Application of dehydroregelma alkaloid in ccne1 amplified ovarian cancer

Abstract

The present application relates to the application of harmine in the preparation of a drug for treating CCNE1 amplified ovarian cancer. The cell line of the CCNE1 amplified ovarian cancer is selected from the SKOV3 cell line, the HEY cell line, the A2780 cell line, the OVCAR8 cell line or the OVCAR3 cell line. The present application finds that harmine has significant specific toxicity to CCNE1 amplified ovarian cancer cells. Cell experiments find that the IC50 value of harmine in CCNE1 high expression ovarian cancer cells is significantly lower than that in CCNE1 low expression ovarian cancer cells. Therefore, Haimine can specifically kill CCNE1 amplified ovarian cancer cells, and is expected to become a precise treatment drug for CCNE1 amplified ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the application of dehydrocamelin in the preparation of drugs for treating CCNE1 amplified ovarian cancer. Background Technology

[0002] Introduction to CCNE1-Amplified Ovarian Cancer and Current Status of Drug Development: Ovarian cancer is the leading cause of death among gynecological malignancies. [1] The latest data shows that there are 19,710 new cases and 13,270 deaths worldwide each year. [2] Currently, the preferred treatment for ovarian cancer is surgery combined with platinum-based chemotherapy. [3] However, the overall 5-year survival rate is only 30-50%. [4] .

[0003] CCNE1 gene amplification has been reported in various malignant tumors. [5] The incidence rate in ovarian cancer is as high as approximately 20%. [6-8] The CCNE1 gene encodes cyclin E1, which is responsible for regulating the transition of the cell cycle from G1 phase to S phase, thereby enabling the cell to enter the DNA synthesis stage (i.e., S phase). [9] Under normal circumstances, cyclin E1 protein levels are tightly regulated.

[10] CCNE1 amplification led to an abnormal increase in cyclin E1 protein levels. [5] This leads to the abolition of the G1-S cell cycle checkpoint and an acceleration of the cell cycle process.

[11] This leads to uncontrolled tumor cell division. Therefore, CCNE1-amplified ovarian cancers are often highly malignant. Meanwhile, there are reports that CCNE1 amplification is associated with resistance to standard platinum-taxane chemotherapy. [8,12] And it is associated with resistance to PARP inhibitors.

[13] These factors make CCNE1 amplified ovarian cancer a recognized refractory type of ovarian cancer. Currently, there is still a lack of effective targeted therapies, and no drugs for precision treatment of CCNE1 amplified ovarian cancer have been developed.

[0004] Currently, a few specific drug molecules targeting CCNE1-amplified ovarian cancer have entered clinical trials, but the results have been less than ideal. To date, only one Phase II clinical trial has been completed: the WEE1 inhibitor adavasertib (AZD1775) as monotherapy for CCNE1-amplified advanced and recurrent malignant solid tumors, including ovarian cancer (NCT03253679). Although the results were effective, the sample size was small, and some patients required additional antiemetic treatment at the drug dosage specified in the study protocol.

[14] Currently, another Phase II clinical trial of AZD1775 monotherapy for CCNE1-overexpressing high-grade serous ovarian cancer is underway (ACTRN12619001185156). Simultaneously, two Phase I clinical trials of CDK2 inhibitors are in progress: one involving BLU-222 monotherapy and combination therapy (NCT05252416), and the other involving INX-315 monotherapy (NCT05735080). However, considering the significant off-target effects of CDK2 inhibitors, their clinical prospects are not optimistic.

[15] In addition, the PKMYT1 inhibitor RP-6306 has recently been used as monotherapy and in combination with the ATR inhibitor RP-3500 to treat solid tumors with CCNE1 amplification.

[16] It has also entered Phase I clinical trials (EUCTR2021-001637-39-DK). In summary, CCNE1 amplified ovarian cancer is a current hot topic in drug development, but an ideal solution has not yet been found.

[0005] Current Status of Harmine as a Drug Molecule: Based on current research on CCNE1-amplified ovarian cancer, inhibiting homologous recombination repair of DNA damage or arresting the G2-M phase transition of the cell cycle may constitute specific cytotoxicity against CCNE1-amplified ovarian cancer cells. Therefore, to find effective drug treatments for CCNE1-amplified ovarian cancer, novel drug molecules can be sought based on the above mechanisms, namely, identifying effective molecules that selectively kill CCNE1-amplified ovarian cancer cells from existing homologous recombination repair inhibitors and G2 / M blockers. Studies have reported that harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) is a drug that inhibits homologous recombination repair... [17,18] With G2 / M cell cycle arrest

[18]

[19] These functional natural product molecules have the potential to become precision drugs for the specific treatment of CCNE1-amplified ovarian cancer.

[0006] Harmine is a natural alkaloid with broad-spectrum anti-inflammatory and antitumor activities. It was originally isolated from the seeds of the medicinal plant *Hymenochaema heliotropium*, which grows in arid regions of the Middle East and China and has long been widely used in folk medicine for its anti-inflammatory properties.

[20] In addition, harmine is also a broad-spectrum antitumor agent, and its antitumor effects have been reported in various malignant tumors, including ovarian cancer. [21-25] .

[0007] Currently, harmine and its derivatives are used as broad-spectrum antitumor agents to inhibit various tumors, including ovarian cancer. Several patents have been granted abroad for applications such as: harmine in combination with isovanillin (US2017042867-A1); harmine in combination with isovanillin and curcumin (US2020147061-A1, US10751330-B2); harmine derivative dihydroharbaline in combination with isovanillin (US2019231756-A1, US10471049-B2); harmine and harmaline derivative combination (US2022033417-A1); and harmine or its salts in combination with cordycepin (WO2022123432-A1). All of these have been granted broad-spectrum antitumor patents. There are patents using harmine as an inhibitor of NF-κB in cancer cells such as ovarian cancer (WO2020097324-A1, US2021393597-A1). In my country, several domestic patents have been approved for preparing various harmine derivatives to enhance their broad-spectrum anti-tumor activity (CN114133390B, CN116514858A, CN115887376A, CN102977096B, CN101429198B, CN101139347B, CN101328175). However, although harmine can be used as a broad-spectrum anti-tumor agent, its efficacy is limited, and no drug has yet been developed, nor have any harmine-related drug molecules entered clinical trials as anti-tumor drugs. Furthermore, there is no research on the differences in the effects of harmine on different types of tumors, or whether it has the potential to become a specific drug for a particular type of tumor.

[0008] Although no drugs with harmine as the active ingredient have yet been launched, and no clinical trials of harmine for anti-tumor purposes have been conducted, harmine has entered the clinical trial stage as a pharmaceutical molecule. Currently, a Phase I clinical trial (NCT05526430) is underway to determine the maximum tolerated dose of harmine in healthy individuals. Because harmine has hallucinogenic effects when used in combination with N,N-dimethyltryptamine (DMT), three Phase I clinical trials are currently underway, respectively investigating the drug metabolism and pharmacokinetics of harmine in combination with DMT in healthy individuals (NCT05829603), its dose-dependent effects on mental consciousness (NCT05780216), and its effects on neural networks and prosocial behavior (NCT04716335). These clinical trials will provide first-hand clinical data support for future research and translation of harmine into pharmaceutical products. In fact, a Phase II clinical trial (IRCT20170311033004N3) is underway to investigate the use of camel worm seed alkaloids (containing harmine) as an adjunct therapy for mild to moderate depression. Simultaneously, another Phase II clinical trial (IRCT20201219049760N1) is underway to investigate the use of camel worm seed decoction (containing harmine) for treating head lice in children. These clinical trials demonstrate the feasibility of harmine as a drug molecule for human application. However, there are currently no reports on the application of dehydrocamel worm alkaloids in CCNE1 amplified ovarian cancer. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of the prior art by providing the application of dehydrocamelin in the preparation of drugs for treating CCNE1 amplified ovarian cancer.

[0010] This invention provides the application of dehydrocamelin in the preparation of drugs for treating CCNE1 amplified ovarian cancer.

[0011] As a preferred example, the CCNE1 amplified ovarian cancer cell line is selected from SKOV3 cell line, HEY cell line, OVCAR8 cell line, A2780 cell line or OVCAR3 cell line.

[0012] The advantages of this invention are as follows: Research has shown that Harmine exhibits significant specific toxicity against CCNE1-amplified ovarian cancer cells. Cellular experiments revealed that the IC50 value of Harmine in CCNE1-overexpressing ovarian cancer cells was significantly lower than that in CCNE1-underexpressing ovarian cancer cells. Therefore, Harmine can specifically kill CCNE1-amplified ovarian cancer cells and holds promise as a precision medicine for CCNE1-amplified ovarian cancer. Attached image description:

[0013] Figure 1 Ovarian cancer cell lines with high CCNE1 expression are more sensitive to harmine.

[0014] Figure 2 Overexpression of CCNE1 enhances the sensitivity of SKOV3 to harmine. Detailed Implementation

[0015] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0016] Example 1

[0017] 2.1 Experimental Materials

[0018] 2.1.1 Cell lines

[0019] Human normal ovarian epithelial cells IOSE80, human ovarian cancer cells A2780, SKOV, OVCAR8, OVCAR3, HEY, and human renal cell carcinoma ovarian epithelial cells HEK-293T were all purchased from the American Type Culture Collection (ATCC).

[0020] 2.1.2 Plasmids

[0021] The overexpression empty vector plasmid Plvx-puro, the knockdown empty vector plasmid Plenti-puro, and the packaging plasmids psPAX2 and pMD2G were donated by Mao Zhiyong's laboratory.

[0022] 2.1.3 Main Experimental Instruments

[0023]

[0024] 2.1.4 Main Reagents

[0025]

[0026]

[0027] 2.1.5 Primer Sequence

[0028]

[0029] 2.2 Experimental Methods

[0030] 2.2.1 Cell Culture

[0031] HEK-293T was cultured in DMEM medium containing 10% fetal bovine serum (heat-inactivated) and 1% penicillin-streptomycin; IOSE80, A2780, SKOV3, OVCAR8, and HEY were cultured in RPMI 1640 medium containing 10% fetal bovine serum (heat-inactivated) and 1% penicillin-streptomycin; OVCAR3 was cultured in RPMI 1640 medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin. All cell lines were cultured in a sterile incubator (37°C, 5% CO2). Cell passage, culture medium changes, and other operations were performed in a sterile environment strictly following aseptic principles.

[0032] Cell resuscitation: Remove cells from the liquid nitrogen container and thaw in a 37°C water bath. Centrifuge at 1200 rpm for 3 min, resuspend cells, and transfer to cell culture dishes containing complete culture medium. Continue culturing in a 5% CO2, 37°C cell culture incubator.

[0033] Cell cryopreservation: Remove cells from the incubator, digest cells with 0.25% trypsin, blow off the cells, and centrifuge at 1200 rpm / min for 3 min. Discard the supernatant and add cell cryopreservation solution. Transfer to labeled cryovials. Incubate overnight at -80°C, and transfer to liquid nitrogen the next day.

[0034] Cell passage: Collect cells in the logarithmic growth phase with high confluence, and digest them with trypsin until the cells become rounded. Discard the trypsin and add 2 mL of complete culture medium to stop digestion. Centrifuge at 1200 rpm for 3 min. Discard the supernatant and resuspend the cells in 1 mL of complete culture medium.

[0035] 2.2.2 Construction of CCNE1 overexpression and knockdown cell lines

[0036] a) PCR amplification of the target gene

[0037] The following PCR reaction system was prepared on ice.

[0038]

[0039] Run the PCR reaction program:

[0040] denaturation at 94℃ for 2 minutes

[0041] ii. Denaturation at 98℃ for 10 seconds

[0042] iii. Annealing at 64℃ for 30 seconds (primer indicator Tm-5)

[0043] iv. Extend at 68℃ for 2 min 30 s (related to the length of the target gene's CDS region, 1 min / kb).

[0044] v.ii.iii.iv. Repeat 35 times.

[0045] vi. Extend at 68℃ for 7 minutes

[0046] Prepare the following enzyme digestion system on ice, add the target empty vectors (Plvx-puro, Plenti-puro) respectively, and incubate at 37 degrees Celsius for 2 hours and 30 minutes.

[0047]

[0048] After agarose gel electrophoresis was used to detect the yield and specificity, the PCR and enzyme digestion products were purified by gel recovery according to the kit instructions. Then, the shRNA and the amplified CCNE1 gene sequence were inserted into the enzyme-digested target empty vectors Plenti-puro and Plvx-puro, respectively.

[0049] b) Transformation and screening of bacteria to obtain plasmids

[0050] Thaw the cloned competent cells on ice (repeated thawing can affect competent cells; it is best to use aliquots for thawing or use immediately after thawing). Add 2 μL of recombinant product to 9 μL of competent cells, gently tap the tube wall to mix (do not shake), and incubate on ice for 30 min. After heat shock in a 42°C water bath for 45 sec, immediately place on ice to cool for 2-3 min. Add LB medium (without antibiotics) and incubate at 37°C for 1 h (200-250 rpm). Transfer the culture medium from step 4 to the center of a preheated agar plate and gently spread it evenly with a sterile spreader. Incubate upside down at 37°C for 12-16 h. Prepare LB medium containing ampicillin resistance (e.g., 40 μL ampicillin + 40 ml LB medium), and add 2 ml to a 15 ml centrifuge tube. Pick a single colony from each sample using a 10 μL pipette tip, add 2 ml of LB medium containing ampicillin, tilt the tube, and incubate overnight at 37°C with a shaker at 225 rpm. Extract plasmids according to the kit instructions.

[0051] c) Lentiviral packaging

[0052] HEK-293T cells were revived and transferred to 10cm culture dishes. When the cell density was observed to be approximately 60-70%, virus packaging was prepared. pMD2G, psPAX2, and the target plasmid were mixed at a DNA content ratio of 1:2:3, resulting in a total DNA content of 16μg. 48µl of PPEI was added to 1.2ml of empty culture medium, mixed thoroughly, and incubated for 20 minutes to form a transfection complex. The complex was then added dropwise to the culture dish, and the dish was gently shaken to distribute it evenly. After culturing under standard conditions for 6 hours, the medium was replaced with fresh complete culture medium. After 72 hours, the virus solution was collected using a 0.45μm sterile filter.

[0053] d) Lentiviral infection and drug resistance screening

[0054] Take an appropriate amount of virus solution and add it to a cell culture dish in the logarithmic growth phase. Replace with ordinary culture medium after 12 hours. 48 hours after infection, select cells with 1 μg / ml puromycin (concentration based on previous experience) for 48-72 hours and then collect the cells.

[0055] 2.2.3 Identification of CCNE1 expression level

[0056] a) qPCR method

[0057] Cellular RNA extraction: Cells in the culture dish were digested with trypsin. After digestion, the cells were centrifuged to lyse the cell mass. The supernatant was discarded, and the cells were washed with PBS. 1 ml of Trizol was added for every 5–106 cells, and the cells were placed on ice for 10 min to lyse them. 2. 200 μL of chloroform (trizol:chloroform = 5:1) was added, vortexed for 15 s, and incubated on ice for 5 min. Centrifuged at 12000 rpm (4℃) for 15 min. The upper aqueous phase was transferred to a new EP tube, and the volume was measured. An equal volume (500 μL) of isopropanol was added, and the tube was inverted to mix thoroughly. The tube was incubated at -20℃ for 15 min to precipitate. Centrifuged at 12000 rpm (4℃) for 15 min. The supernatant was discarded, and the cells were washed with 75% ethanol. The centrifuge tube was gently vortexed, incubated at room temperature for 3–5 min, and then centrifuged at 12000 rpm (4℃) for 5 min. Discard the supernatant, cap and let it air dry until the flocculent precipitate on the EP tube wall is no longer visible or becomes transparent; dissolve the RNA precipitate in DEPC water (15-30ul) and measure the RNA concentration using an instrument.

[0058] Reverse transcription and qPCR reaction: Reverse transcription was performed according to the ABScript Neo RT Master Mix for qPCR with gDNARemove (ABclonal) instructions to convert RNA into cDNA. The steps are as follows:

[0059]

[0060] The reaction procedure is as follows:

[0061]

[0062] Perform real-time quantitative PCR (qRT-PCR) according to the Genious 2XSYBRGreen FastqPCR Mix (ABclonal) instructions. The steps are as follows:

[0063]

[0064] The reaction procedure is as follows:

[0065]

[0066] The relative expression levels of genes in each treatment group were calculated using the 2^-ΔΔCt method.

[0067] b) Western blotting method

[0068] Cell protein extraction: Cells in culture dishes were digested with trypsin. After digestion was terminated, the cells were centrifuged to precipitate the cell mass. The supernatant was discarded, and the cells were washed with PBS. After discarding the supernatant again, an appropriate amount of RIPA protein lysis buffer and protease inhibitor were added. The cells were thoroughly lysed on ice for 30 minutes. The mixture was then centrifuged at 12,000 rpm for 15 minutes in a refrigerated centrifuge, and the supernatant was collected as the tissue protein solution. 5x loading buffer was added to the solution, and the mixture was thoroughly mixed with the protein. The solution was then boiled in a 100°C metal bath for long-term storage.

[0069] Gel electrophoresis: 12% SDS-PAGE gel electrophoresis, stacking gel electrophoresis at 90V for 30 min, and separating gel electrophoresis at 120V for 60 min. Transfer the membrane at 300mA in ice water for 1 second, followed by blocking with 5% skim milk for 1 h. After blocking, wash the membrane three times with TBST, 5 min each time. Add CCNE1 antibody (1:1000) and GAPDH antibody (1:5000) respectively, and incubate overnight at 4℃. Wash three times with TBST buffer, add secondary antibody diluted 1:5000, incubate on a shaker at room temperature for 1 h, wash with TBST, add developer, expose, and photograph. Process the hybridization band images using ImageJ software.

[0070] CCK8 Method: Cells were digested, dissociation was terminated using complete culture medium, centrifuged, resuspended, and counted. Cells were seeded at a density of 2000 cells / well in 96-well plates. The next day, the original culture medium was aspirated and replaced with medium containing the target concentration of harmine, and the cells were cultured in an incubator. After 24h, 48h, and 72h, the culture medium was discarded, and 90μL of RPMI 1640 medium and 10M ICCK8 working solution were added. After incubation for 2h, the optical density (OD) was measured at 450nm using a multi-mode microplate reader, and the inhibition rate was calculated. Six replicates were set for each intervention concentration, and the IC50 was calculated using Graphpad Prism 8.0.1 software.

[0071] Clonalization method: Digest cells, terminate digestion with complete culture medium, centrifuge, resuspend, and count. Seed cells at a density of 1000 cells / well in 6-well plates. The next day, remove the original culture medium and replace it with medium containing the target concentration of harmine, and continue culturing in an incubator. After 2 weeks of culture, discard the culture medium, wash twice with PBS, and fix with 4% paraformaldehyde fixative for 30 min. Gently wash three times with PBS, stain with 0.1% crystal violet for 30 min, then slowly wash away the staining solution with running water and air dry.

[0072] 2.2.4 Results Statistics

[0073] Statistical analysis was performed using GraphPad Prism 8.0.1 software. All experimental procedures were performed in triplicate. For the CCK8 experiment, each biological replicate required five replicates. All data were analyzed using paired t-tests and one-way ANOVA. The IC50 value was calculated using nonlinear regression analysis. p < 0.05 was indicated by *; p < 0.01 was indicated by **; and p < 0.001 was indicated by ***.

[0074] 2.3 Experimental Results

[0075] Previous studies have reported that harmine can inhibit homologous recombination repair of DNA damage. [17,18] This leads to G2-M phase arrest. Given the dual effects of harmine on cell cycle and DNA damage repair, we hypothesized that harmine might have a stronger killing effect on ovarian cancer cells with CCNE1 amplification. To verify this hypothesis, this study selected several ovarian cancer cell lines, determined their CCNE1 expression levels, and used the CCK8 assay to determine the inhibitory effect of harmine on them. The OVCAR3 cell line, with the highest CCNE1 expression level, had the lowest IC50 value for harmine, followed by the OVCAR8 cell line. The HEY and SKOV3 cell lines, with even lower CCNE1 expression levels, had significantly higher IC50 values ​​compared to the former two. These results indicate that ovarian cancer cell lines with higher CCNE1 expression levels are more sensitive to harmine. See [link to study]. Figure 1 (Ovarian cancer cell lines with high CCNE1 expression are more sensitive to harmine).

[0076] Next, this study established a stable CCNE1-overexpressing SKOV3 cell line and compared the killing effect of harmine at different concentrations and time points on CCNE1-overexpressing cell lines. The results showed that CCNE1-overexpressing cells were more sensitive to harmine compared to wild-type cells. These experimental results demonstrate that harmine possesses specific killing activity against CCNE1-overexpressing ovarian cancer cells and has the potential to become a precision medicine for CCNE1-amplified ovarian cancer. See [link to study]. Figure 2 (Overexpression of CCNE1 enhances the sensitivity of SKOV3 to harmine).

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[0106] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Application of dehydrocamelin in the preparation of drugs for treating CCNE1 amplified ovarian cancer.

2. The application according to claim 1, characterized in that, The cell lines for CCNE1 amplified ovarian cancer were selected from SKOV3, HEY, A2780, OVCAR8, or OVCAR3 cell lines.

Images