| Part 1 :Effects of PI3K / AKT / mTOR Signaling Inhibitors on the Biological Function of Tri-negative Breast Cancer CellsBackgroundTriple negative breast cancer (TNBC) is special subtype of breast cancer, estrogen receptor(ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2) are negative. TNBC lacks effective targeted therapy, and is not sensitive to endocrine therapy and conventional chemotherapy. It has high invasiveness and poor prognosis, and its clinical treatment is a very difficult problem. The occurrence and development of tumors is a multi-gene involved, multi-step, multi-stage pathological process, and the imbalance between cell death and proliferation due to abnormalities in signal transduction pathways is one of the important mechanisms of tumorigenesis and progression.PI3K/AKT/mTOR is considered to be one of the most important signaling pathways in-TNBC. At present, there are few studies on the mechanism of PI3K/AKT/mTOR signaling pathway in TNBC. Whether the change of related genes in the pathway is related to the biological behavior of TNBC cell and the molecular inhibitors of the target in the pathway will play an important role in the treatment of breast cancer, which deserves further study.ObjectiveThis study aims to investigate the expression of PI3K/AKT/mTOR mRNA in TNBC.LY294002 and Everolimus, the specific inhibitors of P13K and mTOR, were used to observe whether the anti-tunlor effects on the MDA-MB-231 cell proliferation, cycle distribution and apoptosis of combined inhibitors were a synergistic effect.Methods1 Human samples collection40 pairs of triple negative breast cancer tissues and adjacent normal tissues were collected from routine therapeutic surgery at our department. All samples were obtained with informed consent and approved by the hospital institutional review board.2 RNA extraction and quantitative real-time-PCR (qPCR)Total RNAs from cell culture were extracted from triple negative breast cancer tissues using Trizol Reagent. The purity and concentration of RNAs was evaluated using NanoDrop 2000 spectrophotometer. RNAs were then converted into cDNA using PrimeScript(?) RT Enzyme Mix followed by qPCR using a SuperReal PreMix Plus with the primer sets for PI3K, AKT, mTOR on the 7500 Real-Time PCR systems. GAPDH was used as an internal standard for normalization, and the 2-ACT (ACT=CT mRNA -CT GAPDH) method was used to quantify relative amount of mRNA. The change in mRNA expression was then converted to fold-change,relative to control group.3 Cell cultureThe human breast cancer cell line MDA-MB-231 was obtained from the Cell Resource Center of Peking Union Medical College, and was maintained in 10% fetal bovine serum-supplemented RPMI-1640 medium. All cell cultures were maintained in a humidified incubator at 37? and 5% C02.4 Cell proliferation assaysCell counting kit-8 was used to measure the cell proliferation. MDA-MB-231 cells were seeded in full growth medium, which was replaced with RPMI-1640 24 h later, and the cells were cultured for additional 48 h. Next, approximate 3000 cells in 100?L medium were seeded in a 96-well plate for 24 h and then the medium was replaced by 200?L of full growth medium,which contained LY294002 or/and Everolimus. The cells were cultured for 5 days. Five replicates for each treatment were conducted on the plate. When the cell proliferation assay started, 10?L of CCK-8 solution was added to the medium and then the plate was placed in incubator at 37?. Four hours later, cell numbers were evaluated by measuring the absorbance at 450 nm using an ELx800 Universal Microplate Reader.5 Cell cycleMDA-MB-231 cells (1.5×105/well) were treated with either LY294002 or/andEverolimus in full growth medium for 5 days. Cells were then stained with PI and RnaseA. Cell cycle was further analyzed with flow cytometry.6 Apoptosis testMDA-MB-231 cells (1.5×105/well) were treated with either LY294002 or/and Everolimus in full growth medium for 5 days. Cells were then stained with FITC-conjugated anti-Annexin V antibodies. Cell apoptosis was further analyzed by using the Annexin V-FITC Apoptosis Detection kit with flow cytometry.7 Statistical analysisResults were collected as the average of at least five independent experiments. All the data were presented as means ± standard deviation. The data were analyzed using multi-factor ANOVA followed by Tukey's post hoc test with GraphPad PRISM software package. Data were determined to be statistically different when P<0.05.Results1 Expression levels of PI3K, AKT and mTOR mRNA in triple negative breast cancer tissuesTo determine the function of PI3K, AKT and mTOR in triple negative breast cancer, its expression levels were analyzed in human triple negative breast cancer tissues and pair-matched adjacent normal tissues. Quantitative real-time PCR analysis showed that PI3K, AKT and mTOR mRNA was significantly up-regulated in triple negative breast cancer tissues.2 Effects of LY294002 or/and Everolimus treatment on the proliferation of MDA-MB-231 cellsMDA-MB-231 cells treated with LY294002 exhibited lower proliferation than control group (P <0.05). After treatment with Everolimus, the viabilty of MDA-MB-231 cells did not change significantly (P> 0.05). The viabilty of MDA-MB-231 cells was significantly decreased after co-treatment of LY294002 and Everolimus.3 Effects of LY294002 or/and Everolimus treatment on the cell cycle of MDA-MB-231 cellsCompared with the control group, the ratio of cells in G1 phase increased significantly after LY294002 or Everolimus treatment (P <0.05), the cells were arrested in G1 phase. And the effects of co-treatment was better than the drugs used alone (P <0.05).4 Effects of LY294002 or/and Everolimus treatment on the apoptosis of MDA-MB-231 cellsCompared with the control group, the apoptosis rate increased significantly after LY294002 or Everolimus treatment (P <0.05), and the effects of co-treatment was better than the drugs used alone (P <0.05).Conclusion1 PI3K, AKT and mTOR were significantly up-regulated in human triple negative breast cancer tissues, and may be involved in the development of triple negative breast cancer.2 Inhibition of PI3K/AKT/mTOR pathway inhibited triple negative breast cancer cell proliferation.3 Inhibition of PI3K/AKT/mTOR pathway arrestted triple negative breast cancer cell in G1 phase.4 Inhibition of PI3K/AKT/mTOR pathway promoted triple negative breast cancer cell apoptosis.Part 2:Effect and mechanism of Tanshinone ?A on tamoxifen sensitivity of breast cancer cellsBackgroundAs the second most common type of cancer in women, breast cancer can be classified into three subtypes based on the expression of the estrogen receptor (ER), progesterone receptor (PR),and cell surface receptor of human epidermal growth receptor 2 (HER2), which have been the most commonly used predictive factors in chemotherapy selections for breast cancer patients.However, current chemotherapies for breast cancer often lead to the development of drug resistance. Tamoxifen has long been used for the systemic treatment of patients with breast cancer. The treatment success of tamoxifen is mainly dependent on the expression of the estrogen receptor (ER) in breast carcinoma. However, a large percent of responding patients ultimately develop tamoxifen resistance. Therefore, searching for effective regimens that could prevent or reverse the tamoxifen resistance may bring great benefits in breast cancer treatment.Tanshinone IIA (TSA) is an important lipophilic diterpene extracted from a traditional herbal medicine Salvia miltiorrhiza Bunge (Danshen). TSA has been widely used for the treatment of cardiovascular, cerebrovascular, and postmenopausal syndromes. Previous studies have indicated that it has potent anti-oxidant and anti-inflammatory properties. However,emerging evidence has demonstrated that TSA exhibits great anti-cancer effects on both ER-positive and -negative breast cancer cells. TSA inhibits the growth of breast cancer cells through epigenetic modification of Aurora A expression and function. Furthermore, TSA can also reverse chemotherapy resistance. It has been shown that TSA can block epithelial-mesenchymal transition through HIF-1? down-regulation, reversing hypoxia-induced chemotherapy resistance in breast cancer cell lines. Interestingly, TSA has been shown to alter the expression of various microRNAs in cardiac myocytes. However, little is known about the effects of TSA on miRNA expressions in the breast cancer cell lines.MiRNAs play an important role in the regulation of the expression of various genes. An increasing number of studies have reported that miRNAs are involved in modulating tamoxifen resistance. Specifically, estradiol treatment increases accumulation of miRNA-98 and miRNA-21 in MCF-7 cells. Such a change in miRNA expressions may alter patient response to tamoxifen treatment. Furthermore, estradiol can reduce the expression of miRNA-181a and miRNA-26a,which in turn attenuate cell proliferation. Additionally, more and more miRNAs, including miRNA-375, miRNAs 221/222, miRNA-200, miRNA-342, and miRNA-519a, have been discovered as potential biomarkers for the tamoxifen response.ObjectiveThe present study was undertaken to examine the effects of TSA on tamoxifen resistance.To this end,we derived a tamoxifen-resistant breast cancer cell line (i.e.,MCF-7-TamR) using MCF-7 cells. We evaluated the effects of tamoxifen and TSA treatment on the proliferation and apoptosis of MCF-7 and MCF-7-TamR cells. Meanwhile, the putative mechanisms underlying the effects of TSA on tamoxifen resistance were explored.Methods1 Cell cultureThe human mammary carcinoma cell line MCF-7 was obtained from the Cell Resource Center of Peking Union Medical College, and was maintained in 10% fetal bovine serum-supplemented RPMI-1640 medium. We derived Tamoxifen-resistant MCF-7 cell line (MCF-7-TamR) by continuously exposing it to tamoxifen (1?M; Diluted in 0.1% ethanol) for more than 12 months. All cell cultures were maintained in a humidified incubator at 37? and 5% C02.Consistent with previous studies, MCF-7-TamR cell line exhibited ER positive.2 Cell proliferation assaysCell counting kit-8 was used to measure the cell proliferation. MCF-7 or MCF-7-TamR cells were seeded in full growth medium, which was replaced with RPMI-1640 24 h later, and the cells were cultured for additional 48 h. Next, approximate 3000 cells in 100?L medium were seeded in a 96-well plate for 24 h and then the medium was replaced by 200?L of full growth medium, which contained Tamoxifen or Tanshinone IIA. The cells were cultured for 5 days.Five replicates for each treatment were conducted on the plate. When the cell proliferation assay started, 10?L of CCK-8 solution was added to the medium and then the plate was placed in incubator at 37?. Four hours later, cell numbers were evaluated by measuring the absorbance at 450 nm using an ELx800 Universal Microplate Reader.3 Apoptosis testMCF-7 or MCF-7-TamR cells (1.5×105/well) were treated with either 0.1% ethanol (control)or Tanshinone IIA in full growth medium containing 1?M tamoxifen for 5 days. Cells were then stained with FITC-conjugated anti-Annexin V antibodies. Cell apoptosis was further analyzed by using the Annexin V-FITC Apoptosis Detection kit with flow cytometry.4 Soft agar assayAnchorage-independent soft agar colony formation assay was used to examine the colony formation ability of MCF-7 and MCF-7-TamR cells. Briefly, base agar was formed by adding 1.5 ml FBS supplemented medium containing 0.5% agarose into 35-mm cell culture dishes.Approximate five thousand cells were then seeded in 1.5 mL medium supplemented with 0.35%agarose on top of the bas agar. For tamoxifen and TSA treatments, tamoxifen and/or TSA was then added in 2 ml of liquid medium and disperse on the top of base agar. The cells were then cultured for 10 days at 37? under 5% C02. 0.005% crystal violet was then used to stain the cells in the dishes, the colony formation was further examined under microcamera.5 RNA extraction and quantitative real-time-PCR (qPCR) for miRNATotal miRNA-enriched RNAs from cell culture were extracted from MCF-7 and MCF-7-TamR cells following 0.05?M of TSA treatment for 5 days using Trizol Reagent. The purity and concentration of RNAs was evaluated using NanoDrop 2000 spectrophotometer. RNAs were then converted into cDNA using TaqMan(?) MicroRNA Reverse Transcription Kit followed by qPCR using a TaqMan(?) Small RNA Assay with the miRNA primer sets for miR-22, miR-221,miR-222, miR-200a, miR-200b, or miR-200c on the 7500 Real-Time PCR systems. U6RNA was used as an internal standard for normalization, and the 2-ACT (ACT=CTmicroRNA-CTU6 RNA)method was used to quantify relative amount of miRNA. The change in miRNA expression was then converted to fold-change, relative to control group.6 Western blottingThe expression of c-myc (-65 kDa) was determined by Western blot following 0.05 ?M of TSA treatment for 5 days. MCF-7 or MCF-7-TamR cells were lysed in RIPA buffer containing a EDTA-free protease inhibitor cocktail, 1 mM phenyl methylsulfonyl fluoride, and phosphatase inhibitors. Each sample was then added into 20 ?l2× sample loading buffer. The samples were boiled for 5 min before loading. 10% running gel was utilized. The gel was transferred to a same size Nitrocellulose transfer membrane within transfer buffer under 45 V for 40 min, and probed with the first antibody against c-Myc with a 1/1000 dilution in blocking buffer overnight. The membrane was washed by TTBS for three times before adding secondary antibody with 1/5000 dilution in blocking buffer for 2 hours. Background color was reduced carefully by washing with TTBS. The results were visualized using ECL kit, and protein levels were normalized to GAPDH and quantified using Tanon Gel image system.7 Statistical analysisResults were collected as the average of at least five independent experiments. All the data were presented as means ? standard deviation. The data were analyzed using multi-factor ANOVA followed by Tukey's post hoc test with GraphPad PRISM software package. Data were determined to be statistically different when P<0.05.Results1 Effects of tamoxifen or TSA treatment on the proliferation of MCF-7 and MCF-7-TamR cellsMCF-7-TamR cells exhibited higher proliferation than MCF-7 cells after tamoxifen treatment (P<0.001), and tamoxifen dose-dependently decreased the proliferation of MCF-7 and MCF-7-TamR cells (P<0.001). Furthermore, the lowest effective dose of tamoxifen was 0.5 ?M in MCF-7 cells (P<0.01). but the lowest effective dose of tamoxifen was 5 ?M in MCF-7-TamR cells (P<0.01). Therefore, 1 ?M of tamoxifen was employed as the optimal dosage in further investigation of the effects of TSA on tamoxifen resistance.TSA treatment dose-dependently decreased the proliferation of MCF-7 and MCF-7-TamR cells (P<0.001). However, in contrast to the effects of tamoxifen, higher doses of TSA (i.e., 0.1?M or more) treatment decreased the proliferation of MCF-7 and MCF-7-TamR cells similarly regardless of the cell lines (P>0.05). Specifically, the lowest effective dose of TSA was 0.1?M,and cell proliferation was significantly accelerated (P< 0.01).2 Effects of co-treatment of TSA and tamoxifen on the proliferation, clonogenic potential, and apoptosis of MCF-7 and MCF-7-TamR cellsMCF-7-TamR cells exhibited higher proliferation than MCF-7 cells under the treatment of 0.1% ethanol and 1?M of tamoxifen (P<0.001). Interestingly, 0.05?M of TSA treatment reduced the proliferation in either MCF-7 or MCF-7-TamR cells (P<0.001), as compared to control. This is surprising because 0.05?M of TSA treatment alone failed to alter the proliferation in either MCF-7 or MCF-7-TamR cells. More specifically, 0.05?M of TSA and 1?M of tamoxifen treatment decreased the proliferation of MCF-7-TamR cells to a similar level of MCF-7 cells after 0.1 % ethanol treatment.MCF-7-TamR cells treated with 0.1% ethanol and 1?M of tamoxifen treatment exhibited higher focus number as compared with MCF-7 cells (P<0.01). However, 0.05 ?M of TSA and 1?M of tamoxifen treatment reduced the focus number in either MCF-7 or MCF-7-TamR cells(P<0.001).MCF-7 cells exhibited higher apoptosis than MCF-7-TamR cells under the treatment of 0.1% ethanol and 1?M of tamoxifen (P<0.05). Furthermore, 0.05 ?M of TSA treatment enhanced the apoptosis in either MCF-7 or MCF-7-TamR cells (P<0.001), as compared to control. More specifically, 0.05 ?M of TSA treatment increased the apoptosis of MCF-7-TamR cells to a similar level in MCF-7 cells after 0.1% ethanol treatment.3 Effects of TSA treatment on miRNA expression in MCF-7 and MCF-7-TamR cellsThe expression of miRNA-22 and miRNA 200 families, including 200a, 200b, and 200c was attenuated in MCF-7-TamR cells, but the expression of miRNA 221/222 was enhanced in MCF-7-TamR cells (P<0.001), as compared to MCF-7 cells. However, 0.05?M of TSA treatment only altered the expression of miRNA-22 in either MCF-7 or MCF-7-TamR cells(P<0.001). Specifically, 0.05?M of TSA treatment increased the expression of miRNA-22 in MCF-7-TamR cells to a similar level in MCF-7 cells after 0.1% ethanol treatment.4 Effects of TSA treatment on c-Myc expression in MCF-7 and MCF-7-TamR cellsThe expression of c-Myc was robustly increased in MCF-7-TamR cells, as compared to MCF-7 cells (P<0.01). However, 0.05 ?M of TSA treatment reduced the expression of c-Myc in either MCF-7 or MCF-7-TamR cells (P<0.05). Specifically, 0.05?M of TSA treatment attenuated the expression of c-Myc in MCF-7-TamR cells to a similar level in MCF-7 cells after 0.1% ethanol treatment.Conclusion1 TSA treatment dose-dependently decreased the proliferation of MCF-7 and MCF-7-TamR cell.2 Co-treatment of TSA and tamoxifen reduced the proliferation, clonogenic potential, and enhanced the apoptosis of MCF-7 and MCF-7-TamR cells.3 Compared to MCF-7 cells, the expression of miRNA-22 and miRNA 200 families,including 200a, 200b, and 200c was attenuated in MCF-7-TamR cells, but the expression of miRNA 221/222 was enhanced in MCF-7-TamR cells. TSA treatment increased the expression of miRNA-22 in MCF-7-TamR cells.4 Compared to MCF-7 cells, the expression of c-Myc was robustly increased in MCF-7-TamR cells. TSA treatment attenuated the expression of c-Myc in MCF-7-TamR cells.5 TSA can promote the sensitivity to tamoxifen treatment in tamoxifen-resistant breast cancer cells in vitro, and this phenomenon may involve the miRNA-22 and c-Myc signaling pathways. |
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