CN116942834B - Antitumor drug comprising nucleolus stress inducer - Google Patents

Antitumor drug comprising nucleolus stress inducer Download PDF

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CN116942834B
CN116942834B CN202310692602.7A CN202310692602A CN116942834B CN 116942834 B CN116942834 B CN 116942834B CN 202310692602 A CN202310692602 A CN 202310692602A CN 116942834 B CN116942834 B CN 116942834B
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cells
wdr75
iron death
inducer
nucleolar
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CN116942834A (en
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韩涛
周祥
任文杰
高博
郝茜
张雅琦
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Xinxiang Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids

Abstract

The present invention relates to antitumor agents comprising nucleolus stress inducers. Specifically, the invention discloses a nucleolar stress inducer which can make cancer cells sensitive to iron death induced by Erastin by inhibiting SLC7A11 expression. Furthermore, it is disclosed that the combination of nucleolus stress inducer and iron death inducer produces synergistic inhibition of cancer cell growth and synergistic inhibition of tumor growth in vivo. The invention demonstrates the role of nucleolar stress in promoting iron death and provides a novel combination therapy for treating cancers in which the p53 gene is wild-type.

Description

Antitumor drug comprising nucleolus stress inducer
Technical Field
The invention relates to the technical field of biological medicines, in particular to an anti-tumor drug containing nucleolus stress inducer.
Background
Tumor suppressor p53 prevents tumorigenesis by maintaining genomic stability and eliminates cancer cells by inducing senescence and apoptosis. Recently, there has been increasing evidence that p53 also plays a role in iron death. Initially, p53 was found to limit cystine uptake by inhibiting SLC7a11 expression, thereby promoting iron death. Interestingly, inhibition of SLC7a11 also resulted in activation of lipoxygenase ALOX12, which oxidizes polyunsaturated fatty acids (PUFAs), precursors to PLOOH. In addition, p53 transcription induces expression of SAT1, SAT1 encoding a rate-limiting enzyme in polyamine catabolism to further support ALOX15 mediated lipid peroxidation.
However, p53 activation has also been reported to inhibit iron death under certain conditions and in different cells or tissues. For example, p53 was found to protect Vascular Smooth Muscle Cells (VSMCs) from iron death by activating SLC7a11 in response to metformin therapy. In addition, some iron death inhibitors, such as FSP1 and iPLA2 β, are encoded by p53 induction genes, contributing to p 53-mediated iron death resistance. In addition, p53 is stabilized by Nutlin-3, a selective p53 agonist, inhibiting iron death by inducing the classical target gene CDKN1A (also known as p 21), which subsequently stimulates GSH biosynthesis. These seemingly contradictory findings suggest that p53 is very complex in its function in regulating iron death in a cell-specific and content-specific manner. Accordingly, those skilled in the art are working to study novel inhibition mechanisms that corroborate tumors and develop more efficient antitumor drugs based thereon.
Disclosure of Invention
The invention discloses a nucleolus stress inducer which can make cancer cells sensitive to iron death induced by Erastin by inhibiting the expression of SLC7A 11. Furthermore, it is disclosed that the combination of nucleolus stress inducer and iron death inducer produces synergistic inhibition of cancer cell growth and synergistic inhibition of tumor growth in vivo. The invention demonstrates the role of nucleolar stress in promoting iron death and provides a novel combination therapy for treating cancers in which the p53 gene is wild-type.
In a first aspect of the invention, there is provided a pharmaceutical composition comprising a nucleolar stress inducer and an iron death inducer.
In another preferred embodiment, the molar ratio of the nucleolar stress inducer to the iron death inducer is in the range of 1:5000 to 100:1.
In another preferred embodiment, the molar ratio of the nucleolar stress inducer to the iron death inducer is from 1:2000 to 10:1.
In another preferred embodiment, the pharmaceutical composition is for the treatment of cancer.
In another preferred embodiment, the cancer is a p53 wild-type cancer.
In another preferred embodiment, the nucleolar stress inducer is a compound that specifically inhibits rRNA transcription or synthesis.
In another preferred embodiment, the nucleolar stress inducer comprises CX-3543, CX-5461, BMH-21 and actinomycin D (Act-D).
In another preferred embodiment, the iron death inducer is ellastine (erastin) and derivatives thereof.
In a second aspect of the invention, there is provided a method of preparing a pharmaceutical composition according to the first aspect of the invention, the method comprising admixing a nucleolar stress inducer and an iron death inducer with a pharmaceutically acceptable excipient.
In a third aspect of the invention, there is provided a kit comprising a nucleolar stress inducer and an iron death inducer.
In another preferred embodiment, the nucleolar stress inducer and the iron death inducer are packaged separately in the kit.
In a fourth aspect of the invention there is provided the use of a pharmaceutical composition according to the first aspect of the invention for the manufacture of a medicament for the treatment of cancer.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
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Figure 1 knockdown of wdr75 inhibited cancer cell growth and induced apoptosis. Knockdown of (a-C) WDR75 inhibited growth of a549, H460, and HEYA8 cells. Cells were transfected with control or WDR75siRNA and seeded in 96-well plates before cell viability assays were performed. (D) knock-down of WDR75 does not affect proliferation of H1299 cells. (E) Knockout of WDR75 inhibited the colony forming ability of a549 and H460, but did not inhibit H1299 cells. Cells were transfected with control or WDR75siRNA and then assayed for colony formation. (F) Knock-down of WDR75 induces G1 phase accumulation in a549 and HEYA8, but not H1299 cells. Cells were transfected with control or WDR75siRNA for 48 hours and then flow cytometry analysis was performed. (G) Knock-down of WDR75 promotes apoptosis of a549 and HEYA8 cells, but does not promote apoptosis of H1299 cells. Cells were transfected with control or WDR75siRNA for 48 hours and then subjected to annexin-V-PE/7-AAD staining and flow cytometry analysis.
Figure 2. Knock-down of wdr75 promotes iron death in a549 and HEYA8 cells carrying wild-type p 53. (A, B) WDR75 depletion-induced inhibition of cell growth can be restored by Ferr-1 treatment. A549 and HEYA8 cells transfected with siRNA were seeded in 96-well plates and then treated with DMSO or Ferr-1 (5 μm) for 48 hours. (C) knock-down of WDR75 does not affect iron death of H1299 cells. The knockdown of (D, E) WDR75 increases the level of MDA, which can be restored by Ferr-1 treatment. A549 and HEYA8 cells transfected with the indicated sirnas were treated with DMSO or Ferr-1 (5 μm) for 48 hours, and then MDA assays were performed. The knockdown of (F, G) WDR75 increases the level of ROS, which can be restored by Ferr-1 treatment. A549 and HEYA8 cells transfected with the indicated siRNAs were treated with DMSO or Ferr-1 (5. Mu.M) for 48 hours and then subjected to BODIPY TM 581/591C11 assays and flow cytometry analysis. The knockdown of (H) WDR75 did not affect ROS levels in H1299 cells. (I) Knock-down of WDR75 reduces GSH levels, which can be reversed by Ferr-1 treatment. A549 cells transfected with the indicated sirnas were treated with DMSO or Ferr-1 (5 μm) for 48 hours, and then GSH was determined.
Figure 3 p53 dependent inhibition of SLC7a11 resulted from wdr75 knockdown. The knockdown of (a, B) WDR75 consistently inhibited SLC7a11 mRNA expression. RT-qPCR analysis was performed on A549 and HEYA8 cells transfected with the indicated siRNAs. RT-qPCR also verifies knockdown efficiency. The knockdown of (C, D) WDR75 inhibits SLC7a11 protein expression. IB analysis was performed with designated siRNA transfected a549 and HEYA8 cells. (E) Knock-down of WDR75 does not affect expression of SLC7a11 protein in H1299 cells. Knockdown of (F, G) WDR75 hardly inhibited SLC7a11 expression in p 53-depleted a549 and HEYA8 cells.
Figure 4 knockdown of wdr75 activates p53 by nucleolar stress. (A) Knock-down of WDR75 induces protein levels of p53 and its target gene p 21. (B) knock-down of WDR75 induces mRNA levels of the p53 target gene. (C) knock-down of WDR75 increases the half-life of the p53 protein. Cells transfected with the indicated siRNAs were treated with CHX at the indicated time points and then subjected to IB analysis. Knockdown of (D, E) WDR75 hardly regulates p53 levels in RPL5- (D) or RPL11 depleted cells (E). (F) Knock-down of WDR75 increases the interaction of MDM2 with RPL5 and RPL 11. Cells were transfected with indicated siRNAs or plasmids and co-IP-IB analysis was performed. (G) knock-down of WDR75 disrupts nucleolar localization of B23. Immunofluorescence assays were performed on cells transfected with the indicated siRNA for 48 hours.
FIG. 5 nucleolar stress promotes iron death by the p53-SLC7A11 cascade. (A, B) low dose Act-D induced nucleolar stress mediated p 53-dependent SLC7A11 inhibition. A549 and HEYA8 cells were transfected with indicated sirnas and treated with or without Act-D (5 nM) for 48 hours before IB analysis. (C, D) nucleolar stress mediated inhibition of cell growth can be restored by Ferr-1 treatment. H460 and HEYA8 cells were transfected with indicated siRNA and treated with DMSO/Act-D (5 nM)/Ferr-1 (5. Mu.M) before cell viability assays were performed. (E, F) cisplatin and 5-FU mediated inhibition of cell growth did not involve iron death. H460 and HEYA8 cells were treated with cisplatin (30. Mu.M) or 5-FU (20. Mu.M) together with Ferr-1 (5. Mu.M) for 48 hours, and then subjected to cell viability assay. (G, H) nucleolar stress mediated elevated MDA levels can be restored by Ferr-1 treatment. A549 and HEYA8 cells were transfected with indicated siRNA and treated with DMSO/Act-D (5 nM)/Ferr-1 (5 μm) and then MDA assays were performed. (I, J) nucleolar stress-mediated elevation of ROS levels can be restored by Ferr-1 treatment. (K) Nucleolar stress mediated reduction of GSH levels can be reversed by Ferr-1 treatment.
Fig. 6 nucleolar stress inducer and erastin synergistically promote iron death. The (A, B) WDR75 knockdown enhanced the erastin (10. Mu.M) -mediated inhibition of cell growth. (C, D) Low doses of Act-D (5 nM) enhanced erastin (10. Mu.M) mediated cell growth inhibition. (E, F) WDR75 depletion synergistically increased MDA levels with erastin (10. Mu.M). (G, H) low doses of Act-D (5 nM) synergistically increased MDA levels with erastin (10. Mu.M). (I, J) WDR75 depletion coordinates with erastin (10. Mu.M) to increase ROS levels. (K, L) low doses of Act-D (5 nM) were conjugated with erastin (10. Mu.M) to increase ROS levels. (M, N) nucleolar stress caused by WDR75 depletion (M) or low doses of Act-D (N) synergistically acts with erastin (10. Mu.M) to reduce GSH levels.
Fig. 7. Nucleolar stress inducer and iron death inducer synergistically inhibit cancer cell growth. (A, B) Act-D and erastin synergistically inhibit the growth of A549 cells. Cells were treated with the reagent combinations for 48 hours as indicated, followed by cell viability assays and Chou-Talalay analysis. (C, D) Act-D and erastin synergistically inhibit the growth of HEYA8 cells. (E, F) CX-5461 and erastin synergistically inhibit the growth of A549 cells. (G, H) CX-5461 and erastin synergistically inhibit the growth of HEYA8 cells. (I, J) Act-D and sulfasalazine synergistically inhibit the growth of A549 cells.
Figure 8. Combination of actinomycin D and sulfasalazine is effective in inhibiting tumor growth in vivo. (A) Schematic of xenograft experimental design and drug combination. (B) no weight loss was observed during the treatment period. (C-E) Liproxstatin-1 partially inhibits the anti-cancer effect of Act-D as indicated by the average tumor volume (C), weight (D) and size of A549 cell-derived xenograft tumor (E). (F) Schematic of xenograft experimental design and drug combination. (G) no weight loss was observed during the treatment period. (H) The combination of Act-D with sulfasalazine significantly reduced the average tumor volume of A549 cell-derived xenograft tumors. (I) A significant decrease in tumor weight was observed following combination treatment with Act-D and sulfasalazine. (J) A significant decrease in tumor size was observed following combination treatment with Act-D and sulfasalazine.
FIG. 9 illustrates a working model of nucleolar stress and FIN coordination.
Detailed Description
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.
The inventors have unexpectedly found in the study that nucleolar stress inducers sensitize cancer cells to Erastin-induced iron death by inhibiting the expression of SLC7A 11. More importantly, the combination of nucleolar stress inducer and iron death inducer produces synergistic inhibition of cancer cell growth and synergistic inhibition of tumor growth in vivo. On this basis, the invention discloses the role of nucleolar stress in promoting iron death and provides a novel combination therapy for treating cancers with p53 gene being wild type.
p53 activation promotes or inhibits iron death in a context-dependent manner. The use of p 53-mediated iron death as a tumor suppression strategy remains an active area of research. The inventors found that ablation of WDR75 (a nucleolin that is overexpressed in human cancers and is associated with poor prognosis) increased intracellular MDA and ROS levels and inhibited GSH biosynthesis, resulting in p 53-dependent iron death. Mechanistically, knockout of WDR75 triggers nucleolar stress, activating p53 through RPL5 and RPL11, thus inhibiting expression of SLC7a 11. In addition, low dose actinomycin D induced nucleolus stress, but not genotoxic stress, also promotes iron death via the p53-SLC7A11 axis.
The inventors have found that WDR75, a nucleolin required for rRNA transcription, is overexpressed in human cancers and is associated with poor prognosis and thus can be a biomarker for poor prognosis of cancer. Therefore, the invention discloses the application of detection reagent of WDR75 (such as anti-WDR 75 antibody, reagent for PCR amplification of WDR75 gene, etc.) in preparing auxiliary diagnostic reagent for tumor.
The present invention discloses that ablation of WDR75 induces p53 activation by inducing nucleolar stress, resulting in cancer cell growth arrest and apoptosis. Activation of p53 by WDR75 deficiency or nucleolar stress inducer, rather than by cisplatin and 5-FU, which are genotoxic agents, reduces GSH levels and increases lipid peroxidation, thereby promoting iron death by inhibiting expression of SLC7a 11. Finally, the inventors demonstrate that combinations of nucleolar stress inducers with FINs synergistically inhibit cancer cell growth in vitro and in vivo.
Nucleolus stress inducer
Activation of p53 has been used as an anticancer strategy for wild-type p 53-bearing tumors. One of the methods of activating p53 is to induce nucleolar stress. Ribosomal biogenesis is a multi-step process that is critical to the production of translation mechanisms in cells, including rRNA transcription and processing, and pre-ribosome assembly in nucleoli and ribosome maturation in cytoplasm. The perturbation of this process results in the release of many Ribosomal Proteins (RPs) into the nucleoplasm where they can interact with the MDM2/p53 complex and activate the p53 pathway. Several anticancer agents have been developed, such as CX-3543, CX-5461, BMH-21 and low dose actinomycin D (Act-D), to trigger nucleolar stress by specifically inhibiting rRNA transcription or synthesis.
Iron death inducer (FINs)
Iron death is a non-apoptotic form of regulated cell death driven by iron-dependent phospholipid peroxidation. Phospholipid hydroperoxides (PLOOH) are a lipid-based Reactive Oxygen Species (ROS), which have been identified as effectors of iron death, leading to loss of membrane integrity, rupture of organelles and cell membranes, and fatal oxidative damage. Iron death is driven by membrane remodelling enzymes such as ACSL4 and LPCA T3, several Lipoxygenases (LOXs) and NADPH oxidase. Two cellular components, cystine/glutamate antiporter (system Xc - ) And GPX4, have been found to be critical for the monitoring of iron-death cell death. The cystine/glutamate antiport consists of SLC7a11 and SLC3A2, responsible for the uptake of cystine, which is subsequently reduced to cysteine, resulting in Glutathione (GSH) biosynthesis. GSH is a potent reducing agent that, in conjunction with GPX4, mediates the reduction of PLOOH, thereby preventing lipid peroxidation and iron death. The cystine/glutamate antiport-GPX 4 axis is considered to be the major system for inhibiting iron death in mammalian cells. The widely used iron death inducers (FINs) ellastine (erastin) and RSL3 are capable of inhibiting cystine/glutamate antiporter and GPX4, respectively. In addition, iron death is inhibited by negative regulators such as FSP1, NRF2 and HSPB1, and activated by Hippo and AMPK. Iron death is associated with a variety of pathophysiological conditions, including ischemic organ damage Injury, neurodegeneration, liver and lung fibrosis, immune system disease and cancer.
Pharmaceutical composition
The term "pharmaceutical composition" means a mixture containing a therapeutically effective amount of one or more of the compounds and pharmaceutically acceptable tautomers, solvates, hydrates, or salts thereof, and other pharmaceutically acceptable carriers. The purpose of preparing the compounds into pharmaceutical compositions is to facilitate administration to a subject.
According to one aspect of the present invention there is provided a pharmaceutical composition comprising a nucleolar stress inducer and an iron death inducer.
According to certain embodiments of the invention, the molar concentration ratio of the nucleolar stress inducer to the iron death inducer is about 1:5000-100:1, such as 1:4000-10:1, 1:3000-10:1, 1:2000-10:1, 1:1000-10:1, 1:100-10:1, 1:10-10:1.
According to certain embodiments of the invention, the pharmaceutical composition is for use in the treatment of cancer.
According to certain embodiments of the invention, the cancer comprises prostate cancer, colon cancer, lung cancer and breast cancer.
According to certain embodiments of the present invention, the nucleolar stress inducer and the iron death inducer are present in an amount of 1 to 100% of the pharmaceutical composition, for example, 1 to 99.5%, 1 to 99%, 1 to 90%, 1 to 80%, 1 to 70%, 1 to 60%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 10%, 10 to 100%, 10 to 99.5%, 10 to 99%, 10 to 90%, 10 to 80%, 10 to 70%, 10 to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 100%, 20 to 99.5%, 20 to 99%, 20 to 90%, 20 to 80%, 20 to 70%, 20 to 60%, 20 to 50%, 20 to 40%, 20 to 30%, 30 to 100%, 30 to 99.5%, 30 to 99%, 30 to 90%, 30 to 80%, 30 to 70%, and the like 30-60%, 30-50%, 30-40%, 40-100%, 40-99.5%, 40-99%, 40-90%, 40-80%, 40-70%, 40-60%, 40-50%, 50-100%, 50-99.5%, 50-99%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-99.5%, 60-99%, 60-90%, 60-80%, 60-70%, 70-100%, 70-99.5%, 70-99%, 70-90%, 70-80%, 80-100%, 80-99.5%, 80-99%, 80-90%, 90-100%, 90-99.5%, or 90-99%.
According to certain embodiments of the present application, the nucleolar stress inducer and iron death inducer comprise about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5%, or 100% of the pharmaceutical composition.
Drug synergy: when two or more drugs are combined, the effect of achieving mutual enhancement is called synergy, provided that their directions of action are identical, and the total effect exceeds the sum of the effects of each drug alone. In other words, the combined use of two drugs has an effect greater than the effect of either drug alone, and greater than the additive effect of the two drugs.
The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured. When used herein, "about" is used to modify a numerical value, it is meant that the value can float up or down by within a range of + -10%, + -9%, + -8%, + -7%, + -6%, + -5%, + -4%, + -3%, + -2%, or + -1%.
The term "modulation" includes treatment, prevention or interference.
The term "inducing nucleolar stress" includes triggering nucleolar stress responses.
The term "treatment" refers to the administration of a medicament of the invention to a subject in need of treatment for the purpose of curing, alleviating, ameliorating, alleviating, affecting the disease, symptoms, and constitution of the disease in the subject. Subjects of the invention include mice, rabbits, monkeys, humans, and other mammals.
The term "therapeutically effective amount" refers to an amount of a drug that achieves a therapeutic objective in a subject. It will be appreciated by those of ordinary skill in the art that the "therapeutically effective amount" may vary depending on the route of administration of the drug, the pharmaceutical excipients used, and the combination of the other drugs.
The pharmaceutical compositions of the present invention comprise a safe, effective amount of the present drug (active ingredient) within a range of pharmaceutically acceptable excipients or carriers. Wherein "safe, effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.001-1000mg of active ingredient/agent, preferably 0.05-300mg of active ingredient/agent, more preferably 0.5-200mg of active ingredient/agent.
The active ingredient of the present invention and its pharmacologically acceptable salts can be formulated into various preparations containing the active ingredient of the present invention or its pharmacologically acceptable salts and pharmacologically acceptable excipients or carriers in a safe and effective amount within the range. Wherein "safe, effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the active ingredient is determined according to the specific conditions of the age, illness state, treatment course and the like of the treatment subjects.
"pharmaceutically acceptable excipient or carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable excipients or carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate and the like), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulphate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil and the like), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol and the like), emulsifiers (e.g. humectants (e.g. sodium lauryl sulphate), colorants, flavouring agents, stabilisers, antioxidants, preservatives, pyrogen-free water and the like.
The compositions of the present invention may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically. It can be prepared into any pharmaceutically acceptable dosage form, including but not limited to tablets, oral preparations, medicinal granules, injections, liposomes, targeted administration injection pills, capsules, granules, powders, suppositories, powders, pastes, patches, injection, solutions, suspensions, sprays, lotions, drops, wipes and the like. The pharmaceutical compositions may be formulated as dry powders and mixed with sterile water or buffer to form solutions prior to administration. The pH of the buffer is generally 3 to 11, preferably 5 to 9, more preferably 7 to 8
The compositions of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
Microcapsules containing the compositions of the present invention may be used for sustained release administration of the active ingredients of the present invention. The sustained-release preparation of the active ingredient of the present invention can be prepared with high polymers of lactic-glycolic acid (PLGA) having good biocompatibility and broad biodegradability. The degradation products of PLGA, lactic acid and glycolic acid, can be cleared quickly by the human body. Moreover, the degradability of the polymer can be extended from months to years (Lewis, "Controlled release of bioactive agents form lactide/glycolide polymer," in: M.Chasin and R.Langer (eds.), biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: new York, 1990), pp.1-41)) depending on the molecular weight and composition thereof.
When a pharmaceutical composition is used, a safe and effective amount of the active ingredient of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective administration dosage, and for a human having a body weight of 60kg, the dosage per administration is usually 0.01 to 300mg, preferably 0.5 to 100mg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
Method for preparing pharmaceutical composition
According to one aspect of the present invention there is provided a method of preparing the pharmaceutical composition by mixing a nucleolar stress inducer and an iron death inducer with a pharmaceutically acceptable excipient.
The term "excipient" means a pharmaceutically acceptable ingredient that does not have any pharmacological effect, as is commonly used in pharmaceutical technology for the preparation of granular and/or solid oral dosage forms and/or liquid injection dosage form formulations. Excipients may act as carriers, diluents, or dissolution modifiers, absorption enhancers, stabilizers or adjuvants for preparation, among others. Excipients useful in preparing pharmaceutical compositions are generally safe, non-toxic and acceptable for medical use as well as pharmaceutical use. As used herein, "excipient" or "pharmaceutically acceptable excipient" includes one or more of such excipients.
Medicine box
The terms "kit" or "kit" are used interchangeably herein. Kits comprising a therapeutically effective amount of the therapeutic agent or pharmaceutical composition are disclosed. According to certain embodiments of the present application, the kit further comprises one or more additional therapeutic agents. According to certain embodiments of the present application, the kit further comprises instructions for use. According to certain embodiments of the present application, the kit further comprises means for corresponding modes of administration, such as, but not limited to, needles.
According to one aspect of the present invention there is provided a kit comprising the nucleolar stress inducer and an iron death inducer.
Method for treating disease
According to one aspect of the present invention, there is provided a method of treating cancer with the pharmaceutical composition.
According to certain embodiments of the invention, the pharmaceutical composition comprises a nucleolar stress inducer and an iron death inducer.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the application (including the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," having, "" including, "and" containing "as used herein are to be construed as open-ended terms (i.e., including, but not limited to,") unless otherwise noted herein or clearly contradicted by context. All methods described herein can be performed in any suitable order as would be understood by one skilled in the art unless otherwise indicated herein or clearly contradicted by context.
General method
Cell lines and culture methods
Human non-small cell lung cancer cell lines A549, H460, H1299, ovarian cancer cell line HEY A8, breast cancer cell line CAL51, colorectal cancer cell line HCT116 and embryonic kidney cell line 293T were cultured in DMEM medium supplemented with 10% FBS (Yeasen, shanghai China), 100U/ml penicillin and 100. Mu.g/ml streptomycin (BasalMedia, shanghai China) at 37℃with 5% CO 2 Culturing in an incubator. All cells were mycoplasma free and identified by PCR analysis.
Transient and stable knock-down of WDR75
Cells were seeded at appropriate densities in 6-well plates. The siRNA is transfected into serum-free and antibiotic-free culture medium by liposome transfection reagent, and the siRNA is replaced by complete growth culture medium after 4 to 6 hours. Real-time fluorescent quantitative PCR and immunoblotting experiments were performed 48h after transfection. Since anti-wdr 75 antibody was not obtained, RT-qPCR validated the knockout efficiency of this gene.
The siRNA sequences, primer sequences, etc. used in this study can be designed using routine software in the art. To establish a stable WDR75 knockout cell line, HEK293T cells were co-transfected with WDR75shRNA encoding plasmid and packaging plasmids psPAX2 and PMD2.0G using PEI (Sigma-Aldrich, usa), lentiviral particles were generated and then collected and used to infect target cells for 24h. Cells were selected with puromycin (MedChemExpress, usa) and validated with RT-qPCR.
Cell viability assay
6 hours after transfection, cells were seeded into 96-well plates, 3000 cells per well, and cultured for 24, 48, 72, 96 and 120 hours. At each time point, a cell count kit (CCK-8) reagent (Yeasen) was added to the culture for 2 hours. Cell viability was measured using a microplate reader at an absorbance of 450 nm. To assess cell viability associated with iron and iron death, cells were first seeded three times in 6-well plates and then transfected for 24 hours. Cells were then plated in 96-well plates, 6000 cells/100 ul per well, and treated with the indicated drugs (MedChemExpress) or DMSO for 48 hours. CCK-8 was used to determine cell viability as described above.
Colony formation assay
After 24h of transfection, cells were seeded in 6-well plates with 1000 cells per well and cultured for 14 days. Cells were washed with PBS, fixed with methanol, and stained with a mixture of methanol and crystal violet at room temperature. After staining for 30m, the staining buffer was rinsed with tap water and photographed.
Cell cycle analysis
Cells were collected 48h after transfection, fixed overnight with pre-chilled 70% ethanol, and dark treated with Triton-100 (Sangon, china) and RNase (Sangon) for 30min at room temperature. Cells were then stained with Propidium Iodide (PI) (Vazyme, china) for 30m in the dark. Finally, the cell cycle was examined with a flow cytometer (CytoFLEX S, beckman Coulter, indianapolis, ind., USA).
Apoptosis assay
Apoptosis was detected using an Annexin-V-PE/7-AAD kit (Vazyme) flow cytometer. Briefly, cells were collected 48h after transfection and stained with Annexin-V-PE and 7-AAD for 20m in the dark at room temperature. Apoptosis rates were measured using a flow cytometer (Beckman Coulter).
Real-time quantitative PCR
Using TRIzol (Takara, china) rootTotal RNA was extracted from cells as indicated by the manufacturer. gDNA scavenger mixtures (Vazyme) were used to avoid genomic DNA contamination. The extracted RNA was reverse transcribed into cDNA using Hiscript III qRT SuperMix (Vazyme). Real-time quantitative PCR (RT-qPCR) was performed using SYBR qPCR Master Mix (Vazyme). With 2 -△△Ct The method calculates the relative expression.
Immunoblotting
Immunoblotting (IB) was performed according to conventional procedures. The primary antibodies for IB were as follows, anti-SLC 7A11 antibody (1:100 dilution, cell Signaling Technology, U.S.), anti-vinculin antibody (1:100 dilution, cell Signaling Technology), anti-GAPDH antibody (1:100 dilution, china), anti-p 53 antibody (1:100 dilution, santa Cruz Biotechnology, U.S.), anti-p 21 antibody (1:100 dilution, cell Signaling Technology), anti-RPL 5 antibody (1:100 dilution, abcam, cambridge, U.S.), anti-RPL 11 antibody (1:100 dilution, cell Signaling Technology). The secondary antibodies used were enzyme-labeled affinity pure goat anti-rabbit IgG and anti-mouse IgG (1:10000 dilution, proteontech). Proteins were observed with ECL chemiluminescent reagent (Yeasen).
Immunoprecipitation reaction
A549 cells were transfected with siNC or WDR75 siRNA for 48 hours and harvested after treatment with MG132 for 4-6 hours. Immunoprecipitation (IP) was performed using antibodies shown in the legend. Briefly, 500. Mu.g of protein was incubated with the indicated antibodies for 4 hours at 4 ℃. Protein G beads (Santa Cruz Biotechnology) were then added to the mixture and then incubated at 4 ℃ for 2 hours. The beads were washed 3-5 times with lysis buffer. Protein complexes were detected with IB as described above.
Immunofluorescence
Using Hieff Trans TM Liposome nucleic acid transfection reagent (Yesen) transfects siNC, siWDR75 or siSBDS cells. Immunofluorescent staining assays were performed according to conventional procedures. Briefly, cells were fixed with methanol, incubated with primary antibody B23 (Santa Cruz Biotechnology) overnight at 4 ℃, and then reacted with Alexa Fluor 594 goat anti-rabbit IgG secondary antibody (Yeasen) in a dark environment for 2 hours. After transfection with DAPI (Sigma-Aldrich), cells were observed under an inverted fluorescence microscope (Leica, wetzlar, germany). Disruption of nucleolus integrity or function is increased by B23 translocationAdded to the nuclear mass.
Malondialdehyde assay
Intracellular Malondialdehyde (MDA) levels were measured using Malondialdehyde (MDA) detection kit (DOJINDO, china) according to manufacturer's instructions. Briefly, cells transfected with siRNA for 48 hours and treated with indicator for 24 hours were harvested in antioxidant PBS solution and suspended in lysis buffer and working fluid. The mixture was incubated at 95℃for 15m, then ice-cooled for 5m. The supernatant was collected by centrifugation and the fluorescence intensity was measured at ex540nm and em590nm using a microplate reader.
BODIPY TM 581/591C11 assay
BODIPY TM 581/591C11 dye (Invitrogen, china) was used to detect Reactive Oxygen Species (ROS) in cells and cell membranes. Cells were seeded in 6-well plates for at least 8 hours, then transfected for 48 hours, and treated with the indicated reagents for 24 hours. The cells in each well were then incubated with a cell containing 5. Mu.M BODIPY TM 581/591C11 dye fresh fbs-free medium was incubated for 20m in the dark at 37 ℃. Cells were washed and resuspended in PBS. BODIPY detection by flow cytometry (Beckman Coulter) TM 581/591C11 labeled cells. The fluorescence intensity of living cells was recorded using the grid technique. Partial oxidation of polyunsaturated butadiene of the dye results in a shift of the fluorescence emission peak from-590 nm (FL 2) to-510 nm (FL 1). Each set of data was from 3 independent replicates and analyzed using Flowjo 10.8.1 version.
Glutathione (GSH) and oxidized glutathione disulfide (GSSG) assays
Glutathione levels were determined using GSH and GSSG detection kits (Beyotime, shanghai, china). Cells were seeded in triplicate in 6-well plates for at least 8 hours, then transfected for 48 hours, and treated with the indicated drugs for 24 hours. Cells were collected and treated with a protein remover. The sample was frozen and thawed twice in a liquid nitrogen and 37 ℃ water bath. 50 μl GSH removal was added to each sample. All samples were added with DTNB and glutathione reductase and incubated for 5m at room temperature. Then, 0.5mg/ml NADPH was added to the sample, and incubated at room temperature for 25m. The absorbance at 412nm was measured with a microplate reader. The concentration of total glutathione and GSSG was calculated using a standard curve. Gsh=total glutathione-gssg×2.
Drug combination analysis
To evaluate the efficacy of a combination of nucleolar stress inducer and FINs, nucleolar stress inducer (Act-D, CX-5461) and FINs (erastin, sulfasalazine) were used to treat cancer cells. Cell viability was determined with CCK-8. Fractional impact (FA) values and Combination Index (CI) were analyzed using a Compusyn software based on Chou-Talalay method. CI values represent synergy (CI < 1), additive (ci=1) or antagonistic (CI > 1) of the combination. In particular, CI values of less than 0.6, between 0.6 and 0.8, between 0.8 and 1, respectively represent high, medium and low synergy.
Animal experiment
Mice xenograft experiments were performed as described previously. Animal protocols met ethical guidelines and were approved by the animal welfare committee of the Shanghai cancer center at the double denier university. Female BALB/c nude mice of 5 weeks old were purchased from Shanghai tumor center laboratory animal science center at double denier university and fed in laboratory. Will be 3X 10 6 (Act-D/liproxstatin group-1) or 5X 10 6 A549 cells of (Act-D/sulfasalazine group) were subcutaneously implanted in nude mice. When the tumor grows to about 9 days, physiological saline, act-D (30. Mu.g/kg), liproxstatin-1 (10 mg/kg), sulfasalazine (250 mg/kg) or a combination thereof is injected into the abdominal cavity of the nude mice for 10 days, respectively. Mice were monitored daily and weighed twice a week. Volume = length x width with the formula 2 Tumor volumes were measured and calculated by x 0.52.
Gene expression and prognostic analysis in databases
Comparison of WDR75 expression in various cancer tumor tissues versus normal tissues was analyzed by TCGA and CPTAC databases. And calculating Pearson correlation coefficient, and detecting the expression correlation of SLC7A11 and WDR75 and the expression correlation of p21 and WDR75 in TP53 wild lung adenocarcinoma and ovarian carcinoma samples. The WDR75 expression vs. prognosis is obtained from web resources (http// kmplot. Com/analysis and http:// ualcan. Path. Uab. Edu/index. Html).
Statistical analysis
All in vitro experiments were biological replicates and data were expressed as mean ± Standard Deviation (SD). Statistical significance (P-value) log-rank test was performed using multiplex T-test, or GraphPad Prism 8.2.1 or R-software. * P <0.05, p <0.01, p <0.001, ns, all data were insignificant. All in vitro experimental values were from three independent experiments.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Example 1 overexpression of WDR75 in cancer correlates with poor prognosis in cancer patients
To determine the regulatory factor for p53 activity, the inventors screened for many of the ribosomal biogenesis related factors previously described, and found that consumption of WDR75 could activate p53, as evidenced by up-regulation of the target gene p 21. More recently, WDR75 has been shown to be necessary for rDNA transcription, supporting the function of RPA194, a key subunit of RNA polymerase I. To study the role of WDR75 in cancer, the inventors analyzed TCGA and CPTAC databases and found that WDR75 was expressed at higher levels in different cancers than in normal tissues, such as lung cancer, large intestine adenocarcinoma, hepatocellular carcinoma, breast cancer, ovarian cancer. Notably, the higher the WDR75 level, the worse the prognosis, including lung cancer, renal clear cell carcinoma, ovarian cancer, etc., among the various cancers. These bioinformatic data suggest that WDR75 may play a carcinogenic role.
WDR75 deletion inhibits cancer cell growth and induces apoptosis
Next, the inventors determined whether WDR75 is critical to the growth and survival of cancer cells by depleting WDR75 levels in various cancer cells. Depletion of WDR75 by two independent sirnas significantly inhibited proliferation of wild-type p 53-bearing cancer cells, including lung cancer a549 and H460 cells and ovarian cancer HEY A8 cells (a-C of fig. 1). However, knock-down of WDR75 to p53 deleted H1 299 and HCT116 p53-/- Cells had a marginal effect (D of fig. 1). Consistently, knock-down of WDR75 significantly inhibited colony forming ability of a549 and H460 cells, but for H1299 and HCT116 p53-/- The cells had no effect (E of fig. 1). Then, the inventors performed flow cytometry analysis and found that ablation of WDR75 induced cell cycle arrest in wild-type p53 by increasing G1 phase population and decreasing S phase population (F of fig. 1). In addition, the inventors determined whether down-regulation of WDR75 promotes apoptosis by Annexin V-PE/7-AAD staining apoptotic cells, flow cytometry analysis. As expected, the deletion of WDR75 significantly induced apoptosis of a549 and HEY A8 cells expressing wild-type p53 (G of fig. 1). Notably, under the experimental conditions of the inventors, the inhibition of p 53-wild type cancer cells by WDR75 knockdown appears to be stronger than that of p 53-deleted cancer cells, suggesting that WDR75 function is largely dependent on p53. Taken together, these results indicate that WDR75 is essential for the growth and proliferation of cancer cells, as depletion of it inhibits cell growth and proliferation by inducing G1 arrest and apoptosis.
WDR75 deletion promotes iron death in cancer cells whose p53 gene is wild-type
In addition to testing its role in cancer cell cycle progression and survival, the inventors also determined whether WDR75 is likely to play a role in iron death. For this purpose, the inventors used the potent iron death inhibitor ferrostatin-1 (Ferr-1). Interestingly, by knocking out WDR75 to inhibit the growth of a549 and HEY A8 cells, consistent with the results described above (a-C of fig. 1), the inhibitor partially alleviated the growth of a549 and HEY A8 cells (a and B of fig. 2), suggesting that WDR75 may play a negative role in iron death. This effect on iron death was not observed in p 53-deleted H1299 cells (C of fig. 2), suggesting that wdr 75-regulated iron death may be p 53-dependent. Next, the inventors measured intracellular Malondialdehyde (MDA) levels of WDR75 knockdown cancer cells, as intracellular MDA levels are proportional to the extent of iron death. Consistently, WDR75 was significantly increased by its siRNA consumption in A549 and HEY A8 cells, while these effects were completely eliminated by Ferr-1 treatment (D and E of FIG. 2) and, in addition, BODIPY was used with the fluorescent lipid peroxidation probe TM 581/591C11 the level of ROS in living cells was assessed and then flow cytometry analysis was performed. Again, ablation of WDR75 significantly increased fluorescence intensity in both cell lines and was neutralized by the Ferr-1 treatment (F and G of fig. 2), but not observed in p 53-deleted H1299 cells (H of fig. 2). Glutathione (GSH) is the most abundant intracellular reducing agent that can increase the activity of GPX4, alleviate oxidative damage, and protect cells from iron death. Thus, the inventors tested whether WDR75 depletion would affect glutathione levels in cancer cells. As shown in fig. 2I, knockout of WDR75 significantly reduced GSH levels, while antioxidant Ferr-1 significantly increased GSH levels. Taken together, these results indicate that WDR75 deficiency promotes iron death in a p 53-dependent manner, which will be further studied below.
WDR75 deletion inhibits SLC7A11 expression by activating p53
Since WDR75 depletion mediated iron death appears to be dependent on p53, the inventors have attempted to elucidate its underlying mechanism by screening for iron death-related genes regulated by p 53. By performing a panel of RT-qPCR assays, the inventors found that knock-down WDR75 specifically inhibited SLC7a11 expression in all cancer cell lines examined, while non-uniformly affecting ALOX12, FDXR, PTGS2, GPX4 and DPP4 expression (a and B of fig. 3). Furthermore, knockout of WDR75 reduced the level of SLC7a11 protein in a549 and HEY A8 cells (C and D of fig. 3), but not in H1299 cells (E of fig. 3). It is also noted that a decrease in SLC7A11 expression is accompanied by activation of p53, which can be demonstrated from the induction of p53 and its target gene p21 in response to the consumption of WDR75 (C and D of FIG. 3). Consistent with observations, the inventors found that WDR75 expression correlated negatively with p21 levels and positively with SLC7a11 levels in lung and ovarian cancer samples. Since SLC7a11 is encoded by the p53 inhibitor gene 21, the inventors determined whether the decrease in expression of SLC7a11 was regulated in a p 53-dependent manner. The results indicate that down-regulation of p53 significantly increases expression of SLC7a11 (panels 1vs. 2 in F and G of fig. 3). The p53 deficiency resulted in inhibition of SLC7a11 expression by depletion of WDR75 (panels 2vs. 4 in fig. 3F and 3G). Thus, these results highly suggest that WDR75 deletion may activate p53 which inhibits SLC7a11 expression, thereby causing iron death.
WDR75 loss triggers nucleolar stress mediated p53 activation
To elucidate how a WDR75 deletion leads to p53 activation, the inventors first tested whether WDR75 deletion could enhance the transcriptional activity of p53 by detecting the expression of some p53 target genes. Indeed, consistent with the above results (FIGS. 3C and D), knock-down of WDR75 induces p53 protein levels and expression of its target genes p21, MDM2, BAX and BTG2 at the RNA level (FIGS. 4A and B). Induction of p53 protein levels by depletion of WDR75 was due to the half-life extension of p53 detected by performing a Cycloheximide (CHX) -chase experiment (C of fig. 4). There is sufficient evidence that under nucleolar stress, some RPs are released from the nucleoli into the nucleoplasm where they interact with the MDM2-p53 complex, resulting in p53 activation. Among these MDM 2-bound RPs, RPL5 and RPL11 have been shown to be key participants in the regulation of p53 activity, as disruption of RPL5 and RPL11 binding to MDM2 completely prevents nucleolar stress-induced p53 activation in vitro and in vivo. Thus, the inventors determined whether WDR75 depletion induced p53 activation was dependent on RPL5 and RPL11. As shown in D and E of fig. 4, down-regulation of WDR75 increases p53 expression, while ablation of RPL5 or RPL11 reduces p53 activation caused by WDR75 deletion. Furthermore, ablation of WDR75 enhances the interaction between RPL5/RPL11 and MDM2 (F of fig. 4). In addition, the inventors performed Immunofluorescent (IF) staining of B23, B23 also known as nucleoprotein 1/NPM1, a nucleolar marker necessary for ribosomal biogenesis, to determine IF depletion of WDR75 would compromise nucleolar integrity. As with SBDS ablation as a positive control, knockout of WDR75 disrupts nucleolar localization of B23 (G of fig. 4), indicating nucleolar dysfunction, a prerequisite for nucleolar stress. The inventors' findings indicate that the absence of WDR75 in cancer cells leads to nucleolar stress and thus to p53 activation. Together with the inventors' findings that WDR75 is highly expressed in a variety of human cancers, these results also suggest that WDR75 may be a potential target for developing anti-cancer therapies against wild-type p53 tumors.
Nucleolar stress promotes iron death via the p53-SLC7a11 axis
Since WDR75 loss promotes iron death through nucleolar stress-induced p53 activation, the inventors speculate that nucleolar stress inducers may also promote iron death. Actinomycin D (Act-D) is a common drug for the treatment of cancer, and can selectively inhibit RNA Pol i-dependent transcription at a dose of 10nM or less, thereby inducing nucleolus stress. To verify whether nucleolar stress promotes iron death, the inventors first treated a549 and HEY A8 cells with 5nM Act-D to examine the expression of SLC7a 11. As shown in a and B of fig. 5, act-D significantly induced p53 levels and reduced SLC7a11 levels by IB analysis. The reduction of SLC7A11 is p53 dependent, and the down-regulation of p53 rescues the Act-D induced reduction of SLC7A 11. Interestingly, the inhibition of H460 and HEY A8 cell growth by Act-D was also partially rescued by Ferr-1 (FIGS. 5C and D), consistent with the results of WDR75 knockdown (FIGS. 2A and B). Surprisingly, both genotoxic drugs cisplatin and 5-FU failed to induce iron death, as Ferr-1 failed to restore cell viability when H460 and HEY A8 cells were treated with both drugs (E and F of fig. 5). These results indicate that p 53-activated iron death is a specific nucleolar stress in these cells. In A549 and HEY A8 cells, 5nM Act-D significantly increased MDA levels, while Ferr-1 reversed MDA levels, confirming nucleolar stress-induced iron death (G and H of FIG. 5). Act-D also promoted the production of ROS, and an increase in fluorescence intensity indicated that this was again reversed by Ferr-1 (FIGS. 5I and J). Since SLC7A11 is essential for cystine uptake and GSH biosynthesis as a component of the xc-system, the inventors expect that Act-D will also reduce intracellular GSH levels. This is true (K of fig. 5). Taken together, these results indicate that nucleolar stress reduces expression of SLC7a11 by activating p53 leading to decreased glutathione biosynthesis and induction of iron death.
Nucleolar stress and iron death inducers synergistically inhibit cancer cell growth
It has been reported that both ellastine (erastin) and sulfasalazine (sulfasalazine) used clinically to treat ulcerative colitis inhibit the xc-system, resulting in up-regulation of SLC7a11, which is considered a compensatory feedback control of cell survival. Thus, the inventors speculate that nucleolar stress-mediated reduction of SLC7a11 may sensitize cancer cells to iron death inducers (FINs). To verify this idea, the inventors performed a panel of cell viability assays, using erastin and nucleolar stress inducer separately and in combination to treat cancer cells. Although both erastin and WDR75 depletion reduced cell viability as expected, the combination of the two inhibited the growth of a549 and HEY A8 cells more significantly (a and B of fig. 6). Consistently, the combination of erastin and Act-D also inhibited the growth of cancer cells more significantly than each individual treatment (C and D of fig. 6). To test whether these combined effects are due to increased iron death, the inventors assessed intracellular levels of MDA, ROS, and GSH. Indeed, the combination of erastin with WDR75 consumption or Act-D resulted in a significant increase in MDA (E-H of FIG. 6) and ROS (I-L of FIG. 6) levels, but a more significant decrease in glutathione levels (M and N of FIG. 6), as compared to the results after each individual treatment. These results indicate that nucleolar stress inducers and the FINs synergistically promote iron death.
Next, the inventors tested whether they synergistically inhibited cancer cell growth by calculating the combined index value (CI) of nucleolar stress-inducing agents and FINs when three different drugs Act-d+ erastin, CX-5461+ erastin, and Act-d+ sulfasalazine were combined to treat a549 and HEY A8 cells. Thus, the inventors found that Act-D in combination with erastin significantly inhibited cancer cell growth, exhibiting a stronger synergistic effect, with CI values of less than 0.5 (a-D of fig. 7). The combination of CX-5461 with erastin at different concentrations also showed strong synergy in HEY A8 cells (G and H of FIG. 7), but different synergy in A549 cells (E and F of FIG. 7). Consistently, the combination of Act-D with salazide Liu Qin also shows a more synergistic anticancer effect in cancer cells (I and J of fig. 7). In contrast, nucleolar stress inducers failed to synergistically act with erastin to suppress p 53-deleted H1299 cells, suggesting that this synergy is dependent on p53 activity. Taken together, these results indicate that nucleolar stress inducers and FINs can synergistically inhibit cancer cell growth.
Nucleolus stress and iron death inducer synergistically inhibit tumor growth in vivo
The inventors then established a mouse xenograft model that translated the above cell-based results into more biological relevance. First, the inventors tested in vivo whether Act-D promoted iron death. Nude mice 5 weeks old were inoculated subcutaneously with A549 cells, randomized into four groups, treated with physiological saline, act-D, iproxstatin-1, and Act-D in combination with iproxstatin-1, respectively, for 10 days (FIG. 8A). As expected, act-D reduced the growth rate (C of FIG. 8), weight (D of FIG. 8) and size (E of FIG. 8) of xenograft tumors, while iproxstatin-1 partially restored Act-D mediated tumor growth inhibition (C-E of FIG. 8). Notably, act-D significantly inhibited tumor growth even in the presence of iproxstatin-1 (FIGS. 8C and D), indicating that the anticancer effect of Act-D is also associated with its ability to induce cell cycle arrest and apoptosis. In addition, the inventors generated a mouse xenograft model treated with Act-D and/or sulfasalazine, both drugs having been used clinically. After subcutaneous inoculation of a549 cells, nude mice were randomized into four groups and treated with physiological saline, act-D, sulfasalazine and Act-D in combination with sulfasalazine, respectively, for 10 days (F of fig. 8). The xenograft tumor volume was reduced by about 50% after treatment with either Act-D or sulfasalazine compared to the control group, whereas Act-D in combination with sulfasalazine reduced tumor volume by 80% (H of fig. 8). Act-D, sulfasalazine and combination treatment resulted in 42%, 50% and 71.5% weight loss of tumors, respectively (I of FIG. 8), potential drug-related adverse events were tolerated, as the average body weight of the treated mice was comparable to the control mice (B and G of FIG. 8). Taken together, these results demonstrate that combination therapy with nucleolar stress inducers and FINs can more effectively inhibit tumor growth in vivo (fig. 9).
Discussion of the invention
Iron death disorders are associated with a variety of human diseases including cancer. In this study, the inventors found that WDR75 is a nucleolin that is overexpressed in cancer patients and is associated with poor prognosis, a novel iron death modulator. Although down-regulation of WDR75 inhibited cancer cell growth by inducing apoptosis (fig. 1), studies by the inventors also revealed that down-regulation of WDR75 promoted iron death in a p 53-dependent manner (fig. 2). Mechanistically, down-regulation of WDR75 triggers nucleolar stress, leading to p53 activation by RPL5 and RPL 11 dependent patterns, leading to a decrease in SLC7a11 (fig. 3 and 4). Interestingly, as with the down-regulation of WDR75, nucleolar stress inducer Act-D also promoted iron death via the p53-SLC7a11 axis (a-E of fig. 8). More interestingly, surprisingly, this p 53-dependent iron death occurred only under nucleolar stress, not as a DNA damage inducer (fig. 5e,5 f). Notably, nucleolar stress inducers and the FINs together promote iron death and inhibit cancer growth (fig. 6-8). In summary, the studies presented herein by the inventors revealed a novel role for nucleolar stress-p 53 signaling pathway in promoting iron death (fig. 9), and provided a combination approach with nucleolar stress inducers and FINs, providing a promising approach for future clinical development of effective treatments against wild-type p 53-persistent cancers.
Since rapidly growing and proliferating cancer cells require active nucleolar functions necessary for ribosomal biogenesis and protein translation, they are more susceptible to nucleolar stress-induced cell death. Many nucleolar proteins required for rRNA biosynthesis and processing are found to be up-regulated in human cancers, and thus targeting these proteins provides an opportunity to treat cancer by nucleolar stress. WDR75 was previously identified as a regulator of rRNA biosynthesis by screening 625 nucleolin in HeLa cells. More recently, it was found that WDR75 deletion destabilizes RPA194, thereby blocking RNA Pol i-mediated rDNA transcription and triggering nucleolar stress in U2OS cells. The inventors found that down-regulation of WDR75 induced nucleolar stress in many cancer cell lines, such as relocation of nucleolar marker B23 (G of fig. 4), resulting in activation of p53 in a549, HEY A8, HCT116 and CAL51 cells (C of fig. 3, D of fig. 3, a of fig. 4, B of fig. 4). Notably, WDR75 loss causes cancer cell growth arrest and apoptosis, as well as lipid peroxidation and iron death. Since the high expression of WDR75 in various cancers is associated with poor prognosis of cancer patients, an effective anticancer therapy can be developed against WDR 75.
While it is appreciated that WDR75 loss may promote iron death by causing nucleolar stress, the inventors have further discovered that other nucleolar stress inducers may also cause iron death, for example Act-D. Treatment of cancer cells with 5nM Act-D selectively inhibited RNA pol I activity, triggering nucleolar stress, resulting in reduced SLC7A11 RNA and protein levels by inducing p53 activation (FIGS. 5A and B). Furthermore, the iron death inhibitor Ferr-1 partially rescued the cell growth arrest caused by Act-D, indicating that nucleolar stress inducers did promote iron death (FIGS. 5C and D). The inventors found that Act-D increased intracellular levels of MDA and ROS (D-J of fig. 5) and inhibited GSH levels (K of fig. 5), further supporting the inventors' findings. Furthermore, act-D inhibited tumor growth by promoting iron death in vivo, as l iproxstatin-1 partially inhibited the antitumor activity of the compound (a-E of fig. 8). Thus, these results demonstrate for the first time that nucleolar stress can cause p 53-dependent iron death. Surprisingly, DNA damaging agents, such as cisplatin and 5-FU, did not induce iron death in the several cancer cells tested herein (E and F of fig. 5), although they could also induce p53 activation. It is thought that the elevated p53 levels caused by some DNA damaging agents may not be sufficient to induce iron death. This is supported by a previous study that shows that genotoxic drugs, including cisplatin, 5-FU and doxorubicin, have no or little effect on iron death in various cancer cells. Furthermore, nutlin-3, an MDM2-p53 binding blocker, can induce p53 levels alone without causing any post-translational modification (PTMs) of this tumor suppressor protein, and even demonstrate that iron death can be inhibited by p 21-mediated GSH accumulation. Thus, nucleolar stress inducers may result in certain PTMs of p53 in addition to increasing p53 protein levels. Indeed, studies have shown that nucleolar stress is generally coupled with p 300/CBP-catalyzed p53 acetylation without affecting p53 phosphorylation, which is more related to DNA damage-induced a TM/a TR activation. In particular, acetylation of p53 lysine 101 (K101) by the acetyltransferase CBP has proven to be critical for p 53-mediated transcriptional inhibition of SLC7a 11. In addition, RPL11 or MYBBP1A was recruited to the p53 target gene promoter to enhance p300/cbp mediated p53 acetylation in response to nucleolar stress. Mutation of the K101 residue of p53 may explain why some cancer cells are insensitive to p53-slc7a11 axis mediated iron death. These studies together with the inventors' findings explain why nucleolar stress, rather than DNA damage signaling, can induce p 53-dependent iron death in various cancer cells.
Although both FINs and nucleolar stress inducers promote iron death, their underlying mechanisms are different. Erastin and sulfasalazine induce iron death by inhibiting cystine/glutamate antiporter activity, but conversely increase SLC7A11 expression. As described above, nucleolar stress in cancer cells treated by knockout of WDR75 or Act-D inhibits SLC7a11 transcription in a p53 dependent manner. These nucleolar stressors can enhance iron death induced by Erastin or sulfasalazine, thereby sensitizing cancer cells to these FINs. Indeed, studies by the inventors using these FINs in combination with nucleolar stress inducers to treat cancer cells have shown that they can synergistically induce iron death and inhibit the growth of cancer cells (F-J of FIG. 8) by significantly increasing intracellular MDA and ROS levels, reducing GSH levels (FIG. 6). Nucleolar stress inducers have been shown to disrupt nucleolar integrity and function, in addition to their role in sensitizing cancer cells to iron death, which is critical for cancer cell survival and proliferation and triggers p 53-dependent apoptosis. In summary, these anticancer activities may promote their synergistic effect with the FINs in inhibiting cancer cell growth and proliferation, making the combination strategy an attractive, effective and viable method of eliminating cancer cells.
In summary, studies of the present invention revealed that WDR75 depletion or nucleolar stress induced nucleolar stress promotes iron death via the p53-SLC7A11 axis. Notably, nucleolar stress-mediated inhibition of SLC7a11 sensitizes cancer cells to ns. This provides new opportunities for future development of nucleolar stress inducers and FINs in combination for treating p53 gene for wild type cancer in the cancer field.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (5)

1. A pharmaceutical composition comprising a nucleolar stress inducer CX-5461 and an iron death inducer ellastine; and the molar concentration ratio of the nucleolar stress inducer CX-5461 to the iron death inducer ellastine is 1:100-10:1.
2. Use of a pharmaceutical composition according to claim 1 in the manufacture of a medicament for inhibiting growth of HEYA8 cells cultured in vitro.
3. A process for preparing a pharmaceutical composition according to claim 1, wherein the process comprises mixing nucleolar stress inducer CX-5461 and iron death inducer ellastine with a pharmaceutically acceptable excipient.
4. A kit comprising a nucleolar stress inducer CX-5461 and an iron death inducer ellastine; and the molar concentration ratio of the nucleolar stress inducer CX-5461 to the iron death inducer ellastine is 1:100-10:1.
5. Use of a pharmaceutical composition according to claim 1 for the preparation of a medicament for the treatment of ovarian cancer.
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