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independent of changes in the related mRNA level (Figures 3 and 5). The increased activity of caspase-3 was negated via overexpression of DFF- 45. DFF-45 is the natural inhibitor of DFF-40 (CAD) (Liu et al., 1997). In addition, the increased expression of DFF-45, along with a modest increase (for sulfabenzamide) and a significant decrease (for doxorubicin) in DFF-40 expression (Figure 5), indicated that the increased activity of caspase-3 could be blocked by the increased expression ratio of DFF-45/DFF-40. This r<strong>edu</strong>ces the level of active DFF-40 to trigger DNA fragmentation and appearance of apoptotic symptoms. Furthermore, it has been reported that caspases are activated during autophagy in dying cells and are suppressed for apoptosis induction (Martin et al., 2004; Yu et al., 2004). Therefore, it can be d<strong>edu</strong>ced that during autophagy, the effects of activated caspase-3 on their downstream substrates (like DFF-40) should be suppressed by special factors (e.g. DFF-45 in T-47D cells) only in those cellular routes which are involved in the appearance of apoptosis symptoms (e.g. DNA fragmentation). Cell cycle arrest, an important cellular target affected by sulfabenzamide and doxorubicin, was analyzed using flow cytometry. Incubation of T- 47D cells with 0.33 µM doxorubicin resulted in a significant accumulation of cells in S phase, and to a lesser extent in G2/M phase of the cell cycle (Figure 4B and Table 2). Thus, doxorubicin exerts its antiproliferative action mainly through cell cycle arrest. Induced mitotic catastrophe following increased activation of cyclinB1/Cdc2 may occur while cells are delayed, particularly in G2 phase of the cell cycle (Lindqvist et al., 2007). The induced G2/M arrest along with down regulation of cyclinB1 expression confirmed that anticancer activity of doxorubicin is not via mitotic catastrophe (Figure 5 and Table 2). In contrast to doxorubicin, minimal cell cycle arrest in S and G2/M phases (totally 18%) was observed in T-47D cells incubated with <strong>10</strong>.8 mM sulfabenzamide (Figure 4B and Table2). Thus, cell cycle arrest could not be mainly responsible for a 50% r<strong>edu</strong>ction in cell viability in the presence of sulfabenzamide. As we know, when apoptosis is blocked or delayed autophagy triggered and vice versa. These possibilities are consistent with our findings regarding lack of apoptosis in drug treated cells and induction of autophagy. The induced overexpression of ATG5 supported that autophagy Sulfabenzamide promotes autophagic cell death 51 triggered in the presence of sulfabenzamide (Figure 5). This could be probably occurred through the increase in Bax activity working on mitochondrial membrane to result in activation of caspase-3 for PARP1 and DNA-PK deactivation and autophagy induction. It has been reported that induction of autophagy by PUMA (the p53-inducible BH3-only protein) depends on Bax/Bak and can be reproduced by overexpression of Bax (Yee et al., 2009). Here, in doxorubicin treatment, increase in Bax activity could be occurred in parallel with the increment of Bax transcripts affecting on the cells for caspase-3 activation and changing the cell's destiny toward autophagy (Figure 5). This notion could be also supplied in sulfabenzamide treatment aside from the mild decrease in Bax expression because activation of the existed Bax molecules in the cells could be happened for caspase-3 activation and autophagy induction (Figure 5). It has been also reported that proteolytic cleavage of PARP1, performed by caspase-3, produces specific proteolytic cleavage fragments which are involved in the cell’s decision to change its fate from apoptosis toward autophagy (Munoz-Gamez et al., 2009; Chaitanya et al., 20<strong>10</strong>). Induction of autophagic cell death is dependent on DNA-PK inhibition (Daido et al., 2005). Thus, the increased activity of caspase-3 could finally deactivate PARP1 (has a decreased and constant expression level in sulfabenzamide and doxorubicin treatments, respectively) and DNA-PK (has an increased and invariable expression level in sulfabenzamide and doxorubicin treatments, respectively) until apoptosis was blocked and autophagy induced (Figures 3 and 5). Despite the existence of some controversies regarding the possible role of autophagy in tumor progression by promoting cell survival, autophagy can exist as a backup mechanism promoting cellular death when other mortality mechanisms are not functional. Hyperactivation of autophagy above the threshold point leads to unlimited self-eating of the cells causing autophagy or type II programmed cell death (Hoyer-Hansen et al., 2005; Maiuri et al., 20<strong>10</strong>). Based on our data, downregulation of AKT1 and AKT2 as well as upregulation of PTEN in sulfabenzamide treated cells indicated that cell survival pathways were slowed down (Figure 5). In addition, down regulation of bcl-2 was happened along with the induction of autophagy (Figure 5). It has been reported that targeted silencing of bcl-2 expression (an anti-autophagic gene) in human breast cancer cells with RNA-interference has
52 Raziye MOHAMMADPOUR et al. promoted autophagic cell death and thus presents a therapeutic potential (Akar et al., 2008). p53 is involved in decreasing cell survival potency through inactivation of AKT/mTOR pathways, and stimulation of autophagy via transactivation of DRAM (Maiuri et al., 2007). Thus, the observed increased expression level of DRAM and p53 genes support our conclusion that the repression of AKT/mTOR survival pathway (via p53 overexpression) and autophagy induction (via increased DRAM transcripts) are responsible for r<strong>edu</strong>ced viability of T-47D cells and induction of death inducing autophagy in the presence of sulfabenzamide (Figure 5). T -47D cells contain only a single copy of the p53 missense mutation (Schafer et al., 2000). It has been reported by various studies that mutant p53 may lose its natural antitumor activity (Lim et al., 2009). Interestingly, in the presence of sulfabenzamide the antitumor activities of mutant form of p53 should return to the normal activities of the wild type form to induce autophagic cell death. This is very similar to the mechanism of action for some antitumor drugs reactivating mutant p53 to kill cancerous cells (Lambert et al., 2009). Evidently, checking of the protein expression levels using other supplementary methods such as western blotting could provide us better documents to support the presented real time RT-PCR data. But, in our work, we found that the registered alterations for the level of RNA transcripts were in a good consistence with the expected cellular behaviors when the proteins' expression levels or their activities were theoretically going to become changed in parallel with the RNA levels in the cells. Therefore, regardless of some exceptions, evaluating RNA transcripts could provide us an adequate image illustrating the changes in the proteins’ expression levels in the cells. Conclusions In summary, we showed that cell cycle arrest (and possibly autophagy) may play a role in action of doxorubicin on T-47D cells. However, the contribution of apoptosis and cell cycle arrest antiproliferative effect of sulfabenzamide on T-47D cells is minimal. These observations are in contrast to many reports in which the mechanism of action of sulfonamide derivatives on cancer cells attributed to the conventional processes of apoptosis and cell cycle arrest. We believe that induction of autophagic cell death in T-47D cells is triggered through p53/DRAM pathway (occurred along with decreasing of Akt/mTOR pathway) and this is a reasonable cellular axis to justify our results. Abbreviations AKT: v-akt murine thymoma viral oncogene homolog, mTOR: Mechanistic Target Of Rapamycin, PTEN: Phosphatase and Tensin homolog, DRAM: Damage Regulated Autophagy Modulator, ATG5:Autophagy related gene 5, Beclin1: Bcl2 Interacting protein 1, PARP1: Poly ADP-Ribose Polymerase 1, DFF-40/CAD: DNA Fragmentation Factor 40/ Caspase-Activated DNase, Bax: Bcl2-Associated X protein, Bcl-2: B- Cell Lymphoma 2, AIF: Apoptosis Inducing Factor, DFF-45/iCAD: DNA Fragmentation Factor 45/inhibitor of Caspase-Activated DNase, Cdc2: Cell Division Cycle protein 2, ARF: ADP Ribosylation Factor, GAPDH: Glyceraldehyde-3- Phosphate Dehydrogenase, ACD: Autophagic Cell Death, PCDII: type II Programmed Cell Death, RPMI: Roswell Park Memorial Institute. Conflict of interest The authors declare that they have no competing interest. Authors' contributions SS designed the study and experiments, analyzed and interpreted data and also prepared the manuscript. RM carried out the experiments and participated in data analysis as well as writing the initial draft of the manuscript. SH and SF participated in performing the experiments. NS contributed on giving scientific comments and also carried out final editing of the manuscript. Acknowledgements Iran National Science Foundation (INSF) and Research Council of University of Tehran have been gratefully appreciated by the authors because of their foundational supports for this work. References Akar U, Chaves-Reyez A, Barria M, Tari A, Sanguino A, Kondo Y, et al. Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells. Autophagy. 4: 669–679, 2008. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell.
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