AU2020101830A4

AU2020101830A4 - Doxorubicin combined medicine and its application

Info

Publication number: AU2020101830A4
Application number: AU2020101830A
Authority: AU
Inventors: Zhigang Guo; Lingfeng He; Shiying Zhou
Original assignee: Nanjing Normal University; Nanjing Tech University
Current assignee: Nanjing Normal University
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-09-24
Anticipated expiration: 2028-08-14

Abstract

The invention discloses a Doxorubicin combined medicine, which comprises Doxorubicin, FEN Iprotein inhibitor and a pharmaceutically acceptable carrier. The invention also discloses the application of the combined medicines in preparing medicines for treating cancer, especially colon cancer. Experiments prove that the combination of Doxorubicin and FEN Iinhibitor SC13 can obviously enhance the therapeutic effect of Doxorubicin, obviously inhibit the proliferation of colon cancer cells and induce DNA damage and apoptosis after the combination. More importantly, combined use enhances the anti-tumor effect in mouse model, and reduces the dosage and toxic side effects of Doxorubicin. Therefore, Doxorubicin combined with FEN Iprotein inhibitor has a promising future in the prevention and treatment of colon cancer.

Description

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

Doxorubicin combined medicine and its application

The invention is described in the following statement:-

Doxorubicin combined medicine and its application

TECHNICAL FIELD

The invention belongs to the biomedical technology, and in particular relates to a

Doxorubicin combined medicine and an application of the combined medicine in

preparing an anti-tumor medicine.

BACKGROUND

At present, the incidence and mortality of colon cancer are very high all over the world.

Surgical resection is still the most effective means of colon cancer at present, and

chemotherapy is one of the important adjunctive treatments. Doxorubicin (or

Adriamycin) has been one of the most successful drugs in cancer chemotherapy since its

discovery. It is an anthracycline antibiotic that promotes the death of tumor cells by

inducing DNA damage. Doxorubicin has been proved that it can be inserted into DNA to

make the replication and transcription process of DNA abnormal so as to lead to double

stranded DNA breakage (DSB).However, Doxorubicin has toxic and side effects on

human body, which greatly limits its application in clinical treatment.

DNA fragment endonucleasel FENi is a metal nuclease with structural specificity.

Unlike other restriction endonucleases, its substrate recognized is independent of

sequence, and it only recognizes specific DNA structure. In addition to endonuclease

(FEN) activity, there are exonuclease (EXO) activity and gap endonuclease (GEN)

activity. Because of these three activities, FEN Iplays an extremely important role in

many DNA metabolic pathways, such as DNA fragment degradation, telomere stability maintenance, long patch base excision repair (LP-BER) and subsequent primer removal in the chain. FEN Iplays a key role in maintaining the stability and integrity of genome by participating in DNA replication and repair. There is abnormally high expression of

FEN Iin various tumor cells.

Doxorubicin is a major anticancer drug for colon cancer, which has been used in clinical

treatment for many years, but its therapeutic effect is limited due to drug resistance of

cancer cells. Long-term Doxorubicin treatment usually leads to cancer cells' resistance to

treatment, and can cause serious cardiotoxicity. In the past decades, researchers have

been studying how to overcome chemotherapy tolerance and drug resistance. Other anti

tumor drugs, such as paclitaxel, camptothecin, cisplatin, etc., and radiotherapy also have

side effects in varying degrees.

SUMMARY

Aim of the invention

Aiming at the problems existing in the prior art, the present application provides an

Doxorubicin combined medicine and an application of the combined medicine in

preparing a medicine for treating cancer, especially colon cancer.

Technical scheme

The Doxorubicin combined medicine comprises Doxorubicin, a FEN Iprotein inhibitor

and a pharmaceutically acceptable carrier.

The FEN Iprotein inhibitor is preferably SC13. See patent 201710023851.1 for the

preparation method of SC13.

The application of the Doxorubicin combined medicine in preparing the medicine for

treating cancer is also within the protection scope of the application.

Furthermore, the Doxorubicin combined medicine is preferably used for preparing drugs

for treating colon cancer.

Beneficial effects

The experiments prove that the combined use of Doxorubicin and FENIinhibitor SC13

can obviously enhance the therapeutic effect of Doxorubicin, and obviously inhibit the

proliferation of colon cancer cells and induce DNA damage and apoptosis after combined

use. More importantly, combined use enhanced the anti-tumor effect in mouse model, and

reduced the dosage and toxic side effects of Doxorubicin. Therefore, Doxorubicin

combined with FEN Iprotein inhibitor has a promising future in the prevention and

treatment of colon cancer.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the high expression of FENI in colon cancer cells.

Fig. 2 shows the effect of different chemotherapeutic drugs combined with SC13 on cell

proliferation detected by CCK8. When the cells were treated with drug concentration

gradient, CCK8 detection was performed after dosing 48 hours.

A. detection of the effect of camptothecin alone or combined with SC13 on SW480

proliferation.

B. detection of the effect of paclitaxel alone or combined with SC13 on SW480

proliferation;

C. detection of the effect of carboplatin alone or combined with SC13 on SW480

proliferation;

D. detection of the effect of 5-FU alone or 5-FU combined with SC13 on SW480

proliferation;

E. detection of the effect of Doxorubicin alone or combined with SC13 on the SW480

and NCM460 proliferation;

F. detection of the effect of adding solvent, SC13, Doxorubicin or drug combination on

SW480 proliferation in 5 days;

G. detection of the effect of Doxorubicin alone or Doxorubicin combined with SC13 on

the proliferation of HCT-8.

H. detection of the effect of adding solvent, SC13, Doxorubicin or drug combination on

HCT-8 proliferation in 5 days;

Fig. 3 shows the effect of SC13 combined with Doxorubicin on cell proliferation in

SW480 cells detected by cell imaging and cloning experiments.

A. cell imaging of drug-treated cells for 48 hours;

B. cell clone formation ability under different drug treatments-500 cells were inoculated

into each well of the 6-well plate, and drugs were added after the cells adhered the next

day. After drug treatment for 48 hours, they were replaced with fresh medium without

drugs to continue culture;

C. quantitative map of clone formation;

Fig. 4 shows the influence of drug treatment on SW480 cell proliferation activity detected

by immunofluorescence.

A. The influence of SC13 and Doxorubicin alone or in combination with drugs on cell

proliferation detected by Ki67. The concentration of SC13 and Doxorubicin is 20tM and

0.24M, respectively. After drug treatment for 24 hours, perform the detection. And the

scale in the figure is 20 m.

B. The effects of SC13 and Doxorubicin alone or in combination with drugs on DNA

synthesis of new cells detected by EdU. The concentration of SC13 and Doxorubicin is

tM and 0.24M, respectively. After treatment for 24h, perform the detection. And the

scale in the figure is 20 m.

Fig. 5 shows the detection of cell cycle-related protein expression after different drug

treatments.

A. The concentration of SC13 is 20[M, the concentration of Doxorubicin is 0.2[tM, and

the protein expression is detected by Western Blot after 24 hours of drug treatment, with

Vinculin as the internal reference of protein expression;

B. Immunofluorescence detection of CDK6 expression changes in different drug

treatments, with the scale of 20 m;

C. The expression of Cyclin D1 under different drug treatments was detected by

immunofluorescence, and the scale in the picture was 20 m;

Fig. 6 is the detection of DNA damage under different drug treatments. The concentration

of SC13 is 20[M, of Doxorubicin is 0.2[tM, and it is detected after 24 hours of drug

treatment;

A.Western Blot was used to detect the expression of yH2AX under different drug

treatments, and Tubulin was used as the internal reference of protein expression.

B. Immunofluorescence was used to detect the expression of yH2AX under different drug

treatments. The scale was 20[m,

C. The statistical diagram of yH2AX foci point.

D. The expression of 53BP1 under different drug treatments detected by

immunofluorescence, with the scale of 20 m.

E. The statistical diagram of 53BP1 foci point.

F Chromosome deformity and breakage condition under different drug treatments.

G. The statistical diagram of chromosome breakage.

Fig. 7 shows the apoptosis of SW480 cells treated with different drugs.

A. The apoptosis of cells treated with SC13 and Doxorubicin alone or combined with

drugs detected by FACS;

B. Statistics of apoptosis rate;

C. The expression of several typical apoptosis proteins in cells treated with different drug

groups, detected by Western Blot, with GAPDH protein expression as internal

reference;

D. The expression of Cleaved-caspase3 in cells under different drug treatments detected

by immunofluorescence staining. The scale in the figure is 20 m;

Fig. 8 shows the antitumor effect of Doxorubicin combined with SC13 in nude mice.

A. The growth of tumor volume in nude mice under different drug treatments;

B. The photos of tumor tissues cut off after drug treatment;

C. Weighing records of cut tumor tissues after drug treatment;

D. HE, Ki67, yH2AX, 53BP1, Cleaved-caspase3, TUNEL staining of paraffin sections of

tumors in different drug treatment groups, with the scale of 250 m;

Fig. 9 shows the effect of different drug treatments on homologous recombination repair

of cells and the change of related protein expression. The concentration of SC13 is 20[M,

the concentration of Doxorubicin is 0.2[tM, and the detection is carried out after 24 hours

of drug treatment.

A. Homologous recombination repair ability under different drug treatments detected in

U2OS cells;

B. The expression of Rad51 and BRCA1 protein under different drug treatments detected

in SW480 cells,with the protein expression of Vinculin as an internal reference;

C. BRCA1 expression in SW480 tumor tissue slices under different drug treatments

detected by immunohistochemical, with the scale of 250 m;

D. The protein expression of Rad51 and BRCA1 under different drug treatments

detected in HCT-8 cells, and taking the protein expression of Vinculin as an internal

reference;

Fig. 10 shows the sensitivity of cells to SC13 after BRCA1 knockdown in SW480 and

HCT-8 respectively.

A.Western Blot was used to detect the knock-down efficiency of BRCA1 by lentivirus in

SW480 cells, and the protein expression of Vinculin was used as an internal reference;

B. The effects of SC13 on cell proliferation of wild-type SW480 cells and SW480 cells

with BRCA1 knockdown were detected by CCK8 after 48 hours.

C. Western Blot was used to detect the knock-down efficiency of BRCA1 by lentivirus in

HCT-8 cells, and the protein expression of Vinculin was used as internal reference;

D. The proliferation of wild-type HCT-8 cells and BRCA1-knocked-down HCT-8 cells

was affected by SC13, and was detected by CCK8 after 48 hours.

DESCRIPTION OF THE INVENTION

The application will be described in detail with specific examples below. In this

invention, SC13, a small molecule inhibitor for FEN Iprotein, and Doxorubicin, a

chemotherapy drug, are used. The materials and reagents used in the following examples

can be obtained from commercial sources unless otherwise specified. This example

mainly shows that SC13 can enhance the sensitivity of colon cancer cells to Doxorubicin,

which provides a new example of drug combination for clinical cancer treatment.

Origin of cells: HCT8, SW480 and NCM460 cells are all from American type culture

collection bank (ATCC).

The cells used in this study are human colon cancer SW480 cells and HCT-8 cells, which

are cultured in DMEM medium. In addition, normal human intestinal epithelial cells

NCM460 were used and cultured in 1640 medium. Cell culture media were purchased

from Gibco Company, and 10% fetal bovine serum and 1% penicillin and streptomycin

were added before cell culture.

Drug source: Doxorubicin was purchased from Selleck company; see patent

201710023851.1 for the preparation method of FENI inhibitor SC13.

The FEN Iinhibitor SC13 powder was prepared with DMSO to an initial concentration of

mM, and the Doxorubicin powder purchased from Selleck company was prepared with

physiological saline to an initial concentration of 20mM. When using, the fixed

concentration of SC13 added to the culture medium was 20uM, and the Doxorubicin

concentration gradient (0.luM, 0.2uM, 0.5uM, luM) was used to find the best

combination effect which was combined used with SC13(20uM) to make a time gradient

curve for 1-5 days.

Example 1 Expression of FENI in two kinds of colon cancer cells and normal intestinal

epithelial cells

Select human colon cancer cells SW480, HCT-8 and human normal intestinal epithelial

cells NCM460, culture them respectively, collect samples to extract proteins when the

cells grow well, and detect the expression of FEN Iin the three cells by Western blot,

with Vinculin as the internal reference of protein expression. Results as shown in fig. 1,

we found that the FENI protein expression levels in SW480 and HCT-8 colon cancer

cells were higher than those in NCM460 normal intestinal epithelial cells, which

suggested that FEN Iwas related to the colon cancer occurrence and development.

Example 2 Killing ability of several different chemotherapeutic drugs in combination

with SC13

The FEN Iinhibitor SC13 was added to SW480 and HCT-8 cells respectively, but it was

not found that SC13 alone could significantly inhibit the growth of colon cancer cells (as

shown in fig. 2, fig. 3 and fig. 4).

A series of chemotherapeutic drugs were used in combination with SC13 in colon cancer

SW480 cells, and CCK8 detection was used to detect the effects of different

chemotherapeutic drugs combined with SC13 on cell proliferation. CCK8 detection was

carried out after drug concentration gradient treatment for 48 hours.

The results are shown in fig. 2, wherein A is the effect of camptothecin alone or

camptothecin combined with SC13 on SW480 proliferation. B is the effect of paclitaxel

alone or combined with SC13 on SW480 proliferation. C is the effect of carboplatin alone

or combined with SC13 on SW480 proliferation .And D is the effect of 5-FU alone or 5

FU combined with SC13 on SW480 proliferation. Therefore, SC13 has no obvious

sensitizing effect on camptothecin (Figure.2A), paclitaxel (Figure. 2B), carboplatin

(Figure.2C) and 5-FU (fig. 2D).

When we used SC13 in combination with Doxorubicin, we found that the killing ability

of Doxorubicin in combination with SC13 was obviously stronger than that of

Doxorubicin alone at the same concentration, which indicated that SC13 had a strong

drug sensitizing effect on Doxorubicin in SW480 cells (Figure. 2E). Then, we did the

same drug combination treatment in normal intestinal epithelial cells NCM460, and

found that the sensitizing effect of SC13 on Doxorubicin in NCM460 cells was weaker

than that in SW480 cells (Figure 2E, the difference between two curves of NCM460 cells

was obviously smaller than that of SW480 cells), which indicated that SC13 combined with Doxorubicin had less toxicity to normal cells. In the previous experiments, it was detected after drug addition for 48 hours, in order to verify the effect of drug treatment for a long time, we recorded SW480 cells treated with SC13 and Doxorubicin alone or combined with drugs for 5 days. The results also showed that the survival rate of cells treated with low concentration Doxorubicin alone still exceeded 50% after 5 days, while most of the cells were killed the next day when combined with drugs, and basically all the cells died in 5 days (Figure 2F). In addition, we detected that SC13 also has obvious chemosensitization effect on Doxorubicin in another colon cancer cell HCT-8 (Figure.

2G, Figure. 2H).

Example 3

(1) Inhibitory effect of Doxorubicin combined with SC13 on the growth of SW480 cells

Through previous screening, inoculating cells in 96-well plate, we found a very effective

drug combination scheme of Doxorubicin and SC13 by CCK8 method. We cultured

SW480 cells in a 6-well plate with the same inoculation amount per well, and treated the

cells with SC13 and Doxorubicin alone or in combination with the two drugs. The results

of cell imaging showed that SC13 alone had no obvious killing effect on cells, but at the

same concentration of Doxorubicin, the killing effect of adding SC13 was obviously

stronger than that of Doxorubicin alone (Figure.3A). Clonal formation experiment further

verified that the combination of SC13 and Doxorubicin had an extremely obvious

inhibitory effect on cell growth and clone formation ability (Figure. 3B). Figure. 3C is a

statistical result of clone formation.

(2) The effect of Doxorubicin combined with SC13 on proliferation activity and new

DNA synthesis ability of SW480 cells

Earlier, we found that Doxorubicin combined with SC13 had a strong cell killing effect

on colon cancer cells through CCK8 and clone formation experiments. We suspect that

this is because the cell proliferation was significantly inhibited after drug treatment.

Firstly, we detected Ki67, a cell proliferation marker, by immunofluorescence, and found

that when Doxorubicin alone treated cells, the fluorescence intensity decreased and cell

proliferation was inhibited. However, the fluorescence intensity of Ki67 decreased more

obviously when the drugs were used in combination, and the inhibitory effect on cell

proliferation was stronger (Figure. 4A). EdU staining, another marker of cell

proliferation, showed that Doxorubicin combined with SC13 significantly inhibited the

synthesis of newborn DNA in SW480 cells, and then had a strong inhibitory effect on cell

proliferation (Figure. 4B).

(3) The combined use of Doxorubicin and SC13 can block the cell cycle of SW480 cells

When Doxorubicin is combined with SC13, the inhibition of cell proliferation indicates

that the cell cycle may be blocked. Next, we examined the expression changes of a series

of cell cycle-related proteins under different drug treatments. The results showed that the

expressions of CDK4, CDK6 and Cyclin D1 decreased significantly when Doxorubicin

was used in combination with SC13. The reason was that the expression of p21 protein,

the inhibitor of CDK, increased significantly when drugs were used in combination,

which resulted in the decrease of Rb phosphorylation level and cell cycle arrest (Figure

).

(4) The effect of Doxorubicin combined with SC13 on DNA damage of SW480 cells

Firstly, the protein expression of DNA double-strand break marker yH2AX was detected

by Western Blot under different drug treatments. The results showed that the expression

of yH2AX increased after Doxorubicin treatment, but it increased more significantly

when Doxorubicin and SC13 combined treatment (Fig. 6A).Then, we verified by

immunofluorescence that the fluorescence intensity of yH2AX and another DNA damage

marker, 53BP1, increased significantly when the drugs were used together, indicating that

the degree of DNA damage increased significantly when the drugs were used together

(Figure 6B, Figure 6C, Figure 6D, Figure 6E). Karyotype analysis showed that when

Doxorubicin was used in combination with SC13, more malformations and breaks of

chromosomes would occur (Figure 6F). Fig. 6G is a statistical analysis of chromosome

aberration.

(5) 2.2.6 The effect of Doxorubicin combined with SC13 on apoptosis of SW480 cells

Through the previous research, we know that the combination of Doxorubicin and SC13

can inhibit the proliferation of SW480 cells, block the cell cycle, and aggravate the

degree of DNA damage. We guess that the inhibition of cell proliferation is due to

apoptosis. At first, we used flow cytometry to detect the apoptosis degree of SW480 cells

treated with different drugs. The results showed that SW480 cells were not significantly

apoptotic when treated with two drugs alone, but significantly apoptotic when SC13 was

combined with Doxorubicin (fig. 7A, fig. 7B). By detecting several typical apoptotic

proteins, it was found that the cleavage degree of caspase3 increased obviously, the

expression of pro-apoptotic protein Bax increased obviously, and the expression of anti- apoptotic protein Bcl-2 decreased obviously when the drugs were used together (Figure

7C). The results of immunofluorescence in fig. 7D further prove that the expression of

Cleaved-caspase3-3 increased significantly when combined with drugs.

Example 4 Anti-tumor effect of Doxorubicin combined with SC13 at animal level

Vitro experiments proved that the combination of low concentration Doxorubicin and

SC13 has obvious tumor killing effect on SW480 cells and HCT-8 cells. However, only

vitro cell level experiments are not enough to prove the effect of Doxorubicin combined

with SC13 on tumor tissues in vivo. Therefore, vivo animal level experiments are needed

to study whether the combination of Doxorubicin and SC13 is still effective in animals

vivo. We cultured SW480 cells in large quantities and injected them subcutaneously into

nude mice about 5 weeks old. Each nude mouse was injected subcutaneously with about

3 million cells in a volume of 100 L. In about 10 days, the tumor volume reached about

mm3. At this time, nude mice were randomly divided into 4 groups, which were

injected with drug solvent, SC13, Doxorubicin or the mixture of SC13 and Doxorubicin.

Every time, the injection amount of SC13 is 2mg/kg, and the injection amount of

Doxorubicin is 5mg/kg.The volume of each drug injection is 100L single drug or 200L

mixture of two drugs. The drug was injected every other day for 7 times, and the tumor

volume was recorded every two days. The results in fig. 8A show that with the increase

of drug injection days, the tumor volume of nude mice in the solvent group increased the

fastest, followed by SC13 group and Doxorubicin group, while the tumor volume of the

two drug combination groups hardly increased after 33 days of measurement, indicating

that the drug combination treatment has a good inhibitory effect on the tumor in nude mice .After 33 days of drug treatment, we cut off the tumor tissue of each nude mouse in each group and arranged it according to the size and group. It can be clearly seen that the drug combination group has obvious tumor inhibition effect (Figure 8B). The cut tumor was weighed, and the results showed that the tumor weight of the drug combination group was significantly lighter than that of other groups (Figure 8C). The paraffin section staining of tumor showed that the expression of Ki67 decreased obviously, Cleaved caspase3 increased obviously, the fluorescence intensity of DNA damage markers yH2AX and 53BP1 increased obviously, the fluorescence intensity of TUNEL increased obviously and the apoptosis rate increased (Figure 8D). These results are consistent with the results of cell experiments in vitro.

Example 5 Study on the mechanism of synthetic lethal effect caused by combination of

Doxorubicin and SC13

(1) The effects of Doxorubicin alone and Doxorubicin combined with SC13 on

homologous recombination repair

The above experimental results show that Doxorubicin combined with SC13 has a strong

tumor inhibitory effect both in vitro and in vivo. But why does SC13 and low

concentration Doxorubicin alone have no obvious killing effect on tumor cells, but when

combined with drugs, it has obvious killing effect on cells? With this question, we carried

on the next inquiry. As mentioned earlier, FEN Iplays an important role in the repair of

long base excision, and FEN Iinhibitor SC13 can directly inhibit the process of base

excision and repair (BER) by inhibiting FEN activity in FENI. Colon cancer cells

SW480 and HCT-8 are all DNA repair-free cells. When SC13 is used alone to inhibit

BER pathway in these two kinds of cells, only DNA single strand breaks (SSBs) can not

be repaired in time, while DNA double strand breaks (DSBs) can still be repaired, so

SC13 alone is not enough to kill these two kinds of cells. When SC13 is added in the

presence of low concentration of Doxorubicin, SC13 shows a strong drug sensitizing

effect, and its killing effect on cells is obviously stronger than that of using Doxorubicin

with the same concentration alone. Our guess is that Doxorubicin inhibits the repair of

double strand breaks. Therefore, when SC13 is added to inhibit the repair of single strand

breaks, the DNA repair function of cells is severely hindered, resulting in obvious

synthetic death. Homologous recombination repair (HRR) is an important way to repair

double-strand breaks. We used single cell immunofluorescence analysis to detect the

repair efficiency of HRR by flow cytometry, and found that the repair efficiency of HRR

decreased significantly after cells were treated with Doxorubicin, which proved that HRR

pathway was inhibited after cells were treated with Doxorubicin for some time (Figure

9A). Then, we detected the changes of two important proteins BRCA1 and Rad51 in the

homologous recombination repair pathway in SW480 cells, and found that the expression

of Rad51 decreased when SC13 was combined with Doxorubicin. However, when

Doxorubicin was used alone, the expression of BRCA1 decreased significantly (Figure

9B). We detected BRCA1 by immunohistochemical staining on tumor sections of nude

mice, and found that BRCA1 expression decreased significantly when Doxorubicin was

used alone in vivo (Figure 9C). In HCT-8 cells, the changes of Rad51 and BRCA1 were

consistent with those of SW480 cells (Figure. 9D).

(2) After knocking down BRCA1, the sensitivity of cells to SC13 was detected

We simulated the defects of HRR pathway by knocking down BRCA1 in SW480 (Figure

1OA) and HCT-8 (Figure 1OC) with lentiviral RNAi interference. The result shows that

SW480 (Figure 1OB) and HCT-8 (Figure 1OD), which are originally insensitive to SC13,

become more sensitive to SC13 after knocking down BRCA1. This result further proves

that when FEN Iand BRCA1 can't function normally at the same time, the cells will have

synthetic lethal effect. Combined with the previous experimental results, it can also

explain that one of the reasons for the anti-tumor effect of Doxorubicin combined with

SC13 is that BER and HRR pathways are simultaneously inhibited.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A Doxorubicin combined medicine is characterized by comprising Doxorubicin, FENI

protein inhibitor and a pharmaceutically acceptable carrier.

2. The Doxorubicin combination medicine according to claim lis characterized in that the

FEN1 protein inhibitor is SC13.

3. Application of the Doxorubicin combination medicine according to claim 1 or 2 in

preparing medicines for preventing and treating cancer.

4. Application of the Doxorubicin combination medicine according to claim 1 or 2 in

preparing medicines for preventing and treating colon cancer.

5. Application of FENIinhibitor SC13 combined with chemotherapy drug Doxorubicin

in preparing medicines for preventing and treating colon cancer.

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