A pharmaceutical composition containing huwentoxin-IV and its application

Abstract

The present invention relates to the field of biomedicine, and particularly to a pharmaceutical composition containing huwentoxin-IV and its application. The present invention provides a pharmaceutical composition containing huwentoxin-IV, and the active pharmaceutical ingredients are docetaxel and huwentoxin-IV. The test results confirm that: compared with the group using drugs alone, the combined application of docetaxel and huwentoxin-IV can significantly inhibit the proliferation, invasion and migration of prostate cancer PC3 cells. Its potential molecular mechanism is that docetaxel enhances the inhibitory effect of huwentoxin-IV on the activity of sodium channel Nav1.7 in prostate cancer PC3 cells, and further significantly inhibits the expression of RhoA protein in the cells and blocks the activity of RhoA / Rock2 signaling pathway. In the present invention, the combined use of drugs can significantly reduce the dosage of both, and the anti-tumor effect is significantly enhanced, which is beneficial to reducing the sensitivity and drug resistance of prostate cancer cells to docetaxel, thereby reducing the toxic and side effects caused by high doses to patients, and providing a new way for the treatment of metastatic prostate cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to the application of a pharmaceutical composition containing huwentoxin-IV (HWTX-IV) in the preparation of an anti-prostate cancer drug. Background Art

[0002] Prostate cancer (PCa) is one of the common malignant tumors in adults and the second leading cause of cancer death in men. It is reported that approximately 1.6 million men are diagnosed with prostate cancer each year, and about 366,000 of them die from prostate cancer. Tumor metastasis is the main cause for patients to die from prostate cancer, especially bone metastasis. In the past few decades, surgery, radiotherapy, chemotherapy or medical androgen deprivation therapy has been the main treatment modality for prostate cancer patients. However, when using a single drug for treatment, although it can partially kill tumor cells, it will also damage normal cells in the human body at the same time, and can also cause side effects such as gastrointestinal dysfunction and neuropathy. Moreover, medical androgen deprivation therapy often fails due to the development of metastatic castration-resistant prostate cancer. Therefore, it is of great clinical significance to seek a targeted drug combination that is safe, effective and has less side effects for prostate cancer bone metastasis.

[0003] Domestic and foreign studies have found that taxane drugs play a good role in the treatment of hormone-independent prostate cancer. Especially docetaxel, a semi-synthetic taxol derivative, is the only effective chemotherapy drug that has been proven to prolong the survival period of hormone-independent prostate cancer patients so far and has been approved by the FDA for clinical use. Its mechanism of action is mainly reflected in binding to tubulin in tumor cells, promoting the aggregation of microtubules in cells while inhibiting the depolymerization of microtubules, thereby affecting cell mitosis, making the formation of the spindle in the G 2 / M phase hindered, and the ultimate outcome is to cause cell apoptosis. When docetaxel is used alone, drug resistance often occurs. Although using a high dose of the drug has a certain anti-tumor effect, the side effects produced are obvious. Therefore, it is of great significance to seek a combined drug use mode for docetaxel.

[0004] At present, more and more studies have shown that voltage-gated sodium channels (Navs) are involved in the tumor process. According to the different subunits that make up the sodium channel, sodium channels are divided into 9 subtypes, named Nav1.1 - Nav1.9. It has been reported that the sodium channel subtype Nav1.7 is highly expressed in the highly metastatic rat prostate cancer cell line Mat-lylu, while it is hardly expressed in the low-metastatic rat prostate cancer cell line AT-2. Later, some researchers confirmed that Nav1.7 is highly expressed in the highly metastatic human prostate cancer cell line PC3, while its expression level is very low in the low-metastatic human prostate cancer cell lines DU-145 and LNCap. Therefore, the sodium channel Nav1.7 is expected to become a key potential target for the treatment of prostate cancer metastasis.

[0005] Huwentoxin-IV (HWTX-IV) is a 35-amino acid polypeptide toxin isolated from the venom of the bird spider Selenocosmia huwena, which can inhibit the activity of the sodium channel Nav1.7 by inhibiting its peak current. However, there has been no research report on the role of Huwentoxin-IV (HWTX-IV) in prostate cancer. At present, the combination of drugs for tumors is a research hotspot in anti-tumor treatment. It is of great significance to explore the anti-cancer effect and molecular mechanism of the drug combination of docetaxel and Huwentoxin-IV (HWTX-IV) in human prostate cancer cell lines.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] Aiming at the above deficiencies, the present invention provides a drug composition containing Huwentoxin-IV and its application in the preparation of anti-prostate cancer drugs. The active ingredients of the drug composition include docetaxel and Huwentoxin-IV. Through experimental research, the present invention first found that compared with the use of docetaxel or Huwentoxin-IV (HWTX-IV) alone, the combination of docetaxel and Huwentoxin-IV (HWTX-IV) can significantly inhibit the invasion and metastasis of human prostate cancer cell PC3. In addition, the use of Huwentoxin-IV (HWTX-IV) can reduce the dosage of docetaxel, thereby reducing the sensitivity and drug resistance of prostate cancer cell PC3 to docetaxel. The technical problem solved is to provide a new option for the treatment of metastatic prostate cancer.

[0008] In order to achieve the above invention purpose, the technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides a drug composition containing Huwentoxin-IV (HWTX-IV) and its application in the preparation of anti-prostate cancer drugs.

[0010] Specifically, the main active ingredients of the pharmaceutical composition include docetaxel and huwentoxin-IV (HWTX-IV).

[0011] Specifically, the prostate cancer is bone metastatic prostate cancer.

[0012] Specifically, the molar ratio of docetaxel to huwentoxin-IV (HWTX-IV) is 1-10 μM: 5-20 μM; preferably 2 μM: 5 μM.

[0013] In certain specific embodiments, docetaxel combined with huwentoxin-IV (HWTX-IV) can significantly inhibit the proliferation, invasion and metastasis of prostate cancer cell line PC3.

[0014] On the other hand, the present invention provides that docetaxel combined with huwentoxin-IV (HWTX-IV) has a synergistic effect in regulating the activity of sodium channel Nav1.7 and regulating RhoA / Rock signaling pathway.

[0015] Specifically, the regulation of the activity of sodium channel Nav1.7 is to significantly inhibit the activation of Nav1.7 by affecting the peak current of Nav1.7.

[0016] Preferably, the regulation of RhoA / Rock signaling pathway is to regulate the expression of genes RhoA, p-Rock2, p-LIMK and p-Cofilin.

[0017] Specifically, the regulation of the expression of genes RhoA, p-Rock2, p-LIMK and p-Cofilin is to inhibit the expression of RhoA, p-Rock2, p-LIMK and p-Cofilin.

[0018] The present invention has the following beneficial effects:

[0019] The present invention verifies that docetaxel combined with huwentoxin-IV (HWTX-IV) significantly inhibits the proliferation, invasion and metastasis of prostate cancer cell line PC3 by inhibiting the activation of sodium channel Nav1.7 in PC3 cells, thereby down-regulating the expression of RhoA and blocking the downstream Rock / LIMK / Cofilin phosphorylation cascade reaction, and the two drugs have a synergistic effect. The present invention reveals the antitumor effect of the in vitro combination of a small molecule chemical drug and spider toxin on prostate cancer cell line PC3, provides the possibility of developing docetaxel and huwentoxin-IV (HWTX-IV) into a targeted combination drug for the treatment of prostate cancer, and at the same time provides a new way for the treatment of metastatic prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the result graph of Example 1. Docetaxel combined with huwentoxin-IV (HWTX-IV) significantly inhibits the proliferation of prostate cancer PC3 cells.

[0021] Figure 2 This is the result graph of Example 2. Docetaxel combined with huwentoxin-IV (HWTX-IV) can inhibit the invasion and migration of prostate cancer PC3 cells.

[0022] Figure 3 This is the result graph of Example 3. Docetaxel enhances the inhibitory effect of huwentoxin-IV (HWTX-IV) on the activity of sodium channel Nav1.7 in prostate cancer PC3 cells.

[0023] Figure 4 This is the result graph of Example 4. Docetaxel combined with huwentoxin-IV (HWTX-IV) significantly inhibits the expression of RhoA and the RhoA / ROCK2 signaling pathway in prostate cancer PC3 cells. Detailed implementation methods

[0024] Test materials, instruments and equipment, reagents

[0025] Docetaxel (purchased from Aventis Pharma), huwentoxin-IV (HWTX-IV) (isolated and purified from the venom of Selenocosmia huwena, kindly provided by the College of Life Sciences, Hunan Normal University), human prostate cancer PC3 cell line (purchased from the American Type Culture Collection (ATCC)).

[0026] Fluorescence inverted microscope (Olympus), patch clamp (HEKA), carbon dioxide incubator (Thermo), sterile operating table (Haier), pipette (Eppendorf), inverted optical microscope (COIC), ultrasonic cell disruptor (Ningbo Xinzhi Biology), high-speed centrifuge (Eppendorf), ultracentrifuge (Beckman), microplate reader (Bio-Tek), vertical slab two-dimensional electrophoresis system (Bio-Rad), protein gel imaging system Image ScannerⅢ (GE), water bath (Shanghai Yichang Instrument Factory), constant temperature circulator (Beijing De Tianyou Technology Development Co., Ltd.), ultrapure water system (MILLIPORE), ultra-low temperature refrigerator (Zhongke Meiling), electrophoresis tank (Bio-Rad), power supply (Bio-Rad), PoverPac tmHV high voltage electrophoresis power supply (Bio-Rad), chemiluminescence imager (Bio-Rad), ion acidity meter (METTLER TOLEDO), mini Trans-Blot transfer tank (Bio-Rad), high pressure sterilizer (ZEALWAY), ice maker (GRANT), horizontal shaker (Beijing Liuyi Instrument Factory), electronic balance (METTLER TOLEDO), vortex shaker (Qilin Bell Instrument Manufacturing Co., Ltd.).

[0027] 0.25% Trypsin, fetal bovine serum, glutamine, penicillin and streptomycin (Life), RPMI 1640 culture medium, RhoA, p-Rock2, p-LIMK and p-Cofilin antibodies (purchased from Cell Signaling Technology, USA), CCK-8 cell activity detection kit, BCA protein concentration test kit.

[0028] All the following experiments were repeated three times or more. The data were expressed as mean ± standard deviation and compared with SPSS 18.0 statistical software. The t-test was used to compare the differences between the groups. P value < 0.05 indicated a significant statistical difference.

[0029] Example 1: Docetaxel combined with Huwentoxin-IV (HWTX-IV) significantly inhibited the proliferation of prostate cancer PC3 cells.

[0030] The CCK-8 method was used to detect the effects of different concentrations of docetaxel and Huwentoxin-IV (HWTX-IV) alone and in combination on PC3 cell proliferation and cytotoxicity. The specific operation is as follows: single drug group: 100 μL cell suspension (about 6000-8000 cells) was inoculated into a 96-well plate and placed in an incubator for pre-culture for 24-72 hours to allow the cells to adhere to the wall. Then, 10 μL of 0, 1, 2, 5, 10 μM docetaxel and 10 μL of 0, 5, 10, 15, 20 μM Huwen toxin-IV (HWTX-IV) solution were added to the culture plate, and 10 μL of CCK-8 reagent was added to each well after continued culture for 24 hours. After incubation for 2 hours, the absorbance value (A) at a wavelength of 450 nm was detected with an ELISA instrument to calculate the cell proliferation inhibition rate, and the cell proliferation inhibition rate (R) = [1-(A control group-A experimental group) / (A control group-A blank group)]*100%. 3-5 parallel wells were set for each group. Combination medication group: According to the experimental results of the single medication group, multiple combination medication schemes were set, and the concentration of docetaxel remained unchanged, which was the docetaxel concentration corresponding to the cell inhibition rate of about 30%, that is, 2 μM, and the concentration of Huwen toxin-IV (HWTX-IV) was 5, 10, 15, 20 μM, respectively, and the remaining experimental steps were the same as the single medication group.

[0031] The results showed that docetaxel and huwentoxin-IV (HWTX-IV) at different concentrations could inhibit the proliferation of PC3 cells in a concentration-dependent manner, and the inhibition rate of the combined drug group was significantly higher than that of each drug used alone. Among them, the IC 50 value of docetaxel alone was about 5 μM. When PC3 cells were treated with 2 μM docetaxel, the proliferation inhibition rate was about 30% (Table 1). The IC 50 value of huwentoxin-IV (HWTX-IV) alone was about 10 μM (Table 2). In the combined drug group, when the concentrations of docetaxel and huwentoxin-IV (HWTX-IV) were 2 μM and 5 μM respectively, the inhibition rate of PC3 cell proliferation could reach about 50% (Table 3). Subsequently, in order to further confirm the effect of docetaxel combined with huwentoxin-IV (HWTX-IV) on the proliferation ability of PC3 cells, the CCK-8 method was used to detect the proliferation ability of PC3 cells after 2 μM docetaxel combined with 5 μM huwentoxin-IV (HWTX-IV) acted on PC3 cells for 0, 1, 2, 3, 4, and 5 days. The results further confirmed that docetaxel combined with huwentoxin-IV (HWTX-IV) could significantly inhibit the proliferation of PC3 cells in a time-dependent manner. Compared with the control group, the difference in the drug treatment group was statistically significant (P < 0.001) (Table 4 Figure 1 ).

[0032] Table 1 Proliferation inhibition rate of docetaxel alone on PC3 cells after 24 h (%)

[0033]

[0034] Table 2 Proliferation inhibition rate of huwentoxin-IV (HWTX-IV) alone on PC3 cells after 24 h (%)

[0035]

[0036] Table 3 Proliferation inhibition rate of docetaxel combined with huwentoxin-IV (HWTX-IV) on PC3 cells after 24 h (%)

[0037]

[0038] Table 4 Proliferation inhibition rate of 2 μM docetaxel combined with 5 μM huwentoxin-IV (HWTX-IV) on PC3 cells (%)

[0039]

[0040] Example 2: Docetaxel combined with huwentoxin-IV (HWTX-IV) can inhibit the invasion and migration of prostate cancer PC3 cells.

[0041] The wound-healing assay and Transwell invasion assay were respectively used to detect the effects of docetaxel combined with huwentoxin-IV (HWTX-IV) on the migration and invasion abilities of PC3 cells. The specific operations were as follows: For the wound-healing assay, the cell suspension was inoculated into a 24-well plate. After the cells grew confluently, a pipette tip was used to draw a line along a sterile ruler (the pipette tip should be perpendicular). Subsequently, the cells were washed twice with PBS to remove the scratched cells. Then, the medium containing 1% fetal bovine serum was used as the negative control, and the medium containing 1% fetal bovine serum with 2 μM docetaxel combined with 5, 10, and 20 μM huwentoxin-IV (HWTX-IV) respectively was used as the combined drug stimulation group. Each group was repeated 3 - 5 wells. The 24-well plate was gently shaken to fully mix the drug with the cell culture medium, and then placed in a 37 °C, 5% CO 2 incubator for incubation. Samples were taken at 0 h, 12 h, and 24 h, photographed, and the relative migration rate was calculated. For the Transwell invasion assay, first, PC3 cells were treated with 2 μM docetaxel combined with 5, 10, and 20 μM huwentoxin-IV (HWTX-IV) solutions for 24 h. Then, the PC3 cells were digested with 0.25% trypsin solution for 2 - 3 min. After terminating the digestion, the cells were centrifuged, and the cell pellet was resuspended with fresh medium, and the cell density was adjusted to approximately 1 x 10 6 cells / mL. Subsequently, 100 μL of the cell suspension was added to the upper chamber of the Transwell. The basal membrane on the upper chamber surface of the Transwell needed to be pre-coated with a Matrigel matrix glue diluent (1:5) in advance. 800 μL of cell culture medium was added to the lower chamber of the Transwell, and the culture was continued for 24 h. After 24 h, the Transwell chamber was carefully taken out and fixed with 4% paraformaldehyde for 30 min. After slowly washing with PBS, the cells on the upper chamber surface were removed with a wet cotton swab, and then stained with 0.1% crystal violet solution for 20 min. After air-drying, 5 high-power fields were randomly selected under an inverted microscope for photographing and counting, and the average value was calculated.

[0042] The results showed that docetaxel combined with huwentoxin-IV (HWTX-IV) could significantly inhibit the invasion and migration of PC3 cells. Compared with the control group, the difference in the drug treatment group was statistically significant (P < 0.01)( Figure 2 A). Among them, treating PC3 cells with 2 μM docetaxel combined with 5 μM huwentoxin-IV (HWTX-IV) for 24 h could significantly inhibit the cell migration rate by 46% (P < 0.01)( Figure 2 B). Preferably, 2 μM docetaxel combined with 5 μM huwentoxin-IV (HWTX-IV) was selected to carry out the subsequent experiments.

[0043] Example 3: Docetaxel enhances the inhibitory effect of huwentoxin-IV (HWTX-IV) on the activity of sodium channel Nav1.7 in prostate cancer PC3 cells.

[0044] The patch clamp technique was used to detect the effects of huwentoxin-IV (HWTX-IV) alone and docetaxel combined with huwentoxin-IV (HWTX-IV) on the electrophysiology of PC3 cells. The specific operations are as follows: The glass electrode was pulled in two steps, with the tip diameter of 1.5 - 3.0 μm and the initial resistance controlled at 1.5 - 2.5 MΩ. After forming a high-impedance (GΩ) seal between the electrode and the cell membrane, the fast capacitance was compensated. The cell was clamped at -60 mV, and the cell membrane clamped in the electrode was broken under the action of an instantaneous strong negative pressure. Then the slow capacitance was compensated, and the cell was clamped at -80 mV. After the cell was stable, the current was recorded, and the system series resistance compensation was between 30% - 60%. The extracellular solution (in mM) for measuring the sodium current of PC3 cells: NaCl 140, CaCl 2 1, KCl 3, MgCl 2 1, HEPES 10, pH 7.3; the intracellular solution (in mM): CsF 140, HEPES 10, EGTA 1, NaCl 10, pH 7.3. Both the intracellular and extracellular solutions need to be filtered through a 0.22 μm filter membrane.

[0045] The results showed that when the cell clamping voltage was -80 mV, after treating PC3 cells with 5 μM huwentoxin-IV (HWTX-IV), the activity of Nav1.7 could be inhibited, and the peak current intensity was reduced by about 55% ( Figure 3 A). After treating PC3 cells with 2 μM docetaxel combined with 5 μM huwentoxin-IV (HWTX-IV), the activity of Nav1.7 could be inhibited, and the peak current intensity was reduced by about 85% ( Figure 3 B), and there was a statistically significant difference between the two (P < 0.05).

[0046] Example 4: Docetaxel combined with huwentoxin-IV (HWTX-IV) significantly inhibits the expression of RhoA and the RhoA / ROCK2 signaling pathway in prostate cancer PC3 cells.

[0047] The effects of docetaxel combined with huwentoxin-IV (HWTX-IV) on RhoA and RhoA / ROCK2 signaling pathways in PC3 cells were detected by Western Blotting. The specific operations were as follows: The PC3 cell suspension was inoculated into a 100-mm culture dish. After the cells adhered to the wall, a drug combination of 2 μM docetaxel and 5 μM huwentoxin-IV (HWTX-IV) was added for stimulation treatment for 24 h. Subsequently, total proteins in PC3 cells of each group were extracted with a mixed lysate of RIPA and PMSF, and protein quantification was performed using a BCA protein concentration assay kit. Then, equal amounts of proteins from each group were subjected to 10% SDS-PAGE gel electrophoresis. After electrophoresis, PVDF membrane transfer was carried out for 2 h. The transferred PVDF membrane was blocked with 5% BSA at room temperature for 1-2 h and then primary antibody was added, followed by incubation overnight at 4 °C. It was washed 3 times with 1×TBST, 3 min for each time. Then, secondary antibody was added and incubated at room temperature for 1-2 h, and it was washed 3 times with 1×TBST, 7 min for each time. Finally, developing solution was added and developed in a developing instrument. In this experiment, the group without adding any drug was used as the negative control, and the groups separately adding 2 μM docetaxel and 5 μM huwentoxin-IV (HWTX-IV) were used as the positive controls.

[0048] The results showed that both docetaxel and huwentoxin-IV (HWTX-IV) could inhibit the expression of RhoA, p-Rock2, p-LIMK and p-Cofilin in PC3 cells. At the same time, compared with the negative control group and the single-drug treatment groups, after PC3 cells were treated with 2 μM docetaxel combined with 5 μM huwentoxin-IV (HWTX-IV), the expression levels of RhoA protein and the downstream phosphorylated proteins p-Rock2, p-LIMK and p-Cofilin mediated by RhoA in the cells were significantly down-regulated ( Figure 4 ), indicating that docetaxel combined with huwentoxin-IV (HWTX-IV) could weaken the metastasis of prostate cancer PC3 cells by inhibiting the RhoA / ROCK2 signaling pathway.

[0049] The above embodiments are only preferred embodiments of the present invention, aiming to explain the principles and functions of the present invention, rather than limiting the present invention. Those skilled in the art shall not modify, replace or improve the technical solutions of the present invention without violating the spirit and principles of the present invention. Therefore, the protection scope of this invention patent shall cover the appended claims.

Claims

1. A pharmaceutical composition containing huwentoxin-IV, characterized in that: the active ingredients of the pharmaceutical composition comprise docetaxel and huwentoxin-IV; the molar ratio of docetaxel to huwentoxin-IV is 1-10 μM: 5-20 μM 2. The pharmaceutical composition according to claim 1, wherein, the molar ratio of docetaxel to huwentoxin-IV is 2 μM: 5 μM.

3. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating prostate cancer.

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