A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage

A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage Victor P. Liu, Wai-Ming Li, Jack Lofroth, Mehreen Zeb, Brian O. Patrick, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2701574/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 May, 2023 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Dispiropiperazine compounds are a class of molecules known to confer biological activity, but those that have been studied as cell cycle regulators are few in number. Here, we report the characterization and synthesis of two dispiropiperazine derivatives: the previously synthesized spiro[2’,3]-bis(acenaphthene-1’-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, 1 ), and its previously undescribed isomer, spiro[2’,5’]-bis(acenaphthene-1’-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-5, 2 ). SPOPP-3 ( 1 ), but not SPOPP-5 ( 2 ), was shown to have anti-proliferative activity against a panel of 18 human cancer cell lines with IC 50 values ranging from 0.63-13 µM. Flow cytometry analysis revealed that SPOPP-3 ( 1 ) was able to arrest cell cycle at the G2/M phase in SW480 human cancer cells. Western blot analysis further confirmed the cell cycle arrest is in the M phase. In addition, SPOPP-3 ( 1 ) was shown to induce apoptosis, necrosis, and DNA damage as well as disrupt mitotic spindle positioning in SW480 cells. These results warrant further investigation of SPOPP-3 ( 1 ) as a novel anti-cancer agent, particularly for its potential ability to sensitize cancer cells for radiation-induced cell death, enhance cancer immunotherapy, overcome apoptosis-related drug resistance and for possible use in synthetic lethality cancer treatments. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Chemical biology Biological sciences/Drug discovery Physical sciences/Chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The use of chemicals and radiation to induce DNA damage is the most commonly used method for cancer therapy. Recently, there is an increased interest in manipulating the cell cycle to induce mitotic catastrophe as a novel anti-cancer therapeutic strategy 1 – 5 . Mitotic catastrophe is characterized by cells which would normally be arrested in G2/M phase due to damage in DNA or mitotic spindle but falsely proceed to mitosis due to defective cell cycle checkpoints 6 . The end result is senescence or cell death via either apoptosis, necrosis or autophagy 7 . This strategy relies on the use of DNA damaging agents or radiation in combination with cell cycle checkpoint inhibitors. Indeed, several G2/M phase checkpoint inhibitors 1 – 5 , including irinotecan, a currently used chemotherapeutic drug for metastatic colorectal cancer, have shown potential to sensitize tumor cells to ionizing radiation. As such, the discovery of new compounds which cause G2/M cell cycle arrest remains an important area of cancer research 8 – 10 . There are currently few known biologically active dispiropiperazine derivatives. One of which is prospidium chloride. This compound, also known as prospidine, has cytostatic, anti-inflammatory and immuno-suppressive properties 11 – 13 . It has been classified as an anti-neoplastic compound based on its ability to inhibit T and B cell mitogenesis during lymphoblastic transformation 11 , and to lower tumor volumes of carcinogen-induced mammary tumors in rats 12 . Currently, prospidium chloride is used as an anti-rheumatic drug in refractory rheumatoid arthritis 14 . Despite previous reports of biologically active dispiropiperazine compounds, other chemical derivatives have not been sufficiently explored. Here, we report the discovery of a specific dispiropiperazine derivative, spiro[2’,3]-bis(acenaphthene-1’-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, 1 ) with anti-proliferative activity against a panel of human cancer cell lines and is capable of arresting cell cycle at G2/M phase, and inducing apoptosis, necrosis and DNA damage as well as disrupting mitotic spindle positioning. Results Synthesis of SPOPP-3 (1) and SPOPP-5 (2). A recent report demonstrated the synthesis of two dispiropiperazine derivatives, spiro[2’,3]-bis(acenaphthene-1’-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (referred to here as SPOPP-3, 1 ) and spiro[2’,5]-bis(acenaphthene-1’-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine, through an azomethine ylide cycloaddition reaction using acenaphthenequinone (AcQ) and L-proline as substrates 15 . We performed a similar reaction with slight modifications as described in the Material and Methods to obtain SPOPP-3 ( 1 ) (Fig. 1 ). Surprisingly, we also obtained a small quantity of spiro[2’,5’]-bis(acenaphthene-1’-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (referred to here as SPOPP-5, 2 ) (Fig. 1 and Supplementary Figs. S2, S3, S6-11), an isomer which has not been previously isolated due to its predicted unfavorable formation pathway 15 , 16 . Herein, we report for the first time, the purity (Supplementary Fig. S2) and structure of SPOPP-5 ( 2 ) as determined by FTIR (Supplementary Fig. S3), NMR (Supplementary Figs. S6-11) and X-ray diffraction analyses (Supplementary Fig. S12). SPOPP-3 (1) reduced cell viability in human cancer cell lines. To our knowledge, no bioactivity has been reported for SPOPP-3 ( 1 ). Herein, we show for the first time that SPOPP-3 ( 1 ) significantly reduced cell viability in human colon cancer cells (Fig. 2 ). For SPOPP-3 ( 1 ), the IC 50 was 5.06 ± 1.43 µM, 5.42 ± 0.96 µM and 2.44 ± 0.83 µM in SW480, HT29 and HCT116 human colon cancer cells respectively (Table 1 ). In contrast, its isomer SPOPP-5 ( 2 ) had no significant effect, with IC 50 > 100 µM (Fig. 2 and Table 1 ). We also assessed SPOPP-3 ( 1 ) and SPOPP-5 ( 2 ) on a panel of human cancer cell lines and the summary is shown in Table 1 . Doxorubicin was used as a positive control on the cell lines (Table 1 ). SPOPP-3 ( 1 ) remains inhibitory with IC 50 values ranging from 0.63 ± 0.17 µM in human T lymphoblastoid cell line CEM to 13.0 ± 1.96 µM in human hepatoma cell line HepG2. Again, SPOPP-5 ( 2 ) showed insignificant activity in four additional cancer cell lines (MiaPaca-2, Panc-1, SKOV3 and MDA-MB-231) (Table 1 ). Based on the results that SPOPP-5 ( 2 ) had no significant anti-cell viability effect on seven cancer cell lines, it was not further assessed on the other cell lines. The results showed that SPOPP-3 ( 1 ) has a relatively strong anti-proliferative effect on a panel of human cancer cell lines. Table 1 IC 50 of SPOPP-3 ( 1 ), SPOPP-5 ( 2 ) and doxorubicin against human cancer cell lines. The IC 50 the data shown is an average taken from three independent experiments. ND indicates Not Determined. Cell lines Types IC 50 (µM) of Compounds SPOPP-3 (1) SPOPP-5 (2) Doxorubicin SW480 Human colon cancer 5.06 ± 1.43 > 100 0.22 ± 0.16 HT29 Human colon cancer 5.42 ± 0.96 > 100 0.26 ± 0.02 HCT116 Human colon cancer 2.44 ± 0.83 > 100 0.10 ± 0.05 MiaPaca2 Human pancreatic cancer 8.62 ± 3.18 > 100 0.25 ± 0.24 Panc1 Human pancreatic cancer 9.17 ± 2.67 > 100 0.43 ± 0.14 SKOV3 Human ovarian cancer 6.30 ± 1.35 > 100 0.04 ± 0.01 MDA-MB-231 Human breast cancer 6.17 ± 1.84 > 100 1.06 ± 0.03 MCF-7 Human breast cancer 5.76 ± 0.10 ND 0.03 ± 0.02 MCF-7-Adr Human breast cancer 4.00 ± 0.85 ND ND T47D Human breast cancer 4.76 ± 1.24 ND ND HepG2 Human liver cancer 13.03 ± 1.96 ND 0.36 ± 0.12 HeLa Human cervical cancer 4.23 ± 1.29 ND 0.26 ± 0.12 DU145 Human prostate cancer 9.80 ± 1.94 ND 0.02 ± 0.01 K562 Human leukemia 3.60 ± 1.13 ND ND KG1a Human leukemia 1.76 ± 0.84 ND ND CEM Human leukemia 0.63 ± 0.17 ND ND U251 Human glioblastoma 2.95 ± 0.09 ND 0.01 ± 0.004 U87 Human glioblastoma 6.30 ± 1.72 ND 0.01 ± 0.002 SPOPP-3 (1) arrested cell cycle at G2/M phase. To determine the mechanism whereby SPOPP-3 ( 1 ) decreases cell viability, flow cytometry was performed for cell cycle analysis. As shown in Fig. 3 , treatment with 20 µM SPOPP-3 ( 1 ) caused cell cycle arrest in the G2/M phase (Fig. 3 b). No activity was observed for SPOPP-5 ( 2 ). To further investigate whether SPOPP-3 ( 1 ) induces arrest at G2 or M phase, we performed Western blot analysis to detect phospho-histone H3, an established sensitive mitotic marker 17 . Indeed, phospho-histone H3 level was clearly increased in SW480 cells treated with SPOPP-3 ( 1 ) but not SPOPP-5 ( 2 ) (Fig. 4 ), indicating that SPOPP-3 ( 1 ) arrests SW480 cells at the M phase of the cell cycle. To further investigate the effects of SPOPP-3 ( 1 ) on the cell cycle, we performed immunofluorescence experiments to study cyclin B1 activation. Cyclin B1 is one of the key factors in controlling entry into mitosis 18 , 19 with its expression rapidly increased in G2 phase and peaking at late G2 or early M phase 20 , 21 . As shown in Fig. 5 , the population of tetraploid cells in SPOPP-3 ( 1 )-treated cells as indicated by cells having doubled DAPI signal, was significantly increased when compared with DMSO control. This confirms flow cytometry results that SPOPP-3 ( 1 ) caused cell cycle arrest in the G2/M phase where the cells failed to divide into daughter cells. In control cells, most of the tetraploid cells had significantly higher cyclin B1 staining compared to SPOPP-3-treated tetraploid cells (Fig. 5 b). Thus, our results indicate that SPOPP-3 ( 1 ) treatment is associated with defective cyclin B1 activation. SPOPP-3 (1), but not SPOPP-5 (2), induced cell apoptosis and necrosis. To determine the possible effect of SPOPP-3 ( 1 ) on cell death that led to the observed decrease in cell viability, we used flow cytometry to analyse apoptosis and necrosis. The commonly used stains to detect necrosis and apoptosis are 7-AAD and Annexin V-PE respectively. After treatment with SPOPP-3 ( 1 ), SPOPP-5 ( 2 ) or 2% DMSO for 24 h, cells were double stained with 7-AAD and Annexin V-PE. As shown in Fig. 6 a, cells treated with SPOPP-3 ( 1 ) changed to a more necrotic state (Q1; 32.64%) as compared to the DMSO-treated cells (Q1; 0.5%), and this is statistically significant (Fig. 6 b). SPOPP-3 ( 1 ) also significantly induced apoptosis (Q2 + Q4) in SW480 cells (Fig. 6 ). On the other hand, SPOPP-5 ( 2 ) had no significant effect on apoptosis or necrosis as compared to the control. Effect of SPOPP-3 (1) on mitotic spindle formation. Immunofluorescence studies were carried out to investigate the potential function of SPOPP-3 ( 1 ) as a microtubule toxin. Two drugs, vinblastine and colchicine which are well known to disrupt microtubules, were used as positive controls. Vinblastine, which belongs to the family of vinca alkaloids, binds to tubulin at a specific site and inhibits mitotic spindle formation leading to cell cycle disruption 22 , 23 . When used at high concentrations, vinblastine is known to cause paracrystal formation due to tightly packed tubulin aggregates 24 . Indeed, when SW480 cells were treated with 50 nM vinblastine, paracrystals were observed (Fig. 7 ). Colchicine, on the other hand, disrupted microtubules and caused diffuse a-tubulin staining throughout the cells (Fig. 7 ). In contrast, mitotic spindles could be observed in cells treated with SPOPP-3 ( 1 ). However, the positions of the mitotic spindles appeared to be disrupted when compared with control cells treated with DMSO (Fig. 7 ). Displacement of the mitotic spindles was also associated with the lack of chromosome alignment at the equator of the cell (Fig. 7 ). In summary, these results suggest that although SPOPP-3 ( 1 ) does not disrupt microtubule formation, mitotic spindle positioning appears to be affected. SPOPP-3 (1) induced DNA damage. Based on the findings that SPOPP-3 ( 1 ) causes G2/M arrest associated with cyclin B1 down regulation and mitotic spindle displacement, it may be possible that the activity of SPOPP-3 ( 1 ) is via DNA damage 19 , 25 . To investigate this possibility, we used the qPCR-based method (LORD-Q) to detect DNA lesions 26 . Since this method can detect DNA damage regardless of the type of DNA lesion, we reason that this is the most appropriate method. As shown in Fig. 8 , although the data did not reach statistical significance because of the large variation in data, the effect of SPOPP-3 ( 1 ) on DNA damage can be detected as early as 1 h after treatment. Discussion In this study, we report the synthesis of SPOPP-3 ( 1 ) and show for the first time that it has strong anti-proliferative activity against 18 human cancer cell lines (Table 1 ). To our surprise, using the previously reported synthesis procedure 15 , 16 , we also obtained the structural isomer SPOPP-5 ( 2 ), a novel compound. In contrast to SPOPP-3 ( 1 ), SPOPP-5 ( 2 ) had virtually no anti-proliferative activity (Table 1 ). The anti-proliferative effect of SPOPP-3 ( 1 ) was greatest against human leukemia cells lines (IC 50 from 0.63 to 3.60 µM), human glioblastoma cell lines (IC 50 from 2.95 to 6.30 µM), human colon cancer cell lines (IC 50 from 2.44 to 5.42 µM), human cervical cancer cell lines (IC 50 of 4.23 µM), human ovarian cancer cell lines (IC 50 of 6.30 µM) and human breast cancer cell lines (IC 50 from 4.00 to 6.17 µM). SPOPP-3 ( 1 ) has slightly weaker anti-proliferative activity against human liver cancer cell line (IC 50 of 13.03 µM), human pancreatic cancer cell lines (IC 50 from 8.62 to 9.17 µM) and human prostate cancer cell line (IC 50 of 9.80 µM). The anti-proliferative activity of SPOPP-3 ( 1 ) was associated with its ability to induce apoptosis and necrosis (Fig. 6 ), as well its ability to cause cell cycle arrest in the G2/M phase (Fig. 3 ). Using phosphorylated histone H3 as an M phase marker, it was shown that at least some cells were arrested in M phase (Fig. 4 ). Our results also showed that cyclin B1 expression was drastically reduced upon SPOPP-3 ( 1 ) treatment (Fig. 5 ). Although a rise in cyclin B1 expression in late G2 phase and its translocation to the nucleus is important for the initiation of mitosis, its depletion using siRNA knockdown does not cause cells to be arrested only in G2 phase as this can be explained by the redundant function of cyclin B2 18,27 . We also demonstrated using microscopy that while microtubules seem to be unaffected by SPOPP-3 ( 1 ) treatment, defects in the M phase including mitotic spindle positioning and condensed chromosome alignment, were observed (Fig. 7 ). This may be due to decreased cyclin B1 levels as it is known that cyclin B1 is normally recruited to centrosomes and kinetochores 28 , 29 . We further investigated whether SPOPP-3 ( 1 ) causes DNA damage as it has been documented that cyclin B1 levels are reduced as a result of DNA damage 30 , 31 . Indeed, our results suggest that similar to bleomycin 26 , SPOPP-3 ( 1 ) causes DNA damage at an early stage (Fig. 8 ). As a consequence, such an early DNA damage event could lead to cyclin B1 reduction, cell cycle arrest, apoptosis and necrosis which are cellular processes that occur much later. The anti-proliferative properties of SPOPP-3 ( 1 ) has important implications in cancer therapy. Synthetic lethality is a novel approach in cancer treatment 4 , 5 . Since SPOPP-3 ( 1 ) is a potent inducer of G2/M arrest, it has the potential to be used in combination with G2/M checkpoint inhibitors to trigger mitotic catastrophe which is currently viewed as a favorable treatment strategy to enhance cell death either via apoptosis, necrosis or autophagy 1 – 5 . Particularly, in most of melanoma cases where G1/S transition mediated by the cyclin-CDK4 pathway is defective and has increased dependence on the G2/M checkpoint to induce cell cycle arrest when exposed to DNA damage, SPOPP-3 ( 1 ) may have an added advantage in combination with G2/M inhibitors 4 . Secondly, we found that SPOPP-3 ( 1 ) is an inducer of necrosis. Several lines of investigations have provided evidence to support the role of necrosis in enhancing cancer immunotherapy and as a possible strategy to overcome the resistance of cancer cells to apoptosis 32 , 33 . To this end, it will be important to assess whether SPOPP-3 ( 1 ) has such prowess in inducing pro-inflammatory processes by means of releasing damage-associated molecular patterns such as high mobility group 1 (HMGB1) protein, a marker for necrosis. Release of such factors into the extracellular matrix may lead to activation of CD8 + leukocytes and promote anti-tumor immunity 33 . It is also important to decipher further the mechanism whereby SPOPP-3 ( 1 ) arrests cells at G2/M phase. For instance, piperazine derivatives have been shown to generate reactive oxygen species (ROS) within cells 34 . Therefore, it would be of interest to investigate whether SPOPP-3 ( 1 ) can generate ROS leading to oxidative DNA damage and subsequent arrest at G2/M phase. In terms of necrosis, one of the modes of cell death detected in cells treated with SPOPP-3 ( 1 ), it has become increasingly clear that necrosis may not simply be an uncontrolled cellular process, but rather a regulated pathway commonly known as necroptosis 35 . Necroptosis has also been reported to increase cancer metastasis in certain cell lines 36 . The duality of necroptosis being anti- and pro-tumorigenic still requires investigation to determine in what context is necroptosis beneficial. Since we have found respectable anti-proliferative activity of SPOPP-3 ( 1 ) against a large panel of cancer cell lines, it would be integral to further study the mode of cell death in different cell lines. In conclusion, we describe the synthesis and biochemical characterization of a specific dispiropiperazine derivative called SPOPP-3 ( 1 ) with strong anti-proliferative activity against a wide panel of human cancer cell lines. SPOPP-3 ( 1 ) is able to induce DNA damage, apoptosis, necrosis, arrests cell cycle at G2/M phase and disrupts normal mitotic spindle positioning. This study has laid the foundation for the further development of SPOPP-3 ( 1 ) for possible use as a chemical tool to perturb and understand cellular processes, as well as a potential anti-cancer compound. For the latter, SPOPP-3 ( 1 ) should be explored in synthetic lethality approach and as a necrosis-inducing anti-cancer compound. Methods And Materials Synthesis and purification of SPOPP-3 (1) and SPOPP-5 (2). We adopted the previously described method for synthesizing spiro[2′,3]-bis(acenaphthene-1′-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, 1 ) 15 , 16 . A mixture of acenaphthenequinone (1.822 g, 10 mmol) and L-proline (1.151 g, 10 mmol; Sigma-Aldrich) were dissolved in methanol (200 mL) and heated at 35°C for 3 h. The reaction was monitored by thin layer chromatography (TLC) using ethyl acetate:hexane (1:2; v/v), and the spots were visualized using UV light (254 nm). Typically, an orange precipitate was formed during the first hour which changed to an orange-brown color after an additional hour and to a dark brown color after completion of the reaction. Solvent was removed under reduced pressure leaving a dark brown powder (1.722 g). Crude product, containing both SPOPP-3 ( 1 ) and SPOPP-5 ( 2 ), was mixed with 2 g of normal silica in 200 mL methanol. Solvent was removed under reduced pressure and the silica mixture was dry-loaded onto a column. Purification by flash chromatography, using normal-phase silica (50 g) and a 9:1 mixture of hexanes and ethyl acetate at a flow rate of 12 mL/min, was used to isolate a mixture of SPOPP-3 ( 1 ) and SPOPP-5 ( 2 ). Subsequent purification with three runs of flash chromatography employing a total of 686 mg of crude product were conducted. An orange-coloured band eluting at 530–830 mL corresponding to Rf = 0.14 with 9:1 (hexanes:ethyl acetate) was dried under reduced pressure to produce 280 mg of semi-purified SPOPP-3 ( 1 ). SPOPP-3 ( 1 ) was subsequently purified for biological testing by HPLC using a Phenomenex Luna 5u Phenyl-Hexyl 4.60 mm x 250 mm column. A gradient from 80–92% acetonitrile for 6 min at a flow rate of 2 mL/min was used as the eluent conditions. For the semi-purified SPOPP-3 ( 1 ), a total of 12.5 mg was injected into the HPLC and 5 mg (16%) of purified SPOPP-3 ( 1 ) (at retention of 4.08 min) was obtained. A yellow-coloured band eluting at 320–405 mL corresponding to Rf = 0.24 (9:1 hexanes:ethyl acetate) produced 120 mg of purified SPOPP-5 ( 2 ). SPOPP-5 ( 2 ) was purified using the same HPLC conditions as SPOPP-3 ( 1 ). One hundred twenty mg of semi-purified SPOPP-5 ( 2 ) was injected into HPLC and 21 mg (3%) of purified SPOPP-5 ( 2 ) (at retention of 6.02 min) was obtained. HPLC-MS and NMR analyses were used to confirm the identity of SPOPP-3 ( 1 ) and SPOPP-5 ( 2 ). 1D and 2D NMR spectra were recorded on an Agilent/Varian Inova 400 MHz NMR spectrometer with a 5 mm Kimble NMR tube (Rockwood, TN, USA) at the University of Alberta or on a Bruker 600 MHz NMR with a cryoprobe at the University of British Columbia. All HPLC analyses were performed on Agilent 1260 Infinity Systems with UV detector, and mass spectrometry were done using Agilent 6120 Single Quad MS. X-ray crystallographic analysis. Single orange irregular crystals of SPOPP-5 ( 2 ) (50 mg) were recrystallized from acetonitrile (10 mL) by slow evaporation. Crystals were obtained on day 7. A suitable crystal with dimensions 0.22 × 0.20 × 0.11 mm 3 was selected and mounted on a Bruker APEX II area detector diffractometer. The crystal was kept at a steady T = 90(2) K during data collection. The structure was solved with the ShelXT 37 solution program using dual methods and Olex2 38 as the graphical interface. The model was refined with XL 37 using full matrix least squares minimization on F 2 . Cell culture. All cell lines were obtained from American Type Culture Collection except HT29 (colon adenocarcinoma) which was obtained from Dr. Ranjana Bird at UNBC. All cells were maintained in Eagle’s Minimal Essential Medium (Lonza) except the following: MiaPaca-2 and Panc-1 were maintained in Dulbecco’s Modified Eagle Medium (Lonza), while K562, KG1a and CEM were maintained in RPMI 1640 medium (Lonza). All media were supplemented with 10% fetal bovine serum (Life Technologies Inc.) and antibiotics. Cell viability assay. The cytotoxic MTT assay was used to assess cell viability as previously described 39 . Briefly, cells were plated at a density of 1.5 x 10 3 cells/well in 96-well plates. After 24 h, cells were treated with various concentrations of SPOPP-3 ( 1 ) or SPOPP-5 ( 2 ) for 48 h. All absorbance data were expressed relative to the control, 0.1% DMSO, taken as 100% cell viability. Preparation of cell lysates, immunoblot analysis and antibodies. SW480 cells were seeded in 6-well plates and treated with 20 µM SPOPP-3 ( 1 ), 20 µM SPOPP-5 ( 2 ), or 2% DMSO for 24 h. Cell lysates were prepared as previously described 40 . For immunoblot analysis, protein samples were resolved on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The phospho H3 antibody (Ser10) (D2C8, 1:1,000, Cell Signaling) was used with an overnight incubation at 4°C. Anti-GAPDH (G8795, clone GAPDH-71.1, 1:20,000, Sigma) was also used. Anti-mouse IgM-HRP (sc-2064, 1:4,000, Santa Cruz Biotechnology), anti-mouse IgG-HRP (W402B, 1:4,000, Promega) and anti-rabbit IgG-HRP (W401B, 1:4,000, Promega) were used as secondary antibodies. All blots were visualized with the FluorChem Q system (ProteinSimple). Densitometry analysis was performed using the AlphaView Q software (ProteinSimple). Flow cytometry apoptosis and cell cycle analyses. Cells were plated at 2.5 x 10 5 cells/well in 6-well plates and treated with compounds as described above. Cells were trypsinized followed by centrifugation and washed twice with phosphate-buffered saline. Live cells were stained with PE Annexin V and 7-AAD according to the manufacturer’s instructions for Apoptosis Detection Kit I (BD Pharmingen) and analysed by flow cytometry using a BD FACSMelody cell sorter (BD Biosciences) and BD FACSChorus software (V 1.0). For cell cycle analysis, the BD cycletest plus DNA reagent kit was used to stain for DNA and the data was analysed using the software FlowJo. Immunofluorescence. SW480 cells were plated in 4-well cover glass chambers at a density of 15 x 10 4 cells per well with 0.5 mL EMEM. The cells were treated with the indicated drugs for 24 hours before fixation using 100% methanol (-20°C) for 10 min. Subsequently, the cells were blocked using PBS with 2% BSA and 0.1% Triton-x 100 and then stained with α-tubulin antibody (1:100; Ab4074; Abcam) or cyclin B1 antibody (D5C10, 1:200, cell signaling). Anti-mouse-AF594 and anti-rabbit-AF 594 (1:200; Molecular Probes) were used as secondary antibodies respectively. All antibodies used were diluted in PBS with 0.5% BSA and 0.1% Triton-x 100. All blocking and antibody incubation steps were conducted for 1 h at room temperature. After each antibody incubation step, the cells were washed 3 times (10 min each) with wash buffer containing 0.1% Triton-x-100 in PBS. For cyclin B1 immunofluorescence, cells were fixed in 4% paraformaldehyde for 15 min at room temperature. To stain for DNA, cells were incubated with DAPI dihydrochloride (300 nM diluted in PBS) for 5 min after immunostaining with the secondary antibody. All fluorescence images were taken using an inverted Zeiss Axio Observer Z1 microscope with a motorized stage, Zeiss Axiocam 503 mono camera, Colibri 2 multicolor LED light source, Plan-Apochromat 20 x /0.8 M27 or Plan-Apochromat 63 x /1.4 Oil DIC M27 as objectives. For the quantification of tetraploid cells, DAPI signal in each cell was quantified in each image using the region of interest function in the Zen software. Tetraploid cells were distinguished with a DAPI signal double that of the remaining cells in the same image. Three images (each with at least 36 cells) from each group were quantified. The population of tetraploid cells that were cyclin B1 positive was then determined in each image and presented as an average for each group. DNA damage quantification. To assess whether SPOPP-3 treatment causes DNA damage, a long-run real-time PCR-based method (LORD-Q) was used with modifications 26 . SW480 cells (2.5 x 10 6 ) were treated in 6-well plates with DMSO (control) or 40 mM SPOPP-3 for the indicated time periods before being harvested for total DNA isolation using the DNeasy Blood and Tissue kit (Qiagen). LORD-Q method was carried out using the established primer sets for both the long and short amplicons for the mtDNA gene 26 . PCR efficiencies were calculated using 37.5, 18.75, 9.375 and 4.688 ng DNA as template per PCR reaction. The rtPCR reaction (15 mL total volume) consisted of 0.05 x ResoLight dye, 1x KAPA2G Fast Hot Start ReadyMix, 500 nM of forward and reverse primer, and the aforementioned quantities of isolated DNA as template. For amplification of short amplicons as reference, Quantabio PerfeCTa® SYBR® Green FastMix® was used. Real time PCR analysis was carried out using a Bio-Rad CFX96 system and data analysis was performed using CFX Maestro software. The number of lesions per 10kb was calculated based on the previously established Eq. 2 6,41 . The data presented is averaged from 3 biological replicates ± S.E.M. Statistical analysis. The cytotoxic MTT assays were performed in triplicate (3 wells/treatment) and the absorbance reading from the respective control was taken as 100% cell viability. For immunoblot analysis, the densitometry of phosphor-histone H3 bands were normalized to their respective GAPDH bands and then expressed relative to the DMSO control (taken as 1.0). One-way or two-way ANOVA was performed as indicated. Data are expressed as mean ± SD and were analysed using GraphPad Prism version 8.0.2 (La Jolla, CA) or t-test. Holm-Sidak test was used for post-hoc analysis. A p-value < 0.05 was considered statistically significant. Declarations Acknowledgements This project was supported by funding from the NSERC Discovery Grant (227158) to CHL, the Canada Foundation for Innovation (34711) to CHL, and the BC Knowledge Development Fund (103970) to CHL. VL, MZ and JL were supported by UNBC Research Project Awards. Author contributions statement V.P.L, W.M.L. and C.H.L conceptualized and designed the study. V.P.L conducted the synthesis and experiments to prepare Figures 1, 3, 4, 6 and S1-S11. W.M.L conducted the experiments and prepared Figures 5, 7 and 8. B.O.P. conducted the X-ray crystallography experiments and prepared Figure S12. V.L., J.L. and C.H.L conducted the MTT assays and prepared Figure 2 and Table 1. M.Z. conducted some of the flow cytometry experiments and prepared Figure 6. C.H.L, W.M.L. and T.M.B. provided the supervision. C.H.L. wrote the first draft of the manuscript and managed manuscript revisions. C.H.L provided funding and research facilities. All authors reviewed and approved the final manuscript. Competing interests The authors declare no competing interests. Data Availability The datasets used and/or analysed during the current study are available upon request from the corresponding author. References Visconti, R., Monica, R. D. & Grieco, D. 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Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on cell death 2018. Cell Death Differ. 25 , 486-541 (2018). Sazonova, E. V. et al. A link between mitotic defects and mitotic catastrophe: detection and cell fate. Biol Direct . 16 , 25 (2021). Lu, W. J. et al. #2714, a novel active inhibitor with potent G2/M phase arrest and antitumor efficacy in preclinical models. Cell Death Discovery 4, 24 (2018). Wang, H. et al. Erianin induces G2/M-phase arrest, apoptosis, and autophagy via the ROS/J signaling pathway in human osteosarcoma cells in vitro and in vivo . Cell Death & Disease 7, e2247 (2016). Cabrera, M. et al . G2/M cell cycle arrest and tumor selective apoptosis of acute leukemia cells by a promising benzophenone thiosemicarbazone compound. PLOS ONE 10, 10136878 (2015). Rojo, J. M., Ojeda, G., Portoles, M. P. & Portoles, A. Inhibition of T and B lymphoblastic response by mithramycin, dacabazine, prospidium chloride and peptichemio. Chemotherapy 29, 345-351 (1983). Perez-Lopez, F. R., Urcia, M., Torralba, R., Lafarga, L. & Hergueta, L. Effect of prospidine administered at different times of the day on the development and growth of N-nitrosomethylurea-induced mammary tumors in Wistar rats. Cancer Letts. 24, 111-118 (1984). Benenson, E. V. & Timina, O. B. Prospidine versus methotrexate pulse in highly active rheumatoid arthritis: a controlled 6-month clinical trial. Rheumatol. 13, 54-59 (1994). National Center for Biotechnology Information. PubChem Database. Prospidium chloride, CID=31937, https://pubchem.ncbi.nlm.nih.gov/compound/Prospidium-chloride (accessed on Oct. 31, 2019). Haddad, S. et al . Regio- and stereoselective synthesis of spiropyrrolizidines and piperazines through azomethine ylide cycloaddition reaction Org. Chem. 80, 9064-9075 (2015). Lee, H. X. et al. Regio- and stereoselective synthesis of dispiropyrrolizidines through 1,3-dipolar cycloaddition reaction: inhibition of KRAS expression. Mol. Struct. 1263, 133177 (2022). Hendzel, M. J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348-360 (1997). Huang, Y. et al . The kinetics of G2 and M transitions regulated by B cyclins. PLOS ONE 8, e80861 (2013). Hayward, D. et al . Orchestration of the spindle assembly checkpoint by CDK1-cyclin B1. FEBS Letts. 593, 2889-2907 (2019). Minshull, J. et al . The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 9, 2865-2875 (1990). Hwang, A. et al. Cell cycle-dependent regulation of the Cyclin B1 promoter. Biol. Chem. 270, 28419-28424 (1995). Steinmetz, M. O. & Prota, A. E. Microtubule-targeting agents: Strategies to hijack the cytoskeleton. Trends Cell Biol. 28, 776-792 (2018). Wordeman, L. & Vicente, J. J. Microtubule-targeting agents in disease: Classic drugs, novel roles. Cancers 13, 5650 (2021). Nakamura, Y. & Ishigaki, Y. Immunostaining and time-lapse analysis of Vinblastine-induced paracrystal formation in human A549 cells. Lett. 8, 2387-2392 (2014). Gillis, A. D. et al . p21 Cip1/WAF1 mediates cyclin B1 degradation in response to DNA damage. Cell Cycle 8, 253-256 (2009). Lehle, S. et al . LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis. Nucleic Acids Res. 42, e41 (2014). Soni, D. V. et al. Cyclin B1 is rate limiting but not essential for mitotic entry and progression in mammalian somatic cells. Cell Cycle 7, 1285-1300 (2008). Pines, J. & Hunter, T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. Cell. Biol. 115,1-17 (1991). Bentley, A. M. et al . Distinct sequence elements of cyclin B1 promote localization to chromatin, centrosomes and kinetochores during mitosis. Biol. Cell. 18, 4847-4858 (2007). Nakayama, Y. & Yamaguchi, N. Role of cyclin B1 levels in DNA damage and DNA damage-induced senescence. Rev. Cell Mol. Biol. 305, 303-337 (2013). Kikuchi, I. et al. A decrease in cyclin B1 levels leads to polyploidization in DNA damage-induced senescence. Cell Biol. Int. 34, 645-653 (2014). Zhang, J. et al . Necrosis, and then stress induced necrosis-like cell death, but not apoptosis, should be the preferred cell death mode for chemotherapy: clearance of a few misconceptions. Oncoscience 1, 407-422 (2014). Snyder, A. G. et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Immuno. 4, eaaw2004 (2019). Costa, F. B. et al . The novel piperazine-containing compound LQFM018: necroptosis cell death mechanisms, dopamine D4 receptor binding and toxicological assessment. Pharmacother. 102, 481-493 (2018). Chan, F. K-M. et al . A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. Biol. Chem. 278, 51613-51621 (2003). Strillic, B. et al. Tumour-cell-induced endothelial cells necroptosis via cell death receptor 6 promotes metastasis. Nature 536, 215-218 (2016). Sheldrick, G. M. Crystal structure refinement with SHELXL. Cryst. C71, 3-8 (2015). Bourhis, L. J., Dolomanov, O. V., Gildea, R. J., Howard, J. A. K. & Puschmann, H. The anatomy of a comprehensive constrained, restrained refinement program for the modern computing environment – Olex2 dissected. Acta Crystallogr. A Found Adv. 71, 59-75 (2015). Barad, A. et al . Anti-proliferative activity of a purified polysaccharide isolated from the basidiomycete fungus Paxillus involutus. Polym. 181, 923-930 (2018). Barnes, M. et al . Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains Biol. Chem. 290, 625-639 (2015). Dannenmann, B. et al. Simultaneous quantification of DNA damage and mitochondrial copy number by long-run DNA-damage quantification (LORD-Q). Oncotarget 8, 112417-112425 (2017). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 29 May, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 14 Apr, 2023 Reviews received at journal 30 Mar, 2023 Reviewers agreed at journal 28 Mar, 2023 Reviewers agreed at journal 23 Mar, 2023 Reviewers invited by journal 23 Mar, 2023 Editor assigned by journal 23 Mar, 2023 Editor invited by journal 23 Mar, 2023 Submission checks completed at journal 23 Mar, 2023 First submitted to journal 16 Mar, 2023 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 Advisory Board 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-2701574","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":185863582,"identity":"b9fc2f46-a35c-4cb9-888d-1806cff99a85","order_by":0,"name":"Victor P. Liu","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Victor","middleName":"P.","lastName":"Liu","suffix":""},{"id":185863585,"identity":"7d894fba-d21e-4152-8fd2-ddf33143771e","order_by":1,"name":"Wai-Ming Li","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wai-Ming","middleName":"","lastName":"Li","suffix":""},{"id":185863587,"identity":"0499f1b7-18f1-454d-9ae4-d0b1b58bbc23","order_by":2,"name":"Jack Lofroth","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jack","middleName":"","lastName":"Lofroth","suffix":""},{"id":185863589,"identity":"4248e216-34db-47b1-8201-ea996b5cfb43","order_by":3,"name":"Mehreen Zeb","email":"","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehreen","middleName":"","lastName":"Zeb","suffix":""},{"id":185863590,"identity":"8b608ccc-a0cd-488c-ab9e-4f2f9b8132fc","order_by":4,"name":"Brian O. Patrick","email":"","orcid":"","institution":"University of British Columbia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Brian","middleName":"O.","lastName":"Patrick","suffix":""},{"id":185863592,"identity":"6b089566-1145-4741-ac4f-43f33b262ab6","order_by":5,"name":"Tina M. Bott","email":"","orcid":"","institution":"MacEwan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tina","middleName":"M.","lastName":"Bott","suffix":""},{"id":185863593,"identity":"a1bef117-a1d5-4200-a430-4530deccaacc","order_by":6,"name":"Chow H. Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYDACCRBRwMDAz8DARooWAwYGyQaStRgcIFYL/+zmYxIMBjZyxjdyjz34wWAnT9iSO8fSgFrSjM1u5KUb9jAkGzYQ0mIgkWN2g8HgcOK2Gzlm0gwMBxiJ1fK/fvMMiBZ7YrUcSAAxQFoSCWqRuJGW/iPBINlwxpk35oY9BsnJBLXwz0g+bPChwk6evz3H7MGPCjtbglrAIAHhTqLUj4JRMApGwSggBABjZTU53eB1jwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Northern British Columbia","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chow","middleName":"H.","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2023-03-16 16:29:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2701574/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2701574/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-35927-6","type":"published","date":"2023-05-29T21:03:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":34860075,"identity":"61518cc0-9c80-4fe5-ba53-0de4073bba3e","added_by":"auto","created_at":"2023-03-27 13:55:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136716,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of dispiropiperazine derivatives SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) and SPOPP-5 (\u003cstrong\u003e2\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/1687464e0f57db24710a4aa8.png"},{"id":34860074,"identity":"e37f1f72-a6dd-405e-a6cd-f774fc20f854","added_by":"auto","created_at":"2023-03-27 13:55:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96694,"visible":true,"origin":"","legend":"\u003cp\u003eSPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) inhibited human colon cancer cell viability. Cell viability in SW480, HT29 and HCT116 human colon cancer cells was assessed using the MTT assay. Cells were treated with different concentrations of SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) or SPOPP-5 (\u003cstrong\u003e2\u003c/strong\u003e) for 48 hours. Results shown are representative from three separate experiments. Error bars are S.E.M.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/a4f48f5b8c88749eaa02df2f.png"},{"id":34861845,"identity":"971dcd08-c5c1-4191-a573-2383d6877ce6","added_by":"auto","created_at":"2023-03-27 14:03:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119791,"visible":true,"origin":"","legend":"\u003cp\u003eSPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) arrested cell cycle at G2/M phase in SW480 cells. (a) SW480 cells were treated with 2% DMSO, 20 µM SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e), or 20 µM SPOPP-5 (\u003cstrong\u003e2\u003c/strong\u003e) for 24 h after which cells were harvested and subjected to cell cycle analysis using flow cytometry. (b) The results from (a) and two another additional biological replicates (n = 3) were combined and expressed as shown. The cell cycle percentages were calculated based on the Watson Pragmatic model. Two-way ANOVA was performed: *p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/63f2d59dd25f3dac37339e6b.png"},{"id":34861846,"identity":"630f7857-7956-496b-b0bc-48573e6d4866","added_by":"auto","created_at":"2023-03-27 14:03:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92783,"visible":true,"origin":"","legend":"\u003cp\u003eSPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) induced phospho-histone H3 in SW480 cells. (a) Immunoblots showing phospho-histone H3 and GAPDH expression upon treatment with 2% DMSO, 20 µM SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) or 20 µM SPOPP-5 (\u003cstrong\u003e2\u003c/strong\u003e) for 24 h. (b) The results from (A) and another two biological replicate (n = 3) were averaged and expressed as shown. t-test was used: *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/2de6f2042843d8acf08707f2.png"},{"id":34861849,"identity":"87a223e8-1b45-4a3a-9faf-0be4a885942f","added_by":"auto","created_at":"2023-03-27 14:03:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":849418,"visible":true,"origin":"","legend":"\u003cp\u003eDefective cyclin B1 induction in SPOPP-3 treated cells as shown by immunofluorescence experiments. SW480 cells were treated with 40 µM SPOPP-3 or 2% DMSO for 24 hours before fixation and immunostaining with cyclin B1 antibody and DAPI. (a) Representative image of cells treated with SPOPP-3 (lower images) showing a large population of tetraploid cells (indicated by arrows) without cyclin B1 staining. In contrast, DMSO-treated cells have a relatively small population of tetraploid cells all of which expressed cyclin B1. (b) Tetraploid cells (with doubled DAPI signal) (left) and the percentage of tetraploid cells positive for cyclin B1 (right) were quantified in each group and presented as average ± SD. One-way ANOVA was used: *p \u0026lt; 0.05. Scale bar = 20 mm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/3ef6864055519486ded56cb7.png"},{"id":34863037,"identity":"5d220224-a49c-4ab3-8671-b0dba6e166bf","added_by":"auto","created_at":"2023-03-27 14:11:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":261540,"visible":true,"origin":"","legend":"\u003cp\u003eSPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) induced apoptosis and necrosis in SW480 cells. (a) SW480 cells were treated with 2% DMSO, 20 µM SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e), or 20 µM SPOPP-5 (\u003cstrong\u003e2\u003c/strong\u003e) for 24 h, after which cell lysates were isolated and subjected to cell death analysis using flow cytometry. (b) The results from (a) and two other additional biological replicates (n = 3) were combined and expressed as shown. Two-way ANOVA was used: *p \u0026lt; 0.005,**p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/cda1e333066a706e481cdeda.png"},{"id":34861847,"identity":"93daaef3-098c-496c-b1ea-cd7e610e5ec1","added_by":"auto","created_at":"2023-03-27 14:03:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1033852,"visible":true,"origin":"","legend":"\u003cp\u003eSPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) does not cause microtubule disruption. SW480 cells were treated with 50 nM vinblastine, 1 µM colchicine or 40 µM SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) for 24 hours before fixation and immunostaining with α-tubulin antibody and DAPI. Mitotic spindles were clearly observed in SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) treated cells while colchicine and vinblastine induced microtubule disruption via different mechanisms. Diffuse α-tubulin staining in the cytoplasm and para-crystal formation (arrows) can be observed with colchicine and vinblastine treatment respectively. Scale bar = 5 mm.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/7d6b1fddcea4624e9a74d93d.png"},{"id":34860079,"identity":"86d438ee-9b54-43ae-8200-9d260ec5d041","added_by":"auto","created_at":"2023-03-27 13:55:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":19310,"visible":true,"origin":"","legend":"\u003cp\u003eSPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) induced DNA damage. DNA lesion quantification using LORD-Q in SW480 cells treated with 40 µM SPOPP-3 (\u003cstrong\u003e1\u003c/strong\u003e) for the indicated time periods. Short and long amplicons amplified from the mtDNA gene was used in the quantification of lesions in mitochondrial DNA. Data presented are averaged from three biological replicates ± S.E.M (n = 3)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/d14a5cf8843186e1c88035a8.png"},{"id":44730251,"identity":"3b8e9599-3c1c-4c23-b2da-3977742d7797","added_by":"auto","created_at":"2023-10-16 21:28:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2795032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/89c86997-d3be-418d-be72-819f5db2ad10.pdf"},{"id":34860080,"identity":"45a6cc8d-b95a-4456-8f3b-276720996762","added_by":"auto","created_at":"2023-03-27 13:55:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2309950,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2701574/v1/417c9bb714697b9896b85191.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe use of chemicals and radiation to induce DNA damage is the most commonly used method for cancer therapy. Recently, there is an increased interest in manipulating the cell cycle to induce mitotic catastrophe as a novel anti-cancer therapeutic strategy\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Mitotic catastrophe is characterized by cells which would normally be arrested in G2/M phase due to damage in DNA or mitotic spindle but falsely proceed to mitosis due to defective cell cycle checkpoints\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The end result is senescence or cell death via either apoptosis, necrosis or autophagy\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This strategy relies on the use of DNA damaging agents or radiation in combination with cell cycle checkpoint inhibitors. Indeed, several G2/M phase checkpoint inhibitors\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, including irinotecan, a currently used chemotherapeutic drug for metastatic colorectal cancer, have shown potential to sensitize tumor cells to ionizing radiation. As such, the discovery of new compounds which cause G2/M cell cycle arrest remains an important area of cancer research\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are currently few known biologically active dispiropiperazine derivatives. One of which is prospidium chloride. This compound, also known as prospidine, has cytostatic, anti-inflammatory and immuno-suppressive properties\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. It has been classified as an anti-neoplastic compound based on its ability to inhibit T and B cell mitogenesis during lymphoblastic transformation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and to lower tumor volumes of carcinogen-induced mammary tumors in rats\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Currently, prospidium chloride is used as an anti-rheumatic drug in refractory rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite previous reports of biologically active dispiropiperazine compounds, other chemical derivatives have not been sufficiently explored. Here, we report the discovery of a specific dispiropiperazine derivative, spiro[2\u0026rsquo;,3]-bis(acenaphthene-1\u0026rsquo;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, \u003cb\u003e1\u003c/b\u003e) with anti-proliferative activity against a panel of human cancer cell lines and is capable of arresting cell cycle at G2/M phase, and inducing apoptosis, necrosis and DNA damage as well as disrupting mitotic spindle positioning.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSynthesis of SPOPP-3 (1) and SPOPP-5 (2).\u003c/b\u003e A recent report demonstrated the synthesis of two dispiropiperazine derivatives, spiro[2\u0026rsquo;,3]-bis(acenaphthene-1\u0026rsquo;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (referred to here as SPOPP-3, \u003cb\u003e1\u003c/b\u003e) and spiro[2\u0026rsquo;,5]-bis(acenaphthene-1\u0026rsquo;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine, through an azomethine ylide cycloaddition reaction using acenaphthenequinone (AcQ) and L-proline as substrates\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We performed a similar reaction with slight modifications as described in the Material and Methods to obtain SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Surprisingly, we also obtained a small quantity of spiro[2\u0026rsquo;,5\u0026rsquo;]-bis(acenaphthene-1\u0026rsquo;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (referred to here as SPOPP-5, \u003cb\u003e2\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Figs. S2, S3, S6-11), an isomer which has not been previously isolated due to its predicted unfavorable formation pathway\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Herein, we report for the first time, the purity (Supplementary Fig. S2) and structure of SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) as determined by FTIR (Supplementary Fig. S3), NMR (Supplementary Figs. S6-11) and X-ray diffraction analyses (Supplementary Fig. S12).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSPOPP-3 (1) reduced cell viability in human cancer cell lines.\u003c/b\u003e To our knowledge, no bioactivity has been reported for SPOPP-3 (\u003cb\u003e1\u003c/b\u003e). Herein, we show for the first time that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) significantly reduced cell viability in human colon cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), the IC\u003csub\u003e50\u003c/sub\u003e was 5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43 \u0026micro;M, 5.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 \u0026micro;M and 2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 \u0026micro;M in SW480, HT29 and HCT116 human colon cancer cells respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, its isomer SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) had no significant effect, with IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We also assessed SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) and SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) on a panel of human cancer cell lines and the summary is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Doxorubicin was used as a positive control on the cell lines (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) remains inhibitory with IC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u0026micro;M in human T lymphoblastoid cell line CEM to 13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96 \u0026micro;M in human hepatoma cell line HepG2. Again, SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) showed insignificant activity in four additional cancer cell lines (MiaPaca-2, Panc-1, SKOV3 and MDA-MB-231) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on the results that SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) had no significant anti-cell viability effect on seven cancer cell lines, it was not further assessed on the other cell lines. The results showed that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) has a relatively strong anti-proliferative effect on a panel of human cancer cell lines.\u003c/p\u003e \u003cp\u003e \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\u003eIC\u003csub\u003e50\u003c/sub\u003e of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) and doxorubicin against human cancer cell lines. The IC\u003csub\u003e50\u003c/sub\u003e the data shown is an average taken from three independent experiments. ND indicates Not Determined.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"char\" char=\"\u0026plusmn;\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell lines\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTypes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M) of Compounds\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSPOPP-3 (1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSPOPP-5 (2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDoxorubicin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSW480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman colon cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHT29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman colon cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCT116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman colon cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiaPaca2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman pancreatic cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.62\u0026thinsp;\u0026plusmn;\u0026thinsp;3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePanc1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman pancreatic cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSKOV3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman ovarian cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA-MB-231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman breast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCF-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman breast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCF-7-Adr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman breast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT47D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman breast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman liver cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeLa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman cervical cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDU145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman prostate cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman leukemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKG1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman leukemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman leukemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eU251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman glioblastoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eU87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman glioblastoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\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\u003e \u003cb\u003eSPOPP-3 (1) arrested cell cycle at G2/M phase.\u003c/b\u003e To determine the mechanism whereby SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) decreases cell viability, flow cytometry was performed for cell cycle analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, treatment with 20 \u0026micro;M SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) caused cell cycle arrest in the G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). No activity was observed for SPOPP-5 (\u003cb\u003e2\u003c/b\u003e). To further investigate whether SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) induces arrest at G2 or M phase, we performed Western blot analysis to detect phospho-histone H3, an established sensitive mitotic marker\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Indeed, phospho-histone H3 level was clearly increased in SW480 cells treated with SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) but not SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) arrests SW480 cells at the M phase of the cell cycle. To further investigate the effects of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) on the cell cycle, we performed immunofluorescence experiments to study cyclin B1 activation. Cyclin B1 is one of the key factors in controlling entry into mitosis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e with its expression rapidly increased in G2 phase and peaking at late G2 or early M phase\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the population of tetraploid cells in SPOPP-3 (\u003cb\u003e1\u003c/b\u003e)-treated cells as indicated by cells having doubled DAPI signal, was significantly increased when compared with DMSO control. This confirms flow cytometry results that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) caused cell cycle arrest in the G2/M phase where the cells failed to divide into daughter cells. In control cells, most of the tetraploid cells had significantly higher cyclin B1 staining compared to SPOPP-3-treated tetraploid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Thus, our results indicate that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) treatment is associated with defective cyclin B1 activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSPOPP-3 (1), but not SPOPP-5 (2), induced cell apoptosis and necrosis.\u003c/b\u003e To determine the possible effect of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) on cell death that led to the observed decrease in cell viability, we used flow cytometry to analyse apoptosis and necrosis. The commonly used stains to detect necrosis and apoptosis are 7-AAD and Annexin V-PE respectively. After treatment with SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) or 2% DMSO for 24 h, cells were double stained with 7-AAD and Annexin V-PE. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, cells treated with SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) changed to a more necrotic state (Q1; 32.64%) as compared to the DMSO-treated cells (Q1; 0.5%), and this is statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) also significantly induced apoptosis (Q2\u0026thinsp;+\u0026thinsp;Q4) in SW480 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). On the other hand, SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) had no significant effect on apoptosis or necrosis as compared to the control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of SPOPP-3 (1) on mitotic spindle formation.\u003c/b\u003e Immunofluorescence studies were carried out to investigate the potential function of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) as a microtubule toxin. Two drugs, vinblastine and colchicine which are well known to disrupt microtubules, were used as positive controls. Vinblastine, which belongs to the family of vinca alkaloids, binds to tubulin at a specific site and inhibits mitotic spindle formation leading to cell cycle disruption\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. When used at high concentrations, vinblastine is known to cause paracrystal formation due to tightly packed tubulin aggregates\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Indeed, when SW480 cells were treated with 50 nM vinblastine, paracrystals were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Colchicine, on the other hand, disrupted microtubules and caused diffuse a-tubulin staining throughout the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In contrast, mitotic spindles could be observed in cells treated with SPOPP-3 (\u003cb\u003e1\u003c/b\u003e). However, the positions of the mitotic spindles appeared to be disrupted when compared with control cells treated with DMSO (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Displacement of the mitotic spindles was also associated with the lack of chromosome alignment at the equator of the cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In summary, these results suggest that although SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) does not disrupt microtubule formation, mitotic spindle positioning appears to be affected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSPOPP-3 (1) induced DNA damage.\u003c/b\u003e Based on the findings that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) causes G2/M arrest associated with cyclin B1 down regulation and mitotic spindle displacement, it may be possible that the activity of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) is via DNA damage\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To investigate this possibility, we used the qPCR-based method (LORD-Q) to detect DNA lesions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Since this method can detect DNA damage regardless of the type of DNA lesion, we reason that this is the most appropriate method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, although the data did not reach statistical significance because of the large variation in data, the effect of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) on DNA damage can be detected as early as 1 h after treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we report the synthesis of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) and show for the first time that it has strong anti-proliferative activity against 18 human cancer cell lines (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To our surprise, using the previously reported synthesis procedure\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, we also obtained the structural isomer SPOPP-5 (\u003cb\u003e2\u003c/b\u003e), a novel compound. In contrast to SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) had virtually no anti-proliferative activity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The anti-proliferative effect of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) was greatest against human leukemia cells lines (IC\u003csub\u003e50\u003c/sub\u003e from 0.63 to 3.60 \u0026micro;M), human glioblastoma cell lines (IC\u003csub\u003e50\u003c/sub\u003e from 2.95 to 6.30 \u0026micro;M), human colon cancer cell lines (IC\u003csub\u003e50\u003c/sub\u003e from 2.44 to 5.42 \u0026micro;M), human cervical cancer cell lines (IC\u003csub\u003e50\u003c/sub\u003e of 4.23 \u0026micro;M), human ovarian cancer cell lines (IC\u003csub\u003e50\u003c/sub\u003e of 6.30 \u0026micro;M) and human breast cancer cell lines (IC\u003csub\u003e50\u003c/sub\u003e from 4.00 to 6.17 \u0026micro;M). SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) has slightly weaker anti-proliferative activity against human liver cancer cell line (IC\u003csub\u003e50\u003c/sub\u003e of 13.03 \u0026micro;M), human pancreatic cancer cell lines (IC\u003csub\u003e50\u003c/sub\u003e from 8.62 to 9.17 \u0026micro;M) and human prostate cancer cell line (IC\u003csub\u003e50\u003c/sub\u003e of 9.80 \u0026micro;M).\u003c/p\u003e \u003cp\u003eThe anti-proliferative activity of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) was associated with its ability to induce apoptosis and necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), as well its ability to cause cell cycle arrest in the G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Using phosphorylated histone H3 as an M phase marker, it was shown that at least some cells were arrested in M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Our results also showed that cyclin B1 expression was drastically reduced upon SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although a rise in cyclin B1 expression in late G2 phase and its translocation to the nucleus is important for the initiation of mitosis, its depletion using siRNA knockdown does not cause cells to be arrested only in G2 phase as this can be explained by the redundant function of cyclin B2\u003csup\u003e18,27\u003c/sup\u003e. We also demonstrated using microscopy that while microtubules seem to be unaffected by SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) treatment, defects in the M phase including mitotic spindle positioning and condensed chromosome alignment, were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This may be due to decreased cyclin B1 levels as it is known that cyclin B1 is normally recruited to centrosomes and kinetochores\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We further investigated whether SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) causes DNA damage as it has been documented that cyclin B1 levels are reduced as a result of DNA damage\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Indeed, our results suggest that similar to bleomycin\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) causes DNA damage at an early stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). As a consequence, such an early DNA damage event could lead to cyclin B1 reduction, cell cycle arrest, apoptosis and necrosis which are cellular processes that occur much later.\u003c/p\u003e \u003cp\u003eThe anti-proliferative properties of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) has important implications in cancer therapy. Synthetic lethality is a novel approach in cancer treatment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Since SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) is a potent inducer of G2/M arrest, it has the potential to be used in combination with G2/M checkpoint inhibitors to trigger mitotic catastrophe which is currently viewed as a favorable treatment strategy to enhance cell death either via apoptosis, necrosis or autophagy\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Particularly, in most of melanoma cases where G1/S transition mediated by the cyclin-CDK4 pathway is defective and has increased dependence on the G2/M checkpoint to induce cell cycle arrest when exposed to DNA damage, SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) may have an added advantage in combination with G2/M inhibitors\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Secondly, we found that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) is an inducer of necrosis. Several lines of investigations have provided evidence to support the role of necrosis in enhancing cancer immunotherapy and as a possible strategy to overcome the resistance of cancer cells to apoptosis\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. To this end, it will be important to assess whether SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) has such prowess in inducing pro-inflammatory processes by means of releasing damage-associated molecular patterns such as high mobility group 1 (HMGB1) protein, a marker for necrosis. Release of such factors into the extracellular matrix may lead to activation of CD8\u0026thinsp;+\u0026thinsp;leukocytes and promote anti-tumor immunity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is also important to decipher further the mechanism whereby SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) arrests cells at G2/M phase. For instance, piperazine derivatives have been shown to generate reactive oxygen species (ROS) within cells\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, it would be of interest to investigate whether SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) can generate ROS leading to oxidative DNA damage and subsequent arrest at G2/M phase. In terms of necrosis, one of the modes of cell death detected in cells treated with SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), it has become increasingly clear that necrosis may not simply be an uncontrolled cellular process, but rather a regulated pathway commonly known as necroptosis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Necroptosis has also been reported to increase cancer metastasis in certain cell lines\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The duality of necroptosis being anti- and pro-tumorigenic still requires investigation to determine in what context is necroptosis beneficial. Since we have found respectable anti-proliferative activity of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) against a large panel of cancer cell lines, it would be integral to further study the mode of cell death in different cell lines.\u003c/p\u003e \u003cp\u003eIn conclusion, we describe the synthesis and biochemical characterization of a specific dispiropiperazine derivative called SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) with strong anti-proliferative activity against a wide panel of human cancer cell lines. SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) is able to induce DNA damage, apoptosis, necrosis, arrests cell cycle at G2/M phase and disrupts normal mitotic spindle positioning. This study has laid the foundation for the further development of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) for possible use as a chemical tool to perturb and understand cellular processes, as well as a potential anti-cancer compound. For the latter, SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) should be explored in synthetic lethality approach and as a necrosis-inducing anti-cancer compound.\u003c/p\u003e"},{"header":"Methods And Materials","content":"\u003cp\u003e \u003cb\u003eSynthesis and purification of SPOPP-3 (1) and SPOPP-5 (2).\u003c/b\u003e We adopted the previously described method for synthesizing spiro[2\u0026prime;,3]-bis(acenaphthene-1\u0026prime;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, \u003cb\u003e1\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A mixture of acenaphthenequinone (1.822 g, 10 mmol) and L-proline (1.151 g, 10 mmol; Sigma-Aldrich) were dissolved in methanol (200 mL) and heated at 35\u0026deg;C for 3 h. The reaction was monitored by thin layer chromatography (TLC) using ethyl acetate:hexane (1:2; v/v), and the spots were visualized using UV light (254 nm). Typically, an orange precipitate was formed during the first hour which changed to an orange-brown color after an additional hour and to a dark brown color after completion of the reaction. Solvent was removed under reduced pressure leaving a dark brown powder (1.722 g). Crude product, containing both SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) and SPOPP-5 (\u003cb\u003e2\u003c/b\u003e), was mixed with 2 g of normal silica in 200 mL methanol. Solvent was removed under reduced pressure and the silica mixture was dry-loaded onto a column. Purification by flash chromatography, using normal-phase silica (50 g) and a 9:1 mixture of hexanes and ethyl acetate at a flow rate of 12 mL/min, was used to isolate a mixture of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) and SPOPP-5 (\u003cb\u003e2\u003c/b\u003e). Subsequent purification with three runs of flash chromatography employing a total of 686 mg of crude product were conducted. An orange-coloured band eluting at 530\u0026ndash;830 mL corresponding to Rf\u0026thinsp;=\u0026thinsp;0.14 with 9:1 (hexanes:ethyl acetate) was dried under reduced pressure to produce 280 mg of semi-purified SPOPP-3 (\u003cb\u003e1\u003c/b\u003e). SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) was subsequently purified for biological testing by HPLC using a Phenomenex Luna 5u Phenyl-Hexyl 4.60 mm x 250 mm column. A gradient from 80\u0026ndash;92% acetonitrile for 6 min at a flow rate of 2 mL/min was used as the eluent conditions. For the semi-purified SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), a total of 12.5 mg was injected into the HPLC and 5 mg (16%) of purified SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) (at retention of 4.08 min) was obtained. A yellow-coloured band eluting at 320\u0026ndash;405 mL corresponding to Rf\u0026thinsp;=\u0026thinsp;0.24 (9:1 hexanes:ethyl acetate) produced 120 mg of purified SPOPP-5 (\u003cb\u003e2\u003c/b\u003e). SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) was purified using the same HPLC conditions as SPOPP-3 (\u003cb\u003e1\u003c/b\u003e). One hundred twenty mg of semi-purified SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) was injected into HPLC and 21 mg (3%) of purified SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) (at retention of 6.02 min) was obtained. HPLC-MS and NMR analyses were used to confirm the identity of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) and SPOPP-5 (\u003cb\u003e2\u003c/b\u003e). 1D and 2D NMR spectra were recorded on an Agilent/Varian Inova 400 MHz NMR spectrometer with a 5 mm Kimble NMR tube (Rockwood, TN, USA) at the University of Alberta or on a Bruker 600 MHz NMR with a cryoprobe at the University of British Columbia. All HPLC analyses were performed on Agilent 1260 Infinity Systems with UV detector, and mass spectrometry were done using Agilent 6120 Single Quad MS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eX-ray crystallographic analysis.\u003c/b\u003e Single orange irregular crystals of SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) (50 mg) were recrystallized from acetonitrile (10 mL) by slow evaporation. Crystals were obtained on day 7. A suitable crystal with dimensions 0.22 \u0026times;\u0026ensp;0.20 \u0026times;\u0026ensp;0.11 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e was selected and mounted on a Bruker APEX II area detector diffractometer. The crystal was kept at a steady \u003cem\u003eT\u003c/em\u003e\u0026thinsp;=\u0026thinsp;90(2) K during data collection. The structure was solved with the ShelXT\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e solution program using dual methods and Olex2\u003csup\u003e38\u003c/sup\u003e as the graphical interface. The model was refined with XL\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e using full matrix least squares minimization on \u003cem\u003eF\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture.\u003c/b\u003e All cell lines were obtained from American Type Culture Collection except HT29 (colon adenocarcinoma) which was obtained from Dr. Ranjana Bird at UNBC. All cells were maintained in Eagle\u0026rsquo;s Minimal Essential Medium (Lonza) except the following: MiaPaca-2 and Panc-1 were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (Lonza), while K562, KG1a and CEM were maintained in RPMI 1640 medium (Lonza). All media were supplemented with 10% fetal bovine serum (Life Technologies Inc.) and antibiotics.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability assay.\u003c/b\u003e The cytotoxic MTT assay was used to assess cell viability as previously described\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Briefly, cells were plated at a density of 1.5 x 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates. After 24 h, cells were treated with various concentrations of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) or SPOPP-5 (\u003cb\u003e2\u003c/b\u003e) for 48 h. All absorbance data were expressed relative to the control, 0.1% DMSO, taken as 100% cell viability.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of cell lysates, immunoblot analysis and antibodies.\u003c/b\u003e SW480 cells were seeded in 6-well plates and treated with 20 \u0026micro;M SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), 20 \u0026micro;M SPOPP-5 (\u003cb\u003e2\u003c/b\u003e), or 2% DMSO for 24 h. Cell lysates were prepared as previously described\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. For immunoblot analysis, protein samples were resolved on a 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The phospho H3 antibody (Ser10) (D2C8, 1:1,000, Cell Signaling) was used with an overnight incubation at 4\u0026deg;C. Anti-GAPDH (G8795, clone GAPDH-71.1, 1:20,000, Sigma) was also used. Anti-mouse IgM-HRP (sc-2064, 1:4,000, Santa Cruz Biotechnology), anti-mouse IgG-HRP (W402B, 1:4,000, Promega) and anti-rabbit IgG-HRP (W401B, 1:4,000, Promega) were used as secondary antibodies. All blots were visualized with the FluorChem Q system (ProteinSimple). Densitometry analysis was performed using the AlphaView Q software (ProteinSimple).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry apoptosis and cell cycle analyses.\u003c/b\u003e Cells were plated at 2.5 x 10\u003csup\u003e5\u003c/sup\u003e cells/well in 6-well plates and treated with compounds as described above. Cells were trypsinized followed by centrifugation and washed twice with phosphate-buffered saline. Live cells were stained with PE Annexin V and 7-AAD according to the manufacturer\u0026rsquo;s instructions for Apoptosis Detection Kit I (BD Pharmingen) and analysed by flow cytometry using a BD FACSMelody cell sorter (BD Biosciences) and BD FACSChorus software (V 1.0). For cell cycle analysis, the BD cycletest plus DNA reagent kit was used to stain for DNA and the data was analysed using the software FlowJo.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence.\u003c/b\u003e SW480 cells were plated in 4-well cover glass chambers at a density of 15 x 10\u003csup\u003e4\u003c/sup\u003e cells per well with 0.5 mL EMEM. The cells were treated with the indicated drugs for 24 hours before fixation using 100% methanol (-20\u0026deg;C) for 10 min. Subsequently, the cells were blocked using PBS with 2% BSA and 0.1% Triton-x 100 and then stained with α-tubulin antibody (1:100; Ab4074; Abcam) or cyclin B1 antibody (D5C10, 1:200, cell signaling). Anti-mouse-AF594 and anti-rabbit-AF 594 (1:200; Molecular Probes) were used as secondary antibodies respectively. All antibodies used were diluted in PBS with 0.5% BSA and 0.1% Triton-x 100. All blocking and antibody incubation steps were conducted for 1 h at room temperature. After each antibody incubation step, the cells were washed 3 times (10 min each) with wash buffer containing 0.1% Triton-x-100 in PBS. For cyclin B1 immunofluorescence, cells were fixed in 4% paraformaldehyde for 15 min at room temperature. To stain for DNA, cells were incubated with DAPI dihydrochloride (300 nM diluted in PBS) for 5 min after immunostaining with the secondary antibody. All fluorescence images were taken using an inverted Zeiss Axio Observer Z1 microscope with a motorized stage, Zeiss Axiocam 503 mono camera, Colibri 2 multicolor LED light source, Plan-Apochromat 20 x /0.8 M27 or Plan-Apochromat 63 x /1.4 Oil DIC M27 as objectives. For the quantification of tetraploid cells, DAPI signal in each cell was quantified in each image using the region of interest function in the Zen software. Tetraploid cells were distinguished with a DAPI signal double that of the remaining cells in the same image. Three images (each with at least 36 cells) from each group were quantified. The population of tetraploid cells that were cyclin B1 positive was then determined in each image and presented as an average for each group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA damage quantification.\u003c/b\u003e To assess whether SPOPP-3 treatment causes DNA damage, a long-run real-time PCR-based method (LORD-Q) was used with modifications\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. SW480 cells (2.5 x 10\u003csup\u003e6\u003c/sup\u003e) were treated in 6-well plates with DMSO (control) or 40 mM SPOPP-3 for the indicated time periods before being harvested for total DNA isolation using the DNeasy Blood and Tissue kit (Qiagen). LORD-Q method was carried out using the established primer sets for both the long and short amplicons for the mtDNA gene\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. PCR efficiencies were calculated using 37.5, 18.75, 9.375 and 4.688 ng DNA as template per PCR reaction. The rtPCR reaction (15 mL total volume) consisted of 0.05 x ResoLight dye, 1x KAPA2G Fast Hot Start ReadyMix, 500 nM of forward and reverse primer, and the aforementioned quantities of isolated DNA as template. For amplification of short amplicons as reference, Quantabio PerfeCTa\u0026reg; SYBR\u0026reg; Green FastMix\u0026reg; was used. Real time PCR analysis was carried out using a Bio-Rad CFX96 system and data analysis was performed using CFX Maestro software. The number of lesions per 10kb was calculated based on the previously established Eq.\u0026nbsp;2\u003csup\u003e6,41\u003c/sup\u003e. The data presented is averaged from 3 biological replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e The cytotoxic MTT assays were performed in triplicate (3 wells/treatment) and the absorbance reading from the respective control was taken as 100% cell viability. For immunoblot analysis, the densitometry of phosphor-histone H3 bands were normalized to their respective GAPDH bands and then expressed relative to the DMSO control (taken as 1.0). One-way or two-way ANOVA was performed as indicated. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and were analysed using GraphPad Prism version 8.0.2 (La Jolla, CA) or t-test. Holm-Sidak test was used for post-hoc analysis. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003cbr /\u003e This project was supported by funding from the NSERC Discovery Grant (227158) to CHL, the Canada Foundation for Innovation (34711) to CHL, and the BC Knowledge Development Fund (103970) to CHL. VL, MZ and JL were supported by UNBC Research Project Awards.\u003c/p\u003e\n\u003cp\u003eAuthor contributions statement\u003c/p\u003e\n\u003cp\u003eV.P.L, W.M.L. and C.H.L conceptualized and designed the study. V.P.L conducted the synthesis and experiments to prepare Figures 1, 3, 4, 6 and S1-S11. W.M.L conducted the experiments and prepared Figures 5, 7 and 8. B.O.P. conducted the X-ray crystallography experiments and prepared Figure S12. V.L., J.L. and C.H.L conducted the MTT assays and prepared Figure 2 and Table 1. M.Z. conducted some of the flow cytometry experiments and prepared Figure 6. C.H.L, W.M.L. and T.M.B. provided the supervision. C.H.L. wrote the first draft of the manuscript and managed manuscript revisions. C.H.L provided funding and research facilities. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003cstrong\u003e\u003cbr /\u003e\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003cbr /\u003e\u003c/strong\u003eThe datasets used and/or analysed during the current study are available upon request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVisconti, R., Monica, R. D. \u0026amp; Grieco, D. Cell cycle checkpoint in cancer: a therapeutically targetable double-edged sword. \u003cem\u003e Exp. Clin. Cancer Res. \u003c/em\u003e\u003cstrong\u003e35,\u003c/strong\u003e 153 (2016).\u003c/li\u003e\n\u003cli\u003eDillon, M. T., Good, J. S. \u0026amp; Harrington, K. J. 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Chem.\u003c/em\u003e \u003cstrong\u003e290,\u003c/strong\u003e 625-639 (2015).\u003c/li\u003e\n\u003cli\u003eDannenmann, B. \u003cem\u003eet al. \u003c/em\u003eSimultaneous quantification of DNA damage and mitochondrial copy number by long-run DNA-damage quantification (LORD-Q). \u003cem\u003eOncotarget \u003c/em\u003e\u003cstrong\u003e8,\u003c/strong\u003e 112417-112425 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2701574/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2701574/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDispiropiperazine compounds are a class of molecules known to confer biological activity, but those that have been studied as cell cycle regulators are few in number. Here, we report the characterization and synthesis of two dispiropiperazine derivatives: the previously synthesized spiro[2\u0026rsquo;,3]-bis(acenaphthene-1\u0026rsquo;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-3, \u003cb\u003e1\u003c/b\u003e), and its previously undescribed isomer, spiro[2\u0026rsquo;,5\u0026rsquo;]-bis(acenaphthene-1\u0026rsquo;-one)perhydrodipyrrolo-[1,2-a:1,2-d]-pyrazine (SPOPP-5, \u003cb\u003e2\u003c/b\u003e). SPOPP-3 (\u003cb\u003e1\u003c/b\u003e), but not SPOPP-5 (\u003cb\u003e2\u003c/b\u003e), was shown to have anti-proliferative activity against a panel of 18 human cancer cell lines with IC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.63-13 \u0026micro;M. Flow cytometry analysis revealed that SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) was able to arrest cell cycle at the G2/M phase in SW480 human cancer cells. Western blot analysis further confirmed the cell cycle arrest is in the M phase. In addition, SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) was shown to induce apoptosis, necrosis, and DNA damage as well as disrupt mitotic spindle positioning in SW480 cells. These results warrant further investigation of SPOPP-3 (\u003cb\u003e1\u003c/b\u003e) as a novel anti-cancer agent, particularly for its potential ability to sensitize cancer cells for radiation-induced cell death, enhance cancer immunotherapy, overcome apoptosis-related drug resistance and for possible use in synthetic lethality cancer treatments.\u003c/p\u003e","manuscriptTitle":"A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-27 13:55:22","doi":"10.21203/rs.3.rs-2701574/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-04-14T09:14:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-03-30T18:23:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2e843826-8c9e-4af8-b0ed-1a1ba9444380","date":"2023-03-28T06:51:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4defc5c4-ecaa-436b-89c4-eea841afe2b0","date":"2023-03-23T18:40:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-03-23T11:27:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-23T11:13:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-03-23T09:45:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-03-23T09:42:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2023-03-16T16:27:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5135c880-fdbd-41c9-8e61-8b4bc17eb383","owner":[],"postedDate":"March 27th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":20143682,"name":"Biological sciences/Cancer"},{"id":20143683,"name":"Biological sciences/Cell biology"},{"id":20143684,"name":"Biological sciences/Chemical biology"},{"id":20143685,"name":"Biological sciences/Drug discovery"},{"id":20143686,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2023-10-16T21:12:17+00:00","versionOfRecord":{"articleIdentity":"rs-2701574","link":"https://doi.org/10.1038/s41598-023-35927-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-05-29 21:03:41","publishedOnDateReadable":"May 29th, 2023"},"versionCreatedAt":"2023-03-27 13:55:22","video":"","vorDoi":"10.1038/s41598-023-35927-6","vorDoiUrl":"https://doi.org/10.1038/s41598-023-35927-6","workflowStages":[]},"version":"v1","identity":"rs-2701574","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2701574","identity":"rs-2701574","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}