Application of vitamin K2 in biphasic regulation of ER-alpha36 expression and hepatoma carcinoma cell growth

Abstract

The invention provides application of vitamin K2 in biphasic regulation of ER-alpha36 expression and hepatoma carcinoma cell growth, and belongs to the technical field of medicines.The vitamin K2 has an estrogen-like effect and can biphasic regulate hepatoma carcinoma cell growth through an ER-alpha36 signal transduction pathway, that is, low-dose vitamin K2 up-regulates ER-alpha36 expression and promotes HepG2 cell growth, so that the hepatoma carcinoma cell growth is promoted. A high dose of vitamin K2 down-regulates the expression of ER-alpha 36 and inhibits the growth of HepG2 cells. The new application of the vitamin K2 is found for the first time, and a new strategy is provided for treatment of hepatocellular carcinoma and drug development.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel application of vitamin K2 in biphasic regulation of ER-α36 expression and liver cancer cell growth. Background Technology

[0002] Vitamin K2 (VK2) is a group of compounds with a naphthoquinone ring structure, mainly derived from fermented foods, meat, and dairy products, and can also be synthesized by intestinal bacteria. Traditionally, VK2's primary function is to maintain normal blood clotting. However, with advancements in scientific research, the roles of VK2 have expanded to include bone metabolism regulation, cancer progression, neuroprotection, and bile acid metabolism.

[0003] The liver is considered the primary storage site for vitamin K, with vitamin K2 accounting for approximately 90% of its total vitamin K storage. Therefore, vitamin K metabolism disorders often lead to liver disease. Vitamin K deficiency is common in patients with chronic liver disease, particularly cirrhosis and liver cancer. In female patients with viral cirrhosis, vitamin K2 supplementation significantly reduced the incidence of hepatocellular carcinoma (HCC). Treatment with vitamin K2 analogues in HCC patients after hepatectomy or local ablation significantly reduced tumor recurrence rates and improved 1-, 2-, and 3-year overall survival. This demonstrates that the increased risk of HCC is associated with decreased blood vitamin K2 concentrations due to vitamin K2 deficiency, and high blood vitamin K2 concentrations can prevent / delay the progression of viral hepatitis / cirrhosis to liver cancer.

[0004] Estrogen exerts a wide range of biological effects by binding to the estrogen receptor (ER). Besides regulating the female reproductive system, estrogen is involved in the development of many diseases, such as coronary heart disease, Alzheimer's disease, and various malignant tumors (estrogen-dependent tumors such as breast cancer and estrogen-independent tumors such as lung and liver cancer). ER-α36, a newly discovered splice isoform of ER-α, functions completely differently from ER-α66 (classical ER-α). ER-α36 is primarily located on the cell membrane. Compared to the nuclear presence of ER-α66, the protein structure of ER-α36 lacks the AF-1 and AF-2 domains, which have transcriptional activation functions, but retains the DNA-binding domain and its partial dimerization and ligand-binding domains. Its transcription promoter is also completely different from ER-α66, possessing a unique functional sequence of 27 amino acids at the C-terminus. ER-α36 primarily mediates the estrogen non-classical / rapid pathway and plays a crucial role in ER-α-negative breast cancer, endocrine-resistant breast cancer, lung cancer, liver cancer, and other tumors. Therefore, the research and development of novel ER-α36-targeting products has become a hot research topic. Summary of the Invention

[0005] This invention provides a novel use for vitamin K2, namely, its estrogen-like effects, which can target ER-α36 and regulate ER-α36 expression levels, thereby affecting the growth of liver cancer cells.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides the application of vitamin K2 in the preparation of estrogen-like products.

[0007] In the above applications, the estrogen-like effect of vitamin K2 refers to its ability to regulate ER-α36 expression levels, thereby modulating the growth of liver cancer cells. Specifically, vitamin K2 biphasically regulates ER-α36 expression levels: low-dose vitamin K2 upregulates ER-α36 expression levels and promotes liver cancer cell growth, while high-dose vitamin K2 downregulates ER-α36 expression levels and inhibits liver cancer cell growth.

[0008] In some embodiments of the present invention, the vitamin K2 concentration that upregulates ER-α36 expression and promotes the growth of liver cancer cells is 0.01-1 nM, and the vitamin K2 concentration that downregulates ER-α36 expression and inhibits the growth of liver cancer cells is ≥20 μM.

[0009] Secondly, the present invention provides the use of vitamin K2 in the preparation of drugs for treating hepatocellular carcinoma, wherein the drugs treat hepatocellular carcinoma by downregulating the expression level of ER-α36.

[0010] Preferably, the drug may also contain other active substances used to treat hepatocellular carcinoma, including but not limited to biological agents, small molecule compounds, and traditional Chinese medicine; it is understood that the combined use of vitamin K2 with these active substances can further enhance the treatment effect of hepatocellular carcinoma.

[0011] Preferably, the drug also contains pharmaceutically acceptable excipients, such as carriers, adjuvants, dispersants, binders, disintegrants, etc. The drug can be an oral or topical preparation, and the dosage form includes, but is not limited to, ointments, granules, gels, tablets, liquids, capsules, pills, etc.

[0012] Thirdly, the present invention provides a drug for treating hepatocellular carcinoma, which uses vitamin K2 as one of its active ingredients, and vitamin K2 inhibits the growth of hepatocellular carcinoma cells by downregulating the expression level of ER-α36.

[0013] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0015] Figure 1 This is a comparison of the chemical structures of VK2 and 17β-estradiol (E2) and the predicted binding affinity of VK2 and 17β-estradiol to ER-α36 by computer docking simulation experiments. Figure 2 This is a representative result image of the expression level of ER-α36 in tumor tissue and adjacent tissue of HCC patients detected by Western blot technology, where T is tumor tissue and A is adjacent tissue (1 cm away from the tumor). Figure 3 The figure shows the effects of low concentrations of VK2 and estrogen on the growth of HepG2 and PLC / PRF / 5 cells and the expression of ER-α36. A and D are the results of cell colony formation assay, and B, C and E are the results of Western blot analysis. Figure 4 The figure shows the effects of high concentrations of VK2 and estrogen on the growth of HepG2 cells and the expression of ER-α36. In the figure, A is the result of the cell colony formation assay and B is the result of the Western blot analysis. Figure 5 The figure shows the effect of VK2 treatment on the orthotopic hepatocellular carcinoma xenograft model in Example 4 on tumor and ER-α36 expression. In the figure, A represents the tumor formation in the liver of nude mice, and B represents the results of Western blot detection. Detailed Implementation

[0016] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0018] Vitamin K2 is abbreviated as MK-n, where M represents methylnaphthoquinone, K represents vitamin K, and n represents the number of isoprene side chain residues. There are 14 compounds in vitamin K2 (MK-1 to MK-14), with the most common forms being MK-4 and MK-7. Moreover, the VK2 stored in human organs is mainly MK-4.

[0019] Primary liver cancer is currently the sixth most common malignant tumor worldwide, with HCC accounting for 75%–85% of all cases. HCC has a high incidence, strong heterogeneity, and poor prognosis; therefore, finding and developing novel drugs to inhibit or delay HCC growth is of great significance. To address this technical problem, this invention provides a globally first-discovered novel use for vitamin K2: vitamin K2 exhibits estrogen-like effects and displays a biphasic regulation of low-dose stimulation and high-dose inhibition of liver cancer cell growth.

[0020] Furthermore, the embodiments of the present invention reveal the mechanism by which VK2 biphasically regulates the growth of liver cancer cells via the ER-α36 signaling pathway, namely, low-dose vitamin K2 upregulates ER-α36 expression and promotes HepG2 cell growth, while high-dose vitamin K2 downregulates ER-α36 expression and inhibits HepG2 cell growth.

[0021] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0022] Example 1 This example demonstrates the estrogen-like effects of vitamin K2, which can bind to the ER-α36 protein.

[0023] 17β-estradiol is the most potent endogenous estrogen in living organisms. This study compares the chemical structures of VK2 (MK-4 and MK-7) and 17β-estradiol and uses computer docking simulation experiments to predict the binding affinity of VK2 and 17β-estradiol to ER-α36.

[0024] The results are as follows Figure 1As shown, the chemical core structures of VK2 (MK-4 and MK-7) are similar to those of 17β-estradiol. Furthermore, computer-simulated docking experiments revealed that MK-4, MK-7, and 17β-estradiol can all bind to the ER-α36 protein, with binding energies of -7.554 kcal / mol, -6.684 kcal / mol, and -7.406 kcal / mol, respectively. Both VK2 and 17β-estradiol can form strong polar hydrogen bonds with the ER-α36 protein.

[0025] Example 2 In this study, Western blot was used to detect the expression level of ER-α36 in tumor tissues and adjacent normal tissues of 46 HCC patients. ELISA and chemiluminescence immunoassay were used to detect the serum levels of VK2 and PIVKA-II in 10 HCC patients. The specific experiments are as follows: (1) Western blot experiment.

[0026] Tissue sample preparation: Weigh 50 mg of frozen liver tissue, add 1 mL of RIPA lysis buffer and grind. Place on ice for 30 minutes for lysis, then centrifuge at 12,000 rpm for 20 minutes at 4°C using a low-temperature high-speed centrifuge. The supernatant is then extracted to obtain the total protein of the liver tissue. Protein concentration is quantified by BCA method, and high-temperature protein denaturation is performed.

[0027] Preparation of the gel: Clean and dry the glass plate, and assemble and fix it on the gel holder; after mixing the separating gel, pour it into the glass interlayer, add double distilled water to the top of the liquid surface to press the gel, let it stand at room temperature for 30 minutes, wash the top of the gel with double distilled water, and absorb the liquid on the top with absorbent paper; pour in the stacking gel, insert the comb, and let it stand at room temperature for 30 minutes.

[0028] Sample loading: Adjust the protein concentration according to the protein quantification results. After mixing the sample with the loading buffer, heat to denature the protein. After mixing, add the mixture to the loading wells. The protein loading amount for liver tissue is 15 micrograms.

[0029] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): Add the sample to the electrophoresis well. Initially, set a constant voltage of 40 V for 20 minutes. When the electrophoretic bands approach the interface between the stacking gel and the separating gel, switch to a constant voltage of 120 V to continue electrophoresis. Stop electrophoresis immediately when the electrophoretic bands reach the bottom of the separating gel. Remove the gel and label it.

[0030] Transfer: Prepare a methanol-activated PVDF membrane. Assemble the transfer sandwich in a tray containing transfer buffer, following the order of filter paper, gel, membrane, and filter paper. Place the assembled sandwich into a transfer apparatus and transfer at a constant current of 280 mA for 90 minutes.

[0031] Blocking and elution: After labeling the PVDF membrane, transfer it to a blocking buffer containing 5% skim milk at room temperature for 1 hour; add TBST and elute by shaking for 5 minutes × 3 times at room temperature.

[0032] Primary antibody incubation and elution: Add the corresponding primary antibody according to the required dilution, seal and incubate overnight at 4°C; remove the membrane, add TBST, and rinse with shaking for 5 minutes × 3 times at room temperature.

[0033] Secondary antibody incubation and elution: Add secondary antibody solution diluted with TBST at a ratio of 1:5000, shake at room temperature for 1 hour, and then rinse with TBST for 5 minutes × 6 times.

[0034] ECL chemiluminescence development: Immerse the film in the prepared ECL colorimetric solution (prepared immediately before use) and expose it in the machine.

[0035] Image analysis: Save the image and analyze its grayscale values ​​using ImageJ.

[0036] Representative results of Western blot (see) Figure 2 The results showed that the expression level of ER-α36 protein in the tumor tissue of HCC patients was significantly higher than that in the adjacent normal tissue (36 / 46).

[0037] (2) ELISA experiment.

[0038] The concentration of VK2 in the serum of HCC patients was detected using a human VK2 ELASA kit. The specific procedure is as follows: ① Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant to obtain the sample to be tested; ② Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and return the remaining strips to 4℃ in a resealable bag; ③ Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well; ④ Add 50 μL of the sample to be tested to the sample wells, and do not add any to the blank wells; ⑤ Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 minutes. ⑥ Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times. ⑦ Add 50 μL of substrate A and 50 μL of substrate B to each well, and after 15 minutes, measure the OD value of each well at a wavelength of 45 nm. ⑧ Plot a standard curve on graph paper or using relevant software, with the OD value of the measured standard on the x-axis and the concentration of the standard on the y-axis, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the sample concentration.

[0039] The test results are shown in Table 1. 80% of HCC patients showed a deficiency of VK2 in their serum.

[0040] (3) Magnetic microparticle chemiluminescence immunoassay.

[0041] PIVKA-II is an abnormal prothrombin that is incompletely carboxylated due to vitamin K deficiency. In this case, the concentration of PIVKA-II in the serum of HCC patients was detected using a PIVKA-II assay kit (magnetic microparticle chemiluminescence method). The specific procedure is as follows: ① Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4°C overnight, then centrifuge at 1000× g for 20 minutes, and take the supernatant to obtain the sample to be tested; ② Sample addition and immune reaction: Add 15-50 μL of standard, quality control and test sample to a flat-bottomed test tube, add 45 μL of enzyme-labeled antibody, add 45 μL of coated magnetic bead antibody, add 300 μL of stabilizer, shake for 30 seconds, mix well, and incubate at 37°C for 30 minutes. ③ Washing: Place the flat-bottomed test tube on the magnetic rack for 2 minutes, pour out the supernatant, and invert the test tube and magnetic rack together on absorbent paper to dry; add 300 μL of cleaning agent to each tube, shake for 30 seconds, place the horizontal test tube on the magnetic rack for 2 minutes, pour out the supernatant, and invert the test tube and magnetic rack together on absorbent paper to dry; repeat 5 times; ④ Add luminescent substrate solution: Add 200 μL of luminescent substrate to each tube; ⑤ Read the luminescence value: Measure the luminescence value of each tube using a chemiluminescence analyzer.

[0042] The test results are shown in Table 1. 90% of HCC patients showed a significant increase in serum PIVKA-II.

[0043] Table 1. Serum PIVKA-II, VK and clinicopathological grade of 10 HCC patients

[0044] Example 3 This study examined the changes in HepG2 cell growth and ER-α36 expression in human HCC cells treated with different concentrations of VK2 and estrogen (17β-estradiol).

[0045] (1) Cell clone formation experiment.

[0046] HepG2 cells were digested with trypsin, then thoroughly mixed with complete culture medium to form a uniformly distributed cell suspension. Cell counts in the suspension were then determined using a hemocytometer. The cells were counted at a concentration of 2 × 10⁻⁶ cells / mL. 3 Seed cells at a density of 100 cells / well in 6-well plates. Gently shake the plate to ensure even distribution of cells and prevent cell aggregation. Place the plate in a CO2 incubator, minimizing movement before cell attachment to prevent cell detachment. Administer appropriate drug treatments according to the specific cell experiments required. Change the culture medium every 3-4 days to prevent the accumulation of cell metabolites. Observe and assess cell colony formation every 1 day until clearly visible colonies are observed (usually 10-14 days). Discard the culture medium and wash the cells 1-2 times with PBS buffer. Add 1 mL of 4% paraformaldehyde solution to each well for fixation at room temperature for 15 minutes, discard the fixative, and then add 0.1% crystal violet stain (diluted with 4% paraformaldehyde solution) to each well for staining at room temperature for 30 minutes. Discard the stain, wash with PBS buffer until the bottom of the 6-well plate is colorless, and air dry at room temperature. Finally, scan the colonies using a scanner to image them and calculate the colony formation rate.

[0047] (2) Western blot experiment.

[0048] Cell sample preparation: Adjust and prepare the lysis buffer (RIPA: protease inhibitor: phosphatase inhibitor = 50:1:1) according to the specific number of cells. After vortexing, immediately pre-cool the cells. Remove the cell culture blood and place it on ice. After removing the culture medium, wash the culture dish with pre-cooled PBS buffer. Add an appropriate amount of lysis buffer to the cell culture dish and incubate at 4°C for 20 minutes. Scrape off the lysed proteins and collect the lysis products. Transfer the mixture to a 1.5 mL EP tube using a pipette. Place the EP tube in a centrifuge that has been pre-cooled to 4°C and centrifuge at 12000 rpm for 15 minutes. Collect the supernatant (original cell protein sample solution).

[0049] The other steps are the same as in Example 2.

[0050] (3) Effects of low concentration of VK2 administration.

[0051] When HepG2 human HCC cells were treated with low concentrations of vitamin K2 (0.1 nM and 1 nM) and estrogen (10 nM), colony formation assays showed that, similar to the effects of estrogen, low concentrations of vitamin K2 promoted the growth of HepG2 cells. Figure 3 A). Western blot analysis was performed on HepG2 cells under different treatments, and the results are as follows: Figure 3 As shown in Figure B, after treating HepG2 cells with low concentrations of VK2 and estrogen for 12 hours, both low concentrations of VK2 and estrogen significantly upregulated ER-α36 expression.

[0052] In addition, Western blot analysis of ER-α36 expression in human HCC cells HepG2 and PLC / PRF / 5 revealed that ER-α36 expression was higher in PLC / PRF / 5 cells compared to HepG2 cells. Figure 3 C). Therefore, in this case, human HCC cells PLC / PRF / 5 were further treated with lower concentrations of VK2 (0.01 nM and 0.1 nM). The colony formation assay results showed that, compared with HepG2 cells, 0.01 nM of VK2 significantly promoted the growth of PLC / PRF / 5 cells. Figure 3 D). Treatment of PLC / PRF / 5 cells with different low concentrations of VK2 for 12 hours and Western blot analysis of ER-α36 expression revealed that, compared to HepG2 cells, 0.01 nM of VK2 significantly upregulated ER-α36 expression. Figure 3 E).

[0053] (4) Effects of high concentrations of VK2 administration.

[0054] HepG2 cells were treated with high concentrations of vitamin K2 (10 µM and 50 µM) and estrogen (1 µM). Colony formation assays showed that, similar to estrogen, high concentrations of vitamin K2 inhibited HepG2 cell growth, with a more significant inhibitory effect at a concentration of 50 µM. Figure 4 A).

[0055] Further Western blot analysis was performed on cells under different treatments, and the results are as follows: Figure 4 As shown in Figure B, after treating HepG2 cells with high concentrations of VK2 and estrogen for 12 hours, both high concentrations of VK2 and estrogen (1 µM) downregulated the expression of ER-α36, with the effect being more pronounced at a concentration of 50 µM.

[0056] Example 4 This study, through in vivo animal experiments, further clarified the regulatory role of high concentrations of VK2 in downregulating ER-α36 expression and inhibiting the growth of liver cancer cells.

[0057] The experimental procedure is as follows: Laboratory animals: Six 5-week-old male SPF-grade BALB / c nude mice were purchased from Beijing Weitehe Laboratory Animal Technology Co., Ltd. They were housed under specific pathogen-free conditions with constant temperature (24±2℃) and relative humidity (60%), and this environment was also equipped with a 12-hour circulating light system. The mice were acclimatized for one week. The rules of procedure and experimental protocols for the use of the mice were approved by the Ethics Committee of Jianghan University, and the mice were housed at the School of Medicine of Jianghan University.

[0058] Modeling and processing methods: Use a disposable syringe to draw 5×10 6 50 μL of a HepG2 cell mixture (1:1 mixture of matrix gel and PBS) was slowly and evenly injected into the liver of BALB / c nude mice to construct an orthotopic hepatocellular carcinoma xenograft model. One week later, the nude mice were randomly divided into a control group and a VK2 treatment group. The VK2 treatment group was given VK2 (15 mg / kg) by gavage every two days, while the control group was given DMSO by gavage. After 28 days of feeding, the mice were euthanized by cervical dislocation under anesthesia, and the livers were removed for tumor size analysis and Western blotting experiments (refer to Example 2).

[0059] The experimental results showed that tumors formed in the livers of all nude mice after 28 days, and the tumor volume was smaller in the VK2-treated group compared to the control group. Figure 5 A), and ER-α36 expression was decreased in tumor tissue of the VK2 group ( Figure 5 B). The above results suggest that, at the animal level, VK2 can downregulate the expression of ER-α36, thereby inhibiting HCC tumor growth.

[0060] In summary, vitamin K2 has estrogen-like effects, can biphasically regulate the growth of liver cancer cells through the ER-α36 signaling pathway, and can successfully inhibit the growth of HCC tumors in mice. This is of great significance for the development of novel drugs to inhibit or delay the growth of liver cancer.

[0061] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of Vitamin K2 in the preparation of estrogen-like products.

2. The application according to claim 1, characterized in that, The estrogen-like effects of vitamin K2 regulate the growth of liver cancer cells by modulating the expression level of ER-α36.

3. The application according to claim 2, characterized in that, The vitamin K2 biphasically regulates the expression level of ER-α36, wherein low-dose vitamin K2 upregulates the expression level of ER-α36 and promotes the growth of liver cancer cells, while high-dose vitamin K2 downregulates the expression level of ER-α36 and inhibits the growth of liver cancer cells.

4. The application according to claim 3, characterized in that, The term "low dose" refers to a vitamin K2 concentration of 0.01-1 nM, and the term "high dose" refers to a vitamin K2 concentration of ≥20 μM.

5. The application of vitamin K2 in the preparation of drugs for treating hepatocellular carcinoma, wherein the drugs treat hepatocellular carcinoma by downregulating the expression level of ER-α36.

6. The application according to claim 5, characterized in that, The drug also contains pharmaceutically acceptable excipients.

7. The application according to claim 5, characterized in that, The drug is an oral or topical preparation, and it can be an ointment, granules, gel, tablet, liquid, capsule, or pill.

8. A drug for treating hepatocellular carcinoma, characterized in that, The drug contains vitamin K2 as one of its active ingredients.

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