Modulating tumor reactive stroma by extracorporeal shock waves to control prostate cancer progression.

BACKGROUND: Prostate cancer is the second most common cancer worldwide. Tumor microenvironment is composed of activated fibroblasts, the so called carcinoma-associated fibroblasts (CAFs). They express high levels of α-smooth muscle actin (α-SMA) and type I collagen (COL1), and support proliferation and migration of tumor epithelial cells. Extracorporeal shock waves (ESWs), acoustic waves, are effective in the treatment of hypertrophic scars, due to their ability to modulate fibrosis. Based on this rationale, the study evaluated the effects of ESWs on CAF activation and the influence of ESW-treated CAFs on the growth and migration of epithelial prostatic carcinoma cells. METHODS: Primary cultures of CAFs (n = 10) were prepared from tumors of patients undergoing surgery for high-risk prostate carcinoma. CAFs were treated with ESWs (energy levels: 0.32 mJ/mm2 , 1000 pulses; 0.59 mJ/mm2 , 250 pulses). After treatment, the messenger RNA and protein levels of the stromal activation markers α-SMA and COL1 were determined. Subsequently, two different stabilized cell lines (PC3 and DU145) of androgen-resistant prostate cancer were treated with the conditioned media produced by ESW-treated CAFs. At different times, viability and migration of PC3 and DU145 cells were evaluated. Viability was also assessed by coculture system using CAFs and PC3 or DU145 cells. RESULTS: ESWs reduced gene expression and protein level of α-SMA and COL1 in CAFs. The treatment of PC3 and DU145 with conditioned media of ESW-treated CAFs determined a reduction of their growth and invasive potential. Coculture systems between ESW-treated CAFs and PC3 or DU145 cells confirmed the epithelial cell number reduction. CONCLUSIONS: This in vitro study demonstrates for the first time that ESWs are able to modulate the activation of prostate CAFs in favor of a less "reactive" stroma, with consequent slowing of the growth and migration of prostate cancer epithelial cells. However, only further studies to be performed in vivo will confirm the possibility of using this new therapy in patients with prostate cancer.

Extracorporeal shock waves trigger tenogenic differentiation of human adipose-derived stem cells.

PURPOSES: Incomplete tendon healing impairs the outcome of tendon ruptures and tendinopathies. Human Adipose-derived Stem Cells (hASCs) are promising for tissue engineering applications. Extracorporeal Shock Waves (ESW) are a leading choice for the treatment of several tendinopathies. In this study, we investigated the effects of ESW treatment and tenogenic medium on the differentiation of hASCs into tenoblast-like cells. MATERIALS AND METHODS: hASCs were treated with ESW generated by a piezoelectric device and tenogenic medium. Quantitative real-time PCR was used to check the mRNA expression levels of tenogenic transcription factors, extracellular matrix proteins, and integrins. Western blot and immunofluorescence were used to detect collagen 1 and fibronectin. Collagen fibers were evaluated by Masson staining. Calcium deposition was assessed by Alizarin Red staining. RESULTS: The combined treatment improved the expression of the tendon transcription factors scleraxis and eyes absent 2, and of the extracellular matrix proteins fibronectin, collagen I, and tenomodulin. Cells acquired elongated and spindle shaped fibroblastic morphology; Masson staining revealed the appearance of collagen fibers. Finally, the combined treatment induced the expression of alpha 2, alpha 6, and beta 1 integrin subunits, suggesting a possible role in mediating ESW effects. CONCLUSIONS: ESW in combination with tenogenic medium improved the differentiation of hASCs toward tenoblast-like cells, providing the basis for ESW and hASCs to be used in tendon tissue engineering.

Extracorporeal shock waves modulate myofibroblast differentiation of adipose-derived stem cells.

Mesenchymal stem cells are precursors of myofibroblasts, cells deeply involved in promoting tissue repair and regeneration. However, since myofibroblast persistence is associated with the development of tissue fibrosis, the use of tools that can modulate stem cell differentiation toward myofibroblasts is central. Extracorporeal shock waves are transient short-term acoustic pulses first employed to treat urinary stones. They are a leading choice in the treatment of several orthopedic diseases and, notably, they have been reported as an effective treatment for patients with fibrotic sequels from burn scars. Based on these considerations, the aim of this study is to define the role of shock waves in modulating the differentiation of human adipose-derived stem cells toward myofibroblasts. Shock waves inhibit the development of a myofibroblast phenotype; they down-regulate the expression of the myofibroblast marker alpha smooth muscle actin and the extracellular matrix protein type I collagen. Functionally, stem cells acquire a more fibroblast-like profile characterized by a low contractility and a high migratory ability. Shock wave treatment reduces the expression of integrin alpha 11, a major collagen receptor in fibroblastic cells, involved in myofibroblast differentiation. Mechanistically, the resistance of integrin alpha 11-overexpressing cells to shock waves in terms of alpha smooth muscle actin expression and cell migration and contraction suggests also a role of this integrin in the translation of shock wave signal into stem cell responses. In conclusion, this in vitro study shows that stem cell differentiation toward myofibroblasts can be controlled by shock waves and, consequently, sustains their use as a therapeutic approach in reducing the risk of skin and tissue fibrosis.

Shock waves induce activity of human osteoblast-like cells in bioactive scaffolds.

BACKGROUND: Bone replacement is frequently needed in periodontal, orthopedic, and maxillofacial diseases. To avoid complications with autografts and allografts, artificial grafts (scaffolds) are candidates for stimulating bone regeneration after colonization with osteoblasts. Moreover, osteoblast activity can be induced by biological or physical stimulation. In this research, extracorporeal shock waves were used to improve the ability of human osteoblasts to colonize scaffolds and to induce their osteogenic properties. METHODS: Osteoblasts, treated with shock waves, were seeded on glass-ceramic macroporous scaffolds. Cells in scaffolds were counted after detachment and examined for calcium nodule formation (Alizarin staining), for differentiation markers (real time polymerase chain reaction), and for scaffold colonization (scanning electron microscope). RESULTS: Shock waves initially increased both the number and the activity of osteoblasts in the scaffold, but subsequently increased only osteoblast activity. Moreover, shock waves favored scaffold colonization even in the deeper layers. CONCLUSIONS: The calcium deposits and differentiation markers studied have demonstrated that shock waves increase osteoblast migration and penetration into scaffolds. CLINICAL RELEVANCE: This study may provide an important starting point for the introduction of shock waves to boost bone formation through osteoblast stimulation in diseases characterized by bone defects.

Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients.

Chronic obstructive pulmonary disease (COPD) is due to structural changes and narrowing of small airways and parenchymal destruction (loss of the alveolar attachment as a result of pulmonary emphysema), which all lead to airflow limitation. Extracorporeal shock waves (ESW) increase cell proliferation and differentiation of connective tissue fibroblasts. To date no studies are available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects. We obtained primary bronchial fibroblasts from bronchial biopsies of 3 patients with mild/moderate COPD and 3 control smokers with normal lung function. 16HBE cells were also studied. Cells were treated with a piezoelectric shock wave generator at low energy (0.3 mJ/mm2, 500 pulses). After treatment, viability was evaluated and cells were recultured and followed up for 4, 24, 48, and 72 h. Cell growth (WST-1 test) was assessed, and proliferation markers were analyzed by qRT-PCR in cell lysates and by ELISA tests in cell supernatants and cell lysates. After ESW treatment, we observed a significant increase of cell proliferation in all cell types. C-Kit (CD117) mRNA was significantly increased in 16HBE cells at 4 h. Protein levels were significantly increased for c-Kit (CD117) at 4 h in 16HBE (p < 0.0001) and at 24 h in COPD-fibroblasts (p = 0.037); for PCNA at 4 h in 16HBE (p = 0.046); for Thy1 (CD90) at 24 and 72 h in CS-fibroblasts (p = 0.031 and p = 0.041); for TGFß1 at 72 h in CS-fibroblasts (p = 0.038); for procollagen-1 at 4 h in COPD-fibroblasts (p = 0.020); and for NF-κB-p65 at 4 and 24 h in 16HBE (p = 0.015 and p = 0.0002). In the peripheral lung tissue of a representative COPD patient, alveolar type II epithelial cells (TTF-1+) coexpressing c-Kit (CD117) and PCNA were occasionally observed. These data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in 16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects.

Molecular mechanisms of the D327N SHBG protective role on breast cancer development after estrogen exposure.

Sex Hormone-Binding Globulin, the specific carrier for sex steroids, regulates hormone bioavailable fraction and estrogen signaling system in breast cancer cells. A common single nucleotide polymorphism in the human SHBG gene results in an amino acid substitution (Asp327Asn), which introduces an additional N-glycosylation site, and is associated with reduced breast cancer risk in postmenopausal women. The frequency of this polymorphism was evaluated in a group of patients that developed breast cancer while taking hormonal replacement therapy (HRT) for menopause, an interesting model of estrogen exposure. The polymorphism frequency was significantly higher in women taking HRT that didn't develop any breast cancer than in breast cancer patients (P<0.05). To get insight into the underlying mechanisms, we compared the ability of recombinant wild type and variant (D327N) SHBG to influence estradiol effects in MCF-7 breast cancer cells. D327N SHBG was more effective than wild type protein in inhibiting estradiol-induced cell proliferation and anti-apoptosis. This depended on the fact that D327N SHBG binding to MCF-7 cells was significantly higher than that of wild type protein. As a consequence, D327N caused a larger induction of the second messenger cAMP and a deeper inhibition of the estradiol-induced Erk (1/2) phosphorylation. Our observations, demonstrating the increased efficiency of D327N SHBG in counteracting estradiol action and a significantly higher frequency of Asp327Asn polymorphism in women not developing breast cancer after estrogen exposure, first provide evidence for the mechanism of D327N SHBG protective action.

Extracorporeal shock waves enhance normal fibroblast proliferation in vitro and activate mRNA expression for TGF-beta1 and for collagen types I and III.

BACKGROUND AND PURPOSE: Extracorporeal shock waves (ESWs) are used to good effect in the treatment of soft tissue injuries, but the underlying mechanisms are still unknown. We therefore determined the effects of ESWs on normal fibroblasts in vitro, in order to assess treatment-induced cell response. METHODS: A normal human fibroblast cell line (NHDF-12519) was treated with ESWs generated by a piezoelectric device (Piezoson 100; Richard Wolfe) using different protocols of impulses (300, 1,000, or 2,000 shots) and energy (0.11 or 0.22 mJ/mm(2)). Untreated controls and treated cells were cultivated for 12 days following a single shock-wave treatment. Viability, growth rate, and expression of mRNA for TGFbeta-1 and collagen types I and III were evaluated at days 3, 6, 9, and 12. RESULTS: 1 hour after shock-wave treatment, cell viability showed a decrease related mainly to impulse numbers applied. Fibroblasts treated with energy of 0.22 mJ/mm(2) subsequently showed an increase in proliferation from day 6 to day 9 that was higher than in untreated controls, without interference with the normal cell kinetic profile. mRNA expression was also higher in treated fibroblasts than in untreated controls for TGFbeta-1 on day 6 and day 9, for collagen type I on day 6, and for collagen type III on day 9. INTERPRETATION: These in vitro data confirm that the main factors involved in the repair process of connective tissues are activated by ESWs. The study gives the rationale for, and may provide schedules for, ESW treatment of tendonopathies.

High energy shock waves (HESW) increase paclitaxel efficacy in a syngeneic model of breast cancer.

The combined effect of high energy shock waves (HESW), generated by a piezoelectric device, and paclitaxel on Mat B-III rat breast cancer cells in vitro and in an in vivo animal model is presented. A significant reduction of in vitro Mat B-III cell proliferation versus cells treated with paclitaxel alone was observed with the combined exposure to paclitaxel (0.1, 1, or 10 nM) and HESW (0.22 mJ/mm2, 1000 shots). Moreover earlier induction and enhanced apoptosis occurred in cells subjected to the combined treatment with paclitaxel and HESW at 1 and 10 nM versus paclitaxel alone (p<0.01 and p<0.001, respectively). The percentage of apoptotic cells along with BAD mRNA expression, confirm a significant enhancement of apoptosis in tumor tissues subjected to the combined treatment with paclitaxel (2.5 mg/kg on days 7 and 11) and HESW (0.50 mJ/mm2, 500 shots on day 11) in comparison with paclitaxel alone. In conclusion, these data suggest that HESW enhance paclitaxel cytotoxicity in the Mat B-III syngeneic model of breast cancer.

Fibulin-1 interacts with Sex Hormone Binding Globulin and is linked to less aggressive estrogen-dependent breast cancers.

AIMS: Interaction of Sex Hormone-Binding Globulin (SHBG) with estrogen-sensitive breast cancer cells has a protective role against estrogen exposure. No specific membrane receptor for SHBG had been identified by now, but a putative interaction of SHBG with extracellular matrix associated-proteins (e.g. fibulins) was suggested. In this study we investigated the expression of fibulins, their functional relationship with SHBG and involvement in behavior of estrogen-sensitive breast cancer. MAIN METHODS: Gene expression of fibulins was performed by Real time-PCR on two estrogen-sensitive breast cancer cell lines, MCF-7 and T47D. Fibulin-1 protein expression and localization were determined by Western blot and immunofluorescence. SHBG interaction with-fibulin-1 was assessed by GST-pull down assay. MCF-7 cell growth and gene expression, after fibulin-1 silencing by siRNA, were evaluated. Finally, the expression of fibulin-1 was correlated to clinical and pathological data of 21 breast cancer tissue samples. KEY FINDINGS: Fibulin-1 was expressed in both cell lines and it was increased by estradiol. SHBG interacted with fibulin-1C; proteins co-localized at MCF-7 cell membranes and SHBG localization at membranes disappeared after silencing fibulin-1. Fibulin-1 silencing, moreover, generated MCF-7 cells unresponsive to estradiol and SHBG and characterized by increased proliferation. Finally, in breast cancer tissue samples expressing fibulin-1 the proliferation index was significantly lower than in fibulin-1 negative samples. SIGNIFICANCE: Fibulin-1 interacts with SHBG, it is associated with a less aggressive behavior of breast cancer cells and correlates to a better prognosis of the tumor.

Extracorporeal shockwaves (ESWs) enhance the osteogenic medium-induced differentiation of adipose-derived stem cells into osteoblast-like cells.

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue engineering, given their potential to differentiate into osteoblast-like cells. Interactions among biochemical and mechanical signals result in bone formation and repair. In this process stem cells have a crucial role. Extracorporeal shockwaves (ESWs) are acoustic waves capable of enhancing bone regeneration, suggesting that ESWs may induce some signals for mesenchymal progenitor maturation. The aim of the present work is to investigate the effects of ESW treatment on the differentiation of hASCs into osteoblast-like cells and to better clarify the mechanisms involved. The hASCs were treated with ESWs and osteogenic medium, and the effects in terms of gene expression, alkaline phosphatase (ALP) activity and calcium deposition were then evaluated. Moreover, to investigate the mechanisms of ESW action, reactive oxygen species (ROS) production, extracellular-signal-regulated kinase (ERK) and small 'mothers against' decapentaplegic (Smad) phosphorylation, and bone morphogenetic protein 2 (BMP2) expression were assessed. The ESW treatment increased Runt-related transcription factor 2 (Runx2), ALP and BMP2 expression, as well as ALP activity and calcium deposits with respect to untreated cells. Moreover ESWs induced ROS formation, and both ERK and Smad phosphorylation. The present study shows the effects of ESWs on osteogenic differentiation in an in vitro model using hASCs and defines the mechanisms involved in this process. The observations suggest that the combination of autologous hASCs and ESW treatment may improve bone tissue repair in tissue engineering procedures. Copyright © 2014 John Wiley & Sons, Ltd.

Combining doxorubicin-nanobubbles and shockwaves for anaplastic thyroid cancer treatment: preclinical study in a xenograft mouse model.

Anaplastic thyroid cancer is one of the most lethal diseases, and a curative therapy does not exist. Doxorubicin, the only drug approved for anaplastic thyroid cancer treatment, has a very low response rate and causes numerous side effects among which cardiotoxicity is the most prominent. Thus, doxorubicin delivery to the tumor site could be an import goal aimed to improve the drug efficacy and to reduce its systemic side effects. We recently reported that, in human anaplastic thyroid cancer cell lines, combining doxorubicin-loaded nanobubbles with extracorporeal shock waves, acoustic waves used in lithotripsy and orthopedics without side effects, increased the intracellular drug content and in vitro cytotoxicity. In the present study, we tested the efficacy of this treatment on a human anaplastic thyroid cancer xenograft mouse model. After 21 days, the combined treatment determined the greatest drug accumulation in tumors with consequent reduction of tumor volume and weight, and an extension of the tumor doubling time. Mechanistically, the treatment induced tumor apoptosis and decreased cell proliferation. Finally, although doxorubicin caused the increase of fibrosis markers and oxidative stress in animal hearts, loading doxorubicin into nanobubbles avoided these effects preventing heart damage. The improvement of doxorubicin anti-tumor effects together with the prevention of heart damage suggests that the combination of doxorubicin-loaded nanobubbles with extracorporeal shock waves might be a promising drug delivery system for anaplastic thyroid cancer treatment.

High energy shock waves enhance the cytotoxic effect of doxorubicin and methotrexate to human osteosarcoma cell lines.

Despite recent advances, the prognosis of relapsed osteosarcoma patients remains very poor. Application of high energy shock waves may change the tumour cell growth and increase the cytotoxic effect of in vivo and in vitro chemotherapeutic agents. We studied the effect of their association with doxorubicin or methotrexate on three in vitro osteosarcoma cell lines. The effect of these combinations on SJSA-1, MG-63 and HOS human osteosarcoma cell lines were evaluated through incubation with doxorubicin or methotrexate and high energy shock wave treatment with 1000 shots at 0.22 mJ/mm(2) and an evaluation of the cell number, cell proliferation and apoptosis at days 1, 3 and 6 from the start of treatment. The combination of high energy shock waves and doxorubicin induced a statistically significant advantage in terms of a decrease in cell number on the SJSA-1 and HOS cell lines, a decrease of cell proliferation on all three cell lines and an increase of apoptosis only on the SJSA-1 cell line. The combination of high energy shock waves with methotrexate induced a decrease of the cell number only in the SJSA-1 and in the HOS cell lines, of the cell proliferation in the SJSA-1 and in the MG-63 cell lines, and an increase of apoptosis only on the SJSA-1 cell line. In conclusion, these experiments show an interesting effect of high energy shock waves on osteosarcoma cell lines, which could lead to future studies of the in vivo effects of high energy shock waves in the murine model as well.

High energy shock waves (HESW) for sonodynamic therapy: effects on HT-29 human colon cancer cells.

BACKGROUND: Whether high energy shock waves (HESW), generated by a piezoelectric generator, were able to activate a sonosensitizer, 5-aminolevulinic acid (ALA) and induce inhibition of cell growth in HT-29 human colorectal cancer cells was investigated. MATERIALS AND METHODS: Cell survival and cell death pathways were investigated by cell growth curves, flow cytometry analysis and ELISA nucleosome evaluation. HT-29 cells were exposed to ALA and different HESW treatments: E1 (energy flux density = 0.22 mJ/mm2; 500 and 1000 shots) and E2 (energy flux density = 0.88 mJ/mm2; 500 and 1000 shots). RESULTS: A significant reduction of HT-29 cell growth with respect to untreated cells was observed only after treatment with ALA and HESW E2, 500 shots. In particular, HESWE2, 500 shots, was able to induce an arrest of HT-29 cells exposed to ALA in the G0/G1-phase of the cell cycle. CONCLUSION: HESW is proposed for the sonodynamic treatment of cancer cells.

Feasibility of cord blood stem cell manipulation with high-energy shock waves: an in vitro and in vivo study.

OBJECTIVE: Cord blood CD34+ cells are more uncommitted than their adult counterparts as they can be more easily maintained and expanded in vitro and transduced with lentiviral vectors. The aim of this study was to evaluate whether pretreatment with high-energy shock waves (HESW) could further enhance the expansion of cord blood progenitors and the transduction efficiency with lentiviral vectors. METHODS: Human cord blood CD34+ cells underwent HESW treatment with a wide range of energy and number of shots (from 0.22 mJ/mm2 to 0.43 mJ/mm2 and from 200 to 1500 shots). Cells were then evaluated both for their in vitro expansion ability and in vivo engraftment in primary, secondary, and tertiary NOD/SCID mice. The transduction efficiency with a lentiviral vector (LV) was also evaluated in vitro and in vivo. RESULTS: Cell viability following HESW ranged from 75 to 92%. Pretreatment with HESW significantly improved early progenitor cell expansion after short-term suspension culture. Upon transplantation in primary NOD/SCID mice, the HESW treatment enhanced progenitor cell engraftment (total human CD45(+)CD34+ cells were 10% in controls and 14.5% following HESW, human CD45(+)CD34(+)CD38(-) cells were 0.87% in controls and 1.8% following HESW). HESW treatment enhanced the transduction of a GFP+ lentiviral vector (e.g., at day 42 of culture 6.5% GFP+ cells in LV-treated cell cultures compared to 11.4% of GFP+ cells in HESW-treated cell cultures). The percentage of human GFP+ cell engrafting NOD/SCID mice was similar (34% vs 26.4% in controls); however, the total number of human cells engrafted after HESW was higher (39.6% vs 15%). CONCLUSION: The pretreatment of CD34+ cells with HESW represents a new method to manipulate the CD34+ population without interfering with their ability to both expand and engraft and it might be considered as a tool for genetic approaches.

Doxorubicin-Loaded Nanobubbles Combined with Extracorporeal Shock Waves: Basis for a New Drug Delivery Tool in Anaplastic Thyroid Cancer.

BACKGROUND: No standard chemotherapy is available for anaplastic thyroid cancer (ATC). Drug-loaded nanobubbles (NBs) are a promising innovative anticancer drug formulation, and combining them with an externally applied trigger may further control drug release at the target region. Extracorporeal shock waves (ESWs) are acoustic waves widely used in urology and orthopedics, with no side effects. The aim of the present work was to combine ESWs and new doxorubicin-loaded glycol chitosan NBs in order to target doxorubicin and enhance its antitumor effect in ATC cell lines. METHODS: CAL-62 and 8305C cells were treated with empty NBs, fluorescent NBs, free doxorubicin, and doxorubicin-loaded NBs in the presence or in the absence of ESWs. NB entrance was evaluated by fluorescence microscopy and flow cytofluorimetry. Cell viability was assessed by Trypan Blue exclusion and WST-1 proliferation assays. Doxorubicin intracellular content was measured by high-performance liquid chromatography. RESULTS: Treatment with empty NBs and ESWs, even in combination, was safe, as cell viability and growth were not affected. Loading NBs with doxorubicin and combining them with ESWs generated the highest cytotoxic effect, resulting in drug GI50 reduction of about 40%. Mechanistically, ESWs triggered intracellular drug release from NBs, resulting in the highest nuclear drug content. CONCLUSIONS: Combined treatment with doxorubicin-loaded NBs and ESWs is a promising drug delivery tool for ATC treatment with the possibility of using lower doxorubicin doses and thus limiting its systemic side effects.

Targeting Taxanes to Castration-Resistant Prostate Cancer Cells by Nanobubbles and Extracorporeal Shock Waves.

To target taxanes to castration-resistant prostate cancer cells, glycol-chitosan nanobubbles loaded with paclitaxel and docetaxel were constructed. The loaded nanobubbles were then combined with Extracorporeal Shock Waves, acoustic waves widely used in urology and orthopedics, with no side effects. Nanobubbles, with an average diameter of 353.3 ± 15.5 nm, entered two different castration-resistant prostate cancer cells (PC3 and DU145) as demonstrated by flow cytometry and immunofluorescence. The shock waves applied increased the amount of intracellular nanobubbles. Loading nanobubbles with paclitaxel and docetaxel and combining them with shock waves generated the highest cytotoxic effects, resulting in a paclitaxel GI50 reduction of about 55% and in a docetaxel GI50 reduction of about 45% respectively. Combined treatment also affected cell migration. Paclitaxel-loaded nanobubbles and shock waves reduced cell migration by more than 85% with respect to paclitaxel alone; whereas docetaxel-loaded nanobubbles and shock waves reduced cell migration by more than 82% with respect to docetaxel alone. The present data suggest that nanobubbles can act as a stable taxane reservoir in castration-resistant prostate cancer cells and shock waves can further increase drug release from nanobubbles leading to higher cytotoxic and anti-migration effect.

Sonodynamic treatment as an innovative bimodal anticancer approach: shock wave-mediated tumor growth inhibition in a syngeneic breast cancer model.

Despite the great advances in fighting cancer, many therapies still have serious side effects, thus urging the development of highly selective and safe treatments with a wide range of applicability. Sonodynamic therapy (SDT) is an innovative bimodal anticancer approach in which two normally non-toxic components -- one chemical, a sonosensitizer, and one physical, ultrasound -- selectively combine to cause oxidative damage and subsequent cancer cell death. In this study, we investigate the anticancer effect of SDT using shock waves (SWs) to activate protoporphyrin IX (PpIX) cytotoxicity on a Mat B-III syngeneic rat breast cancer model. The SDT-treated group saw a significant decrease (p<0.001) in magnetic resonance imaging (MRI) tumor size measurements 72 hours after treatment with PpIX precursor 5-aminolevulinic acid (ALA) and SWs. This occurred together with significant increase (p<0.01) in apparent diffusion coefficients between pre- and post-treatment MR tumor maps and strong increase in necrotic and apoptotic histological features 72 hours post-treatment. Moreover, significant HIF1A mRNA expression up-regulation was observed along with the prominent selective cleavage of poly (ADP-ribose) polymerase (PARP) and increased autophagy related protein LC3A/B expression in SDT-treated tumors, as compared to untreated tumors 72 hours post-treatment. Thus, the anticancer effect of SDT can be boosted by SWs, making them a valid technology for furthering investigations into this innovative anticancer approach.

The pan-histone deacetylase inhibitor LBH589 (panobinostat) alters the invasive breast cancer cell phenotype.

Triple-negative breast cancer (TNBC) is a very aggressive type of tumour and its aggressiveness is linked to E-cadherin downregulation. In estrogen-sensitive breast cancer, high levels of E-cadherin fit with high levels of ERα and MTA3 (a component of the transcription Mi-2/NuRD complex with intrinsic DAC activity). In TNBC the E-cadherin downregulation could be due to epigenetic silencing of the CDH1 gene as well as to the lack of a fully functioning ERα-activated pathway. We report that the pan-histone deacetylase inhibitor LBH589, a potent anti-proliferative agent, induced E-cadherin expression on cell membranes of MDA-MB-231 cells (TNBC), determining a reduction of cell invasion and migration. Even though E-cadherin expression in breast cancer is also regulated by estradiol and the ERα/MTA3/Snail/Slug pathway, LBH589 is able to increase E-cadherin without affecting the estrogen pathway. In fact, no expression of ERα, PR and FoxA1 was observed in MDA-MB-231 cells before and after LBH589 treatment; furthermore, the drug caused an increase in Snail and Slug expression with a concomitant reduction of MTA3 levels. Taking into consideration its anti-proliferative and anti-invasive properties, we suggest the use of LBH589 in aggressive breast cancer refractory to hormonal therapy.

Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine.