Application of PRDX1 inhibitor and ferroptosis inducer in preparation of medicines for preventing and treating cancers

Abstract

The invention discloses application of a PRDX1 inhibitor and a ferroptosis inducer in preparation of drugs for preventing and treating cancers, and belongs to the technical field of medicines. It is found that the anti-tumor effect of cinobufagin is closely related to ROS in up-regulated cells of cinobufagin. Mechanism research results show that PRDX1 is an action target of cinobufagin. Molecular docking results show that the PRDX1 has a pocket structure which is mutually combined with cinobufagin and inhibits the PRDX1 from weakening the regulation of the cinobufagin on ROS (reactive oxygen species) and the anti-tumor effect of the cinobufagin. In view that inhibition of PRDX1 can sensitize ferroptosis, the invention evaluates the combined use effect of cinobufagin and ferroptosis inducer in therapeutics. Results show that cinobufagin can effectively promote the anti-tumor effect of the ferroptosis inducer RSL3 on liver cancer, lung cancer and colon cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of a PRDX1 inhibitor and a ferroptosis inducer in preparing a drug for preventing and treating cancer. Background Art

[0002] Liver cancer is the third most deadly malignant cancer in the world. It has the characteristics of unclear early symptoms, rapid malignant progression, easy recurrence after surgery, and easy resistance to chemotherapy. The prognosis and 5-year survival rate of patients are extremely poor.

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

[0004] Peroxidase 1 (PRDX1) is an important member of the peroxidase family and is highly expressed in liver cancer cells. The ROS signaling pathway regulated by PRDX1 plays an important role in tumor chemotherapy resistance and is considered a potential target for tumor treatment. 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 and promotes ferroptosis. However, no PRDX1 inhibitor has entered clinical research as an anti-tumor agent. Summary of the invention

[0005] The purpose of the present invention is to provide the use of a PRDX1 inhibitor and a ferroptosis inducer in the preparation of a drug for preventing and treating cancer, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the use of a PRDX1 inhibitor and a ferroptosis inducer in the preparation of a drug for preventing and treating 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 pharmaceutical 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] The present invention found that the anti-tumor effect of cinobacillus is closely related to its upregulation of intracellular ROS. The results of mechanism studies showed that PRDX1 is the target of cinobacillus. Molecular docking results showed that PRDX1 has a "pocket" structure that binds to cinobacillus, and inhibiting PRDX1 weakens cinobacillus' regulation of ROS and its anti-tumor effect. Given that inhibiting PRDX1 can sensitize ferroptosis, in therapeutics, the present invention evaluated the combined effect of cinobacillus and ferroptosis inducers. The results showed that cinobacillus can effectively promote the anti-tumor effect of ferroptosis inducer RSL3 on liver cancer, lung cancer and colon cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 To inhibit PRDX1 and sensitize cell ferroptosis. Among them, A is that PRDX1 is closely related to the poor prognosis of liver cancer, B is that knocking down PRDX1 significantly increases ROS in cells, C is that knocking down PRDX1 significantly sensitizes RSL3-mediated upregulation of cell LPO levels, and D is that knocking down PRDX1 significantly promotes RSL3-mediated cell death.

[0014] Figure 2 PRDX1 is the target protein of cinobacillus. A is the "pocket" structure of PRDX1 that interacts with the three active ingredients in cinobacillus, B is that cinobacillus upregulates cellular ROS levels, and the upregulation is reversed after knocking down PRDX1, C is that the reactive oxygen scavenger NAC reverses cinobacillus-mediated cell proliferation inhibition, and the inhibition is reversed after knocking down PRDX1, and D is that the reactive oxygen scavenger NAC reverses cinobacillus-mediated cell LDH release, and the promotion is reversed after knocking down PRDX1.

[0015] Figure 3The results show that Cinobufacini sensitizes the ferroptosis-inducing agent RSL3-mediated ferroptosis in cells. A shows that compared with chemotherapy drugs, Cinobufacini can promote RSL3-mediated cell death, B shows that Cinobufacini can upregulate the level of LPO in cells, C shows that Cinobufacini can sensitize the upregulation of LPO in cells mediated by RSL3, D shows that compared with Erastin, Cinobufacini can sensitize the inhibitory effect of RSL3 on cell proliferation, E shows that compared with Erastin, Cinobufacini can promote the release of LDH in cells mediated by RSL3, and F shows that the ferroptosis inhibitor Fer-1 can significantly reverse the ferroptosis of cells mediated by Cinobufacini and RSL3. DETAILED DESCRIPTION

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

[0017] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0018] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0019] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0021] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0022] The embodiments of the present invention provide the use of a PRDX1 inhibitor and a ferroptosis inducer in the preparation of a drug for preventing and treating cancer.

[0023] Cinobufacini is an anticancer Chinese medicine approved for marketing and widely used in China. Clinical studies have shown that Cinobufacini inhibits the occurrence of primary liver cancer and relieves the pain rate of patients. It is often used in combination with sorafenib to treat advanced liver cancer, but the target and mechanism of Cinobufacini in enhancing liver cancer treatment are still unclear. Elucidating the therapeutic targets of Cinobufacini and its mechanism of sensitizing sorafenib will help find new combination strategies and further broaden the scope of Cinobufacini's combination.

[0024] The present invention provides an anti-tumor use of a composition based on inhibiting PRDX1 activity for a specific tumor patient group, and specifically relates to a combination of cinobucoidin and a ferroptosis inducer in a tumor treatment drug and its anti-tumor use. In the present invention, cinobucoidin can enhance ferroptosis mediated by RSL3, a ferroptosis inducer, by inhibiting PRDX1, providing a new strategy for the clinical treatment of liver cancer, lung cancer and colorectal cancer.

[0025] The prevention and treatment of the present invention includes prevention and treatment. The prevention is conventional prevention in the art, preferably refers to preventing or reducing the occurrence of tumors after use when possible tumor factors exist. The treatment is conventional treatment in the art, preferably refers to reducing the degree of tumors, or curing tumors to normalize them, or slowing down the progression of tumors.

[0026] The drug for preventing and treating cancer is an anti-tumor drug based on inhibiting the activity of PRDX1, and the application includes applying the cinobuccal toxin in combination with the ferroptosis inducer RSL3 to treat tumors with high expression of PRDX1.

[0027] In some specific embodiments, the PRDX1 inhibitor is selected from any one of a small molecule compound, a siRNA or a PROTAC molecule.

[0028] In some specific embodiments, the small molecule compound is cinobucini.

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

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

[0031] In some specific embodiments, the cancer includes liver cancer, lung cancer and colon cancer.

[0032] The embodiment of the present invention also provides a drug for preventing and treating cancer, comprising a PRDX1 inhibitor and a ferroptosis inducer.

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

[0034] In some specific embodiments, the small molecule compound is cinobuccal toadflax; and the ferroptosis inducer is RSL3.

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

[0036] Example 1

[0037] The present invention mainly uses human hepatocellular carcinoma cells HepG2, lung cancer cells A549 and colon cancer cells DLD-1 as research objects, uses MTT and cell lactate dehydrogenase (LDH) release level to detect the effects of cinobucoidin and its combination with RSL3 on the inhibition of cell proliferation, and uses flow cytometry to detect cell apoptosis, ROS and LPO content in cells.

[0038] 1 Experimental methods

[0039] 1.1 Active oxygen detection

[0040] HepG2, A549, and DLD-1 cells in the logarithmic growth phase were plated in 6-well plates. The next day, Huachansu (abbreviated as HCS) 4 mg / mL was given for induction treatment for 24 hours. After treatment, the old culture medium was discarded and washed once with PBS. During the detection, the ROS probe was loaded, and the probe was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM, and incubated in a 37°C cell culture incubator in the dark for 30 minutes. After incubation, the cells were washed twice with serum-free culture medium to fully remove the DCFH-DA that did not enter the cells. The cells were digested with trypsin and resuspended in 1 mL PBS to prepare a single cell suspension. The flow cytometer selected an excitation wavelength of 488 nm for detection. FlowJo software was used for data analysis. The higher the fluorescence signal intensity value, the higher the ROS.

[0041] 1.2 Lipid peroxide detection

[0042] HepG2, A549, and DLD-1 cells in the logarithmic growth phase were plated in 6-well plates. The next day, HCS (4 mg / mL) or ferroptosis inducer RSL3 (0.5 μM) was induced for 24 hours and incubated in a 37°C cell culture incubator for the corresponding time. After treatment, the old culture medium was discarded and washed once with PBS. BODIPY 581 / 591C11 stock solution (10 mM) was diluted to a working concentration of 10 μM with serum-free culture medium at a ratio of 1:1000 and incubated at 37°C in the dark for 30 minutes. The negative control was treated with serum-free culture medium. After incubation, the cells were washed 3 times with PBS, digested with trypsin and resuspended in 500 μL PBS. The flow cytometer selected an excitation wavelength of 488 nm for detection. FlowJo software was used for data analysis. The higher the fluorescence signal intensity value, the higher the ROS.

[0043] 1.3PI staining to detect cell death

[0044] To detect whether HCS increases the killing activity of chemotherapy drugs (cisplatin, carboplatin) or ferroptosis inducer (RSL3) on tumor cells, HepG2, A549, and DLD-1 cells in the logarithmic growth phase were plated in 6-well plates. The next day, RSL3 (0.5μM), cisplatin (3μM), carboplatin (10μM), or HCS (4mg / mL) were administered alone as the control group, and RSL3, cisplatin, or carboplatin combined with HCS were used as the experimental group, and incubated in a 37°C cell culture incubator for 24h. After treatment, the old culture medium was discarded and washed once with PBS. Add EDTA-free trypsin to digest the cells, gently blow and collect and transfer to a centrifuge tube, centrifuge at 1000rpm for 5min, and remove the supernatant. Add pre-cooled PBS to resuspend and wash the cells, centrifuge at 1000rpm for 5min, and remove the supernatant. Add 10μL of propidium iodide (PI) solution 5min before flow cytometry detection and gently mix, then perform flow cytometry detection and collect data. To verify whether the 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 the combined treatment of HCS and RSL3. FlowJo software was used for data analysis.

[0045] 1.4MTT

[0046] In order to detect whether HCS increases the inhibitory effect of ferroptosis inducers (RSL3, Erastin) on the proliferation activity of tumor cells, HepG2, A549, and DLD-1 cells in the logarithmic growth phase were plated in 96-well plates. The next day, HCS (4 mg / mL), ferroptosis inducer RSL3 (0.5 μM) or Erastin (1 μM) were administered alone as the control group, and RSL3 or Erastin combined with HCS was used as the experimental group, and incubated in a 37°C cell culture incubator for the corresponding time. Add 10 μL of MTT solution (5 mg / ml) to each well and incubate at 37°C in the dark for 4 hours. After incubation, carefully discard the supernatant, add 150 μL of dimethyl sulfoxide (DMSO) solution to each well, and mix by blowing to fully dissolve the crystals. The enzyme-linked immunosorbent assay was used at OD 490nm and OD 720nm Measure the absorbance of each well, OD 490nm Subtract OD 720nm The absorbance value at the position is the final value. In order 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) 4 h before HCS was administered. GraphPad Prism software was used for data analysis.

[0047] 1.5 Cytotoxic lactate dehydrogenase assay

[0048] To detect whether HCS increases the toxic effect of ferroptosis inducers (RSL3, Erastin) on tumor cells, HepG2, A549, and DLD-1 cells in the logarithmic growth phase were plated in 96-well plates. The next day, HCS (4 mg / mL), ferroptosis inducer RSL3 (0.5 μM) or Erastin (1 μM) were administered alone as the control group, and RSL3 or Erastin combined with HCS were used as the experimental group. The induction treatment was 24 hours and incubated in a 37°C cell culture incubator for the corresponding time. A high control group, a high control blank group, a low control group, and a background blank group were set up respectively. Add 10μL Lysis Solution to each well of the high control group (containing cells and culture medium) to determine the maximum releasable LDH of the cells; add 10μL Lysis Solution to each well of the high control blank group (containing culture medium only) to deduct the background absorbance value of the high control; the low control group (containing cells and culture medium) is not lysed and is used to determine the spontaneous LDH release of untreated normal cells; the background blank group (containing culture medium only) is used to deduct the background absorbance value of the low control and sample wells. Before the test, add 10μL Lysis Solution to the high control group and the high control blank group, and add 10μL culture medium to the low control group, and incubate at 37℃ in the dark for 30min. After the incubation, the direct method or indirect method of cell LDH activity can be used.

[0049] Direct method: aspirate 50μL of culture medium supernatant from each well, add 50μL of Working Solution to each well and shake to mix the remaining culture medium and cells. Indirect method: aspirate 50μL of supernatant from each well into a new 96-well plate, then add 50μL of Working Solution to each well and shake to mix. Incubate at room temperature in the dark for 30 minutes. After incubation, add 50μL of Stop Solution to each well and immediately measure OD using an enzyme-linked immunosorbent assay. 490nm Data analysis was performed using GraphPad Prism software.

[0050] 1.6 Molecular docking

[0051] The online molecular docking platform https: / / www.dockeasy.cn / DockCompound was used to perform molecular docking and obtain binding energy data. PyMOL software was used for image processing.

[0052] 2 Experimental results

[0053] 2.1 Inhibition of PRDX1 enhances cell ferroptosis

[0054] Kaplan-Meier Plotter analysis results suggest that PRDX1, a member of the peroxidase family, is closely associated with poor prognosis of liver cancer ( Figure 1 A). The DCFH-DAROS probe was used to label ROS in cells. The results showed that after siRNA was used to reduce PRDX1 expression, the level of cellular ROS increased significantly ( Figure 1 BODIPY 581 / 591C11 dye was used to label lipid peroxides (LPO) in cells. The results showed that reducing the expression level of PRDX1 could not only increase the basal level of LPO, but also significantly increase the accumulation of LPO mediated by the ferroptosis inducer RSL3 (0.5 μM, 24 h) ( Figure 1 PI staining was used to detect cell death. The results showed that knocking down PRDX1 in human liver cancer 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 The above results all confirmed that PRDX1 is a potential target for sensitizing cellular ferroptosis.

[0055] 2.2PRDX1 is the target protein of Cinobufacini

[0056] Molecular docking results confirmed that PRDX1 has a "pocket" structure that binds to the three active ingredients in HCS (Bufalin, Cinobufagin and Resibufogenin), with low binding energies of -9.403, -9.493 and -9.029 kcal / mol, respectively, indicating that the binding is highly stable ( Figure 2 The DCFH-DAROS probe was used to label ROS in cells. The results showed that HCS significantly upregulated the level of cellular ROS. After the expression of PRDX1 was reduced by small interfering RNA (siRNA), the upregulation of cellular ROS levels mediated by HCS (4 mg / mL, 24 h) was reversed ( Figure 2 (B). N-acetylcysteine ​​(NAC) is a thiol-containing antioxidant that can promote the capture of free radicals in cells and reduce ROS levels. The MTT test results all indicate that when HepG2 cells were pretreated with NAC (50μM, 4h), the inhibitory effect of HCS on cell proliferation was reversed; similarly, when siRNA was used to reduce PRDX1 expression, the effect of HCS (4mg / mL, 24h) was reversed ( Figure 2 Middle C). Further evidence was obtained by detecting the release level of cellular LDH, which showed that HCS-mediated inhibition of cell proliferation was dependent on ROS, and knockdown of PRDX1 reversed the inhibitory effect of HCS (4 mg / mL, 24 h) ( Figure 2 Middle D), suggesting that PRDX1 is a downstream target gene of HCS. In summary, PRDX1 is a target protein regulated by HCS.

[0057] 2.3 Cinobufacini sensitizes ferroptosis-induced cell ferroptosis mediated by RSL3

[0058] Given that knocking down PRDX1 can sensitize ferroptosis, the present invention also evaluated the anti-tumor effect of HCS combined with ferroptosis inducers. The results showed that compared with the single-dose group, the PI positive rate of HepG2 cells in the combined treatment group of HCS (4 mg / mL, 24h) and RSL3 (0.5μM, 24h) increased significantly, indicating that the combined treatment increased cell death, and the combined inhibitory effect of HCS and RSL3 was better than that of its combined effect with cisplatin (3μM, 24h) or carboplatin (10μM, 24h) ( Figure 3 Middle A). BODIPY 581 / 591C11 labeled cell LPO, the results showed that HCS (4mg / mL, 24h) could significantly increase the basal level ( Figure 3 Middle B) and RSL3 (0.5 μM, 24 h)-mediated LPO accumulation ( Figure 3C). MTT was used to detect the proliferation activity of HepG2, A549 and DLD-1 cells. The results showed that HCS (4 mg / mL, 24 h) significantly enhanced the anti-tumor effect of ferroptosis inducer RSL3 (0.5 μM, 24 h) or Erastin (1 μM, 24 h) compared with the single-dose group, and the anti-tumor effect of HCS combined with RSL3 was better than that of HCS combined with Erastin ( Figure 3 D). Further detection of the LDH release level of cells also revealed that HCS (4 mg / mL, 24 h) significantly increased cell death caused by ferroptosis inducer RSL3 (0.5 μM, 24 h) or Erastin (1 μM, 24 h); and compared with Erastin, the combined use of HCS and RSL3 had a better effect in inhibiting tumor growth ( Figure 3 HepG2 cells were pretreated with the classic ferroptosis inhibitor Ferrostain-1 (2 μM, 4 h). The results of cell apoptosis detection showed that Ferrostain-1 could reverse the cell death mediated by the combination of HCS (4 mg / mL, 24 h) and RSL3 (0.5 μM, 24 h) ( Figure 3 Middle F), further suggesting that HCS sensitizes RSL3 mainly by promoting ferroptosis.

[0059] In summary, the present invention found that the anti-tumor effect of Huachan toad is closely related to its upregulation of intracellular ROS. The results of mechanism studies show that PRDX1 is the target of Huachan toad. The molecular docking results show that PRDX1 has a "pocket" structure that binds to Huachan toad, and inhibiting PRDX1 weakens Huachan toad's regulation of ROS and its anti-tumor effect. In view of the fact that inhibiting PRDX1 can sensitize ferroptosis, in therapeutics, the present invention evaluates the combined effect of Huachan toad and ferroptosis inducers. The results showed that Huachan toad can effectively promote the anti-tumor effect of ferroptosis inducer RSL3 on liver cancer, lung cancer and colon cancer cells. Therefore, Huachan toad sensitizes sorafenib in clinical treatment and is related to its activation of ferroptosis. In view of this, the present invention provides a combined application of Huachan toad in tumor treatment in combination with ferroptosis inducers.

[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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of PRDX1 inhibitors and ferroptosis inducers in the preparation of drugs for preventing and treating cancer.

2. The use according to claim 1, characterized in that: The PRDX1 inhibitor is selected from any one of a small molecule compound, siRNA or a PROTAC molecule.

3. The use according to claim 2, characterized in that: The small molecule compound is cinobucoidin.

4. The use according to claim 1, characterized in that: The ferroptosis inducer is selected from RSL3 or Errastin.

5. The use according to claim 4, characterized in that: The ferroptosis inducer is RSL3.

6. The use according to claim 1, characterized in that: The cancers include liver cancer, lung cancer and colon cancer.

7. A drug for preventing and treating cancer, characterized in that: These include PRDX1 inhibitors and ferroptosis inducers.

8. The drug according to claim 7, characterized in that The PRDX1 inhibitor is selected from any one of a small molecule compound, siRNA or a PROTAC molecule; the ferroptosis inducer is selected from RSL3 or Errastin; and the cancer includes liver cancer, lung cancer and intestinal cancer.

9. The drug according to claim 8, characterized in that The small molecule compound is cinobacillus; the ferroptosis inducer is RSL3.

10. A pharmaceutical preparation for preventing and treating cancer, characterized in that: The invention comprises the drug according to claim 7 and pharmaceutically acceptable excipients.

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