Application of Polycarpaurine C in the preparation of drugs for treating ovarian cancer

Abstract

The present invention provides an application of a marine natural product in the preparation of a drug for treating cancer, and for the first time clarifies the anti-tumor effect of the marine natural product Polycarpaurine C. The marine natural product achieves anti-tumor activity by regulating cell autophagy, can significantly inhibit the growth of ovarian cancer cells in vitro and in vivo, and has important value for medical development.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to the application of a marine natural product in preparing a drug for treating ovarian cancer. Background Art

[0002] Ovarian cancer is one of the deadliest cancers of the female reproductive system, and its diagnosis and treatment face many challenges. Ovarian cancer ranks high among female cancer mortality rates worldwide. Due to the lack of obvious early symptoms and effective screening methods, most ovarian cancer patients are already in the late stage when diagnosed, which significantly affects treatment outcomes and survival rates. The five-year survival rate of patients with early-stage ovarian cancer is relatively high, but for patients with advanced ovarian cancer, this rate drops significantly.

[0003] The early symptoms of ovarian cancer are often very general and non-specific, such as abdominal distension, frequent urination, abdominal pain, indigestion, etc. These symptoms are often ignored or misdiagnosed as more common non-fatal diseases. Currently, the diagnosis of ovarian cancer mainly relies on imaging examinations (such as ultrasound, CT and MRI scans) and tumor markers (such as CA-125), but these methods have limited sensitivity and specificity for early ovarian cancer.

[0004] Treatment of ovarian cancer usually includes surgery and chemotherapy. For most patients with ovarian cancer, surgery is the preferred treatment method, which includes removing as much of the tumor as possible (called "cytoreduction") and evaluating the extent of the spread of the disease. After surgery, most patients need to receive chemotherapy to eliminate residual cancer cells. Chemotherapy usually uses platinum drugs and paclitaxel. For specific genetic backgrounds (such as BRCA mutation carriers), PARP inhibitors can be used for treatment. In addition, immune checkpoint inhibitors have also shown therapeutic potential in some trials in recent years.

[0005] Autophagy is an intrinsic cleanup mechanism of cells, by which they degrade and recycle old or damaged organelles, protein aggregates, and other cellular components within them. This process is essential for maintaining the health and function of cells, and also affects cell growth, development, and death. Autophagy plays a complex role in the development and treatment of cancer, and it can either inhibit or promote tumor growth, depending on the type of cancer and the stage of development. In some cases, autophagy can prevent the initial formation of tumors by removing damaged organelles and proteins and limiting inflammatory responses. Researchers are exploring the use of autophagy inhibitors or promoters as cancer treatment strategies. For example, certain chemotherapy drugs can induce autophagic death in cancer cells, while autophagy inhibitors can block cancer cells from using autophagy to evade drug-induced death.

[0006] Marine natural products have become an important source for the discovery and development of anti-tumor drugs due to their unique chemical structures and strong biological activities. The marine environment is an extremely biodiverse ecosystem, in which organisms such as sponges, mollusks, seaweed, corals and microorganisms produce a series of compounds with potential medicinal value. Marine natural products provide new compounds and mechanisms of action in the development of anti-tumor drugs, providing new strategies to combat complex cancer mechanisms. With further research in marine biology and medicinal chemistry, it is expected that more marine natural products with potential anti-cancer activity will be discovered.

[0007] Polycarpaurine C is a Polycarpa aurata A sulfur-containing alkaloid isolated from sphaerocephala (a marine invertebrate). Like other alkaloids, it has potential antibacterial and antiviral properties, but its antitumor activity has been little studied. Summary of the invention

[0008] In order to better develop marine resources and expand the application scope of marine natural products, the present invention attempts for the first time to use sulfur-containing organisms from marine sources.

[0009] To this end, the present invention adopts the following technical solutions:

[0010] In one aspect, the present invention relates to use of Polycarpaurine C in preparing a drug for treating ovarian cancer.

[0011] Furthermore, the Polycarpaurine C treats ovarian cancer by inducing autophagy in ovarian cancer cells.

[0012] Furthermore, the Polycarpaurine C treats ovarian cancer by regulating the LC3-I / LC3-II protein levels of ovarian cancer cells.

[0013] Furthermore, the Polycarpaurine C regulates the conversion of LC3-I protein of ovarian cancer cells to LC3-II protein, thereby inducing autophagy of ovarian cancer cells, thereby treating ovarian cancer.

[0014] In another aspect, the present invention relates to use of Polycarpaurine C in preparing a cell autophagy inducer.

[0015] In another aspect, the present invention relates to a method for inducing ovarian cancer cell autophagy in vitro, characterized in that ovarian cancer cells are contacted with an effective concentration of a cell autophagy inducer in vitro, and the cell autophagy inducer is Polycarpaurine C.

[0016] Furthermore, the effective concentration is 2.5~5 μM.

[0017] Furthermore, the effective concentration is 5 μM.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The present invention provides an application of a marine natural product in the preparation of a drug for treating cancer, and for the first time clarifies the anti-tumor effect of the marine natural product Polycarpaurine C. The marine natural product achieves anti-tumor activity by regulating cell autophagy, can significantly inhibit the growth of ovarian cancer cells in vitro and in vivo, and has important value for medical development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Polycarpaurine C structure

[0021] Figure 2 . Effects of different concentrations of PPC on cell activity

[0022] Figure 3 Effect of CQ on PPC cytotoxicity

[0023] Figure 4 LC3I / LC3II protein expression

[0024] Figure 5 . Effect of PPC on the growth volume changes of ovarian cancer cell xenografts in nude mice

[0025] Figure 6 Effect of PPC on tumor weight of ovarian cancer cell xenografts in nude mice DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or can be obtained by existing known methods.

[0027] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0028] Example 1 In vitro anti-tumor activity test

[0029] 1. Experimental samples

[0030] Reference (Guo P, Wang Z, Li G, et al. First discovery of polycarpine, polycarpaurines A and C, and their derivatives as novel antiviral and antiphytopathogenic fungus agents [J]. Journal of agricultural and food chemistry, 2016, 64 (21): 4264-4272.) Neutralization synthesis method was used to prepare Polycarpaurine C (PCC) compound, the structure of which is shown in FIG. Figure 1 As shown, CAS: 921987-39-5.

[0031] PCC was dissolved in DMSO and prepared as a stock solution for storage. It was diluted with PBS to a working solution of appropriate concentration when used.

[0032] 2. Experimental Methods

[0033] MTT method:

[0034] Human normal liver cells LO2, human ovarian cancer cells A2780, and human ovarian cancer cells OVCAR-8 were cultured in RPMI-1640 medium (containing 10% FBS, 1% PS) at 5% CO2 and 37°C.

[0035] The PCC stock solution was diluted with PBS to obtain working solutions with gradient concentrations.

[0036] Collect cells (LO2, A2780, OVCAR-8) in logarithmic phase growth, adjust the concentration of cell suspension, inoculate the cell suspension in 96-well plates, and plate the cells to be tested to a density of about 10,000 / well. 100 μL of cell suspension was added to each well. Incubate at 5% CO2 and 37°C for 24 hours. Under a microscope, cells can be observed to grow by adhering to the wall. Add 10 μL of PCC to the culture plate, where the final concentrations of PCC are 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM, respectively. Finally, add PBS as a blank control. Place the culture plate in a 5% CO2, 37°C constant temperature incubator for culture. After 24 hours, remove the culture plate, add 10 μl of MTT (5 mg / mL) solution to each well, continue to culture for 4 hours, terminate the culture, and carefully aspirate the culture solution in the well. Then, 100 μl of DMSO was added to each well, and the well was placed on a shaker and shaken at a low speed for 10 min to fully dissolve the purple crystals. The absorbance of each well was measured at 490 nm using an automatic microplate reader.

[0037] 3. Experimental results

[0038] The experimental results are as follows Figure 2 shown.

[0039] The results showed that PCC showed inhibitory activity at different concentrations against A2780 and OVCAR-8 human ovarian cancer cells, and had little toxicity against normal human liver cells LO2.

[0040] Example 2 Study on the anti-tumor mechanism of Polycarpaurine C

[0041] Autophagy is an intracellular degradation process that can occur under a variety of stress conditions, such as organelle damage, the presence of abnormal proteins, and nutrient deprivation. In cancer cells, autophagy can suppress tumorigenesis by inhibiting cancer cell survival and inducing cell death, but it can also promote tumorigenesis by promoting cancer cell proliferation and tumor growth. Therefore, autophagy plays a role in both tumor initiation and suppression, making autophagy a complex but promising area for cancer therapy. We further explored whether Polycarpaurine C produces antitumor activity by inducing cellular autophagy.

[0042] MTT method:

[0043] Human ovarian cancer cells A2780 and OVCAR-8 were cultured in RPMI-1640 medium (containing 10% FBS, 1% PS) at 5% CO2 and 37°C.

[0044] The PCC stock solution was diluted with PBS to obtain working solutions with gradient concentrations.

[0045] Collect cells (A2780, OVCAR-8) growing in the logarithmic phase, adjust the concentration of the cell suspension, inoculate the cell suspension in a 96-well plate, and plate the cells to be tested to a density of about 10,000 / well. 100 μL of cell suspension was added to each well. Incubate at 5% CO2 and 37°C for 24 hours. Under a microscope, cells can be observed to grow by adhering to the wall. Add 10 μL of the autophagy inhibitor chloroquine (CQ) to the culture plate for pretreatment for 1 hour. After pretreatment, discard the inhibitor-containing culture medium, replace with a new culture medium, and add 10 μL of Polycarpaurine C, where the final concentration of Polycarpaurine C is 5 μM. Finally, add PBS as a blank control. Place the culture plate in a 5% CO2, 37°C constant temperature incubator for culture. After 24 hours, remove the culture plate, add 10 μl of MTT (5 mg / mL) solution to each well, continue to culture for 4 hours, terminate the culture, and carefully remove the culture medium in the well. Then, 100 μl of DMSO was added to each well, and the well was placed on a shaker and shaken at a low speed for 10 min to fully dissolve the purple crystals. The absorbance of each well was measured at 490 nm using an automatic microplate reader.

[0046] The experimental results are as follows Figure 3 shown.

[0047] Depend on Figure 3 It can be seen that PPC can inhibit the proliferation of human ovarian cancer cells, and the autophagy inhibitor CQ can significantly reverse the death of ovarian cancer cells caused by PPC, which proves that PPC inhibits the proliferation of ovarian cancer cells by inducing autophagy in ovarian cancer cells.

[0048] Furthermore, when cells undergo autophagy, LC3 (Microtubule-associated proteins 1A / 1B light chain 3) protein undergoes an important transformation from LC3-I to LC3-II. This transformation is a key marker of the autophagy process.

[0049] Western blotting analysis

[0050] After A2780 cells were treated with CQ and different concentrations of PPC, proteins were extracted from the cells using RIPA buffer (Thermo Scientific, Waltham, MA, USA) containing 1% PMSF and 1% protease and phosphatase inhibitor cocktail. After incubation on ice for 20 min, the supernatant was collected by centrifugation at 15,000 rpm for 20 min at 4°C. Then, the protein concentration was quantified using Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). 15 μg of protein was separated by SDS-PAGE and transferred to Immun-Blot® PVDF membrane (Bio-Rad, Hercules, CA, USA). Then, the membrane was blocked with 5% nonfat milk in TBST for 60 min at room temperature. The membrane was probed with primary antibody (1:1000) at 4 °C overnight. The membrane was washed 3 times with TBST for 15 min each time and incubated with secondary antibody (1:5000) for 60 min at room temperature. Afterwards, protein expression was exposed using ECL detection reagent (GE Healthcare, Buckinghamshire, UK), and images were captured using the ChemiDoc™ MP imaging system and Image Lab 5.1 software (Bio-Rad, Hercules, CA, USA).

[0051] The experimental results are as follows Figure 4 shown.

[0052] Depend on Figure 4It can be seen that after PPC treatment of ovarian cancer cells, the LC3-I protein level in cancer cells decreased, the LC3-II protein level increased, and LC3-I was transformed into LC3-II. At the same time, pretreatment with the autophagy inhibitor CQ can inhibit the transformation of LC3-I to LC3-II. It can be seen that Polycarpaurine C induces autophagy in ovarian cancer cells by regulating the transformation of LC3-I protein to LC3-II protein in ovarian cancer cells, thereby inhibiting the proliferation of ovarian cancer.

[0053] Example 3 In vivo anti-tumor activity test

[0054] Test animals: BALB / c (nu / nu) nude mice, weighing 16-18g, female, bred in the experimental animal room of our unit, raised in an SPF-grade environment, with the indoor temperature controlled at 23±2℃, free access to food and water. A total of 20 animals. Adaptive feeding for 7 days before inoculation.

[0055] Drugs and reagents: PPC was dissolved in DMSO and diluted with Tween80 and 5% glucose injection in turn. The groups and dosing schedules are as follows. Model control group: 0.1 mL 5% glucose injection was injected into the tail vein once a week for 3 consecutive weeks. PPC-1 group (5 mg / kg): 0.1 mL PPC (5 mg / kg) was injected into the tail vein once a week for 4 consecutive weeks. PPC-2 group (10 mg / kg): 0.1 mL PPC (10 mg / kg) was injected into the tail vein once a week for 3 consecutive weeks.

[0056] Experimental method: A2780 cells in the logarithmic growth phase were taken and a cell suspension concentration of 5×10 7 The A2780 xenograft model was established by inoculating 0.2 mL / mouse in the right armpit of nude mice. The diameter of the transplanted tumor was measured with a vernier caliper. When the tumor grew to 100 mm 3 The animals were then randomly divided into groups. The anti-tumor effect of the test substance was dynamically observed using the method of measuring tumor diameter. The tumor diameter was measured once every 3 days, and the weight of the mice was checked every 3 days. After 3 weeks, all mice were killed by CO2, and the laparotomy was performed for observation. The tumor was removed, and irrelevant tissues were carefully removed. The mice were washed 2 to 3 times with PBS solution to wash away the blood, drain the water for storage, weigh the weight, and measure the volume. The calculation formula for tumor volume (TV) is: TV = 1 / 2 × a × b 2 , where a is the longest length of the tumor and b is the shortest length of the tumor.

[0057] The relative tumor volume (RTV) was calculated based on the measurement results. The calculation formula is: RTV = V t / V0, where V0 is the tumor volume measured at the time of cage administration (i.e., d0), V t The tumor volume at each measurement. Evaluation index of antitumor activity: relative tumor proliferation rate T / C (%), calculated as follows:

[0058]

[0059] Among them, T RTV : Treatment group R TV ; C RTV : Model group RTV. T RTV / C RTV

[0060] Evaluation index of anti-tumor activity: tumor growth inhibition rate (%), calculated by the following formula:

[0061] Tumor growth inhibition rate = (average tumor weight of model group - average tumor weight of drug-treated group) / average tumor weight of model group × 100%

[0062] The results of the inhibitory effect of PPC on the growth of ovarian cancer cell A2780 xenograft tumors in nude mice are shown in Table 1. Figure 5 and Figure 6 .

[0063] Table 1. Inhibitory effect of PPC on the growth of ovarian cancer cell A2780 xenografts in nude mice

[0064]

[0065] The experimental results show that PPC can inhibit the proliferation of ovarian cancer cells in vivo.

[0066] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. Use of Polycarpaurine C in the preparation of a drug for treating ovarian cancer, wherein: The drug specifically kills ovarian cancer cells.

2. The use according to claim 1, wherein The effective concentration of the Polycarpaurine C in the drug is 5 μM.

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