A compound, composition and application thereof for reducing drug resistance of bladder cancer cells

Abstract

The present invention provides a compound capable of reducing the drug resistance of bladder cancer cells. The structure of the compound is: #imgabs0# where R1 is a saturated alkyl group and R2 is an aromatic ring group containing at least one substituent. PARP inhibitor drugs such as olaparib are mainly applicable to cancer cells with BRCA1 defects and are not applicable to cancer cells without BRCA1 defects. Therefore, how to expand the clinical treatment indications of olaparib is a research hotspot worldwide. The present invention uses a compound with the structure of metformin to inhibit the drug resistance of bladder cancer cells, thereby increasing the sensitivity of bladder cancer cells to therapeutic drugs such as olaparib, and thus promoting the effective treatment of bladder cancer cells.

Description

Technical Field

[0001] The present invention relates to a compound, a composition and an application thereof for treating bladder cancer cells, and in particular to a compound, a composition and an application thereof for reducing the drug resistance of bladder cancer cells. Background Art

[0002] Bladder cancer is the tenth most common tumor in the world, with an age-standardized incidence rate of 9.5 / 100,000 for males and 2.4 / 100,000 for females; and an age-standardized mortality rate of 3.3 / 100,000 for males and 0.86 / 100,000 for females. Current treatments for bladder cancer include: surgical treatment, laser and dynamic therapy, interventional therapy, radiotherapy, etc. However, bladder tumors have a high recurrence rate after surgical treatment, and the pathological grade and clinical stage will worsen after tumor recurrence. Therefore, in addition to other treatments before bladder cancer surgery, bladder cavity chemotherapy is also required immediately after surgery to prevent or reduce the recurrence rate of bladder cancer.

[0003] At present, bladder cancer is mainly treated with intravesical chemotherapy: after the tumor is removed, chemotherapy drugs are directly injected into the bladder through a urinary catheter in order to kill unresected microlesions. This method has fewer side effects, but due to the high recurrence rate of bladder cancer, regular cystoscopy is required.

[0004] Poly ADP ribose polymerase (PARP) is an enzyme involved in the repair of single-strand breaks in DNA. The existing drugs used to treat bladder cancer cells are mainly PARP inhibitors. When PARP is inhibited, it can block the repair of single-strand breaks in DNA in bladder cancer cells. For tumor cells with BRCA1 / 2 (HR repair key protein) defects, the DNA damage induced by PARP inhibitors cannot repair DNA double-strand breaks (DSBs) through the HR repair pathway, resulting in the inability of cells to synthesize DNA normally during DNA chain replication, causing "synthetic lethality" and causing bladder cancer cells to die. However, for tumor cells without BRCA1 / 2 (HR repair key protein) defects, after the double-strand breaks in the DNA of bladder cancer cells, they can be repaired through the homologous recombination (HR) repair pathway to keep the cells alive, which reduces the therapeutic effect.

[0005] Therefore, the current treatment of bladder cancer cells is mainly suitable for cancer cells with BRCA1 / 2 mutations, but not for cancer cells without BRCA1 / 2 mutations (such as BRCA wild-type bladder cancer). Take Olaparib as an example. The drug became the first PARP inhibitor approved by the US FDA in 2014. It has been used to treat patients with recurrent BRCA1 / 2 mutation ovarian cancer with good results. In 2018, Olaparib was first launched in China, becoming the first targeted drug for the treatment of ovarian cancer in China. However, studies have shown that wild-type cancer cells without BRCA1 mutations have an IC50 of more than 10 times higher for Olaparib than ovarian cancer cells with BRCA1 mutations. Therefore, Olaparib is mainly suitable for BRCA1-defective cells, and how to expand the clinical treatment indications of Olaparib is a research hotspot worldwide. Summary of the invention

[0006] The present invention provides a compound, a composition and an application thereof which can be used to reduce the drug resistance of bladder cancer cells, so as to improve the sensitivity of bladder cancer to anticancer drugs.

[0007] The present invention provides a compound that can reduce the drug resistance of bladder cancer cells, and the compound structure is:

[0008] Wherein R1 is a saturated alkyl group, and R2 is an aromatic ring group containing at least one substituent.

[0009] Furthermore, the R2 is a benzene ring group containing at least one substituent.

[0010] Furthermore, the R2 is a para-substituted benzene ring group.

[0011] Furthermore, the substituent of R2 is an electron withdrawing group.

[0012] Furthermore, the substituent of R2 is trifluoromethoxy.

[0013] Furthermore, the R1 is a C2-C6 saturated alkyl group.

[0014] The invention also discloses a composition for treating bladder cancer. The composition comprises the above compound and at least one drug for treating bladder cancer.

[0015] Furthermore, the composition also includes Olaparib.

[0016] The invention also discloses an application of the composition, wherein the composition is used for treating bladder cancer.

[0017] Furthermore, the bladder cancer is BRCA gene mutation type bladder cancer.

[0018] Compared with the prior art, the present invention uses a compound with a metformin structure to inhibit the drug resistance of bladder cancer cells, thereby increasing the sensitivity of bladder cancer cells to therapeutic drugs such as olaparib, thereby promoting effective treatment of bladder cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The inhibitory effect of 4C on RT4 and T24 bladder cancer cell lines at different concentrations;

[0020] Figure 2 MTT was used to detect the combined effect of 4C and olaparib in RT4 bladder cancer cells;

[0021] Figure 3 MTT was used to detect the combined effect of 4C and olaparib in T24 bladder cancer cells;

[0022] Figure 4 This is the cell staining picture of MTT experiment for 4C and olaparib alone and in combination;

[0023] Figure 5 This is the protein expression chart of Cyclin D1 in the single use and combination use of 4C and Olaparib. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0025] The present invention discloses a compound that can reduce the drug resistance of bladder cancer cells. The compound has the structure:

[0026] R1 is a saturated alkyl group, and R2 is an aromatic ring group containing at least one substituent.

[0027] Wherein, R2 can be selected as a monosubstituted or polysubstituted aromatic ring group, and the aromatic ring can be selected as a benzene ring or a heterocyclic ring.

[0028] Optionally, R2 is a benzene ring group containing at least one substituent.

[0029] Particularly, the R2 is a para-substituted benzene ring group.

[0030] Particularly, the substituent of R2 is an electron withdrawing group.

[0031] The substituent of R2 can be selected as a substituent containing F element.

[0032] Particularly, the substituent of R2 is trifluoromethoxy.

[0033] Optionally, R1 is a C2-C5 saturated alkyl group.

[0034] Wherein, in the embodiment of the present invention, R1 is a butane group, and the specific chemical formula is

[0035] The embodiment of the present invention also discloses a composition for treating bladder cancer. The composition comprises the above compound and at least one drug that can be used for treating bladder cancer.

[0036] Optionally, the composition comprises olaparib.

[0037] Among them, those skilled in the art may use other PARP inhibitors besides olaparib based on actual needs, or may use a mixed drug of multiple PARP inhibitors including olaparib.

[0038] The embodiment of the present invention also discloses an application of the above composition, wherein the composition is used for treating bladder cancer.

[0039] Optionally, the bladder cancer is BRCA gene mutation type bladder cancer.

[0040] Among them, bladder cancer is specifically BRCA wild-type bladder cancer, in which there is no mutation in the BRCA1 / 2 gene locus or the mutation does not affect the normal expression of the BRCA1 / 2 gene locus.

[0041] To further illustrate the specific effects of the embodiments of the present invention, (hereinafter referred to as 4C), (hereinafter referred to as 6C) as an example, a comparative experiment is carried out as follows.

[0042] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The Olaparib was purchased from Selleck.

[0043] The compounds 4C and 6C of the present invention were tested on bladder cancer cells. The results are as follows: Figure 1 As shown, 4C has a significant inhibitory effect on RT4 and T24 cancer cells, and the IC50 of 4C for RT4 is 7.8398 μM, and the IC50 for T24 is 11.2469 μM.

[0044] Example 1: In vitro antitumor effects of 4C, 6C and olaparib on BRCA wild-type bladder cancer cells

[0045] The MTT method was used on bladder cancer T24 and RT4 cell lines, and 4C and 6C were combined with olaparib to compare the in vitro anti-tumor effects of single use and combination. CompuSyn software was used to select 4 concentrations of 4C, 4 concentrations of 6C, and 4 concentrations of olaparib, and the CI values ​​were calculated.

[0046] The specific operations are as follows:

[0047] S101. Add 10% fetal bovine serum solution to 5A medium, and seed T24 and RT4 cells in a 96-well plate at a density of 2000 cells / well / 180 μL;

[0048] S102. Cultivate at 37°C and 5% CO2 for 24 h;

[0049] S103. After diluting the drugs of different concentrations with fresh culture medium, the test drug solution was added to the culture plate with cells at an addition volume of 20 μL per well; wherein, a single-use group containing only olaparib, a single-use group of 4C, a single-use group of 6C, a combination group containing olaparib and 4C, and a combination group containing olaparib and 6C were used. The combination group had two major groups, and the specific concentrations of 4C and 6C in the two major groups were 6 μM and 8 μM, respectively. The concentration of olaparib in the solution was adjusted, and at least three replicate wells were set for each concentration to reduce experimental errors, so as to test the inhibitory effects of olaparib on cells in the single-use group and the combination group at different concentrations; a control group (cells were added but no drugs were added) and a blank group (no cells or drugs were added, only culture medium was included);

[0050] S104. After culturing for 72 h at 37°C and 5% CO2, MTT reagent (2 mg / mL) was added at a volume of 50 μL / well and incubated for another 5 h;

[0051] S105. Remove the culture medium, add 150 μL dimethyl sulfoxide, shake and mix for 15 min, and detect the OD value of each well with an ELISA reader at a wavelength of 490 nm. Calculate the cell survival rate based on the OD value readings, and use Grapad Prism 6 to calculate the IC 50 ; CI values ​​were calculated using CompuSyn software.

[0052] The results are as follows Figure 2 , 3 As shown, 4C and olaparib have a strong synergistic inhibitory effect in BRCA wild-type bladder cancer cells, with a CI value of <0.5, indicating a strong synergistic effect.

[0053] Example 2: Comparison of the anti-tumor effects of 4C and 6C alone and in combination with olaparib in BRCA wild-type bladder cancer cells RT4 and T24 using a clone formation assay

[0054] S201. 1500 T24 and RT4 cells were inoculated into 24-well plates respectively, mixed with 0.5 mL of culture medium, and 3 replicate wells were set up for each group;

[0055] S202. After culturing for 24 hours, after the cells adhere to the wall, dilute the drug with fresh culture medium and add 500 μL per well to the culture plate with cells added; the concentration of the single-use group containing only 4C is 6 μM, the concentration of the single-use group containing only 6C is 6 μM, the concentration of the single-use group containing only olaparib is 10 μM, the combination group is composed of 250 μL of 4C with a concentration of 6 μM + 250 μL of olaparib with a concentration of 10 μM, and the combination group is composed of 250 μL of 6C with a concentration of 6 μM + 250 μL of olaparib with a concentration of 10 μM. The drug administration time is set to 7 days;

[0056] S203. Cultivate continuously at 37°C, 5% CO2 for 8 days until the clone grows to a diameter of 1-2 mm, then terminate the culture;

[0057] S204. Discard the supernatant, wash twice with PBS, add 2 mL of 10% paraformaldehyde to each well, and fix for 15 min;

[0058] S205. Remove the fixative and stain with 0.5 mL of crystal violet stain for 15 min;

[0059] S206. Wash off the staining solution with clean water, dry at room temperature, take a photo and use an enzyme-labeled instrument to detect the absorbance at a wavelength of 550nm.

[0060] The results are as follows Figure 4 As shown, compared with 4C or olaparib alone, the combination of the two drugs further inhibited tumor cell proliferation.

[0061] Example 3: Western blot detection of p-ATM and Cyclin D1 expression levels

[0062] The main function of the Cyclin D1 unit is to promote cell proliferation. Cyclin D1 can promote the cell cycle from the G1 phase to the S phase by binding to and activating the cyclin-dependent kinase CDK4, which is unique to the G1 phase. In further mechanism studies, it is shown that Cyclin D1 interacts with damage repair-related proteins such as Rad51 and BRCA2 to form a complex that participates in the DNA repair process, thereby ensuring the survival of tumor cells. Therefore, by inhibiting the content of Cyclin D1, cell cycle arrest and DNA damage repair can be caused, thereby enhancing the sensitivity of Olaparib to BRCA wild-type bladder cancer.

[0063] The specific steps of the test are as follows:

[0064] S301. Four groups were set up (4C alone group, olaparib alone group, 4C + olaparib combination group, and a drug-free group was set up as a control group), the concentration of 4C alone group at the cellular level was 12 μM, the concentration of olaparib alone group was 20 μM, and the 4C + olaparib combination group was prepared by mixing the same volume of 12 μM 4C solution and 20 μM olaparib solution;

[0065] S302. After the drug treatment for an appropriate period of time (adding two drugs at the same time), the cells were collected to extract proteins, and the expression levels of related proteins were detected by western blot.

[0066] The results are as follows Figure 5 As shown, although Olaparib can cause DNA damage, cells will also repair DNA by upregulating DNA damage repair-related proteins p-ATM and Cyclin D1. Under the condition of 4C combination, the upregulation of DNA damage repair-related proteins p-ATM and Cyclin D1 is inhibited, thereby sensitizing Olaparib.

[0067] In summary, Example 4C of the present invention inhibits cyclin D1, thereby inhibiting the repair of DNA damage in bladder cancer cells, thereby enhancing the DNA damage effect induced by olaparib. In order to further clarify the mechanism of 4C sensitizing olaparib, the expression changes of the DNA damage-related protein γH2AX protein after drug treatment were detected, as shown in FIG. Figure 5 As shown in the figure, the results showed that the expression of γH2AX in the combined treatment group was further upregulated compared with the single treatment group, indicating that the DNA damage induced by olaparib was further enhanced in the presence of 4C. At the same time, the combination of 4C caused a significant downregulation of Cyclin D1, while olaparib caused the upregulation of Cyclin D1. The combination of the two inhibited the upregulation of Cyclin D1 caused by olaparib. This shows that 4C inhibits the expression of DNA damage repair-related protein p-ATM by inhibiting Cyclin D1, thereby enhancing the sensitivity of olaparib.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that after reading the specification of this application, the technicians can still modify or replace the specific implementation mode of the present invention with equivalents, but these modifications or changes do not deviate from the scope of protection of the pending claims of the present application.

Claims

1. Use of a composition in the preparation of a drug for treating bladder cancer, characterized in that: The composition consists of a compound that can reduce the drug resistance of bladder cancer cells and a drug that can be used to treat bladder cancer; The compound capable of reducing the drug resistance of bladder cancer cells has the following structural formula: ； The drug that can be used to treat bladder cancer is Olaparib; The bladder cancer is BRCA wild-type bladder cancer.

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