Miconazole Triggers Various Forms of Cell Death in Human Breast Cancer MDA-MB-231 Cells

ORIGINAL ARTICLES

School of Pharmacy1, Scientific Research Center2, Bengbu Medical College, Bengbu, Anhui, China

Miconazole triggers various forms of cell death in human breast cancer MDA-MB-231 cells

CHENGZHU WU1,#,*, MEIJIA GAO1,#, LIN SHEN2, BOHAN LI1, XIANGJIAN BAI1, JIAHUI GUI1, HONGMEI LI1, QIANG HUO1, TAO MA1,*

Received October 21, 2018, accepted November 26, 2018 *Corresponding authors: Cheng-Zhu Wu, Tao Ma, School of Pharmacy, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, Anhui, China [email protected]; [email protected] #These authors contributed equally to this work. Pharmazie 74: 290-294 (2019) doi: 10.1691/ph.2019.8812

In recent years, “drug repurposing” has become an important approach and focus of studies on anti-tumor drug research and development (R&D). As one of the first-generation broad-spectrum imidazole anti-fungal drugs, miconazole (MCZ) exhibits anti-tumor effects in addition to its anti-fungal effect. However, no report has focused on examining the effect of MCZ on the proliferation and cell-death of human breast cancer MDA-MB-231 cells. MCZ significantly inhibited the proliferation of MDA-MB-231 cells in a concentration- and time-dependent manner. We also observed that MCZ induced both apoptosis and necroptosis in MDA-MB-231 cells. Transmission electron microscopy showed submicroscopic structures in these cells, which correspond to necrotic features, in addition to the characteristic features of apoptosis. Pretreatment of cells with z-VAD-fmk, an apoptosis inhibitor or Nec-1, a necroptosis inhibitor, significantly increased their viability compared with MCZ treatment. The initial mechanism of MCZ-mediated cell death in human breast cancer MDA-MB-231 cells involves an increase in the Bax/Bcl-2 ratio, downregulation of apoptosis induced by Akt and p-Akt-473, a simultaneous upregulation of the receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) protein expression, and ROS production to induce necroptosis. Our results suggest that MCZ may be a potential lead compound for the development of anti-breast cancer drugs.

1. Introduction and hence the anti-tumor effects and corresponding mechanisms Results of recently-performed global cancer surveys have shown of metformin, thalidomide, berberine and bilobalide have been that despite advances in therapeutics, the incidence of breast cancer revealed (Matthews et al. 2003; Rizos et al. 2013; Tillhon et al. remains high, which poses a serious threat to women’s health and life; 2012; Zhi et al. 2016). As one of the first-generation broad-spec- about 15% of the cases are of triple-negative breast cancer (TNBC) trum antifungal drugs for treating fungal infections in crops and (Ferlay et al. 2015; Loi et al. 2013; Siegel et al. 2017). TNBC is a humans, miconazole (MCZ) can be administered externally, orally, breast cancer that does not have estrogen receptor (ER), progesterone and intravenously (Fig. 1A) (Garcia-Cuesta et al. 2014; Heeres et receptor (PR), and human epidermal growth factor receptor-2 (Her-2). al. 2010). According to a few reports, MCZ also has been shown TNBC patients generally show an early onset of cancer and are prone to exhibit antitumor effect via the induction of death receptor-me- to recurrence and metastasis. Moreover, there is a lack of effective diated apoptosis in bladder cancer cells (Yuan et al. 2017). In this drugs and standardized treatment guidelines (Hyslop et al. 2013). study, we investigated the effects of MCZ on cell proliferation and Programmed cell death (PCD) is a cellular mechanism operating at cell death to provide a theoretical basis for future research using the crossroads of multiple diseases and investigation into the regula- the TNBC cell line MDA-MB-231 as a model. tion of the same is important for exploring new disease treatments. Studies have shown that in addition to PCD by apoptosis and auto- 2. Investigations and results phagy, necrosis can be also programmed, and this process is termed as necroptosis (Degterev et al. 2005). Although chemotherapeutics 2.1. MCZ inhibits the proliferation of human breast such as paclitaxel and doxorubicin show positive effects in clinical cancer MDA-MB-231 cells application, tumor cells can gradually develop drug resistance, To understand the anti-breast cancer activity of MCZ, we examined which is characterized by the inhibition of the apoptotic pathway, the viability of MDA-MB-231 cells, treated with different concen- eventually reducing the efficacy of these chemotherapeutics (Ateba trations (0–40 μM) of MCZ for 24, 48, and 72 h, using MTT assay. et al. 2018; Hanahan et al. 2011; Holohan et al. 2013). Different The results of the dose-effect curve showed that the viability of cell death pathways operate via different molecular mechanisms, MDA-MB-231 cells gradually decreased with an increase in MCZ and the resistance of tumor cells to apoptosis generally has no effect concentration and a prolonged duration of action, and the maximal on other cell death pathways. Interestingly, the inhibition of apop- inhibitory concentration (IC50) values of MCZ at 24, 48, and 72 h tosis induced by anti-cancer drugs correspondingly increases the were 21.40, 17.62, and 8.71 μM, respectively (Fig. 1B). According proportion of tumor cells that die through non-apoptotic pathways to the results of MTT assay, we selected 1.0, 2.0, and 4.0 μM (Amaravadi et al. 2007; Buytaert et al. 2006). concentrations of MCZ for treatment of MDA-MB-231 cells for 5 Drug repurposing has become a major approach and research focus days and observed that MCZ treatment could significantly inhibit for new drug R&D because of its high safety, shortened develop- the formation of cell colonies at these concentrations (*P <0.05 ment cycle, and clinically confirmed pharmacokinetic properties and **P <0.01) (Fig. 1C and D). These results indicated that MCZ (Chong et al. 2007). In recent years, great progress has been treatment exerted an anti-proliferative effect on MDA-MB-231 achieved by drug repurposing for anti-tumor drug development, cells in a concentration- and time-dependent manner. 290 Pharmazie 74 (2019) ORIGINAL ARTICLES

Fig. 3: MCZ induced apoptosis in MDA-MB-231 cells that is dependent of caspase acitivity. (A) Cell viability following treatment with MCZ (40 μM) with or without 1 h pre-treatment with z-VAD (20 μM), as analyzed by the MTT assay. (B) Annexin V-FITC/PI analysis following treatment with MCZ alone Fig. 1: Anti-proliferation activity of MCZ on human breast cancer MDA-MB-231 or with z-VAD pre-treatment. cells. (A) The structure of miconazole. (B) Cytotoxicity of MCZ was analyzed by the MTT assay. (C) The colony-forming capability was analyzed using a colony fornation assay after treatment with low concentration of transmission electron microscopy. The cells treated with MCZ (40 MCZ. (D) Quantification of colony-forming capability of MDA-MB-231 cells inhibited by MCZ. * P <0.05 and ** P <0.01 compared with the control. μM) exhibited morphological characteristics of apoptosis, such as chromatin margination and nuclear pyknosis in massive cells (Fig. 2C). In addition, with an increase in MCZ concentration, the expression levels of Akt, p-Akt-473 and anti-apoptotic protein 2.2. MCZ induces apoptosis in MDA-MB-231 cells Bcl-2 decreased, and the expression levels of Bax, a pro-apoptotic To further investigate the role of MCZ in inducing MDA-MB- protein, gradually increased in the MCZ-treated cells (Fig. 2D). 231 cell death, we stained the cells with PI and performed flow Thereafter, we compared the differences in viability between the cytometry for detection of cell death. The results of PI staining/ cells subjected to MCZ treatment alone or a combined treatment flow cytometry were consistent with the MTT results; the death with MCZ and a broad-spectrum caspase inhibitor, z-VAD-fmk rate of MDA-MB-231 cells gradually increased with an increase by using MTT assay. As shown in Fig. 3A, the cell viability after 24 h treatment with MCZ (40 μM) alone was 54.48 %, while it increased to 78.9 % after the combined treatment with MCZ and z-VAD-fmk. Next, we verified the above results with Annexin V/PI staining using flow cytometry, which showed that the viability of the cells subjected to the combined treatment of MCZ and z-VAD –fmk combination was significantly higher than that of the cells treated with MCZ alone, indicating that the MCZ-induced cell

Fig. 2: MCZ induced apoptosis in human breast cancer MDA-MB-231 cells. (A) Flow cytometric analysis of cell death after treatment with various concentrations of MCZ using PI staining. (B) MDA-MB-231 cell was treated with MCZ for 24 h, subjected to DAPI staining and visualized using fluorescence microscopy. Red arrowheads indicated apoptotic cells. (C) Electron microscopy of MDA-MB-231 cells treatment with DMSO or MCZ for 24 h. (D) Western blot analyses of Akt, p-Akt, Bax and Bcl-2 levels in MDA-MB-231 cells, using GAPDH as the internal control. in MCZ concentration (Fig. 2A). The cell death rates following the 24 h treatment of MCZ at different concentrations (0–40 μM) were 5.7 %, 13.9 %, and 41.8 %, respectively. Moreover, DAPI staining revealed that with increasing concentrations of MCZ, chromatin showed a higher incidence of pyknosis, as is evident Fig. 4: MCZ induced necroptosis in human breast cancer MDA-MB-231 cells. from increased intensity of DAPI staining, and also some of (A) Eletron microscopy of cells after treating with DMSO or MCZ. (B) the nuclei showed characteristics of apoptosis such as nuclear Cell viability following treatment with MCZ (40 μM) with or without 1 h fragmentation and disintegration (Fig. 2B). To further confirm pre-treatment with Nec-1 (20 μM), as analyzed by the MTT assay. (C) Flow cytometric analysis of cell death after treatment with MCZ with or without the induction of MDA-MB-231 death by MCZ treatment, we 1 h pre-treatment with Nec-l using Annexin V-FITC/ PI staining. (D) The observed the changes in submicroscopic structure of cells by expression of necroptosis-related proteins were analyzed by western blotting. Pharmazie 74 (2019) 291 ORIGINAL ARTICLES death could be protected by z-VAD-fmk (Fig. 3B). These results 3. Discussion demonstrated that MCZ induced apoptosis in MDA-MB-231 cells. Breast cancer ranks first among the malignant tumors in women, posing a serious threat to women’s health and life. In particular, 2.3. MCZ induces necroptosis in MDA-MB-231 cells the failure of chemotherapeutics to deliver targeted treatment due Studies have shown that cell necrosis can be also programmed, to the absence of ER, PR and Her-2 expression in TNBC patients the process of which is called necroptosis, also exists as a mode has proven to be a major difficulty in the field of anti-breast cancer of cell death in addition to apoptosis and autophagy, and can be drug research (Loi et al. 2013). Tumor cells are often defective specifically inhibited by necrostatin-1 (Nec-1) (Degterev et al. in maintaining the dynamic equilibrium between survival and cell 2005; Xie et al. 2013). MDA-MB-231 cells treated with MCZ death. The inhibition of cell death pathways enables the tumors to for 24 h also showed morphological features of typical necrosis, proliferate constantly, and is closely associated with drug resis- characterized by cell membrane rupture and organelle swelling, tance and metastasis in tumors (Lowe et al. 2000; Su et al. 2015). as was observed by transmission electron microscopy (Fig. 4A). In 2005, Professor Yuan from Harvard University officially put As shown in Fig. 4B, the cell viability reduced to 46.71 % after forward the term necroptosis which was observed to be closely 24 h treatment with MCZ (40 μM) alone, which increased up linked to the occurrence and development of tumors (Degterev et to 70.83 % after a combined treatment with Nec-1 and MCZ. al. 2005). Thus, necroptosis has gradually emerged as a potential We obtained similar results with Annexin V/PI staining based “new target” for antitumor drug development (Hitomi et al. 2008; flow cytometry, wherein the viability of the cells pretreated Hu et al. 2007; Kreuzaler et al. 2012). with Nec-1 was higher than that of the cells treated with MCZ Usually, “drugs with known indications” have been clinically alone, demonstrating that Nec-1 had protective effects on the applied for long and hence, have a relatively high safety profile MCZ-induced cell necroptosis (Fig. 4C). Furthermore, western and well-known side effects (DiMasi et al. 2003). Therefore, “drug blotting analysis showed a concentration-dependent increase in repurposing” is a low-risk and high-reward strategy for develop- the expression levels of RIP3 and MLKL upon MCZ treatment, ment of new drugs that can save development time and cost, and while the expression of RIP1 showed a decrease (Fig. 4D). These more importantly, it can also achieve a more rational and efficient results showed that MCZ also induced necroptosis in MDA-MB- utilization of resources. During the early stages of the repurposing 231 cells. study, we found that, MCZ exhibited high cytotoxicity for TNBC MBA-MB-231 cells (the cell viability was 22 % at 5 μg/ml), and therefore, we further examined the effects of MCZ treatment on the proliferation and cell death of breast cancer cells. PCD exists in a variety of diseases, especially in the occurrence, development and treatment of cancer; a selective induction of tumor cell apoptosis can serve as one of the main strategies for cancer treatment (Sun et al. 2009). However, over a prolonged duration of chemotherapy, tumor cells often develop resistance to therapy, which is characterized by suppression of apoptosis. Compared to the well-researched apoptotic cell death pathways, necroptosis is a relatively unexplored mode of cell death which holds the potential of providing new insights and directions for tumor treatment and drug development. For example, induction of necroptosis by chemotherapeutics, such as paclitaxel, 5-FU, shikonin, and magnolol can effectively kill tumor cells, and even overcome multidrug resistance and inhibition of apoptosis in Fig. 5: Effect of MCZ on mitochondrial function in MDA-MB-231 cells. (A) MMPs tumor cells (Grassilli et al. 2013; Han et al. 2007; Jang et al. 2011; were assessed by JC-1 staining and fluorescence microscopy. (B) Effects of Li et al. 2007). cellular ATP levels after treatment with various concentration of MCZ for 3h. There are various factors that induce necroptosis in cells, such as cyto- (C) MCZ induced ROS accumulation. ROS was measured using the dye DCF kines, pathogen-associated model molecules, receptor-interacting at 3 h after stimulation. * P <0.05 and ** P <0.01 compared with the control. proteins (RIP) 1, RIP3, poly (ADP-ribose) polymerase (PARP-1) and ROS; however, most studies on necroptotic signaling pathways focus on the TNF-DR-RIP signaling pathway (Vanlangenakker et 2.4. Effect of MCZ on mitochondrial function in MDA- al. 2012). As an important effector molecule of necroptosis, MLKL MB-231 cells activation upon complex formation with phosphorylated RIP3 is Abnormal mitochondrial function is closely associated with cell a prerequisite for the occurrence of necroptosis (Sun et al. 2012). apoptosis or necrosis. For example, a decline in mitochondrial Our results indicate that MCZ-mediated induction of necroptosis in membrane potential is an important event during the early stages MDA-MB-231 cells was accompanied by a significant increase in of necroptosis. To determine if MCZ treatment had any effect the expression levels of RIP3 and MLKL proteins, which are associ- on the mitochondrial function, we performed a fluorescent ated with the RIP3-MLKL signaling pathway. microscopy-based analysis of mitochondrial function by using Mitochondria are important organelles for cellular energy produc- the calcium indicator JC-1. MDA-MB-231 cells were subjected tion, and also the main target for the production and action of ROS. to MCZ treatment at different concentrations, and we observed The high levels of intracellular ROS cause disequilibrium between a shift in JC-1 fluorescence from red to green. In addition, oxidative stress and antioxidants, leading to cell death. Necroptosis the production of intracellular ATP decreased to 58.37% and results in the loss of membrane integrity, a decline in mitochon- 36.86%, when the cells were treated with 40 and 80 μM MCZ, drial membrane potential, cytochrome release, and an abnormal respectively (Fig. 5B). Since reactive oxygen species (ROS) ATP production (Tsujimoto et al. 2007). Reports suggest that RIP3 are an important factor involved in necroptosis, their intracel- interacts with metabolic enzymes and is involved in the regulation lular production and accumulation are a prerequisite for tumor of cellular energy metabolism and ROS production during necro- necrosis factor-alpha (TNF-α) to induce necroptosis in cells (Ye ptosis (Zhang et al. 2009). MCZ-triggered MDA-MB-231 cell et al. 2012). We detected intracellular ROS levels using the fluo- death is closely associated with similar changes in mitochondrial rescent probe, dichloro- dihydro-fluorescein diacetate (DCFH- function, mainly a decline in the mitochondrial membrane poten- DA), and observed that the fluorescence intensity of oxidized tial, a decrease in ATP levels, and the generation of large amounts dichlorofluorescein (DCF) increased after a 3 h treatment with of ROS. Our results demonstrate that MCZ induces necroptosis MCZ, suggesting that the intracellular ROS levels increased with in MDA-MB-231 cells via up-regulation of the RIP3-MLKL-ROS an increase in MCZ concentration (Fig. 5C). pathway. 292 Pharmazie 74 (2019) ORIGINAL ARTICLES

In conclusion, we explored the effects of MCZ on TNBC subsequent treatment and observation by transmission electron microscopy. Finally, MDA-MB-231 cells, based on the concept of “drug repurposing,” the processed samples were imaged to observe the submicroscopic cellular structures after the administration of MCZ. and observed that MCZ treatment could trigger both, apoptosis and necroptosis in these cells. Therefore, we suggest that MCZ may serve as a promising anti-breast cancer drug, providing a new 4.8. DAPI staining therapeutic strategy for TNBC treatment. MDA-MB-231 cells were seeded in a 12-well as mentioned previously. The cells were cultured for 24 h after treatment with different concentrations of MCZ. After 24 h, the original culture medium was discarded, followed by two washes with PBS, and the 4. Experimental cells were incubated in 500 μl of DAPI dye solution in dark for 5 min, and the changes in the cell nuclei following the MCZ treatment were observed under a fluorescence 4.1. Materials microscope (Olympus, Tokyo, Janpan). Human breast cancer MDA-MB-231 cell line was purchased from the Cell Bank of Chinese Academy of Sciences in Shanghai, China. DMEM and 0.25% trypsin were purchased from Hyclone (UT, USA). Fetal bovine serum was purchased from Hang- 4.9. Western blotting zhou Sijiqing Co., Ltd., China. MTT reagent, JC-1 kit, and reactive oxygen test kits MDA-MB-231 cells were seeded in 60 mm culture dishes at the density of 4 × 105 cells/ were purchased from Beyotime, China. Annexin V/PI staining kit was purchased from dish, and the culture medium was replaced by the medium containing MCZ at different BestBio Tech. Co., South Korea. Primary antibodies used in this study against Bax, concentrations every consecutive day, followed by the transfer to a 37 °C incubator for Bcl-2 and Akt were purchased from Cell Signaling Technology (MA, USA). RIP1, 24 h of culture. After 24 h the cells were harvested, followed by protein extraction and RIP3 and MLKL antibodies were purchased from Abcam, UK. β-Actin antibody, protein quantification by BCA method, and the extracted proteins were separated by goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were SDS-PAGE and transferred to a PVDF membrane. After transfer, the membrane was purchased from Santa Cruz Biotechnology, Inc., CA, USA. MCZ was purchased from blocked, followed by an overnight incubation with primary antibodies (Akt, p-Akt, Bax, China National Institutes for Drug Control (purity > 98%) Bcl-2, RIP1, RIP3 or MLKL) diluted in TBST according to the dilution recommended by the manufacturers at 4 °C. The PVDF membrane was then washed with TBST, followed by incubation with corresponding secondary antibodies at room temperature 4.2. Cell culture for 2 h. After secondary antibody incubation, the PVDF membrane was washed again Human breast cancer MDA-MB-231 cells were cultured in high-glucose DMEM with TBST, followed by developing the blot using enhanced chemiluminescence containing 10 % fetal bovine serum and 100 U/mL penicillin, 100 μg/mL strepto- substrate and were analyzed by Bio-Rad gel imaging system. mycin, in a 37 °C incubator with 5 % CO2 and saturated humidity. 4.10. Detection of mitochondrial membrane potential with JC-1 4.3. MTT assay MDA-MB-231 cells were seeded in 6-well plates at the density of 2 × 105 cells/well The MDA-MB-231 cells in the logarithmic growth phase were trypsinized, centri- overnight for adherence, followed by treatment with different concentrations of MCZ fuged, resuspended, and seeded in a 96-well plate, at the density 6 × 103 cells/ for 24 h. After 24 h, the culture solution was aspirated, followed by the washing of cells well. After adherence, the cells were treated with different concentrations of MCZ with PBS. JC-1 staining solution (1 mL) and culture solution (1 mL) were added in each (0–40 μM) for 24, 48, and 72 h, respectively. After the mentioned time points, 10 μl of well, followed by the well mixing and incubation in a 37 °C incubator for 20 min. During MTT solution (5 μg/ml) was added to each well. After 4 h incubation at 37 °C in the the incubation period, an appropriate amount of JC-1 staining buffer was prepared as per the manufacturer’s instructions and placed on an ice bath. After incubation with JC-1, CO2 incubator, the MTT solution was removed and replaced with 100 μl of DMSO and the absorption was measured at 490 nm using a microplate reader. the supernatant was aspirated from the cells, followed by two washes twice with JC-1 staining buffer. Afterwards, 2 ml of the cell culture medium was added to the wells and the JC-1 staining was observed and imaged by fluorescence microscopy. 4.4. Colony-forming assay MDA-MB-231 cells were seeded in a 6-well plates, at the density of 8 × 103 cells/well. When the cells became adherent and formed colonies, the existing culture medium 4.11. ATP detection was replaced by fresh medium containing MCZ at different concentrations (0, 1, MDA-MB-231 cells were seeded into 12-well plates at previously mentioned density, 2, and 4 μM), and the cells were cultured for 5 days. After 5 days, the cells were followed by treatment with different concentrations of MCZ. After 3 h incubation, washed with phosphate-buffered saline (PBS) and fixed with 4 % paraformaldehyde 100 μl of lysis buffer was added to each well, cells were lysed on ice for 10 min, and for 10 min, followed by staining of cells with 1 ml of crystal violet/well for 10 min at the lysate was centrifuged at 12000 rpm for 5 min. The supernatant was harvested for room temperature, followed by washing with double distilled water until background subsequent examination. An opaque 96-well plate was simultaneously placed on ice. staining completely faded. The cells then were dried at room temperature, photo- 100 μl of ATP test solution was added to each well and was diluted at the ratio 1:9 graphed and quantitatively analyzed by Image J software (NIH, USA). with the dilution buffer prepared on ice, as described in the ATP-test kit instructions, followed by 5 min incubation at room temperature to ensure that the background ATP is completely consumed. From the lysate, 40 μl of the sample was then added 4.5. Detection of cell death using PI staining / fl ow cytometry to 96-well plates accompanied by well mixing, following which ATP levels were determined using a Luminoskan luminometer (Thermo Scientific, Atlanta, GA, USA). MDA-MB-231 cells were seeded into 12-well plates at the density of 1.5 × 105 cells/well. After adherence, the cells were treated with different concentrations of MCZ and were subjected to continuous culture for 24 h. After 24 h, the culture supernatant was transferred to a 5 ml centrifuge tube, followed by washing the cells with PBS and harvesting by 4.12. Detection of reactive oxygen species (ROS) trypsinization, and finally centrifugation at 1500 rpm for 8 min. The supernatant was then MDA-MB-231 cells were seeded in 6-well plates at previously mentioned density discarded, and cells in each tube were fixed by adding 3 ml of pre-cooled 75 % ethanol and overnight for adherence. The cells were treated with MCZ at different concentrations were incubated at 4 °C overnight. After fixation, the cells were centrifuged at 1000 rpm for for 3 h. After 3 h, the original culture solution was removed from each well, followed 10 min and ethanol was removed, followed by a PBS wash, following which 600 μl of PI by the addition of 1 ml of diluted DCFH-DA (DCFH-DA was diluted with the serum- staining solution was added to each tube for staining. After incubation in dark for 2 h, the free medium to a final concentration of 10 μM) and 20 min incubation in the 37 °C percentage of cell death was evaluated in the PI stained cells by Accuri C6 flow cytometry incubator. After the incubation, the cells were washed with PBS twice, followed by (BD Biosciences, Stated of New Jersey, USA) . the analysis of fluorescence intensity of the oxidized substrate, DCF using a fluorescence microscope.

4.6. Detection of cell death using Annexin V-FITC/PI staining MDA-MB-231 cells were cultured in a 12-well dish as mentioned above. After 24 h, 4.13. Statistical analysis 6× 104 cells were harvested from each well by washing with PBS, trypsinization, and The experimental data were analyzed using SPSS v.16.0 software package (SPSS Inc., centrifugation. For Annexin V-FITC staining, 500 μl of buffer and 5 μl of Annexin Chicago, IL,USA), and t-test was performed to compare the data between the two V-FITC staining solution were added to each group of cells, with gentle mixing for 15 groups P*<0.05 and P**<0.01 were considered to be statistically significant differ- min, followed by addition of 5 μl of PI staining solution and subsequent incubation ences between the two test groups. at room temperature in dark for 10 min. The Annexin V-FITC/PI staining was then analyzed by flow cytometry. Acknowledgement: This work was financially supported by the Education Depart- ment of Anhui Natural Science Research Project China under Grant (KJ2018A0232, KJ2018A1009); the Science and Technology Development Fund Project of Bengbu 4.7. Electron microscopy Medical College (BYKF1718); the Foundation of Bengbu Medical College (Byycx Two flasks of MDA-MB-231 cells at about 80 % confluence were selected and 1709). washed twice with PBS. According to the experimental design, DMSO was added to the blank control group, and 40 μM of MCZ was added to the treatment group; Conflicts of interest: None declared and the culture was continued for 24 h. The culture supernatant was removed and the cells were fixed by 2.5% glutaraldehyde for 20 min at room temperature, then cells were harvested. After centrifugation, the supernatant was removed and the cells were References washed twice with pre-cooled PBS. The cells were fixed by 2.5 % glutaraldehyde, Amaravadi RK, Thompson CB (2007) The roles of therapy-induced autophagy and followed by the storage at 4 °C. The fixed samples were sent to Servicebio, China for necrosis in cancer treatment. Clin Cancer Res 13: 7271-7279. Pharmazie 74 (2019) 293 ORIGINAL ARTICLES

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