CN114558019A

CN114558019A - Rab13 gene inhibitor and application thereof

Info

Publication number: CN114558019A
Application number: CN202210304114.XA
Authority: CN
Inventors: 焦保卫; 王慧; 邹丽; 杨钦
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2022-03-26
Filing date: 2022-03-26
Publication date: 2022-05-31
Also published as: CN116036094A; CN116036094B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an inhibitor of Rab13 gene and application thereof. An inhibitor of Rab13 gene can inhibit the expression of Rab13 gene, and the inhibitor for reducing the expression product level of Rab13 gene is bardoxolone methyl. CDDO-Me can directly act on Rab13 promoter and inhibit the promoter activity; and can inhibit the expression of Rab13 at the mRNA and protein level, and enhance the sensitivity of tumors to chemotherapeutic drugs (DOX, PTX, CIS): can reduce the IC50 value of tumor cells treated by chemotherapeutic drugs; increasing the rate of apoptosis of tumor cells after chemotherapeutic treatment; inhibiting tumor stem cell enrichment caused by chemotherapeutic drugs; inhibiting tumor recurrence in situ after chemotherapy treatment; inhibit tumor stem cell dryness (ALDH + stem cell population and tumor sphere formation). The invention has important significance for further improving diseases related to high expression of Rab13 gene.

Description

Rab13 gene inhibitor and application thereof

Technical Field

The invention relates to an inhibitor of Rab13 gene and application of the inhibitor in preparing a medicament for treating diseases related to high expression of Rab13 gene.

Background

The Rab family of GTPases is a major regulator of cell membrane transport. There are approximately 70 Rab members in humans, Rab being involved in all membrane trafficking steps from vesicle formation and trafficking to vesicle docking/tethering and fusion. Vesicular trafficking controls the localization and levels of a large number of proteins, thereby regulating cellular functions, including proliferation, metabolism, cell adhesion, and cell migration. Rab13 has received increased attention in recent years. The existing research proves that: rab13 levels were altered in both glioblastomas and carcinomas, and Rab13 mRNA was found in the membrane processes of breast cancer cells. The previous study shows that: rab13 expression in rat testis tissue and Rab13 expression was found to vary with the seminiferous epithelial cycle; rab13 plays a role in regulating permeability of blood testis barrier; rab13 protein regulates vascular endothelial cell autophagy; rab13 plays a role in extracellular membrane trafficking from TGN to the cell surface by recycling endosomes in polarized epithelial cells; rab13 gene knockout can reduce cancer cell migration and invasion in vitro; rab13 gene knock-down can reduce the spread of cancer cells in vivo; rab13 acts downstream of kinase Mst1, delivering integrin LFA-1 to the cell surface for lymphocyte trafficking. Rab13 gene expression is highly up-regulated during differentiation of human peripheral blood mononuclear cells into osteoclasts and is associated with vesicles.

EGFR is highly expressed in breast cancer and is closely related to poor prognosis of patients^【1】. EGFR targeted therapy has been successfully applied in clinical treatments of lung cancer, colorectal cancer and the like. EGFR antagonists are largely divided into two categories: 1) monoclonal antibodies targeting the EGFR extracellular domain (cetuximab and panitumumab); 2) small molecule inhibitors targeting the intracellular tyrosine kinase domain (TKI): the first generation of non-covalent inhibitors erlotinib and gefitinib; a second generation covalent inhibitor afatinib; oxcetitinib, a third-generation inhibitor, and EAI001 and EAI045, a fourth-generation inhibitor^【2】. However, EGFR antagonists have received little efficacy in the treatment of breast cancer. Metastatic breast cancer patientsClinical trials with cetuximab monotherapy or combined therapy with carboplatin showed that the tumor response rate for cetuximab monotherapy was only about 6%; the response rate of the combined drug group is 18 percent^【3】. Phase II clinical trials with gefitinib in combination with epirubicin and cyclophosphamide treatment in breast cancer patients showed that the pCR (complete remission of pathology) rate in the combination group only exceeded 4.57% in the gefitinib-free group and drug side effects caused the inability of most patients to complete treatment^【4】。

The methoate bardoxolone is an oral antioxidant inflammation regulator which is jointly developed by Reata pharmacy and AbbVie, and is a semi-synthetic triterpenoid compound derived from oleanolic acid. The chemical name of the compound is 2-cyano-3, 12-dioxo olean-1, 9(11) -diene-28-acid methyl ester, also called RTA 402, CDDO-methyl ester and CDDO-Me, and the molecular formula is as follows: c₃₂H₄₃NO₄505.69, chemical structure shown below. It is effective in activating the Keapl-Nrf2 pathway,

and (5) effect. In vitro studies show that: CDDO-Me acts on mouse macrophages and has potent inhibitory activity against interferon-Y induced nitric oxide production with an IC50 of 0.1 nM. CDDO-Me reduced the viability of the leukemia HL-60, KG-1 and NB4 cells with IC50 of 0.4, 0.4, and 0.27. mu.M, respectively. CDDO-Me induces pro-apoptotic Bax protein expression, inhibits activation of ERK1/2, and it inhibits phosphorylation of Bcl-2, which contributes to the induction of apoptosis. CDDO-Me is effective in Inhibiting (IL) -1chemical cookbeta, phorbolester, okadaic acid, dihydrogenperoxide, lipopolysaccharide, and cigarettesmoke activated constitutive and inducible tumor necrosis factor of NF- κ B. In vivo studies: CDDO-Me (60mg/kg) administered in vivo can reduce the number, size and severity of lung tumors. CDDO-Me can also significantly reduce the response of inflammatory factors in vivo under the stimulation of LPS, induce the expression of spleen HO-1 protein, and protect mice against lethal dose of LPS. The CDDO-Me is reported in the literature to inhibit the membrane receptor complex of USP7, LRP6/FZD7, mTOR, Wnt, Hsp90, AKT, Erk, PKB, NF-kappa B, telomerase reverse transcriptase, STAT3, cyclin D1, EGFR2, Nrf2And IKK ovarian cancer, breast cancer, pancreatic cancer, lung cancer, colorectal cancer, acute myeloid leukemia, prostate cancer, melanoma, osteosarcoma, spinal myeloma, glioma, neuroblastoma, oral squamous cell carcinoma, and chronic myeloid leukemia (PMID:27780924, 32015160, 25364233, 29118925,33584286)^【5-9】。

The patent of application number 2009101272661 'application of antagonist of gene Rab13 and medicine containing the antagonist' discloses application of the antagonist of gene Rab13 in preparing medicine for inhibiting tumor cell proliferation and promoting tumor cell apoptosis or enhancing the curative effect of antitumor drugs, wherein the antagonist is siRNA molecule or antisense RNA aiming at mRNA transcribed by gene Rabl3, and a compound capable of inhibiting the expression of gene Rab13 is not found.

The prior art also does not report the effect of bardoxolone methyl on Rab13 gene. The inventor finds that methyldopaxolone (CDDO-Me) inhibits the expression of Rab13 mRNA and protein expression level and inhibits Rab13 promoter activity by screening an FDA approved compound library.

Reference documents:

1.Jacot W,Mollevi C,Fina F,Lopez-Crapez E,Martin PM,Colombo PE,et al.High EGFR protein expression and exon 9PIK3CA mutations are independent prognostic factors in triple negative breast cancers.BMC Cancer 2015；15:986

2.Ayati A,Moghimi S,Salarinejad S,Safavi M,Pouramiri B,Foroumadi A.A review on progression of epidermal growth factor receptor(EGFR)inhibitors as an efficient approach in cancer targeted therapy.Bioorg Chem 2020；99:103811

3.Carey LA,Rugo HS,Marcom PK,Mayer EL,Esteva FJ,Ma CX,et al.TBCRC 001: randomized phase II study of cetuximab in combination with carboplatin in stage IV triple-negative breast cancer.J Clin Oncol 2012；30:2615-23

4.Bernsdorf M,Ingvar C,

L,Tuxen M,Jakobsen E,Saetersdal A,et al.Effect of adding gefitinib to neoadjuvant chemotherapy in estrogen receptor negative early breast cancer in a randomized phase II trial.2011；126:463-70

5.So JY,Lin JJ,Wahler J,Liby KT,Sporn MB,SuhN.A synthetic triterpenoid CDDO-Im inhibits tumorsphere formation by regulating stem cell signaling pathways in triple-negative breast cancer.PLoS One 2014；9:e107616

6.Ball MS,Bhandari R,Torres GM,Martyanov V,ElTanbouly MA,Archambault K,et al. CDDO-Me Alters the Tumor Microenvironment in Estrogen Receptor Negative Breast Cancer. Sci Rep 2020；10:6560

7.Deeb D,Gao X,Dulchavsky SA,Gautam SC.CDDO-me induces apoptosis and inhibits Akt, mTOR and NF-kappaB signaling proteins in prostate cancer cells.Anticancer Res 2007；27:3035-44

8.Qin DJ,Tang CX,Yang L,Lei H,Wei W,Wang YY,et al.Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells.PLoS One 2015；10:e0132337

9.Liby KT,Sporn MB.Synthetic oleanane triterpenoids:multifunctional drugs with a broad range of applications for prevention and treatment of chronic disease.Pharmacol Rev 2012；64:972-1003。

disclosure of Invention

Based on the above technical problems, the present invention intends to provide a Rab13 gene inhibitor, which can inhibit the expression of Rab13 gene to reduce the level of the expression product of Rab13 gene.

The technical scheme of the invention is as follows: an Rab13 gene inhibitor is bardoxolone methyl (CDDO-Me), which can inhibit the expression of Rab13 gene to reduce the level of the expression product of Rab13 gene.

Further, the inhibitor is applied to preparation of a medicine for treating diseases related to high expression of Rab13 gene.

Further, the inhibitor is applied to preparing a chemotherapeutic drug sensitivity medicament related to the high expression of the Rab13 gene.

Further, the inhibitor is applied to preparation of a medicine for enhancing sensitivity of EGFR (epidermal growth factor receptor) targeted medicines.

The invention also protects the application of the inhibitor, namely the bardoxolone methyl, in the preparation of tumor chemotherapy medicaments in combination with other antitumor medicaments.

Furthermore, the inhibitor and chemotherapeutic drugs related to high expression of Rab13 gene are used in combination to prepare antitumor drugs.

Further, the inhibitor and an EGFR targeting drug are combined for use in the preparation of antitumor drugs.

Preferably, the disease drug related to the high expression of Rab13 gene is DOX, PTX, CIS.

Preferably, the EGFR-targeting drug is cetuximab or gefitinib.

The invention proves that the inhibitor CDDO-Me of the screened gene Rab13 can inhibit the horizontal expression of the gene Rab13 in cells and animals, can directly act on the Rab13 promoter and inhibit the promoter activity; the expression of Rab13 can be inhibited at the mRNA and protein levels by inhibiting its promoter activity. CDDO-Me promotes tumor sensitivity to chemotherapeutic drugs (DOX, PTX, CIS) and EGFR-targeted drugs: can reduce the IC50 value of tumor cells treated by chemotherapeutic drugs; increasing the rate of apoptosis of tumor cells after chemotherapeutic treatment; inhibiting tumor stem cell enrichment caused by chemotherapeutic drugs; inhibiting tumor recurrence in situ after chemotherapy treatment. CDDO-Me treatment inhibited tumor stem cell dryness (ALDH + stem cell population and tumor sphere formation). CDDO-Me treatment inhibited TAM/CAF conditioned media-induced tumor stem cell enrichment. CDDO-Me treatment inhibited MMTV-PyMT mouse tumor growth, inhibited MMTV-PyMT mouse tumor dryness (ALDH activity and tomorsphere formation), inhibited TAM/CAF enrichment in tumor tissues. The CDDO-Me can be combined with chemotherapeutic drugs related to the high expression of Rab13 gene and EGFR targeted drugs for use in the preparation of antitumor drugs.

Drawings

FIG. 1 shows that CDDO-Me can dose-dependently inhibit Rab13 promoter activity. Denotes P <0.01, denotes P < 0.001.

FIG. 2A shows that CDDO-Me can dose-dependently inhibit Rab13 mRNA expression. Represents P <0.01, represents P < 0.001.

Fig. 2B shows that CDDO-Me can dose-dependently inhibit Rab13 protein expression.

FIG. 3 shows that CDDO-Me can inhibit Rab13 expression in MMTV-PyMT mouse mammary tumor tissue. Represents P < 0.01.

FIG. 4 shows that the shRNAs effectively knock down Rab13 in HCC1806 and MDA-MB-231 cell lines;

FIG. 5A is a graph showing that Rab13 knockdown inhibits THP-1 mediated increase in the proportion of ALDH-positive breast cancer stem cells;

FIG. 5B is CD44 mediated by the inhibition of CAFs by Rab13 knockdown⁺CD24^-/lowAn increased proportion of breast cancer stem cells;

FIG. 5C shows that Rab13 knockdown inhibits THP-1/CAF-mediated breast cancer tumors formation;

FIG. 6 shows the Rab13 knockdown inhibition of Doxorubicin (DOX), Paclitaxel (PTX) and Carboplatin (CIS) -mediated ALDH positivity and CD44⁺CD24^-/lowAn increased proportion of breast cancer stem cells;

FIG. 7A is a graph showing that Rab13 knockdown promotes Doxorubicin (DOX), Paclitaxel (PTX), and Cisplatin (CIS) -mediated increased apoptosis in breast cancer cells;

FIG. 7B shows that Rab13 knockdown enhances the sensitivity of HCC1806 and MDA-MB-231 breast cancer cells to Doxorubicin (DOX);

FIG. 8A is the change in tumor volume following DOX treatment of control cells or Rab13 knockdown; transforming

FIG. 8B is a tumor profile after DOX treatment of control cells or Rab13 knockdown;

FIG. 9A is a CDDO-Me treatment dose-dependent inhibition of macrophage conditioned medium (RAW-CM) mediated increase in ALDH positive cell proportion;

FIG. 9B shows CDDO-Me treatment dose-dependent inhibition of macrophage conditioned medium (THP-CM) mediated increase in ALDH-positive cell proportion;

FIG. 9C is a CDDO-Me treatment dose-dependent inhibition of tumor associated fibroblast conditioned medium (CAF-CM) mediated increase in the proportion of ALDH positive cells;

FIG. 10A is a graph showing that CDDO-Me treatment inhibits macrophage conditioned medium (RAW-CM) mediated resistance of tumor cells to doxorubicin;

FIG. 10B shows CDDO-Me treatment inhibits macrophage conditioned medium (THP-CM) mediated resistance of tumor cells to doxorubicin;

FIG. 10C shows CDDO-Me treatment inhibits doxorubicin resistance of tumor cells mediated by tumor associated fibroblast conditioned medium (CAF-CM);

FIG. 11 shows that Rab13 knockdown of EGFR expression levels in cells is reduced;

fig. 12 is a graph of Rab13 knockdown enhancing sensitivity of breast cancer cells to cetuximab and gefitinib;

FIG. 13 is a graph of CDDO-Me treatment inhibiting EGFR expression in breast cancer orthotopic transplants;

FIG. 14A is a CDDO-Me treatment increasing the sensitivity of breast cancer cells (MDA-MB-231 and HCC1806) to cetuximab;

figure 14B is a CDDO-Me treatment increasing the sensitivity of HCC1806 to gefitinib.

Detailed Description

The invention provides a new application of methyldopaxolone in inhibiting Rab13 cell and mouse tissue expression.

Example 1 CDDO-Me can dose-dependently inhibit Rab13 promoter Activity

Rab13 promoter-2 kb was cloned into PGL3-Basic vector (PGL3-Rab13-P), and empty load control (PGL3-Basic) and PGL3-Rab13-P were transfected into HEK293T cells according to lipofectamine 2000 transfection reagent (Saimer Feishell technology (China) Co., Ltd.) instructions. 48h after transfection, cells were plated in 96-well plates, 5000 cells per well. Divided into 4 groups of 6 replicate wells, treated as follows:

a first group: negative control group (transfection PGL3-Basic control plasmid);

second group: control group (transfection of PGL3-Rab13-P plasmid), no CDDO-Me treatment;

third, fourth and fifth groups: experimental groups (transfected with PGL3-Rab13-P plasmid) cells were treated at doses of 20, 40 and 80nM, respectively.

The treatment method comprises the following steps: after the cells are transfected for 48 hours, the cells are paved into a 96-well plate, cultured overnight, and directly added with CDDO-Me with the corresponding concentration for 48 hours. Steady-Lumi^TMThe luciferase activity of the firefly luciferase reporter gene detection kit (Biyuntian biotechnology Co., Ltd.) was detected. The results are shown in fig. 1, which shows that CDDO-Me can inhibit luciferase activity (i.e., Rab13 promoter activity) dose-dependently, and the inhibition rates of the third, fourth and fifth groups are 45.2%, 55.9% and 62.2%, respectively (fig. 1).

Example 2CDDO-Me inhibits Rab13 expression at cellular level

2.1Rab13 mRNA expression assay

Selecting good breast cancer cell MDA-MB-231, re-suspending with 10% fetal bovine serum DMEM/F12 culture medium, and adding 2.5 × 10⁵The cells were seeded in a 6cm petri dish and placed in a cell incubator and cultured normally overnight at 37 ℃ with 5% carbon dioxide. To the above cells were added 0 (control, equal volume of dimethyl sulfoxide), 10, 20, 40, and 80nM CDDO-Me, respectively, for 48 h. Total cellular RNA was extracted following standard procedures as described in the RNAioso Plus reagent (from TaKaRa). Using PrimeScript^TMAnd carrying out reverse transcription by using the RT reagent Kit reverse transcription Kit, and carrying out the reaction according to the instruction standard. Rab13 mRNA expression levels were detected using fluorescent quantitative Real-Time PCR. Fluorescent quantitative Real-Time PCR SYBR Premix Ex Taq kit (Applied Biosystem) was used, SYBR Green was used as a fluorescent dye, 20. mu.L of reaction system was used, and the number of reaction cycles was 40.

And (3) PCR running program: denaturation at 95 ℃ for 10 seconds; annealing and extension at 60 ℃, 30 seconds, 20 mu L of reaction system, 40 cycles of reaction cycle number and finally 5 minutes of holding at 72 ℃. The internal reference is beta-Actin. The reaction primers were as follows:

rab13 forward primer (5 '-3'): AGAGCATGGAATCCGATTTTTCG

Rab13 reverse primer (5 '-3'): CTGCTATTTCTCCCCTGCTCA

β -Actin forward primer (5 '-3'): GAGCACAGAGCCTCGCCTTT

beta-Actin reverse primer (5 '-3'): ATCCTTCTGACCCATGCCCA

The reaction primer sequence is shown as SEQID NO. 1-4.

And (3) obtaining a delta Ct value according to a fluorescence map given by an instrument by taking the internal reference as beta-Actin, calculating a relative delta (delta Ct) value, and further calculating the relative change of the mRNA level of Rab 13.

The results show that: CDDO-Me was able to dose-dependently inhibit Rab13 mRNA expression (fig. 2A).

2.2Rab13 protein expression assay

Respectively mixing 2.5X 10⁵(MDA-MB-231) or 5X 10⁵(HCC1806) Breast cancer cells were seeded in 6cm dishes and cultured normally overnight. The cells were treated with 0 (control, equal volume of DMSO), 20, 40, 80 and 160nM CDDO-Me for 48 h. Cells were harvested, lysed with RIPA lysate, and protein concentration was determined with BCA protein quantification kit (purchased from Pierce, #23225) according to the instructions. Western blot was used to measure the expression level of Rab13 in each cell group.

The results show that: CDDO-Me was able to dose-dependently inhibit Rab13 protein expression (fig. 2B).

Example 3 CDDO-Me could inhibit Rab13 expression in MMTV-PyMT mouse mammary tumor tissue

8-10 week old female MMTV-PyMT spontaneous mammary tumor mice (tumor 200-³)4, divided into two groups of 2 (two tumors per mouse), treated as follows:

a first group: a control group;

second group: CDDO-Me treatment group.

The CDDO-Me treatment group was given 5mg/kg CDDO-Me treatment, and the control group was given an equal volume of dimethyl sulfoxide treatment. The preparation is administered by intraperitoneal injection once every 3 days for 5 times. After the experiment is finished, the tumor is dissected and photographed, and meanwhile, the tumor and formalin fixing solution are fixed for 2-7 days, paraffin-embedded sections are carried out, and then immunohistochemical staining is carried out. The method mainly comprises the following steps: after section dewaxing, rehydration and blocking, and the like, the sections are incubated with Anti-Rab13 (Sigma: SAB4200057) antibody at 4 ℃ overnight, the secondary antibody is incubated for 1h at room temperature, hematoxylin staining is carried out after DAB color development, then conventional dehydration and blocking are carried out, the sections are observed by using a conventional optical microscope and photographed for recording, and the IHC staining pictures are quantitatively analyzed.

The results show that: CDDO-Me treatment was able to inhibit Rab13 expression in MMTV-PyMT mouse mammary tumor tissue (figure 3). It is further demonstrated that CDDO-Me is capable of inhibiting Rab13 expression at in vivo levels.

Example 4 effective knock-down of shRNAs of Rab13

shRNAs of Rab13 are cloned into a Plko.1 vector, then the Plko.1-Rab13-shRNA, pMD2G and pSPAX2 plasmids are cotransfected with HEK293T cells, and the lentivirus is packaged. The lentivirus is infected with HCC1806 or MAD-MB-231 cell line, and HCC1806 and MAD-MB-231 cell lines stably knocking down Rab13 are prepared through resistance selection. Control group: transfection of control shRNA (pLKO.1TRC control, Addge Cat # 10879); rab13 knockdown group 1 and Rab13 knockdown group 2shRNA sequences are shown in Table 1 below and SEQ ID NO: 5-10. Protein assays described in 2.2 were performed in Rab13 stable knock-down cell lines to determine whether Rab13 was efficiently knocked down.

Table 1 shRNAs for Rab13 knockdown

The results of the experiment are shown in FIG. 4. The results show that the shRNAs can effectively knock down Rab13 in breast cancer cells.

Example 5 knockdown Rab13 inhibits tumor microenvironment cell-mediated tumor stem cell dryness at the cellular level

5.1 induced differentiation of THP-1 into macrophages

THP-1 cells were treated with 50nM PMA for 3 hours, washed three times with PBS, plated into culture dishes and incubated overnight. The following day, adherent growth of cells was successfully induced into macrophages.

5.2 Co-culture of tumor cells with microenvironment cells (THP-1 macrophages or tumor-associated fibroblasts)

The groups of cells from example 4 were induced with those from 5.1THP-1 differentiated into macrophages was co-cultured. Co-cultivation was carried out using a 0.4. mu.M pore size transwell co-cultivation plate, and 1X 10 plates were laid on the lower layer⁵In example 4, 5X 10 cells were layered on each cell group⁴THP-1 macrophage, after culturing for 48h, carrying out flow detection of stem cell marker and tomorsphere formation experiment.

5.3 flow assay Stem cell markers

ALDH staining was performed according to the ALDH detection kit (Stem Cell Technologies, Cat #01700) instructions. CD44 and CD24 staining were as follows: digesting the cells, taking 1 × 10⁶Each group of cells was washed 3 times with PBS, 100. mu.L of PBS was resuspended in cells, and FITC-labeled CD44 antibody and PE-labeled CD24 antibody (1:100) were added. Incubate on ice for 30min and wash 3 times with PBS. Flow-type machine for respectively detecting ALDH masculinity and CD44⁺CD24^-/lowThe stem cell population ratio varied.

The results show that Rab13 knockdown can inhibit macrophage/tumor-associated fibroblast-mediated ALDH positivity and CD44⁺CD24^-/lowThe stem cell population proportion was increased (fig. 5A and 5B).

5.4 Tumorsephere Formal experiments

Preparing a tomorphere culture medium: 9ml human mammcult basal medium +1ml supplement + 20. mu.l heparin + 5. mu.l hydrocortisone (1 mg/ml). 5.2 after the co-culture of each group of cells, the tumor cells in the lower layer were digested and counted. Resuspension of cells in tomorphere's medium (MDA-MB-231, 2X 10)⁴cell/mL; HCC1806, 1X 10⁴cell/mL), 100. mu.L of the cells were plated in a 96-well plate, the cells were cultured for 7-10 days at 37 ℃ under 5% carbon dioxide, and the cells were counted by photographing.

The results indicate that Rab13 knockdown can inhibit macrophage/tumor associated fibroblast-mediated enhancement of breast cancer cell tumors formation (fig. 5C).

Example 6 knockdown Rab13 inhibits chemotherapeutic-mediated tumor stem cell desiccation at the cellular level

Doxorubicin (DOX, 5nM), paclitaxel (PTX, 1nM) and cisplatin (CIS, 100nM) were treated for 24 hours on each group of cells in 5.2, digested, stained for breast cancer stem cell markers in 5.3, flow tested for ALDH positivity and CD44⁺CD24^-/lowThe stem cell population ratio varied.

The results show that Rab13 knockdown can inhibit macrophage/tumor-associated fibroblast-mediated breast cancer ALDH positive and CD44⁺CD24^-/lowThe stem cell population proportion increased (figure 6).

Example 7 knockdown Rab13 increases sensitivity of breast cancer to chemotherapeutic drugs at the cellular level

7.1 detection of the killing Effect of chemotherapeutic drugs on Breast cancer cells by apoptosis

Cells were digested by doxorubicin (DOX, 5nM), paclitaxel (PTX, 1nM) and cisplatin (CIS, 100nM) for 24 hours in 5.2 groups, and 1X 10 cells were collected per group⁶Cells, washed 3 times with PBS, were double stained with Annexin V and PI following the protocol of the apoptosis detection kit (eBioscience, Cat # 88-8005-74). And (5) detecting apoptosis by flow and counting. The results indicate that Rab13 knockdown enhances apoptosis of breast cancer cells mediated by the chemotherapeutic drugs.

7.2MTS assay of the inhibitory Effect of chemotherapeutic Agents on Breast cancer cells

Each of HCC1806 cells in 5.2 or MDA-MB-231 cells in 5.2 was treated with 0, 0.3125, 0.625, 1.25, 2.5, and 5. mu.M Doxorubicin (DOX) for 48 hours, or each of MDA-MB-231 cells in 5.2 was treated with 0, 1, 2, 4, 8. mu.M DOX. Then, the cell activity was measured by MTS, and the MTS assay kit (CellTiter) was used

AQueous Non-Radioactive Cell promotion Assay, Promega, G3581). The formula for calculating the cell inhibition rate is as follows: inhibition (%) - (DOX untreated group (0 μ M) -DOX treated group)/DOX untreated group (0 μ M) × 100%. And (5) drawing a tumor cell inhibition curve. The results show that Rab13 knockdown enhances the inhibition of breast cancer cell activity mediated by the chemotherapeutic drugs.

Example 8 knockdown Rab13 in inhibiting chemotherapeutic drug-mediated Breast cancer recurrence

8.1 establishment of model of breast cancer orthotopic transplantation tumor

Respectively mixing 5 × 10⁶Control cells and Rab13 Stable knockdown cells were resuspended in 100. mu.L medium and matrigel 1:1 in a mixed solution. After the anesthetic is injected into the abdominal cavity of the nude mouse, 75% alcohol disinfection is carried out on the abdominal cavity, the skin is cut open to expose the fourth pair of mammary glands, 100 mu L of the cell suspension is injected into the left mammary fat pad, and the skin is sutured by a suturing device.

8.2 Doxorubicin (DOX) administration treatment

About 100-200mm long after 3 weeks of tumor transplantation³And carrying out DOX administration treatment. The grouping is as follows:

a first group: a control group, wherein the knock-down control group is injected with PBS with the same volume;

second group: control group + DOX, control group (Rab13 not knocked down) given DOX treatment, injection dose of 5mg/kg/3 days, total 3 times;

third group: rab13 knockdown group + DOX, Rab13 knockdown mice were given the same DOX treatment as the second group.

The measurement of the volume of the mice was performed from the start of the transplantation after the tumor cell transplantation for 7 weeks. After the experiment was completed, the tumor was dissected and photographed. The formula for calculating the tumor volume is as follows: tumor size 0.52 x (width)²X is long.

The results show that tumor volume continued to increase after discontinuation of Rab13 knockdown dosing, eventually making no difference from the control group that did not, while tumor growth remained inhibited after the Rab13 knockdown dosing was over. This demonstrates that Rab13 is effective in inhibiting recurrence after administration of breast cancer chemotherapy (fig. 8A and 8B).

Example 9 CDDO-Me treatment inhibits microenvironment cell-mediated sternness of breast cancer stem cells

9.1 microenvironment cell Conditioned Medium (CM) preparation

Growing the tumor-associated fibroblast (CAF) to 70-80% confluence, replacing a serum-free culture medium for further culturing for 24 hours, collecting a culture supernatant, centrifuging and taking dead cells to obtain the tumor-associated fibroblast conditioned medium (CAF-CM).

RAW264.7 or THP-1 macrophage and MDA-MB-231 after induced differentiation according to 5.1 are co-cultured for 48h by a method in 5.2, then tumor cells are removed and continuously cultured for 24h by a serum-free culture medium, culture supernatant is collected and centrifuged to take dead cells, and macrophage conditioned medium (RAW-CM and THP-CM) is obtained respectively.

9.2CDDO-Me and CM co-processing cells to detect the stem dryness of the breast cancer stem cells

MDA-MB-231 cells were treated with CDDO-Me or CM, respectively, and the treatment groups were as follows: control, cells treated with an equal volume of DMSO; experimental group, 0, 20, 40, 80 and 160nM CDDO-Me were suspended in 20% macrophage conditioned medium or 100% CAF-CM to treat cells, and after 24 hours, experiments for flow detection of breast cancer stem cell population ratio and tomorsphere formation were performed according to methods in 5.3 and 5.4, respectively.

The results show that CDDO-Me treatment can inhibit microenvironment cell-mediated breast cancer ALDH positive and CD44⁺CD24^-/lowIncreased proportion of stem cell population (FIGS. 9A and 9B) and tumors sphere formation (FIG. 9C).

Example 10 CDDO-Me treatment inhibits tumor microenvironment cell-mediated chemotherapy resistance

MDA-MB-231 cells were treated with Doxorubicin (DOX) or/and CDDO-Me or and CM, respectively, and the treatment groups were as follows:

a first group: DOX treatment at concentrations of 0.17625, 0.3125, 0.625, 1.25, 2.5 and 5 μ M;

second group: DOX + RAW-CM/THP-CM/CAF-CM treated group, cells were given 20% RAW-CM or THP-CM treatment or 100% CAF-CM treatment on the basis of the first group

Third group: DOX + RAW-CM/THP-CM/CAF-CM + CDDO-Me treatment group, 160nM CDDO-Me treatment was given on the second group basis.

After 24 hours of cell treatment, MTS assay was performed according to MTS kit instructions to evaluate cell activity. The results show that conditioned medium treatment with macrophages and tumor-associated fibroblasts can enhance the resistance of breast cancer cells to DOX, while the cell activity is further reduced by this resistance to CDDO-Me treatment inhibitors.

The experimental results are shown in fig. 10A, 10B, and 10C. The results show that: indicating that CDDO-Me treatment can inhibit DOX drug resistance mediated by tumor as environmental cells. Therefore, the CDDO-Me can be combined with tumor chemotherapy drugs for use in preparing antitumor drugs.

Example 11 knockdown Rab13 inhibition of EGFR protein expression

The protein assay described in 2.2 was performed in Rab13 stable knock-down cell lines (see example 4) to determine the effect of knock-down Rab13 on EGFR protein levels. The results of the experiment are shown in FIG. 11. The results show that protein expression of EGFR can be inhibited after Rab13 knock-down.

Example 12 knockdown Rab13 enhancement of Breast cancer sensitivity to EGFR-targeting drugs

Rab13 stable knockdown HCC1806 cell line and its corresponding control cell line (see example 4) were plated at 5000 cells/well cell concentration into 96-well plates and after overnight cell growth, treated with cetuximab or gefitinib. The treatment concentration is as follows: cetuximab, 0 or 300 nM; gefitinib, 0 or 1 μ M. After 72 hours of cell treatment, MTS assay was performed according to the MTS kit protocol to evaluate cell activity.

The results of the experiment are shown in fig. 12 (left) and 12 (right). The result shows that cetuximab has no inhibition effect on Rab13 non-knock-down control cells, and gefitinib can inhibit the activity of the cells. The inhibition effect of cetuximab and gefitinib on Rab13 knockdown cells is obviously stronger than that of Rab13 knockdown cells. The results show that knockdown Rab13 can increase the effectiveness of breast cancer cells on EGFR targeting drugs cetuximab and gefitinib.

Example 13 CDDO-Me could inhibit EGFR expression in breast cancer orthotopic transplanted tumor tissue

And (3) establishing a breast cancer orthotopic transplantation tumor model by using MDA-MBA-231 cells according to 8.1. Two weeks after tumorigenesis, randomly dividing the nude mice into two groups, 10 mice in each group, and injecting equal volume of DMSO into the abdominal cavity of one group without a control group; another group was administered with CDDO-Me (2mg/kg/3 days) intraperitoneally. Tumors were dissected 10 times after administration and IHC staining was performed with Anti-EGFR (Sigma: SAB4200057) antibody (same as in example 3).

The results show that: CDDO-Me treatment was able to inhibit EGFR expression in breast cancer transplantable tumor tissues (fig. 13).

Example 14 CDDO-Me can enhance the sensitivity of breast cancer to EGFR-targeting drugs

MDA-MB-231 or HCC1806 cells were plated into 96-well plates at 5000 cells/well cell concentration and after overnight cell growth, treated with cetuximab or gefitinib. Cetuximab or gefitinib treatment were grouped as follows:

a first group: control, treated with equal volumes of PBS and DMSO;

second group: respectively treating cetuximab or gefitinib with concentrations of 300nM or 2 μ M;

third group: CDDO-Me single drug treatment at 160 nM;

and a fourth group: cetuximab or gefitinib is co-treated with CDDO-Me at the same concentrations as in the second and third groups.

After MDA-MB-231 cells were treated for 48 hours or HCC1806 cells were treated for 72 hours, MTS assay was performed according to the MTS kit protocol to evaluate cell viability.

The results show that cetuximab has no obvious inhibition effect on breast cancer cells, gefitinib and CDDO-Me single drugs can inhibit the breast cancer cells, and the inhibition effect of the combined treatment of the cetuximab or the gefitinib and the CDDO-Me on the breast cancer cells is obviously higher than that of a single drug treatment group (fig. 14A and 14B). This suggests that CDDO-Me may increase the effectiveness of breast cancer cells on EGFR targeting drugs cetuximab and gefitinib. That is to say, the CDDO-Me and the EGFR targeting drug are used together to be applied to the preparation of the antitumor drugs.

Claims

1. An inhibitor of the Rab13 gene, characterized in that: the inhibitor is bardoxolone methyl, and can inhibit the expression of Rab13 gene and reduce the expression product level of Rab13 gene.

2. The use of claim 1, wherein: the inhibitor is used for preparing a medicament for treating diseases related to the high expression of Rab13 gene.

3. Use according to claim 2, characterized in that: the inhibitor is used for preparing a medicine for enhancing the sensitivity of chemotherapeutic drugs related to the high expression of Rab13 gene.

4. Use according to claim 2, characterized in that: the inhibitor is applied to the preparation of the medicine for enhancing the sensitivity of the EGFR targeting medicine.

5. Use according to claim 3, characterized in that: the inhibitor and chemotherapeutic drugs related to the high expression of Rab13 gene are used together to prepare the antitumor drugs.

6. The use of claim 4, wherein: the inhibitor and an EGFR targeting drug are combined for use in the preparation of antitumor drugs.

7. The use of claim 5, wherein: the medicines for diseases related to the high expression of Rab13 gene are DOX, PTX and CIS.

8. The use of claim 6, wherein: the EGFR targeting drug is cetuximab or gefitinib.