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A novel cancer stem-like cell line (3AB-OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG-63 cells by long-term treatment (100 days) with 3-aminobenzamide (3AB). 3AB-OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle-shaped, polygonal-shaped, and rounded-shaped. With respect to MG-63 cells, 3AB-OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self-renewal ability and much higher levels of cell cycle markers which account for G1-S/G2-M phases progression. Differently from MG-63 cells, 3AB-OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP-binding cassette transporter ABCG2-dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG-63 cells. 3AB-OS cells are much less committed to osteogenic and adipogenic differentiation than MG-63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF-1alpha, FLIP-L, Bcl-2, XIAP, IAPs, and survivin). 3AB-OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting.

The present study shows that nuclear factor erythroid 2-related factor 2 (NRF2) and miR-29b-1-5p are two opposite forces which could regulate the fate of MDA-MB-231 cells, the most studied triple-negative breast cancer (TNBC) cell line. We show that NRF2 activation stimulates cell growth and markedly reduces reactive oxygen species (ROS) generation, whereas miR-29b-1-5p overexpression increases ROS generation and reduces cell proliferation. Moreover, NRF2 downregulates miR-29b-1-5p expression, whereas miR-29b-1-5p overexpression decreases p-AKT and p-NRF2. Furthermore, miR-29b-1-5p overexpression induces both inhibition of DNA N-methyltransferases (DNMT1, DNMT3A, and DNMT3B) expression and re-expression of HIN1, RASSF1A and CCND2. Conversely, NRF2 activation induces opposite effects. We also show that parthenolide, a naturally occurring small molecule, induces the expression of miR-29b-1-5p which could suppress NRF2 activation via AKT inhibition. Overall, this study uncovers a novel NRF2/miR-29b-1-5p/AKT regulatory loop that can regulate the fate (life/death) of MDA-MB-231 cells and suggests this loop as therapeutic target for TNBC.

Triple-negative breast cancer (TNBC) is a form of BC characterized by highaggressiveness and therapy resistance probably determined by cancer stem cells. MCL1 is an antiapoptotic Bcl-2 family member that could limit the efficacy of anticancer agents as recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL). Here, we investigated MCL1 expression in TNBC tissues and cells. We found MCL1 differentially expressed (upregulated or downregulated) in TNBC tissues. Furthermore, in comparison to the human mammary epithelial cells, we found that MDA-MB-231 cells show similar messenger RNA levels but higher MCL1 protein levels, whereas it resulted downregulated in MDA-MB-436 and BT-20 cells. We evaluated the effects of rh-TRAIL and A-1210477, a selective MCL1 inhibitor, on cell viability and growth of MDA-MB-231 cells. We demonstrated that the drug combination reduced the cell growth and activated the apoptotic pathway. Similar effects were observed on three-dimensional cultures and tertiary mammospheres of MDA-MB-231 cells. In MDA-MB-231 cells, after MCL1 silencing, rh-TRAIL confined the cell population in the sub-G0/G1 phase and induced a drop in the mitochondrial transmembrane potential. To understand themolecular mechanism by which the loss of MCL1 function sensitizes the MDA-MB-231 cells to rh-TRAIL, we analyzed by real-time reverse transcription polymerase chain reaction, the expression of genes related to apoptosis, stemness, cell cycle, and those involved in epigenetic regulation. Interestingly, among the upregulated genes through MCL1 silencing or inhibition, there was TNFRSF10A (DR4). Moreover, MCL1 inhibition increased DR4 protein levels and its cell surface expression. Finally, we demonstrated MCL1-DR4 interaction and dissociation of this complex after A-1210477 treatment. Overall, our findings highlight the potential MCL1-roles in MDA-MB-231 cells and suggest that MCL1 targeting could be an effective strategy to overcome TNBC's rh-TRAIL resistance.

MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR- 29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b-1 ectopic overexpression decreased TNBC cell growth, self-renewal, migration, invasiveness and paclitaxel resistance repressing WNT/βcatenin and AKT signaling pathways and stemness regulators. We identified SPINDLIN1 (SPIN1) among predicted miR-29b- 1-5p targets. Consistently, SPIN1 was overexpressed in most TNBC tissues and cell lines and negatively correlated with miR-29b-1-5p. Target site inhibition showed that SPIN1 seems to be directly controlled by miR-29b-1-5p. Silencing SPIN1 mirrored the effects triggered by miR-29b-1 overexpression, whereas SPIN1 rescue by SPIN1miScript protector, determined the reversal of the molecular effects produced by the mimic-miR-29b-1-5p. Overall, we show that miR-29b-1 deregulation impacts on multiple oncogenic features of TNBC cells and their renewal potential, acting, at least partly, through SPIN1, and suggest that both these factors should be evaluated as new possible therapeutic targets against TNBC., This study was partially funded by the European Regional Development Fund, European Territorial Cooperation 2007-2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007-2013; the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2013; R. Di Fiore and R. Drago-Ferrante were recipients of fellowships granted by the European Regional Development Fund, European Territorial Cooperation 2007-2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007-2013; D. Carlisi was a recipient of a fellowship granted by MIUR (contract no. 82, January 23, 2014)., peer-reviewed

Osteosarcoma is a highly metastatic tumor affecting adolescents, for which there is no second-line chemotherapy. As suggested for most tumors, its capability to overgrow is probably driven by cancer stem cells (CSCs), and finding new targets to kill CSCs may be critical for improving patient survival. TP53 is the most frequently mutated tumor suppressor gene in cancers and mutant p53 protein (mutp53) can acquire gain of function (GOF) strongly contributing to malignancy. Studies thus far have not shown p53-GOF in osteosarcoma. Here, we investigated TP53 gene status/role in 3AB-OS cells-a highly aggressive CSC line previously selected from human osteosarcoma MG63 cells-to evaluate its involvement in promoting proliferation, invasiveness, resistance to apoptosis and stemness. By RT-PCR, methylation-specific PCR, fluorescent in situ hybridization, DNA sequence, western blot and immunofluorescence analyses, we have shown that-in comparison with parental MG63 cells where TP53 gene is hypermethylated, rearranged and in single copy-in 3AB-OS cells, TP53 is unmethylated, rearranged and in multiple copies, and mutp53 (p53-R248W/P72R) is post-translationally modified and with nuclear localization. p53-R248W/P72R-knockdown by short-interfering RNA reduced the growth and replication rate of 3AB-OS cells, markedly increasing cell cycle inhibitor levels and sensitized 3AB-OS cells to TRAIL-induced apoptosis by DR5 up-regulation; moreover, it strongly decreased the levels of stemness and invasiveness genes. We have also found that the ectopic expression of p53-R248W/P72R in MG63 cells promoted cancer stem-like features, as high proliferation rate, sphere formation, clonogenic growth, high migration and invasive ability; furthermore, it strongly increased the levels of stemness proteins. Overall, the findings suggest the involvement of p53-R248W/P72R at the origin of the aberrant characters of the 3AB-OS cells with the hypothesis that its GOF can be at the root of the dedifferentiation of MG63 cells into CSCs.

The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1 h of incubation with 25 µM PN and reached a peak at 8-16 h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3 h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16 h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells.

The mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. This article shows that parthenolide stimulates in human osteosarcoma MG63 and melanoma SK-MEL-28 cells a mechanism of cell death, which is not prevented by z-VAD-fmk and other caspase inhibitors. In particular treatment with parthenolide rapidly stimulated (1-2 h) reactive oxygen species (ROS) generation by inducing activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, cell detachment from the matrix, and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca(2+) also favored dissipation of Δψm, which seemed primarily determined by permeability transition pore opening, since Δψm loss was partially prevented by the inhibitor cyclosporin A. Staining with Hoechst 33342 revealed in most cells, at 3-5 h of treatment, chromatin condensation, and fragmentation, while only few cells were propidium iodide (PI)-positive. In addition, at this stage apoptosis inducing factor (AIF) translocated to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15 h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by N-acetylcysteine, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF.

Osteosarcoma (OS), an aggressive highly invasive and metastatic bone-malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self-renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let-7d microRNA was downregulated in the 3AB-OS-CSCs, derived from the human OS-MG63 cells. Here, we aimed to assess whether let-7d modulation affected tumorigenic and stemness properties of these OS-CSCs. We found that let-7d-overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 and p27 CDK-inhibitors. Let-7d also decreased sarcosphere-and-colony forming ability, two features associated with self-renewing, and it reduced the expression of stemness genes, including Oct3/4, Sox2, Nanog, Lin28B, and HMGA2. Moreover, let-7d induced mesenchymal-to-epithelial-transition, as shown by both N-Cadherin-E-cadherin-switch and decrease in vimentin. Surprisingly, such switch was accompanied by enhanced migratory/invasive capacities, with a strong increase in MMP9, CXCR4 and VersicanV1. Let-7d- overexpression also reduced cell sensitivity to apoptosis induced by both serum-starvation and various chemotherapy drugs, concomitant with decrease in caspase-3 and increase in BCL2 expression. Our data suggest that let-7d in 3AB-OS-CSCs could induce plastic-transitions from CSCs-to-non-CSCs and vice-versa. To our knowledge this is the first study to comprehensively examine the expression and functions of let-7d in OS-CSCs. By showing that let-7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let-7d modulation, it is urgent to better define its multiple functions. J. Cell. Physiol. 231: 1832-1841, 2016. © 2015 Wiley Periodicals, Inc.

Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non‑apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypo-phosphorylated/active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB‑231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB‑231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target.

The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we suggest that parthenolide rapidly stimulated NOX activity with production of superoxide anion (O2•-), which was converted by superoxide dismutase 1 (SOD1) into hydrogen peroxide (H2O2). In the second phase of treatment (8-16 h), parthenolide increased the number of positive cells to DHE signal. Since this event was not prevented by apocynin and DPI and was associated with positivity of cells to MitoSox Red, a fluorochrome used to detect mitochondrial production of O2•-, we suggest that parthenolide induced production of O2•- at the mitochondrial level independently by NOX activity in the second phase of treatment. Finally, in this phase, most cells became positive to hydroxyphenyl fluorescein (HPF) signal, a fluorescent probe to detect highly reactive oxygen species (hROS), such as hydroxyl radical and peroxynitrite. Therefore, parthenolide between 8-16 h of treatment induced generation of O2•- and hROS, in close correlation with a marked reduction in cell viability.

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR‑29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel therapeutic agent might offer benefits for OS treatment.

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5–20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs.

Retinoblastoma is the most common intraocular malignancy of childhood. In developing countries, treatment is limited, long-term survival rates are low and current chemotherapy causes significant morbidity to pediatric patients and significantly limits dosing. Therefore there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of patients with retinoblastoma. here, we investigated the effects of two natural compounds okadaic acid (OKa) and parthenolide (PN) on human retinoblastoma Y79 cells. For the first time we showed that OKa/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by lowering in p-akt levels, increasing in the stabilized forms of p53 and potent decrease in ps166-Mdm2. We also showed the key involvement of PTeN which, after OKa/PN treatment, potently increased before p53, thus suggesting that p53 activation was under PTeN action. Moreover, after PTEN-knockdown p-akt/ ps166Mdm2 increased over basal levels and p53 significantly lowered, while OKa/PN treatment failed both to lower p-akt and ps166-Mdm2 and to increase p53 below/over their basal levels respectively. OKa/PN treatment potently increased ROs levels whereas decreased those of Gsh. Reducing cellular Gsh by l-butathionine-[s,R]-sulfoximine treatment significantly anticipated the cytotoxic effect exerted by OKa/ PN. Furthermore, the effects of OKa/PN treatment on both Gsh content and cell viability were less pronounced in PTeN silenced cells than in control cells. The results provide strong suggestion for combining a treatment approach that targets the PTeN/akt/Mdm2/p53 pathway.

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second-line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB-OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB-OS cells have hypertriploid karyotype with 71-82 chromosomes. By comparing 3AB-OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let-7/98 and miR-29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets.

Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. In this paper, we report the production of mammospheres from three lines of TNBC cells and demonstrate that both parthenolide (PN) and its soluble analog dimethylaminoparthenolide (DMAPT) suppressed this production and induced cytotoxic effects in breast cancer stem-like cells, derived from dissociation of mammospheres. In particular, the drugs exerted a remarkable inhibitory effect on viability of stem-like cells. Such an effect was suppressed by N-acetylcysteine, suggesting a role of reactive oxygen species (ROS) generation in the cytotoxic effect. Instead z-VAD, a general inhibitor of caspase activity, was ineffective. Analysis of ROS generation, performed using fluorescent probes, showed that both the drugs stimulated in the first hours of treatment a very high production of hydrogen peroxide. This event was, at least in part, a consequence of activation of NADPH oxidases (NOXs), as it was reduced by apocynin and diphenylene iodinium, two inhibitors of NOXs. Moreover, both the drugs caused downregulation of Nrf2 (nuclear factor erythroid 2-related factor 2), which is a critical regulator of the intracellular antioxidant response. Prolonging the treatment with PN or DMAPT we observed between 12 and 24 h that the levels of both superoxide anion and hROS increased in concomitance with the downregulation of manganese superoxide dismutase and catalase. In addition, during this phase dissipation of mitochondrial membrane potential occurred together with necrosis of stem-like cells. Finally, our results suggested that the effect on ROS generation found in the first hours of treatment was, in part, responsible for the cytotoxic events observed in the successive phase. In conclusion, PN and DMAPT markedly inhibited viability of stem-like cells derived from three lines of TNBCs by inducing ROS generation, mitochondrial dysfunction and cell necrosis., peer-reviewed

Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM2, the phosphorylated-MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt-MDM2-p53 complex and increased nuclear p53 levels. Our results suggest that phospho-Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho-Akt and inhibitor of apoptosis proteins.

Paclitaxel (PTX) is an anticancer drug currently in phase II clinical trials. This study shows for the first time that low doses of PTX (5 nM) potently induce apoptosis in human retinoblastoma Y79 cells. The effect of PTX is accompanied by a potent induction of E2F1 which appears to play a critical role in the effects induced by PTX. PTX induced a dose- and time-dependent effect, with G2/M arrest, cyclines A, E and B1 accumulation and a marked modification in the status of Cdc2-cyclin B1 complex, the major player of the G2/M checkpoint. Apoptosis followed G2/M arrest. An early and prolonged increase in p53 expression with its stabilization by phosphorylation and acetylation and its nuclear translocation occurred. Consistently, PTX increased p21WAF1, bax and MDM2 levels, suggesting that p53 is transcriptionally active. p53 accumulated following both E2F1 up-regulation and increase in the levels of p14ARF which interacts with MDM2 preventing ubiquitination and proteosomal degradation of p53. Both extrinsic (E2F1/Fas/JNK/caspase-2 activation) and intrinsic (Bcl-2 phosphorylation, Bid fragmentation and Bax increase) pathways seemed to be involved. Loss of mitochondrial potential and activation of apoptosome and executive caspase-3,-6 and-7 was shown. Incubation with either the irreversible pan-caspase inhibitors Z-VAD-FMK, or SP600125, a selective inhibitor of JNK, or pifithrin alpha, a potent p53 inhibitor, significantly inhibited the effects induced by PTX.