Composition capable of inducing HCT116 cell iron autophagy and application

Abstract

The invention discloses a composition capable of inducing HCT116 cell iron autophagy and application thereof.The composition is composed of berberine with the concentration being 10-80 [mu] M and curcumin with the concentration being 10-80 [mu] M. The composition composed of berberine and curcumin has a remarkable synergistic effect on inhibition of HCT116 cell proliferation; according to the present invention, the ROS level can be increased to participate in the regulation of the proliferation, the cell cycle and the autophagy of HCT116 cells, such that the targeting drug for treating CRC can be prepared, and the treatment effect on colorectal cancer can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical formulations, and particularly to the technical field of pharmaceutical compositions for inhibiting cancer cell proliferation and treating malignant tumors. Specifically, the present invention relates to a composition capable of inducing ferroptosis in HCT116 cells and its use for inhibiting cancer cell proliferation, and for preparing CRC-targeted drugs by inducing ferroptosis in cells for treating malignant tumors of the colon or rectum. Background Art

[0002] Colorectal cancer includes colon cancer and rectal cancer, and is the third most common malignant tumor globally and the second leading cause of cancer-related deaths. In recent years, although the incidence and mortality rates have been significantly reduced in developed countries through the promotion of screening, such as colonoscopy and fecal occult blood testing, in developing countries, due to the westernization of diet, the increasing obesity rate, the increasing intake of red meat, and the lack of early screening, the incidence of colorectal cancer has shown a rapid growth trend, and the trend of younger age is obvious. Among them, the incidence rate in people under 50 years old has been increasing year by year. Molecular biology research has revealed the heterogeneity of colorectal cancer. Approximately 85% is chromosomally unstable, such as APC, KRAS, TP53 gene mutations, and 15% is microsatellite instability (MSI-H / dMMR), and the latter is related to Lynch syndrome and is sensitive to immunotherapy. In addition, molecular characteristics such as BRAF V600E mutation and HER2 amplification have been confirmed to be closely related to prognosis and treatment options. The significant improvement in the early diagnosis rate has significantly improved the survival of patients, but the 5-year survival rate of advanced metastatic colorectal cancer (mCRC) is still less than 15%. There are significant regional differences. In European and American countries, the early diagnosis rate has been increased to 40% through national screening, while the proportion of early cases in Asian countries is less than 30%. The research also found that intestinal microbiota dysbiosis and chronic inflammation, such as ulcerative colitis, are closely related to the occurrence of colorectal cancer, providing new directions for prevention and targeted intervention.

[0003] The treatment of colorectal cancer focuses on surgery. Early-stage patients can achieve radical cure through laparoscopic or robotic-assisted surgery, and the progress of anal-preserving techniques has significantly improved the quality of life of rectal cancer patients. Neoadjuvant chemoradiotherapy is used for locally advanced rectal cancer to downstage and improve the success rate of surgery. Advanced-stage patients are mainly treated systemically. Based on the chemotherapy regimens FOLFOX and FOLFIRI, the combination of targeted drugs can significantly extend survival: anti-angiogenic drugs are applicable to a wide range of patients, while EGFR inhibitors are only used for patients with wild-type RAS / BRAF. Immunotherapy has completely changed the prognosis of dMMR / MSI-H patients, and the objective response rate of the PD-1 inhibitor pembrolizumab as first-line treatment reaches 45%. Dual-targeted therapy for BRAF V600E mutations and the trastuzumab + pertuzumab regimen for HER2-positive patients also show potential. Current research hotspots include KRAS G12C inhibitors, personalized vaccines, and CAR-T cell therapy. At the same time, the minimal residual disease monitoring technology based on circulating tumor DNA is promoting precise decision-making in postoperative adjuvant therapy. The popularization of the multidisciplinary comprehensive treatment model has further optimized the balance between efficacy and quality of life. Summary of the Invention

[0004] Targeted drugs have always been one of the indispensable important means for treating cancer. The present invention aims to provide and prove that the combination of Berberine and Curcumin can jointly regulate iron in the tumor microenvironment, providing a key target and an effective pair combination mode for the development of new anti-CRC drugs. Specifically, the present invention provides a composition that can induce ferroptosis in HCT116 cells, which can significantly synergistically inhibit the proliferation of colon cancer cells under the action of the composition of the present invention, providing a new solution for the preparation of targeted drugs for treating colorectal cancer, further enhancing the effectiveness of colorectal cancer treatment, thereby prolonging the patient's life and reducing the patient's mortality.

[0005] In order to achieve the above object, the technical solution adopted in this application is as follows: The present invention provides a composition that can induce ferroptosis in HCT116 cells, and the composition is composed of berberine at a concentration of 10 μM - 80 μM and curcumin at a concentration of 10 μM - 80 μM. Through the research of the applicant, it is found that multiple combinations within the above concentration range have inhibitory effects on the proliferation of various colon cancer cells including HT-29 and HCT116, and the significance of the inhibitory effect depends on the concentration and mixing ratio of berberine (BER) and curcumin (CUR). Single berberine (BER) or curcumin (CUR) cannot induce a significant accumulation of ROS in HCT116 cells, but when berberine (BER) and curcumin (CUR) act on HCT116 cells together, they can effectively trigger the production of ROS in colorectal cancer cells, thereby inducing apoptosis.

[0006] As a preferred ratio setting, the concentrations of both berberine and curcumin are 20 μM.

[0007] Based on the fact that berberine and curcumin have effective inhibitory effects on the proliferation of HCT116 cells, the above composition that can induce ferroptosis in HCT116 cells can be used as an active ingredient to inhibit the proliferation of cancer cells in the manufacture of targeted drugs for treating CRC, which has a positive effect on the treatment of cancer.

[0008] Through research by the applicant, it is found that the composition composed of berberine and curcumin activates ferroptosis mediated by ROS through enhancing NCOA4 / Ferritin, and can participate in regulating the proliferation, cell cycle and autophagy of HCT116 cells by increasing the ROS level.

[0009] In order to further broaden the treatment methods of the present invention, the CRC targeted drug is an injection or an oral preparation.

[0010] Beneficial effects: The composition composed of berberine and curcumin provided by the present invention has a significant synergistic effect on inhibiting the proliferation of HCT116 cells, and can participate in regulating the proliferation, cell cycle and autophagy of HCT116 cells by increasing the ROS level, so that a targeted drug for treating CRC can be manufactured, effectively improving the treatment efficacy of colorectal cancer. Description of the drawings

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0012] Figure 1 It is the chemical structure of berberine (BER).

[0013] Figure 2 It is the chemical structure of curcumin (CUR).

[0014] Figure 3 It is the survival rate curve of HT29 cells treated with different concentrations of berberine.

[0015] Figure 4 It is the survival rate curve of HT29 cells treated with different concentrations of curcumin.

[0016] Figure 5 It is the survival rate curve of HCT116 cells treated with different concentrations of berberine.

[0017] Figure 6 It is a curve graph of the survival rate of HCT116 cells after treatment with curcumin at different concentrations.

[0018] Figure 7 It is a graph of cross - determination of the survival rate of HCT116 cells using Cell Counting Kit - 8.

[0019] Figure 8 It is a graph of cytoplasmic vacuoles of HCT116 cells in the control group and experimental groups 1 - 3 in Example 2.

[0020] Figure 9 It is Figure 8 a graph of the quantitative data of the fluorescence intensity of each group.

[0021] Figure 10 It is a graph showing the expression level of ROS in HCT116 cells detected using the Reactive Oxygen Species Assay Kit in Example 3.

[0022] Figure 11 It is Figure 10 the quantitative data of ROS accumulation in each group Figure 12 It is a graph showing the detection of iron ion levels in the control group and experimental groups 4 - 6 by Ferro Orange in Example 4.

[0023] Figure 13 It is Figure 12 the quantitative data of iron ions in each group.

[0024] Figure 14 It is a graph of the changes in NCOA4 / ferritin pathway proteins and LC3 when treated with the ROS inhibitor NAC (2 mM) or in combination with berberine and curcumin.

[0025] Figure 15 It is Figure 14 a graph of the quantitative data of NCOA4 / ferritin pathway proteins and LC3 in each group.

[0026] Figure 16 It is a graph showing the detection of the expression level of iron ions in HCT116 cells in the control group and experimental groups 7 - 9 using Ferro Orange.

[0027] Figure 17 It is Figure 16 the quantitative data of iron ions in the control group and experimental groups 7 - 9.

[0028] Figure 18 It is an image of the subcutaneous tumor tissue of mice in Example 5.

[0029] Figure 19It is the curve graph of the body weight of mice measured after the experiment in the subcutaneous tumor transplantation mouse model in Example 5.

[0030] Figure 20 It is the curve graph of the tumor weight of mice measured after the experiment in the subcutaneous tumor transplantation mouse model in Example 5.

[0031] Figure 21 It is the curve graph of the tumor volume growth of mice measured after the experiment in the subcutaneous tumor transplantation mouse model in Example 5.

[0032] Figure 22 It is the HE staining image of the subcutaneous tumor tissue.

[0033] Figure 23 It is the Ki-67 diagram of the subcutaneous tumor tissue.

[0034] Figure 24 It is to evaluate the expression level of Ki67 by immunofluorescence. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. Usually, the components of the embodiments of the present application described and shown in the accompanying drawings here can be arranged and designed in various different configurations.

[0036] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application claimed, but merely represents the selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

[0037] Example 1: To better illustrate that the composition composed of berberine and curcumin provided by the present invention has a significant synergistic inhibitory effect on cancer cells including HT-29 and HCT116 cells, this example will be described with detailed experimental procedures and data.

[0038] Before elaborating in detail that berberine BER or curcumin CUR (hereinafter referred to as B&C) can effectively enhance ferritin autophagy mediated by NCOA4 / ferritin by activating ROS, thereby playing an anti-colorectal cancer role, first, the relevant terms involved in the present invention will be explained, and the relevant materials and chemical reagents used in the experiment will be described to facilitate a better understanding of the present invention.

[0039] Term explanation: Autophagy is a mechanism of cellular "self-digestion" and plays an indispensable role in many pathological conditions such as cancer, aging, metabolic disorders, and infections. Studies have found that the increase in autophagy substrates during autophagy activation usually enables tumor cell survival and growth, and regulating autophagy is a promising cancer treatment strategy at present.

[0040] Ferroptophagy is a process in which the nuclear receptor coactivator 4 (NCOA4) mediates the autophagic degradation pathway of intracellular ferritin to achieve dynamic regulation of intracellular iron storage and release, thereby maintaining intracellular iron homeostasis.

[0041] Cell culture: The cell lines HCT116 and HT-29 were provided by Sevier Biotechnology Co., Ltd. in Wuhan, China. All cells were stored in DMEM medium (Gibco, USA), supplemented with 10% fetal bovine serum (Gibio, USA), and cultured in an incubator at 37°C with 5% CO2.

[0042] Chemical reagents: Berberine (CAS: 453-37-7, purity ≥96.62%) was purchased from MedChemExpress (New Jersey, USA). Curcumin (CAS: 633-65-8, purity ≥96.62%) was purchased from MedChemExpress (New Jersey, USA). The primary antibodies including p62, p-mTOR, CD133, OCT4, LC3, NCOA4, GAPDH were provided by Proteintech (Wuhan, China). P21, CDK4, CDK6, KEAP1, Ferritin, LGR5, CD44, SOX2, ATG5, NRF2, HO-1 were provided by Beyotime (Shanghai, China). BCL2 and AKT were provided by Abcam (Shanghai, China). NAC and SC79 were purchased from MedChemExpress (New Jersey, USA).

[0043] Cell viability assay: The cell viability of HCT116 and HT-29 was measured using the Cell Counting Kit-8 (CCK-8, APExBio, USA). Cells (5 × 103) were seeded in 96-well plates overnight, treated with different concentrations of BER and CUR for 24 hours, and then incubated with 10% CCK-8 at 37°C for 1 hour. The results were measured with an enzyme-linked immunosorbent assay (ELISA) reader (bibase, Shandong, China) at an absorbance of 450 nm.

[0044] In order to fully demonstrate the unexpected technical effects of the present invention compared with the prior art, in this embodiment, the synergistic effects of the composition are compared with each component separately. Before the description, in order to ensure the objectivity of the unexpected technical effects obtained in the present invention, it is necessary to introduce the IC50 parameter to illustrate the sensitivity of the antibody, that is, the strength of the specificity of the antibody. Among them, in this embodiment, IC50 refers to the concentration of BER, CUR or B&C when inducing apoptosis of 50% of tumor cells, such as HT-29 and HCT116 cells, that is, the concentration corresponding to when the ratio of the number of apoptotic HT-29 / HCT116 cells to the total number of cells is exactly 50%. Thus, the ability of BER, CUR or B&C to induce apoptosis of tumor cells is measured by the value of IC50. The smaller the obtained IC50 value, the stronger the induction ability of the antibody, indicating a better anti-cancer effect, and the better the therapeutic effect of the CRC-targeted drug prepared therefrom on colorectal cancer; on the contrary, the worse the effect, the less significant the therapeutic effect. Similarly, it can also be used to illustrate the strength of the tolerance of tumor cells to antibody drugs. The stronger the inhibitory ability, the worse the tolerance of tumor cells to the antibody; the weaker the inhibitory ability, the stronger the tolerance of tumor cells to the antibody.

[0045] The first group of experimental antibodies: The chemical structure of berberine (BER) is as Figure 1 shown Berberine was grouped according to concentrations of 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, 20 μM, 40 μM, 60 μM and 80 μM, and HT-29 and HCT116 cells were treated respectively for 24 hours; at the same time, a control group was established; the cell survival rates obtained at the above different concentrations were compared with the control group. The results showed that for HT-29 cells, IC50 could not be effectively measured in the range of 1 μM - 80 μM, that is, it indicated that HT-29 cancer cells had a very strong tolerance to berberine, and the inhibitory effect of berberine on HT-29 cancer cells was not significant. See Figure 3 shown. According to the same method, the survival rate of HCT116 cancer cells obtained by the experiment was compared with the control group. Berberine inhibited the proliferation of colorectal cancer cells in a concentration-dependent manner, and the half-maximal inhibitory concentration (IC50) value of 24-hour BER treatment on HCT116 cells was 52.6 μM. See Figure 5 shown.

[0046] The second group of experimental antibodies: The chemical structure of curcumin (CUR) is as Figure 2 shown Curcumin was grouped at concentrations of 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, 20 μM, 40 μM, 60 μM and 80 μM respectively, and HT-29 and HCT116 cells were treated respectively for 24 hours; at the same time, a control group was established; the cell survival rates obtained at the above different concentrations were compared with the control group. The results showed that curcumin exhibited inhibitory effects on both HT-29 and HCT116 cancer cells, and the IC50 values of 24-hour CUR treatment for HT-29 and HCT116 cells were 58.6 μM and 56.7 μM respectively, see Figure 4 , Figure 6 as shown, and when the concentration of curcumin reached about 60 μM, the best inhibitory effect was achieved for HT-29 cells. Subsequently, a linear inhibitory effect was not exhibited due to the increase in concentration.

[0047] The third group of experimental antibodies: a composition composed of berberine (BER) and curcumin (CUR) (i.e., the present invention, hereinafter referred to as B&C) From the first and second groups of the above experiments, it can be found that the inhibition of single berberine (BER) and curcumin (CUR) on different cancer cells HT-29 and HCT116 is not uniform and consistent. Even for HT-29 cancer cells, a single drug cannot achieve a good inhibitory effect, and there are also significant differences in the sensitivity shown for different cells. In order to explore the cross-concentration gradient of the two drugs berberine (BER) and curcumin (CUR), the aim is to obtain the optimal concentration value for their synergistic inhibition, so as to maximize the inhibitory effect on cancer cells. In order to better and intuitively reflect the technical effects produced by drug synergy, this group of experiments selected HCT116 cells with higher sensitivity as the research object.

[0048] The experimental method of the cross-experiment used in this group of experiments is the same as that of the first and second groups of the above experiments, except that the combined concentrations of berberine (BER) and curcumin (CUR) are different, but the concentration of any drug is controlled within the range of 1 μM - 60 μM to obtain a cell survival rate chart as Figure 7 shown. As can be seen from Figure 7 it, when berberine (BER) and curcumin (CUR) are used in combination at a concentration of 20 μM or 30 μM, the inhibitory effect on HCT116 cells reaches 50%, which is significantly more remarkable than that of a single drug, and the concentration value is reduced by 50% - 60% compared with a single drug, and the inhibitory effect has been improved by leaps and bounds.

[0049] In summary, the composition formed by berberine and curcumin at a concentration of 20 μM or 30 μM has a significant inhibitory effect on HCT116 cells, and the inhibitory effect far exceeds that of single berberine or curcumin on HCT116 cells.

[0050] Example 2: In this example, based on Example 1, the pathway by which the composition formed by berberine and curcumin can significantly induce apoptosis of HCT116 cells was analyzed and experimented, so as to deeply explore the targets of the composition formed by berberine and curcumin acting on various cancer cells including HCT116 cells, thereby providing a basis and possibility for the manufacture of anti-cancer targeted drugs.

[0051] In this example, four groups were established with DMSO, 20 μM berberine (BER), 20 μM curcumin (CUR), and 20 μM berberine + curcumin respectively, namely the control group and three experimental groups 1 - 3. At the same time, HCT116 cells were treated for 24 hours, and the above control group and experimental groups 1 - 3 were analyzed by fluorescence immunology to obtain Figure 8 the shown fluorescently labeled cytoplasmic vacuoles and Figure 9 the quantitative data of the fluorescence intensity shown. As can be seen from Figure 8 it, the formation of fluorescently labeled cytoplasmic vacuoles increased, and compared with the single-drug groups, the fluorescence intensity of the B&C group was the most significantly enhanced. This indicates that B&C can trigger the production of ROS in colorectal cancer cells, thereby inducing apoptosis. As can be seen from Figure 9 it, the fluorescence intensity of 20 μM berberine + curcumin was significantly higher than that of other groups. It can be seen that 20 μM berberine + curcumin can effectively induce the accumulation of intracellular ROS.

[0052] Example 3: In order to further confirm that 20 μM berberine + curcumin has a current inducing effect on the accumulation of intracellular ROS, in this example, as described in Example 2, four groups were also established with DMSO, 20 μM berberine (BER), 20 μM curcumin (CUR), and 20 μM berberine + curcumin respectively, namely the control group and three experimental groups 4 - 6. At the same time, HCT116 cells were treated for 24 hours. In this example, the above control group and experimental groups 4 - 6 were detected by flow cytometry respectively to obtain Figures 10 - 11, Detection revealed that under the action of the drug, the ROS level in HCT116 cells increased. Among them, in experimental group 6, that is, the B&C group, the ROS level was significantly higher than that in the single-drug group and the control group. Thus, it was concluded that B&C might participate in regulating the proliferation, cell cycle and autophagy of HCT116 cells by significantly increasing the ROS level. The detection method in this example was to use the Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) to detect the ROS content in HCT116 cells. Cells (3 × 105) were seeded in 6-well plates overnight and treated with BER (20 μM) and CUR (20 μM) for 24 h. After washing 3 times with PBS, the cells were collected. Then, the cells were incubated with fresh medium supplemented with 10 μM DCFH-DA (Beyotime, Shanghai, China) at 37 °C for 20 min. The ROS level was detected using a flow cytometer (Beckman Coulter, USA), and quantification was performed using FlowJo 7.6.2 software.

[0053] Cells were evenly seeded in 12-well plates (1 × 105 cells / well) and cultured for 24 hours without using drugs. Next, they were treated with different drug concentrations for 24 hours and evaluated using a Reactive Oxygen Species (ROS) Assay Kit (Beyotime Biotechnology, Shanghai, China). Finally, the samples were imaged and analyzed using a fluorescence microscope. ROS-positive cells emitted green fluorescence when using a fluorescence microscope. The fluorescence intensity was quantified using ImageJ software.

[0054] Example 4: Based on the finding in Example 2 and Example 3 that B&C could induce an increase in ROS, this example will further conduct experimental research on the root cause of the increase in ROS. In this example, the ferrous ion Fe in cells was analyzed by conventional flow cytometry 2+ for changes, obtaining the ferrous ion Fe as shown in Figures 12 - 13 The ferrous ion Fe in the control group and experimental groups 4 - 6 2+ levels, as well as the ferrous ion Fe 2+ And the ferrous ion Fe 2+Quantitative data. It should be noted that since the experimental method used in this example is conventional flow cytometry, it mainly includes inoculating HCT-116 cells in a 96-well plate and growing overnight. After treatment with BER and CUR for 12 hours, the cells were co-stained with FerroOrange (1 μM) (MKbio, China) in serum-free medium at 37°C for 30 minutes, washed with PBS, and then the cells were analyzed by flow cytometry; this method is prior art and is well-known in the art, so it will not be elaborated here.

[0055] First, it can be intuitively found from Figure 13 that experimental group 6 is the B&C composition group. Under the induction of the B&C composition, the content of ferrous ion Fe 2+ significantly increases, and as can be seen in 4E, under the induction of the B&C composition, the level of ferrous ion Fe 2+ is significantly higher than that of other experimental groups and the control group. It can be seen that the B&C composition at the same concentration has a significantly higher degree of inducing the release of intracellular ferrous ion Fe 2+ and promoting the occurrence of ferritin autophagy in cells than berberine or curcumin alone at the same concentration, far higher than the control group. That is to say, B&C can induce and enhance iron autophagy in cells.

[0056] However, since mitochondria are the source of ROS production and are closely related to the content of ferrous ion Fe 2+ , ferritin autophagy and ferroptosis, in order to further improve the reliability of the experimental results, this example also carried out a reverse experimental demonstration, that is, using the ROS inhibitor NAC to treat HCT116 cells. A new control group was established, and experimental groups 7-9 were established by adding only 2 mM of NAC, 20 μM of the B&C composition, and 2 mM of NAC + 20 μM of the B&C composition. According to the results of Western blot, experimental group 8: the B&C group significantly enhanced ferritin autophagy, manifested as a significant increase in the levels of NCOA4 and LC3-II, and at the same time, the ferritin expression decreased. On the contrary, experimental group 9: the combination of 2 mM of NAC and B&C weakened this effect, see Figures 14 - 15 . The results of flow cytometry showed that, contrary to the increase in Fe²⁺ in HCT116 cells treated with B&C in experimental group 8, the Fe²⁺ level was significantly inhibited after the combined treatment of NAC and B&C in experimental group 9, specifically see Figures 16 - 17 . These results prove that B&C can play an anti-colorectal cancer role by promoting ROS accumulation and then enhancing NCOA4 / ferritin-mediated ferritin autophagy.

[0057] Example 5: This example is based on the conclusion that B&C promotes ferroptosis and exerts significant anti-tumor activity through the ROS / NCOA4 / ferritin pathway obtained from the in vitro experiments of the above Examples 1-4, and a xenograft tumor growth experiment is carried out.

[0058] In this example, BALB / c nude mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Sichuan, China). All mice were housed in a comfortable environment with fresh air, sufficient water and food. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Chengdu University of Traditional Chinese Medicine Hospital (2023003). The method of establishing the model was based on the guidance of xenografts. HCT116 cells were injected subcutaneously into the axilla of nude mice. Approximately 1 week later, the mice with successful tumors were randomly divided into 4 groups: the blank group, the BER (20 mg / kg) group, the CUR (20 mg / kg) group, and the BER (20 mg / kg) + CUR (20 mg / kg) group. Then, the mice were intraperitoneally injected with the corresponding reagent or an equal volume of solvent once a day for 3 weeks. The tumor size and body weight were measured and recorded every 2 days for 10 days. Finally, the mice were euthanized and the tumors were isolated. The tumor tissues of the mice are as Figure 18 shown. The results showed that the weight of the tumor tissues of the mice under the action of B&C was significantly lower than that of the control group and the single-drug groups, as shown in Figure 19 shown. The average body weight and the corresponding tumor volume of the mice showed a similar trend, as shown in Figures 20 - 21As shown, the tumor volume of the B&C group was smaller than that of the control group and the single-drug group. Obviously, B&C has a significant synergistic effect in inhibiting the growth of colorectal cancer in vivo. Subsequently, the colorectal mucosal tissues of the mice were fixed in paraformaldehyde, dehydrated, embedded in paraffin and cut into slides. After dewaxing and rehydration, the slides were stained with hematoxylin and eosin. Then, immunohistochemical (IHC) staining was performed on the slides of mouse colorectal tissues. H2O2 (3%) was used to eliminate the endogenous peroxidase activity on the slides after antigen repair. After blocking with bovine serum albumin, the slides were incubated with the corresponding primary antibody, and then incubated with a secondary antibody labeled with horseradish peroxidase (HRP); using a 3,3′-diaminobenzidine kit (Servicebio, China), after counterstaining, dehydration and sealing, the slides were detected and analyzed using an optical microscope; for immunofluorescence (IF) detection, the slides were incubated with the microtubule-associated protein 1A / 1B light chain 3 (LC3) antibody at 4°C overnight, and then incubated with a secondary antibody conjugated with HRP at room temperature for 50 minutes. CY3-tyramide (Servicebio, China) was used for fluorescence signal amplification. For the second labeling step, the slides were incubated with the Tom20 antibody at 4°C overnight, and then incubated with a secondary antibody. The resulting images were captured using a Nikon Eclipse C1 fluorescence microscope, and the results were analyzed using ImageJ software. Hematoxylin-eosin staining and Ki67 immunohistochemistry were performed on the tumor tissues, see Figures 22 - 23 as shown. At the same time, compared with the control group and the single-drug group, the expression of Ki67, an important marker of cell proliferation, was significantly reduced in the B&C group, see Figure 24 as shown. To sum up, it is sufficient to confirm that B&C has a strong ability to inhibit the growth of colorectal cancer in vivo. Thus, drugs containing B&C have a predictable significant effect on the treatment of colon cancer and rectal cancer.

[0059] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composition capable of inducing iron autophagy in HCT116 cells, characterized in that: It is composed of berberine at a concentration of 10μM-80μM and curcumin at a concentration of 10μM-80μM.

2. A composition capable of inducing iron autophagy in HCT116 cells according to claim 1, characterized in that: The concentrations of berberine and curcumin are both 20 μM or 30 μM.

3. Use of a composition capable of inducing iron autophagy in HCT116 cells according to claim 1 in the manufacture of a targeted drug for treating CRC.

4. The use according to claim 3, characterized in that The composition enhances NCOA4 / Ferritin-mediated iron autophagy by activating ROS.

5. The use according to claim 3, characterized in that: The CRC targeted drug is an injection or an oral preparation.

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