CN115300512B - Use of ATR inhibitor VE-822 in the treatment of lung adenocarcinoma - Google Patents
Use of ATR inhibitor VE-822 in the treatment of lung adenocarcinoma Download PDFInfo
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Abstract
The present invention relates to the use of ATR inhibitor VE-822 in the treatment of lung adenocarcinoma. The invention discusses the specific action mechanism of ATR inhibitor VE-822 in inhibiting lung adenocarcinoma from the level of molecules, cells, animals and the like. And it is proposed that ATR inhibitor VE-822 can activate deubiquitinase OTUD1 besides being used as an inhibitor of ATR signal path, inhibit proliferation, migration and invasion of lung adenocarcinoma cells and inhibit growth of subcutaneous lung adenocarcinoma of nude mice, so that VE-822 can activate deubiquitinase OTUD1 for treating lung adenocarcinoma.
Description
Technical field:
the invention relates to the technical field of biological medicines, in particular to application of an ATR inhibitor VE-822 in treating lung adenocarcinoma.
The background technology is as follows:
cancer is a leading cause of death worldwide. According to the report of the world health organization (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, cancer is increasingly prominent as the leading cause of death.
At present, great progress is made in the development of antitumor drugs, particularly in the fields of immunotherapy and targeted therapy. Targeted therapies for many cancers are small molecule targeted drugs or monoclonal antibodies that inhibit the activity of proteins that promote tumor growth. However, tumor cells develop resistance by over-expression of the target protein and/or by obtaining new mutations in the target protein. Accordingly, researchers have begun to explore alternative therapies that modulate protein function by modulating the expression levels of proteins rather than protein activity. Among them, ubiquitin-protease system (UPS) plays an important role in regulating protein expression level, and deubiquitinase is an important component of UPS.
VE-822 is an ATR inhibitor, which is usually activated for single strand breaks or in response to general replication stress. ATR inhibitors consume nucleotides, interfering with DNA replication by promoting cleavage of the arrested replication fork and/or by inhibiting its repair. VE-822 inhibits DNA damage repair, inhibiting tumor progression by inducing apoptosis. Phase I clinical experiments 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, there has been no study on the mechanism of action of VE-822 on deubiquitinase. The invention illustrates a molecular mechanism of VE-822 targeting deubiquitinase OTUD1 for inhibiting lung adenocarcinoma, and provides a novel molecular target and a treatment strategy for clinically preventing or treating lung adenocarcinoma.
The invention comprises the following steps:
(one) solving the technical problems
Against the background, the invention explores the specific action mechanism of ATR inhibitor VE-822 for inhibiting lung adenocarcinoma from the level of molecules, cells, animals and the like. Besides being used as an inhibitor of an ATR signal path, the ATR inhibitor VE-822 can activate the deubiquitinase OTUD1, inhibit proliferation, migration and invasion of lung adenocarcinoma cells and inhibit growth of subcutaneous lung adenocarcinoma of nude mice, and the molecular mechanism of VE-822 targeting the deubiquitinase OTUD1 for inhibiting lung adenocarcinoma is clarified, so that VE-822 can activate the deubiquitinase OTUD1 for preparing medicines for treating lung adenocarcinoma, and a novel molecular target and a treatment strategy are provided for clinical prevention or treatment of lung adenocarcinoma.
(II) technical scheme
In order to solve the technical problems, the invention adopts the following technical scheme: use of ATR inhibitor VE-822 in treating lung adenocarcinoma, wherein 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 deubiquitinase OTUD 1. The VE-822 has the following structural formula:
further, the deubiquitinase OTUD1 inhibits lung adenocarcinoma cell proliferation, migration and invasion by modulating the stability of FHL1 protein.
Further, the deubiquitinase OTUD1 is a deubiquitinase of FHL1 protein in lung adenocarcinoma, interacts with FHL1 protein through OTU domain, and deubiquitinates FHL1 protein through enzyme active site C320, stabilizing expression of FHL1 protein.
Further, the FHL1 protein is expressed down in lung adenocarcinoma patient samples, the expression level of the FHL1 protein is positively correlated with the survival rate of lung adenocarcinoma patients, and the FHL1 protein deficiency can promote proliferation, migration and invasion of lung adenocarcinoma cells.
The invention also provides application of the deubiquitinase OTUD1 as a target molecule in screening lung adenocarcinoma prevention and treatment drugs, wherein the drugs comprise gene therapy drugs for over-expression of the OTUD1 or activators or analogues of the OTUD1
Further, the compound of which the drug is VE-822, or a pharmaceutically acceptable salt thereof, is combined with an additional therapeutic agent.
Further, the medicament is formulated into gel, transdermal patch, injectable fluid, pill, capsule.
In addition, the invention also provides application of the deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnostic marker.
The application of deubiquitinase OTUD1 as a target molecule for screening lung adenocarcinoma molecular diagnostic markers.
Furthermore, the invention also provides application of the deubiquitinase OTUD1 serving as a detection index in lung adenocarcinoma treatment.
The invention discusses the effect and specific mechanism of VE-822 in treating lung adenocarcinoma from multiple levels such as molecular, cellular and animal levels. Studies have shown that VE-822 activates the deubiquitinase OTUD1, which acts as a deubiquitinase for FHL1 protein in lung adenocarcinoma, and through interaction of the OTU domain with FHL1 protein and through enzyme active site C320 deubiquitination of FHL1 protein, the expression of FHL1 protein is stabilized, thereby achieving inhibition of lung adenocarcinoma cell proliferation, migration and invasion. In addition, the invention also provides application of the deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnostic marker, or application of the deubiquitinase OTUD1 as a target molecule screening lung adenocarcinoma molecular diagnostic 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 screening lung adenocarcinoma prevention and treatment drug.
(III) beneficial effects
The invention has the beneficial effects that: the invention discovers that VE-822 can treat lung adenocarcinoma by targeting and activating the deubiquitinase OTUD1, and discusses the specific action mechanism of the ATR inhibitor VE-822 for inhibiting lung adenocarcinoma from the levels of molecules, cells, animals and the like. Besides being used as an inhibitor of an ATR signal path, the ATR inhibitor VE-822 can activate the deubiquitinase OTUD1, inhibit proliferation, migration and invasion of lung adenocarcinoma cells and inhibit growth of subcutaneous lung adenocarcinoma of nude mice, and the molecular mechanism of VE-822 targeting the deubiquitinase OTUD1 for inhibiting lung adenocarcinoma is clarified, so that VE-822 can activate the deubiquitinase OTUD1 for preparing medicaments for treating lung adenocarcinoma. In addition, the invention also provides the application of the deubiquitinase OTUD1 as a lung adenocarcinoma molecular diagnosis marker, or the application of the deubiquitinase OTUD1 as a target molecule screening lung adenocarcinoma molecular diagnosis marker, and the application of the deubiquitinase OTUD1 as a detection index in evaluating lung adenocarcinoma, or the application of the deubiquitinase OTUD1 as a target molecule screening lung adenocarcinoma prevention and treatment drug, thereby providing a new molecular target and treatment strategy for clinical prevention or treatment of lung adenocarcinoma.
Description of the drawings:
in order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows that VE-822 is capable of up-regulating the expression of OTUD1 and FHL1 in lung adenocarcinoma cell lines. Wherein A is the protein level change of OTUD1 and FHL1 after H827 cells are treated for 48 hours with VE-822 at different concentrations; mRNA levels of OTUD1 were varied 48 hours after H827 cells were treated with different concentrations of VE-822; protein levels of OTUD1 and FHL1 were varied 48 hours after H2030 cells were treated with different concentrations of VE-822; mRNA levels of OTUD1 were varied 48 hours after treatment of H2030 cells with different concentrations of VE-822.
FIG. 2 shows that VE-822 inhibits proliferation of lung adenocarcinoma cell lines. Wherein A is the change in cell number after H827 cells were treated for 48 hours at different concentrations of VE-822; b is the change in cell number 48 hours after treatment of H2030 cells with different concentrations of VE-822.
FIG. 3 shows that VE-822 and OTUD1 can inhibit proliferation of lung adenocarcinoma cells, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped. Wherein A is the construction of an OTUD1 over-expression cell line and a control cell line Vector thereof on H827 cells, and the proliferation capacity of the cells is detected by applying 0.1 mu M of VE-822 stimulation; b is the construction of an OTUD1 over-expression cell line and a control cell line Vector thereof on H2030 cells, and the proliferation capacity of the cells is tested by applying 0.1 mu M of VE-822 stimulation.
Figure 4 shows that VE-822 and OTUD1 can inhibit migration and invasion of lung adenocarcinoma cells H827, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped. Wherein A is H827-Vector cells, and a scratch experiment proves that the migration capacity of the cells is reduced under the stimulation of VE-822; b is H827-OTUD1 cells under the stimulation of VE-822, and scratch experiments prove that the migration capacity of the cells is reduced and weaker than that of the cells in A; the migration capacity of H827-Vector cells is reduced by a transwell migration experiment under the stimulation of VE-822; d is H827-OTUD1 cells under the stimulation of VE-822, and a transwell migration experiment proves that the migration capacity of the cells is reduced and weaker than that of the cells in A; e is H827-Vector cells under the stimulation of VE-822, and a transwell invasion experiment proves that the invasion capacity of the cells is reduced; f is H827-OTUD1 cells under the stimulation of VE-822, transwell invasion experiments prove that the invasion capacity of the cells is reduced and weaker than that of A.
Figure 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 the OTUD1 are overlapped. Wherein, A is H2030-Vector cells, and a scratch experiment proves that the migration capacity of the cells is reduced under the stimulation of VE-822; b is H2030-OTUD1 cells, under the stimulation of VE-822, the scratch experiment proves that the migration capacity of the cells is reduced and weaker than that of the cells in A; the migration capacity of the cells is reduced as proved by a transwell migration experiment of the cells with the C of H2030-Vector under the stimulation of VE-822; the migration test of the cells with D of H2030-OTUD1 under the stimulation of VE-822 proves that the migration capacity of the cells is weakened and weaker than that of the cells in A; e is H2030-Vector cells under the stimulation of VE-822, and a transwell invasion experiment proves that the invasion capacity of the cells is reduced. F is H2030-OTUD1 cells under the stimulation of VE-822, transwell invasion experiments prove that the invasion capacity of the cells is reduced and weaker than that of A.
FIG. 6 shows that VE-822 inhibits proliferation of lung adenocarcinoma cell lines in nude mice. Nude mice were subcutaneously injected 5x10 6 The cells of H827-Vector or H827-OTUD1 lung adenocarcinoma were given by gavage administration VE-822 (60 mg/kg) after two weeks of injection, four days per week, three days of withdrawal, three weeks of administration, and five weeks of injection. Wherein A is a general photograph of nude mice; b is a tumor volume photograph of the nude mice; c is a tumor volume statistical graph of the nude mice; d is a line graph of the tumor volume change process of the nude mice.
FIG. 7 shows that mRNA expression of OTUD1 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 the oncom database, which synthesizes different database results, and in different lung adenocarcinoma databases, compared with normal tissues, the OTUD1 mRNA expression quantity in various lung adenocarcinoma tissues is changed; e is a survival curve of the relation between the OTUD1 expression quantity and the survival rate in a kmlot database.
Figure 8 shows down-regulation of OTUD1 protein expression in patient samples and human lung adenocarcinoma cell lines. Wherein A is the change of the OTUD1 protein expression level in lung adenocarcinoma tissues compared with the normal control tissues in a patient sample detected by Western Blot experiments; b is Western Blot experiment to detect the change of OTUD1 protein expression in various lung adenocarcinoma cell lines compared with normal bronchial epithelial cells and 293T cells.
Figure 9 shows that FHL1 expression is down-regulated in lung adenocarcinoma. Wherein A is the result of IP-MS through OTUD1, the gene names of all proteins are searched one by one in the oncomine tumor database, wherein the proteins which are down-regulated in lung adenocarcinoma and the reduction times thereof are obtained; B. c, D is FHL1 mRNA expression level change of lung adenocarcinoma tissues and normal tissues in each lung adenocarcinoma database of oncomine; e is a survival curve of the relation between the expression quantity and the survival rate of FHL1 in a kmlot database.
FIG. 10 shows that OTUD1 stabilizes FHL1 protein expression. Wherein A is 293T cells transfected with FHL1, simultaneously, OTUD1 or CS is not transfected or transfected, MG132 is added after 36h of transfection, and corresponding protein quantity change is detected through Western Blot after 48h of transfection; b is FHL1 transfected into 293T cells, a group of transfected control plasmids (Vector), a group of transfected OTUD1, CHX is added at a designated time before sample collection, and Western Bloy detects the corresponding protein amount change. C is 293T cell transfected vector or OTUD1, cycloheximide CHX is added before collecting cells, and the influence of OTUD1 on FHL1 protein half-life is detected by a western blot experiment.
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, one group of two holes are transfected with or not transfected with OTUD1-Flag, the other group of two holes are transfected with or not transfected with OTUD1-Flag, the samples are collected after 48 hours for co-IP experiments, and protein immunoprecipitation is carried out by using Flag or Myc antibodies respectively, so that the corresponding protein amount change is detected; b is a complete OTUD1 structure and schematic diagram of each truncated structure; c is that in 293T cells, FHL1-Myc and OTUD1 and truncated plasmids thereof are transfected, samples are collected after 48 hours for carrying out a co-IP experiment, FHL1-Myc is immunoprecipitated by using Flag antibodies, and the corresponding protein amount change is detected; d is that Ub-HA and FHL1-Myc are transfected in 293T cells, OTUD1 or CS is not transfected or transfected, MG132 (5 mu M) is added for 12 hours after 36 hours of transfection, and the ubiquitination level is detected in a sampling way.
The specific embodiment is as follows:
in order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail 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 up-regulating the expression of OTUD1 and FHL1 in lung adenocarcinoma cell lines.
Experimental grouping: VE-822:O, 0.1, 1, 5, 10, 15. Mu.M (total amount of DMSO was used for each group)
The log phase H827 (FIGS. 1A-B) or H2030 (FIGS. 1C-D) lung adenocarcinoma cell line was inoculated in 6-well plates, VE-822 was added at corresponding concentrations after 12 hours, total cellular proteins and total RNA were collected after 48 hours, and protein levels of OTUD1 and FHL1 were detected by western blot experiments (FIGS. 1A and C), respectively, and mRNA levels of OTUD1 were detected by qPCR experiments (FIGS. 1B and D). In summary, VE-822 is capable of up-regulating the mRNA and protein levels of OTUD1 and of up-regulating the protein levels of FHL 1.
2. VE-822 inhibits proliferation of lung adenocarcinoma cell lines.
Experimental grouping: VE-822:O, 0.1, 1, 5, 10, 15. Mu.M (total amount of DMSO was used for each group)
Log phase H827 or H2030 lung adenocarcinoma cell lines were seeded in 96-well plates with 5000 cells per well, and after 12 hours, VE-822 was added at the corresponding concentrations, respectively, and after 48 hours CCK-8 (ablronal, RM 02823) experiments were performed to examine cell viability. Mixing CCK-8 reagent and cell culture medium at a ratio of 1:10, discarding the culture medium in 96-well plates, adding 100 μl of CCK-8 mixed solution into each well, incubating in a 37 ℃ incubator for 1 hr, detecting absorbance at 450nm with a microplate reader, and detecting cell viability= (OD Experimental hole -OD Blank hole )/(OD Control wells -OD Blank hole ) VE-822 was able to inhibit H827 cell proliferation as shown in FIG. 2A, and the higher the concentration, the more inhibitory activity, and FIG. 2B indicated that VE-822 was able to inhibit H2030 cell proliferation, and the higher the concentration, the more inhibitory activity.
3. VE-822 and OTUD1 can inhibit proliferation of lung adenocarcinoma cells, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped. OTUD1 overexpressing cell lines and their control cell lines 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 with 1000 cells per well and after 12 hours 0.1 μm VE-822 was added, after which cell viability was measured every 24 hours, as described above. The results showed that over-expression of OTUD1 inhibited cell proliferation and VE-822 inhibited cell proliferation within 5 days, with greater inhibition when added as shown in figures 3A-B.
4. Scratch experiments show that VE-822 and OTUD1 can inhibit migration capacity of H827 and H2030 lung adenocarcinoma cell lines, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped.
Experimental grouping: H827-vector+O.mu.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
(the total amount was made up with solvent DMSO for each group)
Inoculating cells in the logarithmic phase to a 6-pore plate, when the cell density reaches about 90%, marking out cross scratches from left to right and from top to bottom at the bottom of the pore plate by using a gun head, photographing the width of the scratches at the same position, photographing at the same position after 1 day, and photographing at the same position after 2 days. As shown in fig. 4A, ve-822 can inhibit H827 cell migration, as shown in fig. 4A and B, OTUD1 can inhibit H827 cell migration, and the inhibition effect is stronger when the two are overlapped. As shown in FIGS. 5A and B, VE-822 and OTUD1 can inhibit migration of H2030 cells, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped.
5. transwell migration experiments show that VE-822 and OTUD1 can inhibit migration capacity of H827 and H2030 lung adenocarcinoma cell lines, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped.
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
(the total amount was made up with solvent DMSO for each group)
The log phase cells were resuspended in serum-free medium and seeded in the upper chamber of a transwell 24-well plate with H827 cell density of 5X10 4 200. Mu.L/well H2030 cell density of 2X 10 4 200. Mu.L/well. 600 μl of complete medium was added to the lower chamber of the transwell 24-well plate. After culturing at 37℃for 48 hours, the medium in the upper and lower chambers was aspirated, and the cells not migrated in the upper chamber were gently scraped with a cotton swab. The cells were replaced, rinsed twice with PBS and the 4% paraformaldehyde solution was fixed for 30 minutes. Sucking formaldehyde, airing at room temperature, adding 600 μl of crystal violet dye solution into each hole, and dyeing at room temperature for 30 minutes. ddH2O is washed 3 times, is observed and photographed under a microscope after being dried at room temperature, and is counted. It was observed that VE-822 inhibited H827 cell migration as in FIG. 4C, and OTUD1 inhibited H827 cell migration as in FIGS. 4C and D, with greater inhibition when the two were added. As shown in fig. 5C and D, both VE-822 and OTUD1 can inhibit H2030 cell migration, and the inhibition effect is stronger when the two are superimposed.
6. transwell invasion experiments show that VE-822 and OTUD1 can inhibit invasion capacity of H827 and H2030 lung adenocarcinoma cell lines, and the inhibition effect is stronger when the VE-822 and the OTUD1 are overlapped.
Experimental grouping: as above
Taking out matrigel from-80deg.C, adding ice in refrigerator at 4deg.C overnight to melt, diluting matrigel with serum-free culture medium to 300 μl/ml, adding 100ul onto upper chamber filter membrane (8 um filter membrane), standing at 37deg.C for 1 hr, inoculating cells, and culturing at H827 cell density of 5×10 4 200. Mu.L/well H2030 cell density of 3X 10 4 200. Mu.L/well, followed by the same procedure. As a result, as shown in fig. 4E, ve-822 can inhibit H827 cell invasion, as shown in fig. 4E and F, OTUD1 can inhibit H827 cell invasion, and the inhibition effect is stronger when the two are superimposed. As shown in FIGS. 5E and F, VE-822 and OTUD1 both inhibit H2030 cell invasion, and the inhibition effect is stronger when the two are overlapped.
7. The nude mouse tumorigenesis experiment shows that VE-822 can inhibit proliferation of lung adenocarcinoma cell lines in the nude mouse.
Experimental grouping: h827-vector+0mg/kg; H827-Vector +60mg/kg;
H827-OTUD1+0mg/kg；H827-OTUD1+60mg/kg
selecting 4-6 week old male nude mice, and subcutaneously injecting H827-Vector or H827-OTUD1 lung adenocarcinoma cells 5x10 into the middle and rear parts of the axilla on both sides 6 Mu.l/200. Mu.l, annotateVE-822 (60 mg/kg) was administered by gavage two weeks later, four days per week, three days off, three weeks on, five weeks after injection. The length and width of the subcutaneous tumor of nude mice (the longest axis is long, and the length is measured as width at the uniform position in the vertical direction) were measured by a vernier caliper, the tumor volume was calculated, and v=1/2×length×width 2 Measurements were taken twice a week. The experiment was ended around 5 weeks (or when the tumor volume was greater than 2000mm 3 Or the weight reduction exceeds 20% to end the experiment), the mice are photographed after anesthesia, subcutaneous tumors are separated, and photographing is performed after grouping placement. See fig. 6, wherein fig. a is a general photograph of a nude mouse, B is a photograph of a tumor volume of the nude mouse at the end, C is a statistical graph of the tumor volume of the nude mouse, and D is a line graph of the tumor volume change process of the nude mouse. The results show that VE-833 or OTUD1 can inhibit proliferation of tumor cells in nude mice, and the inhibition effect is stronger when the VE-833 or OTUD1 is overlapped.
8. OTUD1 expression is down-regulated in lung adenocarcinoma, and OTUD1 expression level is positively correlated with survival rate.
(1) The expression of OTUD1 in TCAG databases was analyzed in the UALCAN website (http:// UALCAN. Path. Uab. Edu/index. Html) and down-regulated expression in various types of tumors, as shown in fig. 7a, OTUD1 was most significantly expressed in most tumors, especially lung adenocarcinoma and lung squamous carcinoma. Three lung adenocarcinoma databases were further found in the oncomine tumor database (http:// www.oncomine.org/resource/logic. Html), as shown in fig. 7B-D, the expression level of OTUD1 in lung adenocarcinoma was significantly lower than in the normal group. To further investigate the relationship between OTUD1 and prognosis, the relationship between OTUD1 expression level and survival rate of lung adenocarcinoma patients was found in a kmlot database (https:// kmlot. Com/analysis /), as shown in fig. 7e, OTUD1 expression level and survival rate were positively correlated. Taken together, the results of the database analysis indicate that OTUD1 expression is down-regulated in lung adenocarcinoma, and that OTUD1 expression is higher than that of the lung adenocarcinoma, the prognosis is better.
(2) 12 lung adenocarcinoma clinical samples were collected, and a beside-cancer tissue (N) and a cancer tissue (T) were taken for each sample, and the protein expression amount of OTUD1 was detected by western blot, 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 protein expression amount of OTUD1 was detected by western blot experiment, as in fig. 8B, protein expression of OTUD1 in lung adenocarcinoma cell lines was significantly lower than normal bronchial epithelial cells or embryonic kidney cells. In conclusion, the expression level of OTUD1 in lung adenocarcinoma was significantly reduced at the tissue and cell line level in patients.
9. FHL1 expression is down-regulated in lung adenocarcinoma, and FHL1 expression quantity is positively correlated with survival rate.
(1) Protein immunoprecipitation and mass spectrometry (immunoprecipitation and mass spectrometry, IP-MS).
The 293T tool cells in log phase were inoculated into 10cm cell culture dishes, the OTUD1-Flag plasmid was transfected after 12 hours, fresh medium was changed after 6 hours of transfection, and cells were collected for IP experiments after 48 hours of transfection. The cell pellet was collected and resuspended in 1ml IP buffer and lysed on ice for 15 min, sonicated on ice (4 s/1s,25%,1 min) and lysed on ice for another 15 min. Followed by centrifugation at 12000rpm at 4℃for 15 minutes. The supernatant was taken, 60. Mu.l was taken as input, and 100ul of beads and 1. Mu.l of Flag antibody were added to the remaining supernatant, and incubated overnight at 4℃with windmill rotation. Then, the mixture was centrifuged at 1000rpm at 4℃for 1 minute, and the supernatant was discarded, and 1ml of IP buffer was added to wash the beads 3 times. The final centrifugation at 1000rpm at 4℃for 1 minute, the supernatant was discarded, and 75. Mu.l of loading buffer was added to the beads as IP. And (3) carrying out SDS-page gel electrophoresis on input and IP samples, and cutting the gel to carry out mass spectrum detection when bromophenol blue is electrophoresed to about 1 cm below the separation gel. Mass spectrometry detected 122 proteins, we counted the changes in lung adenocarcinoma for these proteins in the oncomine database, as shown in fig. 9A, where FHL1 is the protein that was most significantly reduced in lung adenocarcinoma.
(2) The database analyzes the expression change of FHL1 in lung adenocarcinoma. Further, three lung adenocarcinoma databases were found in the oncom database, as shown in fig. 9B-D, the expression level of FHL1 in lung adenocarcinoma was significantly lower than in the normal group. To further explore the relationship between FHL1 and prognosis, the relationship between FHL1 expression and survival rate of lung adenocarcinoma patients was found in a kmlot database, as shown in FIG. 9E, the FHL1 expression and survival rate were positively correlated. Taken together, the database analysis results show that FHL1 expression is down-regulated in lung adenocarcinoma, and the prognosis of the patient with high FHL1 expression is better.
10. OTUD1 stable FHL1 frontal protein expression
(1) OTUD1 has no effect on FHL1 mRNA levels
The 293T tool cells in log phase were inoculated into 6-well plates, vector, OTUD1 or OTUD1-CS (deubiquitination enzyme active site mutant) were transfected after 6 hours, fresh medium was changed after 6 hours of transfection, cells were collected after 48 hours of transfection, total RNA was extracted and reverse transcribed into cDNA for qPCR experiments to examine the effect of OTUD1 on mRNA levels of FHL1, as in fig. 10a, OTUD1 and its enzyme active site mutant OTUD1-CS had no effect on mRNA levels of FHL 1.
(2) OTUD1 is capable of stabilizing the protein level of FHL1
The 293T tool cells in log phase were inoculated into 6-well plates, vector, OTUD1 or OTUD1-CS (deubiquitinase active site mutant) or MG132 (5 μm 12 hours) was added after 6 hours of transfection, fresh medium was changed after 6 hours of transfection, cells were collected after 48 hours of transfection, total proteins were extracted and subjected to western blot experiments to examine the effect of OTUD1 on the protein level of FHL1, as in fig. 10b, OTUD1 was able to stabilize the protein expression of FHL1, whereas OTUD1-CS lost the ability to stabilize FHL 1.
(3) OTUD1 can prolong half-life of FHL1 protein
The 293T tool cells in log phase were seeded in 6-well plates, vector or OTUD1 was transfected after 6 hours, fresh medium was changed after 6 hours of transfection, cells were collected after 48 hours of transfection, and cycloheximide CHX (50 μm treatment 0, 30, 60, 90 minutes, respectively) was added before the collection to inhibit protein synthesis. The influence of OTUD1 on the half-life of FHL1 protein is detected by extracting total protein for western blot experiment, as shown in figure 10C, the half-life of FHL1 protein can be prolonged by OTUD 1. In conclusion, OTUD1 is able to stabilize the protein level of FHL1 and this ability depends on its deubiquitinase activity.
11. OTUD1 enables the deubiquitination modification of FHL1
(1) Interaction of OTUD1 with FHL1 protein
Experimental grouping: transfection Vector-flag+FHL1-Myc, IP: flag
Transfection OTUD1-flag+FHL1-Myc, IP: flag
Transfection OTUD1-flag+vector-Myc, IP: myc
Transfection OTUD1-flag+FHL1-Myc, IP: myc
The 293T tool cells in log phase were seeded into 6cm cell culture dishes and transfected according to the above groups after 12 hours, fresh medium was changed after 6 hours of transfection and cells were collected for IP experiments after 48 hours of transfection. The cell pellet was collected and resuspended in 500ul IP buffer, and lysed on ice for 15 min, sonicated on ice (4 s/1s,25%,1 min) and further lysed on ice for 15 min. Followed by centrifugation at 12000rpm at 4℃for 15 minutes. The supernatant was taken, 50ul was taken as input, 40. Mu.l of beads and 0.5. Mu.l of Flag antibody were added to the first and second sets of supernatant, 40. Mu.l of beads and 0.5. Mu.l of Myc antibody were added to the third and fourth sets of supernatant, and the mixture was placed on a windmill at 4℃for incubation overnight. Then, the mixture was centrifuged at 1000rpm at 4℃for 1 minute, and the supernatant was discarded, and 1ml of IP buffer was added to wash the beads 3 times. The final centrifugation at 1000rpm at 4℃for 1 minute, the supernatant was discarded, and 50. Mu.l of loading buffer was added to the beads as IP. The input and IP samples were subjected to western blot experiments to detect the presence of interactions between OTUD1 and FHL1 as shown in FIG. 11A.
(2) OTUD1 interacts with FHL1 via its OUT domain
As shown in FIG. 11B, OTUD1 consists of four domains, ala-rich, linker, OTU, UIM. We constructed the truncations OTUD1-A, B, C, D deleted in each domain respectively, and explored the interaction relationship between the OTUD1 truncations and FHL 1.
Experimental grouping: transfection Vector-flag+FHL1-Myc, IP: flag
Transfection OTUD1-WT-flag+FHL1-Myc, IP: flag
Transfection OTUD1-A-flag+FHL1-Myc, IP: flag
Transfection TUD1-B-flag+FHL1-Myc, IP: flag
Transfection OTUD1-C-flag+FHL1-Myc, IP: flag
Transfection OTUD1-D-flag+FHL1-Myc, IP: flag
The 293T tool cells in log phase were seeded into 6cm cell culture dishes and transfected according to the above groupings after 12 hours, fresh medium was changed after 6 hours of transfection, and cells were collected after 48 hours of transfection for IP experiments according to the above protocol. The results are shown in FIG. 11C, where the OTUD1-C truncate did not interact with FHL1, indicating that OTUD1 caused interactions with FHL1 through its OUT structure.
(3) OTUD1 enables the deubiquitination modification of FHL1
Experimental grouping: transfection Vector-flag+FHL1-Myc+Ub-HA+MG 1325. Mu.M 12h, IP:Myc
Transfection OTUD1-WT-flag+FHL1-Myc+Ub-HA+MG 1325. Mu.M 12h, IP:Myc
Transfection OTUD1-CS-flag+FHL1-Myc+Ub-HA+MG 1325. Mu.M 12h, IP:Myc
The 293T tool cells in log phase were seeded in 6cm cell culture dishes, transfected according to the above groups after 12 hours, fresh medium was changed after 6 hours of transfection, 5. Mu.M MG132 was added after 36 hours of transfection, and cells were collected after 12 hours for ubiquitination experiments. The cell pellet was collected and resuspended in 50. Mu.l of IP buffer, and 6. Mu.l of 10% SDS was added thereto and mixed, and the mixture was denatured by heating at 95℃for 30 minutes. Subsequently, 500. Mu.l of IP buffer was added and the mixture was sonicated on ice (4 s/1s,25% for 1 min). Followed by centrifugation at 12000rpm at 4℃for 15 minutes. 60 μl of the supernatant was taken as input, 60 μl of beads and 1 μl Myc antibody were added to the remaining supernatant, and incubated overnight at 4deg.C with windmill. Then, the mixture was centrifuged at 1000rpm at 4℃for 1 minute, and the supernatant was discarded, and 1ml of IP buffer was added to wash the beads 3 times. The final centrifugation at 1000rpm at 4℃for 1 minute, the supernatant was discarded, and 75. Mu.l of loading buffer was added to the beads as IP. The ubiquitination level of FHL1-Myc was detected by western blot experiments with input and IP samples, as shown in FIG. 11D, OTUD1 was able to reduce the ubiquitination level of FHL1 and its deubiquitination enzyme active site mutant lost this ability.
In summary, the invention discusses the specific action mechanism of the ATR inhibitor VE-822 for inhibiting lung adenocarcinoma from the levels of molecules, cells, animals and the like, and proposes that the ATR inhibitor VE-822 can activate the deubiquitinase OTUD1, inhibit proliferation, migration and invasion of lung adenocarcinoma cells and inhibit growth of subcutaneous lung adenocarcinoma of nude mice besides being used as an inhibitor of an ATR signal path, and elucidates the molecular mechanism of VE-822 for activating the deubiquitinase OTUD1 to stabilize FHL1 and further inhibit lung adenocarcinoma, thus indicating that VE-822 can activate the deubiquitinase OTUD1 for preparing medicaments 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 used as a target molecule for screening lung adenocarcinoma molecular diagnosis markers, and the deubiquitinase OTUD1 can be used as a detection index for evaluating lung adenocarcinoma or used as a target molecule for screening lung adenocarcinoma prevention and treatment drugs, so that a novel molecular target and a treatment strategy are provided for clinical prevention or treatment of lung adenocarcinoma.
Finally, it should be noted that the above embodiments are only for illustrating the invention and not for limiting the scope of the invention. Further, after reading the technical content of the present invention, those skilled in the art may make various changes, modifications or variations to the present invention, and all such equivalent forms are also within the scope of protection defined by the claims of the present application.
Claims (1)
- Application of VE-822 and OTUD1 in preparation of medicines for inhibiting lung adenocarcinoma, wherein the CAS number of VE-822 is 1232416-25-9, and the molecular formula is C24H25N5O3S.
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| "The deubiquitinase OTUD1 inhibits non-small cell lung cancer progression by deubiquitinating and stabilizing KLF4";Xiaoyan Ma et al.;《Thorac Cancer.》;第13卷(第5期);第761-770页 * |
| "VE-822 upregulates the deubiquitinase OTUD1 to stabilize FHL1 to inhibit the progression of lung adenocarcinoma";Qi Zhang et al.;《Cellular Oncology》;第46卷(第4期);第1-27页 * |
| "去泛素化酶USP35促进非小细胞肺癌细胞迁移和侵袭";陈兆波等;《山东大学学报》;第60卷(第4期);第30-37页 * |
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