CN115300512A - Use of ATR inhibitor VE-822 for treating lung adenocarcinoma - Google Patents
Use of ATR inhibitor VE-822 for treating lung adenocarcinoma Download PDFInfo
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Abstract
The invention relates to the use of an ATR inhibitor VE-822 in the treatment of lung adenocarcinoma. The invention discusses the specific action mechanism of the ATR inhibitor VE-822 in inhibiting lung adenocarcinoma from the levels of molecules, cells, animals and the like. And the ATR inhibitor VE-822 can activate deubiquitinase OTUD1 besides being used as an inhibitor of ATR signal channel, inhibit the proliferation, migration and invasion of lung adenocarcinoma cells and inhibit the growth of nude mouse subcutaneous lung adenocarcinoma, which shows that VE-822 can activate deubiquitinase OTUD1 for treating lung adenocarcinoma.
Description
The technical field is as follows:
the invention relates to the technical field of biological medicines, in particular to application of an ATR inhibitor VE-822 in treatment of lung adenocarcinoma.
The background art comprises the following steps:
cancer is the leading cause of death worldwide. According to the report of the World Health Organization (WHO) 2019, cancer is the first or second leading cause of death in the population under 70 years of age in 112 countries, and the third or fourth leading cause of death in 23 countries, with cancer as the main cause of death becoming increasingly prominent.
At present, great progress is made in developing antitumor drugs, particularly in the fields of immunotherapy and targeted therapy. Many cancer targeted therapies are small molecule targeted drugs or monoclonal antibodies that inhibit the activity of proteins that promote tumor growth. However, tumor cells develop resistance by overexpression of the target protein and/or by acquiring new mutations in the target protein. Thus, researchers have begun exploring alternative therapies that modulate protein function by modulating the expression level of the protein rather than the activity of the protein. Among them, ubiquitin-proteasome system (UPS) plays an important role in regulating protein expression level, and deubiquitinase is an important component of UPS.
VE-822 is an inhibitor of ATR, which is normally activated for single strand breaks or in response to general replicative stress. ATR inhibitors consume nucleotides and interfere with DNA replication by promoting cleavage of the arrested replication fork and/or by inhibiting its repair. VE-822 inhibits DNA damage repair, and inhibits tumor progression by inducing apoptosis. Phase i clinical trials in advanced solid tumors have been completed, demonstrating that VE-822 is safe, tolerable in humans, and that target participation and primary anti-tumor responses are observed. However, the mechanism of action of VE-822 on deubiquitinating enzyme has not been studied. The invention clarifies a molecular mechanism of VE-822 targeting deubiquitinase OTUD1 for inhibiting lung adenocarcinoma, and provides a new molecular target and a treatment strategy for clinically preventing or treating lung adenocarcinoma.
The invention content is as follows:
technical problem to be solved
Against the background, the present invention discusses the specific mechanism of action of ATR inhibitor VE-822 in inhibiting lung adenocarcinoma at the molecular, cellular and animal levels. And the ATR inhibitor VE-822 can be used as an inhibitor of ATR signal channel, activate deubiquitinase OTUD1, inhibit proliferation, migration and invasion of lung adenocarcinoma cells, inhibit growth of nude mouse subcutaneous lung adenocarcinoma, and clarify a molecular mechanism of VE-822 targeting deubiquitinase OTUD1 for inhibiting lung adenocarcinoma, so that VE-822 can activate deubiquitinase OTUD1 for preparing a medicament for treating lung adenocarcinoma, and a new molecular target and a new treatment strategy are provided for clinical prevention or treatment of lung adenocarcinoma.
(II) technical scheme
In order to solve the technical problem, the invention adopts the following technical scheme: the application of the ATR inhibitor VE-822 in treating lung adenocarcinoma is characterized in that the CAS number of VE-822 is 1232416-25-9, the molecular formula is C24H25N5O3S, the administration target of VE-822 is deubiquitinase OTUD1, and proliferation, migration and invasion of lung adenocarcinoma cells are inhibited by activating the deubiquitinase OTUD 1. The VE-822 has the following structural formula:
further, the deubiquitinase OTUD1 inhibits the proliferation, migration and invasion of lung adenocarcinoma cells by regulating the stability of FHL1 protein.
Further, the deubiquitinase OTUD1 is deubiquitinase of FHL1 protein in lung adenocarcinoma, interacts with FHL1 protein through an OTU structural domain, and stabilizes the expression of the FHL1 protein through the active site C320 deubiquitination FHL1 protein.
Furthermore, the expression of the FHL1 protein is down-regulated in a sample of a patient with lung adenocarcinoma, the expression quantity of the FHL1 protein is positively correlated with the survival rate of the patient with lung adenocarcinoma, and the deletion of the FHL1 protein can promote the proliferation, migration and invasion of lung adenocarcinoma cells.
The invention also provides application of the deubiquitinase OTUD1 as a target molecule to screening drugs for preventing and treating lung adenocarcinoma, wherein the drugs comprise gene therapy drugs of OTUD1 overexpression or activators or analogues of OTUD1
Further, the drug is a compound of VE-822, or a pharmaceutically acceptable salt thereof, in combination with an additional therapeutic agent.
Further, the medicament is configured into gels, transdermal patches, injectable fluids, pills, capsules.
In addition, the invention also provides application of the deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnostic marker.
And the application of deubiquitinase OTUD1 as a target molecule for screening molecular diagnostic markers of lung adenocarcinoma.
Furthermore, the invention also provides application of the deubiquitinase OTUD1 as a detection index in treatment of lung adenocarcinoma.
The invention discusses the effect and specific mechanism of VE-822 in treating lung adenocarcinoma at multiple levels of molecule, cell and animal level. Research shows that the VE-822 can activate a deubiquitinase OTUD1, the deubiquitinase OTUD1 is used as deubiquitinase of FHL1 protein in lung adenocarcinoma, the OTU structural domain interacts with the FHL1 protein, the enzyme activity site C320 deubiquitinase FHL1 protein stabilizes the expression of the FHL1 protein, and therefore proliferation, migration and invasion of lung adenocarcinoma cells are inhibited. In addition, the invention also provides application of the deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnosis marker, or application of the deubiquitinase OTUD1 as a target molecule for screening the lung adenocarcinoma molecular diagnosis marker, and application of the deubiquitinase OTUD1 as a detection index in evaluating lung adenocarcinoma, or application of the deubiquitinase OTUD1 as a target molecule for screening lung adenocarcinoma prevention and treatment medicines.
(III) advantageous effects
The invention has the following beneficial effects: according to the invention, the research finds that the VE-822 can treat the lung adenocarcinoma by targeting activation of the deubiquitinating enzyme OTUD1, and the specific action mechanism of the ATR inhibitor VE-822 for inhibiting the lung adenocarcinoma is discussed from the levels of molecules, cells, animals and the like. And the ATR inhibitor VE-822 can be used as an inhibitor of ATR signal channel, activate deubiquitinase OTUD1, inhibit proliferation, migration and invasion of lung adenocarcinoma cells, and inhibit growth of subcutaneous lung adenocarcinoma of nude mice, so that a molecular mechanism of VE-822 targeting deubiquitinase OTUD1 for inhibiting lung adenocarcinoma is elucidated, and VE-822 can activate deubiquitinase OTUD1 for preparing a medicine for treating lung adenocarcinoma. In addition, the invention also provides application of the deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnosis marker, or application of the deubiquitinase OTUD1 as a target molecule for screening lung adenocarcinoma molecular diagnosis markers, and application of the deubiquitinase OTUD1 as a detection index in evaluating lung adenocarcinoma, or application of the deubiquitinase OTUD1 as a target molecule for screening lung adenocarcinoma prevention and treatment medicines, and provides a new molecular target and a treatment strategy for clinically preventing or treating lung adenocarcinoma.
Description of the drawings:
in order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.
FIG. 1 shows that VE-822 is able to up-regulate the expression of OTUD1 and FHL1 in lung adenocarcinoma cell lines. Wherein A is the protein level change of OTUD1 and FHL1 after the H827 cells are treated by VE-822 with different concentrations for 48 hours; b is the change of the mRNA level of OTUD1 after treating H827 cells with different concentrations of VE-822 for 48 hours; c is the protein level change of OTUD1 and FHL1 after H2030 cells are treated by VE-822 with different concentrations for 48 hours; d is the change of the mRNA level of OTUD1 after H2030 cells are treated by VE-822 with different concentrations for 48 hours.
FIG. 2 shows VE-822 inhibits proliferation of lung adenocarcinoma cell lines. Wherein A is the change of cell number after treating H827 cells with different concentrations of VE-822 for 48 hours; b is the change in cell number 48 hours after treating H2030 cells with different concentrations of VE-822.
FIG. 3 shows that VE-822 and OTUD1 both inhibit the proliferation of lung adenocarcinoma cells, and when the two are added, the inhibition effect is stronger. Wherein A is the OTUD1 overexpression cell line constructed on H827 cells and a control cell line Vector thereof, 0.1 mu M VE-822 stimulation is given, and the cell proliferation capacity is detected; b, an OTUD1 overexpression cell line and a control cell line Vector thereof are constructed on H2030 cells, and the cell proliferation capacity is tested by giving 0.1 mu M VE-822 stimulation.
FIG. 4 shows that VE-822 and OTUD1 can inhibit migration and invasion of lung adenocarcinoma cell H827, and the inhibition effect is stronger when the VE-822 and OTUD1 are added. Wherein A is H827-Vector cells, and the scratch test proves that the migration capacity of the cells is weakened under the stimulation of VE-822; b is H827-OTUD1 cells, and the scratch experiment proves that the migration capability of the cells is weakened and weaker than that in A under the stimulation of VE-822; c is H827-Vector cells, and transwell migration experiments prove that the migration capacity of the cells is weakened under the stimulation of VE-822; d is H827-OTUD1 cells, and transwell migration experiments prove that the migration capacity of the cells is weakened and weaker than that in A under the stimulation of VE-822; e is H827-Vector cells, and the invasion capacity of the cells is weakened through a transwell invasion experiment under the stimulation of VE-822; f is H827-OTUD1 cells under the stimulation of VE-822, and the transwell invasion experiment proves that the invasion capacity of the cells is weakened and weaker than that in A.
FIG. 5 shows that VE-822 and OTUD1 can inhibit migration and invasion of lung adenocarcinoma cells H2030, and the inhibition effect is stronger when the VE-822 and OTUD1 are added. Wherein A is H2030-Vector cells, and the scratch experiment proves that the migration capacity of the cells is weakened under the stimulation of VE-822; b is H2030-OTUD1 cells, and the scratch experiment proves that the migration capability of the cells is weakened and weaker than that in A under the stimulation of VE-822; c is H2030-Vector cells, and transwell migration experiments prove that the migration capacity of the cells is weakened under the stimulation of VE-822; d is H2030-OTUD1 cells, and transwell migration experiments prove that the migration capacity of the cells is weakened and weaker than that in A under the stimulation of VE-822; e is H2030-Vector cells, and transwell invasion experiments prove that the invasion capacity of the cells is weakened under the stimulation of VE-822. F is H2030-OTUD1 cells, and transwell invasion experiments prove that the invasion capacity of the cells is weakened and weaker than that of the cells in A under the stimulation of VE-822.
FIG. 6 shows that VE-822 inhibits the proliferation of lung adenocarcinoma cell lines in nude mice. Nude mice subcutaneous injection of 5x10 6 H827-Vector or H827-OTUD1 lung adenocarcinoma cells were injected two weeks later with administration of VE-822 (60 mg/kg) by gavage, four days per week, three days off, three weeks for three weeks, and five weeks after injection. Wherein A is a gross photo of a nude mouse; b is a photograph of the tumor volume of the nude mice; c is a statistical graph of tumor volumes of nude mice; d is a line graph of the change process of the tumor volume of the nude mice.
Figure 7 shows that OTUD1 mRNA expression is down-regulated in lung adenocarcinoma. Wherein A is the expression quantity change of OTUD1 mRNA in various tumors in a TCGA database; B-D is an oncomine database, different database results are integrated, and in different lung adenocarcinoma databases, compared with normal tissues, the OTUD1 mRNA expression quantity in various lung adenocarcinoma tissues changes; e is a survival curve of the relationship between the OTUD1 expression quantity and the survival rate in the kmplot database.
FIG. 8 shows that OTUD1 protein is down-regulated in patient samples and human lung adenocarcinoma cell lines. Wherein A is the change of OTUD1 protein expression quantity in lung adenocarcinoma tissues in a patient sample detected by a Western Blot experiment compared with self normal control tissues; and B, a Western Blot experiment is used for detecting the change of the OTUD1 protein expression quantity in various lung adenocarcinoma cell lines compared with normal bronchial epithelial cells and 293T cells.
Figure 9 demonstrates that FHL1 expression is down-regulated in lung adenocarcinoma. Wherein A is an IP-MS result of OTUD1, the gene names of all proteins are searched one by one in an onconine tumor database, and the proteins which are down-regulated in lung adenocarcinoma and the reduction times thereof are obtained; B. c and D are changes of FHL1 mRNA expression quantity of lung adenocarcinoma tissues and normal tissues in each lung adenocarcinoma database of oncomine; e is a survival curve of the relationship between the expression level and the survival rate of FHL1 in the kmplot database.
Figure 10 shows that OTUD1 stabilizes FHL1 protein expression. Wherein A is 293T cells transfected FHL1, and at the same time OTUD1 or CS is not transfected or transfected, MG132 is added after 36h of transfection, and the corresponding protein amount change is detected by Western Blot after 48h of transfection; b is 293T cells transfected FHL1, one group is transfected with a control plasmid (Vector), one group is transfected with OTUD1, CHX is added at a designated time before collection, and Western Bloy is used for detecting the corresponding protein quantity change. C is 293T cell transfection vector or OTUD1, cycloheximide CHX is added before cell collection, and a western blot experiment detects the influence of OTUD1 on the half-life of FHL1 protein.
Figure 11 shows that OTUD1 reduces ubiquitination of FHL 1. Wherein A is in 293T cells, one group of two holes are transfected with FHL1-Myc, and are transfected or not transfected with OTUD1-Flag, the other group is transfected with OTUD1-Flag, and are transfected or not transfected with FHL1-Myc, samples are collected after 48h for carrying out a co-IP experiment, and the Flag or Myc antibodies are respectively used for immunoprecipitating protein, and the change of the corresponding protein amount is detected; b is a schematic diagram of a complete OTUD1 structure and structures of various truncated bodies; c is in 293T cell, transfection FHL1-Myc and OTUD1 and truncation body plasmid thereof, collecting sample after 48h and carrying out co-IP experiment, immunoprecipitating FHL1-Myc by using Flag antibody, and detecting the corresponding protein amount change; d is transfected Ub-HA and FHL1-Myc in 293T cells, and OTUD1 or CS is not transfected or transfected, MG132 (5 mu M) is added after 36h of transfection for 12h, and the ubiquitination level is detected by collecting samples.
The specific implementation mode is as follows:
in order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
VE-822 inhibits proliferation, migration and invasion of lung adenocarcinoma cells by activating OTUD 1. 1. VE-822 is capable of upregulating the expression of OTUD1 and FHL1 in lung adenocarcinoma cell lines.
Grouping experiments: VE-822, O, 0.1, 1, 5, 10, 15. Mu.M (total amount of each group supplemented with solvent DMSO)
H827 (figure 1A-B) or H2030 (figure 1C-D) lung adenocarcinoma cell lines in log phase are inoculated in 6-well plates, VE-822 with corresponding concentration is added after 12 hours, total cell protein and total RNA are respectively collected after 48 hours, western blot experiment is carried out to detect protein levels of OTUD1 and FHL1 (figure 1A and C), and qPCR experiment is carried out to detect mRNA level of OTUD1 (figure 1B and D). In conclusion, VE-822 can up-regulate the mRNA and protein levels of OTUD1 and can up-regulate the protein level of FHL 1.
2. VE-822 inhibits the proliferation of lung adenocarcinoma cell lines.
Grouping experiments: VE-822, O, 0.1, 1, 5, 10, 15. Mu.M (total amount of each group supplemented with solvent DMSO)
The log-phase H827 or H2030 lung adenocarcinoma cell lines were seeded in 96-well plates at 5000 cells per well, corresponding concentrations of VE-822 were added after 12 hours, and CCK-8 (Abclonal, RM 02823) experiments were performed after 48 hours to check cell viability. Mixing the CCK-8 reagent with a cell culture medium according to a ratio of 1 Experiment hole -OD Blank hole )/(OD Control well -OD Blank hole ) As shown in fig. 2A, VE-822 can inhibit the proliferation of H827 cells, and the higher the concentration is, the stronger the inhibitory ability is, and fig. 2B shows that VE-822 can inhibit the proliferation of H2030 cells, and the higher the concentration is, the stronger the inhibitory ability is.
3. VE-822 and OTUD1 can inhibit proliferation of lung adenocarcinoma cells, and the inhibition effect is stronger when the two are added. An OTUD1 over-expressing cell line and its control cell line Vector were constructed in H827 or H2030 lung adenocarcinoma cell lines, respectively.
Experimental grouping: vector; vector-VE-822; OTUD1; OTUD1-VE-822
Log phase cells were seeded in 96-well plates at 1000 cells per well, and after 12 hours 0.1 μ M VE-822 was added, after which cell viability was measured every 24 hours, as above. The results show that the cell viability is increased every day within 5 days, the OTUD1 overexpression can inhibit the cell proliferation, the VE-822 can also inhibit the cell proliferation, and the inhibition effect is stronger when the two are superposed, as shown in figures 3A-B.
4. Scratch experiments show that both VE-822 and OTUD1 can inhibit the migration capacity of H827 and H2030 lung adenocarcinoma cell lines, and the inhibition effect is stronger when the two are superposed.
Grouping experiments: H827-Vector + O μ M; H827-Vector + 0.1. Mu.M; H827-Vector + O.5. Mu.M;
H827-OTUD1+OμM；H827-OTUD1+0.1μM；H827-OTUD1+O.5μM
H2030-Vector+OμM；H2030-Vector+5μM；H2030-Vector+10μM；
H2030-OTUD1+OμM；H2030-OTUD1+5μM；H2030-OTUD1+10μM
(total amount of each group supplemented with DMSO solvent)
Inoculating logarithmic phase cells into a 6-hole plate, when the cell density reaches about 90%, scratching a cross-shaped scratch from left to right at the bottom of the hole plate from top to bottom by using a gun head, taking a scratch width picture at the same position, taking a picture at the same position after 1 day, and taking a picture at the same position after 2 days. For example, FIG. 4A, VE-822 can inhibit H827 cell migration, and for example, for FIGS. 4A and B, OTUD1 can inhibit H827 cell migration, and the inhibition effect is stronger when the two are added. As shown in fig. 5A and B, VE-822 and OTUD1 both inhibited H2030 cell migration, and when the two were added, the inhibition effect was stronger.
5. transwell migration experiments show that both VE-822 and OTUD1 can inhibit the migration capacity of H827 and H2030 lung adenocarcinoma cell lines, and the inhibition effect is stronger when the two are superposed.
Experimental grouping: H827-Vector + O.mu.M; H827-Vector + 0.1. Mu.M;
H827-OTUD1+OμM；H827-OTUD1+0.1μM
H2030-Vector+OμM；H2030-Vector+5μM；
H2030-OTUD1+OμM；H2030-OTUD1+5μM
(total amount of each group supplemented with DMSO solvent)
The log phase cells were resuspended in serum-free medium and seeded in the upper chamber of a transwell 24-well plate at a H827 cell density of 5X10 4 200 μ L/well, cell density of H2030 is 2X 10 4 Per 200. Mu.L/well. 600. Mu.l of complete medium was added to the lower chamber of a transwell 24-well plate. After culturing at 37 ℃ for 48 hours, the medium in the upper and lower chambers was aspirated, and the cells that did not migrate in the upper chamber were gently wiped off with a cotton swab. The chamber was returned, rinsed twice with PBS and fixed for 30 minutes in 4% paraformaldehyde solution. Absorbing formaldehyde, drying at room temperature, adding 600 mu l of crystal violet dye solution into each hole, and dyeing for 30 minutes at room temperature. And (5) cleaning by ddH2O for 3 times, airing at room temperature, observing and photographing under a microscope, and counting. It was observed that e.g. FIG. 4C, VE-822 were able to inhibit migration of H827 cells, e.g. FIGS. 4C and D, OTUD1 was able to inhibit migration of H827 cells, and the inhibition was stronger when the two were added. As shown in fig. 5C and D, VE-822 and OTUD1 both inhibited H2030 cell migration, and when the two were added, the inhibition effect was stronger.
6. transwell invasion experiments show that both VE-822 and OTUD1 can inhibit the invasion capacity of H827 and H2030 lung adenocarcinoma cell lines, and the inhibition effect is stronger when the two are added.
Experimental grouping: same as above
Taking out matrigel from-80 deg.C, thawing overnight in refrigerator at 4 deg.C, diluting matrigel with serum-free medium to 300 μ l/ml, adding 100ul of the matrigel onto the upper filter membrane (8 um filter membrane), standing in incubator at 37 deg.C for 1 hr, inoculating cells, and culturing at H827 cell density of 5 × 10 4 At a cell density of 3X 10, at a concentration of/200. Mu.L/well and H2030 4 200 μ L/well, the subsequent steps are as above. As a result, for example, FIG. 4E, VE-822 can inhibit the invasion of H827 cells, and for example, for FIGS. 4E and F, OTUD1 can inhibit the invasion of H827 cells, and the inhibition effect is stronger when the two are superposed. As shown in FIGS. 5E and F, VE-822 and OTUD1 both inhibited H2030 cell invasion, and when the two were added, the inhibition effect was stronger.
7. The nude mouse tumorigenesis experiment shows that the VE-822 can inhibit the proliferation of the lung adenocarcinoma cell line in the nude mouse.
Grouping experiments: H827-Vector +0mg/kg; H827-Vector +60mg/kg;
H827-OTUD1+0mg/kg；H827-OTUD1+60mg/kg
selecting 4-6 week old male nude mice, injecting H827-Vector or H827-OTUD1 lung adenocarcinoma cells 5x10 subcutaneously in the middle and back of two side armpits 6 One dose per 200 μ l, VE-822 (60 mg/kg) was administered by intragastric administration two weeks after injection, four days per week, three days after withdrawal, three weeks for three weeks, five weeks after injection and material selection. During the period, the length and width of the subcutaneous tumor (the longest axis is long, and the width is measured at the place where the length is uniform in the vertical direction) of the nude mouse were measured with a vernier caliper, and the tumor volume was calculated, V =1/2 × length × width 2 Measured twice a week. The experiment is ended about 5 weeks (or when the tumor volume is greater than 2000 mm) 3 Or the experiment is finished when the weight loss exceeds 20%), the mice are anesthetized and photographed, subcutaneous tumors are separated, and the mice are placed in groups and photographed. Referring to FIG. 6, the graph A is a gross photo of the nude mice, B is a photo of the tumor volume of the nude mice at the end, C is a statistical graph of the tumor volume of the nude mice, and D is a line graph of the change process of the tumor volume of the nude mice. The results show that both VE-833 and OTUD1 can inhibit the proliferation of tumor cells in nude mice, and the inhibition effect is stronger when the VE-833 and OTUD1 are added.
8. The OTUD1 expression is down-regulated in lung adenocarcinoma, and the OTUD1 expression quantity is positively correlated with the survival rate.
(1) The expression of OTUD1 in various types of tumors in the TCAG database was analyzed in the UALCAN website (http:// UALCAN. Path. Uab. Edu/index. Html), as shown in FIG. 7A, the expression of OTUD1 was downregulated in most tumors, especially in lung adenocarcinoma and lung squamous carcinoma. Three lung adenocarcinoma databases were further found in the onconine tumor database (http:// www. Onconine. Org/resource/logic. Html), as shown in fig. 7B-D, the expression level of OTUD1 in lung adenocarcinoma was significantly lower than that in the normal group. In order to further explore the relationship between OTUD1 and prognosis, the relationship between the OTUD1 expression level and the survival rate of lung adenocarcinoma patients is searched in a kmplot database (https:// kmplot. Com/analysis /), as shown in figure 7E, the OTUD1 expression level is positively correlated with the survival rate. In conclusion, the database analysis result shows that the OTUD1 expression is down-regulated in lung adenocarcinoma, and the prognosis is better when the OTUD1 expression is high.
(2) 12 clinical lung adenocarcinoma samples were collected, and in each case, a para-carcinoma tissue (N) and a carcinoma tissue (T) were taken, and western blot analysis was performed to detect the protein expression level of OTUD1, as shown in fig. 8a, the expression of OTUD1 in lung adenocarcinoma tissues was significantly reduced. In addition, 8 lung adenocarcinoma cell lines (H460, a549, SPCA1, H1299, H2030, H827, H1975, H292), 1 bronchial epithelial cell (BEAS-2B) and 1 embryonic kidney cell (293T) were collected, and the protein expression amount of OTUD1 was examined by western blot assay, as shown in fig. 8B, the protein expression of OTUD1 in the lung adenocarcinoma cell lines was significantly lower than that of normal bronchial epithelial cells or embryonic kidney cells. In conclusion, at the level of tissues and cell lines of patients, the expression level of OTUD1 in lung adenocarcinoma is significantly reduced.
9. FHL1 is down-regulated in lung adenocarcinoma, and the expression quantity of the FHL1 is positively correlated with the survival rate.
(1) Protein immunoprecipitation and mass spectrometry (IP-MS).
The 293T tool cells in the logarithmic phase are inoculated to a 10cm cell culture dish, the OTUD1-Flag plasmid is transfected after 12 hours, the fresh culture medium is replaced after 6 hours of transfection, and the cells are collected after 48 hours of transfection for IP experiment. The cell pellet was collected and the cells resuspended in 1ml IP buffer, lysed on ice for 15 min, sonicated on ice (4 s/1s,25%,1 min) and lysed on ice for 15 min. Followed by centrifugation at 12000rpm for 15 minutes at 4 ℃. The supernatant was collected, 60. Mu.l of the supernatant was used as input, and the remaining supernatant was added with 100. Mu.l of beads and 1. Mu.l of Flag antibody, and incubated overnight at 4 ℃ with windmill rotation. After centrifugation at 1000rpm for 1 minute at 4 ℃ the supernatant was discarded and 1ml of IP buffer was added to wash the beads 3 times. The final 4 ℃ 1000rpm centrifugation for 1 minutes, abandoning the supernatant, to the beads added in the loading buffer 75 u l, as IP. And (3) carrying out SDS-page gel electrophoresis on the input and IP samples, and cutting the gel when the bromophenol blue electrophoresis is about 1 cm below the separation gel for mass spectrometry detection. The mass spectrum detected 122 proteins, which we counted in the oncomine database for changes in lung adenocarcinoma, as shown in fig. 9A, where FHL1 is the most significantly reduced protein in lung adenocarcinoma.
(2) The database analyzed the changes in FHL1 expression in lung adenocarcinoma. Three lung adenocarcinoma databases were further found in the oncomine database, as shown in fig. 9B-D, with FHL1 expressed in lung adenocarcinoma at significantly lower levels than in the normal group. To further explore the relationship between FHL1 and prognosis, a Kmplot database was searched for the relationship between the expression level of FHL1 and the survival rate of patients with lung adenocarcinoma, as shown in FIG. 9E, the expression level of FHL1 was positively correlated with the survival rate. In conclusion, the database analysis result shows that the expression of FHL1 is down-regulated in lung adenocarcinoma, and the prognosis is better for patients with high FHL1 expression.
10. OTUD1 Stable FHL1 protein expression
(1) OTUD1 has no effect on mRNA level of FHL1
The 293T tool cells in the logarithmic phase are inoculated to a 6-well plate, vector, OTUD1 or OTUD1-CS (deubiquitinating enzyme activity site mutant) is transfected after 6 hours, fresh culture medium is replaced after 6 hours of transfection, cells are collected after 48 hours of transfection, total RNA is extracted and is reversely transcribed into cDNA to carry out qPCR experiment to detect the influence of OTUD1 on the mRNA level of FHL1, for example, the graph is shown in figure 10A, OTUD1 and the enzyme activity site mutant OTUD1-CS thereof have no influence on the mRNA level of FHL 1.
(2) OTUD1 stabilizes protein levels of FHL1
293T tool cells in log phase are inoculated to a 6-well plate, vector, OTUD1 or OTUD1-CS (deubiquitinase activity site mutant) or MG132 (5 mu M for 12 hours) is added after 6 hours of transfection, fresh culture medium is replaced after 6 hours of transfection, cells are collected after 48 hours of transfection, and total protein is extracted to carry out western blot experiment to detect the influence of OTUD1 on the protein level of FHL1, for example, as shown in FIG. 10B, OTUD1 can stabilize the protein expression of FHL1, and OTUD1-CS loses the capability of stabilizing FHL 1.
(3) OTUD1 can prolong half-life of FHL1 protein
293T tool cells in log phase are inoculated into a 6-well plate, vector or OTUD1 is transfected after 6 hours, fresh culture medium is replaced after 6 hours of transfection, cells are collected after 48 hours of transfection, and cycloheximide CHX (50 mu M treatment for 0, 30, 60 and 90 minutes respectively) is added before collection of cells to inhibit protein synthesis. Total protein was extracted and subjected to western blot experiment to detect the effect of OTUD1 on half-life of FHL1 protein, as shown in FIG. 10C, OTUD1 can prolong half-life of FHL1 protein. Taken together, OTUD1 was able to stabilize protein levels of FHL1, and this ability was dependent on its deubiquitinating enzyme activity.
11. OTUD1 can enable FHL1 to be subjected to deubiquitination modification
(1) Interaction between OTUD1 and FHL1 protein
Experimental grouping: transfection of Vector-Flag + FHL1-Myc, IP Flag
Transfection of OTUD1-Flag + FHL1-Myc, IP: flag
Transfection of OTUD1-Flag + Vector-Myc, IP: myc
Transfection of OTUD1-Flag + FHL1-Myc, IP: myc
And (3) inoculating 293T tool cells in a logarithmic phase to a 6cm cell culture dish, transfecting after 12 hours according to the group, replacing a fresh culture medium after 6 hours of transfection, and collecting the cells after 48 hours of transfection for IP experiment. The cell pellet was collected and resuspended in 500ul IP buffer, lysed on ice for 15 min, sonicated on ice (4 s/1s,25%,1 min) and lysed on ice for 15 min. Followed by centrifugation at 12000rpm for 15 minutes at 4 ℃. Taking the supernatant, taking 50ul as input, adding 40 mu l of beads and 0.5 mu l of Flag antibody into the first group and the second group of the residual supernatant, adding 40 mu l of beads and 0.5 mu l of Myc antibody into the third group and the fourth group of the residual supernatant, and placing the mixture at 4 ℃ for windmill-rotary incubation overnight. After centrifugation at 1000rpm for 1 minute at 4 ℃ the supernatant was discarded and 1ml of IP buffer was added to wash the beads 3 times. The final centrifugation at 1000rpm for 1min at 4 ℃ was performed, the supernatant was discarded, and 50. Mu.l of loading buffer was added to the beads as IP. The input and IP samples were tested in a western blot assay, as shown in FIG. 11A, for the presence of interaction between OTUD1 and FHL 1.
(2) OTUD1 interacts with FHL1 via its OUT domain
As shown in FIG. 11B, OTUD1 consists of four domains of Ala-rich, linker, OTU and UIM. We respectively construct truncation bodies OTUD1-A, B, C and D with deletion of each structural domain, and explore the interaction relation between the OTUD1 truncation body and FHL 1.
Experimental grouping: transfection of Vector-Flag + FHL1-Myc, IP Flag
Transfection of OTUD1-WT-Flag + FHL1-Myc, IP Flag
Transfection of OTUD1-A-Flag + FHL1-Myc, IP: flag
Transfection of TUD1-B-Flag + FHL1-Myc, IP Flag
Transfection of OTUD1-C-Flag + FHL1-Myc, IP: flag
Transfection of OTUD1-D-Flag + FHL1-Myc, IP: flag
And (3) inoculating 293T tool cells in a logarithmic phase to a 6cm cell culture dish, transfecting according to the grouping after 12 hours, replacing a fresh culture medium after 6 hours of transfection, and collecting the cells after 48 hours of transfection to perform an IP (Internet protocol) experiment according to the scheme. The results are shown in FIG. 11C, where OTUD1-C truncation does not interact with FHL1, indicating that OTUD1 allows interaction with FHL1 through its OUT structure.
(3) OTUD1 can enable FHL1 to be subjected to deubiquitination modification
Experimental grouping: transfection of Vector-Flag + FHL1-Myc + Ub-HA + MG 1325. Mu.M 12h, IP
Transfection of OTUD1-WT-Flag + FHL1-Myc + Ub-HA + MG 1325. Mu.M 12h, IP
Transfection of OTUD1-CS-Flag + FHL1-Myc + Ub-HA + MG 1325. Mu.M 12h, IP
And (3) inoculating 293T tool cells in a logarithmic phase to a 6cm cell culture dish, transfecting according to the grouping after 12 hours, replacing a fresh culture medium after 6 hours of transfection, adding 5 mu M MG132 after 36 hours of transfection, and collecting the cells after 12 hours to perform an ubiquitination experiment. The cell pellet was collected, the cells were resuspended in 50. Mu.l of IP buffer, 6. Mu.l of 10% SDS was added thereto and the mixture was homogenized, followed by denaturation at 95 ℃ for 30 minutes. Subsequently 500. Mu.l of IP buffer was added and sonicated on ice (4 s/1s,25%,1 min). Followed by centrifugation at 12000rpm for 15 minutes at 4 ℃. The supernatant was collected, 60. Mu.l was used as input, and the remaining supernatant was added with 60. Mu.l of beads and 1. Mu.l of Myc antibody, and incubated overnight at 4 ℃ with windmill rotation. After centrifugation at 1000rpm for 1 minute at 4 ℃ the supernatant was discarded and 1ml of IP buffer was added to wash the beads 3 times. The final centrifugation at 4 ℃ for 1min at 1000rpm was performed, the supernatant was discarded, and 75. Mu.l of loading buffer was added to the beads as IP. The ubiquitination level of FHL1-Myc is detected by carrying out a western blot experiment on input and IP samples, as shown in figure 11D, OTUD1 can reduce the ubiquitination level of FHL1, and the mutant of the active site of deubiquitinating enzyme loses the ability.
In conclusion, the invention discusses the specific action mechanism of ATR inhibitor VE-822 for inhibiting lung adenocarcinoma from levels of molecules, cells, animals and the like, provides that ATR inhibitor VE-822 can activate deubiquitinase OTUD1 besides being used as an inhibitor of ATR signal channel, inhibit proliferation, migration and invasion of lung adenocarcinoma cells, inhibit growth of nude mouse subcutaneous lung adenocarcinoma, clarifies the molecular mechanism that VE-822 activates deubiquitinase OTUD1 so as to deubiquitize and stabilize FHL1 and further inhibit lung adenocarcinoma, and indicates that VE-822 can activate deubiquitinase OTUD1 for preparing the medicine for treating lung adenocarcinoma; in addition, the invention also provides that the deubiquitinase OTUD1 can be used as a lung adenocarcinoma molecular diagnosis marker or a target molecule screening lung adenocarcinoma molecular diagnosis marker, and the deubiquitinase OTUD1 can be used as a detection index for evaluating lung adenocarcinoma or used as a target molecule screening lung adenocarcinoma prevention and treatment medicine, so that a new molecular target and a treatment strategy are provided for clinically preventing or treating lung adenocarcinoma.
Finally, it should be noted that the above examples are only used for illustrating the present invention and do not limit the protection scope of the present invention. Further, various alterations, modifications and variations may be made by those skilled in the art after reading the technical content of the present invention, and all such equivalents may fall within the scope of the protection defined by the claims of the present application.
Claims (10)
- The application of the ATR inhibitor VE-822 in treating lung adenocarcinoma is characterized in that the drug target of the ATR inhibitor VE-822 is deubiquitinase OTUD1, and proliferation, migration and invasion of lung adenocarcinoma cells are inhibited by activating deubiquitinase OTUD 1.
- 2. The use according to claim 1, wherein the deubiquitinase OTUD1 inhibits lung adenocarcinoma cell proliferation, migration and invasion by modulating the stability of FHL1 protein.
- 3. The use according to claim 1, wherein the deubiquitinase OTUD1 is a deubiquitinase of FHL1 protein in lung adenocarcinoma, which interacts with FHL1 protein through OTU structural domain and stabilizes the expression of FHL1 protein through enzyme active site C320 deubiquitinating FHL1 protein.
- 4. The use of claim 3, wherein the FHL1 protein is expressed in a sample of a patient with lung adenocarcinoma in a down-regulated manner, the expression level of the FHL1 protein is positively correlated with the survival rate of the patient with lung adenocarcinoma, and the deubiquitinase OTUD1 is involved in the occurrence and development of lung adenocarcinoma by stabilizing the expression of the FHL1 protein.
- 5. The application of the deubiquitinase OTUD1 as a target molecule for screening lung adenocarcinoma prevention and treatment medicines is characterized in that the medicines comprise gene therapy medicines of over-expression of the deubiquitinase OTUD1 or activators or analogues of the deubiquitinase OTUD 1.
- 6. The use of claim 5, wherein the drug is a compound of VE-822, or a pharmaceutically acceptable salt thereof, in combination with an additional therapeutic agent.
- 7. The use of claim 6, wherein the medicament is configured as a gel, transdermal patch, injectable fluid, pill, capsule.
- 8. Application of deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnostic marker.
- 9. Application of deubiquitinase OTUD1 as a target molecule for screening molecular diagnostic markers of lung adenocarcinoma.
- 10. The deubiquitinase OTUD1 is used as a detection index to evaluate lung adenocarcinoma.
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| CN114686461A (en) * | 2022-04-08 | 2022-07-01 | 南阳理工学院 | Application of deubiquitinase USP45 in preparation of lung squamous carcinoma treatment drug |
| CN114686461B (en) * | 2022-04-08 | 2023-07-18 | 南阳理工学院 | Application of deubiquitinase USP45 in preparation of medicines for treating lung squamous carcinoma |
| CN116173218A (en) * | 2023-04-28 | 2023-05-30 | 北京大学口腔医学院 | Use of protein transport inhibitors for the preparation of a medicament for the treatment or alleviation of periodontitis |
| CN116173218B (en) * | 2023-04-28 | 2023-08-04 | 北京大学口腔医学院 | Use of protein transport inhibitors for the preparation of a medicament for the treatment or alleviation of periodontitis |
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