A Novel Anticancer Agent, 8-Methoxypyrimido4 `,5 `: 4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and Independent Apoptotic Pathways

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido 4 `,5 `: 4,5] thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly ( ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways

MPTQ-mediated cell death is associated with increased phosphorylation of p53 at Ser15.

<p>A) Western blot analysis of phospho-p53 (Ser15), p53 and GAPDH. Neuro 2a cells were either treated with 30 µM of MPTQ or DMSO alone for 24 hours. Three independent isolates were obtained and 60 µg of total proteins were size fractionated in 12% SDS-PAGE and western blotted either with anti-phospho-p53 (ser15) or with anti-p53 antibody. The blots were stripped and hybridized with anti-GAPDH antibody to normalize any loading difference. B) Immunocytochemistry of phopho-p53 (Ser15). Images represent three independent experiments C) Nuclear phospho-p53 (Ser15) intensity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066430#pone-0066430-g006" target="_blank">figure 6</a>. Histograms represent mean integrated nuclear phopho-p53 (Ser15) intensity±SD of three independent experiments. p value calculated by Student’s t-test is displayed which indicates significant increased phosphorylation of p53 at Ser15 in MPTQ treated neuroblastoma cells.</p

Increased proteolysis of PARP in MPTQ treated neuro 2a neuroblastoma cells.

<p>A) Neuro 2a cells were cultured and treated with 30 µM of MPTQ for 24 hours and lysates were prepared from three independent treatments. 60 µg of total protein were size fractionated in 12% SDS-polyacrylamide gel and immunoblotted with anti-PARP antibody that detects both full length PARP and cleaved PARP. Cleaved PARP is seen only in MPTQ treated N2a cells but not at all in control cells indicating the proteolysis of PARP, a hallmark feature in apoptotic cells. B) Immunocytochemistry of PARP using an antibody specific for cleaved PARP. Detection was done using Alexa fluor 594 labelled secondary antibodies. Nuclei were stained with DAPI. Nuclei with cleaved PARP were seen only in MPTQ treated cells. The figure represents at least three independent experiments.</p

MPTQ treatment increases Bax protein expression and redistribution in neuro 2a neuroblastoma cells.

<p>A) Neuro 2a cells were cultured and treated with 30 µM of MPTQ for 24 hours followed by immunocytochemistry for Bax expression by an anti-Bax antibody. Detection was done using Alexa 594 labelled secondary antibody. Nuclei were stained with DAPI. B) Cytoplasmic level of Bax immunosignal was obtained using multi-cell scoring module of MetaMorph software and mean of three random images were displayed as histograms. Error bar indicates standard deviation. p value was calculated by Student t-test and is displayed which strongly indicates the overexpression of Bax protein in MPTQ treated neuro 2a cells.</p

Working model of MPTQ-mediated apoptosis in neuro 2a neuroblastoma cells.

<p>MPTQ activates ATM (an indicator of DNA double strand breaks) and p53. MPTQ treatment also upregulates Bax protein level which activates caspase-dependent intrinsic apoptosis pathway by activating caspase-9 followed by caspase-3 and -7 which in turn inactivates PARP. Caspase-independent intrinsic apoptosis pathway was also activated by nuclear translocation of AIF. MOMP = mitochondrial outer membrane permeabilization.</p

MPTQ-mediated cell death is associated with activation of caspases of intrinsic apoptosis pathway but not of extrinsic pathway.

<p>A) Neuro 2a cells were cultured and treated with 30 µM of MPTQ for 24 hours and lysates were prepared. 60 µg of total proteins were resolved in 12% SDS-PAGE and immunoblotted with anti-caspase-8 or anti-caspase-2 or anti-caspase-9 or anti-caspase-3 or anti-caspase-7 antibody. Blots were stripped and immunoblotted with anti-GAPDH antibody. The results clearly indicate the activation of caspase-9, -3 and-7 but not caspase-8 and -2 in MPTQ treated cells. B) Immunocytochemistry of caspase-3 protein was performed as described earlier. Increased caspase-3 level was observed in the nucleus of MPTQ treated neuro 2a cells but not in control cells. C) Nuclear level of caspase-3 immunosignal was obtained using multi-cell scoring module and mean of three random images of two independent experiments were displayed as histograms. Error bar indicates standard deviation. D) Western blot analysis of cleaved caspase-3 level in cytosolic and nuclear fraction of MPTQ treated or untreated neuro 2a cells. Blots were also immunoblotted with anti-GAPDH and anti-histone H3 antibodies for normalization. E) Densitometric analysis of procaspase-3 and cleaved caspase-3 bands were made from cytosolic as well as from nuclear fractions. Cleaved caspase-3 to procaspase-3 ratio was obtained. Mean and standard deviation from three independent isolates were obtained and plotted as histograms. p value was calculated by Student’s t-test and is displayed which indicates significant increased mobilization of cleaved caspase-3 from cytoplasm to nucleus in MPTQ treated neuro 2a cells.</p

MPTQ induced nuclear DNA breaks in neuro 2a cells are positive for TUNEL staining, an indicator of apoptosis.

<p>Neuro 2a cells were cultured and treated with 30 µM of MPTQ for 48 hrs. Control cells were treated with equal amount of DMSO. A) Fluorescent images of DAPI and fluorescein-12-dUTP were captured from multiple random fields using multi-dimension acquisition module of MetaMorph using identical settings and images were displayed with equal pixel intensity. Images display increased TUNEL positive neuro 2a cells in MPTQ treated cells than control cells B) Number of TUNEL positive cells were calculated using multi-cell scoring module of MetaMorph software and mean of three independent experiments are presented as histograms with standard deviation as error bars. Results indicated more than 80% cells were positive for TUNEL in MPTQ treated cells where as only 13% in control cells. p value was calculated by Student’s t-test and displayed. p≤0.05 is considered statistically significant.</p

MPTQ-mediated neuro 2a neuroblastoma cell death is associated with increased phosphorylation of ATM.

<p>A) 30 µM of MPTQ treated or untreated cells were fixed after 24 hours of treatment and immunolabelled with an antibody specific for phosphorylated ATM (ser1983). Detection was done using Alexa fluor 594 labelled secondary antibodies. Nuclei were stained by DAPI. Z-stack images were captured from multiple random fields, processed and displayed as described in methods. B) Intensity of phospho-ATM was measured in cytoplasmic and nuclear compartment. Histograms represent mean±standard deviation of three independent images from two independent experiments. C) Western blot analysis of phosphorylated ATM on cytosolic and nuclear fraction of MPTQ treated or untreated neuro 2a cells. Blots were also immunoblotted with anti-GAPDH and anti histone H3 antibodies for normalization. The results indicate increased phosphorylated ATM level in nuclear compartment of MPTQ treated cells than untreated cells. D) Densitometric analysis of phosphorylated ATM bands was performed and the values are plotted as mean±standard deviation of three independent isolates. Statistical analysis was made by Student’s t-test and p value is displayed. p value ≤0.05 is considered significant.</p

MPTQ induces nuclear DNA fragments of oligonucleosomal size in neuro 2a cells.

<p>A) Agarose gel electrophoresis of genomic DNA isolated from untreated and 2.5, 5, 10, 20 or 30 µM MPTQ treated cells for 48 hours indicated dose-dependent increased DNA fragmentation. B) Intensity of fragmented DNA was calculated and mean with standard deviation was plotted. Data represents three independent experiments. p value displayed for each duration of treatment was calculated by comparing with control sample using Student’s t-test. p value ≤5×10⁻² is considered significant. AU = Arbitrary Units.</p

Cytotoxic and antiproliferative effect of MPTQ on human SH-SY5Y neuroblastoma cells.

<p>A) Bright field images after 6 days of 30 µM MPTQ treated cells exhibit gross morphological disintegration compared to untreated cells. SH-SY5Y cells demonstrate plasma membrane blebbing with cytoplasmic oozing (yellow arrow head) and irregular nuclear compaction and fragmentation (red arrow head) only in MPTQ treated cells but not in normal cells. B) Antiproliferative effect of MPTQ on SH-SY5Y cells by MTT assay. Cells were treated with 30, 60 or 90 µM of MPTQ or with equal amount of DMSO (control) for 2, 4 and 6 days. After each time point, MTT assay was performed as described earlier. Graphs were plotted as mean±standard deviation of three independent isolates. * indicates the differences between untreated and treated samples are statistically significant (p<0.05; Student’s t-test). C) Live-Dead assay on MPTQ (90 µM) treated SH-SY5Y cells show increased number of dead cells after 4 days over untreated cells. D) Number of dead cells were counted from three random fields and from three separate experiments and plotted as histogram (mean±standard deviation). p-value ≤0.05 represents statistical significance between the mean of untreated and MPTQ treated SH-SY5Y cells. DAT = days after treatment.</p