5 apos; application of-methylthioadenosine in preparation of radiotherapy sensitizing drug

Abstract

The invention discloses application of 5 '-methylthioadenosine in preparation of a radiotherapy sensitizing drug, and belongs to the technical field of radiotherapy drugs. The invention provides an application of 5 '-methylthioadenosine in preparation of a prostatic cancer radiotherapy sensitization drug. Specifically, by adding 5 '-methylthioadenosine into prostatic cancer radiotherapy-resistant cells, it is found that the 5'-methylthioadenosine can inhibit growth of the prostatic cancer radiotherapy-resistant cells, induce apoptosis, retard the cell cycle in a radiotherapy-sensitive G2 / M period and improve the radiosensitivity of prostatic cancer cells, so that a new prostatic cancer radiotherapy sensitizer can be provided. Therefore, the radiotherapy effect is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of radiotherapy drugs, and particularly relates to the application of 5'-methylthioadenosine in the preparation of radiotherapy sensitizing drugs. Background Art

[0002] Prostate cancer (PCa) is one of the most common malignant tumors in the male urogenital system. According to the statistics of the World Health Organization (WHO), the incidence of prostate cancer ranks second among male malignant tumors globally, and the mortality rate ranks fifth. Although early-stage localized prostate cancer can achieve a relatively high cure rate through radical surgery or radiotherapy, about 20%-30% of patients will still experience biochemical recurrence or distant metastasis after treatment, and the treatment effect of metastatic prostate cancer or high-risk locally advanced prostate cancer is more limited. As one of the core treatment methods for prostate cancer, the main mechanism of radiotherapy is to induce DNA damage and apoptosis of tumor cells through ionizing radiation, but its efficacy is limited by the complex microenvironmental characteristics in tumor tissues, such as the activation of molecular mechanisms related to radiotherapy resistance and tumor heterogeneity. In addition, since the prostate is adjacent to normal organs such as the bladder and rectum, increasing the radiotherapy dose may increase the risk of complications such as urinary incontinence and rectal bleeding, limiting the further optimization of clinical efficacy.

[0003] In recent years, the research and development of radiotherapy sensitizers has become an important strategy to improve the radiotherapy effect of prostate cancer. Radiotherapy sensitizers selectively enhance the killing effect of radiation on cancer cells by targeting specific molecular pathways of tumor cells or improving the tumor microenvironment, while reducing damage to normal tissues. Its mechanisms of action include: (1) regulating the cell cycle distribution to cause tumor cells to arrest in the radiation-sensitive G2 / M phase; (2) inhibiting the DNA damage repair pathway (such as targeting key proteins such as PARP, ATM / ATR, etc.); (3) improving the tumor hypoxic microenvironment by inhibiting hypoxia-inducible factor (HIF-1α) or delivering oxygen carriers to enhance the free radical effect generated by radiation; (4) combining with immunotherapy to activate the anti-tumor immune response by releasing tumor antigens. At present, various sensitizers have shown potential in preclinical studies, but their translational applications still face challenges, including insufficient tumor targeting, systemic toxicity, and the molecular mechanisms of radiotherapy resistance have not been fully elucidated.

[0004] 5'-Methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (SAM) during polyamine synthesis. There has been no relevant report on the radiotherapy resistance effect of 5'-methylthioadenosine in prostate cancer. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the application of 5'-methylthioadenosine in the preparation of radiotherapy sensitizing drugs, which solves the problems in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions: The application of 5'-methylthioadenosine in the preparation of radiotherapy sensitizing drugs for prostate cancer.

[0007] Further, the radiotherapy sensitization for prostate cancer includes: inhibiting the proliferation of radiotherapy-resistant prostate cancer cells.

[0008] Further, the radiotherapy sensitization for prostate cancer includes: arresting the cell cycle of radiotherapy-resistant prostate cancer cells at the G2 / M phase.

[0009] Further, the radiotherapy-resistant prostate cancer cells are cells that survive after multiple irradiations of prostate cancer cells with electron rays and can be passaged; The irradiation process is as follows: prostate cancer cells are cultured in vitro. When the cell density grows to 80%, they are irradiated with an electron beam dose of 2GY, and then placed in a constant temperature incubator for continued culture; after the surviving cells grow confluently, they are digested and passaged; multiple irradiations, cultures, and passages are continued; the irradiation doses are respectively: 2GY for 2 times, 4GY for 2 times, 6GY for 2 times, 8GY for 2 times, 10GY for 2 times, with a total dose of 60GY. After stopping irradiation, continue to culture for 2 - 3 weeks.

[0010] A radiotherapy sensitizing drug for prostate cancer, which includes the only active ingredient: 5'-methylthioadenosine.

[0011] A nano drug delivery system, which includes a nanoparticle carrier, and 5'-methylthioadenosine is encapsulated in the nanoparticle carrier.

[0012] Further, the nanoparticle carrier is a liposome or a polymer-based nanoparticle.

[0013] Further, the polymer-based nanoparticle is: PLGA, PCL or chitosan.

[0014] The application of 5'-methylthioadenosine in the preparation of drugs for the treatment of prostate cancer.

[0015] Further, the treatment of prostate cancer includes: inhibiting the proliferation of radiotherapy-resistant prostate cancer cells.

[0016] The beneficial effects of the present invention: By adding 5'-methylthioadenosine into prostate cancer radioresistant cells, the present invention discovers that it can inhibit the growth of prostate cancer radioresistant cells, induce apoptosis, arrest the cell cycle at the G2 / M phase sensitive to radiotherapy, and improve the radiosensitivity of prostate cancer cells, thereby being able to provide a radiosensitizer for prostate cancer to increase the radiotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 is the molecular structural formula of 5'-methylthioadenosine of the present invention; Figure 2 is the analysis diagram of constructing prostate cancer radioresistant cells by the method of gradually increasing the radiotherapy dose in the present invention; Figure 3 is the result diagram of non-targeted metabolomics determination of prostate cancer parental cell DU145 and radioresistant cell DU145R in the present invention; Figure 4 is the result diagram of detecting the proliferation level of DU145R cells treated with different MTA for 1 - 3 days by the CCK-8 method in the present invention; Figure 5 is the analysis diagram of detecting the inhibitory effect of MTA on the proliferation of DU145R cells by the colony formation assay in the present invention; Figure 6 is the result diagram of detecting the inhibitory effect of MTA combined with radiotherapy on the proliferation of DU145R cells by the CCK-8 method and the colony formation assay in the present invention; Figure 7 is the result diagram of detecting the inhibitory effect of MTA combined with radiotherapy on the proliferation of DU145R cells by the EdU assay in the present invention; Figure 8 is the analysis diagram of MTA combined with radiotherapy increasing apoptosis of radioresistant prostate DU145R cells in the present invention; Figure 9 is the result diagram of MTA combined with radiotherapy increasing the cell cycle arrest at the G2 / M phase of DU145R cells in the present invention; Figure 10 is the analysis diagram of MTA enhancing the radiosensitivity of DU145R cell xenografts in vivo in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0020] Example 1 The prostate cancer cell line (DU145 cells) used in this example was purchased from Wuhan Punosai Life Science Co., Ltd. The DU145R cell line is a radiotherapy-resistant cell line constructed in the present invention. The construction method includes: Cultivate DU145 cells in vitro. When the cell density grows to 80%, irradiate them with an electron beam of 2GY dose, and then place the cells in a constant temperature incubator for continued cultivation. Observe under an inverted microscope that some cells die, and remove the dead cells in the supernatant. Digest and passage the surviving cells when they grow confluent. Continue to irradiate, cultivate, and passage according to the above method. The irradiation doses are 2GY irradiated 2 times, 4GY irradiated 2 times, 6GY irradiated 2 times, 8GY irradiated 2 times, and 10GY irradiated 2 times, with a total dose of 60GY. After stopping irradiation, continue to cultivate for 2 - 3 weeks.

[0021] In this example, the radiotherapy resistance phenotype of DU145R cells was also carried out. The experimental method is as follows: Seed 1000 DU145 and DU145R cells into 6-well plates respectively, and then irradiate them with different radiation doses of 0Gy, 2Gy, 4Gy, 6Gy, and 8Gy respectively. After 10 days, colony formation occurs. Count the number of colony formations and perform a single-hit multi-target model analysis to evaluate the radiotherapy resistance of DU145R cells. The results are as Figure 2 shown, Figure 2 in A of which shows the effects of different radiotherapy doses on the colony formation ability of prostate cancer parental cells DU145 and prostate cancer radiotherapy-resistant cells DU145R, Figure 2 and B in which shows that the single-hit multi-target model shows that DU145R has obvious radiotherapy resistance.

[0022] Example 2 In this example, non-targeted metabolomics determination was performed on prostate cancer parental cells DU145 and radiotherapy-resistant cells DU145R. As Figure 3 shown, Figure 3 it shows in which that S-adenosylmethionine (the structural diagram is as Figure 1 shown) is significantly reduced in prostate cancer radiotherapy-resistant cells DU145R; Among them, the process of cell non-targeted metabolomics determination is as follows: DU145 and DU145R cells are cultured to the logarithmic growth phase (70%-80% confluence), with 6 biological replicates in each group. After washing the cells with PBS, they are digested with trypsin and the cells are collected. After centrifugation, the supernatant is discarded. The cells are quickly frozen with liquid nitrogen and stored in an -80°C refrigerator until extraction. The metabolites are extracted using the methanol-water-chloroform method, vacuum centrifugally concentrated or dried with nitrogen, and stored at -80°C. An equal amount of each experimental group is mixed as a quality control sample (QC) for monitoring the instrument stability. Liquid chromatography-mass spectrometry (LC-MS) is used for metabolite detection.

[0023] Example 3 In this example, the CCK-8 method was used to detect the effect of different concentrations of MTA on the proliferation level of DU145R cells.

[0024] DU145R cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5000 cells / well. After 24 h, the cells were treated with different concentrations of MTA. At 24 h, 48 h, and 72 h, 10 μL of CCK-8 solution was added to the cell culture medium, and then the cells were placed back in the incubator and incubated for another 2 h. The wavelength of the microplate reader was set to 450 nm to measure the absorbance value.

[0025] The measurement results are as Figure 4 shown, Figure 4 It can be seen that: regardless of whether it is 24 h, 48 h, or 72 h, the absorbance of the cells at 450 nm gradually decreases as the MTA concentration increases; in addition, at the same MTA concentration, the absorbance of the cells at 72 h is lower than that at 48 h, and the absorbance at 48 h is lower than that at 24 h. The above results show that MTA inhibits the proliferation of DU145R cells in a time- and drug concentration-dependent manner.

[0026] Example 4 In this example, a colony formation assay was used to detect the inhibitory effect of MTA on the proliferation of DU145R cells.

[0027] DU145R cells in the logarithmic growth phase were made into a single-cell suspension, and 1000 cells were seeded in 6-well plates. After the cells adhered overnight, MTA at concentrations of 0, 12.5 μM, 25 μM, and 50 μM was added. After treatment, the cells were placed in the incubator and cultured for 10 - 14 days until visible cell colonies appeared in the control group. After drawing grid lines on the back of the 6-well plate, the number of colonies with ≥50 cells was counted under a microscope. After fixing with 4% paraformaldehyde for 10 min, the plate was washed 2 - 3 times with PBS, then stained with crystal violet staining solution for 10 min, the plate was washed with deionized water, air-dried, and photographed.

[0028] The experimental results are as Figure 5As shown, Figure 5 A in Figure 5 shows the effect of different concentrations of MTA on the colony formation ability of DU145R cells, Figure 5 and B in Figure 5 is the quantitative analysis of Figure 5 A in Figure 5 . It can be seen that as the concentration of MTA gradually increases, the number of cell colonies gradually decreases. This result indicates that MTA inhibits the colony formation of DU145R cells in a drug concentration-dependent manner.

[0029] Example 5 In this example, the CCK-8 method and colony formation assay were used to detect the inhibitory effect of MTA combined with radiotherapy on the proliferation of DU145R cells. 1) CCK-8 method: DU145R cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5000 cells / well. After 24 h, the cells were treated in different ways (25 µM MTA or 6 Gy radiotherapy, or the combination of both). After the cells were cultured for another 72 h, 10 µL of CCK-8 solution was added to the cell culture medium, and then the cells were placed in an incubator and incubated for another 2 h. The wavelength of the microplate reader was set to 450 nm, and the absorbance value was measured. The inhibitory effect of MTA (25 µM) combined with radiotherapy (6 Gy) on the proliferation level of DU145R cells was detected by the CCK-8 method, and the results are shown in Figure 6 A in Figure 6 . It can be seen that the combination of MTA and radiotherapy significantly inhibits the proliferation of DU145R cells.

[0030] 2) Colony formation assay: DU145R cells in the logarithmic growth phase were made into a single cell suspension, and 1000 cells were seeded in 6-well plates. After the cells adhered overnight, they were treated in different ways (25 µM MTA or 6 Gy radiotherapy, or the combination of both). After treatment, the cells were placed in an incubator and cultured for 10 - 14 days until visible cell colonies appeared in the control group. After drawing grid lines on the back of the 6-well plate, the number of colonies with ≥50 cells was counted under a microscope. After fixing with 4% paraformaldehyde for 10 min, the wells were washed 2 - 3 times with PBS, then stained with crystal violet staining solution for 10 min, the well plate was washed with deionized water, dried and photographed. The experimental results of the cell colony formation assay are shown in Figure 6 B in Figure 6 , Figure 6 and C in Figure 6 is the quantitative analysis of Figure 6 B in Figure 6 . It can be seen that the combination of MTA and radiotherapy can significantly reduce the formation of cell colonies.

[0031] Example 6 In this example, an ethynyl-2'-deoxyuridine (EdU-594) cell proliferation detection kit was used to detect the inhibition of DU145R cell proliferation by MTA combined with radiotherapy. DU145R cells in the logarithmic growth phase were seeded in 24-well plates at a density of 2×10 4 cells / well. After 24 h, the cells were treated in different ways (25 μM MTA or 6 Gy radiotherapy, or the combination of both). After the cells were cultured for another 48 h, EdU working solution was added. The cells were incubated in the incubator for another 2 h, and then the medium was removed and the cells were fixed with 4% paraformaldehyde for 15 min. The fixing solution was removed, and the cells were washed with washing solution. Subsequently, the cells were permeabilized with cell permeabilization solution for 10 min. The cell permeabilization solution was removed, and the cells were washed with cell washing solution. Click reaction solution was added to the wells and incubated for 15 min. After washing 3 times with cell washing solution, the nuclei were stained with a blue fluorescent dye Hoechst 33342 solution that can penetrate the cell membrane. It emits strong blue fluorescence after embedding into double-stranded DNA. Subsequently, fluorescence detection was performed using a fluorescence microscope.

[0032] The results of detecting the inhibition of DU145R cell proliferation by MTA combined with radiotherapy using the EdU experiment are shown in Figure 7 A as shown in Figure 7 ; B in Figure 6 is the quantitative analysis of A in

[0033] Example 7 In this example, flow cytometry was used to detect the increase in DU145R cell apoptosis by MTA combined with radiotherapy.

[0034] DU145R cells in the logarithmic growth phase were seeded in 96-well plates at a density of 25×10 4 cells / well. After the cells adhered overnight, they were treated in different ways (25 μM MTA or 6 Gy radiotherapy, or the combination of both). After culturing for another 48 h, the supernatant cells were collected, and the cells were digested with trypsin. The cells (including the cells in the supernatant) were collected and resuspended and washed with PBS. After centrifugation, the PBS was removed, and the cell pellet was resuspended with binding buffer solution. A fluorescent dye propidium iodide that can embed into double-stranded DNA and release red fluorescence and a green fluorescent dye Annexin V-fluorescein isothiocyanate (Annexin V-FITC) were added, and the cells were incubated in the dark for 15 min. Cell apoptosis was detected by flow cytometry.

[0035] The experimental results are shown in Figure 8 as Figure 8 A in Figure 8 shows the effect of MTA combined with radiotherapy on the apoptosis level of DU145R cells detected by flow cytometry, and B in Figure 8Quantitative analysis of A in; it can be seen that the combined use of MTA and radiotherapy can significantly increase the apoptosis level of DU145R cells.

[0036] Example 8 In this example, flow cytometry was used to detect the increase in G2 / M phase cell cycle arrest of DU145R cells by MTA. The previous operation steps were the same as those in the apoptosis experiment (Example 7). After collecting the cells, pre-cooled 70% ethanol was added and fixed at 4°C for more than 4 h. Centrifuge at 1500 rpm for 5 min, wash once with PBS, add propidium iodide (PI, 50 μg / mL) and ribonuclease A (100 μg / mL), and incubate at 4°C in the dark for 30 min. The cell cycle was detected by flow cytometry, and the results were analyzed using FlowJo software.

[0037] The experimental results are as Figure 9 shown, Figure 9 A in shows the effect of flow cytometry in detecting the cell cycle arrest of DU145R cells by the combination of MTA and radiotherapy, Figure 9 B and C in are the quantitative analysis of Figure 9 A in, and it can be seen that the combined use of MTA and radiotherapy can significantly arrest the cell cycle in the G2 / M phase sensitive to radiotherapy.

[0038] Example 9 In this example, the radiosensitivity of MTA in enhancing the in vivo xenograft tumors of DU145R cells was verified in mouse in vivo experiments; Among them, 5-6-week-old Balb / c male nude mice with an average body weight of 20 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and raised in a specific pathogen-free (SPF) facility. 1×10 6 cells / 100 μL of DU145R cells were injected into the groin of each mouse, and xenograft tumors were formed in about 7 days. When the average tumor volume reached 50 mm 3 , the mice were randomly divided into four groups (5 mice in each group). 0.1% DMSO was used as the blank control, MTA (200 mg / kg, once a day, intraperitoneal injection), local radiotherapy or radiotherapy combined with MTA. The length and width of the tumor were measured every 5 days, and the tumor volume was calculated according to the following formula: length × width × width × 0.52. During radiotherapy, a lead shield was used to protect the rest of the mouse. The animal experiment was approved by the Ethics Review Committee of Soochow University.

[0039] As Figure 10 shown in A, the treatment mode of subcutaneous xenograft tumors of DU145R cells in nude mice was as follows: A total of 1×10 6 cells / 100 μL of DU145R cells were injected into the groin of nude mice to form xenograft tumors (n = 20). When the average volume of subcutaneous xenograft tumors reached 50 mm 3When the nude mice were randomly divided into four groups of 5 mice each, the groups were as follows: (1) negative control group (0.5% dimethyl sulfoxide DMSO); (2) MTA group; (3) local radiotherapy group; (4) MTA and local radiotherapy combined group. When the average tumor volume reached 50 mm 3 treatment was started. The negative control group and the MTA drug were injected into the nude mice by intraperitoneal injection. The nude mice were treated with MTA at a dose of 200 mg / kg per day from day 7 to day 20 (a total of 14 days) after tumor inoculation. On the 4th and 10th days of drug administration, the tumor sites of the nude mice were irradiated with a dose of 8 Gy. On the 25th day, the nude mice were sacrificed by cervical dislocation, and the tumor samples of the nude mice were collected for photographing and weighing.

[0040] After sacrificing the nude mice by cervical dislocation, the tumors of the mice were dissected and photographed as shown in Figure 10 B in; Figure 10 C in is a statistical chart of the tumor mass of nude mice; Figure 10 D in is the tumor volume growth curve of nude mice. It can be seen that although both MTA and ionizing radiation can inhibit the growth of subcutaneous transplanted tumors in nude mice, the effect is not significant, while the MTA combined with ionizing radiation group can significantly inhibit the growth of transplanted tumors.

[0041] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. Application of 5'-methylthioadenosine in the preparation of radiosensitizing drugs for prostate cancer radiotherapy.

2. Use of 5'-methylthioadenosine according to claim 1 in the preparation of a radiosensitizing drug for prostate cancer radiotherapy, characterized in that, The radiosensitization of prostate cancer radiotherapy includes: inhibiting the proliferation of prostate cancer radioresistant cells.

3. Use of 5'-methylthioadenosine according to claim 2 in the preparation of a radiosensitizing drug for prostate cancer radiotherapy, characterized in that, The radiosensitization of prostate cancer radiotherapy includes: arresting the cell cycle of prostate cancer radioresistant cells at the G2 / M phase.

4. Use of 5'-methylthioadenosine according to claim 2 or 3 in the preparation of a radiosensitizing drug for prostate cancer radiotherapy, characterized in that, The prostate cancer radioresistant cells are cells that survive and can be passaged after multiple irradiations of prostate cancer cells with electron rays; The irradiation process is as follows: prostate cancer cells are cultured in vitro. When the cell density reaches 80% growth, they are irradiated with an electron beam dose of 2 Gy, and then placed in a constant temperature incubator for continued culture; when the surviving cells grow confluently, they are digested and passaged; multiple irradiations, cultures, and passages are continued; the irradiation doses are respectively: 2 Gy for 2 times, 4 Gy for 2 times, 6 Gy for 2 times, 8 Gy for 2 times, 10 Gy for 2 times, with a total dose of 60 Gy. After stopping irradiation, the cells are cultured for 2 - 3 weeks.

5. A radiosensitizing drug for prostate cancer radiotherapy, characterized in that, It includes the only active ingredient: 5'-methylthioadenosine.

6. A nano drug delivery system, comprising a nanoparticle carrier, characterized in that, The nanoparticle carrier encapsulates 5'-methylthioadenosine.

7. A nanomedicine delivery system according to claim 6, wherein The nanoparticle carrier is a liposome or a polymer-based nanoparticle.

8. A nanomedicine delivery system according to claim 7, characterized in that, The polymer-based nanoparticle is: PLGA, PCL, or chitosan.

9. Application of 5'-methylthioadenosine in the preparation of drugs for the treatment of prostate cancer.

10. Use of 5'-methylthioadenosine according to claim 9 in the preparation of a therapeutic drug for prostate cancer, characterized in that, The treatment of prostate cancer includes: inhibiting the proliferation of prostate cancer radioresistant cells.

Images