Application of ursodesoxycholic acid UDCA in inhibiting Skp2 protein and resisting non-small cell lung cancer

Abstract

The invention discloses an application of ursodesoxycholic acid UDCA in inhibition of Skp2 protein and resistance to non-small cell lung cancer, and particularly discloses an application of ursodesoxycholic acid UDCA in preparation of an Skp2 protein inhibitor, the ursodesoxycholic acid UDCA inhibits Skp2 to enable a cell cycle to be stagnated in a G0 / G1 phase, so that degradation of p21Cip1 and p27Kip1 is reduced. Meanwhile, the invention discloses application of ursodesoxycholic acid UDCA in preparation of a non-small cell lung cancer cell proliferation activity inhibitor. The ursodesoxycholic acid UDCA is reported to have relatively strong Skp2 protein inhibition capability and non-small cell lung cancer inhibition capability for the first time, is simple in structure, is one of main components of bile acid in bear gall powder, is also a chemical drug capable of being artificially synthesized, can be stably separated and extracted, and has a good application prospect. The compound is a natural antitumor compound which is efficient and easy to obtain.

Description

Technical Field

[0001] This invention belongs to the field of new pharmaceutical applications of ursodeoxycholic acid (UDCA), specifically relating to the application of UDCA in inhibiting Skp2 protein and in the treatment of non-small cell lung cancer. Background Technology

[0002] Bile acids (BAs) are the main components of human bile, formed from cholesterol through liver conversion. They can be divided into primary and secondary bile acids. Primary bile acids include cholic acid (CA) and chenodeoxycholic acid, while secondary bile acids include deoxycholic acid (DA), lithocholic acid (LA), and ursodeoxycholic acid (UDCA). Abnormal metabolic characteristics in the body's metabolic network, including bile component composition, bile acid metabolic pathways, cholesterol metabolism, and intestinal absorption mechanisms, are all associated with the occurrence and development of cancer. From a metabolic perspective, abnormal regulatory patterns in these metabolic pathways can be considered potential biomarkers for cancer, providing a new perspective for early tumor identification and mechanistic research.

[0003] Ursodeoxycholic acid (UDCA), chemically named 3α,7β-dihydroxy-5β-cholestane-24-acid, has the molecular formula C2. 24 H 40 O4, with a molecular weight of 392.56, has the following molecular structure:

[0004]

[0005] UDCA affects cancer cell migration, invades cancer stem cells, induces ecological imbalance, and reduces damage from anticancer drugs by inhibiting cell growth and inducing cell death, thus exhibiting certain antitumor activity (JFGoossens, C. Bailly, Ursodeoxycholic acid and cancer: From chemoprevention to chemotherapy, Pharmacology & Therapeutics, 203(2019)107396). Lee et al. (J.Lee, EMHong, JHKim, JHKim, JHJung, SWPark, DHKoh, HJJang, Ursodeoxycholic acid shows antineoplastic effects in bile duct cancer cells via apoptosis induction; p53 activation; and EGFR-ERK, COX-2, and PI3K-AKT pathway inhibition, Molecular Biology Reports, 48(2021)6231-6240) have shown that UDCA inhibits the proliferation and invasion of bile duct cancer cells by inducing apoptosis, activating p53, and inhibiting the EGFR-ERK, COX-2, and PI3K-AKT pathways. Studies by Kovács et al. (P. Kovács, S. Schwarcz, P. Nyerges, TIBíró, G. Ujlaki, P. Bai, E. Mikó, Anticarcinogenic effects of ursodeoxycholic acid in pancreatic adenocarcinoma cell models, Frontiers in Cell and Developmental Biology, 12(2024)1487685) have shown that low concentrations of UDCA, close to the serum reference range, have beneficial effects on pancreatic ductal adenocarcinoma cells, inhibiting cell proliferation, reducing cancer stem cell characteristics, inducing mitochondrial oxidative metabolism, and not interfering with chemotherapy drugs. UDCA can inhibit the proliferation of various cancer cell lines and induce apoptosis, with the mechanism varying depending on the cancer cell type. Currently, UDCA is widely used in the treatment of primary cholangitis and primary sclerosing cholangitis, approved by the US Food and Drug Administration, and has good safety and relatively mild adverse reactions even at high doses. Although there are tumor-related studies, the mechanism of action of the drug is not yet clearly understood.

[0006] Skp2 protein is a member of the F-box protein family. It binds to the conserved parts of the SCF complex, Skp1, Cullin-1, and Rbx1, to form an E3 ubiquitination ligase. It plays an important role in regulating cell cycle progression and cell survival by promoting the destruction of various tumor suppressor proteins (including p27, p21, p57, p130, and FOXO1), thereby promoting tumor development (Z.Cai, A.Moten, D.Peng, C.H. Su, B.S. Pan, R.Manne, HY.Li, HK.Lin, The Skp2 Pathway: A Critical Target for Cancer Therapy, Seminars in Cancer Biology, 67(2020)16-33). The pro-tumor properties of Skp2 protein have been confirmed in various cancers, and it is currently a hot topic in research on potential targets for cancer therapy (Z.Cai, A.Moten, D.Peng, C.H. Su, B.S. Pan, R.Manne, HY.Li, HK.Lin, The Skp2 Pathway: A Critical Target for Cancer Therapy, Seminars in Cancer Biology, 67(2020)16-33; J.R.S. Kaar, J.K. Pagan, M. Pagano, SCF ubiquitin ligase-targeted therapies, Nature Reviews. Drug). Discovery, 13(2014)889-903), the mechanisms involved include cell cycle, proliferation, apoptosis, autophagy and ferroptosis, especially in NSCLC (non-small cell lung cancer) where abnormal DNA copy number led to abnormally high expression of Skp2 protein in 44% of NSCLC patient samples (S. Yokoi, K. Yasui, M. Mori, T. Iizasa, T. Fujisawa, J. Inazawa, Amplification and overexpression of SKP2 are associated with metastasis of non-small-cell lung cancers to lymph nodes, The American Journal of Pathology, 165(2004)175-180).For NSCLC patients with KRas mutations, increased Skp2 protein expression can serve as an independent diagnostic marker for poor survival (CQZhu, FHBlackhall, M. Pintilie, P. Iyengar, N. Liu, J. Ho, T. Chomiak, D. Lau, T. Winton, FA. Shepherd, MSTsao, Skp2 genecopy number aberrations are common in non-small cell lung carcinoma, and its overexpression in tumors with ras mutation is a poor prognostic marker, Clinical Cancer Research: an official journal of the American Association for Cancer Research, 10(2004) 1984-1991). Given its crucial role in controlling tumor development and progression, Skp2 protein has become a potential pharmacological target for anticancer therapy.

[0007] Inhibiting the activity of Skp2-related proteins through drugs is the primary approach. There are three forms of inhibition: the first is by suppressing the expression or degradation of upstream proteins or genes of Skp2 in the cell cycle; the second is by accelerating the degradation of Skp2 protein while simultaneously inhibiting the formation of the Skp2 complex; the third is by directly acting on Skp2 protein without affecting its degradation, but by altering the levels of downstream proteins in the Skp2 pathway, reducing the expression of proteins such as p27 and p21, thereby changing the growth, proliferation, and metastasis capabilities of tumor cells.

[0008] Bile acids have brought increasing surprises in the field of cancer, including angiogenesis, cancer cell proliferation and death, tumor invasion and metastasis, inflammation, and immune regulation. Due to their unique amphiphilicity and broad class, UDCAs play diametrically opposed roles in different cancers, and even in the same type of cancer. This contradictory effect imbues bile acids with a mysterious quality. The cytotoxic properties of bile acids enable researchers to further explore and develop potential cancer treatments.

[0009] Patent document CN202211275190.9 discloses the application of a traditional Chinese medicine composition in the preparation of a drug for treating bacterial prostatitis. This composition comprises cholic acid, ursodeoxycholic acid, buffalo horn, isatis root, honeysuckle, gardenia, baicalin, and mother-of-pearl. This composition shows significant improvement in bacterial prostatitis, reducing pathogen infection and alleviating inflammatory responses. Furthermore, research data shows that this composition can significantly enhance the therapeutic effect of cephalosporin drugs on bacterial prostatitis, thus it can be used as a synergist in combination with these drugs for the treatment of bacterial prostatitis. However, this patent document does not mention the application of ursodeoxycholic acid in inhibiting Skp2 protein and in the treatment of non-small cell lung cancer.

[0010] Patent document CN202411417415.9 discloses a kit and detection method for simultaneously detecting 15 bile acid profiles and 3 bile acid precursors in serum. The 15 bile acid profiles include cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, taurocholic acid, and taurocholic acid. The 3 bile acid precursors include 3α,7α-dihydroxycobalamin prostaglandin, 3α,7α,12α-trihydroxycholestannic acid, and 7α-hydroxy-4-cholesterol-3-one. The kit and detection method can simultaneously and accurately detect the aforementioned 15 bile acid profiles and 3 bile acid precursors, with short detection time and high accuracy, thus addressing to some extent the clinical need for assessing the enterohepatic cycle composed of the biliary system, intestine, portal circulation, and hepatocytes. However, this patent literature does not mention any related applications of ursodeoxycholic acid in inhibiting Skp2 protein and in combating non-small cell lung cancer.

[0011] Patent document CN202510852092.4 discloses the application of a small molecule compound, rye ketoacid B, in inhibiting Skp2 protein and combating prostate cancer cell metastasis. Studies have found that rye ketoacid B can significantly reduce the half-life of Skp2 by enhancing the polyubiquitination process on the Skp2 protein, thereby accelerating its degradation and reducing the protein level of Skp2 in DU145 cells. Rye ketoacid B induces proteasome degradation of Skp2 through the polyubiquitination pathway of the K48 chain, thereby inhibiting tumor cell metastasis and achieving an anti-prostate cancer effect. However, this patent document only discloses the application of the small molecule compound rye ketoacid B in inhibiting Skp2 protein and combating prostate cancer cell metastasis, and does not mention the application of ursodeoxycholic acid in inhibiting Skp2 protein and combating non-small cell lung cancer. Summary of the Invention

[0012] The purpose of this invention is to provide the application of ursodeoxycholic acid (UDCA) in inhibiting Skp2 protein and in combating non-small cell lung cancer.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] One of the objectives of this invention is the application of ursodeoxycholic acid (UDCA) in the preparation of Skp2 protein inhibitors, wherein UDCA inhibits Skp2, causing cell cycle arrest at the G0 / G1 phase, thereby reducing the degradation of p21Cip1 and p27Kip1.

[0015] Another objective of this invention is the application of ursodeoxycholic acid (UDCA) in the preparation of an inhibitor of non-small cell lung cancer cell proliferation activity. UDCA inhibits Skp2, causing cell cycle arrest at the G0 / G1 phase, thereby reducing the degradation of p21Cip1 and p27Kip1 and effectively inhibiting the excessive proliferation of non-small cell lung cancer cells.

[0016] Furthermore, the non-small cell lung cancer cells are A549 cells and / or H1975 cells.

[0017] Furthermore, the effective concentration of ursodeoxycholic acid (UDCA) for inhibiting the proliferation activity of non-small cell lung cancer cells is 100–500 μM.

[0018] Furthermore, the ursodeoxycholic acid (UDCA) is used in combination with the Skp2 inhibitor Skp2-IN-4 to synergistically inhibit the proliferation activity of non-small cell lung cancer cells.

[0019] The significant advantages of this invention are: This invention is the first to report that ursodeoxycholic acid (UDCA) has strong inhibitory ability on Skp2 protein and non-small cell lung cancer. Its structure is simple. It is not only one of the main components of bile acids in bear bile powder, but also a chemical drug that can be artificially synthesized. It can be stably separated and extracted, and is a highly efficient and easily obtainable natural antitumor compound.

[0020] This invention is the first to discover that ursodeoxycholic acid (UDCA) can induce cell cycle arrest in A549 cells by inhibiting Skp2, thereby affecting cell proliferation. Specifically, UDCA inhibits Skp2, causing cell cycle arrest at the G0 / G1 phase, thus reducing the degradation of p21Cip1 and p27Kip1. The tumor suppressor protein p27 can effectively control the excessive proliferation of cancer cells. Therefore, UDCA, by inhibiting Skp2 protein expression, can regulate its downstream pathways, thereby promoting cell cycle arrest in A549 non-small cell lung cancer cells and effectively inhibiting their excessive proliferation, thus achieving the goal of suppressing the proliferative activity of non-small cell lung cancer cells. Attached Figure Description

[0021] Figure 1 This study demonstrates the inhibitory effect of UDCA on the growth of A549 and H1975 cell lines in vitro.

[0022] Figure 2 The proportion of cells treated with UDCA and the proportion of cells in the G0 / G1, S, and G2 phases of the cell cycle.

[0023] Figure 3 A comparative diagram showing the effect of UDCA on Skp2 expression in A549 and H1975 cells.

[0024] Figure 4 A comparative diagram showing the effects of UDCA on the expression of related proteins in A549 and H1975 cells under the action of proteasome inhibitors (A); Western Bolt plot showing the effect of UDCA on the expression of related proteins in A549 cells and stability curves of related proteins (B).

[0025] Figure 5 Treatment with UDCA alters the distribution of cell cycle stages in A549 cells.

[0026] Figure 6 UDCA showed a dose-dependent promoting effect on Skp2-IN-4-induced cell death.

[0027] Figure 7 The combined use of UDCA and the Skp2 inhibitor (Skp2-IN-4) in xenograft models showed synergistic lethal effects both in vivo and in vitro. Detailed Implementation

[0028] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0029] Both A549 and H1975 cells were obtained from the American Type Culture Collection Center (ATCC). They were cultured in RPMI-1640 medium (C22400500BT, GIBCO) supplemented with 2 mM L-glutamine (25030, Life Technologies) and 10 wt% FBS. All cell lines were identified. Cell line validation was performed using short tandem repeat (STR) analysis in a PowerPlex 21 system. STR data for both A549 and H1975 cell lines were found to be consistent with those from the ATCC.

[0030] Example 1

[0031] Ursodeoxycholic acid (UDCA) inhibits the proliferation of A549 and H1975 cells.

[0032] Cell proliferation was assessed using CCK8 assay. The effects of different concentrations of ursodeoxycholic acid (UDCA) on the viability of A549 and H1975 cells at 24 h, 48 h, and 72 h were investigated.

[0033] 5×10 3 Two A549 and H1975 cells were seeded into 96-well plates, with six replicates per group. The plates were incubated at 37°C with 5% CO2 for 24 hours, and a ring of PBS buffer was added to the blank wells at the edges.

[0034] Remove the 96-well plate and place it under a microscope for observation. After the cells adhere to the plate, first aspirate the culture medium and add different concentrations of the same volume of ursodeoxycholic acid (UDCA) to the experimental group. Add the same volume of PBS buffer to the control group and incubate for 24h, 48h and 72h respectively.

[0035] Add CCK8 solution: Take out the 96-well plate and observe its state under a microscope. Add 10 μL of CCK8 solution to each well, shake gently, and incubate in an incubator for 2 hours. Formazan will form in the cells. Place the plate on a vibrator at low speed for 10 seconds.

[0036] The absorbance was measured at 450 nm, and the OD values ​​of A549 cells and H1975 cells after drug administration were calculated. Different time periods of 24 h, 48 h and 72 h were set, and then the experiment was repeated.

[0037]

[0038] Formula for calculating cell viability:

[0039] Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0040] The results are as follows Figure 1 As shown in the figure, UDCA inhibited the growth of both A549 and H1975 cell lines, and the inhibitory effect increased with increasing concentration. Compared with the control group, the proliferation rate of A549 and H1975 cells was significantly reduced at UDCA concentrations of 250 μM and 500 μM. Significant differences in cell growth rates were observed between the experimental and control groups after 48 h and 72 h of stimulation. This indicates that ursodeoxycholic acid (UDCA) can effectively inhibit the proliferation of NSCLC cells.

[0041] Example 2

[0042] Effects of ursodeoxycholic acid (UDCA) on the A549 cell cycle

[0043] Cells treated with different concentrations of UDCA were collected from 6-well plates and fixed overnight at 4°C with 70% ethanol (v / v), followed by washing twice with phosphate-buffered saline (PBS). Cells were stained for 1 h in a solution containing 50 μM PI and 5 μg / mL ribonuclease A, and then analyzed using a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA). Cell cycle data were analyzed using ModFit LT(3.1) software.

[0044] The results are as follows Figure 2 As shown, flow cytometry analysis indicated that ursodeoxycholic acid (UDCA) treatment resulted in a dose-dependent increase in the G1 phase cell population of NSCLC cells.

[0045] Example 3

[0046] Effects of different concentrations of ursodeoxycholic acid (UDCA) on SKP2 expression levels in A549 and H1975 cells

[0047] A549 and H1975 cell lines were selected and divided into a blank group, a 250 μM UDCA group, and a 500 μM UDCA group. An equal volume of DMSO was added to the blank group. After incubating the cells in each test group for 48 h, the expression level of Skp2 protein in each group was detected, with β-actin as the internal control protein.

[0048] Standard Western blot procedures were used to detect protein expression in cells during Western blot analysis using the following antibodies: anti-actin (Cat.#AF7018, Affinity Biosciences, 1:1000) and anti-Skp2 (Cat.#AF0505, Affinity Biosciences, 1:1000). Protein signals were detected using an ECL kit (Thermo Fischer Scientific, Waltham, MA, USA).

[0049] The results are as follows Figure 3As shown in the figure, Western blot analysis of Skp2 expression levels in A549 and H1975 cells revealed that, compared to the control group, Skp2 protein expression levels were significantly lower in both the 250 μM and 500 μM UDCA treatment groups. This indicates that UDCA effectively inhibits Skp2 protein expression in A549 and H1975 cells. Therefore, it can be inferred that ursodeoxycholic acid (UDCA) effectively inhibits NSCLC cell proliferation by suppressing Skp2 protein expression.

[0050] Example 4

[0051] Effect of ursodeoxycholic acid (UDCA) on Skp2 stability

[0052] 1. Select A549 and H1975 cell lines and divide them into a blank group, a 250 μM UDCA group, a 500 μM UDCA treatment group, and a 500 μM UDCA + protease inhibitor MG132 group. The blank group was treated with an equal volume of DMSO. After incubating the cells in each test group for 48 h, the expression level of Skp2 protein in each group was detected, with β-actin as the internal control protein.

[0053] Standard Western blot procedures were used to detect protein expression in cells during Western blot analysis using the following antibodies: anti-actin (Cat.#AF7018, Affinity Biosciences, 1:1000) and anti-Skp2 (Cat.#AF0505, Affinity Biosciences, 1:1000). Protein signals were detected using an ECL kit (Thermo Fischer Scientific, Waltham, MA, USA).

[0054] Results: Combined treatment with the protease inhibitor MG-132 did not completely restore the expression level of Skp2 protein in the two non-small cell lung cancer cell lines to the level observed in the untreated control group.

[0055] 2. The A549 cell line was selected and divided into two groups: a blank group and a group treated with ursodeoxycholic acid (UDCA) at a concentration of 500 μM. Cell proteins were collected at different times (0 h, 8 h, 16 h and 24 h) after CHX stimulation. The expression level of Skp2 protein in each group was detected, with β-actin as an internal reference protein.

[0056] Standard Western blot procedures were used to detect protein expression in cells during Western blot analysis using the following antibodies: anti-actin (Cat.#AF7018, Affinity Biosciences, 1:1000) and anti-Skp2 (Cat.#AF0505, Affinity Biosciences, 1:1000). Protein signals were detected using an ECL kit (Thermo Fischer Scientific, Waltham, MA, USA).

[0057] The results are as follows Figure 4 As shown in the figure, the degradation rate of Skp2 protein was only slightly affected in the A549 cell line treated with UDCA. These results confirm that the downregulation of Skp2 protein expression induced by ursodeoxycholic acid (UDCA) is mainly achieved by reducing its transcriptional level.

[0058] Example 5

[0059] Treatment with ursodeoxycholic acid (UDCA) altered the distribution of cell cycle stages in A549 cells.

[0060] The Flag-Skp2 plasmid was obtained from Addgene (#89142). The plasmid was transfected with Lipofectamine 2000 (1662298, Invitrogen). A549 cells were seeded into 6-well plates and transfected with either the Flag-Skp2 or Flag vector at 0.5 μg. After culturing for a specific time, further post-transfection assays were performed.

[0061] Cells treated with different concentrations of UDCA were collected from 6-well plates and fixed overnight at 4°C with 70% ethanol (v / v), followed by washing twice with phosphate-buffered saline (PBS). Cells were stained for 1 h in a solution containing 50 μM PI and 5 μg / mL ribonuclease A, and then analyzed using a FACScan flow cytometer (Beckman Coulter, Fullerton, CA, USA). Cell cycle data were analyzed using ModFit LT(3.1) software.

[0062] The results are as follows Figure 5 As shown in the figure, Skp2 altered the distribution of cell cycle phases in A549 cells after treatment with ursodeoxycholic acid (UDCA).

[0063] Example 6

[0064] In vitro cell proliferation experiment - synergistic inhibitory effect of ursodeoxycholic acid (UDCA) and Skp2 inhibitor

[0065] The inhibitory effects of the Skp2 inhibitor Skp2-IN-4 (HY-174346, MedChemExpress) and / or UDCA on the proliferation of A549 and H1975 cells were detected using the CCK8 assay.

[0066] 5×10 3 Two A549 and H1975 cells were seeded into 96-well plates, with six replicates per group. The plates were incubated at 37°C with 5% CO2 for 24 hours, and a ring of PBS buffer was added to the blank wells at the edges.

[0067] Remove the 96-well plate, wait for the cells to adhere, first aspirate the culture medium, and add different concentrations of the same volume of culture medium containing SKP2-IN-4 and / or UDCA to the experimental group, and add the same volume of PBS to the control group. Incubate for 48 hours in a carbon incubator.

[0068] Add CCK8 solution: Take out the 96-well plate and observe its state under a microscope. Add 10 μL of CCK8 solution to each well, shake gently, and incubate in an incubator for 2 hours. Formazan is formed in the cells. Place the plate on a vibrator at low speed for 10 seconds.

[0069] The absorbance was measured at 450 nm, and the OD values ​​of A549 cells and H1975 cells after drug administration were calculated. The experiment was repeated after 48 h.

[0070] Formula for calculating cell viability:

[0071] Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0072] The results are as follows Figure 6 As shown in the figure, ursodeoxycholic acid (UDCA) exhibits a dose-dependent promoting effect on Skp2-IN-4-induced cell death.

[0073] Example 7

[0074] In vivo tumorigenesis experiment - synergistic inhibitory effect of UDCA and Skp2 inhibitors in a xenograft mouse model

[0075] 100 μL of LA549 cell suspension (1 × 10⁶ cells per mouse) was prepared. 7 A mouse model of lung cancer was established by subcutaneous inoculation of the right axilla with 10 cells (15-30 mm²) into 5-week-old female BALB / c nude mice. Seven days later, all tumors were measured. 3Mice were randomly divided into three groups (n=6 per group): DMSO group, 250 mg / kg UDCA group, and 250 mg / kg UDCA + 25 mg / kg Skp2-IN-4 group. UDCA and SKP2-IN-4 were administered intraperitoneally every two days for 14 days. Tumor growth was measured every two days, and tumor volume V was calculated using the following formula: V = (L × W) / (L × W) 2 )×0.52, where L is the length of the xenograft tumor and W is the width. At the end of treatment, all nude mice were euthanized, and their tumors were then isolated and weighed.

[0076] The results are shown in Figure 7. As can be seen from the figure, UDCA significantly inhibited tumor growth compared to the control group. The combination of UDCA and Skp2-IN-4 showed a stronger inhibitory effect on tumor growth than UDCA alone. Furthermore, the combination of Skp2-IN-4 and UDCA demonstrated a significant and sustained effect in a mouse model of tumor transplantation, with no signs of toxicity observed. The combination of Skp2-IN-4 and UDCA achieved a tumor growth inhibition rate of 69.63%, exceeding the 55.10% inhibition rate of UDCA alone. After the combined use of ursodeoxycholic acid (UDCA) and the Skp2 inhibitor Skp2-IN-4, there was no significant change in body weight between the Skp2-IN-4+UDCA group and the UDCA group.

[0077] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. Use of ursodeoxycholic acid (UDCA) in the preparation of an inhibitor of Skp2 protein, wherein the ursodeoxycholic acid (UDCA) causes cell cycle arrest at G0 / G1 phase by inhibiting Skp2, thereby reducing the degradation of p21Cip1 and p27Kip1.

2. Use of ursodeoxycholic acid (UDCA) in the preparation of an inhibitor of the proliferation activity of non-small cell lung cancer cells, wherein the ursodeoxycholic acid (UDCA) causes cell cycle arrest at G0 / G1 phase by inhibiting Skp2, thereby reducing the degradation of p21Cip1 and p27Kip1, thereby effectively inhibiting the excessive proliferation of non-small cell lung cancer cells.

3. Use according to claim 2, characterized in that: The non-small cell lung cancer cells are A549 cells or / and H1975 cells.

4. Use according to claim 2, characterized in that: The effective concentration of the ursodeoxycholic acid (UDCA) in inhibiting the proliferation activity of non-small cell lung cancer cells is 100-500 μM.

5. Use according to claim 2, characterized in that: The ursodeoxycholic acid (UDCA) is used in combination with Skp2 inhibitor Skp2-IN-4 to achieve synergistic inhibition of the proliferation activity of non-small cell lung cancer cells.

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