determining selenocysteine according to claim 13, wherein, in the step of determining selenocysteine contained in the cytoplasm, the fluorescent dye represented by the following formula (1) is specifically reacted with selenocysteine under a condition of a pH of 5.5 to 6.5, ##STR00033## wherein R.sup.11 represents a hydrogen atom or a functional group represented by the following formula (2), and R.sup.42 represents a hydrogen atom or a methyl group, ##STR00034## wherein R.sup.13 represents a hydrogen atom or a methyl group.

15. The method for determining selenocysteine according to claim 14, wherein the fluorescent dye is represented by the following formula: ##STR00035##

16. The method for determining selenocysteine according to claim 12, wherein the reducing agent is tris(carboxyethyl)phosphine.

17. A kit for determining selenocysteine, comprising: a fluorescent dye represented by the following general formula (I) and a buffer solution at a pH of 5.5 to 6.5, ##STR00036## wherein A represents an acryloyl group or a methacryloyl group, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently an atom or an atomic group selected from the group consisting of a hydrogen atom, a hydroxyl group, a thiol group, a halogen atom, an amino group, a sulfonamide group, an azido group, and a cyano group; and a linear alkyl group, a branched alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group in which one or more hydrogen atoms are optionally replaced with other atoms or functional groups and that optionally contain one or more of an amino group, a carbonyl group, an oxygen atom, and a sulfur atom in the carbon skeletons, and among R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6, any two adjacent ones share atoms, forming a cyclic fluorescent moiety where the fluorescence wavelength and fluorescence intensity change one or both, due to the conversion of the 0-A group to an OH group.

18. The kit for determining selenocysteine according to claim 17, wherein the fluorescent dye is represented by following formula (1): ##STR00037## wherein R.sup.11 represents a hydrogen atom or a functional group represented by the following formula (2), and R.sup.12 represents a hydrogen atom or a methyl group, ##STR00038## wherein R.sup.13 represents a hydrogen atom or a methyl group.

19. The kit for determining selenocysteine according to claim 18, wherein the fluorescent dye is represented by the following formula: ##STR00039##

20. The kit for determining selenocysteine according to claim 17, wherein the reducing agent is tris(carboxyethyl) phosphine.

21. The kit for determining selenocysteine according to claim 17, wherein the buffer solution is any of acetate buffer solution, phosphate buffer solution, citrate buffer solution, MES buffer solution, and Bis-Tris buffer solution.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0048] FIG. 1 is a graph showing the results of Example 1.

[0049] FIG. 2 is a graph showing the results of Example 2.

[0050] FIG. 3 is a graph showing the results of Example 3.

[0051] FIG. 4 is a graph showing the results of Example 3.

DESCRIPTION OF EMBODIMENTS

[0052] A method for evaluating the cystine uptake ability of cells according to the first embodiment of the present invention (hereinafter occasionally abbreviated as a method for evaluating the cystine uptake ability of cells or an evaluation method) has the steps of contacting cells with selenocystine to allow the cells to take up selenocystine, washing away selenocystine not taken up by the cells, and crushing the cells and determining selenocystine contained in the cytoplasm.

[0053] The evaluation object in the evaluation method is any cells in which cystine transporter is expressed, and specific examples thereof include A549 (cells derived from human pulmonary alveoli basal epithelial gland cancer), HeLa (cells derived from human cervical cancer), HepG2 (cells derived from human liver cancer), HL60 (human leukemia cell line), MOLT-4 (cells derived from a human acute lymphoblastic tumor), U-251 (malignant human glioma cell line), U-87 MG (cells derived from human glioblastoma), BxPC-3 (cells derived from human pancreas cancer), THP-1 (cells derived from human acute monocytic leukemia), SK-MEL-30 (cells derived from human skin cancer), EFO-21 (cells derived from human serous cystoma), GAMG (cells derived from a human brain tumor), Karpas-707 (cells derived from human multiple myeloma), OE-19 (cells derived from human esophagus cancer), U266/70 (cells derived from human multiple myeloma), RT4 (cells derived from human bladder epithelial papilloma), and U-2 OS (cells derived from human osteosarcoma).

[0054] Cells such as the cells mentioned above as an evaluation object are first contacted with selenocystine to allow the cells to take up selenocystine. Examples of the method for taking selenocystine into cells include a method involving dissolving selenocystine in culture medium or a buffer solution not containing cystine (for example, cystine-free DMEM, HBSS, PBS, or the like), then adding the culture medium or the buffer solution containing selenocystine to cells, and leaving the cells to stand in a CO.sub.2 incubator at 37 C. for around 5 minutes to 1 hour.

[0055] Selenocystine not taken up by cells is removed before selenocystine taken up by cells is determined. Examples of the removal method include, but are not particularly limited to, a method for washing the cells with a buffer solution, liquid culture medium, or the like not containing selenocystine.

[0056] The cells are then crushed, the cytoplasm is lysed, and selenocystine contained in the cytoplasm is determined to evaluate the selenocystine uptake ability. The method for crushing cells is not particularly limited, and any well-known method can be used. Examples include a method for lysing cells with a water-soluble organic solvent such as an alcohol, DMSO or a buffer solution containing a surfactant.

[0057] Selenocystine in the eluted cytoplasm is then determined. Specific examples of the method for determining selenocysteine include a method involving reducing selenocystine to produce selenocysteine; contacting the selenocysteine with a fluorescent dye that specifically reacts with selenocysteine to emit fluorescence or reacting a fluorescent dye with selenocysteine under the condition that the fluorescent dye specifically reacts with selenocysteine to emit fluorescence; and measuring the fluorescence intensity to determine selenocystine.

[0058] Specific examples of a preferable reducing agent to be used for reducing selenocystine include tris(2-carboxyethyl) phosphine (TCEP), having many advantages such as having no smell or no toxicity and being excellent in hydrophilicity and stability.

[0059] Selenocysteine is then contacted with a fluorescent dye that specifically reacts with selenocysteine to change in one or both of the fluorescence wavelength and the fluorescence intensity even in the presence of cysteine, or selenocysteine is reacted with a fluorescent dye under the condition that the fluorescent dye specifically reacts with selenocysteine to change in one or both of the fluorescence wavelength and the fluorescence intensity even in the presence of cysteine; and the fluorescence intensity is measured to determine selenocystine.

[0060] Examples of the preferable dye and the condition of the specific reaction include specifically reacting a fluorescent dye represented by the following general formula (I) with selenocysteine under the condition of a pH of 5.5 to 6.5,

##STR00017##

[0061] wherein A represents an acryloyl group or a methacryloyl group, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each independently an atom or an atomic group selected from the group consisting of a hydrogen atom, a hydroxyl group, a thiol group, a halogen atom, an amino group, a sulfonamide group, an azido group, and a cyano group; and a linear alkyl group, a branched alkyl group, a cycloalkyl group, an aryl group, and a heteroaryl group in which one or more hydrogen atoms are optionally replaced with other atoms or functional groups and that optionally contain one or more of an amino group, a carbonyl group, an oxygen atom, and a sulfur atom in the carbon skeletons, and among R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6, any two adjacent ones share atoms, forming a cyclic fluorescent moiety where the fluorescence wavelength and fluorescence intensity change one or both, due to the conversion of the 0-A group to an OH group.

[0062] Specific examples of the fluorescent dye represented by general formula (I) to be specifically reacted with selenocysteine under the condition of a pH of 5.5 to 6.5 include fluorescent dyes represented by the following formulae:

##STR00018## ##STR00019##

[0063] Examples of a particularly preferable fluorescent dye include a substance represented by the following formula (1):

##STR00020##

[0064] wherein R.sup.11 represents a hydrogen atom or a functional group represented by the following formula (2), and R.sup.12 represents a hydrogen atom or a methyl group,

##STR00021##

[0065] wherein R.sup.13 represents a hydrogen atom or a methyl group.

[0066] In the case of the fluorescent dye represented by the formula mentioned above (1), the specific reactions of fluorescent dye with selenocysteine and the reaction mechanism thereof are represented, for example, by the following formula.

##STR00022## ##STR00023##

[0067] Examples of preferable buffer solution include acetate buffer solution, phosphate buffer solution, citrate buffer solution, MES buffer solution, and Bis-Tris buffer solution.

[0068] The kit for evaluating the cystine uptake ability of cells according to the second embodiment of the present invention contains a reducing agent that reduces selenocystine to selenocysteine, a fluorescent dye represented by the following general formula (I), and a buffer solution at a pH of 5.5 to 6.5. Since specific examples of the reducing agent, the fluorescent dye, and the buffer solution are as described in the above-mentioned first embodiment of the present invention, a detailed description thereof is omitted.

[0069] The method for determining selenocysteine according to the third embodiment of the present invention has a step of contacting a fluorescent dye that specifically reacts with selenocysteine to change in one or both of the fluorescence wavelength and the fluorescence intensity or reacting a fluorescent dye with selenocysteine under the condition that the fluorescent dye specifically reacts with selenocysteine to change in one or both of the fluorescence wavelength and the fluorescence intensity; and measuring the fluorescence intensity to determine selenocysteine. Since the steps are as described in the above-mentioned first embodiment of the present invention, a detailed description thereof is omitted.

EXAMPLES

[0070] The Examples performed to confirm the function and the effect of the present invention will then be described.

Example 1: Examination of Reactivity of Fluorescein O, O-Diacrylate (FOdA) with Selenocystine

[0071] Fluorescein O,O-diacrylate (FOdA, 10 M) was mixed with TCEP (200 M) and any of 10 M selenocystine, 10 M cystine, and 20 M glutathione in buffers (100 mM acetate buffers at pHs of 5 and 5.5, 100 mM MES buffers at pHs of 6 and 6.5, and 100 mM phosphate buffer at a pH of 7), and the mixture was incubated at 37 C. for 30 minutes. The fluorescence intensity of each solution was measured with a plate reader (Infinite(R) 200 PRO, excitation wavelength: 485 nm, fluorescence wavelength: 535 nm).

[0072] As shown in FIG. 1, it was confirmed that FOdA was hardly reacted with cystine or glutathione in the buffers at pH 6 and 6.5 and selectively reacted with selenocystine to emit fluorescence.

Example 2: Examination of Ability to Take Selenocystine into Cells

[0073] DMEM (cystine-free) was added to HeLa cells, and the cells were incubated in a CO.sub.2 incubator (37 C.) for 5 minutes. After the removal of the supernatant, DMEM (cystine-free) containing selenocystine at concentrations was added, and the cells were incubated in the CO 2 incubator (37 C.) for 30 minutes. After removing the supernatant, the cells were washed with PBS three times and then lysed with methanol. Then, 100 mM MES at a pH of 6 containing 10 M FOdA and 200 M TCEP was added, and the mixture was incubated at 37 C. for 30 minutes. The fluorescence intensity of each solution was then measured with a plate reader (Infinite(R) 200 PRO, excitation wavelength: 485 nm, fluorescence wavelength: 535 nm).

[0074] As shown in FIG. 2, it was confirmed that the fluorescence intensity increased depending on the concentration of selenocystine to be added to the cells. This result shows that selenocystine is taken up by the cells, and selenocystine taken up by the cells can be subjected to fluorescence measurement by the reaction using FOdA and TCEP.

Example 3: Examination of Inhibition of Selenocystine Uptake by Cystine Transporter Inhibitor

[0075] DMEM (cystine-free) containing a cystine transporter inhibitor (sulfasalazine or erastin) was added to HeLa cells, and the cells were incubated in the CO.sub.2 incubator (37 C.) for 5 minutes. DMEM (cystine-free) containing 200 M selenocystine was then added, and the cells were incubated in the CO.sub.2 incubator (37 C.) for 30 minutes. After removing the supernatant, the cells were washed with PBS three times and lysed with methanol. Then, 100 mM MES at a pH of 6 containing 10 M FOdA and 200 M TCEP was added, and the cells were incubated at 37 C. for 30 minutes. The fluorescence intensity of each solution was then measured with a plate reader (Infinite(R) 200 PRO, excitation wavelength: 485 nm, fluorescence wavelength: 535 nm).

[0076] FIG. 3 shows the results. Since the fluorescence intensity significantly decreased in the cells to which the cystine transporter inhibitor was added, it was confirmed that selenocystine was taken through cystine transporter.

[0077] As shown in FIG. 4, the cystine transporter inhibitor concentration-dependent inhibitory activity was able to be calculated from this decrease in fluorescence intensity.

[0078] The present invention enables various embodiments and modifications without departing from the broad spirit and the scope of the present invention. The above-mentioned embodiments are for describing the present invention, and do not limit the scope of the present invention. That is, the scope of the present invention is shown not by the embodiments but by Claims. Various modifications made within Claims and the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0079] The present application is based on Japan Patent Application 2021-34870, filed on Mar. 5, 2021, and includes Description, Claims, Drawings, and Abstract of thereof. The disclosure in the Japan Patent Application mentioned above is incorporated herein in its entirety as a reference.