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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.

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in m, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company

Breast cancer (BC) is the most common endocrine cancer and the second leading cause of cancer-related death in women. About 75% of BCs expresses high levels of estrogen receptors that sustain the tumor growth. Moreover, in BC estrogens prevent apoptosis induced by granzyme B released by cytotoxic T lymphocytes and NK cells through the production of the granzyme B inhibitor PI-9. As a consequence, cancer cells acquire the ability to escape immune surveillance’s signaling. Although some studies explored the role of PI-9 in BC cells, its presence has not been investigated in cancer stem cells so far. In this research, tertiary tumorspheres were obtained from estrogen receptor-alfa positive (ERalfa+) BC MCF7 cells and studies were performed to evaluate their stem identity. These tumorspheres showed high levels of stemness markers (Nanog, Oct3/4 and Sox2) and self-renewal ability. The exposure to estrogens (17-beta estradiol and genistein) increased the number and sizes of tumorspheres as well as the level of the proliferating cell nuclear antigen (PCNA). The analysis of the three isoforms (66, 46 and 36 kDa) of ERalfa disclosed that tertiary tumorspheres exhibit a marked increase in ERalfa36, while the level of ERalfa66, which is highly expressed in MCF7 cells, dropped. Then, we analyzed the granzyme B inhibitor PI-9, which is transcriptionally regulated by ERalfa66. Surprisingly, we found that tertiary tumorspheres express a higher level of both PI-9 protein and mRNA than MCF7 cells, despite the reduced level of ERalfa66. The high content of PI-9 might be ascribed to the activation of proliferative CXCR4/phospho-p38 axis which was observed only in tertiary tumorspheres. Taken together, these events could supply a selective advantage to BC stem cells by interfering with immune surveillance systems and open the way to new possible targets for BC treatment.

Introduction: Osteosarcoma (OS), an aggressive tumor a!ecting adolescents, shows therapy resistance and recurrence which can depend on cancer stem cells (CSCs), the source for tissue renewal and hold malignant potential; thus, OS treatment requires their eradication. Here, using 3AB-OS CSCs previously obtained from the OS-MG63 cells, we focused on the role of let-7d in managing stemness properties of 3AB-OS CSCs. Methods: 3AB-OS CSCs were stably transfected with pCDomH-plasmid, containing mir-let7-d, or empty vector as a control. Cell proliferation and viability were evaluated by EdU and Trypan blue assays, respectively. Sarcosphere and colony assays and wound healing and transwell invasion assays were performed to evaluate let-7d e!ects on self-renewal and migratory/invasive abilities of 3AB-OS cells, respectively. Publicly available databases were used to identify let-7d predicted target genes. RT-PCR and western blot analyses were employed to validate the targets of let-7d. Results: We have found that let-7d-overexpression reduces cell proliferation, lowers CCND2 and E2F2, increases p21 and p27. Let-7d also decreases sarcosphere- and colony-forming ability and decreases Oct3/4, Sox2, Nanog, Lin28B and HMGA2. Moreover, let-7d induces mesenchymal-to-epithelium-transition, as shown by both N-Cadherin-E-cadherin-switch and vimentin decrease. "is switch is accompanied by enhanced migratory/invasive capacities and by MMP9, CXCR4 and VersicanV1 increases. Resistance to serum starvation and chemotherapy, decrease in caspase-3 and increase in Bcl-2 were also observed. Conclusions: "is study shows that let-7d displays both suppressor and oncogenic functions, suggesting that, before prospecting new therapeutic strategies by let-7d use, it is urgent to better understand its functions.

Introduction: Breast cancers (BC) are the major cause of death in women. More than 70% of BCs express high levels of estrogen receptor-α (ERα) and are sustained for their growth by the hormone. Estrogens seem to protect BC cells from apoptosis mediated by immunosurveillance associated with cytotoxic T lymphocytes and NK cells granzyme B release. However, the production of granzyme B inhibitor PI-9 by tumor cells causes a short-circuit in immunosurveillance’s signalling. Although it has been shown the role of PI-9 in BC cells, its presence has not been investigated in tumor stem cells so far. Methods: Cell viability was evaluated by MTT, cell cycle by propidium iodide staining; mRNA and protein levels by qPCR and western blotting. Tumorspheres from ERα+BC MCF7 cells were isolated in ultra-low attachment conditions. The higher expression of stemness markers (Nanog, Oct3/4 and Sox2) was found in tertiary tumorspheres which were used in our study. Results: Low doses (10 nM-10 μM) of 17-β estradiol consistently increased the number of MCF7 cells more than tumorspheres, while higher doses (50-100 μM) reduced cell number as a consequence of G2/M cell cycle arrest. The analysis of ERα disclosed the presence of three different isoforms (66, 46 and 36 kDa) in MCF7 cells. In contrast, tumorspheres exhibited an increase in ERα36, which lacks transcriptional activity, while the level of ERα66 was undetectable. Then, we analyzed the level of PI-9, which is transcriptionally regulated by ERα66. Surprisingly, we found that tertiary tumorspheres, express higher levels of both PI-9 protein and mRNA than MCF7 cells. Conclusions: Our data provided evidence that the high level of PI-9 in ER+ tertiary tumorspheres could supply a selective advantage to BC stem cells by interfering with immune-surveillance systems. Ongoing studies aim to elucidate the relationship between the levels of different ERα isoforms and PI-9 high expression in BC-stem cells.

Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. It is known that a small proportion of tumour cells, termed cancer stem cells (CSCs), is responsible for tumour formation, progression and recurrence. The sesquiterpene lactone parthenolide (PN) was identified as the first small molecule capable of killing CSCs.1 Previously we have shown2 that PN and its soluble analogue DMAPT induce a strong cytotoxic effect in MDA-MB231 cells, the most studied line of TNBCs. In the present research we investigated about the effects exerted by both PN and DMAPT on breast cancer stem-like cells derived from three lines of TNBCs (MDA-MB231, BT20 and MDA-MB436). The two compounds inhibited both the production of mammospheres from the three lines of cells and the viability of breast cancer stem-like cells derived from dissociation of mammospheres. This effect was suppressed by NAC, while z-VAD, a general inhibitor of caspase activity, was ineffective. PN and DMAPT induced in stem-like cells, in the first hours of treatment, a strong production of hydrogen peroxide. Prolonging the time of treatment (12-24h) the levels of both superoxide anion and hROS (hydroxyl radicals and peroxynitrite) increased in concomitance with down-regulation of MnSOD and catalase, dissipation of mitochondrial membrane potential and cell necrosis. It is noteworthy that treatment with PN and DMAPT also caused a rapid and remarkable decrement of the level of Nrf-2, which is a critical regulator of the intracellular antioxidant response. 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.

Osteosarcoma (OS) is the most common malignancy of bone in children and adolescent. It is a highly invasive and metastatic bone-malignancy because of which, despite therapeutic advances, 30%-50% of patients still die of pulmonary metastasis. As a consequence, there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of the patients. Advances in OS treatment are inconceivable without better understanding of molecular mechanism of osteosarmagenesis and, especially, metastatic processes. Growing evidence suggests that cancer stem cells (CSCs), which have self-renewing and malignant potential, are at the root of tumor growth and relapse. Thus, a challenge for innovative therapy is their identification and eradication. Here, we have used the 3AB-OS CSCs, a cell line previously produced in our laboratory from the OS-MG63 cells, which was genetically, molecularly and functionally characterized. This study was focused on the role of let-7d miRNA-previously found by us to be downregulated in 3AB-OS-CSCs- in managing their stemness properties. We have found that let-7d-overesperession reduces cell proliferation by both decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 and p27 CDK-inhibitors. Let-7d also reduces sarcosphere and colony-forming ability and the expression of Oct3/4, Sox2, Nanog, Lin28B and HMGA2, key regulators of cancer cell stemness. Moreover, let-7d induces mesenchymal-to-epithelium-transition, as shown by both N-Cadherin-E-cadherin-switch and vimentin decrease. Surprisingly, this swich was accompanied by enhanced migratory/invasive capacities and by increases in MMP9, CXCR4 and VersicanV1. Let-7d also reduced the resistance to serum starvation and chemotherapy. A decrease in caspase-3 with an increase in Bcl-2 was also observed. Overall, this study shows that let-7d displaying both suppressor and oncogenic functions behaves as a Janus-Faced miRNA. Thus, we suggest that, before prospecting new therapeutic strategies by let-7d modulation, it is urgent to better understand its functions.

A new polycylic compound was synthesized and evaluated for its antiproliferative activity against the NCI panel of human tumoral cell lines showing IC50 values in the range 0.47-5.43 µM. The compound induced a decrease of the level of cyclin B1, whereas increased the level of the inhibitor p21 WAF1

We showed that the sesquiterpene lactone Parthenolide (PN) exerts strong cytotoxic effects on triple negative breast cancer MDA-MB231 cells. Our recent results suggest that PN exerts in these cells a cytoprotective effect, which is due to the activation of mTOR pathway. To inhibit this protective response we employ the HDAC inhibitor SAHA, which is known to prevent AKT/mTOR pathway. We show that PN activates Akt, mTOR, p70S6kinase and NRF2 while SAHA abolishes these effects. Further cell pretreatment with SAHA synergistically sensitizes the cells to the cytotoxic effect of PN. Moreover SAHA alone activates the autophagic process. The addition of PN to SAHA reduces this effect and induces apoptosis. SAHA/PN combination also inhibits DNMT1 and produces hyperacetylation of histones. Epigenetic changes caused by these effects are responsible for the increased expression of oncosuppressor gene products, such as p21 and p27 and for the decrease of Bcl2 and p65 levels. In conclusion SAHA suppresses protective response exerted by PN; PN inhibits SAHA effect on autophagy and finally SAHA/PN combination induces epigenetic modifications with changes in gene expression.

Retinoblastoma is the most common intraocular malignancy afflicting children. The incidence is higher in developing countries, where treatment is limited and long-term survival rates are low. Vincristine, etoposide, and carboplatin -the agents commonly used in the treatment of retinoblastoma- determine side effects causing significant morbidity to pediatric patients and significantly limiting dosing. Thus, identifying new drugs and molecular targets to facilitate the development of novel therapeutics, and finding natural drug combinations to kill cancer cells by synergistically acting at subtoxic doses, may be a good goal. Here, we investigated the effects of two natural compounds, okadaic acid (OKA) and parthenolide (PN), in human retinoblastoma Y79 cells. We showed that OKA/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by decrease in p-Akt, increase in the stabilized p53 forms and potent decrease in pS166–Mdm2. We also showed the key involvement of PTEN which, after OKA/PN treatment, potently increased before p53, suggesting that p53 activation was under PTEN action. PTEN-knockdown increased p-Akt/ pS166Mdm2 over basal levels and significantly lowered p53, 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 while decreased those of GSH. Reducing cellular GSH by butathionine-sulfoximine treatment significantly anticipated the cytotoxic effect exerted by OKA/PN. The effects of OKA/PN treatment on both GSH content and cell viability were less pronounced in PTEN silenced cells than in control cells. Our study reports for the first time both a synergistic apoptotic action between OKA and PN and the involvement of PTEN as key player in the apoptotic mechanism in human retinoblastoma Y79 cells. The results provide strong suggestion for combined inhibition of the PTEN/Akt/Mdm2/p53 pathway.

Parthenolide, a sesquiterpene lactone found in European feverfew, is used in traditional medicine for its anti-inflammatory activity. In addition, parthenolide has been considered as a novel and effective anti-tumor agent because it induces cytotoxic effects in several tumor cell lines. Our studies demonstrated that parthenolide exerted strong cytotoxic effects in osteosarcoma MG63 and melanoma SK-Mel28 cells in culture. Staining with Hoechst 33342 revealed in most cells after brief periods of treatments (3-5h) chromatin condensation and fragmentation, while only few cells were PI-positive. Prolonging the treatment (5-14h) PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective, thus suggesting a caspase-independent cell death. The study of the mechanism of action provided evidence that treatment with parthenolide rapidly stimulated (1-2 h) ROS generation, in particular by inducing activation of extracellular signal-regulated kinase1/2 and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, JNK activation and cell detachment from the matrix. ROS generation together with mitochondrial accumulation of Ca2+ favoured dissipation of Δψm, which appeared primarily determined by the opening of the permeability transition pore (PTP), since Δψm loss was partially prevented by cyclosporin A, an inhibitor of PTP opening. Recently, we focused our attention on MDA-MB231 cells, a very aggressive and poorly differentiated breast cancer cell line, which is negative for estrogen receptor alpha. Preliminary results suggested that parthenolide induced cell death in these cells with a mechanism similar to that demonstrated in osteosarcoma and melanoma cells. Interestingly, we demonstrated that in MDA-MB231 cells the effect of parthenolide was potentiated by the addition of z-VAD-fmk, a general inhibitor of caspases. Studies are in progress to elucidate the mechanism of this interaction which could suggest new strategies for the treatment of ER-α negative breast cancer.