Use of nipagin propyl in the preparation of a medicament for the treatment of prostate cancer

Abstract

This invention, entitled "Use of Propylparaben in the Preparation of Drugs for Treating Prostate Cancer," belongs to the field of pharmaceutical technology. The technical problem to be solved is to provide a novel application of propylparaben in the preparation of products for treating prostate cancer. This invention systematically evaluates the biological effects of propylparaben in prostate cancer through cellular level experiments. For the first time, it was discovered that under specific experimental conditions, propylparaben can inhibit the proliferation and migration of androgen-sensitive prostate cancer cells (LNCaP) and promote apoptosis, while not inhibiting the growth of normal prostate cells within a certain concentration range. These results indicate that propylparaben and its structural characteristics can serve as candidate research objects for the development of prostate cancer-related drugs, providing a reference experimental basis for subsequent structural optimization and derivative design.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of propylparaben in the preparation of drugs for treating prostate cancer. Background Technology

[0002] Among the global spectrum of male malignant tumors, prostate cancer caused by abnormal prostate proliferation is one of the most common male malignant tumors worldwide, ranking second in incidence and fifth in mortality. These high incidence and mortality rates seriously threaten patients' lives and health. Currently, the main clinical treatment for localized prostate cancer is radical prostatectomy, but postoperative complications such as urinary incontinence, vas deferens injury, and erectile dysfunction are common, significantly reducing patients' quality of life. For advanced or metastatic cases, although androgen deprivation therapy (ADT) is the first-line treatment, most patients eventually develop refractory castration-resistant prostate cancer (CRPC). Existing chemotherapy drugs have significant side effects and are prone to drug resistance, necessitating the search for new pathogenic mechanisms and therapeutic targets.

[0003] Meanwhile, environmental factors, especially long-term cumulative exposure to exogenous chemicals, are considered to play a significant role in the occurrence and progression of prostate cancer. Propylparaben (PP), a representative member of the paraben preservative class, is widely used in food, pharmaceuticals, and personal care products. Individuals can be continuously exposed to this compound through various routes, including ingestion, inhalation, and skin contact. Its molecular structure consists of a lipid-soluble aromatic ring and a relatively polar ester side chain. This physicochemical characteristic endows it with strong biomembrane permeability, giving it a certain potential for tissue distribution and accumulation in vivo, including in prostate tissue. Epidemiological studies have reported that the detection levels of propylparaben in the body fluids or tissue samples of prostate cancer patients are higher than in non-tumor controls, and there is a correlation between exposure levels and disease progression. These findings suggest that propylparaben may participate in the pathological processes associated with prostate cancer and influence the biological behavior of tumor cells.

[0004] However, although existing studies have revealed the association between propylparaben exposure and prostate cancer risk at the population level, its specific functions and molecular mechanisms of action remain unclear. Furthermore, the existing research framework for prostate cancer drugs and mechanisms includes relatively limited functional studies of this type of environmental chemical, lacking direct evidence regarding its effects on tumor cell proliferation, migration, and invasion under different exposure doses. This gap in understanding, to some extent, restricts a deeper understanding of the pathogenic mechanisms associated with propylparaben and limits the possibility of developing intervention strategies based on its molecular characteristics. Summary of the Invention

[0005] The purpose of this invention is to provide: The use of propylparaben in the preparation of drugs for treating prostate cancer and related technologies, in order to solve technical problems such as new applications of propylparaben in the preparation of products for treating prostate cancer, or combinations thereof.

[0006] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0007] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0008] Definitions of terms related to raw materials of traditional Chinese medicine can be found in the reference "Pharmacopoeia of the People's Republic of China (2020 Edition): China Medical Science and Technology Press: May 2020: 1st Edition".

[0009] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0010] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0011] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0012] The term "propylparaben" used in this article refers to an organic compound with CAS number 94-13-3 and molecular formula C64-13-3. 10 H 12 O3 is mainly used as a preservative in food, cosmetics, and animal feed.

[0013] In a first aspect, the present invention provides the use of propylparaben in the preparation of a medicament for treating prostate cancer.

[0014] In some embodiments, the drug has the effect of inhibiting the proliferation and migration of androgen-sensitive prostate cancer cells LNCaP, and / or promoting apoptosis.

[0015] In some embodiments, the dosage of propylparaben is 15.63-1000 μM.

[0016] In some embodiments, the dosage form of the drug is powder, granules, or capsules.

[0017] In some embodiments, the dosage form of the drug is a powder.

[0018] In some embodiments, the drug may further include pharmaceutically acceptable excipients.

[0019] In some embodiments, the excipients are selected from at least one of fillers, lubricants, binders, flow aids, disintegrants, and solubilizers.

[0020] The present invention has at least the following beneficial effects: This invention systematically evaluated the biological effects of propylparaben in prostate cancer through cellular-level experiments. For the first time, it was discovered that under specific experimental conditions, propylparaben can inhibit the proliferation and migration of androgen-sensitive prostate cancer cells (LNCaP) and promote apoptosis. These results indicate that propylparaben and its structural characteristics can serve as candidate research subjects for the development of prostate cancer-related drugs, providing a reference experimental basis for subsequent structural optimization and derivative design. Attached Figure Description

[0021] Figure 1 Results of the detection of the effects of different types of parabens on LNCaP activity in prostate cancer cells; Figure 2 Results of the detection of the effects of different types of parabens on the viability of prostate RWPE-1 cells; Figure 3 Results of the detection of the effects of different types of parabens on the proliferation of prostate cancer cells LNCaP; Figure 4 Effects of different types of parabens on the cell proliferation of RWPE-1 prostate cells; Figure 5 Representative microscopic images and cell migration results from the cell scratch assay; Figure 6 Flow cytometry image of cells undergoing Annexin V-FITC / PI double staining for apoptosis detection; Figure 7Representative images and results of the number of migrating cells from the Transwell migration assay. Detailed Implementation

[0022] To make the objectives, technical solutions, and beneficial effects of this invention clearer, detailed explanations are provided below through specific embodiments. It should be noted that these embodiments are for illustrative purposes only and do not constitute any limitation on the scope of the invention. Unless otherwise stated, the experimental methods described in the embodiments are conventional techniques in the art, and the materials and reagents used are commercially available.

[0023] The LNCaP cells in the following experimental example were cultured as follows: LNCaP cells were placed in RPMI-1640 medium (Gibco, C22400500CP) containing 10% serum (Gibco, 10091148) and cultured at 37°C with 5% CO2 introduced.

[0024] The following experimental example shows the culture method of RWPE-1 cells: RWPE-1 cells were placed in RWPE-1 special medium (Procell, CM-0200) and cultured at 37°C with 5% CO2 introduced.

[0025] Experimental Example 1: Effects on Cell Viability Cell viability was assessed using a CCK-8 assay (Bioroyee, CK04-500) according to the manufacturer's protocol. LNCaP cells and RWPE-1 cells were cultured at 5 × 10⁶ cells per well. 3 The cells were seeded into 96-well plates. After culturing at 37°C with 5% CO2 for 24 h, the medium was replaced with serum-free 1640 medium containing different concentration gradients of methylparaben, ethylparaben, propylparaben, and butylparaben. After culturing for another 24 h, 10 µL CCK-8 was added, and the absorbance of each well was assessed at 450 nm using a microplate reader (Tecan Infinite M200 microplate reader; Tecan Group, Ltd., Männedorf, Switzerland). The cell viability assay results for LNCaP and RWPE-1 are as follows: Figure 1 , Figure 2 As shown. Data are expressed as mean ± standard deviation (Mean ± SD). Compared with the control group, ns represents no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0026] according to Figure 1 , Figure 2The results showed that methylparaben, ethylparaben, propylparaben, and butylparaben all exhibited cytotoxicity against LNCaP cells at different concentrations. However, since methylparaben and butylparaben also caused cytotoxicity to RWPE-1 cells, subsequent experiments primarily focused on ethylparaben and propylparaben. Propylparaben showed a dose-dependent effect; with increasing propylparaben concentration, the survival rate of LNCaP cells significantly decreased (P < 0.0001 for all concentration groups). No cytotoxicity was observed in RWPE-1 cells within the 15-1000 μM range; and a slight increase in cell activity was even observed at intermediate concentrations (125-500 μM). Therefore, propylparaben selectively inhibits LNCaP prostate cancer cells, exhibits almost no toxicity to normal prostate epithelial cells RWPE-1, and at intermediate concentrations, it can slightly enhance RWPE-1 cell activity, demonstrating its ability to differentiate tumor cells from normal cells.

[0027] Experimental Example 2: Effects on Cell Proliferation The effects of adding methylparaben, ethylparaben, propylparaben, and butylparaben on the proliferation of LNCaP and RWPE-1 cells were investigated.

[0028] 2×10 per hole 3 LNCaP cells were seeded at high density in 96-well plates. After 24 hours of culture, three concentrations of 125, 250, and 500 μM were added, with four replicates per group. A blank control well was also included. Cells were cultured for 3 and 6 days, respectively, according to CY-Quant... ® The OD value was measured according to the instructions of the Invitrogen Cell Proliferation Detection Kit (C7026). Values ​​with an OD value between 0.2 and 1.0 were selected as valid values. LNCaP and RWPE-1 cells were used in the experiment, and the results are as follows: Figure 3 , Figure 4 As shown. Data are expressed as mean ± standard deviation (Mean ± SD). Compared with the control group, ns represents no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0029] according to Figure 3 , 4 The results showed that in LNCaP cells, the fluorescence intensity of the methylparaben and ethylparaben treatment groups (125, 250, and 500 μM) at the 6-day time point was not significantly different from that of the control group, indicating that cell proliferation was not persistently inhibited. Only propylparaben could persistently inhibit LNCaP cell proliferation. In RWPE-1 cells, the fluorescence intensity of the propylparaben treatment groups (125 and 250 μM) was mostly not significantly different from that of the control group (ns), indicating that it did not inhibit RWPE-1 cell growth.

[0030] Therefore, propylparaben has the best anticancer effect among the four drugs, but it does not inhibit the growth of normal prostate cells within a certain concentration.

[0031] Experiment Example 3: Cell Scratch Test To detect cancer cell migration after propylparaben stimulation. LNCaP cells in logarithmic growth phase were digested with trypsin, centrifuged, and collected at a density of 2.5 × 10⁶ cells per well. 5 Cells were evenly seeded into 12-well plates and cultured in a 37°C incubator containing 5% CO2. When the cell density reached 90-100%, scratching was performed. After scratching, the cells were washed with PBS. In the control group, serum-free RMPI-1640 medium containing the drug solvent was added. In the experimental groups, serum-free RMPI-1640 medium containing 125 μM and 250 μM propylparaben (94-13-3) were added, respectively, and the cells were treated at 37°C for 24 h and 48 h. Images of the samples were taken at 0 h, 24 h, and 48 h after scratching. Scale bar = 500 μm. Scratch healing was observed under an inverted microscope at a magnification of ×200. The scratch area at four different locations in each group was measured using ImageJ, and the cell migration rate of each group was calculated. Experimental results are as follows: Figure 5 As shown in the figures, Figure A illustrates the migration of LNCaP cells under different concentrations of propylparaben treatment, and Figure B presents the quantitative statistical analysis of LNCaP cell migration rate. Data are expressed as mean ± standard deviation (Mean ± SD). Compared with the control group, **** P < 0.0001. Figure A shows the migration of LNCaP cells under different concentrations of propylparaben (Control, 125 μM, 250 μM), with the yellow curve outlining the edge of the scratched area to define the region not covered by cells. Figure B represents the quantitative statistical analysis of LNCaP cell migration rate (Migration Rate %). The results indicate that with increasing propylparaben concentration, the wound healing ability of LNCaP cells is significantly inhibited.

[0032] Experiment 4: Flow Cytometry Annexin V / PI double staining method was used to detect the apoptosis rate of cells treated with propylparaben. Cells were centrifuged for 5 min, the supernatant was discarded, cells were collected, washed once with PBS, gently resuspended, and counted; 1-5 × 10⁶ cells were collected. 5Resuspended cells were centrifuged at 300 g for 5 min, and the supernatant was discarded. Cells were washed once with PBS, centrifuged again, and the supernatant was discarded. Cells were resuspended in 500 μL of diluted 1×Annexin V Binding Buffer. 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added to the cell suspension. The mixture was gently vortexed and incubated at room temperature in the dark for 15-20 min. The cells were immediately analyzed after the reaction. LNCaP / RWPE-1 cells were used in the experiment, and the results are as follows: Figure 6 As shown in the figure. The results showed that the proportion of early apoptotic (lower right quadrant) and late apoptotic (upper right quadrant) cells in the LNCaP cell 250μM treatment group was significantly increased, while the proportion of early apoptotic and late apoptotic cells in the RWPE-1 cell 250μM treatment group was basically the same as that in the control group.

[0033] Experiment Example 5: Transwell migration experiment LNCaP cells that have been pre-starved for 12-24 hours were fed at a rate of 8 × 10⁻⁶ cells / year. 4 Cells were resuspended in serum-free 1640 medium containing 125, 250, and 500 μM propylparaben, respectively. 600 μL of 1640 medium containing the same concentration of the drug and 20% FBS was added to the lower chamber. After incubation at 37°C with 5% CO2 for 48 hours, the chamber was removed, and cells were fixed with 4% paraformaldehyde at 4°C for 20 minutes, followed by a wash with PBS. Cells were then stained with 0.1% crystal violet at room temperature for 10 minutes, washed with PBS, and observed under a microscope. Cell images were taken at ×10 magnification, and the number of cells passing through the filter membrane was counted in five fields of view. The number of cells in each field of view was calculated, and the experiment was repeated three times. The results are shown below. Figure 7 As shown.

[0034] Compared with the Control group, the number of migrating cells began to decrease significantly in the 125 μM drug treatment group; the number further decreased in the 250 μM group; and the 500 μM group showed very few residual cells on the filter membrane, resulting in a sparse field of view, demonstrating a significant anti-migration effect. Quantitative counting results showed that the number of migrating cells was significantly lower in the 125 μM, 250 μM, and 500 μM groups compared with the Control group, with extremely significant differences (****P<0.0001). The cell membrane-penetrating ability gradually weakened with increasing drug concentration, suggesting that the drug directly inhibits cell movement and transmembrane migration at the cell morphology level, and the inhibitory effect increases with increasing concentration.

[0035] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. Use of propylparaben in the preparation of drugs for treating prostate cancer.

2. The use according to claim 1, characterized in that, The drug has the effect of inhibiting the proliferation and migration of androgen-sensitive prostate cancer cells LNCaP, and / or promoting apoptosis.

3. The use according to claim 1, characterized in that, The dosage of propylparaben is 15.63-1000 μM.

4. The use according to claim 1, characterized in that, The drug is available in the form of powder, granules, or capsules.

5. The use according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 1, characterized in that, The excipients are selected from at least one of fillers, lubricants, binders, flow aids, disintegrants, and solubilizers.

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