Isothiocyanates Potentiate Tazemetostat-Induced Apoptosis by Modulating the Expression of Apoptotic Genes, Members of Polycomb Repressive Complex 2, and Levels of Tri-Methylating Lysine 27 at Histone 3 in Human Malignant Melanoma Cells.

In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.

Effectiveness of Hyperthermia as Monotherapy and Adjuvant Therapy Approaches Against an In Vitro Model of Colorectal Carcinoma.

BACKGROUND/AIM: Despite improvement in current therapies, the 5-year overall survival rate of colorectal carcinoma is still low especially in its metastatic form. On the other hand, hyperthermia has been utilized as a cancer treatment approach to improve overall therapeutic efficacy. In the present study, we have aimed to develop an optimized hyperthermic protocol against an in vitro model of human colon carcinoma, as a single and/or adjuvant treatment approach. MATERIALS AND METHODS: We have utilized an in vitro model of human colorectal carcinoma consisting of colorectal carcinoma (HT29, CaCo2) and normal colon epithelial (CCD841CoN) cell lines. Cells were exposed to 45°C, over 120 min, in the presence or absence of chemotherapeutic (5-Fluorouracil, Capecitabine) and targeted (Bevacizumab, Cetuximab) drugs. Cell viability levels were determined by the Alamar-blue assay while determination of cell death, reactive oxygen species (ROS) production, mitochondrial membrane depolarization (ΔΨµ) levels and cell cycle progression were performed by flow cytometry. RESULTS: CaCo2 and HT29 cells showed a differential response towards i) cell viability, ii) cell death, iii) ROS and ΔΨµ levels as well as iv) cell cycle distribution, in the presence of hyperthermia alone (monotherapy) or in combination with the above-mentioned drugs (adjuvant therapy). Finally, normal colon epithelial (CCD841CoN) cells remained minimally affected. CONCLUSION: We have developed an optimized experimental hyperthermic protocol, as a promising monotherapy and/or adjuvant therapy approach, with the capacity to potentiate chemotherapeutic as well as targeted drug-induced cytotoxicity against a model of colorectal carcinoma, to a variable degree.

Assessment of the Immunomodulatory Properties of the Probiotic Strain Lactobacillus paracasei K5 in vitro and In Vivo.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1α (IL-1α), ΙL-1ß, IL-6, tumor necrosis factor-alpha (TNF-α), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character.

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.

Hyperthermia Suppresses Post - In Vitro Proliferation and Tumor Growth in Murine Malignant Melanoma and Colon Carcinoma.

BACKGROUND: Several studies have highlighted hyperthermia's ability to enhance the effectiveness of radiation and chemotherapy in various in vitro and in vivo cancer models. MATERIALS AND METHODS: In vivo murine models of malignant melanoma and colon carcinoma were utilized for demonstrating hyperthermia's therapeutic effectiveness by examining levels of caspase 3, COX-2 and phospho-H2A.X (Ser139) as endpoints of apoptosis, proliferation and DNA damage respectively. RESULTS: Hyperthermia induced in vitro cytotoxicity in malignant melanoma (B16-F10) and colon carcinoma (CT26) cell lines. In addition, it reduced post-in vitro proliferation and suppression of tumor growth by inducing the expression of caspase-3 and phospho-H2A.X (Ser139) while reducing the expression of COX-2 in both murine cancer models. CONCLUSION: Hyperthermia can exert therapeutic effectiveness against melanoma and colon carcinoma by inhibiting a number of critical cellular cascades including apoptosis, proliferation and DNA damage.

The Role of Isothiocyanates as Cancer Chemo-Preventive, Chemo-Therapeutic and Anti-Melanoma Agents.

Many studies have shown evidence in support of the beneficial effects of phytochemicals in preventing chronic diseases, including cancer. Among such phytochemicals, sulphur-containing compounds (e.g., isothiocyanates (ITCs)) have raised scientific interest by exerting unique chemo-preventive properties against cancer pathogenesis. ITCs are the major biologically active compounds capable of mediating the anticancer effect of cruciferous vegetables. Recently, many studies have shown that a higher intake of cruciferous vegetables is associated with reduced risk of developing various forms of cancers primarily due to a plurality of effects, including (i) metabolic activation and detoxification, (ii) inflammation, (iii) angiogenesis, (iv) metastasis and (v) regulation of the epigenetic machinery. In the context of human malignant melanoma, a number of studies suggest that ITCs can cause cell cycle growth arrest and also induce apoptosis in human malignant melanoma cells. On such basis, ITCs could serve as promising chemo-therapeutic agents that could be used in the clinical setting to potentiate the efficacy of existing therapies.

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.

Effects of hyperthermia as a mitigation strategy in DNA damage-based cancer therapies.

Utilization of thermal therapy (hyperthermia) is defined as the application of exogenous heat induction and represents a concept that is far from new as it goes back to ancient times when heat was used for treating various diseases, including malignancies. Such therapeutic strategy has gained even more popularity (over the last few decades) since various studies have shed light into understanding hyperthermia's underlying molecular mechanism(s) of action. In general, hyperthermia is applied as complementary (adjuvant) means in therapeutic protocols combining chemotherapy and/or irradiation both of which can induce irreversible cellular DNA damage. Furthermore, according to a number of in vitro, in vivo and clinical studies, hyperthermia has been shown to enhance the beneficial effects of DNA targeting therapeutic strategies by interfering with DNA repair response cascades. Therefore, the continuously growing evidence supporting hyperthermia's beneficial role in cancer treatment can also encourage its application as a DNA repair mitigation strategy. In this review article, we aim to provide detailed information on how hyperthermia acts on DNA damage and repair pathways and thus potentially contributing to various adjuvant therapeutic protocols relevant to more efficient cancer treatment strategies.

Peroral transgastric endoscopic procedures in pigs: feasibility, survival, questionings, and pitfalls.

BACKGROUND: Multiple studies have demonstrated the feasibility of peroral transgastric endoscopic procedures in porcine models. We present our experience with these procedures in a porcine survival model and expose the technical difficulties and worries about their application in humans. METHODS: We performed the following transgastric procedures in nine small pigs (20-30 kg) under general anesthesia using a double-channel therapeutic endoscope: peritoneoscopy (nine pigs), cholecystectomy (six pigs), gastrojejunal anastomosis (two pigs), and fallopian tube excision (one pig). Postsurgical complications and survival of the pigs were recorded during a 3-week follow-up. RESULTS: In six pigs, the gallbladder was successfully resected. The time of the procedure ranged from 2.5 to 3.5 hours. In one case, severe bleeding occurred from the cystic artery during dissection and was successfully managed by placement of endoscopic clips. Five pigs survived for 3 weeks after cholecystectomy. In one pig, severe peritonitis developed because of gastric contents leakage due to failure of gastric closure and the animal was euthanized 3 days after the procedure. Endoscopic transgastric gastrojejunostomy was successfully performed in two pigs without major complications. The time required for the procedure averaged about 50 min. Fallopian tube excision was also successful in one pig without any complications. The entire procedure was performed within 60 min. CONCLUSIONS: This study reports the feasibility of natural orifice transluminal endoscopic surgery (NOTES) procedures in a porcine survival model and impresses the efficacy, the safety, the complications, and some technical difficulties of transgastric NOTES procedures, which merit further evaluation in future studies.

Comparison of angiogenic activities after urethral reconstruction using free grafts in rabbits.

OBJECTIVE: To determine the most suitable type of graft-free penile skin grafts or mucosal grafts from bladder or buccal regions - for urethral reconstruction in an animal model, as evaluated on the basis of angiogenic activity. METHODS: Twenty-two male White New Zealand rabbits were randomly divided into four groups. In the control group (group O, n=4) a simple urethrotomy and closure was performed, whereas a ventral urethral defect was created in groups A, B, and C and then bridged using the following onlay patches: free penile skin (group A, n=6), buccal mucosal graft (group B, n=6), and bladder mucosal graft (group C, n=6). On the 21st postoperative day, the animals were sacrificed and the retrieved implants were subjected to macroscopic and microscopic analysis. The angiogenic activity was assessed with immunohistochemistry, using the anti-CD31 MoAb and the phosphatase antialkaline phosphatase procedure. The native vascularity of penile skin as well as buccal and bladder mucosa was assessed in rabbits from group O (n=3). Statistical analysis was performed using the one-way ANOVA. RESULTS: The angiogenesis in a magnification of x200 in groups O, A, B, and C was 34.1+/-4.1 (mean+/-SD), 61.7+/-6.4, 94.3+/-6.4, and 91.5+/-7.2 vessels per optical field, respectively. There were, statistically significant differences (p<0.001) between groups A and B and between groups A and C, but not (p>0.05) between groups B and C. The native vascularity of penile skin, buccal mucosa and bladder mucosa was 23.3+/-3.0, 24.6+/-3.7 and 17.0+/-2.6 vessels per optical field, respectively. CONCLUSION: The viability of mucosal grafts from bladder or buccal regions is better than that of a free penile graft because of higher angiogenic activity. Although the mucosal grafts showed the same angiogenic activity, the buccal mucosa graft is preferable because of its easier harvesting.