A method for enhancing the anticancer activity of salinomycin and its application

Abstract

The present invention discloses a method and application for enhancing the anticancer activity of salinomycin. Telomerase, as a reverse transcriptase, can use TERC as a template and the catalytic function of TERT to add the TTAGGG repeat sequence to the end of the telomere, and prevent telomere shortening through the telomere DNA synthesis function, so as to immortalize cancer cells. In addition to the traditional telomere extension function, TERT is also closely related to gene transcription, DNA damage repair, stemness maintenance and ROS regulation in cancer cells. The present invention uses the telomerase inhibitor BIBR1532 to inhibit telomerase activity, thereby improving the anticancer activity of salinomycin. Targeting telomerase is an effective strategy to enhance the anticancer activity of salinomycin with little effect on normal cells.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to a method for enhancing the anticancer activity of salinomycin and its application. Background Art

[0002] Salinomycin is a monocarboxyl polyether antibiotic extracted from the culture medium of Streptomyces albus in the early 1970s. For a long time, salinomycin was only used as an anticoccidial drug for the treatment of livestock diseases. It was not until 2009 that Weinberg's team discovered that salinomycin has anti-cancer function. Subsequent studies have shown that salinomycin can inhibit the activity of a variety of cancer cells. Moreover, unlike traditional chemotherapy drugs such as paclitaxel, doxorubicin, cisplatin, and temozolomide, salinomycin can not only kill cancer cells, but also eliminate tumor stem-like cells and multidrug-resistant cells. In view of these advantages, salinomycin has been highly valued by scientists and is considered to have the potential to be developed as a highly effective anticancer drug. However, high concentrations of salinomycin can cause severe systemic adverse reactions in mammals, thus hindering its application in the treatment of human diseases. Although many methods have been found to increase the anticancer activity of salinomycin and thus reduce the amount of salinomycin used, such as combining salinomycin with compounds targeting histone deacetylase, pyruvate dehydrogenase kinase, or autophagy, these methods themselves can cause damage to normal cells. Therefore, finding more effective methods to enhance the anticancer activity of salinomycin with little effect on normal cells is an urgent problem to be solved. Summary of the invention

[0003] The purpose of the present invention is to provide a method and application for enhancing the anticancer activity of salinomycin in order to solve the above problems.

[0004] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0005] A method and application for enhancing the anti-cancer activity of salinomycin, using a telomerase inhibitor to inhibit telomerase activity and improve the anti-cancer activity of salinomycin;

[0006] The mechanism is: telomerase is a reverse transcriptase, composed of RNA template TERC and protein catalytic subunit TERT. Telomerase can use TERC as a template and the catalytic function of TERT to add the TTAGGG repeat sequence to the end of the telomere. In cancer cells, telomerase can prevent telomere shortening through telomeric DNA synthesis function, thereby immortalizing cancer cells. TERT is also closely related to gene transcription, DNA damage repair, stemness maintenance and ROS regulation in cancer cells. Targeting telomerase is an effective method to enhance the anti-cancer activity of salinomycin with little effect on normal cells.

[0007] Wherein, the telomerase inhibitor is BIBR1532.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The telomerase inhibitor BIBR1532 is used to inhibit telomerase activity, thereby enhancing the anti-cancer activity of salinomycin. Targeting telomerase is an effective strategy to enhance the anti-cancer activity of salinomycin with little effect on normal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the structural formula of BIBR1532 and salinomycin;

[0011] Figure 2 is the effect of simultaneous treatment of BIBR1532 and salinomycin on the viability of MCF-7 cells;

[0012] Figure 3 is the effect of simultaneous treatment of BIBR1532 and salinomycin on the viability of MDA-MB-231 cells;

[0013] Figure 4 is the effect of simultaneous treatment of BIBR1532 and salinomycin on the morphology of microspheres;

[0014] Figure 5 is the effect of simultaneous treatment with BIBR1532 and salinomycin on the number of mammospheres;

[0015] Figure 6 is the effect of simultaneous treatment of BIBR1532 and salinomycin on ROS production;

[0016] Figure 7 is the effect of simultaneous treatment with BIBR1532, salinomycin, and ROS scavenger NAC on the viability of MCF-7 cells;

[0017] Figure 8 is the effect of simultaneous treatment of BIBR1532 and salinomycin on tumor growth;

[0018] Fig. 9 The effect of simultaneous treatment with BIBR1532 and salinomycin on tumor tissue morphology. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings:

[0020] Example 1

[0021] Effects of simultaneous treatment with BIBR1532 and salinomycin on the viability of MCF-7 cells

[0022] MCF-7 cells were cultured at 1×10 4The cells were inoculated into a 96-well culture plate at a density of 100 μL / well. After inoculation, the culture plate was placed in a humidified incubator at 37°C and 5% CO2 for 24 hours. BIBR1532 and salinomycin were added to make the final concentration of BIBR1532 15 μM and the concentrations of salinomycin 1, 2, 4, 8, and 16 μM, respectively. The culture plate was returned to the incubator and continued to be cultured for 72 hours. CCK-8 solution (10 μL / well) was added and cultured in the incubator for 2 hours. Finally, the culture plate was placed in an ELISA reader and the absorbance was measured at 450 nm. The relative cell viability was calculated using the absorbance value. Figure 2 The effect of simultaneous treatment with BIBR1532 and salinomycin on the viability of MCF-7 cells. Figure 2 The results showed that BIBR1532 could enhance the inhibitory effect of different concentrations of salinomycin on MCF-7 cell viability, indicating that BIBR1532 could enhance the inhibitory effect of salinomycin on breast cancer.

[0023] Example 2

[0024] Effects of simultaneous treatment with BIBR1532 and salinomycin on the viability of MDA-MB-231 cells

[0025] MDA-MB-231 cells were cultured at 1×10 4 The cells were inoculated into a 96-well culture plate at a density of 100 μL / well. After inoculation, the culture plate was placed in a humidified incubator at 37°C and 5% CO2 for 24 hours. BIBR1532 and salinomycin were added to make the final concentration of BIBR1532 15 μM and the concentrations of salinomycin 1, 2, 4, 8, and 16 μM, respectively. The culture plate was returned to the incubator and continued to be cultured for 72 hours. CCK-8 solution (10 μL / well) was added and cultured in the incubator for 2 hours. Finally, the culture plate was placed in an ELISA reader and the absorbance was measured at 450 nm. The relative cell viability was calculated using the absorbance value. Figure 3 The effect of simultaneous treatment with BIBR1532 and salinomycin on the viability of MDA-MB-231 cells. Figure 3 The results showed that BIBR1532 could enhance the inhibitory effect of different concentrations of salinomycin on the viability of MDA-MB-231 cells, indicating that BIBR1532 could enhance the inhibitory effect of salinomycin on breast cancer.

[0026] Example 3

[0027] Effects of simultaneous treatment with BIBR1532 and salinomycin on mammosphere formation

[0028] MCF-7 cells were seeded into DMEM / F12 medium at a density of 5000 cells / well, and 20ng / mL basic fibroblast growth factor bFGF, 20ng / mL human recombinant epithelial growth factor EGF, 4μg / mL heparin, 1% streptomycin and penicillin were added, and cultured in a 6-well ultra-low adhesion culture plate. BIBR1532 and salinomycin were added at the same time, so that the concentration of BIBR1532 was 15μM and the concentration of salinomycin was 4μM. After 7 days, the morphology and number of mammospheres were observed under a microscope and photographed. Figure 4 The effect of simultaneous treatment with BIBR1532 and salinomycin on the morphology of mammospheres. Figure 5 The effect of simultaneous treatment with BIBR1532 and salinomycin on the number of mammospheres. Figure 4 and Figure 5 It can be seen that BIBR1532 can enhance the inhibitory effect of salinomycin on the formation of mammospheres, indicating that BIBR1532 can enhance the inhibitory effect of salinomycin on tumor stem-like cells.

[0029] Example 4

[0030] Effects of simultaneous treatment with BIBR1532 and salinomycin on ROS production

[0031] MCF-7 cells were cultured at 1×10 6 The cells were inoculated into 6-well culture plates at a density of 100 μg / well. After inoculation, the culture plates were placed in a humidified incubator at 37°C and 5% CO2 for 24 hours. BIBR1532 and salinomycin were added to make the final concentration of BIBR1532 15 μM and the concentration of salinomycin 8 μM, and the culture plates were returned to the incubator for 72 hours. DCFH-DA (10 μM) was added and incubated for 30 minutes, and the intracellular ROS content was detected by flow cytometry. Figure 6 It is the level of ROS in MCF-7 cells after simultaneous treatment with BIBR1532 and salinomycin. Figure 6 BIBR1532 was shown to enhance salinomycin-induced ROS production in breast cancer cells.

[0032] Example 5

[0033] Effects of simultaneous treatment with BIBR1532, salinomycin and ROS scavenger (N-acetyl-L-cysteine, NAC) on the viability of MCF-7 cells

[0034] MCF-7 cells were cultured at 1×10 4The cells were inoculated into a 96-well culture plate at a density of 100 μL / well. After inoculation, the culture plate was placed in a humidified incubator at 37°C and 5% CO2 for 24 hours. BIBR1532, salinomycin and NAC were added to make the final concentration of BIBR1532 15 μM, salinomycin 8 μM and NAC 10 mM. After 48 hours or 72 hours, CCK-8 solution (10 μL / well) was added and incubated for 2 hours. Finally, the culture plate was placed in an ELISA reader and the absorbance was measured at 450 nm. The relative cell viability was calculated using the absorbance value. Figure 7 The effect of simultaneous treatment with BIBR1532, salinomycin and NAC on the viability of MCF-7 cells. Figure 7 This indicates that clearing ROS can partially reverse the inhibition of MCF-7 cell viability induced by BIBR1532 and salinomycin.

[0035] Example 6

[0036] Effects of simultaneous treatment with BIBR1532 and salinomycin on tumor growth

[0037] 5×10 6 MCF-7 cells were injected subcutaneously into BALB / c immunodeficient mice. 3 BIBR1532 (1.5 mg / kg) and salinomycin (2 mg / kg) were injected once every 3 days, and the tumor volume was measured and recorded once every 3 days with a vernier caliper. The tumor volume calculation formula is: length × width 2 / 2(mm 3 ). After 36 days, the tumor tissues were removed, embedded in paraffin, sliced, stained with HE, and observed under an optical microscope. Figure 8 is the effect of simultaneous treatment of BRBI1532 and salinomycin on tumor growth; Fig. 9 The effect of simultaneous treatment of BIBR1532 and salinomycin on tumor tissue. Figure 8 and Fig. 9 It can be seen that BIBR1532 can enhance the inhibitory effect of salinomycin on tumor growth and enhance the destructive effect of salinomycin on tumor tissue.

Claims

1. The use of salinomycin and telomerase inhibitor in the preparation of a substance with enhanced anticancer activity, characterized in that: The telomerase inhibitor inhibits telomerase activity, the telomerase inhibitor is BIBR1532, and the final concentration of BIBR1532 is 15 μM; The cancer is breast cancer.

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