A method for inducing differentiation of cancer cells into adipocytes

Abstract

The present application relates to the technical field of induced transformation of cancer cells, and provides a method for inducing differentiation of cancer cells into adipocytes, which comprises the following steps: (1) culturing cancer cells in a basic medium, and when the confluence of the cells reaches 80-100%, inducing differentiation; (2) replacing the basic medium in step (1) with an induction differentiation medium 1, and culturing for 4-8 days; (3) replacing the induction differentiation medium 1 with an induction differentiation medium 2, and culturing for 1-4 days; (4) replacing with the basic medium, and culturing for 6-12 days, so that the cancer cells are successfully induced to differentiate into adipocytes; the method of the present application can transform malignant proliferative and metastatic cancer cells into mature adipocytes, and the adipocytes formed by differentiation contain fat droplets and highly express marker genes of mature adipocytes. The method of the present application can effectively inhibit the proliferation and metastasis of cancer cells, and provides a new idea and method for clinical cancer treatment, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of cancer cell induction and transformation technology, and specifically provides a method for inducing cancer cells to differentiate into adipocytes. Background Technology

[0002] In the development of cancer, there is a stage called epithelial-mesenchymal transition (EMT). During this stage, cancer cells possess characteristics similar to stem cells, with the potential to transform into various cell types. Epithelial cells can transform into mesenchymal cells, leading to loss of cell polarity, decreased adhesion, and changes in the cytoskeleton, thereby enhancing cell migration and motility. Utilizing EMT, these cancer cells can detach from the initial tumor and metastasize to distant sites. Research has confirmed that EMT is a key event involved in cancer invasion and metastasis. After tumor cells spread through the bloodstream, they undergo mesenchymal-epithelial transition (MET), forming secondary metastatic lesions. MET is the reverse process of EMT; cancer cells transform from mesenchymal cells back into epithelial cells, with enhanced proliferative capacity and faster growth.

[0003] Scientists used rosiglitazone in combination with the MEK inhibitor tramitinib in mice to induce breast cancer cells to differentiate into adipocytes, reducing tumor invasiveness and inhibiting tumor metastasis. The main method employed was as follows: breast cancer cells were seeded into culture dishes, cultured overnight, and then treated with 200 ng / ml of recombinant human BMP2 protein for 3 days, followed by treatment with 200 ng / ml BMP2 and 2 μM rosiglitazone for 4 days, and then treated with 2 μM rosiglitazone for 3 days. The culture medium used was DMEM containing 10% fetal bovine serum. The results showed that after induction with rosiglitazone and bone morphogenetic protein-2 (BMP-2), breast cancer cells undergoing EMT transformed into adipocytes, expressing various adipocyte markers and responding to isoproterenol and insulin in the same way as adipocytes. Furthermore, nine days after induction, the researchers removed the induction culture medium and replaced it with ordinary culture medium. They observed that these differentiated adipocytes maintained adipocyte characteristics and did not revert to mesenchymal-like cancer cells, confirming that this induced differentiation was irreversible. However, this method of inducing cancer cell differentiation has a significant limitation: it can only induce EMT-transformed cancer cells into adipocytes, that is, it can only induce differentiation of cancer cells in a mesenchymal state, not cancer cells in an epithelial state. The use of BMP2 in this method is specifically to promote EMT transformation of cancer cells. Therefore, this method has stringent conditions for inducing cancer cell differentiation and is highly limited, only inducing differentiation of cancer cells in a mesenchymal state, and lacks universality.

[0004] In their previous research, the inventors successfully induced normal human skin fibroblasts to differentiate into adipocytes (see Chinese Patent Application No. 2019108760496). However, normal human skin fibroblasts and cancer cells differ significantly in growth, development, and physiological mechanisms, primarily in the following ways: First, human skin fibroblasts have a limited number of passages and do not possess the ability to proliferate or metastasize indefinitely, nor do they undergo EMT or MET transformation. Cancer cells, on the other hand, are infinitely proliferating cells with the ability to metastasize malignantly and undergo EMT or MET transformation. Second, human skin fibroblasts are generally in a resting state, typically proliferating rapidly during wound repair and secreting collagen fibers and extracellular matrix to participate in wound healing. Cancer cells, however, are constantly proliferating or metastasizing and almost never secrete collagen fibers or extracellular matrix. Third, human skin fibroblasts differentiate from mesenchymal cells during the embryonic period and are generally found in the connective tissue of the skin, existing in a mesenchymal state. Cancer cells, however, are diverse in type and origin, with some existing in an epithelial state and others in a mesenchymal state. Therefore, the induction of cancer cell differentiation is complex and difficult, and there is a need to study simpler, more universal and effective methods for inducing cancer cells to differentiate into adipocytes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for inducing cancer cells to differentiate into adipocytes, comprising the following steps:

[0006] (1) The cancer cells were cultured in a basic culture medium and induced to differentiate when the cell confluence reached 80-100%.

[0007] (2) Replace the basic culture medium in step (1) with the differentiation induction medium ① and culture for 4-8 days;

[0008] (3) Replace the induction differentiation medium ① with the induction differentiation medium ② and culture for 1-4 days;

[0009] (4) Replace with basal culture medium and incubate for 6-12 days;

[0010] The basal culture medium is a high-glucose DMEM (Dulbecco's modified Eagle medium) containing fetal bovine serum and antibiotics.

[0011] The induction differentiation medium ① is a basal medium containing dexamethasone, insulin, rosiglitazone and 1-methyl-3-isobutylxanthine;

[0012] The differentiation-inducing medium ② is a basal medium containing insulin.

[0013] Preferably, the basal culture medium is a high-glucose DMEM medium containing 5%-20% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0014] Preferably, the induction differentiation medium ① is a basal medium containing 0.5-1 μM dexamethasone, 5-20 μg / mL insulin, 1-2 μM rosiglitazone, and 0.2-0.5 mM 1-methyl-3-isobutylxanthine.

[0015] More preferably, the induction differentiation medium ① is a basal medium containing 1 μM dexamethasone, 10 μg / mL insulin, 2 μM rosiglitazone, and 0.5 mM 1-methyl-3-isobutylxanthine.

[0016] In the process of inducing differentiation of cancer cells using the induction differentiation medium①, the medium is changed regularly according to the conventional cell culture method. In this embodiment of the invention, the medium is changed once every two days.

[0017] Preferably, the differentiation-inducing medium ② is a basal medium containing 5-20 μg / mL insulin.

[0018] More preferably, the differentiation-inducing medium ② is a basal medium containing 10 μg / mL insulin.

[0019] In the above method of the present invention, the environmental conditions for inducing differentiation are 35℃~37℃ and 5% CO2.

[0020] A specific differentiation-inducing scheme of the present invention is as follows:

[0021] Cancer cells were passaged into 35mm culture dishes with coverslips, 3mL of basal culture medium was added, and the dishes were placed in a cell culture incubator containing 5% CO2 and cultured at 37℃. When the cell confluence reached 80-100%, the basal culture medium was removed, and induction differentiation medium ① was added. The cells were cultured continuously for 4-8 days, with the medium changed every two days. Induction differentiation medium ① was removed, and induction differentiation medium ② was added. The cells were cultured for 1-4 days. Induction differentiation medium ② was removed, and basal culture medium was added. The cells were cultured for another 6-12 days, with the medium changed every two days.

[0022] The adipocytes obtained by the above method are within the scope of protection of this invention.

[0023] The application of the above method in inhibiting cancer cell proliferation and metastasis by inducing cancer cells to differentiate into adipocytes falls within the scope of protection of this invention.

[0024] This invention provides the use of a composition containing dexamethasone, insulin, rosiglitazone, and 1-methyl-3-isobutylxanthine in the preparation of a drug that induces cancer cells to differentiate into adipocytes or in the preparation of a drug that inhibits the proliferation and metastasis of cancer cells.

[0025] In the above application, cancer cells were first induced to differentiate using a composition containing dexamethasone, insulin, rosiglitazone, and 1-methyl-3-isobutylxanthine, and then induced to differentiate using insulin.

[0026] Specifically, a differentiation-inducing medium ① is prepared by a composition containing dexamethasone, insulin, rosiglitazone, and 1-methyl-3-isobutylxanthine. The differentiation-inducing medium ① is the above-mentioned basal medium containing 0.5-1 μM dexamethasone, 5-20 μg / mL insulin, 1-2 μM rosiglitazone, and 0.2-0.5 mM 1-methyl-3-isobutylxanthine. Insulin is added to the above-mentioned basal medium to make the final insulin concentration 5-20 μg / mL, thus preparing the differentiation-inducing medium ②.

[0027] The cancer cells applicable to this method do not require EMT transformation and are not affected by their state; they can be cancer cells in an epithelial state or a mesenchymal state. Therefore, this method has universality. Applicable cancer cells include, but are not limited to, HeLa (human cervical cancer cells), U2OS (human osteosarcoma cells), SW620 (human colon cancer cells), SW480 (human colon cancer cells), HCT116 (human colon cancer cells), HT29 (human colon cancer cells), A549 (human non-small cell lung cancer cells), MDA-MB-231 (human breast cancer cells), HepG2 (human liver cancer cells), T47D (human breast cancer cells) cell lines, and isolated primary cancer cells.

[0028] This invention successfully induced various cancer cells to differentiate into adipocytes using the aforementioned method. Taking HeLa cells, U2OS cells, A549 cells, and SW620 cells as examples, a "4+2+10 day" induction differentiation model was adopted. Cancer cells were successfully induced to differentiate into adipocytes. It was found that the treatment time of induction differentiation medium ① was at least 4 days, and the treatment time of induction differentiation medium ② was at least 1 day; otherwise, the induction differentiation efficiency was low. Oil Red staining results showed that the method of this invention could differentiate cancer cells into adipocytes with an efficiency of approximately 25-70%. This method transforms malignant proliferating and metastatic cancer cells into mature adipocytes. The differentiated adipocytes contain both lipid droplets and highly express marker genes of mature adipocytes, significantly reducing the proliferation and metastasis of cancer cells. This method effectively inhibits the proliferation and metastasis of cancer cells, providing new ideas and methods for clinical cancer treatment, and has promising application prospects. Attached Figure Description

[0029] Figure 1 This is an image showing the Oil Red staining results of cells after the induction of differentiation in Example 1.

[0030] Figure 2 This is a diagram showing the results of the detection of mature adipocyte marker genes in adipocytes obtained through induced differentiation in Example 1.

[0031] Figure 3 This is an image showing the Oil Red staining results of cells after the induction of differentiation in Example 2.

[0032] Figure 4 This is a graph showing the results of the detection of mature adipocyte marker genes in adipocytes obtained by induced differentiation in Example 2.

[0033] Figure 5 This is an image showing the Oil Red staining results of cells after the induction of differentiation in Example 3.

[0034] Figure 6 This is a graph showing the results of the detection of mature adipocyte marker genes in adipocytes obtained by induced differentiation in Example 3.

[0035] Figure 7 This is an image showing the Oil Red staining results of cells after the induction of differentiation in Example 4. Detailed Implementation

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, and unless otherwise specified, the reagents used in the present invention are commercially available.

[0037] In this embodiment of the invention, after the differentiation induction was completed, the coverslips in the culture dish were removed and stained with Oil Red to detect the differentiation induction effect; in addition, RNA was extracted from the remaining cells in the culture dish and subjected to real-time PCR to detect the expression of the marker genes Adiponectin, Fapp4 and Leptin of mature adipocytes, to further verify the results of the differentiation induction.

[0038] (1) Oil Red staining

[0039] Oil Red O is a strong lipid solvent and staining agent that can bind to triglycerides, and is therefore frequently used for fat staining. This invention uses Oil Red staining on induced differentiated cells to detect fat droplets within mature adipocytes. The specific procedure is as follows:

[0040] ① Wash the coverslip three times with PBS, then fix it with 4% paraformaldehyde (PFA) for 20 minutes;

[0041] ② After washing 3 times with PBS, equilibrate with 60% isopropanol for 5 minutes;

[0042] ③ Stain with 3 g / L Oil Red O (dissolved in 60% isopropanol) in a sealed container for 10 minutes;

[0043] ④ Wash three times with 60% isopropanol;

[0044] ⑤ Stain with hematoxylin for 2 minutes;

[0045] ⑥ Wash 3 times with PBS, air dry, mount the slide and perform imaging;

[0046] ⑦ Count the number of cells that are positive for Oil Red staining;

[0047] (2) Detection of marker genes for mature adipocytes: Adiponectin, Fapp4, and Leptin are marker genes for mature adipocytes and are often used to measure the effectiveness of adipocyte differentiation induction. In this invention, total RNA was extracted from differentiated cells using Trizol, reverse transcribed, and then subjected to quantitative real-time PCR to detect the expression levels of these three genes: Adiponectin, Fapp4, and Leptin. The primers used for quantitative PCR detection of the three marker genes for mature adipocytes, Adiponectin, Fapp4, and Leptin, were as follows:

[0048] Adiponectin:5'-GATGGCAGAGATGGCAC-3'

[0049] 5'-GCTGAGCGGTATACATAGG-3'

[0050] Fabp4:5'-ACGAGAGGATGATAAACTGGTGG-3'

[0051] 5'-GCGAACTTCAGTCCAGGTCAAC-3'

[0052] Leptin:5'-CACCAAAACCCTCATCAAGACA-3'

[0053] 5'-CTTTCTGTTTTGGAGGAGACTGACT-3'

[0054] In this embodiment of the invention, *** indicates P<0.001, ****P<0.0001, and the differences between different treatment groups are statistically significant.

[0055] Example 1: A method for inducing cancer cells to differentiate into adipocytes (1)

[0056] This embodiment uses HeLa cells in an epithelial state as an example to illustrate the results of inducing the differentiation of HeLa cells into adipocytes using the method of the present invention.

[0057] 1. The culture media involved in the induction process are as follows:

[0058] Basic culture medium: high glucose Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin;

[0059] Differentiation induction medium ①: Dexamethasone, insulin, rosiglitazone, and 1-methyl-3-isobutylxanthine were added to the basal medium to make their final concentrations 1 μM, 10 μg / mL, 2 μM, and 0.5 mM, respectively.

[0060] Differentiation induction medium ②: Add insulin to the basal medium to a final concentration of 10 μg / mL.

[0061] 2. Induction process:

[0062] (1) Pass HeLa cells into a 35 mm culture dish covered with a coverslip, add 3 mL of basal culture medium, and place in a cell culture incubator containing 5% CO2 at 37°C.

[0063] (2) After the cell density reaches 80-100%, remove the basal culture medium, add the induction differentiation medium ①, and continuously induce culture for 4 days, replacing the culture medium with fresh medium every two days.

[0064] (3) Remove the induction differentiation medium ①, add the induction differentiation medium ②, and induce culture for 2 days;

[0065] (4) Remove the induction differentiation medium ②, add the basic medium, and continue to culture for 10 days, changing the medium with fresh medium every two days.

[0066] (5) After the induction of differentiation was completed, the results were detected by Oil Red staining and detection of mature adipocyte marker genes. The Oil Red staining results are shown below. Figure 1 The stained areas are stained fat (appearing red after Oil Red staining). Uninduced HeLa cells showed no fat accumulation, while HeLa cells induced for 16 days showed significant fat accumulation. The number of uninduced HeLa cells that stained positive for Oil Red was 0; while the differentiation efficiency of HeLa cells induced for 16 days into adipocytes was approximately 70%.

[0067] Results of mature adipocyte marker gene detection are shown below Figure 2 Quantitative PCR was used to detect three marker genes of mature adipocytes: adiponectin, Fapp4, and leptin. These three genes were not detected in uninduced HeLa cells, but high levels of expression were detected in HeLa cells induced for differentiation for 16 days.

[0068] The method in this embodiment successfully induced HeLa cells to differentiate into adipocytes in vitro.

[0069] Example 2: Method for inducing cancer cells to differentiate into adipocytes (2)

[0070] This embodiment uses U2OS cells in a mesenchymal state as an example to illustrate the results of inducing the differentiation of U2OS cells into adipocytes using the method of the present invention.

[0071] The culture medium and specific experimental steps involved in this embodiment are the same as in Example 1, and the "4+2+10 day" induction differentiation mode is also used. The difference is that the cancer cells are U2OS cells.

[0072] After differentiation induction was completed, the results were assessed using Oil Red staining and detection of mature adipocyte marker genes. The results are as follows:

[0073] (1) Results of Oil Red staining are as follows Figure 3 As shown, the stained areas are stained fat (appearing red after Oil Red staining). Uninduced U2OS cells showed no fat accumulation, while U2OS cells induced for 16 days showed significant fat accumulation. The number of uninduced U2OS cells showing positive Oil Red staining was 0; while the differentiation efficiency of U2OS cells induced for 16 days into adipocytes was approximately 25%.

[0074] (2) Detection of marker genes for mature adipocytes

[0075] like Figure 4 As shown, three marker genes of mature adipocytes—Adiponectin, Fapp4, and Leptin—were detected by quantitative PCR. Low-level expression of these three genes was detected in uninduced U2OS cells, while high-level expression was detected in U2OS cells induced for differentiation for 16 days.

[0076] Example 3: Method for inducing cancer cells to differentiate into adipocytes (3)

[0077] This embodiment uses SW620 cells in an epithelial state as an example to illustrate the results of inducing SW620 cells to differentiate into adipocytes using the method of the present invention.

[0078] The culture medium and specific experimental steps involved in this embodiment are the same as in Example 1, and the "4+2+10 day" induction differentiation mode is also used. The difference is that the cancer cells are SW620 cells.

[0079] After differentiation induction was completed, the results were assessed using Oil Red staining and detection of mature adipocyte marker genes. The results are as follows:

[0080] (1) Results of Oil Red staining are as follows Figure 5 As shown, the stained areas are stained fat (appearing red after Oil Red staining). Uninduced SW620 cells showed no fat accumulation, while SW620 cells induced for 16 days showed significant fat accumulation. The number of uninduced SW620 cells showing positive Oil Red staining was 0; while the differentiation efficiency of SW620 cells induced for 16 days into adipocytes was approximately 40%.

[0081] (2) Detection of marker genes for mature adipocytes

[0082] like Figure 6 As shown, three marker genes of mature adipocytes—Adiponectin, Fapp4, and Leptin—were detected by quantitative PCR. Low-level expression of these three genes was detected in uninduced SW620 cells, while high-level expression was detected in SW620 cells induced for differentiation for 16 days.

[0083] Example 4: Method for inducing cancer cells to differentiate into adipocytes (4)

[0084] This embodiment uses A549 cells in an epithelial state as an example to illustrate the results of inducing A549 cells to differentiate into adipocytes using the method of the present invention.

[0085] The culture medium and specific experimental steps involved in this embodiment are the same as in Example 1, and the "4+2+10 day" induction differentiation mode is also used. The difference is that the cancer cells are A549 cells.

[0086] After differentiation induction was completed, the results were detected using Oil Red staining. The results are as follows:

[0087] like Figure 7 As shown, the stained areas are stained fat (appearing red after Oil Red staining). Uninduced A549 cells showed no fat accumulation, while A549 cells induced for 16 days showed significant fat accumulation. The number of uninduced A549 cells showing positive Oil Red staining was 0; while the differentiation efficiency of A549 cells induced for 16 days into adipocytes was approximately 38%.

[0088] This invention also induced differentiation of various cancer cells, including MDA-MB-231, HepG2, and T47D, using the method of this invention. Results showed that the method of this invention could induce differentiation of various cancer cells, including MDA-MB-231, HepG2, and T47D, into adipocytes, with differentiation rates ranging from 15% to 80%. This indicates that the method of this invention can transform malignantly proliferating and metastatic cancer cells into mature adipocytes. The differentiated adipocytes contain lipid droplets and highly express marker genes of mature adipocytes. This method of this invention effectively inhibits the proliferation and metastasis of cancer cells, providing new ideas and methods for clinical cancer treatment, and has promising application prospects.

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for inducing cancer cells to differentiate into adipocytes, characterized in that, Includes the following steps: (1) The cancer cells were cultured in a basic culture medium, and when the cell confluence reached 80-100%, differentiation was induced. (2) Replace the basal culture medium in step (1) with the induction differentiation medium ① and culture for 4 days; (3) Replace the induction differentiation medium ① with the induction differentiation medium ② and culture for 2 days; (4) Replace with basal culture medium and culture for 10 days; The cancer cells described in step (1) do not require CoCl2 treatment before culture; The basal culture medium is a high-glucose DMEM medium containing fetal bovine serum and antibiotics; The induction differentiation medium ① is a basal medium containing only 0.5-1 μM dexamethasone, 5-20 μg / mL insulin, 1-2 μM rosiglitazone, and 0.2-0.5 mM 1-methyl-3-isobutylxanthine; The differentiation-inducing medium ② is a basal medium containing insulin; The method does not require EMT transformation; the cancer cells are HeLa cells, SW620 cells, or A549 cells in an epithelial state.

2. The method according to claim 1, characterized in that, The basal culture medium is a high-glucose DMEM medium containing 5%-20% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

3. The method according to claim 1, characterized in that, The induction differentiation medium ① is a basal medium containing only 1 μM dexamethasone, 10 μg / mL insulin, 2 μM rosiglitazone, and 0.5 mM 1-methyl-3-isobutylxanthine.

4. The method according to claim 1, characterized in that, The induction differentiation medium ② is a basal medium containing only 5-20 μg / mL insulin.

5. The method according to claim 4, characterized in that, The induction differentiation medium ② is a basal medium containing only 10 μg / mL insulin.

6. The method according to any one of claims 1-5, characterized in that, The environmental conditions for inducing differentiation were 35℃~37℃ and 5% CO2.

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