YD277:the Study Of A Novel Small Compound That Inhibits Proliferation Of TNBC

BACKGROUD AND PURPOSEBreast cancer is a complex malignant disease caused by mammary duct and breast lobular dysplasia, including many kinds of subtypes, with different therapies and diagnosis. Breast cancer mainly happens in females, seldom occurs in males, and presents as a sporadic carcinoma. There are about one million new cases of breast cancer every year, which is becoming the most common carcinoma in female. In developed countries, breast cancer mortality rate were 10% in all cancer and up to 23% among female who die of cancer. At present, females were diagnosed breast cancer with a double attack rate and those who were higher with a family history. Although in the past 20 years, the mortality of breast cancer was decreased, some subtypes of breast cancer are still ineffective with conventional therapy, especially triple-negative breast cancer (TNBC), which refers to any breast cancer does not express the genes for estrogen receptor (ER), progesterone receptor (PR) and Her2/neu. About 15-25% breast cancer are TNBC, with a poorest diagnosis in all kinds of breast cancer. The bad diagnosis is mainly due to the resistance of routine therapies and HER2 targeted therapy. Moreover, the 3-5years recurrent rate was high in TNBC. Consider all above, more and more researchers are looking for other treatments for TNBC therapies.Because of the fact that the research progress of TNBC therapy is still at the primary stage, and the diagnosis is poor, it is urgently needed to look for a new and effective treatment for TNBC. ML264 is the inhibitor of KLF5 and EGR1, effectively reduces the growth of colorectal cancer in vivo and in vitro. But ML264 is ineffective in the treatment of breast cancer. Professor Jia Zhou from Department of Pharmacology and Toxicology, University of Texas Medical Branch, changing the structure of ML264, forming 24 derivatives(YD-2-1 to YD-2-100).we found that the ML264 derivative YD277 is effective reducing the growth many kinds of breast cancer cell lines in vivo and in vitro, but not inhibits the proliferation of normal mammary epithelial cell line MCF 10A. Animal experiment shows that 15mg/Kg YD277 effectively reduces the growth of tμMor xenografts in null mice, with low toxicity and safe, can hardly affect the major organs of the mice. We also found that 2-3μM YD277 can inhibit cancer cell lines by ER-STRESS pathway and finally activate caspase 3 and cause apoptosis in vitro.Methods1. SRB assay to verify cell viability. In order to find out the potential anti-cancer compounds, breast cancer cell lines Hct 116, MDA-MB-231, HCC 1806 were planted in 96 well plates at 2000 cells/well, incubate with 10μM of 24 derivatives and ML264, with DMSO as negative control, after 48h, the cells were then fixed with 150μl 10% trichloro acetic acid for lh,wash 5times with deionized water. Then the cells were stained with 50ul 0.4%(W/V)SRB (Sulforhodamine B assay, Sigma) for 10min, then the plates were washed 5 times with 1% acetic acid and dried. Finally, 100μ1 10mM Tris base was added to each well. Optical densities at 530nm were measured at a spectrophotometric plate reader. The cell viability values at different drug dosages were plotted in EXCEL and IC50 values were obtained from the graphs.2. Cell cycle analysis After treated with YD277 with different concentrations for 36hr, Adherent and detached MDA-MB-231 and MDA-MB-468cells were digested, harvested, and wash twice with PBS. About 6X105 cells were resuspended in 200ul PBS with 0.6% NP-40,2mg/ml RNaseA, 0.1mg/ml propidiμM iodide, incubated for 30 min at 37℃ in dark. The cells were then analyzed on an Accuri C6 flow cytometer (BD).3. Apoptosis analysis MDA-MB-231 and MDA-MB-468 cells were treated with different concentrations of YD277 for 36hr, cells were harvested and stained with anti-Annexin V antibody (eBioscience) and propidiμM iodide, and then analyzed by an Accuri C6 flow cytometer(BD)4. DNA synthesis assay DNA synthesis of MDA-MB-231 and MDA-MB-468 were tested with the Click-iT EdU microplate assay kit (Invitrogen, Carlsbad. CA) according to the manufacturer’s protocols. Finally,10 fields were randomly captured, and EdU positive cells and total cell nμMber were counted for each sample. The EdU positive cell were divided by total cell nμMber as results for analysis.5. Western blotting analysis After incubating with YD277 with the concentrations of 0,1,3,10 μM for 36hr, discard the culture mediμM, MDA-MB-231 and MDA-MB-468 were collected using lysis buffer (with P8340),and then the cell lysate was centrifuged and the resulting supernatant was collected. Then mixed with the loading buffer and subjected to SDS-PAGE and transferred onto polyinylidene fluoride (PVDF) membranes. The membrances were incubated with diluted primary antibodies,horseradish peroxidase(HRP) conjugated secondary antibodies. After Western lighting chemilμMinescence reagent plus added, viewed on an ImageQuant LAS4000 Biocompound imager to visualize the expression levels of specific proteins.6. ER-STRESS Pathway study. According to the significant increased expression of ER-STRESS specific proteins Bip, IRE1α, p-Jnk, C1-C3, YD277 was probably causing apoptosis by ER-STRESS. In order to nail down the suggestion, siBip, silREla, siJnk were designed to rescue the cytotocicity of YD277.7. Xenografts All mice studies were approved by the institutional ethics committee of Kunming Institute of zoology. CAS. Nude mice were purchased from SJA Lab Animal. Animal were house under specific pathogen-free conditions in ventilated and filtered cages under positive pressure. Xenograft tμMor were generated by injecting subcutaneously 1X106 MDA-MB-231 hμMan mammary gland breast cancer cells into left and right flanks of 6 week old female nude mice. TμMor volμMe was determined by caliper measurement and calculated by established methods. When the tμMors reached a volμMe of about 10 mm3, mice were divided into 3 groups, varying by 15mg/kg group, the vehicle solution was used as negative group, and the Epirubicin hydrochloride was used as positive group. All mice were treated intraperitoneally (i.p.) every other day. Mice were weighted and the tμMor volμMe were measured every 4 days. Experiments were terminated when the tμMor’s greatest measurement reached about 1cm, tμMors were peeled, weighted and fixated for further analysis.Results1. YD277 is the latent small compound inhibits breast cancer. YD277was confirmed as an effective compound that suppresses hμMan breast cancer cell lines Hct116, MDA-MB-231 and HCC 1806 from 24 derivatives of ML 264 at the concentration of 10μM for 48hr, DMSO as the negative control.YD277 shows its potential to inhibit 10 different breast cancer cell lines, including Hela, MCF 7, T-47D, SUM 149PT, MDA-MB-231, MDA-MB-453, Hs 578t, HCC 1806, HCC 1937, in terms of IC50. It appears that YD277 can wildly suppress most of the breast cancer cell lines, and at the concentration of 2μM can effectively inhibit the growth of MDA-MB-231 and MDA-MB-468.2. YD277 leads to the cell cycle G2/M arrest in MDA-MB-231 and MDA-MB-468 TNBC cell lines. When tested with different dosages of YD277(0,1,3,10 μM) for 36hr, MDA-MB-231 and MDA-MB-468 were measured cell cycle distribution. It shows that YD277 can significantly increase the percentage of G1 in MDA-MB-231, and in MDA-MB-468.3. YD277 results in cell apoptosis in MDA-MB-231 and MDA-MB-468. After treatment with different dosages of YD277(0, 1,3,10μM)for 36hr, MDA-MB-231 and MDA-MB-468 became round, floated, and formed apoptotic bodies. Flow cytometry with AnnexinV and PI staining were used to identify apoptosis degree. We found that 10μM YD277 can significantly induce cell apoptosis in TNBC.4. YD277 significantly inhibits DNA synthesis in MDA-MB-231 and MDA-MB-468. EdU assay was used to test whether YD277 blocked DNA synthesis in TNBC cell lines MDA-MB-231 and MDA-MB-468. It shows a large decrease proportion of proliferation in both cell lines in the concentration of 3μM and 10μM.5. YD277 regulates the expression levels of cell cycle, apoptosis, autophagy genes in TNBC cell lines. Base on the fact that YD277 causes G1 arrest, cell apoptosis, and reduces DNA synthesis, we further examined protein change of cell cycle, apoptosis, and autophagy(Figure5 a). After treated with a dosages of 0,1,3, 10μM in the cell lines of MDA-MB-231 and MDA-MB-468, YD277 can dramatically reduce the protein levels of cyclin D1, and increase expression of p21 and p27. Consistent with the result of cell apoptosis in flow cytometry, it turns out an increase of apoptotic proteins cleave PARP1, cleave caspase 3 and autophagy protein LC3B and a deregulation of anti-apoptotic proteins Bcl 2, Bcl-xl and Survivin. We further detected ER-STRESS pathway and found that proteins in the pathway were activated, such as Bip, IRE1α, p-Jnk, cleave-caspase3. YD277 was probably causing cell apoptosis by inducing ER-STRESS.6. Knockdown the proteins of ER-STRESS can partly rescue the damage of YD277. At the present of siBip and siIREla, YD277 inhibits TBNC cell lines MDA-MB-231 and MDA-MB-468 with a higher ICso, shows that blocking the ER-STRESS pathway can partly rescue the injuries of YD277. The result proves that the mechanism YD277 inhibits TNBC partly due to ER-STRESS.7. YD277 inhibits the growth of MDA-MB-231 tμMor xenografts in nude mice. Subsequent to find that YD277 inhibits proliferation of TNBC cell lines in vitro, we next measured its effectiveness in suppressing growth of tμMor xenografts in nude mice. As shown in Figure 7A, every two days injections of YD-2-77 could significantly affect tμMor growth compared with sesame oil as vehicle. Continuous monitoring of tμMor volμMe shows that tμMor in YD277 group grew slower than vehicle group (Figure 7b), and at the end of 28 days treatment, we found that it is indistinctly YD277 affected the weight of the mice.YD277 inhibits the growth of TNBC in vivo but harmless to the body.8. The result of protein pulldown is ongoing.ConclusionYD277 is a new small compound that effectively inhibits TNBC cell lines proliferation. In the present study, YD277 at the concentration of 2-3μM can wildly suppresses most of breast cancer cell lines in vitro. It causes cell apoptosis by cell cycle arrest, induces apoptotic protein and autophagy. Our results shows that YD277 finally activates ER-STRESS, which causes apoptosis. Our further in vivo experiment shows 15mg/kg YD277 can significant inhibits tμMor xenograft, but do no harm to the nude mice bodies. YD277 maybe become a prefer treatment for chemotherapy. Ongoing studies in our lab aim to identify the direct targets of YD277.