CN116942815A - Use of proton pump inhibitors in tumor immunotherapy - Google Patents

Abstract

The present invention provides the use of proton pump inhibitors in tumor immunotherapy.

Description

Use of proton pump inhibitors in tumor immunotherapy

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a proton pump inhibitor in tumor immunotherapy.

Background

Proton pump inhibitors (proton pump inhibitors, PPIs) include Omeprazole (Omeprazole), lansoprazole (Lansoprazole), pantoprazole (Pantoprazole)) Rabeprazole (Rabeprazole) and the like are widely used in clinic for treating gastric acid related diseases due to remarkable acid resistance and good safety ^[1,2] . PPIs as a weakly basic compound which is not easily dissociated in alkaline environment, is inactive, and can enter into parietal cell secretion tube via cell membrane, and can be converted into sulfenate and hypoflavin amide compounds, and H after encountering acidic environment with pH less than 2 ⁺ /K ⁺ The thiol group on the cysteine residue in ATPase reacts covalently to form a disulfide bond, which causes H ⁺ /K ⁺ ATPase is inactivated, thereby inhibiting gastric acid secretion.

ADP ribosylating factor 1 (ARF 1), one of the small G proteins, is widely distributed in eukaryotic cells and is involved in many intracellular physiological processes including substance transport ^[3] . As an important molecular switch in cell signaling, ARF1 circulates between an inactive GDP-bound form and an active GTP-bound form, and this circulation is regulated by two regulatory factors, guanylate exchange factors (guanine nucleotide exchange factors, GEFs) and gtpase-activated proteins (GTPase activating proteins, GAPs), respectively, which promote release of GDP from small G proteins, bind GTP, transduce signals to downstream effector proteins, a positive regulator; GAPs can enhance GTPase activity of small G protein, promote hydrolysis of GTP and inactivation, and are negative regulator ^[4-6] 。

In recent years, a great deal of research shows that the high expression and abnormal activation of ARF1 are involved in the development and progression of tumors, such as proliferation, invasion, migration and the like ^[7-10] . Recent studies have revealed a relationship between ARF1 and tumor stem cell survival ^[11,12] . Tumor stem cells are a subpopulation of cells in the tumor that are in the state of and characteristic of stem cells, which can lead to therapeutic resistance, tumor metastasis, tumor immune escape, disease recurrence, and ultimately patient death. The ultimate goal of tumor stem cell research is to find pathways that can selectively regulate such cells, and then target these pathways to kill such cells. Studies indicate that tumor stem cells are metabolically unique, and that regulation of ARF1 is required for their lipid metabolism; knockout in miceARF1 causes a blockage of lipid metabolism, accumulation of lipid droplets, and further causes metabolic stress including mitochondrial defects and endoplasmic reticulum stress, which selectively kills tumor stem cells, and the dying tumor stem cells release pathogen-associated molecular patterns, activate dendritic cells, which further enhance infiltration and activation of T cells, thereby stimulating anti-tumor immunity ^[12] . The ARF1 pathway knockdown has multiple functions, not only can kill tumor stem cells, but also can activate tumor-specific immune response, destroy solid tumors, and obtain lasting therapeutic effect ^[12] . Therefore, the development of the ARF1 inhibitor has great scientific significance and wide application prospect for treating various cancers.

Because the small G protein surface is relatively smooth, there is a lack of stable and easy small molecule binding pockets in addition to guanylate binding pockets ^[5,6] And small G proteins have a picomolar affinity for guanylic acid, which limits the development of inhibitors that target small G proteins directly ^[13] . Nevertheless, in recent years, researchers have made much effort in finding ARF1 inhibitors. BFA has been reported as an ARF1 inhibitor ^[14] 、LM11 ^[15] 、SecinH3 ^[16] 、Golgicide A ^[17] 、AMF-26 ^[18] For example, many of these inhibitors exert their effects of inhibiting ARF1 activity by interfering with the normal function of GEF or disrupting the interaction of ARF1 with GEF, and their findings are of interest for studying the function of ARF1, but so far, the modes of action of these inhibitors are relatively single, the related activity studies are not comprehensive enough, and their clinical effects and safety are yet to be studied further. Therefore, the development of safe and effective ARF1 inhibitors has great scientific significance and wide application prospect for various cancer treatments.

Disclosure of Invention

The inventors have found that proton pump inhibitors (rabeprazole, lansoprazole, omeprazole, pantoprazole, PPIs) are capable of covalently binding to C159 of ARF 1; in vivo and in vitro studies jointly show that rabeprazole can remarkably inhibit the activity level of ARF1 and exert the anti-tumor immunity effect by regulating and regulating the lipolysis pathway of tumor stem cells. Based on this, the present invention has been completed.

Accordingly, in one aspect, the present invention provides the use of a proton pump inhibitor in the preparation of an ARF1 inhibitor.

In the present invention, the proton pump inhibitor means a proton pump inhibitor which inhibits H in cells of the stomach wall ⁺ /K ⁺ Drugs that inhibit gastric acid secretion by atpase (proton pump), examples of which include, but are not limited to, rabeprazole, lansoprazole, omeprazole, pantoprazole, and the like.

In one embodiment, the proton pump inhibitor may be one or more selected from rabeprazole, lansoprazole, omeprazole, and pantoprazole. In a preferred embodiment, the proton pump inhibitor may be rabeprazole.

In another aspect, the invention provides the use of a proton pump inhibitor in the manufacture of a medicament for the treatment of a tumor.

In the present invention, the tumor refers to a tumor containing tumor stem cells.

In one embodiment, the tumor may be selected from tumors containing tumor stem cells, such as leukemia, breast cancer, colon cancer, and the like.

In one embodiment, the tumor may be colon cancer.

In one embodiment, the proton pump inhibitor may be one or more selected from rabeprazole, lansoprazole, omeprazole, and pantoprazole. In a preferred embodiment, the proton pump inhibitor may be rabeprazole.

In such uses, the proton pump inhibitor acts as an ARF1 inhibitor.

In one embodiment, the proton pump inhibitor is capable of significantly reducing the level of ARF1 activity in colon cancer cells and significantly inducing lipid droplet accumulation.

In one embodiment, the proton pump inhibitor is capable of significantly inhibiting tumor growth.

In one embodiment, the proton pump inhibitor is capable of significantly enhancing infiltration of immune cells of tumor tissue, increasing the number of killer T cells, and reducing the number of depleting T cells.

According to the invention, firstly, through a protein thermomigration experiment, PPIs can obviously reduce the thermal stability of ARF1 protein in a concentration-dependent manner; due to PPIs and H ⁺ /K ⁺ The covalent binding properties of ATPase, the unique cysteine C159 of ARF1 was used for binding site validation, and the protein thermomigration experiments and primary mass spectrometry experiments, secondary mass spectrometry experiments of wild-type ARF1 and point mutant ARF1 (C159) together demonstrated covalent binding of PPIs to ARF1 and confirmed the binding site as C159. Rabeprazole has the greatest effect on ARF1 thermal stability and is used for subsequent activity studies.

Next, in vitro guanylate exchange experiments found a significant inhibition of ARF1 guanylate exchange by rabeprazole. Cell level studies have found that rabeprazole can significantly reduce the level of ARF1 activity in CT26 colon cancer cells and significantly induce lipid droplet accumulation. Animal level research shows that rabeprazole can obviously inhibit the growth of CT26 transplanted tumor, and after flow analysis of immune cell subgroup of tumor tissue, rabeprazole has obvious immune activation effect and is expressed as CD3 ⁺ CD8 ⁺ Significant upregulation of T cells and CD3 ⁺ CD8 ⁺ PD1 ⁺ T cells, CD3 ⁺ CD8 ⁺ TIM3 ⁺ T cells, CD3 ⁺ CD8 ⁺ PD1 ⁺ TIM3 ⁺ The significant down-regulation of T cells was consistent with the results of immunohistochemical staining.

The invention has the following beneficial effects:

1. the present invention has found that Proton Pump Inhibitors (PPIs) are capable of covalently binding to C159 of ARF 1;

2. the invention discovers that the rabeprazole can obviously inhibit ARF1 activity, plays an anti-tumor immunity effect by regulating a tumor stem cell lipolysis pathway, and provides a new treatment means for anti-tumor immunity.

Drawings

Fig. 1 is a graph showing that PPIs (rabeprazole (a), lansoprazole (b), omeprazole (c), pantoprazole (d)) in example 1 significantly reduce the thermal stability of ARF1 in a protein thermomigration experiment.

FIG. 2 showsPTS experiment and ARF1 after addition of DTT in example 2 ^C159A The results of PTS experiments (rabeprazole (a), lansoprazole (b), omeprazole (c), pantoprazole (d)) of the mutants were summarized as the results of protein thermomigration experiments of PPIs (e).

FIG. 3 shows the determination of PPIs and ARF1 by Q-TOF mass spectrometry in example 3 ^WT (a) Or ARF1/ARF1 ^C159A (b) Effect of pre-incubation on ARF1 protein molecular weight.

FIG. 4 shows the results of Q-exact mass spectrometry (rabeprazole (a), lansoprazole (b), omeprazole (c), pantoprazole (d)) in example 4, showing PPIs modified ARF1 peptide fragments.

FIG. 5 shows the inhibition of the guanylate exchange process of ARF1 by rabeprazole in example 5.

FIG. 6 shows the results of cell level activity verification of rabeprazole in example 6. (a) Rabeprazole significantly reduces the level of ARF1 activity in CT26 colon cancer cells, where n.s. represents no significant difference, represents P <0.01, and represents P <0.001; (b) Rabeprazole can obviously induce lipid drop accumulation in CT26 colon cancer cells.

Fig. 7 shows the inhibition of tumor growth in CT26 mice by rabeprazole in example 7, wherein P <0.001 is represented.

Fig. 8 shows the effect of flow cytometry (a) and immunohistochemical staining (b) of example 8 on immune cell subsets in CT26 mouse engrafted tumor tissue, wherein P <0.05 represents P <0.01 and P <0.001.

Detailed Description

The experimental methods used in the examples below are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Materials and reagents

The main reagent comprises: naCl (SCRC, 10019318), tris-HCl (Meinauguration, MB 6025), anhydrous MgCl ₂ (Ourchem, 7786-30-3), SYPRO Orange dye (Sigma, S5692), DMSO (Sigma, D8418-1L), ARF 1G-LISA Activation Assay Kit (Cytoskeleton, BK 132), PBS (Menlengen)MA 0015), immunostaining fixative (Beyotime, P0098), immunostaining permeabilizer (Triton X-100) (Beyotime, P0096), nile red (MedChemExpress, HY-D0718), anti-fluorescence quenching fixative (DAPI-containing) (Beyotime, P0131), red cell lysate (Beyotime, C3702), fixable Viablity Stain dye (BD Horizan, 564997), anti-mouse CD16/32 anti-body (Invitrogen, 14-0161-82), anti-CD3 anti-body (Invitrogen, 11-0032-82), anti-CD8 anti-body (Biolegend, 100738), anti-PD1 anti-body (Biolegend, 135219), anti-TIM3 anti-body (Invitrogen, 12-5870-82).

The main materials are as follows: compounds were purchased from MedChemExpress company (rabeprazole, HY-B0656; lansoprazole, HY-13662; omeprazole, HY-B0113; pantoprazole, HY-17507), DTT (MedChemExpress, HY-15917), GDP (MedChemExpress, HY-113066A), MANT-GTP (Invitrogen, M12415), 0.1mL 96-well skirtless PCR tube (dwarf tube) (DN Biotech, 5371012), heat seal membrane (Thermo Scientific, AB-1107), 384 Kong Heiban (Corning, 3575), lab-Tek 8 well plate (Thermo, 154534).

Instrument for measuring and controlling the intensity of light

Real-time fluorescence quantitative PCR instrument (Bio-Rad), nLC-1000-Q-Exactive (ThermoFisher), spark multifunctional enzyme-labeled instrument (Tecan), OLYMPUS IX73 fluorescence microscope, bio-Rad ZE5 flow analyzer.

Example 1 influence of PPIs on the thermal stability of ARF1 in protein thermomigration experiments

The experimental method comprises the following steps:

the effect of compounds on protein thermostability was evaluated using a protein thermomigration assay in which wild-type recombinant human Δ17arf1 protein (soluble truncated form lacking the first 17 amino acids, ARF1 ^WT ). 20. Mu.L of the reaction system, 2.5. Mu.M ARF1 protein, 5 XSYPRO Orange (Sigma, S5692) and test compounds (PPIs: rabeprazole, lansoprazole, omeprazole, pantoprazole) of different concentrations (50. Mu.M (20X), 12.5. Mu.M (5X)) were added to a white 96-well plate, and the sample wells were sealed with a Absolute qPCR Plate Seals heat-sealing film, and real-time fluorescent signals were collected with a real-time fluorescent quantitative PCR instrument (Bio-Rad) at a temperature of from 25℃to 95 ℃. After the end of the experiment, use CFX Connect EvaluationSystem analysis fitting T _m Values. The exported data was reconstructed using Graphpad Prism 8.0.

Experimental results:

as shown in fig. 1a-d, rabeprazole, lansoprazole, omeprazole, pantoprazole all significantly reduced the thermal stability of ARF1 protein.

Example 2 PTS experiment and ARF1 after DTT addition ^C159A PTS experiments of mutants.

The experimental method comprises the following steps: protein thermomigration assay was used to evaluate whether DTT, a reducing agent capable of breaking disulfide bonds, would affect PTS assay results, by adding 1mM DTT to the reaction system in the same manner as in example 1; evaluation of PPIs against ARF1 Using protein thermomigration experiments ^C159A Influence of the thermal stability of muteins the procedure was as in example 1, substituting ARF1 for the wild-type ARF1 protein ^C159A And (3) protein.

Experimental results: as shown in fig. 2, DTT is added/replaced with ARF1 ^C159A The effect of the protein, PPIs (rabeprazole, lansoprazole, omeprazole, pantoprazole) on the thermal stability of ARF1 was all absent, suggesting that covalent binding of PPIs to cysteine 159 of ARF1 may occur.

Example 3Q-TOF Mass Spectrometry of PPIs and ARF1/ARF1 ^C159A ARF1 protein molecular weight before and after pre-incubation.

The experimental method comprises the following steps:

the experiment is used for detecting the molecular weight of protein and is divided into a control group and a compound group, wherein an incubation system of the compound group is as follows: 20mM Tris (pH 8.0), 100mM NaCl,5mM MgCl ₂ ，100μM ARF1 ^WT /ARF1 ^C159A Protein, 1mM compound (rabeprazole, lansoprazole, omeprazole, pantoprazole). Control groups were added with equal proportions of DMSO. The control group and the compound group were placed in a refrigerator at 4 ℃ for incubation overnight, and then sent to a mass spectrometry detection service platform of Shanghai pharmaceutical research institute of academy of sciences of China for protein molecular weight determination.

Experimental results:

as shown in FIGS. 3a-b, PPIs (rabeprazole, lansoprazole, omeprazole, pantoprazole) and ARF1 ^WT After pre-incubation, the increase in ARF1 molecular weight was suggestedPPIs react covalently with cysteines. PPIs and ARF1 ^C159A After pre-incubation, ARF1 molecular weight was unchanged confirming that the covalent binding site was at C159.

Example 4Q-actual secondary mass spectrometry results show PPIs modified ARF1 peptide fragments.

The experimental method comprises the following steps: the sample incubation system is: 20mM Tris (pH 8.0), 100mM NaCl,5mM MgCl ₂ ，100μM ARF1 ^WT Protein, 1mM compound (rabeprazole, lansoprazole, omeprazole, pantoprazole), was prepared and incubated overnight at 4 ℃. The detection process is as follows: sample addition of 100. Mu.L of 100mM NH ₄ HCO ₃ Trypsin (10 ng/. Mu.L) was enzymatically reacted at 37℃for 17 hours. The next day, the sample was centrifuged at 12000 Xg for 30 minutes, and the supernatant was lyophilized, desalted and then lyophilized to obtain peptide lyophilized powder. Then adding 15 mu L of 0.1% FA (formic acid) solution to dissolve peptide fragment freeze-dried powder, centrifuging for 5 minutes at 12000 Xg, taking the supernatant, adding the supernatant into a sample loading bottle, detecting by mass spectrum (Q-exact), searching by MaxQuant (1.6.5.0), and controlling the false positive rate of protein identification to be less than 1%.

Experimental results: as shown in fig. 4, the secondary mass spectrometry results further demonstrate that PPIs (rabeprazole, lansoprazole, omeprazole, pantoprazole) covalently modify C159 of ARF 1.

Example 5 influence of rabeprazole on the process of exchange of ARF1 guanylate

The experimental method comprises the following steps: the ARF1 protein is labeled with GDP in advance to obtain ARF1 ^GDP The experimental buffer system is 20mM HEPES pH 7.5, 150mM NaCl,1mM MgCl ₂ ，1mM DTT，ARF1 ^GDP 、MANT-GTP、ARNO ^Sec7 Final concentrations of 20. Mu.M, 10. Mu.M, 1. Mu.M, respectively, ARF1 was first prepared by 384 well blackboard ^GDP Mixing MANT-GTP and different concentrations of compounds, incubating at room temperature for 15 min in dark place, and adding ARNO ^Sec7 The exchange reaction was initiated and fluorescence signals were continuously monitored using Spark multifunctional enzyme-labeled instrument (Tecan) until the plateau was reached. The derived data was re-mapped with Graphpad Prism 8.0.

Experimental results: as shown in fig. 5, rabeprazole has a significant inhibitory effect on the ARF1 guanylate exchange process and is concentration dependent.

EXAMPLE 6 verification of the cellular level Activity of rabeprazole

The experimental method comprises the following steps:

GLISA experiment: ARF1 activity levels in CT26 cells were determined using Cytoskeleton ARF 1G-LISA Activation Assay Kit (BK 132) according to the methods described herein.

Lipid drop staining experiments:

when CT26 cells are in good growth state, they are inoculated into a Thermo Lab-Tek 8 well plate one day and night in advance, and each well is 2×10 ⁴ After the next day of cell attachment, the cells are treated with compound or DMSO. After the treatment is finished, the dyeing steps are as follows:

(1) After removal of the medium, the wells were rinsed 3 times with 200 μl of PBS for 5 minutes each;

(2) Fixing with immunostaining fixing solution for 30 min;

(3) Washing with PBS 3 times, 200 μl per well, 5 minutes each;

(4) Permeabilizing the cells with immunostaining permeabilization solution (Triton X-100) for 30 minutes;

(5) Washing with PBS 3 times, 200 μl per well, 5 minutes each;

(6) The PBS is sucked off, nile red staining solution (2 mu M) is prepared in a dark place, 200 mu L of staining solution is added to each hole, and the mixture is incubated for 20 minutes in a dark place; incubation procedure cover slips were cleaned (after sterilization with 75% alcohol, ddH was used) ₂ O-washing three times).

(7) Under the dark condition, PBS is washed for 3 times, 200 mu L of each hole is washed for 5 minutes;

(8) Sealing with anti-fluorescence quenching sealing liquid, dripping a proper amount of sealing liquid onto an 8-hole plate, slowly and reversely buckling a cover glass, coating a circle of the edge of the cover glass with nail polish without generating bubbles, air-drying, and then preserving in a refrigerator at 4 ℃, wherein the photographing is completed by using an OLYMPUS IX73 fluorescence microscope.

Experimental results: as shown in fig. 6a, rabeprazole can significantly reduce the level of ARF1 activity in CT26 colon cancer cells; as shown in fig. 6b, rabeprazole was able to significantly induce lipid droplet accumulation in CT26 colon cancer cells.

EXAMPLE 7 Effect of rabeprazole on the growth of CT26 mouse transplantable tumors

The experimental method comprises the following steps: all animal experiments were approved and validated by the institutional animal care committee of Shanghai pharmaceutical research, national academy of sciences, according to the prescribed guidelines (IACUC Issue No. 2021-03-JHL-22). The subjects were female Balb/c mice of 6-8 weeks of age. CT26 colon cancer cells (2.5X10 each) were injected subcutaneously ⁵ Individual cells) to establish a model of colon cancer engraftment. When the tumor grows to a volume of about 100mm ³ At this time, mice were randomly divided into control and dosing groups, and mice were intraperitoneally injected with solvent blank or compound (40 mg/kg/day). After the end of the experiment, tumor tissue of the mice was taken and analyzed.

Experimental results: as shown in fig. 7, rabeprazole has a significant inhibitory effect on the growth of the transplanted tumor of CT26 mice.

Example 8 flow cytometry and immunohistochemical staining analysis of the Effect of rabeprazole on immune cell subsets in CT26 mouse engrafted tumor tissue

1. Flow cytometry experimental method:

(1) Single cell suspension preparation of tumor tissue

Preparing digestive juice in advance: to 100mL of RPM 1640 was added 0.1% collagenase, 0.001% hyaluronidase, 0.002% DNase, since DNase is Ca ²⁺ /Mg ²⁺ Depending on the enzyme, 12. Mu.L of 1M CaCl is added into the digestion solution ₂ And 12. Mu.L of 1M MgCl ₂ 。

Tumor tissue digestion: after euthanasia of the mice, tumor tissue was removed, about 500mg of the portion near the outer edge where no erosion was present was placed in the EP tube, 300 μl of digestive juice was added, and minced with scissors. 1mL of the digested solution was supplemented and the mixture was subjected to digestion in a shaker at 37℃and 180rpm for 60 minutes.

And (3) filtering: 5mL of PBS was previously added to a 15mL centrifuge tube, digested tumor tissue was filtered into the centrifuge tube with 200 mesh gauze, the resulting filtrate was a single cell suspension, which was then centrifuged (1000 rpm,3 minutes) and the supernatant removed.

Lysing erythrocytes: cells were resuspended by adding 500. Mu.L of ammonium chloride erythrocyte lysate and after 1 minute, lysis was stopped by adding 3mL of PBS. Centrifuging and removing the supernatant.

And (3) filtering: the EP tube was prepared in advance, 1mL of PBS was added to the centrifuge tube of the previous step, and the mixture was resuspended, filtered into the EP tube with 200 mesh gauze, centrifuged, and the supernatant was removed. Adding PBS for resuspension to obtain single cell suspension of tumor tissue.

(2) Cell surface marker staining

Dead-living staining: appropriate amounts of single cell suspension of tumor tissue were taken in EP tubes and dead and live cells were differentiated using Fixable Viablity Stain dye (BD Horizan, 564997) and stained on ice for 10 minutes. At the same time, one EP tube was added to 200. Mu.L of cell sample and only stained alive. After the end of staining, the staining was terminated with PBS containing 2% FBS.

Blocking Fc receptors: this step was performed in PBS solution of 2% fbs. The Fc receptor on the cell surface was blocked using anti-mouse CD16/32 anti-body and incubated on ice for 10 minutes.

Dyeing: after the end of blocking, the flow antibody Mix was directly added for incubation, and CD3, CD8, PD-1, TIM3 antigen molecules located on the cell surface were stained together. After staining, the cells were resuspended in 400. Mu.L of 2% FBS in PBS and detected on-machine.

2. Immunohistochemical staining: the isolated fresh CT26 tumor tissue was sent to Whansaiville Biotechnology Inc. for paraffin section and immunohistochemical staining procedures.

Experimental results: as shown in fig. 8 (above), flow analysis shows that rabeprazole can significantly enhance infiltration of immune cells of tumor tissues of mice, increase the number of killer T cells, and reduce the number of depleting T cells. Is shown as CD3 ⁺ CD8 ⁺ Significant upregulation of T cells and CD3 ⁺ CD8 ⁺ PD1 ⁺ T cells, CD3 ⁺ CD8 ⁺ TIM3 ⁺ T cells, CD3 ⁺ CD8 ⁺ PD1 ⁺ TIM3 ⁺ Significant down-regulation of T cells. As shown in fig. 8 (bottom), the immunohistochemical staining results for CD8, PD1 were consistent with the flow results.

The invention discovers that PPIs can be covalently combined with ARF1, wherein rabeprazole can obviously inhibit ARF1 activity, and the anti-tumor immunity effect is exerted by regulating a CT26 colon cancer cell lipolysis pathway, so that a novel treatment means is provided for anti-tumor immunity.

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Claims (9)

1. Use of a proton pump inhibitor in the preparation of an ARF1 inhibitor.

2. The use according to claim 1, wherein the proton pump inhibitor is one or more selected from rabeprazole, lansoprazole, omeprazole, and pantoprazole.

3. The use according to claim 1, wherein the proton pump inhibitor is rabeprazole.

4. Use of a proton pump inhibitor in the manufacture of a medicament for the treatment of a tumor.

5. The use according to claim 4, wherein the tumor is a tumor containing tumor stem cells.

6. The use according to claim 4, wherein the tumor comprises leukemia, breast cancer, colon cancer.

7. The use according to any one of claims 4-6, wherein the proton pump inhibitor is one or more selected from rabeprazole, lansoprazole, omeprazole, and pantoprazole.

8. The use according to any one of claims 4-6, wherein the proton pump inhibitor is rabeprazole.

9. Use according to any one of claims 4-6, wherein the proton pump inhibitor is an ARF1 inhibitor.