Application of bifidobacterium in colon cancer radiotherapy

Abstract

The invention relates to application of bifidobacteria in colon cancer radiotherapy. The application comprises the following steps: specifically planting bifidobacteria in a hypoxic region of a tumor tissue for proliferation; the bifidobacterium is used for inducing macrophages in the area to generate high-concentration NO to expand blood vessels, blood perfusion is increased, oxygen is increased locally, and tumor hypoxia is relieved; the hypoxia-induced radiotherapy tolerance is reversed, and the radiotherapy is sensitized. The bifidobacterium induces macrophages to generate high-concentration NO, and the NO can be used for regulating a tumor hypoxia microenvironment and overcoming hypoxia-induced radiotherapy tolerance. The bifidobacterium is proved to have no pathogenicity or immune side effect after entering blood as a physiological probiotic, and has good biological safety. Due to the obligate anaerobic characteristic of bifidobacterium, bifidobacterium can specifically colonize and proliferate in a hypoxic region after entering blood, and shows targeting to solid tumors.

Description

Technical Field

[0001] The present invention relates to the field of radiotherapy for colon cancer, and in particular to the application of bifidobacteria in radiotherapy for colon cancer. Background Art

[0002] Cancer is one of the major diseases that threaten human health worldwide. Currently, the main methods of cancer treatment in clinical practice include surgery, chemotherapy, radiotherapy and immunotherapy. Although traditional methods are constantly improving, they still have their own limitations. Surgical treatment is difficult to completely eliminate cancer cells, which are prone to recurrence and metastasis, and cannot cure metastatic tumors. Immunotherapy is an emerging and effective cancer treatment method, but it may cause cytokine storms. Radiotherapy and chemotherapy can effectively kill cancer cells to a certain extent, but at the same time they will damage normal tissues and cause serious side effects of radiotherapy and chemotherapy. In addition, due to the complex microenvironment of the tumor, cancer cells will develop a certain tolerance to long-term chemotherapy and radiotherapy, which will affect the effect of treatment.

[0003] Radiotherapy is one of the main means of cancer treatment and has been widely used in the treatment of many types of cancers in clinical practice. However, due to the excessive growth of tumor cells during the growth of malignant tumors, abnormal angiogenesis and poor blood flow in solid tumors can lead to an imbalance between oxygen supply and consumption. The imbalance between energy supply and energy consumption causes tumor hypoxia and forms a tumor hypoxic microenvironment. Since oxygen is a key factor in the process of radiotherapy-induced tumor cell killing, the hypoxic areas in the tumor have stronger resistance than the normal oxygen areas, which can lead to hypoxia-related radioresistance in many types of solid malignant tumors. Hypoxia-induced radioresistance is the main cause of tumor radiotherapy failure, such as Figure 2 shown.

[0004] Under hypoxic conditions, to achieve the same level of tumor cell killing effect, the required radiation dose is three times that of normal oxygen state. Increasing the dose of ionizing radiation can enhance the therapeutic effect of tumors, but it is also easy to cause serious damage to normal tissues adjacent to the tumor. Studies have found that changes in the tumor microenvironment after ionizing radiation are closely related to the ability to resist radiotherapy. Since hypoxia weakens the fixation of DNA damage, hypoxia-inducible factor 1α (HIF-1α)-mediated cell survival is reduced, so it plays a key role in radiation resistance. Increasing oxygen delivery, normalizing tumor blood vessels, inhibiting HIF-1α, and preventing the recruitment of bone marrow-derived cells required for angiogenesis are all used to reduce tumor hypoxia, improve radiotherapy response, and prevent tumor recurrence after treatment. Therefore, how to overcome hypoxia-induced radiotherapy tolerance by regulating the tumor microenvironment and enhance the therapeutic effect of radiotherapy on hypoxic tumors has attracted much attention. With the development of nanotechnology, different strategies based on nanoparticles have been developed to regulate the tumor microenvironment, overcome hypoxia-related radiation resistance, and enhance the therapeutic effect of RT. For example: polymer nanoparticles, metal or magnetic nanoparticles, etc. They achieve the regulation of tumor microenvironment through the following strategies: (i) specific delivery of oxygen to hypoxic tumor areas; (ii) production of large amounts of oxygen, nitric oxide (NO) and reactive oxygen (ROS) in hypoxic tumor sites. Generally, these nanoparticles achieve their complex functions through complex design, synthesis and construction processes, and achieve radiosensitization by optimizing their physicochemical properties. However, these nanoparticles face many important problems, such as technical challenges, high costs and low efficiency brought about by complex synthesis processes. In addition, these artificially synthesized substances also face serious limitations such as low biodegradability, poor stability and potential toxicity in clinical applications. Therefore, it is very necessary to discover a new tumor reoxygenation strategy for regulating the tumor microenvironment and enhancing radiotherapy sensitivity. Summary of the invention

[0005] To solve the above problems, the present invention provides the use of bifidobacteria in radiotherapy for colon cancer, using the characteristics of bifidobacteria inducing macrophages to produce NO and targeted colonization and proliferation in hypoxic microenvironment as RT sensitizers to better achieve the radiotherapy effect. Specifically, the purpose of the present invention is achieved as follows:

[0006] The application of bifidobacteria in radiotherapy for colon cancer includes: specifically colonizing bifidobacteria in the hypoxic area of ​​tumor tissue for proliferation; using bifidobacteria to induce macrophages in the area to produce high concentrations of NO to dilate blood vessels, increase blood perfusion, increase local oxygen, and relieve tumor hypoxia; reversing hypoxia-induced radiotherapy tolerance and sensitizing radiotherapy.

[0007] Furthermore, the application of establishing a subcutaneous colon cancer tumor model in mice comprises the following steps:

[0008] S1. Cell culture and tumor model establishment: CT26 colon cancer cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics at 37°C and 5% CO2; the antibiotics were penicillin-streptomycin at a concentration of 10,000 U / mL; in an SPF-grade laboratory sterile environment, CT26 cells with a confluence of 60-70% were digested with trypsin and prepared into a cell suspension at a cell concentration of 4×10 7 / mL; select 26-30 days old, 20-30 grams of Balb / c male mice; take 0.1mL of the prepared cell suspension and inoculate it subcutaneously on the back of the selected nude mice. When the tumor volume reaches 70-100mm 3 , a subcutaneous colon cancer animal model was successfully established;

[0009] S2. Cultivation of bifidobacteria; Bifidobacteria were cultured in BBL liquid medium and placed in an anaerobic tank together with an anaerobic bag to provide an anaerobic environment required for bacterial growth and reproduction, and incubated in a constant temperature incubator at 37°C with 5% CO2;

[0010] S3. Gavage; prepare bifidobacterium suspension, 5*10 9 CFU colony counts were used to continuously gavage Bifidobacterium to subcutaneous colon cancer-bearing mice for 3 days, once a day, 200uL each time.

[0011] S4. Irradiation: Tumor-bearing mice were irradiated with a small animal irradiator 48 hours after the last oral gavage, with a single dose of 12 Gy;

[0012] S5. Group observation, the mice were divided into a short-term treatment group and a long-term treatment group; the short-term treatment group was killed on the fifth day after irradiation and the tumor was taken to observe the effect of short-term tumor treatment; the long-term treatment group was observed every other day after irradiation. The observation lasted for 31 days, and the mice were killed after the 31st day and the tumor was taken to observe the effect of long-term tumor treatment.

[0013] Furthermore, HIF-1α is highly expressed in the tumor tissue before treatment; and the expression ability of the hypoxia-inducible factor HIF-1α in the tumor tissue is reduced after radiotherapy.

[0014] The beneficial effects of the present invention are as follows:

[0015] (I) Bifidobacterium induces macrophages to produce high concentrations of NO, which can be used to regulate the tumor hypoxic microenvironment and overcome hypoxia-induced radiotherapy tolerance.

[0016] (ii) Bifidobacterium, as a physiological probiotic, has been proven to have no pathogenicity or immune side effects after entering the blood and has good biosafety.

[0017] (III) Due to their obligate anaerobic properties, bifidobacteria can specifically colonize and proliferate in hypoxic areas after entering the blood, showing targeting properties against solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the application flow chart of the present invention;

[0019] Figure 2 Schematic diagram of hypoxia-induced radioresistance as the main cause of tumor radiotherapy failure;

[0020] Figure 3 The bifidobacterium is administered intragastrically in the verification step of the safety and targeting of the bifidobacterium in the embodiment of the present invention.

[0021] The distribution of Bifidobacterium colonies in the heart, liver, spleen, lung, kidney, blood and tumor tissues at different times after inoculation is a schematic diagram;

[0022] Figure 4 The step of verifying the safety and targeting of Bifidobacterium in the embodiment of the present invention is

[0023] Schematic diagram of the Gram staining results of bifidobacterium culture colonies after intragastric administration;

[0024] Figure 5 The step of verifying the safety and targeting of Bifidobacterium in the embodiment of the present invention is

[0025] Schematic diagram of Gram staining of paraffin sections of tumor tissue after intragastric administration of bacteria;

[0026] Figure 6 This is a schematic diagram of HE staining results of normal tissues and organs of mice in the verification step of the safety and targeting of Bifidobacterium described in the examples of the present invention;

[0027] Figure 7 A schematic diagram of NO content in the detection of NO content in tumor tissue in the steps of an embodiment of the present invention;

[0029] Figure 8 These are comparison photos of tumor sizes of mice in each group after short-term treatment according to the examples of the present invention;

[0030] Fig. 9 This is a statistical chart comparing the tumor masses of mice in each group after long-term treatment according to the embodiment of the present invention;

[0031] Fig.10 This is a schematic diagram of the TUNEL staining results of mouse tumor tissues described in the examples of the present invention;

[0032] Fig.11 This is a schematic diagram of PCNA staining results of mouse tumor tissues according to an embodiment of the present invention;

[0033] Fig.12 This is a schematic diagram of the HIF-1α staining results of mouse tumor tissue according to an embodiment of the present invention; DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features and objectives of the present invention easy to understand, the technical solution of the present invention is further explained below in conjunction with one embodiment and a specific implementation method of the use of bifidobacteria in colon cancer radiotherapy given by the present invention.

[0035] like Figure 1-12 As shown, the specific embodiments given for the present invention are as follows:

[0036] In the figures, Bifidobacterium is represented by BBM.

[0037] (I) Cultivation of Bifidobacterium and detection of its basic properties

[0038] ① Cultivation of Bifidobacterium

[0039] Bifidobacterium was cultured in BBL liquid medium and placed in an anaerobic tank together with an anaerobic bag to provide an anaerobic environment required for bacterial growth and reproduction, and incubated in a constant temperature incubator at 37° C. containing 5% CO 2 .

[0040] ② Bacterial colony count

[0041] Add 2% agar to the BBL liquid medium to prepare a solid medium, dilute the bifidobacterium culture solution after a certain period of culture, take 10 μL of the liquid and vertically drip it into the solid medium, so that the droplets vertically pass through the solid culture plate. Then put the solid culture plate and the anaerobic bag into an anaerobic tank, incubate it in a constant temperature incubator at 37°C and 5% CO2, and count the colonies on the fixed culture plate after incubation for 24 hours.

[0042] ③Draw the growth curve of Bifidobacterium

[0043] The bifidobacterium was inoculated into BBL liquid culture medium and cultured in a sterile anaerobic environment for 48 hours. A small amount of bacterial liquid was taken every 2 hours, and the absorbance at 450 nm was measured with an ultra-micro spectrophotometer to draw a bifidobacterium growth curve of culture time-absorbance.

[0044] ④ Bifidobacterium performance test

[0045] The bifidobacterium culture medium in the logarithmic growth phase was collected, centrifuged at 1000 rpm for 10 min, and rinsed twice with PBS.

[0046] Resuspend in PBS and adjust the bacterial concentration to 3 × 10 8 cfu / mL, and Gram staining was used to observe bacterial morphology and distribution.

[0047] (II) Verification of the targeting and safety of Bifidobacterium and detection of NO content in tumor tissue

[0048] ①Cell culture and tumor model establishment

[0049] Select Balb / c male tumor-bearing mice of about 4 weeks old and weighing 20-30 grams;

[0050] CT26 human colon cancer cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics (here penicillin-streptomycin, 10000U / mL) at 37°C and 5% CO2. In a sterile environment of an SPF laboratory, CT26 cells with a confluence of 60-70% were digested with trypsin and prepared into a cell suspension with a cell concentration of 4×10 7 Take 0.1mL of cell suspension and inoculate it subcutaneously on the back of nude mice. When the tumor diameter reaches 10mm and the volume reaches 70-100mm 3 , the subcutaneous colon cancer animal model was successfully established.

[0051] ②Verification of the safety and targeting of Bifidobacterium

[0052] The mice were gavaged, and a normal saline group was set up as a control. At different time points after gavage, the mice's and representative normal organs (heart, liver, spleen, lung, kidney and blood) were collected, and the tissues were divided into two parts, one part was homogenized by a tissue homogenizer, and the colonies were observed after the homogenate was cultured, and the other half of the tissue was fixed with 4% paraformaldehyde solution and then paraffin sectioned, stained with Gram stain, and the distribution of bifidobacteria in various organs was observed under a microscope to verify the safety and targeting of bifidobacteria.

[0053] like Figure 3 As shown, mice were gavaged with bifidobacteria at different time points, and normal tissues, blood and tumor tissues were taken to observe the distribution of bifidobacteria in different parts. The results showed that 24 hours after gavage, bacteria were distributed in the normal tissues of heart, liver, spleen, lung and kidney. 96 hours after gavage, bifidobacteria were only distributed in tumor tissues, and basically not distributed in other normal tissues and organs, indicating that bifidobacteria have good tumor targeting and biosafety.

[0054] like Figure 4 As shown in the figure, after oral administration of bifidobacteria, the tumor tissue was ground and cultured for bifidobacterium colonies for Gram staining to compare the colony morphology before and after oral administration. The results showed that the bacterial colony morphology was consistent before and after oral administration, indicating that bifidobacteria successfully colonized the tumor area. The tumor targeting of bifidobacteria was verified.

[0055] like Figure 5As shown, a schematic diagram of Gram staining of paraffin sections of tumor tissue was used to observe the distribution of bifidobacteria in tumor tissue. The blue area within the red oval represents the bifidobacteria in the tumor tissue, indicating that bifidobacteria are targeted and colonized in the tumor area.

[0056] like Figure 6 As shown, after oral administration of bifidobacterium, heart, liver, spleen, lung and kidney tissues were taken for HE staining to observe the safety of bifidobacterium on mice. The results showed that no toxic reactions occurred in the normal tissues and organs of mice, proving that bifidobacterium has good biosafety for mice.

[0057] (III) Detection of NO content in tumor tissue

[0058] After bifidobacteria were orally administered to tumor-bearing mice, tumor tissues were collected and homogenized in an ice bath at a ratio of 1:5 to 10 for tissue mass (unit: g): extract volume (unit: mL). Centrifuge at 10,000 g, 4°C for 15 min, take the supernatant, place on ice, and use a nitric oxide (NO) quantitative determination kit to detect the NO content in the tissue fluid.

[0059] like Figure 7 The figure shows the effect of NO content on tumor mass; low NO concentration (less than 100 nM) promotes proliferation and metastasis; high NO concentration (greater than 400-500 nM) inhibits tumor growth.

[0060] (IV) Analysis of tumor blood flow and vascular changes

[0061] In order to quantitatively and qualitatively evaluate the changes in tumor blood flow in tumor-bearing mice after treatment with bifidobacteria, a three-dimensional ultrasound power Doppler imaging system was used for quantitative analysis. At the predetermined time points after intravenous injection, 3D images of the tumor mass were acquired with a z-slice thickness of 0.05 mm, and the blood flow changes were calculated using a volumetric analysis function. (V) Study on the effect of bifidobacteria on sensitizing colon cancer to radiotherapy

[0062] The tumor-bearing mice were divided into groups for investigation: a. saline group; b. bifidobacterium group; cfX-ray group; dfX-ray + bifidobacterium group. The tumor-bearing mice were then divided into a short-term treatment group and a long-term treatment group; the survival vitality of each group of mice receiving treatment was observed, and the tumor volume was observed and measured. The short-term treatment group was killed on the fifth day after irradiation and the tumor was taken to observe the effect of short-term tumor treatment; the long-term treatment group was observed every other day after irradiation. The observation lasted for 31 days, and the mice were killed after the 31st day and the tumor was taken to observe the effect of long-term tumor treatment. Figure 8 The following are the comparison photos of the tumor sizes of the four groups in the short-term treatment group. It can be seen from the figure that compared with the X-ray group alone, the tumors in the X-ray group + Bifidobacterium group were significantly reduced, which has a better tumor inhibition effect; Fig. 9The figure shows the statistical graph of tumor mass in the four groups in the long-term group. It can be observed from the figure that compared with the X-ray group alone, the tumor in the X-ray group + Bifidobacterium group was significantly reduced, which has a better tumor inhibition effect.

[0063] The degree of cell apoptosis was detected by TUNEL method, and the expression of PCNA in tumor tissue was detected by immunofluorescence. Fig.10 The figure shows the results of TUNEL staining of mouse tumor tissue. The green fluorescence in the figure represents TUNEL fluorescence. The stronger the green fluorescence, the more tumor cells are undergoing apoptosis. It can be observed from the figure that the green fluorescence intensity of the X-ray+Bifidobacterium group is the strongest, and the more tumor cells are undergoing apoptosis.

[0064] like Fig.11 The figure shows the PCNA staining results of mouse tumor tissue. The green fluorescence in the figure represents PCNA fluorescence. The stronger the green fluorescence, the more tumor cells proliferate. It can be observed from the figure that the green fluorescence intensity of the X-ray+Bifidobacterium group is the weakest, and the tumor cells proliferate less.

[0065] The hypoxic microenvironment of tumor tissue is one of the important factors causing radiotherapy resistance. In the hypoxic area, the cell hypoxia-inducible factor HIF-1α is highly expressed. Improving the hypoxic microenvironment of the tumor can reduce the expression of HIF-1α, thereby reversing radiotherapy resistance and sensitizing radiotherapy. Therefore, we performed HIF-1α immunofluorescence observation on the tumor tissue of mice after treatment. The results are as follows Fig.12 As shown, green fluorescence represents the expression level of HIF-1α. From the figure, it can be observed that in the group of bifidobacteria gavage followed by radiotherapy, HIF-1α was highly expressed in the tumor tissue before treatment; after radiotherapy, the expression ability of the hypoxia-inducible factor HIF-1α in the tumor tissue was reduced.

[0066] The above results all indicate that after Bifidobacterium was gavaged into tumor-bearing mice, Bifidobacterium could be targeted and colonized in tumor tissues, but not distributed in other normal tissues or organs; after gavage and radiotherapy, tumor growth was well inhibited and the expression of HIF-1α in tumor tissues was greatly reduced. It can be considered that Bifidobacterium can sensitize colon cancer radiotherapy.

[0067] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. Application of Bifidobacterium in radiotherapy of colon cancer.

2. The use of bifidobacteria in radiotherapy of colon cancer according to claim 1, characterized in that: Bifidobacterium is specifically colonized in the hypoxic area of ​​tumor tissue for proliferation. The bifidobacterium induces macrophages in the area to produce high concentrations of NO to dilate blood vessels and increase blood perfusion, thereby increasing local oxygen to relieve tumor hypoxia, thereby reversing hypoxia-induced radiotherapy tolerance and achieving the purpose of sensitizing radiotherapy.

3. The use of bifidobacteria in radiotherapy of colon cancer according to claim 1 or 2, characterized in that: After entering the blood, the bifidobacteria can specifically colonize and proliferate in the hypoxic area, showing targeting to solid tumors.

4. The use of bifidobacteria in radiotherapy of colon cancer according to claim 1 or 2, characterized in that: Before treatment, HIF-1α is highly expressed in the tumor tissue; after radiotherapy, the expression ability of the hypoxia-inducible factor HIF-1α in the tumor tissue is reduced.

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