benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Skin-Neoplasms

Ursodeoxycholic acid (UDCA) is a type of hydrophilic bile acid extracted from animal bile with a wide range of biological functions. The present results demonstrated that UDCA could effectively inhibit the proliferation of two human melanoma cell line (M14 and A375) with time‑ and concentration‑dependence. Following exposure to various concentrations of UDCA, M14 cells exhibited typical morphological changes and weaker ability of colony forming. Flow cytometry analysis demonstrated that UDCA could induce a decrease of mitochondrial membrane potential and an increase in reactive oxygen species (ROS) levels in M14 cells. The cell cycle was arrested in the G2/M phase, which was confirmed by the decrease of cyclin‑dependent kinase 1 and cyclinB1 at the protein level. However, when M14 cells were treated with UDCA and Z‑VAD‑FMK (caspase inhibitor) synchronously, the apoptosis rate of the cells was reduced significantly. In addition, it was demonstrated that UDCA induced apoptosis of human melanoma M14 cells through the ROS‑triggered mitochondrial‑associated pathway, which was indicated by the increased expression of cleaved‑caspase‑3, cleaved‑caspase‑9, apoptotic protease activating factor‑1, cleaved‑poly (ADP‑ribose) polymerase 1 and the elevation of B cell lymphoma‑2 (Bcl‑2) associated X protein/Bcl‑2 ratio associated with apoptosis. Therefore, UDCA may be a potential drug for the treatment of human melanoma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Humans; Melanoma; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Reactive Oxygen Species; Signal Transduction; Skin Neoplasms; Ursodeoxycholic Acid

L929 fibroblastoma cells (L929-A) and L929 fibrosarcoma cells (L929-N) are different cell lines that are commonly used to study the cytotoxicity of tumor necrosis factor alpha (TNFα). TNFα has been reported to induce necrosis in both of these cell lines. However, comparing the TNFα-induced cell death in these two cell lines, we found that, unlike the L929-N cells that show typical RIP3-dependent necrosis, TNFα-induced cell death in L929-A cells is pan-caspase inhibitor Z-VAD-FMK (Z-VAD)-sensitive, which does not depend on RIP3. We also confirmed that the cell death signal in the L929-A cells was initiated through cytosol-preassembled ripoptosome and that the knockdown of either Caspase-8 or RIP1 protein blocked cell death. Compared with the L929-N cells, the L929-A cell line had lower levels of constitutive and inducible TNFα autocrine production, and the pan-caspase inhibitors Z-VAD or Q-VD did not kill the L929-A cells as they affect the L929-N cells. Moreover, the L929-A cells expressed less RIP3 protein than the L929-N cells; therefore, TNFα failed to induce RIP3-dependent necroptosis. In addition, the ripoptosome-mediated cell death signal was transduced to the mitochondria through Caspase-8-mediated and RIP1 kinase activity-dependent Bid cleavage. The RIP1 kinase inhibitor Necrostatin-1 (Nec-1) or Caspase-8 knockdown completely blocked Bid cleavage, and the knockdown of Bid or Bax/Bak prevented TNFα-induced cell death in the L929-A cells. Although the activation of Bax/Bak decreased the mitochondrial membrane potential, the levels of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-C) and Smac, declined, and western blotting and immunofluorescence staining analysis did not determine whether these proteins were redistributed to the cytosol. In addition, the mitochondrial outer membrane protein Tom20 was also reduced, indicating that the reduced mitochondria proteins may be induced by the reduced mitochondria numbers. No efficient cyt-C release was observed; therefore, the limited activation and cleavage of downstream caspases, including Caspase-9, Caspase-7, Caspase-6 and Caspase-3, was insufficient to kill the cells. The Caspase-9, Caspase-6 and Caspase-3/7 inhibitors or Caspase-9 and -3 knockdown also failed to block cell death, and the overexpression of Bcl-2 also did not abrogate cell death. Moreover, the dead cells showed necrotic-like but not apoptotic characteristics under transmission electronmicroscopy, and these featu

Topics: Amino Acid Chloromethyl Ketones; Animals; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 8; Caspase Inhibitors; Cell Death; Cell Line, Tumor; Dermatofibrosarcoma; GTPase-Activating Proteins; Mice; Mitochondria; Receptor-Interacting Protein Serine-Threonine Kinases; Skin Neoplasms; Tumor Necrosis Factor-alpha

Merkel cell carcinoma (MCC) is an aggressive skin cancer with rising incidence. In this study, we demonstrate that mTOR activation and suppressed autophagy is common in MCCs. mTOR inhibition in two primary human MCC cell lines induces autophagy and cell death that is independent of caspase activation but can be attenuated by autophagy inhibition. This is the first study to evaluate mTOR and autophagy in MCC. Our data suggests a potential role of autophagic cell death upon mTOR inhibition and thus uncovers a previously underappreciated role of mTOR signaling and cell survival, and merits further studies for potential therapeutic targets.

Topics: Amino Acid Chloromethyl Ketones; Carcinoma, Merkel Cell; Caspases; Cell Line, Tumor; Cell Survival; Enzyme Activation; Formaldehyde; Humans; Paraffin Embedding; Phagosomes; Protein Kinase Inhibitors; Skin Neoplasms; Tissue Fixation; TOR Serine-Threonine Kinases; Up-Regulation

Search for new substances with antiproliferative activity towards melanoma cells is important since malignant melanoma is notoriously resistant to conventional chemotherapy. Benzo[c]phenanthridine alkaloids (BAs) are natural products with significant anti-proliferative activities, therefore they are considered as agents promising for cancer therapy.. The effects of five BAs (sanguinarine, chelerythrine, chelidonine, sanguilutine, and chelilutine) on human malignant melanoma cell lines were compared. The study focused on BAs effects on DNA, anti-apoptotic and p53 protein levels; and the involvement of p53 in cellular responses to alkaloids treatment.. Melanoma cell lines, two wild types and two with dysfunctional p53 derived from one of them were used. The mechanism of anti-proliferative and pro-apoptotic effects and the effect on DNA was investigated using MTT assay, flow cytometry, Western blot analysis, fluorescence and electron microscopy.. All tested alkaloids exhibit strong anti-proliferative activity. CHL, CHE and SA induced apoptosis, which was probably mediated by decreasing levels of anti-apoptotic proteins (Bcl-xL, Mcl-1, XIAP) and was accompanied by mitochondrial membrane potential decrease as well as caspase-3 and PARP cleavage. Although all alkaloids caused DNA damage, which was demonstrated by induction of H2AX phosphorylation, none of the tested alkaloids stabilised p53 and their toxicity in cells with non-functional p53 was comparable to wild type cells.. Despite the profound similarity of BAs molecular structures, it is clear that the mechanism of cell death induction is different for each alkaloid. Our results indicate that BAs could be effective in malignant melanoma treatment, including tumours which have lost wild type p53.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Apoptosis; Benzophenanthridines; Caspases; Cell Proliferation; Cell Survival; DNA; DNA Damage; Genes, p53; Humans; Melanoma; Models, Biological; Models, Chemical; Skin Neoplasms; Tumor Suppressor Protein p53

Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinases; Cyclins; Cysteine Proteinase Inhibitors; Enzyme Activation; G1 Phase; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proanthocyanidins; Signal Transduction; Skin Neoplasms; Vitis

Early assessment of the efficacy of anticancer agents is a highly desirable and an unmet need in clinical oncology. Clinical imaging of cell-death may be useful in addressing this need, as induction of tumor cell-death is the primary mechanism of action of most anticancer drugs. In this study, we examined the performance of N,N'-Didansyl-L-cystine (DDC), a member of the ApoSense family of novel small molecule detectors of cell-death, as a potential tool for monitoring cell-death in cancer models. Detection of cell-death by DDC was examined in fluorescent studies on B16 melanoma cells both in vitro and ex vivo following its in vivo administration. In vitro, DDC manifested selective uptake and accumulation within apoptotic cells that was highly correlated with Annexin-V binding, changes in mitochondrial membrane potential, and caspase activation. Uptake was not ATP-dependent, and was inducible by calcium mobilization. In vivo, DDC selectively targeted cells undergoing cell-death in melanoma tumors, while not binding to viable tumor cells. Chemotherapy caused marked tumor cell-death, evidenced by increased DDC uptake, which occurred before a detectable change in tumor size and was associated with increased animal survival. These data confirm the usefulness of imaging of cell-death by DDC as a tool for early monitoring of tumor response to anti-cancer therapy.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Calcium; Caspase Inhibitors; Caspases; Cell Death; Cell Proliferation; Cysteine Proteinase Inhibitors; Cystine; Dansyl Compounds; Female; Fluorescent Dyes; Humans; Male; Melanoma, Experimental; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Molecular Probe Techniques; Molecular Probes; Skin Neoplasms; Treatment Outcome

Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P < 0.05-0.001) and induced cell death (3-51%, P < 0.01-0.001) in a dose (5-75 microM)- and time (12-72 h)-dependent manner, which was associated with an increase in G(1) arrest. G(0)/G(1) phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin kinase inhibitors (Cdki) and cyclins. Our western blot analysis showed that berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P < 0.05-0.001) than non-berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Berberine; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytochromes c; G1 Phase; Humans; Keratinocytes; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms