Application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for cancer prevention and treatment

Abstract

This invention discloses the application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for cancer prevention and treatment, belonging to the field of pharmaceutical technology. This invention discovers that the antitumor effect of bufalin is closely related to its upregulation of intracellular ROS. Mechanistic studies indicate that PRDX1 is the target of bufalin. Molecular docking results show that PRDX1 has a "pocket" structure that binds to bufalin; inhibiting PRDX1 weakens the regulation of ROS and the antitumor effect of bufalin. Given that inhibiting PRDX1 can enhance ferroptosis, this invention evaluates the therapeutic effect of the combination of bufalin and ferroptosis inducers. Results show that bufalin effectively promotes the antitumor effect of the ferroptosis inducer RSL3 in liver cancer, lung cancer, and colon cancer cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for the prevention and treatment of cancer. Background Technology

[0002] Liver cancer is the third leading cause of death from malignant cancer worldwide. It is characterized by inconspicuous early symptoms, rapid malignant progression, easy recurrence after surgery, and easy resistance to chemotherapy. Patients have extremely poor prognosis and 5-year survival.

[0003] The imbalance of reactive oxygen species (ROS) generation and clearance plays a crucial role in regulating cell proliferation and survival, and is a fundamental mechanism of tumor drug resistance. Intracellular ROS overload triggers various cell death mechanisms, including apoptosis and ferroptosis. Unlike conventional drug-induced apoptosis, ferroptosis is a cell death pathway characterized by the accumulation of iron ions and lipid peroxidation (LPO), representing an emerging strategy for reversing tumor drug resistance. The mechanism driving ferroptosis involves the inhibition of the antioxidant system. The classic ferroptosis inducer RSL3 directly inhibits glutathione peroxidase 4 (GPX4), leading to ROS accumulation and cell death. Targeting the antioxidant system to induce ferroptosis has attracted considerable attention as a potential strategy for tumor treatment and overcoming drug resistance.

[0004] Peroxidase 1 (PRDX1) is an important member of the peroxidase family, highly expressed in liver cancer cells. The ROS signaling pathway it regulates plays a crucial role in tumor chemotherapy resistance and is considered a potential target for cancer therapy. Recent studies have reported that PRDX1 is closely related to ferroptosis; silencing PRDX1 expression in the human liver cancer cell line HepG2 increases iron and LPO accumulation, promoting ferroptosis. However, currently, no PRDX1 inhibitors have entered clinical trials as anti-tumor agents. Summary of the Invention

[0005] The purpose of this invention is to provide the application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for the prevention and treatment of cancer, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is the application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for the prevention and treatment of cancer.

[0008] The second technical solution of the present invention is a drug for preventing and treating cancer, comprising a PRDX1 inhibitor and a ferroptosis inducer.

[0009] The third technical solution of the present invention is a drug preparation for preventing and treating cancer, comprising the drug and pharmaceutically acceptable excipients.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] This invention reveals that the antitumor effect of bufalin is closely related to its upregulation of intracellular ROS. Mechanistic studies indicate that PRDX1 is the target of bufalin. Molecular docking results show that PRDX1 has a "pocket" structure that binds to bufalin, and inhibiting PRDX1 weakens the regulation of ROS and the antitumor effect of bufalin. Given that inhibiting PRDX1 can enhance ferroptosis, this invention evaluates the therapeutic effects of combining bufalin with ferroptosis inducers. Results show that bufalin effectively promotes the antitumor effect of the ferroptosis inducer RSL3 in liver cancer, lung cancer, and colon cancer cells. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 To inhibit PRDX1-sensitized ferroptosis. A shows that PRDX1 is closely associated with poor prognosis in liver cancer; B shows that PRDX1 knockdown significantly increases ROS levels in cells; C shows that PRDX1 knockdown significantly sensitizes RSL3-mediated upregulation of LPO levels in cells; and D shows that PRDX1 knockdown significantly promotes RSL3-mediated cell death.

[0014] Figure 2 PRDX1 is a target protein of bufotalin. Specifically, A represents the existence of a "pocket" structure in which PRDX1 interacts with the three active components of bufotalin; B represents bufotalin upregulating cellular ROS levels, and this upregulation is reversed upon PRDX1 knockdown; C represents the reversal of bufotalin-mediated cell proliferation inhibition by the reactive oxygen species scavenger NAC, and this inhibition is reversed upon PRDX1 knockdown; and D represents the reversal of bufotalin-mediated cellular LDH release by the reactive oxygen species scavenger NAC, and this promoting effect is reversed upon PRDX1 knockdown.

[0015] Figure 3This study describes the ferroptosis mediated by the ferroptosis inducer RSL3, as sensitized by bufalin. Specifically, A indicates that bufalin promotes RSL3-mediated cell death more effectively than chemotherapeutic drugs; B indicates that bufalin upregulates cellular LPO levels; C indicates that bufalin sensitizes the upregulation of cellular LPO levels mediated by RSL3; D indicates that bufalin sensitizes the inhibitory effect of RSL3 on cell proliferation more effectively than Erastin; E indicates that bufalin promotes the release of cellular LDH more effectively than Erastin; and F indicates that the ferroptosis inhibitor Fer-1 significantly reverses ferroptosis mediated by both bufalin and RSL3. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0022] This invention provides the application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for the prevention and treatment of cancer.

[0023] Huachansu is an approved and widely used traditional Chinese medicine preparation for cancer treatment in China. Clinical studies have shown that Huachansu inhibits the occurrence of primary liver cancer and alleviates patient pain rates. It is often used in combination with sorafenib to treat intermediate and advanced liver cancer. However, the therapeutic targets and mechanisms of Huachansu in sensitizing liver cancer are still unclear. Elucidating the therapeutic targets of Huachansu and its mechanism of sensitizing sorafenib will help in finding new combination strategies and further broaden the scope of Huachansu's combination applications.

[0024] This invention addresses a specific cancer patient population by providing an antitumor use of a composition based on inhibiting PRDX1 activity. Specifically, it relates to the combined use of bufalin in a cancer treatment drug and a ferroptosis inducer, and its antitumor application. In this invention, bufalin can provide a novel strategy for the clinical treatment of liver cancer, lung cancer, and colorectal cancer by inhibiting ferroptosis mediated by the PRDX1-sensitizing ferroptosis inducer RSL3.

[0025] The prevention and treatment described in this invention include both prevention and treatment. The prevention refers to conventional prevention in the art, preferably referring to preventing or reducing tumor development when potential tumor-causing factors are present. The treatment refers to conventional treatment in the art, preferably referring to reducing the severity of the tumor, curing the tumor to normalize it, or slowing the progression of the tumor.

[0026] The cancer prevention and treatment drug is an anti-tumor drug based on inhibiting PRDX1 activity, and the application includes using the bufalin in combination with the ferroptosis inducer RSL3 to treat tumors that highly express PRDX1.

[0027] In some specific implementations, the PRDX1 inhibitor is selected from any one of small molecule compounds, siRNA, or PROTAC molecules.

[0028] In some specific implementations, the small molecule compound is bufotin.

[0029] In some specific implementations, the ferroptosis inducer is selected from RSL3 or Errastin.

[0030] In some specific implementations, the ferroptosis inducer is RSL3.

[0031] In some specific implementations, the cancers include liver cancer, lung cancer, and colorectal cancer.

[0032] This invention also provides a drug for preventing and treating cancer, including a PRDX1 inhibitor and a ferroptosis inducer.

[0033] In some specific embodiments, the PRDX1 inhibitor is selected from any one of small molecule compounds, siRNA, or PROTAC molecules; the ferroptosis inducer is selected from RSL3 or Errastin; and the cancer includes liver cancer, lung cancer, and colorectal cancer.

[0034] In some specific implementations, the small molecule compound is bufotin; the ferroptosis inducer is RSL3.

[0035] This invention also provides a pharmaceutical preparation for preventing and treating cancer, comprising the drug and pharmaceutically acceptable excipients.

[0036] Example 1

[0037] This invention mainly focuses on human hepatocellular carcinoma cells HepG2, lung cancer cells A549, and colon cancer cells DLD-1. The effects of Huachansu and its combination with RSL3 on cell proliferation inhibition were detected by MTT assay and cellular lactate dehydrogenase (LDH) release level. Flow cytometry was used to detect cell apoptosis, ROS and LPO content in cells, etc.

[0038] 1 Experimental Methods

[0039] 1.1 Reactive Oxygen Detection

[0040] HepG2, A549, and DLD-1 cells in logarithmic growth phase were seeded in 6-well plates. The following day, cells were induced with 4 mg / mL HCS for 24 h. After treatment, the old culture medium was discarded, and the cells were washed once with PBS. For detection, ROS probes were loaded. The probes were diluted with serum-free medium at a ratio of 1:1000 with DCFH-DA to a final concentration of 10 μM, and incubated at 37°C in the dark for 30 min. After incubation, the cells were washed twice with serum-free medium to remove any unfilled DCFH-DA. Cells were digested with trypsin and resuspended in 1 mL PBS to prepare a single-cell suspension. Flow cytometry was performed at an excitation wavelength of 488 nm. Data analysis was performed using FlowJo software; higher fluorescence signal intensity values ​​indicate higher ROS levels.

[0041] 1.2 Detection of lipid peroxides

[0042] HepG2, A549, and DLD-1 cells in logarithmic growth phase were seeded into 6-well plates. The following day, cells were induced with HCS (4 mg / mL) or ferroptosis inducer RSL3 (0.5 μM) for 24 h and incubated at 37°C for the corresponding time. After treatment, the old culture medium was discarded, and the cells were washed once with PBS. BODIPY 581 / 591C11 stock solution (10 mM) was diluted 1:1000 with serum-free medium to a working concentration of 10 μM, and incubated at 37°C in the dark for 30 min. The negative control was treated with serum-free medium. After incubation, the cells were washed three times with PBS, digested with trypsin, and resuspended in 500 μL PBS. Flow cytometry was performed at an excitation wavelength of 488 nm. Data analysis was performed using FlowJo software; higher fluorescence signal intensity values ​​indicate higher ROS.

[0043] 1.3 PI staining to detect cell death

[0044] To investigate whether HCS increases the cytotoxic activity of chemotherapy drugs (cisplatin, carboplatin) or ferroptosis inducer (RSL3) on tumor cells, HepG2, A549, and DLD-1 cells in logarithmic growth phase were seeded in 6-well plates. The following day, control groups received RSL3 (0.5 μM), cisplatin (3 μM), carboplatin (10 μM), or HCS (4 mg / mL) as a single drug, while experimental groups received RSL3, cisplatin, or carboplatin combined with HCS. Cells were incubated at 37°C for 24 h. After treatment, the old culture medium was discarded, and the cells were washed once with PBS. Cells were digested with trypsin without EDTA, gently pipetted, and transferred to centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated. Cells were resuspended in pre-chilled PBS, washed, centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated. Five minutes before flow cytometry analysis, 10 μL of propidium iodide (PI) solution was added and gently mixed before data collection. To verify whether cell death induced by the combined treatment of HCS and RSL3 is related to ferroptosis, HepG2 cells were pretreated with the classic ferroptosis inhibitor Ferrostain-1 (2 μM) 4 h before receiving the combined treatment of HCS and RSL3. Data analysis was performed using FlowJo software.

[0045] 1.4MTT

[0046] To investigate whether HCS enhances the inhibitory effect of ferroptosis inducers (RSL3, Erastin) on tumor cell proliferation, HepG2, A549, and DLD-1 cells in logarithmic growth phase were seeded in 96-well plates. The following day, HCS (4 mg / mL), ferroptosis inducer RSL3 (0.5 μM), or Erastin (1 μM) as single-drug treatment served as the control group, while RSL3 or Erastin combined with HCS served as the experimental group. Cells were incubated at 37°C for the corresponding time. 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated at 37°C in the dark for 4 hours. After incubation, the supernatant was carefully discarded, and 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well and mixed thoroughly by pipetting to dissolve any crystals. The cells were then analyzed using an ELISA reader at OD... 490nm and OD 720nm The absorbance and OD of each well were measured. 490nm Subtract OD 720nm The absorbance value at the specified point is the final value. To verify whether the inhibitory activity of HCS on cell proliferation is related to its regulation of ROS, HepG2 cells were pretreated with NAC (50 μM) for 4 hours before HCS administration. Data analysis was performed using GraphPad Prism software.

[0047] 1.5 Detection of cytotoxic lactate dehydrogenase

[0048] To investigate whether HCS increases the cytotoxicity of ferroptosis inducers (RSL3, Erastin) on tumor cells, HepG2, A549, and DLD-1 cells in logarithmic growth phase were seeded in 96-well plates. The following day, control groups received HCS (4 mg / mL), ferroptosis inducer RSL3 (0.5 μM), or Erastin (1 μM) alone; experimental groups received a combination of RSL3 or Erastin and HCS. Induction treatment lasted 24 h, followed by incubation at 37°C for the corresponding time. High-dose control, high-dose blank control, low-dose control, and background blank groups were also established. 10 μL of Lysis Solution was added to each well of the high-control group (containing cells and culture medium) to determine the maximum releaseable LDH from cells. 10 μL of Lysis Solution was added to each well of the high-control blank group (containing culture medium only) to subtract the background absorbance value of the high-control group. The low-control group (containing cells and culture medium) was not lysed and was used to determine the spontaneous LDH release from untreated normal cells. The background blank group (containing culture medium only) was used to subtract the background absorbance values ​​of the low-control and sample wells. Before detection, 10 μL of Lysis Solution was added to the high-control and high-control blank groups, and 10 μL of culture medium was added to the low-control group. The mixtures were incubated at 37°C in the dark for 30 min. After incubation, cell LDH activity could be measured using either a direct or indirect method.

[0049] Direct method: Aspirate 50 μL of culture medium supernatant from each well, and using the remaining culture medium and cells as the test subjects, add 50 μL of Working Solution to each well and vortex to mix. Indirect method: Aspirate 50 μL of supernatant from each well into a new 96-well plate, and then add 50 μL of Working Solution to each well and vortex to mix. Incubate at room temperature in the dark for 30 min. After incubation, add 50 μL of Stop Solution to each well, and immediately measure the OD using an ELISA reader. 490nm The absorbance at a given location was measured. Data analysis was performed using GraphPad Prism software.

[0050] 1.6 Molecular docking

[0051] Molecular docking was performed and binding energy data were obtained using the online molecular docking platform https: / / www.dockeasy.cn / DockCompound. Image processing was performed using PyMOL software.

[0052] 2 Experimental Results

[0053] 2.1 Inhibition of PRDX1-sensitized ferroptosis

[0054] Kaplan-Meier Plotter analysis suggests that PRDX1, a member of the peroxidase family, is closely associated with poor prognosis in liver cancer. Figure 1 (A). Using the DCFH-DAROS probe to label ROS in cells, the results showed that after reducing PRDX1 expression with siRNA, cellular ROS levels significantly increased. Figure 1 B). BODIPY 581 / 591C11 dye labeling of lipid peroxides (LPO) in cells showed that reducing PRDX1 expression not only increased basal LPO levels but also significantly increased LPO accumulation mediated by the ferroptosis inducer RSL3 (0.5 μM, 24 h). Figure 1 (C). PI staining was used to detect cell death. The results showed that knocking down PRDX1 in human hepatocellular carcinoma cell line HepG2, human non-small cell lung cancer cell line A549, and human colon cancer cell line DLD-1 significantly increased RSL3 (0.5 μM, 24 h)-mediated cell death. Figure 1 (D). The above results all confirm that PRDX1 is a potential target for sensitizing ferroptosis.

[0055] 2.2PRDX1 is a target protein of bufotoxin.

[0056] Molecular docking results confirmed that PRDX1 possesses a "pocket" structure that binds to the three active components (Bufalin, Cinobufagin, and Resibufogenin) in HCS, with low binding energies of -9.403, -9.493, and -9.029 kcal / mol, respectively, indicating highly stable binding. Figure 2 (A) Using the DCFH-DAROS probe to label ROS in cells, the results showed that HCS significantly upregulated cellular ROS levels, and the upregulation of cellular ROS levels mediated by HCS (4 mg / mL, 24 h) was reversed after reducing PRDX1 expression with small interfering RNA (siRNA). Figure 2 (B) N-acetylcysteine ​​(NAC) is a thiol-containing antioxidant that promotes free radical scavenging in cells and reduces ROS levels. MTT assay results all indicated that when HepG2 cells were pretreated with NAC (50 μM, 4 h), the inhibitory effect of HCS on cell proliferation was reversed; similarly, reducing PRDX1 expression with siRNA reversed the effect of HCS (4 mg / mL, 24 h). Figure 2 (C). Further evidence from detecting cellular LDH release levels revealed that HCS-mediated cell proliferation inhibition is ROS-dependent, and PRDX1 knockdown reversed the inhibitory effect of HCS (4 mg / mL, 24 h). Figure 2 The results (D) suggest that PRDX1 is a downstream target gene of HCS. In conclusion, PRDX1 is a target protein regulated by HCS.

[0057] 2.3 Ferroplastosis mediated by the ferroptosis-sensitizing agent RSL3 (bufotin)

[0058] Given that knocking down PRDX1 can sensitize ferroptosis, this invention also evaluated the antitumor effect of HCS combined with a ferroptosis inducer. The results showed that compared to the single-drug groups, the combined HCS (4 mg / mL, 24 h) and RSL3 (0.5 μM, 24 h) group significantly increased the PI positivity rate of HepG2 cells, suggesting that the combined treatment increased cell death. Furthermore, the inhibitory effect of the combination of HCS and RSL3 was superior to that of its combination with cisplatin (3 μM, 24 h) or carboplatin (10 μM, 24 h). Figure 3 (A). BODIPY 581 / 591C11 labeled cells with LPO, and the results indicated that HCS (4 mg / mL, 24 h) significantly increased basal levels ( Figure 3 LPO accumulation mediated by B) and RSL3 (0.5 μM, 24 h) Figure 3(C). The proliferation activity of HepG2, A549, and DLD-1 cells was detected using the MTT assay. The results showed that compared to the single-drug groups, HCS (4 mg / mL, 24 h) significantly enhanced the antitumor effects of the ferroptosis inducers RSL3 (0.5 μM, 24 h) or Erastin (1 μM, 24 h), and the antitumor effect of HCS combined with RSL3 was better than that of HCS combined with Erastin. Figure 3 (D). Further examination of cellular LDH release levels revealed that HCS (4 mg / mL, 24 h) significantly increased cell death induced by the ferroptosis inducers RSL3 (0.5 μM, 24 h) or Erastin (1 μM, 24 h); and compared with Erastin, the combined use of HCS and RSL3 showed better inhibitory effects on tumor growth. Figure 3 HepG2 cells were pretreated with the classic ferroptosis inhibitor Ferrosen-1 (2 μM, 4 h). Apoptosis assays showed that Ferrosen-1 could reverse cell death mediated by the combination of HCS (4 mg / mL, 24 h) and RSL3 (0.5 μM, 24 h). Figure 3 The results from the study (F) further suggest that HCS primarily works by promoting ferroptosis and sensitizing RSL3.

[0059] In summary, this invention reveals that the antitumor effect of bufalin is closely related to its upregulation of intracellular ROS. Mechanistic studies indicate that PRDX1 is the target of bufalin. Molecular docking results show that PRDX1 has a "pocket" structure that binds to bufalin, and inhibiting PRDX1 weakens the regulation of ROS and the antitumor effect of bufalin. Given that inhibiting PRDX1 can enhance ferroptosis, this invention evaluates the therapeutic effect of combining bufalin with a ferroptosis inducer. Results show that bufalin effectively promotes the antitumor effect of the ferroptosis inducer RSL3 in liver cancer, lung cancer, and colon cancer cells. Therefore, the sensitization of sorafenib by bufalin in clinical treatment is related to its activated ferroptosis. In view of this, this invention provides a combined application of bufalin with a ferroptosis inducer in tumor treatment.

[0060] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for the prevention and treatment of cancer, characterized in that, The PRDX1 inhibitor is a small molecule compound; the small molecule compound is bufalin. The ferroptosis inducer is RSL3; The cancers mentioned include liver cancer, non-small cell lung cancer, and colon cancer.

2. A drug for preventing and treating cancer, characterized in that, Including PRDX1 inhibitors and ferroptosis inducers; The PRDX1 inhibitor is a small molecule compound; the cancers include liver cancer, non-small cell lung cancer, and colon cancer; The small molecule compound is bufotin; the ferroptosis inducer is RSL3.

3. A pharmaceutical preparation for the prevention and treatment of cancer, characterized in that, Includes the drug as described in claim 2 and pharmaceutically acceptable excipients.

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