LU505196B1

LU505196B1 - Carvedilol in the Preparation of Drugs to Reverse Leukaemia Resistance

Info

Publication number: LU505196B1
Application number: LU505196A
Authority: LU
Inventors: Yanpeng Xu
Original assignee: Affiliated Hospital Of Zunyi Medical Univ
Priority date: 2023-01-13
Filing date: 2023-09-27
Publication date: 2024-03-28
Also published as: CN116270614A

Abstract

The present invention discloses the use of carvedilol in the preparation of drugs to reverse leukaemia resistance, and belongs to the field of pharmaceutical technology, wherein said reversing leukaemia resistance drug means reversing leukaemia resistance to glucocorticoid drugs. The carvedilol in the preparation of drugs to reverse leukaemia resistance is provided by the present invention, through which it has been found that carvedilol up-regulates the expression of TopoIIα by inhibiting β3-AR/cAMP/PKA/STAT3, thereby reversing the resistance of T-ALL to dexamethasone.

Description

Carvedilol in the Preparation of Drugs to Reverse Leukaemia Resistance 1000168

Technical Field

The present invention relates to the field of pharmaceutical technology, and specifically to the application of carvedilol in the preparation of drugs to reverse leukaemia resistance.

Background Technology

Acute lymphoblastic leukaemia (ALL) is a haematological disease characterised by abnormal differentiation and malignant proliferation of bone marrow haematopoietic stem cells or cells of the lymphoid lineage. Currently, glucocorticosteroids, such as dexamethasone (DEX), are one of the most important drugs in the treatment of acute T-lymphoblastic leukaemia (T-ALL), but 20% of children develop resistance to this drug, which is the main cause of treatment failure and relapse. Therefore, reversing T-ALL resistance to dexamethasone would be of great clinical significance in the treatment of leukaemia.

B-Adrenergic receptors (B-ARs), categorised as B1-AR, B2-AR and B3-AR, have been found to be closely associated with cancer development and progression. In addition, most studies have reported the relationship between B-ARs and tumour proliferation, apoptosis, invasion and angiogenesis. However, there are limited reports on the relationship between B-ARs and tumour drug resistance. Calvani et al. found that B3-AR was associated with adriamycin resistance in malignant tumours, but the exact mechanism is unknown. In addition, Feijun et al. showed that

B2-ARs play an important role in cisplatin resistance in ovarian cancer. This suggests that B-ARs may be associated with cancer drug resistance. According to the Kyoto Encyclopedia of Genes and

Genomes (KEGG), it has been recognised that activated B-ARs directly or indirectly stimulate a variety of signalling pathways, including the ras, PI3K/AKT/MTOR, and JAK2/STAT3 signalling pathways, via cAMP/PKA or cAMP/Epac. Many studies have also shown that these signalling pathways are closely associated with drug resistance in cancer. Our previous studies 215079196 confirmed that B-AR and STAT3 are highly expressed in T-ALL glucocorticoid-resistant cell lines, so the B3-AR/cAMP/PKA/STAT3 signalling pathway plays an important role in T-ALL resistance to DEX by regulating the expression of downstream resistance-related molecules, and B-ARs blockers reverse T-ALL drug resistance to DEX by blocking this signalling pathway.

Carvedilol (CVD), a third-generation B-adrenergic receptor blocker, is used ubiquitously in the treatment of heart failure and hypertension. It has a non-selective blocking effect on

B-adrenergic receptors (B1-AR, B2-AR and B3-AR). Currently, research on CVD focuses on its protective effect against chemotherapy-induced heart failure. However, several studies have found that CVD can reverse tumour multidrug resistance (MDR). Takara K and Kakumoto suggested that CVD may act as a reversal agent of MDR in cervical cancer, while Jonsson O et al. suggested that it may act as an inhibitor of the resistance-associated transmembrane transporter protein, p-glycoprotein (P-gp), to reverse adriamycin resistance in breast cancer. They also reported that CVD reversed adriamycin resistance in bladder cancer cells. Thus, it is well known that CVD can reverse drug resistance in tumours. However, whether CVD can reverse T-ALL resistance to DEX has not been reported. To this end, the present invention provides the application of carvedilol in the preparation of drugs to reverse leukaemia resistance.

Contents of the Invention

The present invention provides the application of carvedilol in the preparation of drugs to reverse leukaemia resistance, where carvedilol up-regulates the expression of Topolla by inhibiting B3-AR/cAMP/PKA/STAT3, thereby reversing T-ALL resistance to DEX.

In order to achieve the above objectives, the technical solutions adopted in the present invention are:

The present invention provides the use of carvedilol in the preparation of drugs to reve/#385196 leukaemia resistance.

Preferably, said reversing leukaemia resistance drug means reversing leukaemia resistance to glucocorticoid drugs.

Preferably, said glucocorticoid drug include dexamethasone and its derivatives, prednisone and its derivatives, hydrocortisone and its derivatives.

Preferably, said leukaemia is acute T-lymphoblastic leukaemia.

Preferably, said carvedilol reverses the resistance of acute T lymphoblastic leukaemia to glucocorticoid drugs by inhibiting B3-AR/cAMP/PKA/STAT3 to upregulate the expression of

Topolla

Preferably, said drug is in the form of a solid, semi-solid or liquid.

Preferably, the formulation of said drug comprises an aqueous solution, a non-aqueous solution, a suspension, an ingot, a capsule, a tablet, a granule, a pill or a powder.

Preferably, said drug is administered by the route of injection administration or oral administration.

The beneficial effects of the present invention compared to the existing technology are:

The present invention provides the application of carvedilol in the preparation of drugs to reverse leukaemia resistance, through which it was found that carvedilol up-regulates the expression of Topolla by inhibiting B3-AR/cAMP/PKA/STAT3, thereby reversing the resistance of

T-ALL to the glucocorticoid dexamethasone.

Brief Description of the Drawings

FIG.1: the viability of DEX treated CEM-C1 and CEM-C7 cells of the present invention;

FIG.2: the induction of apoptosis in CEM-C1 and CEM-C7 cells by DEX of the present invention;

FIG.3: the resistance of CEM-C1 cells treated with CVD plus different concentrations of DEX of the present invention;

FIG.4: the difference in expression of the B-AR of the present invention in CEM-C1 and

CEM-C7 cells;

FIG.5: the expression differences of JAK2/STAT3 of the present invention on CEM-C1 and

CEM-C7 cells;

FIG.6: the differences in the expression of different drug resistance proteins of the present invention on CEM-C1 and CEM-C7 cells;

FIG.7: the viability of CEM-C1 and CEM-C7 cell lines treated with SR59230A of the present invention;

FIG.8: the viability of CEM-C1 and CEM-C7 cell lines treated with sodium BRL 37344 of the present invention;

FIG.9: the expression differences between CEM-C1 and CEM-C7 treated with 20uSR59230A of the present invention;

FIG.10: the viability of CEM-C1 and CEM-C7 cell lines treated with Static, the STAT3 inhibitor of the present invention;

FIG.11: the viability of Colivelin TFA treated CEM-C1 and CEM-C7 cell lines of the present invention;

FIG.12: the expression differences between Colivelin TFA and Static of the present invention treated with CEM-C1, CEM-C7 respectively;

FIG.13: the viability of two cell lines, CEM-C1 and CEM-C7, treated with bisoprolol hemifumarate, ICI 118551 hydrochloride and Amonafide of the present invention;

FIG.14: the resistance of DEX of the present invention to bisoprolol hemifumarate, hydrochloride, SR59230A and sodium BRL 37344 treatment of CEM-C1 cells;

FIG.15: the resistance of DEX of the present invention to Static and ColivelinTFA treated cells;

FIG.16: the resistance of DEX of the present invention to Amonafide treated cells;

FIG.17: the differences in the expression of B3-AR, cAMP, PKA, STAT3 and p-STAT3 after CVD treatment of CEM-C1 and CEM-C7 of the present invention;

FIG. 18: a plot of xenograft tumour volume and weight for the DEX-C7 group and the CVD + 5 DEX-C1 group of the present invention;

FIG.19: the results of H&E staining of some xenograft cells in the DEX- C7 group and DEX +

CVD-C1 group of the present invention;

FIG.20: the bone marrow leukaemia infiltration in the DEX-C7 group and CVD + DEX-C1 group of mice of the present invention;

FIG.21: a graph of the expression of the CDs antigen of the present invention;

FIG.22: a flow cytometry analysis of the present invention.

Specific Embodiments

In order to enable the technical personnel in the field to better understand the technical solution of the present invention to be implemented, the following combines specific embodiments and the accompanying drawings to further illustrate the present invention, but the cited embodiments are not to be taken as a limitation of the present invention. The following test methods and detection methods are conventional methods if not otherwise specified; the reagents and raw materials mentioned are commercially available if not otherwise specified. example of implementation

Materials and methods

Materials

Drugs and Reagents:

Carvedilol (CVD) was purchased from TopScience (Shanghai, China) CAS No.: 72956-0953" 0 with the following structural formula: a ;

Bisoprolol hemifumarate (B1-AR blocker), ICI 118551 hydrochloride (B2-AR blocker),

SR59230A (B3-AR blocker), BRL 37344 sodium (B3-AR activator), Stattic (STAT3 inhibitor), Colivelin

TFA (STAT3 activator), Amonafide (Topoll inhibitor) and dexamethasone (DEX) were purchased from MCE (Shanghai, China);

RPMI-1640 medium was purchased from Gibco (Grande Island, NY, USA);

Fetal bovine serum (FBS) was obtained from Livning Biotech (Beijing, China);

Cell-Counting Kit-8 (CCK8) was purchased from Dojindo (Beijing, China);

Quantitative analysis of DNA content (cell cycle) was purchased from Solarbio (Beijing,

China);

Apoptosis kits were purchased from US EVERBRIGHT (Suzhou, China);

B1-AR, B2-AR and B3-AR antibodies were obtained from Abbkine Scientific Co.,Ltd (Santa

Cruz, CA, USA);

Antibodies to cAMP, JAK2, STAT3, p-STAT3 and B-actin were purchased from Abcam (Cambridge, MA, USA);

PKA, P-gp, MRP, Topolla, B-Tubulin and GAPDH antibodies were obtained from Huabio (Hangzhou, China).

Experimental Methods

Drug preparation: DEX and other drugs were dissolved in a small amount of DMSO and then diluted in RPMI-1640 medium and stored at -80°C. Before use, the above drugs were diluted in the medium separately and made into a concentration of 10~80 uM. 17008198

Cell culture: Human T-ALL cell lines CEM-C1 and CEM-C7 were obtained from West China

Hospital of Sichuan University and were preserved in RPMI-1640 medium containing 10% fetal bovine serum, respectively, at 37°C in a 5% CO» incubator.

Cell viability assays:

The inhibitory effect of carvedilol on leukaemia cell viability was detected by CCK8 assay in 96-well plates (20,000 cells per well) were used separately:

CVD (0, 10, 20, 40, 60, and 80 uM) acted for 24 h and 48 h.

DEX (0, 0.5, 1, 5, 10, 20, 50, 100, and 200 pM) acted for 24 h, 48 h, and 72 h.

Bisoprolol hemifumarate, ICI 118551 hydrochloride, and SR59230A (0, 10, 20, 40, 60, and 80 uM) acted for 24 h and 48 h.

BRL 37344 sodium (0, 2.5, 5, 10, 20, and 50 uM) acted for 6 h and 24 h.

Stattic and Colivelin TFA (0, 0.5, 1, 5, 10, and 20 uM) acted for 24 h and 48 h.

Amonafide (0, 1, 5, 10, 20, and 50 uM) acted for 24 h and 48 h.

Then 10 uLCCK8 was added to each well and incubated at 37°C for 2 h under light-avoidance conditions, and then the 96-well plate was placed in an enzyme labeller (BIO-BRI Chengdu, China) and detected at 450 nm.

Methods of apoptosis experiments:

The effect of dexamethasone (1 pM and 5 pM) on apoptosis of leukaemia cells was detected by fluorescent staining with Annexin V/PI. Cells were inoculated in 12-well plates at a density of 2x10° cells/ml, and after incubation with dexamethasone (1 uM and 5 pM) for 24 h and 48 h, the cells were collected and gently washed three times with PBS, and then, 2x10° cells were collected and incubated with 100 pl of 1xAnnexin V binding buffer was resuspended, and then 5 pL of

YFR647A-Annexin V and 5 pl of propidium iodide (PI) were incubated with the cells at room temperature and protected from light for 15 min. Apoptosis was detected using flow cytometry

(BD FACSCanto, San Jose, CA, USA). 505796

Immunoblot analysis method (Western blot):

CEM-C1 and CEM-C7 cells were treated with 20 uM CVD, 20 pM SR59230, 20 UM BRL 37344sodium, 0.5 pM Static, and 5 pM Colivelin TFA, respectively, for 24 h. The cells were then centrifuged at 1200 rpm for 5 min, the supernatant was removed, and the supernatant was added to a PMSF(100:1) RIPA lysis enhancement buffer; then centrifuged at 12000 g for 30 min at 4°C, the supernatant was removed, and the protein samples were quantified by BCA method, adjusted to 2.3 ug/ul, and added to the uploading buffer.

Equal amounts of protein lysates were disaggregated by 10% PAGE and transferred to electrophoresis membranes using the BIO-RAD semi-dry transfer system; the membranes were then closed with 5% skimmed milk for 60 min at room temperature and incubated overnight at 4°C with appropriate concentrations of primary antibodies (1:1000 GAPDH, B-actin, B-tubulin,

B1-AR, B2-AR, B3-AR, cAMP, PKA, Topolla, P-gp, MRP; 1:2000 JAK2, STAT3, and P-STAT3) after which the membranes were washed three times with TBST and incubated for 60 min with the secondary antibody (1:10,000) three times. The membranes were placed in ECL luminescent reagent for 10 s and scanned with a gel imager (Bio-Rad, Hercules, CA, USA).

Construction of an in vivo model of T-ALL:

The animal experiment protocol was approved by Zunyi Medical University (Ethical Approval

No. ZMU21-2204-001) and was conducted in accordance with the UK Animals (Scientific

Procedures) Act 1986 and related guidelines, European Union Directive 2010/63/EU on animal experimentation.

Four-week-old female nude mice, weighing 17.0~ 20.0 g, were purchased from Changzhou

Cavens Laboratory Animal Co. Ltd (strain: BALB/c-nu, batch number: 202210375, licence: SCXK (Su) 2021-0013). These mice were kept in the SPF laboratory of Zunyi Medical University for one week, and the logarithmic growth phase CEM-C1 and CEM-C7 cells were washed twice with PBS,

the cell concentration was adjusted to 5x10’ cells/mL, and 100 pl of cell suspension was injected >98 subcutaneously into the posterior side of the right axilla of each mouse. Xenograft tumours were formed 1 week after transplantation. Nude mice injected with CEM-C1 cells were randomly divided into four groups (3 mice in each group): control group (CON-C1 group), carvedilol group (CVD-C1 group), dexamethasone group (DEX-C1 group), and combination group (CVD + DEX-C1 group). Nude mice injected with CEM-C7 cells were randomly divided into two groups (3 mice per group): control group (CON-C7 group) and dexamethasone group (DEX-C7 group). When the leukaemia transplant tumour grew to approximately 0.5 cm, CVD was administered: 1 mg/kg/d

CVD by gavage; DEX was administered: 20 mg/kg/d DEX by intraperitoneal injection. The control group was given an equal amount of saline. Two weeks after administration, we compared the size and weight of the leukaemic transplant tumours, the percentage rates of CEM-C1 and

CEM-C7 cells in the peripheral blood, and the total number of white blood cells.

Statistical analyses:

Statistical analyses were performed using SPSS 18.0 (Zunyi Medical University, China),

Compusyn software was used for the statistical semi-inhibitory concentration of drugs, data were expressed as mean + standard deviation, and one-way ANOVA or unpaired t-tests were used to assess the differences. p<0.05, the difference was statistically significant.

Experimental results 1. Identification of DEX resistance in CEM-C1 and CEM-C7 cell lines

CEM-C1 and CEM-C7 cells were treated with DEX at concentrations ranging from 0.5 ~ 200

UM for 24 h, 48 h, and 72 h, respectively. As shown in FIG. 1, CCK8 analysis showed that dexamethasone significantly inhibited the activity of CEM-C7 cells compared with CEM-C1 cells (P < 0.05). The induction of apoptosis in CEM-C1 and CEM-C7 cells by 1 uM and 5 uM DEX was also analysed by flow cytometry, and the induction of apoptosis in CEM-C1 and CEM-C7 cells by dexamethasone is illustrated in FIG. 2, where a is the induction of 24 h and b is the induction of 2° 48 h for both CEM-C1 and CEM-C7. As shown in FIG. 2, a and b shown, DEX could not significantly induce apoptosis in CEM-C1 cells but significantly promoted apoptosis in CEM-C7 cells compared with CEM-C1 cells (P < 0.05). Thus, CEM-C1 cells were resistant to dexamethasone, whereas

CEM-C7 cells were sensitive to dexamethasone. 2. CVD reverses DEX resistance in CEM-C1 cells

The two cell lines were treated with CVD at concentrations of 10~ 80 uM for 24 h and 48 h, respectively, and it was found that CVD reduced the cellular activity, and the semi-inhibitory concentrations (IC50) of CVD were 16.80+4.51 pM and 12.35+1.42 pM for CEM-C1 and CEM-C7 cells at 24 h, and at 48 h, respectively, the semi-inhibitory concentrations (IC50) were 7.68+1.35

UM and 7.27+1.29 uM, respectively (shown as c in FIG. 2).

Then CEM-C1 cells were treated with non-inhibitory concentrations (less than IC50) of CVD combined with different concentrations of DEX for 24 h and 48 h. FIG.3 shows the effect of 2 uM and 5 uM CVD combined with different concentrations of DEX on the activity of CEM-C1 cells after treatment with 2 uM and 5 uM of CVD by CCK8 method, and FIG.3 shows that a is the reversed resistance folds of CVD+DEX treated for 24 h, and b is the 24-h reversal of CVD resistance multiplicity, c is the CVD+DEX treatment for 48h, and d is the reversed resistance multiplicity of CVD at 48h. The results showed that both 2 uM and 5 uM CVD could reverse the resistance of CEM-C1 to DEX, with the reversal multiples of 3.71 and 2.24 at 24 h and 2.32 and 2.28 at 48 h, respectively. 3. Differential levels of B3-AR, STAT3 and Topolla proteins between CEM-C1 and CEM-C7 cells

In order to further explore the mechanisms related to the reversal of DEX resistance in

CEM-C1 cells by CVD, the present invention initially examined the differences in the expression of

PB- AR proteins in CEM-C1 and CEM-C7 cells, and FIG. 4 shows the differences in the expression of

B-AR in CEM-C1 and CEM-C7 cells. As shown in FIG. 4, B3-AR protein was significantly highly expressed in CEM-C1 cells compared to CEM-C7 cells (P < 0.05), whereas the expression of B1-AR and B2-AR proteins was not significantly different on these two cells (P > 0.05).

The levels of JAK2 and STAT3 proteins were examined, and FIG.5 shows the difference in

JAK2/STAT3 expression on CEM-C1 and CEM-C7 cells. As shown in FIG.5, STAT3 protein was significantly highly expressed in CEM-C1 cells compared to CEM-C7 cells (P < 0.05), whereas JAK2 protein was not significantly different in the two cell lines (P > 0.05).

In addition, we also examined the expression levels of drug resistance-related proteins, and

FIG.6 shows the expression differences of different drug resistance proteins on CEM-C1 and

CEM-C7 cells. As shown in FIG.6, compared with CEM-C7 cells, we found that only Topolla protein expression was significantly reduced on CEM-C1 cells (P < 0.05), while MRP and P-gp proteins were not significantly different between the two cell lines (P > 0.05). 4. Validation of the B3-AR/STAT3/Topolla signalling pathway

Firstly, to determine whether B3-AR could regulate STAT3, CEM-C1 and CEM-C7 cell lines were treated with SR59230A (B3-AR blocker) at a concentration of 10 uM ~ 80 uM for 24 h and 48 h, respectively, while BRL 37344 sodium (B3-AR activator) at a concentration of 2.5 pM ~ 50 uM was applied for treatment, respectively. CEM-C1 and CEM-C7 cell lines for 6 h and 24 h. FIG.7 shows the activity graphs of SR59230A-treated CEM-C1 and CEM-C7 cell lines; in FIG.7, a is

CEM-C1 and b is CEM-C7; and FIG.8 shows the activity graphs of BRL 37344 sodium-treated

CEM-C1 and CEM-C7 cell lines; in FIG.8, a is CEM-C1 and b is CEM -C7. As shown in FIG.7 and 8,

CCK8 analysis indicated that SR59230A decreased the viability of both CEM-C1 and CEM-C7 cell lines, while BRL 37344 sodium promoted the viability of both CEM-C1 and CEM-C7 cells. After that, the changes of STAT3 protein were detected after applying 20 uM SR59230A and BRL 37344sodium to treat CEM-C1 and CEM-C7 for 24h, respectively. As shown in FIG.9, where a is in — CEM-C1 cells, STAT3 was significantly decreased in the SR59230A group compared with the control group (P < 0.05); while b is in CEM-C7 cells, STAT3 was elevated in the BRL 37344 sodium 9 group compared with the control group (P < 0.05). This suggests that B3-AR positively regulates

STAT3 expression.

Next, CEM-C1 and CEM-C7 cell lines were treated with the STAT3 inhibitor Static at concentrations of 0.5 uM ~ 20 uM for 24 h and 48 h, and the two cell lines were treated with

Colivelin TFA (STAT3 activator) at concentrations of 0.5 pM ~ 20 uM for 6 h and 24 h. FIG. 10 shows the activity graphs of STAT3 inhibitor Static treatment of the CEM-C1 and CEM-C7 cell lines activity graphs, in FIG.10, a is CEM-C1 and b is CEM-C7; FIG.11 shows the activity graphs of

Colivelin TFA treatment of CEM-C1 and CEM-C7 cell lines, in FIG.11, a is CEM-C1 and b is CEM-C7.

As shown in FIG.10 and 11, the analysis of CCK8 assay showed that Static inhibited the viability of both cell lines, while Colivelin TFA promoted the viability. Afterwards, changes in Topolla protein were detected after applying 5 uM Colivelin TFA and Static to treat CEM-C1 and CEM-C7 for 24 h, respectively. As shown in FIG.12, protein blotting analysis showed that Topolla was significantly lower (P < 0.05) in the Colivelin TFA group and significantly higher (P < 0.05) in the Static group compared with the control group. This indicates that STAT3 can negatively regulate the expression of Topolla.

In summary, B3-AR positively regulates STAT3 and then negatively regulates Topolla. 5. CVD upregulates Topoll through inhibition of B3-AR/cAMP/PKA/STAT3, which in turn reverses CEM-C1 resistance to DEX

Firstly, two cell lines, CEM-C1 and CEM-C7, were treated with bisoprolol hemifumarate (B1-AR blocker) and ICI 118551 hydrochloride (B2-AR blocker) at concentrations ranging from 10 ~ 80 uM, and Amonafide (Topoll inhibitor) at concentrations ranging from 1 ~ 50 uM for 24 h and 48 h, respectively. FIG. 13 shows the activity diagrams of the two cell lines of CEM-C1 and CEM-C7 treated with bisoprolol hemifumarate, ICI 118551 hydrochloride and Amonafide, respectively.

Where a is bisoprolol hemifumarate, b is ICI 118551 hydrochloride, and c is Amonafide in FIG. 13.

As shown in FIG. 13, the CCK8 results demonstrated that ICI 118551 hydrochloride and

Amonafide inhibited the viability of both cell lines, while bisoprolol hemifumarate did not alter cell viability.

Secondly, the CCK8 method was applied to detect the changes of DEX resistance in CEM-C1 cells after the intervention of three B-AR blockers respectively, while the changes of DEX sensitivity in CEM-C7 cells after the intervention of B3-AR activator were also applied. In FIG.14, a is the B1-AR blocker bisoprolol hemifumarate, b is the B2-AR blocker ICI 118551 hydrochloride, c is the B3-AR blocker SR59230A, and d is the B3-AR activator BRL 37344 sodium. As shown in

FIG.14 a-c, the B3-AR activator BRL 37344 sodium was applied to CEM-C7 cells with 20 uM bisoprolol hemifumarate, 20 pM ICI 118551 hydrochloride and 20 uM SR59230A after treating

CEM-C1 cells for 24, followed by 10 uM DEX for 24 h and 48 h, respectively, CCK8 results showed that only SR59230A treatment decreased the resistance of CEM-C1 cells to DEX (P < 0.05), while bisoprolol hemifumarate and ICI 118551 HCI did not reduce the resistance of CEM-C1 cells to DEX; meanwhile, as shown in FIG.14 d, the sensitivity of CEM-C7 cells to DEX was slightly reduced after

BRL 37344 sodium (B3-AR activator) treatment. This indicates that B3-AR is positively correlated with the resistance of CEM-C1 to DEX.

Again, after treating CEM-C1 with 0.5 uM Static (STAT3 inhibitor) and CEM-C7 cells with 1 uM

Colivelin TFA (STAT3 activator) for 24 h, respectively, the two cell lines were again treated with 10

UM DEX, and FIG. 15 shows the changes in resistance and sensitivity to Static and ColivelinTFA after treating these two cell lines, respectively. DEX resistance and sensitivity changes, in FIG. 15, a is Static treated CEM-C1 and b is ColivelinTFA treated CEM-C7. As shown in FIG. 15, the resistance of CEM-C1 cells to DEX was reduced after Static treatment (P < 0.05), while Colivelin

TFA treated CEM-C7 cells were showed a slight decrease in sensitivity to DEX. This suggests that

STAT3 is also positively correlated with the resistance of CEM-C1 to DEX.

Then, CEM-C1 and CEM-C7 cells were treated with 5 uM Amonafide (Topoll inhibitor) for 24 h, respectively, and then treated with 10 uM DEX again. FIG.16 shows the changes of resistance "96 and sensitivity to DEX in CEM-C1 and CEM-C7 cells after treatment with Amonafide. In FIG. 16, a is the Amonafide treatment of CEM-C1, and b is Amonafide-treated CEM-C7. As shown in FIG. 16,

CEM-C1 cells were significantly more resistant to DEX (P < 0.05), and CEM-C7 cells were significantly less sensitive to DEX (P < 0.05), and had become resistant to DEX. This suggests that

Topoll is a key target for CEM-C1 resistance to DEX.

Finally, after applying 20 pM CVD and BRL 37344 Sodium to intervene in CEM-C1 and

CEM-C7 cells, respectively, Western blot was performed to detect the expression of key proteins on the B3-AR/cAMP/PKA/STAT3/Topoll signalling pathway. FIG.17 shows the differences in the expression of B3AR, cAMP, PKA, STAT3 and p-STAT3 after CVD treatment of CEM-C1 and CEM-C7.

In FIG.17, a is CVD-intervened CEM-C1 cells and b is BRL 37344 sodium-intervened CEM-C7 cells.

As shown in FIG.17, B3-AR, cAMP, PKA, STAT3 and p -STAT3 were significantly decreased (P < 0.05) and Topolla was significantly increased (P < 0.05) in CEM-C1 cells in CVD group, compared with control group. Meanwhile, B3-AR, cAMP, PKA, STAT3 and P -STAT3 were significantly higher (P < 0.05) and Topolla was significantly lower (P < 0.05) in CEM-C7 cells in the BRL 37344 sodium group as compared to the control group.

In summary, the B3-AR/cAMP/PKA/STAT3/Topoll signalling pathway plays an important role in CEM-C1 on DEX, and CVD reverses CEM-C1 resistance to DEX by the mechanism that CVD upregulates Topoll expression after inhibiting B3-AR/cAMP/PKA/STAT3, which in turn reverses

CEM-C1 resistance to DEX resistance. 6. In vivo CVD in mice reverses CEM-C1 resistance to DEX

Firstly, CVD reversal of CEM-C1 resistance to DEX was investigated by in vivo experiments; after leukaemia transplantation tumour formation (one week post-transplantation), mice injected with CEM-C7 cells were randomly divided into the control group (CON-C7 group) and DEX (DEX-

C7 group), and mice injected with CEM-C1 cells were randomly divided into the control group

(CON-C1 group), CVD (CVD- C1 group), DEX (DEX- C1 group) and combined drug (CVD + DEX- ei groups. After 2 weeks of drug administration, the graft tumour volume and weight of each group were plotted in FIG. 18, as shown in FIG. 18, the xenograft volume and weight increased rapidly in the CON-C7 group compared with the DEX-C7 group (P < 0.05), and in the CON-C1, CVD- C1 and

DEX-C1 groups compared with the CVD + DEX-C1 group (P < 0.05). In contrast, there was no difference between CON-C1, CVD-C1, and DEX-C1 groups (P > 0.05). Secondly, FIG.19 shows the

H&E staining results of transplant tumours in each group. As shown in FIG.19, apoptosis and coagulative necrosis occurred in some xenograft cells in the DEX- C7 group and DEX + CVD-C1 group. FIG.20 shows the leukaemic infiltration of mouse bone marrow (BM) in each group. As shown in FIG.20, the bone marrow leukaemic infiltration was lower in mice in the DEX-C7 group and the CVD + DEX-C1 group.

Finally, FIG. 21 shows the expression of CDs antigens, as shown in FIG. 21, flow cytometry detected the expression of CDs antigens on both cell lines, suggesting that CD4 was the highest expressed surface marker. The flow cytometry analysis is shown in FIG.22, where a is mouse peripheral blood and b is mouse bone marrow, as shown in FIG.22, the flow cytometry analysis also confirmed that leukaemia had infiltrated into mouse peripheral blood (Blood) and bone marrow (BM), and that the percentage of CEM-C7 cells in the DEX-C7 group was significantly lower than that in the CON-C7 group (P < 0.05), and that in the CVD + DEX-C1 group, the percentage of CEM-C1 cells was significantly lower than that in all other groups (P < 0.05).

However, there was no significant difference in the percentage of leukaemia cells between the

DEX-C7 and CVD + DEX-C1 groups.

In summary, this suggests that in mice CVD has a role in reversing CEM-C1 resistance to DEX.

Obviously, those skilled in the technology can make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus,

if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their technical equivalents, the present invention is intended to encompass these modifications and variations as well.

Claims

1. Carvedilol in the preparation of drugs to reverse leukaemia resistance.

2. The application according to claim 1, characterised in that said reversing leukaemia resistance drug means reversing leukaemia resistance to glucocorticoid drugs.

3. The application according to claim 1, characterised in that said glucocorticoid drug comprises dexamethasone and its derivatives, prednisone and its derivatives, hydrocortisone and its derivatives.

4. The application according to claim 3, characterised in that said leukaemia is acute T-lymphoblastic leukaemia.

5. The application according to claim 4, characterised in that said carvedilol reverses the resistance of acute T lymphoblastic leukaemia to glucocorticoid drugs by inhibiting B3-AR/cAMP/PKA/STAT3 to upregulate the expression of Topolla.

6. An application according to claim 1, characterised in that said drug is in the form of a solid, semi-solid or liquid.

7. The application according to claim 1, characterised in that the formulation of said drug comprises an aqueous solution, a non-aqueous solution, a suspension, an ingot, a capsule, a tablet, a granule, a pill or a powder.

8. The application according to claim 1, characterised in that said drug is administered by the route of injection administration or oral administration.