Application of loranoside C in preparation of medicine for treating prostatic cancer

Abstract

The invention provides an application of loranoside C in preparation of a medicine for treating prostatic cancer. Experiments prove that the erioside C can obviously inhibit the growth and survival of prostate cancer cells, has certain selectivity on the effects of the prostate cancer cells and normal prostate cells, can obviously inhibit the growth of prostate tumors, and shows a dose-effect relationship.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to the application of lanatoside C in the preparation of a medicine for treating prostate cancer. Background Art

[0002] The prostate is a unique male sexual organ located between the bladder and the urogenital diaphragm. With the advent of a serious aging era around the world, the incidence of prostate-related diseases has increased significantly, which has become a major issue affecting the health and quality of life of middle-aged and elderly men. Among them, benign prostatic hyperplasia, prostate cancer and prostatitis are the most common lesions of the prostate. Prostate cancer is one of the most common malignant tumors of the urinary male reproductive system.

[0003] Currently, the main treatment options for prostate cancer are active monitoring, chemotherapy, radiotherapy, hormone therapy and surgical treatment. Among them, surgical treatment is the main treatment method for patients with localized and some locally advanced stages. However, for patients with advanced, locally invasive or metastatic prostate cancer, the treatment strategy usually turns to chemotherapy and androgen deprivation therapy. Commonly used chemotherapy drugs include docetaxel and cabazitaxel. After intravenous injection of chemotherapy drugs, while killing cancer cells in the body, it also damages normal cells in the body, causing related toxic side effects.

[0004] Lanatoside C (Lan C, molecular formula: C 49 H 76 O 20 ) is a cardiac glycoside with the structural formula shown below:

[0005]

[0006] Lanatin C is commonly used for congestive heart failure and arrhythmia. In recent years, studies have reported the therapeutic effects of lanatin C on some tumor diseases, but there have been no reports on its treatment of prostate cancer. Summary of the invention

[0007] One object of the present invention is to provide the use of lanatoside C in the preparation of a medicament for treating prostate cancer.

[0008] Another object of the present invention is to provide use of a pharmaceutical composition containing lanatoside C in the preparation of a drug for treating prostate cancer.

[0009] In one aspect, the present invention provides the use of lanatoside C in the preparation of a medicament for treating prostate cancer.

[0010] In the present invention, the lanatoside C has the following structure:

[0011]

[0012] In some embodiments, lanatoside C can inhibit the growth and survival of prostate cancer cells. In some embodiments, the prostate cancer cells include, but are not limited to, human prostate cancer cells PC-3, DU145, and LNCAP.

[0013] In another aspect, the present invention provides use of a pharmaceutical composition containing lanatoside C in the preparation of a medicament for treating prostate cancer.

[0014] In some embodiments, the pharmaceutical composition containing lanatoside C comprises: lanatoside C; and optionally one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutically acceptable carriers include, for example, fillers, diluents, solvents, sweeteners, surfactants, odorants, flavoring agents, lubricants, etc.

[0015] In some embodiments, the pharmaceutical composition can be prepared into various dosage forms, such as tablets, capsules, powders, syrups, solutions, suspensions, and aerosols.

[0016] The present invention verifies through experiments that lanatoside C can significantly inhibit the growth and survival of prostate cancer cells, and that lanatoside C has a certain selectivity for the effect on prostate cancer cells and normal prostate cells. In addition, lanatoside C can significantly inhibit the growth of prostate tumors and present a dose-effect relationship. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows the morphological effects of Lan C administration for 48 h on human prostate cancer cells PC-3, DU145, and LNCAP (Scale bar = 50 μm).

[0018] Figure 2 The figure shows the effect of Lan C administration for 24h, 48h and 72h on the survival rate of human prostate cancer cells PC-3 (A), DU145 (B), and LNCAP (C) cells (compared with the vehicle control group: *P<0.05, **P<0.01, ***P<0.001, N=5).

[0019] Figure 3 The figure shows the inhibitory effect of Lan C administration for 48 h on the proliferation of human prostate cancer cells PC-3, DU145, LNCAP (A) and human normal prostate stromal immortalized cells WPMY-1 and human prostate fibroblasts HPRF (B).

[0020] Figure 4The experimental figure (A) and statistical bar graph (B) show the effect of Lan C on the colony-forming ability of human prostate cancer cells PC-3 and DU145 (compared with the vehicle control group: **P<0.01, ***P<0.001, N=3).

[0021] Figure 5 The scatter plots (A) and the statistical bar graphs (B) of cell apoptosis ratios in each group after Lan C treatment of human prostate cancer cell PC-3 for 48 hours (compared with the vehicle control group: **P<0.01, ***P<0.001, N=3).

[0022] Figure 6 The scatter plot (A) and the bar graph (B) of cell apoptosis statistics in each group after Lan C treatment of human prostate cancer cell DU145 for 48 hours (compared with the vehicle control group: ***P<0.001, N=3).

[0023] Figure 7 Lan C inhibits the growth of xenograft mouse tumor models induced by subcutaneous injection of PC-3 cells. (A) Body weight changes of Balb / c nude mice during administration; (B) Tumor volume change curve; (C) Tumor weight (compared with the control group: *P<0.05, **P<0.01, ***P<0.001, N=9).

[0024] Figure 8 Shown are the appearance of tumor growth in each group of nude mice after four weeks of Lan C treatment. DETAILED DESCRIPTION

[0025] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0026] Example

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] 1. Experimental Methods

[0030] 1.1 Cell and drug sources

[0031] Prostate cancer cell lines PC-3, DU145 and LNCAP were purchased from the National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences; human normal prostate stromal immortalized cells WPMY-1 were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.; human prostate fibroblasts HPRF were purchased from Sciencell.

[0032] Human prostate cancer cells PC-3 were cultured using F12K medium supplemented with fetal bovine serum at a final concentration of 10% and cultured in a 37°C constant temperature, 5% carbon dioxide incubator.

[0033] Human prostate cancer cell DU145 was cultured using MEM medium supplemented with fetal bovine serum at a final concentration of 10%, and cultured in a 37°C constant temperature, 5% carbon dioxide incubator.

[0034] Human prostate cancer cells LNCAP were cultured using RPMI-1640 medium supplemented with fetal bovine serum at a final concentration of 10%, and cultured in a 37°C constant temperature, 5% carbon dioxide incubator.

[0035] The human normal prostate stromal immortalized cells WPMY-1 were cultured using DMEM high-glucose medium supplemented with fetal bovine serum at a final concentration of 10%, and cultured in a 37°C constant temperature, 5% carbon dioxide incubator.

[0036] Human prostate fibroblasts HPRF were cultured using FM medium supplemented with 2% fetal bovine serum and 1% FGS at a final concentration, and cultured in a 37°C constant temperature, 5% carbon dioxide incubator.

[0037] Lan C was purchased from Hepeng (Shanghai) Biotechnology Co., Ltd., CAS No.: 17575-22-3, molecular formula: C 49 H 76 O 20 .

[0038] 1.2 Morphological observation

[0039] Human prostate cancer cells PC-3, DU145 and LNCAP cells were placed in a 37°C, 5% CO2 constant temperature incubator for culture until the cells grew to the logarithmic growth phase. Subsequently, 96-well plates were inoculated according to the corresponding target cell concentration (PC-3 inoculation number was 8000 / well; DU145 inoculation number was 6000 / well; LNCAP inoculation number was 10000 / well), 100 μL per well, and 5 replicate wells were set for each cell group. After the cells adhered to the wall, they were divided into a solvent control group (1‰ dimethyl sulfoxide (DMSO)) and a Lan C experimental group (50-400nM). After 48 hours of treatment, the culture plate was removed, and the changes in cell morphology were observed and photographed under an inverted microscope.

[0040] 1.3 CCK-8 experiment

[0041] Cells with good growth status, no contamination and cell confluence of more than 90% were taken for CCK-8 experiment to determine the effect of different concentrations of Lan C on the cell survival rate of the above five cell lines. First, the adherent cells were digested and prepared into a cell suspension. The cell concentration was adjusted after counting. Then, according to the corresponding target cell concentration (PC-3 inoculation number was 10,000 cells / well in the 24h group, 8,000 cells / well in the 48h group, and 6,000 cells / well in the 72h group. DU145 inoculation number was 8,000 cells / well in the 24h group, 6,000 cells / well in the 48h group, and 4,000 cells / well in the 72h group. LNCAP inoculation number was 12,000 cells / well in the 24h group, 10,000 cells / well in the 48h group, and 8,000 cells / well in the 72h group. WPMY-1 inoculation number was 6,000 cells / well in the 48h group. HPRF inoculation number was 6,000 cells / well in the 48h group.) 96-well plates were inoculated, 100 μL per well, and a series of doses of Lan were set. C group (25-400nM) and solvent control group (1‰ DMSO), each cell group was set up with 5 replicates. On the second day of culture, each group was given corresponding Lan C or solvent treatment, and after 24h, 48h and 72h of culture, the 96-well plate was taken out, 10μL CCK-8 solution was added to each well, and after incubation for 2-4h, the absorbance value at a wavelength of 450nm was detected with an enzyme marker. Cell survival rate = [(OD value of experimental group - OD value of blank group) / (OD value of solvent group - OD value of blank group)] × 100%.

[0042] 1.4 Colony formation assay

[0043] The cell models of this experiment are two types of prostate cancer cells, PC-3 and DU145 cells. Cells with good growth status, no contamination and cell confluence of more than 90% were selected for cell colony formation experiment to determine the inhibition of Lan C on colony formation of the above two types of prostate cancer cells. After cell digestion, the cell concentration was adjusted to 1×102 -5×10 2 / mL, inoculated into 6-well plates at 2mL / well, and cultured in a CO2 constant temperature incubator for about 24h until the cells adhered to the wall. After the adhesion was completed, Lan C with a final concentration of 50nM, 100nM, and 200nM and the corresponding volume of solvent (1‰ DMSO) were added, and 3 replicates were set up for each group. After 10-12 days of culture, the culture plate was taken out, and 4% paraformaldehyde was added to each well for fixation, and crystal violet staining was used for photography and analysis.

[0044] 1.5 Cell apoptosis experiment

[0045] The cell models of this experiment are two types of prostate cancer cells, PC-3 and DU145 cells. Cells with good growth status, no contamination and cell confluence of more than 90% were selected for cell apoptosis experiment to determine the effect of Lan C on the apoptosis of the above two types of prostate cancer cells. According to the cell digestion steps described above, the adherent cells were made into cell suspension, manually counted and diluted, and the cell concentration was controlled at 20-25×10 4 The next day, Lan C was added at final concentrations of 25nM, 50nM, 100nM, 200nM, and 400nM, respectively. The control group was given an equal volume of solvent (1‰ DMSO). After 48h of culture, the cells were digested with EDTA-free trypsin and then centrifuged at 300g and 4℃ for 5min to collect the cells. After digestion, the cells were washed twice with pre-cooled PBS, each time centrifuged at 300g and 4℃ for 5min. 1-5×10 5 Cells. Aspirate PBS and add 100μL 1×BindingBuffer to resuspend the cells. Add 5μL Annexin V-FITC and 10μL PI Staining Solution and mix gently. Incubate at room temperature for 10-15min in a dark place. Add 400μL 1×Binding Buffer, mix well and place on ice. Samples were detected by flow cytometry within 1 hour.

[0046] 1.6 Nude mouse tumor formation experiment

[0047] 36 male BALB / C nude mice were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., and the experimental animal production license number is SCXK (Zhejiang) 2024-0001. Animal specifications at the time of receipt: body weight range 10-16g, age 28-34 days. The breeding conditions all met the SPF animal breeding standards: 12 hours of light and 12 hours of darkness per day, indoor temperature 20℃-26℃, relative humidity 40%-70%. After 3 days of adaptive breeding, PC-3 cells were resuspended in F12K medium and the cell concentration was adjusted to 1.5×10 7 / mL, 200 μL (3×10 6 cells) were injected subcutaneously into the lower right back. When the tumor grew to 50-150 mm 3 At 14:00, the nude mice were randomly divided into a control group, a Lan C low-dose group (5 mg / kg), a Lan C medium-dose group (10 mg / kg), and a Lan C high-dose group (20 mg / kg) according to the tumor volume, with 9 animals in each group. The animals were given corresponding doses of Lan C and solvent (1‰ DMSO) by gavage at a volume of 10 ml / kg, once a day for four consecutive weeks. The tumor volume was measured twice a week and the weight of the mice was recorded. If the tumor volume of any mouse reached 2000 mm 3 The experiment was terminated and the tumor was removed.

[0048] 1.7 Statistical analysis

[0049] The experimental results were expressed as mean ± standard deviation (mean ± SD), and SPSS26.0 software was used for statistical analysis. When the experimental results met the normality test, one-way analysis of variance was used for inter-group comparison. When the variance was homogeneous, the least significant difference (LSD) method was used for analysis; when the variance was unequal, Dunnet's T3 analysis method was used. P < 0.05 indicated that the difference was statistically significant.

[0050] 2. Experimental results

[0051] 2.1 Effect of Lan C on the morphology of prostate cancer cells

[0052] In vitro, the effects of different doses of Lan C (50nM, 100nM, 200nM, 400nM) on the growth state and cell morphology of prostate cancer PC-3, DU145, and LNCAP cells were observed after 48 hours of action. With the increase of Lan C dose, the cell growth of the three prostate cancer cells was significantly inhibited, the cell outline was blurred, and the cell adhesion ability decreased. When the Lan C concentration reached 200nM, it was observed under the microscope that the growth of the three prostate cancer cells was almost completely inhibited, and the cell morphology was severely damaged. There was a certain difference in the sensitivity of the three prostate cancer cells to Lan C, and the sensitivity was PC-3>DU145>LNCAP.

[0053] (See Figure 1 )

[0054] 2.2 Effects of Lan C on the growth of prostate cancer cells and normal prostate cells

[0055] CCK-8 assay was used to detect the effect of Lan C (25-400 nM) on the survival rate of PC-3, DU145, LNCAP, WPMY-1 and HPRF cells after 24 h, 48 h and 72 h of in vitro administration, so as to evaluate the growth inhibitory effect of Lan C on prostate cancer cells and its effect on the growth of normal prostate cells. Figure 2 The results show the effect of Lan C administration for 24h, 48h and 72h on the survival rate of human prostate cancer cells PC-3 (A), DU145 (B), and LNCAP (C) (compared with the vehicle control group: *P < 0.05, **P < 0.01, ***P < 0.001, N = 5). The results of CCK-8 experiment show that Figure 2 As shown, after 48h and 72h of treatment, all doses of Lan C could significantly inhibit the survival of PC-3 cells (P<0.01); after 48 and 72h of treatment, 100-400nM Lan C could significantly inhibit the survival of DU145 cells (P<0.01); 400nM Lan C could significantly inhibit the survival of LNCAP cells at 24h, 48h and 72h (P<0.001).

[0056] After 24 hours of Lan C treatment, the half-inhibition concentration (IC 50 ) were 208.10nM, 151.30nM and 565.50nM respectively; after 48h of action, the IC 50 were 79.72nM, 96.62nM and 344.80nM respectively; after 72h of action, the IC 50 They are 45.43nM, 96.43nM and 304.60nM respectively (see Table 1).

[0057] Table 1. IC values ​​of Lan C against human prostate cancer cells PC-3, DU145, and LNCAP 50 Value (nM)

[0058]

[0059] Figure 3 The figure shows the inhibitory effect of Lan C administration for 48 hours on the proliferation of human prostate cancer cells PC-3, DU145, LNCAP (A) and human normal prostate stromal immortalized cells WPMY-1 and human prostate fibroblasts HPRF (B). Figure 3As shown, human normal prostate stromal cells WPMY-1 and human prostate fibroblasts HPRF were treated with Lan C for 48 hours. The IC values ​​of Lan C on HPRF cells were 50 The concentration was about 426nM, which was significantly greater than 79.72nM of PC-3 and 96.62nM of DU145, indicating that HPRF cells were less sensitive to Lan C than DU145 and PC-3, two non-hormone-dependent prostate cancer cells. Lan C had no significant effect on the growth of human normal prostate stromal immortalized cells WPMY-1, indicating that Lan C had a certain selectivity for prostate cancer cells and normal prostate cells.

[0060] 2.3 Inhibitory effect of Lan C on colony formation of prostate cancer cells

[0061] The results of colony formation experiments showed that Lan C could significantly inhibit the colony formation of PC-3 and DU145. Compared with the vehicle control group, the colonies formed by the two prostate cancer cells decreased significantly with the increase of Lan C concentration (P < 0.01), indicating that Lan C has a strong anti-proliferation and colony formation inhibitory effect on PC-3 and DU145 (see Figure 4 , Table 2).

[0062] Table 2. Effect of Lan C on cell clones of human prostate cancer cells PC-3 and DU145

[0063]

[0064] Compared with the vehicle control group: **P<0.01, ***P<0.001, N=3.

[0065] 2.4 Effect of Lan C on apoptosis of prostate cancer cells

[0066] The results of flow cytometry showed that compared with the solvent control group, 25-400nM Lan C promoted the increase of PC-3 cell apoptosis rate, among which 200nM and 400nM Lan C groups induced significant apoptosis of PC-3 cells (P<0.01); after DU145 cells were given Lan C for 48h, the apoptosis of cells in the 200nM and 400nM Lan C groups was significant (P<0.001) (see Table 3-4, Figure 5-6 ).

[0067] Table 3. Lan C-induced apoptosis rate of human prostate cancer cell PC-3

[0068]

[0069] Compared with the vehicle control group: **P<0.01, ***P<0.001, N=3.

[0070] Table 4. Lan C-induced apoptosis rate of human prostate cancer cell DU145

[0071]

[0072] Compared with the vehicle control group: **P<0.01, ***P<0.001, N=3.

[0073] 2.5Lan C inhibits subcutaneous tumors in nude mice

[0074] In the nude mouse tumor formation experiment, the weight change data showed that the weight of the nude mice in the control group first increased and then decreased, and the weight of the Lan C dose groups showed a slow downward trend after treatment. This change may be related to the rapid growth of the tumor in the control group, while the growth of the tumor in the Lan C group was inhibited. The tumor volume detection results showed that compared with the control group, the tumor growth rate of each Lan C dose group was significantly slowed down after 1 week of administration (W). As the administration time increased, the tumors in the Lan C groups were significantly reduced. Among them, the growth inhibition effect of the tumor in the high-dose Lan C group was the strongest (P < 0.001). The anatomical results showed that the tumors in each Lan C dose group were significantly smaller than those in the control group, and the tumors in the high-dose group were more significantly reduced (P < 0.001). The above results show that 5-20 mg / kg of Lan C can significantly inhibit the growth of prostate tumors and show a dose-effect relationship (see Table 5-Table 7, Figure 7-Figure 8 ).

[0075] Table 5. Effect of Lan C administration for four weeks on the body weight of nude mice (g)

[0076]

[0077] Compared with the control group: *P<0.05, **P<0.01, ***P<0.001, N=9.

[0078] Table 6. Effect of Lan C administration for 4 weeks on tumor volume in nude mice (mm 3 )

[0079]

[0080] Compared with the control group: *P<0.05, **P<0.01, ***P<0.001, N=9.

[0081] Table 7. Effect of Lan C administration on tumor weight in nude mice after 4 weeks

[0082]

[0083] Compared with the control group: *P<0.05, **P<0.01, ***P<0.001, N=9.

Claims

1. Application of lanatoside C in the preparation of drugs for treating prostate cancer.

2. Use of a pharmaceutical composition containing lanatoside C in the preparation of a drug for treating prostate cancer.

3. The use according to claim 2, wherein: The pharmaceutical composition containing lanatoside C comprises: lanatoside C; and optionally one or more pharmaceutically acceptable carriers.

4. The use according to claim 3, wherein: The pharmaceutically acceptable carrier includes fillers, diluents, solvents, sweeteners, surfactants, odorants, flavors, and lubricants.

5. The use according to claim 3, wherein: The pharmaceutical composition is prepared into tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

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