Development of a novel experimental In vitro model of isothiocyanate-induced apoptosis in human malignant melanoma cells

Background: Isothiocyanates are constituents of cruciferous vegetables which have been associated with reduced cancer risk partially through their ability to induce apoptosis in malignant cells including melanoma. Materials and Methods: We have utilized human malignant melanoma (A375), epidermoid carcinoma (A431) and immortalized keratinocyte (HaCaT) cells exposed to various isothiocyanates, under different experimental conditions. Results: An experimental in vitro model utilizing low isothiocyanate concentrations (0.1-5 μM for 48 h with all treatments being refreshed after 24h) was shown to be (i) most efficient in exerting an anti-cancer effect when compared to higher concentrations (5-100 μM for 24 or 48 h added as a single bolus) and (ii) specific to A375 cells while A431 and HaCaT cells remained unaffected. Such effect involved the activation of several caspases including (iii) initiator caspases 8, 9, 4 (indicating the involvement of intrinsic, extrinsic and endoplasmic reticulum-based pathways) and (iv) effector caspases 3, 7 and 6. Conclusion: Utilization of low isothiocyanate concentrations (under the conditions described herein) exerts an anti-cancer effect specific to human malignant melanoma cells thus providing a therapeutic basis for their utilization in management of the disease

Hyperthermia induces therapeutic effectiveness and potentiates adjuvant therapy with nontargeted and targeted drugs in an in vitro model of human malignant melanoma

In the present study, we have aimed to characterize the intrinsic, extrinsic and ER-mediated apoptotic induction by hyperthermia in an in vitro model of human malignant melanoma and furthermore, to evaluate its therapeutic effectiveness in an adjuvant therapeutic setting characterized by combinational treatments with non-targeted (Dacarbazine & Temozolomide) and targeted (Dabrafenib & Vemurafenib) drugs. Overall, our data showed that both low (43 °C) and high (45 °C) hyperthermic exposures were capable of inducing cell death by activating all apoptotic pathways but in a rather distinct manner. More specifically, low hyperthermia induced extrinsic and intrinsic apoptotic pathways both of which activated caspase 6 only as opposed to high hyperthermia which was mediated by the combined effects of caspases 3, 7 and 6. Furthermore, significant involvement of the ER was evident (under both hyperthermic conditions) suggesting its role in regulating apoptosis via activation of CHOP. Our data revealed that while low hyperthermia activated IRE-1 and ATF6 only, high hyperthermia induced activation of PERK as well suggesting that ultimately these ER stress sensors can lead to the induction of CHOP via different pathways of transmitted signals. Finally, combinational treatment protocols revealed an effect of hyperthermia in potentiating the therapeutic effectiveness of nontargeted as well as targeted drugs utilized in the clinical setting. Overall, our findings support evidence into hyperthermia’s therapeutic potential in treating human malignant melanoma by elucidating the underlying mechanisms of its complex apoptotic induction

Novel docosahexaenoic acid ester of phloridzin inhibits proliferation and triggers apoptosis in an in vitro model of skin cancer

Skin cancer is among the most common cancer types accompanied by rapidly increasing incidence rates, thus making the development of more efficient therapeutic approaches a necessity. Recent studies have revealed the potential role of decosahexaenoic acid ester of phloridzin (PZDHA) in suppressing proliferation of liver, breast, and blood cancer cell lines. In the present study, we investigated the cytotoxic potential of PZDHA in an in vitro model of skin cancer consisting of melanoma (A375), epidermoid carcinoma (A431), and non-tumorigenic (HaCaT) cell lines. Decosahexaenoic acid ester of phloridzin led to increased cytotoxicity in all cell lines as revealed by cell viability assays. However, growth inhibition and induction of both apoptosis and necrosis was more evident in melanoma (A375) and epidermoid carcinoma (A431) cells, whereas non-tumorigenic keratinocytes (HaCaT) appeared to be more resistant as detected by flow cytometry. More specifically, PZDHA-induced cell cycle growth arrest at the G2/M phase in A375 and A431 cells in contrast to HaCaT cells, which were growth arrested at the G0/G1 phase. Elevated intracellular generation of reactive oxygen species ROS was detected in all cell lines. Overall, our findings support the potential of PZDHA as a novel therapeutic means against human skin cancer. © 2018 by the authors

The effect of platelet-rich plasma gel in the early phase of patellar tendon healing

Introduction: The aim of this study is to assess if an application of platelet-rich plasma (PRP) gel would improve the mechanical properties of rabbit's patellar tendon after resecting its central portion. Materials and methods: Forty skeletally mature New Zealand White rabbits were used. Two groups ten rabbits each (PRP and control group) were used to evaluate mechanical properties and histology after 14 days and two groups ten rabbits each (PRP and control groups) were used to evaluate mechanical properties and histology after 28 days. Results: At 14 days, PRP group showed a 72.2% increase in force at failure, a 39.1% increase in ultimate stress, and a 53.1% increase in stiffness, as compared with controls. These changes were statistically significant (P < 0.05). At 28 days, there was no longer any significant difference between PRP and control groups (P > 0.05). Discussion: In our study, the mechanical properties of the regenerated tendon in the PRP group were significantly improved in relation to the control group. It appears that PRP has a strong effect in the early phase of tendon healing. This effect is probably due to the growth factors that are released from the platelets during activation. © Springer-Verlag 2009

Temporal and spatial expression of TGF-β1 in an Achilles tendon section model after application of platelet-rich plasma

Background: To investigate the effect of platelet-rich plasma (PRP) on TGF-β1 expression during tendon healing. Methods: We used 48 skeletally mature New Zealand White rabbits. 24 rabbits received the PRP, and 24 rabbits served as an untreated control group. Equal numbers of animals were sacrificed at 1st, 2nd, 3rd, and 4th week. The surgical procedure involved a transverse incision to transect the Achilles tendon. A volume of 1. ml of PRP was then injected into the tendon mass in the PRP group. Histological and immunohistochemical evaluations with an anti-TGF-β primary antibody were performed. Results: The pattern of expression of TGF-β1 in the PRP group was characterized by a significant upregulation during the first 2 weeks and subsequently significant downregulation in the 3rd and 4th week in comparison with the controls. Conclusions: Our results suggest that PRP may affect the tendon healing process by altering the expression of TGF-β1. © 2009 European Foot and Ankle Society

Allyl isothiocyanate regulates lysine acetylation and methylation marks in an experimental model of malignant melanoma

Objective(s): Isothiocyanates (ITCs) are biologically active plant secondary metabolites capable of mediating various biological effects including modulation of the epigenome. Our aim was to characterize the effect of allyl isothiocyanate (AITC) on lysine acetylation and methylation marks as a potential epigenetic-induced anti-melanoma strategy. Methods: Our malignant melanoma model consisted of (1) human (A375) and murine (B16-F10) malignant melanoma as well as of human; (2) brain (VMM1) and lymph node (Hs 294T) metastatic melanoma; (3) non-melanoma epidermoid carcinoma (A431) and (4) immortalized keratinocyte (HaCaT) cells subjected to AITC. Cell viability, histone deacetylases (HDACs) and acetyltransferases (HATs) activities were evaluated by the Alamar blue, Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively, while their expression levels together with those of lysine acetylation and methylation marks by western immunoblotting. Finally, apoptotic gene expression was assessed by an RT-PCR-based gene expression profiling methodology. Results: AITC reduces cell viability, decreases HDACs and HATs activities and causes changes in protein expression levels of various HDACs, HATs, and histone methyl transferases (HMTs) all of which have a profound effect on specific lysine acetylation and methylation marks. Moreover, AITC regulates the expression of a number of genes participating in various apoptotic cascades thus indicating its involvement in apoptotic induction. Conclusions: AITC exerts a potent epigenetic effect suggesting its potential involvement as a promising epigenetic-induced bioactive for the treatment of malignant melanoma. © 2019, The Author(s)

Application of PRP gel alone or in combination with guided bone regeneration does not enhance bone healing process: An experimental study in rabbits

Introduction: The aim of this study was to assess the hypothesis that application of platelet-rich plasma (PRP) gel in mandibular defects in rabbits, alone or in combination with guided bone regeneration (GBR) techniques, could enhance the bone healing process. Materials and methods: Thirty New Zealand white rabbits were used. Three groups of 10 animals each were assigned, and the animals were sacrificed after 12 weeks. During the operation, a rotating trephine bur was used to create circular defects 10-mm in diameter in the region anterior to the jaw angles. In group human fascia lata (HFL), a human fascia lata membrane was used. In group PRP, PRP gel was used to fill the defect, and in group HFL + PRP, PRP was used to fill the defect which after that was covered with a human fascia lata membrane. Results: In general, none of the control sides and the PRP treated sides had full development of bone or filling of the defect through bone bridging. Conversely, the sides on which the fascia lata membrane or the combination of membrane and PRP had been applied were characterized mostly by development of newly formed bone that bridged the gap. Conclusions: Our results suggest that the application of PRP gel alone or in combination with GBR does not enhance bone healing process. © 2010 European Association for Cranio-Maxillo-Facial Surgery