Cytotoxic effect of DEBIO 1143 on Triple Negative Mouse Breast Cancer Cell Lines by Non-Apoptosis Death Mechanisms

Cytotoxic effect of DEBIO 1143 on Triple Negative Mouse Breast Cancer Cell Lines by Non-Apoptosis Death Mechanisms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytotoxic effect of DEBIO 1143 on Triple Negative Mouse Breast Cancer Cell Lines by Non-Apoptosis Death Mechanisms Berrin Tuğrul, Meral Alp, Erdal Balcan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3543602/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Triple negative breast cancer (TNBC) is a lethal cancer lacking therapeutic targets. In this project, we aimed to investigate the cytotoxic effect of DEBIO 1143, a SMAC mimetic, on 4T1 and 4T1-HER2 mouse TNBC cell lines and the cell death pathway through which this effect is mediated. Methods and results MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used to determine cell viability, flow cytometry analysis was performed to determine apoptosis, and MDC (monodansylcadaverine) was used to determine autophagic vesicles. In addition, LC3II, Beclin 1, RIP3 and cIAP1 proteins were analyzed by western blot. In both cell lines, it was determined that DEBIO 1143 IC 50 doses caused predominantly non-apoptotic cell death in the 48-h drug-treated group. In western blot analysis, LC3II level was significantly increased in 4T1-HER2 cells treated with DEBIO 1143 for 24 and 48 hours compared to control. Beclin 1 expression was significantly elevated in the drug treatment groups of both cell lines. The expression level of RIP3 in 4T1 cells was relatively increased after 24 and 48 h treatment of DEBIO 1143 compared to control. cIAP1 protein bands were detected in both control and 24- h treatment groups of both cell lines, but not in 48- h treatment groups. Conclusions Our findings revealed that DEBIO 1143 showed a time-dependent cytotoxic effect in mouse TNBC cell lines. This effect may be predominantly mediated through non-apoptosis cell death mechanisms such as autophagy and necrosis. Further research is recommended to clarify the issue fully. 4T1 4T1-HER2 DEBIO 1143 Cell death mechanisms SMAC mimetic Triple negative breast cancer Figures Figure 1 Figure 2 Figure 3 1.Introduction Breast cancer (BC) is the most common type of cancer in women, which is the second most common cause of cancer-related deaths and constitutes 29% of all cancers. Breast tumors are defined in six different groups as luminal A, luminal B, human epidermal growth factor receptor (HER2) positive, triple negative, basal-like, and claudin-low [ 1 ]. Among the subtypes, triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancers [ 2 ]. TNBC is a type of cancer that is deficient in estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The treatment is poor, the risk of metastasis is high, and it results in death within five years [ 3 ]. The traditional treatment approach has been the use of cytotoxic therapeutics since the 1970s [ 4 ]. There are currently limitations in the response of TNBC to conventional chemotherapeutic therapy due to their lack of molecular targets [ 5 ]. Due to the lack of specificity of therapeutic targets, it is clear that new targeted therapies and alternative strategies are needed [ 2 ]. Endogenous second mitochondria-derived activator of caspase (SMAC) mimetics are a new class of drugs targeted in cancer therapy. Small molecule inhibitors of apoptosis inhibitor protein, which mimics endogenous apoptosis inhibitor protein antagonist second mitochondrial caspase activators, are called SMAC mimetics. In recent years, studies on the effect of SMAC mimetics on cancer cells have gained momentum [ 6 ]. SMACs are mitochondrial pro-apoptotic proteins that suppress apoptosis inhibitory proteins (IAPs). IAPs are highly expressed in many human cancers. SMAC mimetics bind to IAP proteins and abolish the inhibitory activity of IAPs by disrupting the IAP-caspase and IAP-SMAC interaction [ 7 ]. DEBIO 1143 (AT-406) is an orally active SMAC mimetic that effectively targets XIAP and cIAP-1/2 [ 8 ]. In various human cancer cell lines, it is stated that it suppresses cancer cell growth with high efficiency [ 9 , 10 ]. It has been shown to induce apoptosis in vivo xenograft tumor studies in mice, rats, dogs, and non-human primates [ 11 , 12 ]. Currently, DEBIO 1143 is being investigated in phase I/II clinical trials for human cancer therapy [ 13 ]. SMAC mimetics have been shown to promote different cell death pathways other than apoptosis in different cancer cell types [ 9 , 14 , 15 ]. The effect of SMAC mimetics on different types of cell death depends on the type of cancer, its stage, and the molecules with which they interact. However, these mechanisms are not fully elucidated. In a previous study, we found that DEBIO 1143 co-administration with Tamoxifen had a synergistic cytotoxic effect in MCF-7 and BT-474 estrogen receptor positive (ER+) human breast cancer cell lines. These findings demonstrated that treatment with both the drug and the SMAC mimetic, alone or in combination, induced cell death other than apoptosis, which may be associated with autophagy or necroptosis. In this study, it is recommended to evaluate the expression levels of key proteins associated with apoptosis, necroptosis and autophagy to determine which type of cell death mediates the cytotoxic effect [ 16 ]. Here, the cytotoxic effect of DEBIO 1143 on 4T1 and 4T1-HER2 triple negative mouse breast cancer cell lines was investigated through which of the programmed cell death types. In addition, the expression levels of specific key proteins (LC3II, BECN1, RIP3, and cIAP1) in cell death pathways of apoptosis, autophagy and necroptosis were also evaluated. 2.Materials and methods 2.1. Materials The triple negative mouse 4T1 and 4T1-HER2 mouse breast cancer cell lines were kindly provided by Dr. M. H. Kershaw, Immune Innovation Laboratory Peter MacCallum Cancer Centre, Melbourne, Australia. The 4T1 cell line does not express ER, PR and HER2 proteins (triple negative). The 4T1-HER2 cell line has ER (-), PR (-) and HER2 (+) molecular features. 1 millimolar (mM) stock solution was prepared by dissolving 5 mg of powder DEBIO 1143 (ApexBio, Taiwan) in 8.901 ml of dimethyl sulfoxide (DMSO) in a dark bottle. The prepared stock solution was filtered by 0.22 µm filter and stored at -20˚C. A new stock solution was used for each experiment. 2.2. Cell culture 4T1 and 4T1-HER2 cells were cultured in RPMI-1640 medium by adding 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin/Streptomycin, in incubators containing 5% CO 2 and humidity at 37 o C. The viability, proliferation and infection status of the cells were checked daily with an inverted microscope (Zeiss, Germany). Passaging was performed when it was determined that the cell density reached above 80%. Excess cell lines that would not be used for the experiment were stored in freezing medium in a deep freezer at -80 o C. 2.3. Addition of DEBIO 1143 to cell lines The final concentration of DMSO in the culture medium was set to < 0.1%. DEBIO 1143 was added to 4T1 and 4T1-HER2 mouse breast cancer cell lines in increasing doses (3 micromolar (µM), 5µM, 10µM, 15µM, 20 µM, 25µM). The cell lines were kept in an incubator with 5% CO 2 and humidity at 37 o C for 24 and 48 hours. 2.4. Counting cells by Trypan Blue method After DEBIO 1143 was added to the cell cultures, cell counting was performed using the trypan blue staining method to determine the number of viable cells per ml. Cells attached to the flask were removed with Trypsin/EDTA and centrifuged at 1000 rpm for 10 minutes. The supernatant was then discarded, and the cell pellet was diluted with 10 ml of medium. This suspension and Trypan blue were mixed in a 1:1 ratio. Cells in this mixture were transferred to a Neubeuer slide and counted under a light microscope. 2.5. Cell viability measurement The Methylthiazolyldiphenyl-tetrazolium bromide (MTT) viability assay was used to determine the inhibitory concentration of DEBIO 1143 that caused a 50% reduction in cell viability (IC 50 ). 1 x 10 4 cell per ml were seeded into 96-well plates. Experiments were repeated 3 times for all doses (3µM, 5µM, 10µM, 15µM, 20µM, 25µM). For each dose added to the treatment groups, 3 wells were used. According to the manufacturer’s protocol of the MTT viability assay (M5655, Sigma), the procedures were performed for each cell line for 24 and 48 hours. The results were evaluated with the GraphPad Prism software (Ver. 6.0; GraphPad Software, La Jolla, CA, USA). To calculate the percent viability, the following formula was used: Percent Viability = (Experimental Absorbance – Background Absorbance) / (Positive Control Absorbance – Background Absorbance) x 100% 2.6. FITC annexin V/dead cell apoptosis kit 4T1 and 4T1-HER2 cells were seeded to 6-well plates at 5x10 6 cells/ml per well. DEBIO 1143 was added to the cultures at the IC 50 dose determined for each cell line. Two wells with only antibiotic-free media were designated as negative controls. 4T1 and 4T1-HER2 cells were incubated at 37 o C and 5% CO 2 for 24 and 48 hours. Then, Annexin V and PI staining was performed according to the manufacturer's protocol. Stained cells were analyzed by flow cytometry measuring fluorescence emission at 530 nm (eg FL1) and > 575 nm (eg FL3). 2.7. Monodansylcadaverine (MDC) staining Autofluorescent monodansylcadaverine (MDC) (30432, Sigma) staining was performed to visualize autophagic vacuoles after DEBIO 1143 treatments. Sterile coverslips were placed in 24-well plates before cell culture. Cells were seeded at 1 x 10 5 in each well in 24 well plates (two of the wells are positive control) and incubated for 24 hours. Then, DEBIO 1143 at the IC 50 dose determined for each cell line was applied for 24 and 48 hours. At the end of the periods, the wells were washed 3 times for 5 minutes with cold phosphate buffered saline (PBS). Then the cells were fixed in cold methanol at -20 ºC for 10 minutes and washed 3 times with PBS. Cells fixed on coverslips were stained with 0.005 mM MDC for 10 minutes. Then it was washed with PBS. Autophagic vacuoles were visualized under a fluorescent microscope (Olympus, Japan). Vacuole index was calculated by counting cells with and without vacuoles from different areas. (Vacuole index = number of vacuole (+) cells/ total number of cells x 100). 2.8. Western blotting Western blotting protocol was applied for 4 proteins involved in pathways associated with regulated cell death mechanisms. These proteins are Microtubule-associated protein 1A/1B-light chain 3II (LC3II) with a molecular weight of about 17 kDa (kilodalton), Beclin1 (BECN1) of 52kDa, receptor-interacting protein kinase 3 (RIP3/RIPK3) of 57 kDa and cellular inhibitor of apoptosis protein 1 (cIAP1) of 70kDa. Beta actin (β-actin) of 50 kDa was used as the positive control protein. For SDS-PAGE, appropriate gel concentrations were prepared for proteins isolated from the 4T1 and 4T1-HER2 control and DEBIO 1143 treatment groups at 24 and 48 hours. LC3II, BECN1, RIP3 and cIAP1 proteins separated by SDS-PAGE were transferred from the gel to Polyvinyldifluoride (PVDF) membranes. For this, PVDF was kept in 100% methanol for 1 minute, then passed through bidistilled water and equilibrated with the gel in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 15 minutes. The proteins were transferred to PVDF membranes in a blotting tank with transfer buffer for 1 hour at 350 mA constant current. The PVDF membrane was then removed and blocked with 5% (w/v) non-fat dried milk in Tris-buffered saline-Tween 20 (TBST: 1.0 M Tris (Sigma, T6066), 1.5 M NaCl, 0.05% Tween 20) for 30 minutes. The membrane was washed with TBST 3 times for 5 minutes. The primary antibodies indicated in Table 1 were applied at a concentration of 0.5 µg/ml in pH 7.4 PBS overnight at + 4°C. After the membranes were washed with TBST, the appropriately labeled secondary antibody (Table 1 ) was applied at a concentration of 1 µg/ml in pH 7.4 PBS for 1 hour at 22°C. Band intensities were developed by 3,3′- diaminobenzidine (DAB; Roche-11718096001) as chemiluminescence peroxidase substrate. The reaction was stopped by washing with ultra distilled water and bands were scanned at 600 dpi resolution. Table 1 Properties and dilution rates of primary and secondary antibodies used in Western blotting. Brand /Catalog Host Reactivity Clonality Dilution Signal Primer Antibodies LC3II Invitrogen PA5-115501 Rabbit Hu, Mo, Rt Polyclonal 1:200 HRP Beclin 1 Invitrogen PA5-96649 Rabbit Hu, Mo, Rt Polyclonal 1:500 HRP RIP3 Invitrogen PA5-19956 Rabbit Hu, Mo, Rt Polyclonal 1:500 HRP cIAP1 Invitrogen PA5-20066 Rabbit Hu, Mo Polyclonal 1:250 HRP Seconder Antibodies Anti-Rabbit Thermo Scientific 32460 Go Rb Polyclonal 1:1000 HRP Go: goat, Hu: human, Mo: mouse, Rb: rabbit, Rt: rat. 2.9. Statistical analysis After MDC application to determine autophagic vesicles, the vesicle index was extracted to determine the possible autophagic effect of DEBIO 1143. To do this, the vesicles in 10 randomly selected cells from the control and treatment groups were used in the “Cell Counter” tab in the ImageJ software (version 1.46c, NIH, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/ ). The obtained values were compared statistically in the control and treatment groups using SPSS (ver 15.0. SPSS Inc., Chicago, IL, USA) application. Student's t test was used to compare the groups. The data were taken as mean ± SD. A p value less than 0.05 was considered as a significant difference between the groups. 3.Results All data obtained from the study are given in Table 2 . Table 2 All data obtained from the study CELL LINE RESULTS MTT (IC 50 µM) Flow Cytometry MDC Western Blotting (Band image densities) 1 Viable cells (%) Early apoptotic (%) Late apoptotic (%) Non-apototic (%) Vesicle densities LC3II BECN1 RIP3 cIAP1 4T1 24-h C 99,6 0,0 0,0 0,4 p>0.05 ++ + ++ +++++ D- 1143 20.49 96,7 0,1 0,1 3,2 p = 0.016 +++ ++++ +++ +++++ 4T1-HER2 24-h C 99,6 0,3 0,0 0,1 p>0.05 + ⸺ +++ +++++ D- 1143 19.56 98,1 1,1 0,0 0,8 p = 0.001 ++++ ++++ +++ +++++ 4T1 48-h C 99,3 0,1 0,0 0,6 p>0.05 ++ + +++ +++++ D- 1143 36,2 51,9 0,8 3,6 43,7 p 0.05 + ⸺ +++ +++++ D- 1143 22.45 64,7 3,4 4,8 27,1 p = 0.001 ++++ ++++ +++ ⸺ 1 ⸺ no band; +: band density is very very low; ++: very low band density; +++: low band density; ++++: high band density; +++++: band density more; MTT: Methylthiazolyldiphenyl-tetrazolium bromide MDC: Monodansylcadaverine; C: Control; D-1143: DEBIO 1143 Cell viability measurement The IC 50 values of DEBIO 1143 on 4T1 and 4T1-HER2 cells were calculated as 20.49 µM and 19.56 µM at 24 hours, and 36.2 µM and 22.45 µM at 48 hours, respectively. Flow cytometry findings The flow cytometric results showed that treatment of DEBIO 1143 (20.49 µM, 19.56 µM, respectively) to 4T1 and 4T1-HER2 cells for 24 hours had no significant effect when each cell line was compared to its control group (Fig. 1 ). It was observed that 48 hours of DEBIO 1143 (36.2 µM) application to 4T1 cells stimulated apoptotic cell death by 4.4% and non-apoptosis cell death by 43.7%. When compared to controls, it was determined that the drug had a significant effect on cell death other than apoptosis (Fig. 1 ) Application of DEBIO 1143 (22.45 µM) for 48 hours in the 4T1-HER2 cell line had a cytotoxic effect with 8.2% apoptotic cell death and 27.1% non-apoptosis mechanism(s). When compared with the control group (0.4% dead cell), drug administration causes cell death by both apoptotic (8.2%) and non-apoptotic (27.1%) mechanisms. In this cell line, the cytotoxic effect of the drug triggers the non-apoptotic cell death mechanism (Fig. 1 ). MDC findings In both cell lines, it was determined that DEBIO 1143 application significantly increased the number of vesicles in both 24 and 48 hours. After 24 and 48 hours of application in 4T1 cells, p = 0.016 and p < 0.001, respectively (Fig. 2 a,b), while p = 0.001 in 4T1-HER2 cells (Fig. 2 a,b). However, no significant relationship was found between the vesicle densities in cell lines of 24 and 48 hours DEBIO 1143 applications. Western blotting LC3II protein level was moderately increased after 24- and 48-h of DEBIO 1143 treatments in 4T1 cells compared to control. However, in 4T1-HER2 cell line, LC3II protein bands obtained from both 24 and 48 hours of DEBIO 1143-treatment groups were more intense than controls (Fig. 3 ). No clear bands for BECN1 protein were observed in the control groups. However, the expression level of BECN1 was dramatically increased in both cell lines after 24- and 48-h DEBIO 1143 treatment. RIP3 protein bands were similar in control and treatment groups at 24 and 48 hours in both cell lines (Fig. 3 ). A prominent expression of cIAP1 protein was observed in both 4T1 or 4T1-HER2 cells in control and 24-h DEBIO 1143 treatment. However, no bands of this protein were seen in both cells after 48 h treatment of DEBIO 1143 (Fig. 3 ). 4.Discussion SMAC mimetics is a new treatment strategy developed for different types of cancer. They act as antagonists of apoptosis inhibitors. In recent studies, it has been associated with autophagy and necroptosis, which are cell death mechanisms apart from apoptosis in different cancer types. DEBIO 1143 (AT-406) is an SMAC mimetic that has been investigated in in vitro , in vivo and phase I/II clinical studies on various cancers [ 13 ]. Since TNBC lacks ER, PR and HER 2 expressions, it does not respond to any treatment strategy targeting these receptors. Therefore, there are currently limits to the response to conventional chemotherapeutic treatment [ 17 , 18 ]. DEBIO 1143 is SMAC mimetic of interest among therapeutic agents that can be targeted on cellular functions important in the formation and progression of TNBC [ 19 ]. The IC 50 doses of DEBIO 1143 at 24 and 48 hours were 20.49 µM and 36.2 µM in 4T1 cell line and 19.56 µM and 22.45 µM in 4T1-HER2 cell line, respectively. In both cell lines, cytotoxic effect of DEBIO 1143 was determined to be through non-apoptotic cell death at 48 hours. According to these data, it was determined that DEBIO 1143 had a time-dependent cytotoxic effect on both cell lines. Bellaye et al. [ 20 ] evaluated the effects of DEBIO 1143 on tumor cell death and metabolism in an animal model based on TNBC cell line human MDA-MB-231 and reported that DEBIO 1143 induced early apoptosis both in vitro and in vivo 6 hours after treatment. Another finding from the same study was that DEBIO 1143 showed a dose-dependent increase in late apoptosis/necrosis of MDA-MB-231 cells. The genetic characteristics of this cell line are similar to the 4T1 cell line in our study. However, in our 48 hours cell death finding in this cell line, the higher level of cell death type was non-apoptotic death, and the lower level was early and late apoptosis. In the 48 hours group of 4T1-HER2 cells, similar results were obtained. Moreover, DEBIO 1143 showed a time-dependent effect on cell death in both mouse breast TNBC cell lines. Many cancer therapies, including breast cancer, rely on the activation of the apoptotic pathway leading to cell death. Nevertheless, there is a complex relationship between intracellular apoptosis and autophagy. On the one hand, activation of apoptosis can lead to activation or suppression of autophagy. On the other hand, inactivation of apoptosis may result in either activation or suppression of autophagy. The role of proteins such as Beclin-1 and Bcl-2 in regulating the relationship between these two cell death mechanisms has been emphasized. Various stimuli can determine which of these molecules will play a role in cell fate. The complexity is that activated autophagy may act in the direction of cell death or survival [ 21 ]. SMAC mimetics have been shown to promote different cell death pathways other than apoptosis in different cancer types [ 9 , 14 , 15 ], In our study, administration of the SMAC mimetic DEBIO 1143 to 4T1 and 4T1-HER2 cell lines at IC 50 doses for 48 hours significantly reduced cell viability compared to controls of each. However, the predominant cytotoxic effect in both cell lines appears to be mediated by non-apoptosis death mechanisms. This suggests that SMAC mimetics may activate different types of cell death depending on the cancer type, stage, and molecules they interact with. However, these mechanisms have not been fully elucidated. We recently reported that cell death in ER + breast cancer cell lines caused by DEBIO 1143 alone or in combination with Tamoxifen and SMAC mimetic was not associated with apoptosis but may be associated with other types of cell death [ 16 ]. Our findings from TNBC study are similar to the cell death mechanism findings of DEBIO 1143 in ER + breast cancer cell lines. LC3 is a soluble protein with a molecular mass of ∼17 kDa that is ubiquitous in mammalian tissues and cells in vitro . Autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles, during autophagy. Simultaneously, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form the LC3-phosphatidylethanolamine conjugate (LC3-II), which is taken up into autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosome components are degraded by lysosomal hydrolases. At the same time, LC3-II in the autolysosomal lumen is degraded. Therefore, the autophagosomal marker LC3-II reflects autophagic activity [ 22 ]. LC3 family members are one of the major players in the autophagy pathway and their expression is not associated with a specific tumor type or stage [ 23 ]. In our literature search, we determined that studies on LC3 breast cancer association are very limited. In our study, LC3-II expression was determined in both control and treatment groups in both cell lines at 24 and 48 hours. In 4T1-HER2 cell line, LC3II protein bands obtained from both 24 and 48 hours of DEBIO 1143-treatment groups were more intense than controls. This finding suggests that DEBIO 1143 may exert cytotoxic effects by inducing LC3 expression and autophagy in TNBC. Western blot analysis revealed that Beclin-1 expression was dramatically increased in both cell lines in both 24 and 48 hours drug treated groups. On the other hand, there is a very weak band in the 4T1 control group, while there is no band in the 4T1-HER2 cell line (Table 2 ). These findings suggest that in both cell lines, SMAC mimetic may have an effect via autophagy mechanism, one of the non-apotosis regulated cell death types, in the 24 and 48 hour groups. Consistently, Jung et al. [ 24 ] reported that Beclin-1 is expressed in breast cancer and its subtypes and that may play a role in both breast cancer suppression and tumor progression; therefore, the decision to use Beclin-1 inducer or inhibitor should be made according to breast cancer stage and subtype. In our study, the significant level of BECN1 expression in DEBIO 1143-treated groups in both cell lines suggests that it may inhibit the progression of TNBC breast cancer. Loss of the BECN1 gene increases the risk of aggressive cancers, including HER2 (+) breast cancer. However, it is not clear whether there is a benefit of inducing autophagy in preventing the development of HER2 (+) breast cancer. One study in breast cancer suggests that HER2 interacts with Beclin-1 and suppresses autophagy [ 25 ]. However, low BECN1 mRNA level has been reported to lead to poor prognosis in TNBC [ 26 ]. In our study, it was found that the cytotoxic effect of the drug was mediated by non-apoptosis death mechanism and Beclin-1 was expressed in both cell lines at 24 and 48 hours. This finding suggests that DEBIO 1143 may induce HER2-independent autophagy in cancer cells. Furthermore, MDC staining of autophagic vacuoles showed a significant difference in vesicle density in 4T1 and 4T1-HER2 cells treated with DEBIO 1143 for 24 and 48 hours compared to controls. In addition, the lack of a significant difference in vesicle density between cell lines with differential HER2 expression suggests the possibility that DEBIO 1143 may drive cells to autophagy independently of HER2. RIP3 is a key signaling molecule involved in the necroptosis (programmed necrosis) pathway [ 27 ]. The formation of the RIP1/RIP3 complex is required for the necrosome formation step of the necroptosis mechanism. RIP3 activation is repressed by cIAP1 and cIAP2. IAP antagonists such as SMAC mimetics trigger autoubiquitination and proteasomal degradation of cIAP proteins. This leads to a decrease in RIP1 ubiquitination. Thus, when caspase activation is suppressed, disruption of SMAC mimetic interaction with RIP3 or cIAPs leads to facilitation of non-canonical NFκB signaling for necrosome formation in the cytosol resulting increased cell death. RIP1-dependent complex IIb formation can induce cell necroptosis in the absence of caspase-8 and apoptosis in its presence [ 28 ]. DEBIO 1143 binds to XIAP, cIAP1 and cIAP2 proteins, causes rapid degradation and inhibits cancer cell growth in various human cancer cell lines [ 29 ]. Our western blot analysis results show that RIP3 was expressed in both cell lines treated with DEBIO 1143 for 24 and 48 hours, and cIAP1 was expressed in both cell lines in control and 24 hours groups, but not in 48 hours cells. According to flow cytometry data, cell viability in 4T1 and 4T1-HER2 cell lines was 99.6% and 99.6% in controls and 96.7% and 98.1% in 24-h DEBIO 1143 treated groups, respectively. This suggests that DEBIO 1143 does not inhibit cIAP1s effectively in the 24-h experimental groups. In both cell lines, DEBIO 1143 had a significant percentage of non-apoptosis cell death at 48 hours (Table 2 ). In addition, the presence of RIP3 and absence of cIAP1 in 48 hours-treated 4T1 and 4T1-HER2 cells points out that DEBIO 1143 may also promote necroptosis, one of the cell death types, by inhibiting cIAPs. Due to some technical problems, we could not perform western blot analysis for at least three replicates. Therefore, statistical data for western blotting could not be obtained. We considered this as a limitation of the study. 5.Conclusion In our study, DEBIO 1143 IC 50 dose showed its cytotoxic effect in TNBC mouse breast cancer 4T1 and 4T1-HER2 cell lines within 48 hours in a time-dependent manner. In 48 hours treatments, the cytotoxic effect of DEBIO 1143 on both cell lines may be through non-apoptotic cell death mechanisms. We determined that DEBIO 1143 caused cell death by suppressing cIAP1s. The presence of Beclin 1 expression supports the possibility that the non-apoptotic effect can be induced by autophagy and that this effect directs cells to HER2-independent autophagy. In conclusion, this study revealed that DEBIO 1143 exerts its effect on TNBC in vitro through non-apoptotic cell death mechanisms. In the light of the data obtained from this study, further in vivo investigations and combined drug trials may contribute to the development of new treatment strategies for TNBC, which is lacking in terms of therapeutic targets. Declarations Acknowledgements We thank to Scientific Research Projects Committee of Manisa Celal Bayar University for their financial supports. Author contributions Berrin Tugrul: Conceptualization, Methodology, Writing – original draft, Funding acquisition. Meral Alp: Methodology. Erdal Balcan: Methodology. Funding This work supported by the Scientific Research Projects Committee of Manisa Celal Bayar University, Project No. 2019- 006. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. 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Clin Cancer Res 23:1379-1387. https://doi:10.1158/1078-0432.CCR-16-2172 Lecis D, De Cesare M, Perego P, Conti A, Corna E, Drago C et al (2013) Smac mimetics induce inflammation and necrotic tumour cell death by modulating macrophage activity. Cell Death Dis 4:e920. https://doi.org/10.1038/cddis.2013.449 Bai L, Smith DC, Wang S (2014) Small-molecule SMAC mimetics as new cancer therapeutics. Pharmacol Ther 144:82-95. https://doi: 10.1016/j.pharmthera.2014.05.007 İşseven M, Tuğrul B (2020) The effect of DEBIO 1143 usage alone or in combination with tamoxifen on estrogen receptor positive breast cancer cell lines. Pamukkale Medical J 13:9-18. https://doi.org/10.31362/patd.623005 Lehmann BD, Pietenpol JA (2014) Identification and use of biomarkers in treatment strategies for triple-negative breast cancer subtypes. J Pathol 232:142-150. https://doi:10.1002/path.4280 Abramson VG, Lehmann BD, Ballinger TJ, Pietenpol JA (2015) Subtyping of triple-negative breast cancer: implications for therapy. Cancer 121:8-16. https://doi: 10.1002/cncr.28914. Epub 2014 Jul 16. Ocana A, Pandiella A (2017) Targeting oncogenic vulnerabilities in triple negative breast cancer: biological bases and ongoing clinical studies. Oncotarget 8:22218-22234. https://doi:10.18632/oncotarget.14731 Bellaye PS, Oudot A, Vrigneaud JM, Raguin O, Bichat F, Vaslin A et al (2018) Nuclear Imaging Study of the Pharmacodynamic Effects of Debio 1143, an Antagonist of Multiple Inhibitor of Apoptosis Proteins (IAPs), in a Triple-Negative Breast Cancer Model. Contrast Media Mol Imaging 2018:8494031. https://doi.org/10.1155/2018/8494031 Zambrano J, Yeh ES (2016) Autophagy and Apoptotic Crosstalk: Mechanism of Therapeutic Resistance in HER2-Positive Breast Cancer. Breast Cancer (Auckl) 10:13-23. https://doi.org/10.4137/BCBCR.S32791 Tanida I, Ueno T, Kominami E (2008) LC3 and Autophagy. Methods Mol Biol 445:77-88. https://doi.org/10.1007/978-1-59745-157-4_4 Jacquet M, Guittaut M, Fraichard A, Despouy G (2021) The functions of Atg8-family proteins in autophagy and cancer: linked or unrelated? Autophagy 17:599-611. https://doi:10.1080/15548627.2020.1749367 Jung YY, Lee YK, Koo JS (2016) The potential of Beclin 1 as a therapeutic target for the treatment of breast cancer. Expert Opin Ther Targets 20:167-178. https://doi.org/10.1517/14728222.2016.108597 Vega-Rubin-de-Celis S, Zou Z, Fernandez AF, Ci B, Kim M, Xiao G, Xie Y, Levine B (2018) Increased autophagy blocks HER2-mediated breast tumorigenesis. Proc Natl Acad Sci U S A 115:4176-4181. https://doi.org/10.1073/pnas.1717800115 Cicchini M, Chakrabarti R, Kongara S, Price S, Nahar R, Lozy F et al (2014) Autophagy regulator BECN1 suppresses mammary tumorigenesis driven by WNT1 activation and following parity. Autophagy 10:2036-2052. https://doi:10.4161/auto.34398 Sun, L, Wang H, Wang Z, He S, Chen S, Liao D et al (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148:213-227. https://doi.org/10.1016/j.cell.2011.11.031 Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P (2014) Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol 15:135-147. https://doi.org/10.1038/nrm3737 Casimiro S, Alho I, Bettencourt M, Pires R, Lipton A, Costa L (2013) RANKL enhances the effect of an antagonist of inhibitor of apoptosis proteins (cIAPs) in RANK-positive breast cancer cells. J Bone Oncol 2:116-122. https://doi.org/10.1016/j.jbo.2013.07.001 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3543602","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":245299050,"identity":"56d58596-970d-4229-92c9-d5b56dd36fd6","order_by":0,"name":"Berrin Tuğrul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACAxCRAOM9qICwJYjRAlGUcAauxQC/Fri5iW1EaDFn73324eEOhjqD82ufSSTOs8szOMB88DYPw598XFose44bz0g8wyBhcOO5mUTituRigwNsydY8DAaWDbgcdiONGeQeoJZjbEAtBxI3HOAxkwZqwekyg/vPkLXMAWnh/4Zfyw02qJbzbUAtDWBb2PBqsewBO0xCciZQr0XCseRiycNsxpZzDIxxajFnP8bM+LPNhp/v/DHGGx9q7PL4jjc/vPGmQg53KEMAMCIkEqBsZrCDCWgAA/4DxKgaBaNgFIyCkQgACO5OyvJ0wUsAAAAASUVORK5CYII=","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Berrin","middleName":"","lastName":"Tuğrul","suffix":""},{"id":245299051,"identity":"84a2bb09-bdd7-4765-ac9e-a627ec14f4d3","order_by":1,"name":"Meral Alp","email":"","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meral","middleName":"","lastName":"Alp","suffix":""},{"id":245299052,"identity":"d7880d0d-f93a-4516-9d80-b0445381361a","order_by":2,"name":"Erdal Balcan","email":"","orcid":"","institution":"Manisa Celal Bayar University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erdal","middleName":"","lastName":"Balcan","suffix":""}],"badges":[],"createdAt":"2023-11-02 11:29:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3543602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3543602/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46029083,"identity":"4e302c17-f14b-416c-adf5-b517037b5cf2","added_by":"auto","created_at":"2023-11-07 17:47:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":931227,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry analysis images of 4T1 and 4T1-HER2 cells stained with FITC Annexin V after 24 and 48 hours of drug treatment.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3543602/v1/cbf1273e2c60f91afe5079e3.png"},{"id":46027138,"identity":"33675a86-9a39-4283-ac2a-27ade66ca7f9","added_by":"auto","created_at":"2023-11-07 17:39:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1319629,"visible":true,"origin":"","legend":"\u003cp\u003eMDC fluorescence microscope images in 4 T1 and 4T1-HER2 cells treated with 24 (a) and 48 (b) hours DEBIO 1143 and the distribution graph of the number of vesicles according to drug doses in the cells.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3543602/v1/2aa4c0fd184dda31d4e8dcd9.png"},{"id":46027136,"identity":"11f4871d-219c-4867-9e5c-f43bd6023b8b","added_by":"auto","created_at":"2023-11-07 17:39:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246884,"visible":true,"origin":"","legend":"\u003cp\u003eAdministration and control groups western blot findings. β-actin was used as positive control protein. Cont; control group, 24-h; 24 hours cell line, 48 h; 48 hours cell line.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3543602/v1/2dbc180d5227304b500bba74.png"},{"id":46031101,"identity":"c3163468-299a-457c-859a-ac731ddb4bd2","added_by":"auto","created_at":"2023-11-07 18:03:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2559557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3543602/v1/3f474aa1-3a83-4127-bf36-a1806b7a4e0e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxic effect of DEBIO 1143 on Triple Negative Mouse Breast Cancer Cell Lines by Non-Apoptosis Death Mechanisms","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most common type of cancer in women, which is the second most common cause of cancer-related deaths and constitutes 29% of all cancers. Breast tumors are defined in six different groups as luminal A, luminal B, human epidermal growth factor receptor (HER2) positive, triple negative, basal-like, and claudin-low [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the subtypes, triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. TNBC is a type of cancer that is deficient in estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The treatment is poor, the risk of metastasis is high, and it results in death within five years [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The traditional treatment approach has been the use of cytotoxic therapeutics since the 1970s [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. There are currently limitations in the response of TNBC to conventional chemotherapeutic therapy due to their lack of molecular targets [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to the lack of specificity of therapeutic targets, it is clear that new targeted therapies and alternative strategies are needed [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEndogenous second mitochondria-derived activator of caspase (SMAC) mimetics are a new class of drugs targeted in cancer therapy. Small molecule inhibitors of apoptosis inhibitor protein, which mimics endogenous apoptosis inhibitor protein antagonist second mitochondrial caspase activators, are called SMAC mimetics. In recent years, studies on the effect of SMAC mimetics on cancer cells have gained momentum [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. SMACs are mitochondrial pro-apoptotic proteins that suppress apoptosis inhibitory proteins (IAPs). IAPs are highly expressed in many human cancers. SMAC mimetics bind to IAP proteins and abolish the inhibitory activity of IAPs by disrupting the IAP-caspase and IAP-SMAC interaction [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDEBIO 1143 (AT-406) is an orally active SMAC mimetic that effectively targets XIAP and cIAP-1/2 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In various human cancer cell lines, it is stated that it suppresses cancer cell growth with high efficiency [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It has been shown to induce apoptosis \u003cem\u003ein vivo\u003c/em\u003e xenograft tumor studies in mice, rats, dogs, and non-human primates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Currently, DEBIO 1143 is being investigated in phase I/II clinical trials for human cancer therapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSMAC mimetics have been shown to promote different cell death pathways other than apoptosis in different cancer cell types [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The effect of SMAC mimetics on different types of cell death depends on the type of cancer, its stage, and the molecules with which they interact. However, these mechanisms are not fully elucidated.\u003c/p\u003e \u003cp\u003eIn a previous study, we found that DEBIO 1143 co-administration with Tamoxifen had a synergistic cytotoxic effect in MCF-7 and BT-474 estrogen receptor positive (ER+) human breast cancer cell lines. These findings demonstrated that treatment with both the drug and the SMAC mimetic, alone or in combination, induced cell death other than apoptosis, which may be associated with autophagy or necroptosis. In this study, it is recommended to evaluate the expression levels of key proteins associated with apoptosis, necroptosis and autophagy to determine which type of cell death mediates the cytotoxic effect [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, the cytotoxic effect of DEBIO 1143 on 4T1 and 4T1-HER2 triple negative mouse breast cancer cell lines was investigated through which of the programmed cell death types. In addition, the expression levels of specific key proteins (LC3II, BECN1, RIP3, and cIAP1) in cell death pathways of apoptosis, autophagy and necroptosis were also evaluated.\u003c/p\u003e"},{"header":"2.Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe triple negative mouse 4T1 and 4T1-HER2 mouse breast cancer cell lines were kindly provided by Dr. M. H. Kershaw, Immune Innovation Laboratory Peter MacCallum Cancer Centre, Melbourne, Australia. The 4T1 cell line does not express ER, PR and HER2 proteins (triple negative). The 4T1-HER2 cell line has ER (-), PR (-) and HER2 (+) molecular features.\u003c/p\u003e \u003cp\u003e1 millimolar (mM) stock solution was prepared by dissolving 5 mg of powder DEBIO 1143 (ApexBio, Taiwan) in 8.901 ml of dimethyl sulfoxide (DMSO) in a dark bottle. The prepared stock solution was filtered by 0.22 \u0026micro;m filter and stored at -20˚C. A new stock solution was used for each experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell culture\u003c/h2\u003e \u003cp\u003e4T1 and 4T1-HER2 cells were cultured in RPMI-1640 medium by adding 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin/Streptomycin, in incubators containing 5% CO\u003csub\u003e2\u003c/sub\u003e and humidity at 37\u003csup\u003eo\u003c/sup\u003eC. The viability, proliferation and infection status of the cells were checked daily with an inverted microscope (Zeiss, Germany). Passaging was performed when it was determined that the cell density reached above 80%. Excess cell lines that would not be used for the experiment were stored in freezing medium in a deep freezer at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Addition of DEBIO 1143 to cell lines\u003c/h2\u003e \u003cp\u003eThe final concentration of DMSO in the culture medium was set to \u0026lt;\u0026thinsp;0.1%. DEBIO 1143 was added to 4T1 and 4T1-HER2 mouse breast cancer cell lines in increasing doses (3 micromolar (\u0026micro;M), 5\u0026micro;M, 10\u0026micro;M, 15\u0026micro;M, 20 \u0026micro;M, 25\u0026micro;M). The cell lines were kept in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e and humidity at 37\u003csup\u003eo\u003c/sup\u003eC for 24 and 48 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Counting cells by Trypan Blue method\u003c/h2\u003e \u003cp\u003eAfter DEBIO 1143 was added to the cell cultures, cell counting was performed using the trypan blue staining method to determine the number of viable cells per ml. Cells attached to the flask were removed with Trypsin/EDTA and centrifuged at 1000 rpm for 10 minutes. The supernatant was then discarded, and the cell pellet was diluted with 10 ml of medium. This suspension and Trypan blue were mixed in a 1:1 ratio. Cells in this mixture were transferred to a Neubeuer slide and counted under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell viability measurement\u003c/h2\u003e \u003cp\u003eThe Methylthiazolyldiphenyl-tetrazolium bromide (MTT) viability assay was used to determine the inhibitory concentration of DEBIO 1143 that caused a 50% reduction in cell viability (IC\u003csub\u003e50\u003c/sub\u003e). 1 x 10\u003csup\u003e4\u003c/sup\u003e cell per ml were seeded into 96-well plates. Experiments were repeated 3 times for all doses (3\u0026micro;M, 5\u0026micro;M, 10\u0026micro;M, 15\u0026micro;M, 20\u0026micro;M, 25\u0026micro;M). For each dose added to the treatment groups, 3 wells were used. According to the manufacturer\u0026rsquo;s protocol of the MTT viability assay (M5655, Sigma), the procedures were performed for each cell line for 24 and 48 hours. The results were evaluated with the GraphPad Prism software (Ver. 6.0; GraphPad Software, La Jolla, CA, USA). To calculate the percent viability, the following formula was used:\u003c/p\u003e \u003cp\u003ePercent Viability = (Experimental Absorbance \u0026ndash; Background Absorbance) / (Positive Control Absorbance \u0026ndash; Background Absorbance) x 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. FITC annexin V/dead cell apoptosis kit\u003c/h2\u003e \u003cp\u003e4T1 and 4T1-HER2 cells were seeded to 6-well plates at 5x10\u003csup\u003e6\u003c/sup\u003e cells/ml per well. DEBIO 1143 was added to the cultures at the IC\u003csub\u003e50\u003c/sub\u003e dose determined for each cell line. Two wells with only antibiotic-free media were designated as negative controls. 4T1 and 4T1-HER2 cells were incubated at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 and 48 hours. Then, Annexin V and PI staining was performed according to the manufacturer's protocol. Stained cells were analyzed by flow cytometry measuring fluorescence emission at 530 nm (eg FL1) and \u0026gt;\u0026thinsp;575 nm (eg FL3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Monodansylcadaverine (MDC) staining\u003c/h2\u003e \u003cp\u003eAutofluorescent monodansylcadaverine (MDC) (30432, Sigma) staining was performed to visualize autophagic vacuoles after DEBIO 1143 treatments. Sterile coverslips were placed in 24-well plates before cell culture. Cells were seeded at 1 x 10\u003csup\u003e5\u003c/sup\u003e in each well in 24 well plates (two of the wells are positive control) and incubated for 24 hours. Then, DEBIO 1143 at the IC\u003csub\u003e50\u003c/sub\u003e dose determined for each cell line was applied for 24 and 48 hours. At the end of the periods, the wells were washed 3 times for 5 minutes with cold phosphate buffered saline (PBS). Then the cells were fixed in cold methanol at -20 \u0026ordm;C for 10 minutes and washed 3 times with PBS. Cells fixed on coverslips were stained with 0.005 mM MDC for 10 minutes. Then it was washed with PBS. Autophagic vacuoles were visualized under a fluorescent microscope (Olympus, Japan). Vacuole index was calculated by counting cells with and without vacuoles from different areas. (Vacuole index\u0026thinsp;=\u0026thinsp;number of vacuole (+) cells/ total number of cells x 100).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blotting\u003c/h2\u003e \u003cp\u003eWestern blotting protocol was applied for 4 proteins involved in pathways associated with regulated cell death mechanisms. These proteins are Microtubule-associated protein 1A/1B-light chain 3II (LC3II) with a molecular weight of about 17 kDa (kilodalton), Beclin1 (BECN1) of 52kDa, receptor-interacting protein kinase 3 (RIP3/RIPK3) of 57 kDa and cellular inhibitor of apoptosis protein 1 (cIAP1) of 70kDa. Beta actin (β-actin) of 50 kDa was used as the positive control protein.\u003c/p\u003e \u003cp\u003eFor SDS-PAGE, appropriate gel concentrations were prepared for proteins isolated from the 4T1 and 4T1-HER2 control and DEBIO 1143 treatment groups at 24 and 48 hours. LC3II, BECN1, RIP3 and cIAP1 proteins separated by SDS-PAGE were transferred from the gel to Polyvinyldifluoride (PVDF) membranes. For this, PVDF was kept in 100% methanol for 1 minute, then passed through bidistilled water and equilibrated with the gel in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 15 minutes. The proteins were transferred to PVDF membranes in a blotting tank with transfer buffer for 1 hour at 350 mA constant current. The PVDF membrane was then removed and blocked with 5% (w/v) non-fat dried milk in Tris-buffered saline-Tween 20 (TBST: 1.0 M Tris (Sigma, T6066), 1.5 M NaCl, 0.05% Tween 20) for 30 minutes. The membrane was washed with TBST 3 times for 5 minutes. The primary antibodies indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were applied at a concentration of 0.5 \u0026micro;g/ml in pH 7.4 PBS overnight at +\u0026thinsp;4\u0026deg;C. After the membranes were washed with TBST, the appropriately labeled secondary antibody (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was applied at a concentration of 1 \u0026micro;g/ml in pH 7.4 PBS for 1 hour at 22\u0026deg;C. Band intensities were developed by 3,3\u0026prime;- diaminobenzidine (DAB; Roche-11718096001) as chemiluminescence peroxidase substrate. The reaction was stopped by washing with ultra distilled water and bands were scanned at 600 dpi resolution.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProperties and dilution rates of primary and secondary antibodies used in Western blotting.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrand\u003c/p\u003e \u003cp\u003e/Catalog\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReactivity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eClonality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSignal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003ePrimer Antibodies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLC3II\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen PA5-115501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHu, Mo, Rt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1:200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBeclin 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen PA5-96649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHu, Mo, Rt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRIP3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen PA5-19956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHu, Mo, Rt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1:500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ecIAP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInvitrogen PA5-20066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHu, Mo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1:250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSeconder Antibodies\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAnti-Rabbit\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThermo Scientific 32460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePolyclonal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHRP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGo: goat, Hu: human, Mo: mouse, Rb: rabbit, Rt: rat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eAfter MDC application to determine autophagic vesicles, the vesicle index was extracted to determine the possible autophagic effect of DEBIO 1143. To do this, the vesicles in 10 randomly selected cells from the control and treatment groups were used in the \u0026ldquo;Cell Counter\u0026rdquo; tab in the ImageJ software (version 1.46c, NIH, Bethesda, MD, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsb.info.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"http://rsb.info.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The obtained values were compared statistically in the control and treatment groups using SPSS (ver 15.0. SPSS Inc., Chicago, IL, USA) application. Student's \u003cem\u003et\u003c/em\u003e test was used to compare the groups. The data were taken as mean \u0026plusmn; SD. A p value less than 0.05 was considered as a significant difference between the groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3.Results","content":"\u003cp\u003eAll data obtained from the study are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e All data obtained from the study\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"756\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.26984126984127%\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eCELL LINE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"83.73015873015873%\" colspan=\"10\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRESULTS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.424960505529226%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eMTT\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"44.707740916271725%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.006319115323855%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.8609794628752%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eWestern Blotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Band image densities)\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.693877551020408%\"\u003e\n \u003cp\u003e\u003cstrong\u003eViable cells (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.92517006802721%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEarly apoptotic (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.92517006802721%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLate apoptotic (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.92517006802721%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNon-apototic (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.92517006802721%\"\u003e\n \u003cp\u003e\u003cstrong\u003eVesicle densities\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.693877551020408%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLC3II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBECN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.993197278911564%\"\u003e\n \u003cp\u003e\u003cstrong\u003eRIP3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.693877551020408%\"\u003e\n \u003cp\u003e\u003cstrong\u003ecIAP1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.397622192866579%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e4T1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e24-h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e99,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003ep\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.4151212553495%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD- 1143\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e20.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e96,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e0,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e0,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e3,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003ep = 0.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.4151212553495%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.397622192866579%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e4T1-HER2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e24-h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e99,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003ep\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\"\u003e\n \u003cp\u003e\u003cstrong\u003e⸺\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.4151212553495%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD- 1143\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e19.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e98,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e1,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e0,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e0,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003ep = 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.4151212553495%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.397622192866579%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e4T1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e48-h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e99,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003ep\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.4151212553495%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eD- 1143\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e36,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e51,9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e0,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e3,6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e43,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003ep \u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.4151212553495%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e⸺\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"7.397622192866579%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4T1-HER2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e48-h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e99,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003e0,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.039630118890356%\"\u003e\n \u003cp\u003ep\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.718626155878468%\"\u003e\n \u003cp\u003e\u003cstrong\u003e⸺\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.208718626155878%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.5297225891677675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.4151212553495%\"\u003e\n \u003cp\u003e\u003cstrong\u003eD- 1143\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e22.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e64,7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e3,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e4,8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003e27,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.841654778887303%\"\u003e\n \u003cp\u003ep = 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.4151212553495%\"\u003e\n \u003cp\u003e\u003cstrong\u003e++++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.704707560627675%\"\u003e\n \u003cp\u003e\u003cstrong\u003e+++\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131241084165477%\"\u003e\n \u003cp\u003e\u003cstrong\u003e⸺\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e⸺ no band; +: band density is very very low; ++: very low band density; +++: low band density; ++++: high band density; +++++: band density more; MTT: Methylthiazolyldiphenyl-tetrazolium bromide MDC:\u0026nbsp;Monodansylcadaverine; C: Control; D-1143: DEBIO 1143\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e values of DEBIO 1143 on 4T1 and 4T1-HER2 cells were calculated as 20.49 \u0026micro;M and 19.56 \u0026micro;M at 24 hours, and 36.2 \u0026micro;M and 22.45 \u0026micro;M at 48 hours, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry findings\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe flow cytometric results showed that treatment of DEBIO 1143 (20.49 \u0026micro;M, 19.56 \u0026micro;M, respectively) to 4T1 and 4T1-HER2 cells for 24 hours had no significant effect when each cell line was compared to its control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It was observed that 48 hours of DEBIO 1143 (36.2 \u0026micro;M) application to 4T1 cells stimulated apoptotic cell death by 4.4% and non-apoptosis cell death by 43.7%. When compared to controls, it was determined that the drug had a significant effect on cell death other than apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApplication of DEBIO 1143 (22.45 \u0026micro;M) for 48 hours in the 4T1-HER2 cell line had a cytotoxic effect with 8.2% apoptotic cell death and 27.1% non-apoptosis mechanism(s). When compared with the control group (0.4% dead cell), drug administration causes cell death by both apoptotic (8.2%) and non-apoptotic (27.1%) mechanisms. In this cell line, the cytotoxic effect of the drug triggers the non-apoptotic cell death mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMDC findings\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn both cell lines, it was determined that DEBIO 1143 application significantly increased the number of vesicles in both 24 and 48 hours. After 24 and 48 hours of application in 4T1 cells, p\u0026thinsp;=\u0026thinsp;0.016 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b), while p\u0026thinsp;=\u0026thinsp;0.001 in 4T1-HER2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). However, no significant relationship was found between the vesicle densities in cell lines of 24 and 48 hours DEBIO 1143 applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLC3II protein level was moderately increased after 24- and 48-h of DEBIO 1143 treatments in 4T1 cells compared to control. However, in 4T1-HER2 cell line, LC3II protein bands obtained from both 24 and 48 hours of DEBIO 1143-treatment groups were more intense than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo clear bands for BECN1 protein were observed in the control groups. However, the expression level of BECN1 was dramatically increased in both cell lines after 24- and 48-h DEBIO 1143 treatment.\u003c/p\u003e \u003cp\u003eRIP3 protein bands were similar in control and treatment groups at 24 and 48 hours in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA prominent expression of cIAP1 protein was observed in both 4T1 or 4T1-HER2 cells in control and 24-h DEBIO 1143 treatment. However, no bands of this protein were seen in both cells after 48 h treatment of DEBIO 1143 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"4.Discussion","content":"\u003cp\u003eSMAC mimetics is a new treatment strategy developed for different types of cancer. They act as antagonists of apoptosis inhibitors. In recent studies, it has been associated with autophagy and necroptosis, which are cell death mechanisms apart from apoptosis in different cancer types. DEBIO 1143 (AT-406) is an SMAC mimetic that has been investigated in \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003ein vivo\u003c/em\u003e and phase I/II clinical studies on various cancers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince TNBC lacks ER, PR and HER 2 expressions, it does not respond to any treatment strategy targeting these receptors. Therefore, there are currently limits to the response to conventional chemotherapeutic treatment [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. DEBIO 1143 is SMAC mimetic of interest among therapeutic agents that can be targeted on cellular functions important in the formation and progression of TNBC [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e doses of DEBIO 1143 at 24 and 48 hours were 20.49 \u0026micro;M and 36.2 \u0026micro;M in 4T1 cell line and 19.56 \u0026micro;M and 22.45 \u0026micro;M in 4T1-HER2 cell line, respectively. In both cell lines, cytotoxic effect of DEBIO 1143 was determined to be through non-apoptotic cell death at 48 hours. According to these data, it was determined that DEBIO 1143 had a time-dependent cytotoxic effect on both cell lines. Bellaye et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] evaluated the effects of DEBIO 1143 on tumor cell death and metabolism in an animal model based on TNBC cell line human MDA-MB-231 and reported that DEBIO 1143 induced early apoptosis both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e 6 hours after treatment. Another finding from the same study was that DEBIO 1143 showed a dose-dependent increase in late apoptosis/necrosis of MDA-MB-231 cells. The genetic characteristics of this cell line are similar to the 4T1 cell line in our study. However, in our 48 hours cell death finding in this cell line, the higher level of cell death type was non-apoptotic death, and the lower level was early and late apoptosis. In the 48 hours group of 4T1-HER2 cells, similar results were obtained. Moreover, DEBIO 1143 showed a time-dependent effect on cell death in both mouse breast TNBC cell lines.\u003c/p\u003e \u003cp\u003eMany cancer therapies, including breast cancer, rely on the activation of the apoptotic pathway leading to cell death. Nevertheless, there is a complex relationship between intracellular apoptosis and autophagy. On the one hand, activation of apoptosis can lead to activation or suppression of autophagy. On the other hand, inactivation of apoptosis may result in either activation or suppression of autophagy. The role of proteins such as Beclin-1 and Bcl-2 in regulating the relationship between these two cell death mechanisms has been emphasized. Various stimuli can determine which of these molecules will play a role in cell fate. The complexity is that activated autophagy may act in the direction of cell death or survival [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSMAC mimetics have been shown to promote different cell death pathways other than apoptosis in different cancer types [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], In our study, administration of the SMAC mimetic DEBIO 1143 to 4T1 and 4T1-HER2 cell lines at IC\u003csub\u003e50\u003c/sub\u003e doses for 48 hours significantly reduced cell viability compared to controls of each. However, the predominant cytotoxic effect in both cell lines appears to be mediated by non-apoptosis death mechanisms. This suggests that SMAC mimetics may activate different types of cell death depending on the cancer type, stage, and molecules they interact with. However, these mechanisms have not been fully elucidated. We recently reported that cell death in ER\u0026thinsp;+\u0026thinsp;breast cancer cell lines caused by DEBIO 1143 alone or in combination with Tamoxifen and SMAC mimetic was not associated with apoptosis but may be associated with other types of cell death [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our findings from TNBC study are similar to the cell death mechanism findings of DEBIO 1143 in ER\u0026thinsp;+\u0026thinsp;breast cancer cell lines.\u003c/p\u003e \u003cp\u003eLC3 is a soluble protein with a molecular mass of \u0026sim;17 kDa that is ubiquitous in mammalian tissues and cells \u003cem\u003ein vitro\u003c/em\u003e. Autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles, during autophagy. Simultaneously, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form the LC3-phosphatidylethanolamine conjugate (LC3-II), which is taken up into autophagosomal membranes. Autophagosomes fuse with lysosomes to form autolysosomes, and intra-autophagosome components are degraded by lysosomal hydrolases. At the same time, LC3-II in the autolysosomal lumen is degraded. Therefore, the autophagosomal marker LC3-II reflects autophagic activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. LC3 family members are one of the major players in the autophagy pathway and their expression is not associated with a specific tumor type or stage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In our literature search, we determined that studies on LC3 breast cancer association are very limited. In our study, LC3-II expression was determined in both control and treatment groups in both cell lines at 24 and 48 hours. In 4T1-HER2 cell line, LC3II protein bands obtained from both 24 and 48 hours of DEBIO 1143-treatment groups were more intense than controls. This finding suggests that DEBIO 1143 may exert cytotoxic effects by inducing LC3 expression and autophagy in TNBC.\u003c/p\u003e \u003cp\u003eWestern blot analysis revealed that Beclin-1 expression was dramatically increased in both cell lines in both 24 and 48 hours drug treated groups. On the other hand, there is a very weak band in the 4T1 control group, while there is no band in the 4T1-HER2 cell line (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings suggest that in both cell lines, SMAC mimetic may have an effect via autophagy mechanism, one of the non-apotosis regulated cell death types, in the 24 and 48 hour groups. Consistently, Jung et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] reported that Beclin-1 is expressed in breast cancer and its subtypes and that may play a role in both breast cancer suppression and tumor progression; therefore, the decision to use Beclin-1 inducer or inhibitor should be made according to breast cancer stage and subtype. In our study, the significant level of BECN1 expression in DEBIO 1143-treated groups in both cell lines suggests that it may inhibit the progression of TNBC breast cancer.\u003c/p\u003e \u003cp\u003eLoss of the BECN1 gene increases the risk of aggressive cancers, including HER2 (+) breast cancer. However, it is not clear whether there is a benefit of inducing autophagy in preventing the development of HER2 (+) breast cancer. One study in breast cancer suggests that HER2 interacts with Beclin-1 and suppresses autophagy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, low BECN1 mRNA level has been reported to lead to poor prognosis in TNBC [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, it was found that the cytotoxic effect of the drug was mediated by non-apoptosis death mechanism and Beclin-1 was expressed in both cell lines at 24 and 48 hours. This finding suggests that DEBIO 1143 may induce HER2-independent autophagy in cancer cells. Furthermore, MDC staining of autophagic vacuoles showed a significant difference in vesicle density in 4T1 and 4T1-HER2 cells treated with DEBIO 1143 for 24 and 48 hours compared to controls. In addition, the lack of a significant difference in vesicle density between cell lines with differential HER2 expression suggests the possibility that DEBIO 1143 may drive cells to autophagy independently of HER2.\u003c/p\u003e \u003cp\u003eRIP3 is a key signaling molecule involved in the necroptosis (programmed necrosis) pathway [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The formation of the RIP1/RIP3 complex is required for the necrosome formation step of the necroptosis mechanism. RIP3 activation is repressed by cIAP1 and cIAP2. IAP antagonists such as SMAC mimetics trigger autoubiquitination and proteasomal degradation of cIAP proteins. This leads to a decrease in RIP1 ubiquitination. Thus, when caspase activation is suppressed, disruption of SMAC mimetic interaction with RIP3 or cIAPs leads to facilitation of non-canonical NFκB signaling for necrosome formation in the cytosol resulting increased cell death. RIP1-dependent complex IIb formation can induce cell necroptosis in the absence of caspase-8 and apoptosis in its presence [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDEBIO 1143 binds to XIAP, cIAP1 and cIAP2 proteins, causes rapid degradation and inhibits cancer cell growth in various human cancer cell lines [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our western blot analysis results show that RIP3 was expressed in both cell lines treated with DEBIO 1143 for 24 and 48 hours, and cIAP1 was expressed in both cell lines in control and 24 hours groups, but not in 48 hours cells. According to flow cytometry data, cell viability in 4T1 and 4T1-HER2 cell lines was 99.6% and 99.6% in controls and 96.7% and 98.1% in 24-h DEBIO 1143 treated groups, respectively. This suggests that DEBIO 1143 does not inhibit cIAP1s effectively in the 24-h experimental groups. In both cell lines, DEBIO 1143 had a significant percentage of non-apoptosis cell death at 48 hours (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the presence of RIP3 and absence of cIAP1 in 48 hours-treated 4T1 and 4T1-HER2 cells points out that DEBIO 1143 may also promote necroptosis, one of the cell death types, by inhibiting cIAPs.\u003c/p\u003e \u003cp\u003eDue to some technical problems, we could not perform western blot analysis for at least three replicates. Therefore, statistical data for western blotting could not be obtained. We considered this as a limitation of the study.\u003c/p\u003e"},{"header":"5.Conclusion","content":"\u003cp\u003eIn our study, DEBIO 1143 IC\u003csub\u003e50\u003c/sub\u003e dose showed its cytotoxic effect in TNBC mouse breast cancer 4T1 and 4T1-HER2 cell lines within 48 hours in a time-dependent manner. In 48 hours treatments, the cytotoxic effect of DEBIO 1143 on both cell lines may be through non-apoptotic cell death mechanisms. We determined that DEBIO 1143 caused cell death by suppressing cIAP1s. The presence of Beclin 1 expression supports the possibility that the non-apoptotic effect can be induced by autophagy and that this effect directs cells to HER2-independent autophagy.\u003c/p\u003e \u003cp\u003eIn conclusion, this study revealed that DEBIO 1143 exerts its effect on TNBC \u003cem\u003ein vitro\u003c/em\u003e through non-apoptotic cell death mechanisms. In the light of the data obtained from this study, further \u003cem\u003ein vivo\u003c/em\u003e investigations and combined drug trials may contribute to the development of new treatment strategies for TNBC, which is lacking in terms of therapeutic targets.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe thank to Scientific Research Projects Committee of Manisa Celal Bayar University for their financial supports.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eBerrin Tugrul: Conceptualization, Methodology, Writing \u0026ndash; original draft, Funding acquisition. Meral Alp: Methodology. Erdal Balcan: Methodology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work supported by the Scientific Research Projects Committee of Manisa Celal Bayar University, Project No. 2019- 006.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study, which include molecular biologic, immunophenotypic and computational data are inclued in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiarte S, Alonso-Romero JL, Nicolas FJ (2018) SIRT1 and Estrogen Signaling Cooperation for Breast Cancer Onset and Progression. 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Cell Death Dis 4:e920. https://doi.org/10.1038/cddis.2013.449 \u003c/li\u003e\n\u003cli\u003eBai L, Smith DC, Wang S (2014) Small-molecule SMAC mimetics as new cancer therapeutics. Pharmacol Ther 144:82-95. https://doi: 10.1016/j.pharmthera.2014.05.007\u003c/li\u003e\n\u003cli\u003eİşseven M, Tuğrul B (2020) The effect of DEBIO 1143 usage alone or in combination with tamoxifen on estrogen receptor positive breast cancer cell lines. Pamukkale Medical J 13:9-18. https://doi.org/10.31362/patd.623005\u003c/li\u003e\n\u003cli\u003eLehmann BD, Pietenpol JA (2014) Identification and use of biomarkers in treatment strategies for triple-negative breast cancer subtypes. J Pathol 232:142-150. https://doi:10.1002/path.4280\u003c/li\u003e\n\u003cli\u003eAbramson VG, Lehmann BD, Ballinger TJ, Pietenpol JA (2015) Subtyping of triple-negative breast cancer: implications for therapy. Cancer 121:8-16. https://doi: 10.1002/cncr.28914. 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Proc Natl Acad Sci U S A 115:4176-4181. https://doi.org/10.1073/pnas.1717800115 \u003c/li\u003e\n\u003cli\u003eCicchini M, Chakrabarti R, Kongara S, Price S, Nahar R, Lozy F et al (2014) Autophagy regulator BECN1 suppresses mammary tumorigenesis driven by WNT1 activation and following parity. Autophagy 10:2036-2052. https://doi:10.4161/auto.34398\u003c/li\u003e\n\u003cli\u003eSun, L, Wang H, Wang Z, He S, Chen S, Liao D et al (2012) Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148:213-227. https://doi.org/10.1016/j.cell.2011.11.031 \u003c/li\u003e\n\u003cli\u003eVanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P (2014) Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol 15:135-147. https://doi.org/10.1038/nrm3737 \u003c/li\u003e\n\u003cli\u003eCasimiro S, Alho I, Bettencourt M, Pires R, Lipton A, Costa L (2013) RANKL enhances the effect of an antagonist of inhibitor of apoptosis proteins (cIAPs) in RANK-positive breast cancer cells. J Bone Oncol 2:116-122. https://doi.org/10.1016/j.jbo.2013.07.001 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"4T1, 4T1-HER2, DEBIO 1143, Cell death mechanisms, SMAC mimetic, Triple negative breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-3543602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3543602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTriple negative breast cancer (TNBC) is a lethal cancer lacking therapeutic targets. In this project, we aimed to investigate the cytotoxic effect of DEBIO 1143, a SMAC mimetic, on 4T1 and 4T1-HER2 mouse TNBC cell lines and the cell death pathway through which this effect is mediated.\u003c/p\u003e\u003ch2\u003eMethods and results\u003c/h2\u003e \u003cp\u003eMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used to determine cell viability, flow cytometry analysis was performed to determine apoptosis, and MDC (monodansylcadaverine) was used to determine autophagic vesicles. In addition, LC3II, Beclin 1, RIP3 and cIAP1 proteins were analyzed by western blot. In both cell lines, it was determined that DEBIO 1143 IC\u003csub\u003e50\u003c/sub\u003e doses caused predominantly non-apoptotic cell death in the 48-h drug-treated group. In western blot analysis, LC3II level was significantly increased in 4T1-HER2 cells treated with DEBIO 1143 for 24 and 48 hours compared to control. Beclin 1 expression was significantly elevated in the drug treatment groups of both cell lines. The expression level of RIP3 in 4T1 cells was relatively increased after 24 and 48 h treatment of DEBIO 1143 compared to control. cIAP1 protein bands were detected in both control and 24- h treatment groups of both cell lines, but not in 48- h treatment groups.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings revealed that DEBIO 1143 showed a time-dependent cytotoxic effect in mouse TNBC cell lines. This effect may be predominantly mediated through non-apoptosis cell death mechanisms such as autophagy and necrosis. Further research is recommended to clarify the issue fully.\u003c/p\u003e","manuscriptTitle":"Cytotoxic effect of DEBIO 1143 on Triple Negative Mouse Breast Cancer Cell Lines by Non-Apoptosis Death Mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-07 17:39:42","doi":"10.21203/rs.3.rs-3543602/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c2c385d-ae3e-49cb-b370-6fb22dee7493","owner":[],"postedDate":"November 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-07T17:39:44+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-07 17:39:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3543602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3543602","identity":"rs-3543602","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}