cytochrome-c-t and Skin-Neoplasms

The concept of "vasculogenic mimicry" (VM) was introduced to describe the unique ability of highly aggressive tumor cells to form capillary-like structure (CLS) and matrix-rich patterned network in three-dimensional cultures that mimic embryonic vasculogenic network. Here, we provide the experimental evidence that CLS structure formation requires apoptotic cell death through activation of caspase-dependent mechanism. Our results indicate that the formation of CLS is also related to the reactive oxygen species (ROS) levels.

Topics: Antioxidants; Apoptosis; Capillaries; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Melanoma; Models, Biological; Neovascularization, Pathologic; Reactive Oxygen Species; Skin Neoplasms; Time Factors

Melanoma is a malignant tumor that originates from the skin's melanocytes and has the highest death rate from skin cancer. Developing more efficacious anticancer medications with fewer adverse effects is the key to effective cancer management. Natural products are considered relevant and cost-effective sources of treatment. The plant (Polypodium vulgare) is a small and evergreen fern. One of the most important chemical compounds in the extract of this herb is flavonoids, which are thought to have beneficial effects in the treatment of melanoma through antioxidant properties.. Due to the limitations of current cancer management and cytotoxic drugs available in the country, the need to study drugs of natural origin has become more prominent. In this regard, the present study aims to investigate the cytotoxic effects of the ethanolic extract of Polypodium vulgare on A375 melanoma cells.. Polypodium vulgare was extracted in 80% ethanol by the maceration. Then, its effects on the cell death of the melanoma cell line A375 compared to the AGO-1522 cell line as control were measured using the MTT-assay technique. The amount of cellular lipid peroxidation was estimated by TBARS assay. The amount of cellular ROS was calculated by fluorescent reagent 2,7-dichlorofluorescein diacetate. Cytochrome c concentration was measured by a cytochrome c immunoassay kit.. In this experiment, the anticancer effects of Polypodium vulgare ethanolic extract on human melanoma cell lines were investigated for the first time. Herb extract with a concentration of 0.123 mg/ml significantly increased the death of A375 melanoma cells (p < 0.001), lipid peroxidation (p < 0.01), and reactive oxygen species (ROS) (p < 0.01) and cytochrome c concentration (p < 0.001). Meanwhile, the same amount was ineffective and safe on AGO-1522 normal fibroblast cells.. A 0.123 mg/ml concentration of Polypodium vulgare increases apoptosis in melanoma cells. Meanwhile, the same amount was safe on healthy cells. So, it could be considered an effective treatment without side effects in human melanoma.

Topics: Antineoplastic Agents; Cell Line; Cell Line, Tumor; Cytochromes c; Ethanol; Humans; Melanoma; Melanoma, Cutaneous Malignant; Plant Extracts; Polypodium; Reactive Oxygen Species; Skin Neoplasms

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.

Topics: Animals; Caspase 3; Cytochromes c; Fibroblasts; Gene Knockout Techniques; HEK293 Cells; HeLa Cells; Humans; Inflammasomes; Macrophages; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Membranes; Mutation; Phosphorylation; Primary Cell Culture; Protein Domains; Pyroptosis; Receptors, Estrogen; Skin Neoplasms; Threonine

Melanoma is a highly aggressive and deadly cancer with a poor prognosis given its drug resistance. A defect in apoptosis is one of the key mechanisms that contribute to drug resistance in Melanoma. An important sea marine animal is the Holothuria parva, also known as the sea cucumber, which has various pharmacological activities. Compounds obtained from sea cucumbers have shown to have anticancer activity through induction of apoptosis singling.. In the present study, selective toxicity and apoptotic effect of three extracts of H. parva were assessed on skin mitochondria isolated from mouse animal models of melanoma. The mitochondria was isolated from melanoma cells via differential centrifuges and treated with various concentrations (250, 500 and 1000 µg/ml) of metanolic, diethyl ether and n-hexane extracts of H. parva.. All the applied concentrations (250, 500 and 1000 µg/ml) of three extracts of H. parva increased the reactive oxygen species (ROS) generation only in the skin mitochondria isolated from melanoma cells group (in comparison to the control group). Additionally, all three extracts (250, 500 and 1000 µg/ml) induced swelling within the mitochondria, the collapse of the mitochondrial membrane potential (MMP) and the release of cytochrome c from the mitochondria. Flow-cytometry analysis demonstrated that n-hexane and diethyl ether extracts of H. parva selectively and progressively induced apoptosis only on melanoma but not healthy control skin cells group.. Given these results, the potentially bioactive compounds found in H. parva render it a strong candidate for further research in molecular identification and confirmatory in vivo studies. Clinical trials are also warranted in the general process of novel drug discovery for the treatment of melanoma cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biological Products; Cytochromes c; Drug Screening Assays, Antitumor; Holothuria; Humans; Indian Ocean; Melanoma, Experimental; Membrane Potential, Mitochondrial; Mice; Mitochondria; Reactive Oxygen Species; Skin; Skin Neoplasms

Urea Transporter B (UT-B) is a membrane channel protein that mediates the rapid transmembrane transport of urea and participates in urine concentration. Urea Transporter B is expressed in skin, but we found that there is little expression in human melanoma tissue. In this study, we examined the effects of UT-B overexpression in melanoma. The results indicated that there is no UT-B mRNA expression in B16 cells, and UT-B overexpression repressed B16 cell proliferation and induced apoptosis in vitro. We show that UT-B overexpression causes increased reactive oxygen species production, which may be caused by mitochondria dysfunction. The mitochondrial membrane potential (ΨΔm) was lower in UT-B-overexpressing B16 cells. The proteins involved in complexes I, III, IV and V of the respiratory chain were clearly downregulated in UT-B-overexpressing B16 cells, which would strongly reduce the activity of the electron transport chain. We found that mitochondrial release of cytochrome C into the cytoplasm also increased, indicating that apoptosis had been activated. In addition, UT-B overexpression reduced AKT phosphorylation and MDM2 expression and increased p53 expression; p53 activation may be involved in the anticancer effects of UT-B overexpression. Urea Transporter B overexpression also inhibited tumor growth in vivo. In conclusion, we demonstrated that UT-B may be related to the occurrence of melanoma and play a role in tumor development.

Topics: Aged; Animals; Cell Death; Cell Line, Tumor; Cytochromes c; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Melanoma; Melanoma, Experimental; Membrane Potential, Mitochondrial; Membrane Transport Proteins; Mice; Middle Aged; Mitochondria; Signal Transduction; Skin Neoplasms; Tumor Suppressor Protein p53; Urea Transporters

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Curcumin; Cytochromes c; Diarylheptanoids; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

The currently available data suggest that natural products may exert significant cytotoxic and immunomodulatory effects. Plant-derived chemotherapeutic agents such as taxol, etoposide or vincristine, currently used in cancer therapy, are prominent examples in this regard. However, there is a need for new and nat- ural anticancer compounds with low or without toxicity to normal cells. One of the active compounds responsible for the immune effects is β-glucan derived from cereals, fungi, seaweeds, yeasts and bacteria. The recent data suggest that β-glucans are potent immunomodulators with anticancer properties. Antitumor properties of fungi and yeast derived β-glucans have been widely recognized, but those polysaccharides are mostly insoluble, creating several problems especially in topical formulation. To overcome the issue of low water solubility, in the current study a more soluble β-glucan type from oats was chosen for the investigation of its antitumor activities. Cytotoxic effects were studied using a human melanoma cell line (Me45). The effect of electroporation on the antitumor activity of oat β-glucan was investigated as well. Cellular viability assessment, immuno-cytochemistry and immunofluochemistry were employed to evaluate biologic effects. Our results indicate strong anticancer properties of oat β-glucan, enhanced by electroporation.

Topics: Adult; Antineoplastic Agents, Phytogenic; Apoptosis; Avena; beta-Glucans; Caspase 12; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Electrochemotherapy; Humans; Male; Melanoma; Phytotherapy; Plants, Medicinal; Skin Neoplasms; Solubility

Topics: Animals; Antineoplastic Agents; Apoptosis; Cantharidin; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cell Survival; Cyclin D; Cytochromes c; DNA Fragmentation; G1 Phase Cell Cycle Checkpoints; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Reactive Oxygen Species; Receptors, Death Domain; Signal Transduction; Skin Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Transplantation, Heterologous

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.

Topics: Adenosine Triphosphate; Animals; Antimicrobial Cationic Peptides; Antineoplastic Agents; beta-Alanine; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytotoxins; Erythrocytes; Female; Hemolysis; HMGB1 Protein; Humans; Inhibitory Concentration 50; Injections, Intralesional; Lysosomes; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Rats; Remission Induction; Skin Neoplasms; Vacuoles

A mild and efficient synthetic development of 2-arylbenzothiazoles 5 mediated by ceric ammonium nitrate (CAN) via intramolecular cyclization of N-phenyl-thiobenzamides 4 was achieved. Further compounds 5 were reduced to corresponding amines 6, and their photodynamic therapy (PDT) effect was evaluated on malignant human melanoma A375 cells. Amine 6l plus ultraviolet A (UVA) induced caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 positive CytoDeath staining, and subsequent apoptotic cell death. Our data disclosed that treatment of A375 cells with 6l plus UVA resulted in a decrease in mitochondrial membrane potential (ΔΨmt), oxidative phosphorylation system (OXPHOS) subunits, and adenosine triphosphate (ATP) but an increase in mitochondrial DNA 4977-bp deletion via reactive oxygen species (ROS) generation. Transmission electron microscopy (TEM) observations also showed major ultrastructural alterations of mitochondria. Additionally, 6l plus UVA was also shown to reduce murine melanoma size in a mouse model. The present study supports the hypothesis that 6l-PDT may serve as a potential ancillary modality for the treatment of melanoma.

Topics: Animals; Apoptosis; Benzothiazoles; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; Humans; Melanoma, Experimental; Membrane Potential, Mitochondrial; Mice, Inbred ICR; Mitochondria; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Skin Neoplasms; Tumor Burden; Ultraviolet Rays

Previous studies suggest that tazarotene, a new member of the acetylenic class of RARβ/γ selective retinoids which is approved to treat a variety of skin diseases, exhibits an anti-proliferative effect in human basal cell carcinoma (BCC) by triggering caspase-dependent apoptosis. However, the detailed molecular mechanisms underlying the anti-tumor activity of tazarotene are poorly understood. This study aims at investigating the molecular mechanisms of tazarotene-induced apoptosis in human BCC cells. Our results are the first to demonstrate that tazarotene induces mitochondria-dependent cleavage of caspase-9 and -3 and PARP in BCC cells by producing reactive oxygen species (ROS) and activating caspase-8 through both ROS and death receptor signaling. These events are accompanied by a decrease in BCL-2 and BCL-xl anti-apoptotic proteins as well as by survivin and XIAP, two IAP family members. Furthermore, our results presented for the first time that tazarotene triggers a convergence of the intrinsic and extrinsic apoptotic pathways via the caspase-8-truncated Bid signaling pathway. Collectively, these data provide insights into the molecular mechanisms underlying tazarotene-induced apoptosis in human BCC cells, suggesting that this compound is a potential anti-skin cancer drug.

Topics: Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma, Basal Cell; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Down-Regulation; Enzyme Activation; Fas-Associated Death Domain Protein; G1 Phase Cell Cycle Checkpoints; Humans; In Situ Nick-End Labeling; Inhibitor of Apoptosis Proteins; Keratolytic Agents; Membrane Potential, Mitochondrial; Mitochondria; Nicotinic Acids; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Death Domain; RNA Interference; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Survivin; X-Linked Inhibitor of Apoptosis Protein

Centratherum anthelminticum (L.) Kuntze (scientific synonyms: Vernonia anthelmintica; black cumin) is one of the ingredients of an Ayurvedic preparation, called "Kayakalp", commonly applied to treat skin disorders in India and Southeast Asia. Despite its well known anti-inflammatory property on skin diseases, the anti-cancer effect of C. anthelminticum seeds on skin cancer is less documented. The present study aims to investigate the anti-cancer effect of Centratherum anthelminticum (L.) seeds chloroform fraction (CACF) on human melanoma cells and to elucidate the molecular mechanism involved.. A chloroform fraction was extracted from C. anthelminticum (CACF). Bioactive compounds of the CACF were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human melanoma cell line A375 was treated with CACF in vitro. Effects of CACF on growth inhibition, morphology, stress and survival of the cell were examined with MTT, high content screening (HSC) array scan and flow cytometry analyses. Involvement of intrinsic or extrinsic pathways in the CACF-induced A375 cell death mechanism was examined using a caspase luminescence assay. The results were further verified with different caspase inhibitors. In addition, Western blot analysis was performed to elucidate the changes in apoptosis-associated molecules. Finally, the effect of CACF on the NF-κB nuclear translocation ability was assayed.. The MTT assay showed that CACF dose-dependently inhibited cell growth of A375, while exerted less cytotoxic effect on normal primary epithelial melanocytes. We demonstrated that CACF induced cell growth inhibition through apoptosis, as evidenced by cell shrinkage, increased annexin V staining and formation of membrane blebs. CACF treatment also resulted in higher reactive oxygen species (ROS) production and lower Bcl-2 expression, leading to decrease mitochondrial membrane potential (MMP). Disruption of the MMP facilitated the release of mitochondrial cytochrome c, which activates caspase-9 and downstream caspase-3/7, resulting in DNA fragmentation and up-regulation of p53 in melanoma cells. Moreover, CACF prevented TNF-α-induced NF-κB nuclear translocation, which further committed A375 cells toward apoptosis.. Together, our findings suggest CACF as a potential therapeutic agent against human melanoma malignancy.

Topics: Apoptosis; Asteraceae; Caspases; Cell Proliferation; Cytochromes c; Female; Humans; Melanoma; Middle Aged; Mitochondria; NF-kappa B; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Seeds; Signal Transduction; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal microscopies. Circular dichroic spectral analysis and stability curve for Gibb's free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photostability studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formulations to have better ameliorative potentials in combating skin melanoma.

Topics: Apigenin; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Chemistry, Pharmaceutical; Cytochromes c; DNA Fragmentation; Humans; Lactic Acid; Melanoma; Melanoma, Cutaneous Malignant; Mitochondria; Nanoparticles; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Reactive Oxygen Species; Skin Neoplasms

Despite findings that photodynamic treatment with bis (3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine initiated tumor regression in mice skin, queries regarding its mode of action - answers to which will be functional to design clinical trials on squaraine based photodynamic therapy - remain unanswered. Our investigation reveals the in vivo mechanism of action of the photosensitizer.. Skin tumor was induced in Swiss albino mice using 7,12-dimethyl benzanthacene. After the intraperitoneal administration of the dye in tumor induced mice, its concentration in subcellular fractions of the tumor tissue was determined fluorimetrically. Cytochrome c release from the mitochondrial membrane after the photodynamic treatment was analyzed. The observations stemming from this part lead to histopathological examination of tumor tissues. Apoptotic markers like caspase-3, Bcl-2 and Bax were also studied.. Major portion of the dye accumulated in the mitochondria. Cytochrome c leakage from mitochondria after squaraine PDT suggests loss of mitochondrial membrane integrity, which was further confirmed by the results of histopathological analysis. The activity of caspase-3 was elevated, expression of Bcl-2 diminished and that of Bax increased - all these results show enhancement of apoptosis in the tumor region after the treatment.. The results lead to the elucidation of mechanism of tumor destruction which proves to be mitochondria mediated apoptotic damage of tumor tissue. The study assumes significance since it defines the in vivo mode of action of a photosensitizer. Also, the query of how a squaraine based photosensitizer evokes tumor response is being dealt with here, for the first time.

Topics: Animals; Cyclobutanes; Cytochromes c; Male; Mice; Mitochondria; Phenols; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms; Treatment Outcome

Centipedegrass extract (CGE) is mainly composed of maysin and its derivatives, which are recognized internationally as natural compounds. Compared to other flavonoids, maysin has a unique structure in that mannose is bound to the flavonoid backbone. CGE exhibits some biological properties in that it can function as an anti-oxidant, anti-inflammatory, anti-adipogenic, and insecticidal. Whether CGE has other biological functions, such as anti-cancer activity, is unknown.. B16F1 (mouse) and SKMEL-5 (human) cells were treated with CGE, and their subsequent survival was determined using MTT assay. We performed a cell cycle analysis using propidium iodide (PI), and detected apoptosis using double staining with annexin V-FITC/PI. In addition, we examined mitochondrial membrane potentials using flow cytometry, as well as signaling mechanisms with an immunoblotting analysis.. CGE inhibited skin cancer cell growth by arresting the cell cycle in the G2/M phase, and increased both early and late apoptotic cell populations without affecting normal cells. Furthermore, we observed mitochondrial transmembrane depolarization, increased cytochrome-c release, caspase-3 and caspase-7 activation, and increased poly ADP-ribose polymerase degradation. CGE also downregulated activation of p-AKT, p-glycogen synthase kinase-3β (GSK-3β), and p-BAD in a time-dependent manner. LY294002 inhibition of phosphoinositide 3-kinase (PI3K) significantly sensitized skin cancer cells, which led to an increase in CGE-induced apoptosis.. CGE controlled skin cancer cell growth by inhibiting the PI3K/AKT/GSK-3β signaling pathway and activating the effector caspases. This study is the first to demonstrate anti-cancer properties for CGE, and that CGE may be an effective therapeutic agent for treating skin cancer.

Topics: Animals; Apoptosis; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Chromones; Cytochromes c; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Plant Extracts; Poaceae; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Neoplasms

Skin is generally damaged through genetic and environmental factors such as smoking, exposure to xenobiotics, heat, hormonal changes, and ultraviolet light. These factors can cause skin diseases. Cissus quadrangularis Linn. (CQ) has been used in folk medicine for the treatment of skin diseases since ancient times. Taking in to consideration the medicinal properties exhibited by this genus, it was decided to investigate the anti-cancer activity of CQ. Extracts obtained from CQ and their phenolic contents were subjected to in vitro evaluation of anticancer activity by using A431 (skin epidermoid carcinoma, human) cell line. The A431 cells were treated with different extracts of CQ in a dose dependent manner. Out of five extracts, the acetone extract demonstrated significant anti-cancer activity in the A431 cell line. Hexane, chloroform, ethyl acetate and methanol extracts also exhibited cytotoxicity but to a comparatively lesser extent than the acetone extract. The GI(50) value of the acetone extract was found to be 8 μg mL(-1), whereas GI(50) value of purified fraction of acetone extract, termed as AFCQ (active acetone fraction of CQ) with respect to A431 cells, was found to be 4.8 μg mL(-1). Furthermore, the mechanism of anticancer activity exhibited by AFCQ was investigated by comparing its effect with the standard anticancer drug Doxorubicin (DOX) by evaluating the status of apoptotic markers after treatment of A431 cells with AFCQ and DOX. Bax-Bcl-2 ratio along with the release of cytochrome c from mitochondria to cytoplasm, which is a hallmark of apoptosis, was also evaluated. Cleavage of PARP revealed that AFCQ induces apoptosis in A431 cells with reference to DOX.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cissus; Cytochromes c; Humans; Mitochondria; Plant Extracts; Plant Stems; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

Several acridine derivatives have been screened for their therapeutic potential and some are already established as antiprotozoan, antibacterial or anticancer agents. However, phenyl derivative at C-9 position of acridine had remained unexplored for their biological activity so far. In this report, we present our findings with 9-phenyl acridine (ACPH) as an anticancer agent. Both A375 and HeLa, two human cancer cell lines, were more sensitive to ACPH than normal cells namely human lymphocytes and also Chinese hamster V79 cells. ACPH also led to regression of tumour volume in mice. In A375 cells, we have shown that it caused DNA damage and blocked cell cycle progression at G(2)-M phase. Treatment with ACPH led to lowering of mitochondrial potential, upregulation of bax, release of cytochrome C and activation of caspase 3. As a new agent showing lower toxicity to normal cells and greater sensitivity towards cancerous cell line, ACPH shows promise as an effective cancer chemotherapeutic agent. ACPH treatment resulted in apoptotic death of cells through mitochondria-mediated caspase-dependent pathway.

Topics: Acridines; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cricetinae; Cricetulus; Cytochromes c; DNA Damage; Drug Screening Assays, Antitumor; G2 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Membrane Potential, Mitochondrial; Mice; Skin Neoplasms; Tumor Burden

Actinic keratosis (AK) is characterized by high prevalence and the risk to proceed to squamous cell carcinoma (SCC). Cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE (2) ) synthesis has been reported in AK and SCC, and the COX inhibitor diclofenac in hyaluronic acid (diclofenac/HA) was approved for AK therapy. Its mode of action, however, remained to be unravelled. In the present study, diclofenac resulted in reduced PGE (2) levels in apoptosis-sensitive cutaneous SCC cell lines (SCL-II, SCC-12, SCC-13) whereas no PGE (2) and no COX-2 expression was detectable in a SCC cell line resistant to apoptosis induction (SCL-I). Activation of mitochondrial apoptosis pathways was evident in SCC cells owing to loss of the mitochondrial membrane potential and release of the mitochondrial factors cytochrome c and apoptosis-inducing factor. Characteristic proapoptotic changes at the level of Bcl-2 proteins occurred in sensitive cells, as upregulation of Bad and downregulation of Mcl-1 and Bcl-w. In contrast, Bad was already high, and Mcl-1 and Bcl-w were already low in resistant SCL-I, even without treatment, which may be explained by the lack of PGE (2) . An antiapoptotic downregulation of proapoptotic Bcl-2 proteins Noxa and Puma was, however, also seen in SCL-I, suggesting here pathways independent of COX-2. The regulations of Mcl-1 and Bad were also reproduced in SCC cells by the more selective COX-2 inhibitor celecoxib, thus further underlining the specific role of COX-2. The findings illuminate the mode of action of diclofenac/HA in SCC cells as well as principles of their resistance, which may allow further adaptation and improvement of the new therapy.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; Carcinoma, Squamous Cell; Celecoxib; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cytochromes c; Diclofenac; Dinoprostone; Down-Regulation; Humans; Hyaluronic Acid; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Skin Neoplasms; Sulfonamides; Up-Regulation

Expression of Axl, a receptor tyrosine kinase, is increased in cutaneous squamous cell carcinoma (SCC). Examination of a series of cutaneous SCC tumors revealed positive phospho-Akt (P-Akt) staining accompanied by weak TUNEL staining in Axl-positive tumors, suggesting an anti-apoptotic role for Axl in SCC survival. The role of Axl in UV-induced apoptosis was investigated in a cutaneous SCC cell line using retroviral short hairpin RNA sequences enabling stable Axl knock-down. We show that, although Axl knock-down has no effect on cell proliferation, it sensitizes cells to UV-induced apoptosis through increased activation of the pro-apoptotic protein Bad, a change in the conformation of Bax and Bak, release of cytochrome c into the cytosol, and activation of caspases. These events are accompanied by faster Akt dephosphorylation in UV-treated Axl knock-down cells and correlate with the degree of Axl knock-down. Treatment with the pan-caspase inhibitor zVAD-fmk partially rescued cells from UV-induced apoptosis but did not affect Bid cleavage or cytochrome c release, suggesting that cells die via the mitochondrial-mediated pathway. Thus, Axl confers resistance of SCC cells to apoptosis and displays potential as a target for therapeutic intervention in cutaneous SCC.

Topics: Apoptosis; Axl Receptor Tyrosine Kinase; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-Associated Death Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Humans; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Receptor Protein-Tyrosine Kinases; Signal Transduction; Skin Neoplasms; Ultraviolet Rays

Photodynamic therapy was found to be an effective therapy for local malignant tumors. This study demonstrated that 80 μg/ml Hedyotis corymbosa extracts with 0.8 J/cm(2) fluence dose caused M21 skin cancer cell death. Photoactivated H. corymbosa-induced M21 cell death is a typical apoptosis that is accompanied by nuclear condensation, externalization of phosphatidylserine and the changes in protein expression of apoptosis-related proteins, such as Bcl-2 and caspase family members. This study applied 2D electrophoresis to analyse the proteins involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We found 12 proteins to be markedly changed. According to the results of protein sequence analysis of these altered protein spots, we identified that the expression of cytoskeletal proteins and chaperones were involved in the photoactivated H. corymbosa-induced M21 cell apoptosis. We further demonstrated that photoactivated H. corymbosa caused a significant effect on the cytoskeleton distribution and mitochondrial activity in M21 cells. Based on the above findings, this study characterized the effects and mechanisms of the photoactivated H. corymbosa-induced apoptosis in M21 skin cancer cells.

Topics: Actin Cytoskeleton; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; Cytoskeletal Proteins; Drugs, Chinese Herbal; Hedyotis; Humans; Melanoma; Mitochondria; Molecular Chaperones; Phalloidine; Photochemotherapy; Proteomics; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

The Solanum species herbs have been used to treat cancer for centuries; however, the underlying mechanisms and effectiveness in vivo remain unclear.. SR-T100, extracted from the Solanum incanum, contains solamargine alkaloid as the main active ingredient. Here, we investigated the apoptosis-inducing effects of SR-T100 for targeting squamous cell carcinoma (SCC) in vitro and in vivo.. We elucidated the mechanism by which SR-T100 induces apoptosis of human SCCs (A431, SCC4, SCC9, and SCC25) cells. The efficacy and safety issues were addressed regarding topical treatment of SR-T100 on UVB-induced cutaneous SCC of hairless mice and actinic keratoses (AKs) of human.. SR-T100 induces apoptosis in human SCCs cell lines by up-regulating the expressions of tumor necrosis factor receptors (TNFRs) and Fas, and downstream adaptors FADD/TRADD of the TNF-α and Fas ligand signaling cascades. SR-T100 also triggered the mitochondrial apoptotic pathway, as up-regulated cytochrome c and Bax, down-regulated Bcl-X(L). Animal experiments showed that all papillomas (35/35) and 27 of 30 UVB-induced microinvasive SCCs in hairless mice disappeared within 10 weeks after once-daily application of topical SR-T100. Furthermore, 13 patients, who suffered with 14 AKs, were treated with once-daily topical SR-T100 gel and 10 AKs cured after 16 weeks, showing negligible discomforts.. Our studies indicate that SR-T100 induces apoptosis of SCC cells via death receptors and the mitochondrial death pathway. The high efficacy of SR-T100 in our preclinical trial suggests that SR-T100 is a highly promising herb for AKs and related disorders.

Topics: Aged; Aged, 80 and over; Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cytochromes c; Female; Humans; Male; Mice; Mice, Hairless; Mitochondria; Papilloma; Plant Extracts; Receptors, Tumor Necrosis Factor; Signal Transduction; Skin Neoplasms; Solanaceous Alkaloids; Solanum; Ultraviolet Rays

In this study, we have investigated the chemotherapeutic potential of a purple violet pigment (PVP), which was isolated from a previously undescribed Antarctic Janthinobacterium sp. (Ant5-2), against murine UV-induced 2237 fibrosarcoma and B16F10 melanoma cells.. The 2237, B16F10, C50, and NIH3T3 cells were treated with PVP at different doses and for different times, and their proliferation and viability were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle arrest induced by PVP in 2237 fibrosarcoma cells was assessed by flow cytometry and expression analysis of cell cycle regulatory proteins were done by Western blot. Apoptosis induced by PVP in 2237 cells was observed by annexin-V/propidium iodide double staining flow cytometry assay and fluorescence microscopy. To further determine the molecular mechanism of apoptosis induced by PVP, the changes in expression of Bcl-2, Bax and cytochrome c were detected by Western blot. The loss of mitochondrial membrane potential in PVP treated 2237 cells was assessed by staining with JC-1 dye following flow cytometry. Caspase-3, Caspase-9 and PARP cleavage were analyzed by Western blot and Caspase-3 and -9 activities were measured by colorimetric assays.. In vitro treatment of murine 2237 cells with the PVP resulted in decreased cell viability (13-79%) in a time (24-72 h) and dose (0.1-1 μM)-dependent manner. The PVP-induced growth inhibition in 2237 cells was associated with both G0/G1 and G2/M phase arrest accompanied with decrease in the expression of cyclin dependent kinases (Cdks) and simultaneous increase in the expression of cyclin dependent kinase inhibitors (Cdki) - Cip1/p21 and Kip1/p27. Further, we observed a significant increase in the apoptosis of the 2237 fibrosarcoma cells which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2, disruption of mitochondrial membrane potential, cytochrome c release, activation of caspase-3, caspase-9 and poly-ADP-ribose-polymerase (PARP) cleavage.. We describe the anti-cancer mechanism of the PVP for the first time from an Antarctic bacterium and suggest that the PVP could be used as a potent chemotherapeutic agent against nonmelanoma skin cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Cytochromes c; Fibrosarcoma; Indoles; Melanoma; Melanoma, Experimental; Membrane Potential, Mitochondrial; Mice; Oxalobacteraceae; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

To unravel the molecular mechanisms underlying the chemopreventive potential of [6]-gingerol, a pungent ingredient of ginger rhizome (Zingiber officinale Roscoe, Zingiberaceae), against benzo[a]pyrene (B[a]P)-induced mouse skin tumorigenesis.. Topical treatment of [6]-gingerol (2.5 muM/animal) was given to the animals 30 min prior and post to B[a]P (5 mug/animal) for 32 weeks. At the end of the study period, the skin tumors/tissues were dissected out and examined histopathologically. Flow cytometry was employed for cell cycle analysis. Further immunohistochemical localization of p53 and regulation of related apoptogenic proteins were determined by Western blotting.. Chemopreventive properties of [6]-gingerol were reflected by delay in onset of tumorigenesis, reduced cumulative number of tumors, and reduction in tumor volume. Cell cycle analysis revealed that the appearance of sub-G1 peak was significantly elevated in [6]-gingerol treated animals with post treatment showing higher efficacy in preventing tumorigenesis induced by B[a]P. Moreover, elevated apoptotic propensity was observed in tumor tissues than the corresponding non-tumor tissues. Western blot analysis also showed the same pattern of chemoprevention with [6]-gingerol treatment increasing the B[a]P suppressed p53 levels, also evident by immunohistochemistry, and Bax while decreasing the expression of Bcl-2 and Survivin. Further, [6]-gingerol treatment resulted in release of Cytochrome c, Caspases activation, increase in apoptotic protease-activating factor-1 (Apaf-1) as mechanism of apoptosis induction.. On the basis of the results we conclude that [6]-gingerol possesses apoptotic potential in mouse skin tumors as mechanism of chemoprevention hence deserves further investigation.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Benzo(a)pyrene; Blotting, Western; Caspases; Catechols; Cytochromes c; Fatty Alcohols; Flow Cytometry; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Kaplan-Meier Estimate; Male; Mice; Microtubule-Associated Proteins; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; Signal Transduction; Skin; Skin Neoplasms; Survivin; Time Factors; Treatment Outcome; Tumor Suppressor Protein p53

To investigate the chemopreventive potential of resveratrol, a phytoalexin found in seeds and skin of grapes, berries and peanuts in 7,12 dimethyl benz(a)anthracene (DMBA) induced mouse skin tumorigenesis.. Topical treatment of resveratrol was given to the animals 1 h prior to DMBA for 28 weeks. At the end of the study period, the skin tumors were dissected out and western blotting was carried out to examine the regulation of proteins involved in anti-tumorigenesis in response to resveratrol.. Chemopreventive properties of resveratrol were reflected by delay in onset of tumorigenesis, reduced cumulative number of tumors, and reduction in tumor volume. Results of the western blotting showed that resveratrol treatment increased the DMBA suppressed p53 and Bax while decreased the expression of Bcl-2 and Survivin. Further, resveratrol supplementation resulted in release of cytochrome C, caspases activation, increase in apoptotic protease-activating factor-1 (Apaf-1) as mechanism of apoptosis induction. Resveratrol was also found to inhibit skin tumorigenesis through regulation of Phosphatidylinositol-3-kinase (PI3K)/ and AKT proteins which are implicated in cancer progression because it stimulates proliferation and suppresses apoptosis.. Based on the results we can conclude that resveratrol regulates apoptosis and cell survival in mouse skin tumors as mechanism of chemoprevention hence deserve to be a chemopreventive agent.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinogens; Caspases; Cytochromes c; Cytosol; Down-Regulation; Enzyme Activation; Female; Inhibitor of Apoptosis Proteins; Mice; Microtubule-Associated Proteins; Mitochondria; Phosphatidylinositol 3-Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Repressor Proteins; Resveratrol; Signal Transduction; Skin Neoplasms; Stilbenes; Survivin; Tumor Suppressor Protein p53

Many naturally occurring phytochemicals have shown cancer chemopreventive potential in a variety of bioassay systems. One such naturally occurring biologically active compound is tea Camellia sinensis, which is the most consumed beverage in the world after water. The most abundant and active constituents of tea are polyphenols (epigallocatechin gallate and theaflavins). In the present study, cancer chemopreventive properties of both black tea polyphenols (BTP) and green tea polyphenols (GTP) on 7,12-dimethylbenz[a]anthracene (DMBA) induced mouse skin carcinogenesis were studied. BTP and GTP treatment showed delay in onset of tumorigenesis, reduction in cumulative number of tumors and increased tumor free survival. Both BTP and GTP were found to modulate the expression of proteins involved in apoptotic pathway. Tea polyphenols treatment along with DMBA exposure resulted in upregulation of p53, and proapoptotic protein Bax, whereas enhanced expression of antiapoptotic proteins, Bcl-2 and survivin by DMBA were downregulated. Further, we showed that tea polyphenols supplementation resulted in release of cytochrome c, caspases activation, and increase in apoptotic protease activating factor and poly (ADP-ribose) polymerase cleavage as mechanism of apoptosis induction. The results also provide strong evidence that BTP is a better chemopreventive agent against skin tumorigenesis as compared to GTP at the tested dose levels. Thus, we can conclude that the polyphenolic constituents present in black tea and green tea induce mitochondrial pathway of apoptosis and hence can be used as a potential chemopreventive agents against skin cancer.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspases; Cytochromes c; Enzyme Activation; Flavonoids; Male; Mice; Mitochondria; Models, Biological; Phenols; Phytotherapy; Poly(ADP-ribose) Polymerases; Polyphenols; Signal Transduction; Skin; Skin Neoplasms; Tea; Tumor Suppressor Protein p53

In this study, aloe-emodin (AE) was less cytotoxic to human noncancerous skin cells (premalignant keratinocytic HaCaT and fibroblast Hs68) than to nonmelanoma cancer cells (epidermoid carcinoma A431 and head and neck squamous cell carcinoma SCC25). Notably, AE induced apoptosis by up-regulating tumor necrosis factor-alpha and Fas ligand and their cognate receptors, downstream adaptor TNF-R1-associated death domain and Fas-associated death domain, and activated caspase-8 in A431 and SCC25 cells. Moreover, AE up-regulated p53, increased intracellular reactive oxygen species levels, depleted intracellular-reduced GSH, up-regulated cytochrome c and Bax, down-regulated Bcl-2, and activated caspase-9 and -3. The combinatory use of AE and 5-fluorouracil (5-Fu) achieved significantly more cell death in A431 and SCC25 cells than only the use of AE or 5-Fu, likely via regulation of caspase-8, -9, and -3 expressions. Incorporating AE into the liposomal formulation accelerated cell death of A431 and SCC25 cells within a short time. Furthermore, skin permeation profiles of drug suggest that the liposomal formulation enhances transdermal delivery of AE. Experimental data demonstrate the feasibility of applying liposome to deliver AE in clinical therapy.

Topics: Administration, Cutaneous; Anthraquinones; Antineoplastic Agents; Apoptosis; Caspase 8; Caspase 9; Cell Line; Cytochromes c; Fas Ligand Protein; Fas-Associated Death Domain Protein; Fluorouracil; Humans; Liposomes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Skin; Skin Neoplasms; TNF Receptor-Associated Death Domain Protein; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Although diallyl trisulphide (DATS) has been found to induce apoptosis in various tumour cells, its cytotoxicity in melanoma cells has not yet been defined and the molecular pathway by which DATS induces apoptosis is not well understood.. To determine growth inhibition of DATS in human melanoma cells (A375 and M14) by inducing apoptosis, and to investigate the mechanism underlying such effects.. Growth inhibition by DATS was estimated by the tetrazolium assay. Apoptosis induction in DATS-treated cells was assessed by staining with 4',6-diamidino-2-phenylindole (DAPI) and double staining with annexin V and propidium iodide. Expression of Bcl-2, Bax, Bcl-xL/Bcl-xS, cytochrome c release, activation of caspase-9 and poly(ADP-ribose) polymerase (PARP) were determined by western blotting. The activity of caspase-3 was measured using a colorimetric assay.. DATS exerted its cytotoxic effect in a time-dependent and dose-dependent manner by inducing apoptosis in A375 and M14 cells. Expression of Bcl-2 and Bcl-xL was downregulated. Release of cytochrome c and activation of the downstream effectors caspase-3, caspase-9 and PARP were detected after DATS sensitization.. DATS inhibits growth of melanoma cells by inducing apoptosis in association with downregulation of Bcl-2 and Bcl-xL and activation of caspases.

Topics: Allyl Compounds; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Caspases; Cell Line, Tumor; Cytochromes c; Down-Regulation; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Skin Neoplasms; Sulfides

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).\ This article has been retracted at the request of the Editor-in-Chief.\ Multiple figures in this article appear to be falsified/fabricated, and can not be verified as the corresponding author does not have the original data.\ Figure 2. It appears that data has been duplicated in panels V and VI.\ Figure 3A. Lanes II and VI in the p53 wild band appear to be duplicated.\ Figure 4A. Lanes I, II, V and VI of the Beta-actin blot appear to be the same data replicated.\ Figure 4B. The representative blots in the Bcl-2 band, lanes V and VI are identical, as are all lanes in the Beta-actin band.\ Figure 5B. Lanes III and IV of the Apaf 1 band, when rotated and vertically stretched, are duplicated and appear in Figure 3D as lanes III and IV of the Cytochrome C blot in “Chemopreventive potential of resveratrol in mouse skin tumors through regulation of mitochondrial and PI3K/AKT signaling pathways.” Pharmaceutical Research (2009). Doi: 10.1007/s11095-008-9723-z.\ Figure 5C. Lanes II and V of the Caspase 9 band appear to be duplicated.\ Figure 5E. The bands in lane V and VI of the Beta-actin blot are duplicated.\ Figure 5B and 5C. The Beta-actin lane IV band in 5B and lane IV in 5C appear to be duplicated from Figure 6B in “Hepatoprotective effects of lupeol and mango pulp extract of carcinogen induced alteration in Swiss albino mice.” Molecular Nutrition & Food Research (2007). Doi: 10.1002/mnfr.200600113.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anticarcinogenic Agents; Antioxidants; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Carcinogens; Chemoprevention; Cytochromes c; Disease Models, Animal; In Situ Nick-End Labeling; Male; Mice; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Skin; Skin Neoplasms; Stilbenes; Tetradecanoylphorbol Acetate

Imiquimod is an immune response modifier currently used as a topical treatment of genital warts, basal cell carcinoma, cutaneous metastasis of malignant melanoma, and vascular tumors. We developed more efficient killers from the same family of compounds that can induce apoptosis without the prominent pro-inflammatory response associated with imiquimod. Among these new products, tk;4EAPB0203, a member of the imidazo[1,2-a]quinoxalines, exhibits an important cytotoxic activity in vitro. HTLV-I-associated adult T-cell leukemia (ATL) and HTLV-I-negative peripheral T-cell lymphomas are associated with poor prognosis. Using potentially achievable concentrations of EAPB0203, we demonstrate inhibition of cell proliferation, G2/M cell- cycle arrest, and induction of apoptosis in HTLV-I-transformed and HTLV-I-negative malignant T cells and fresh ATL cells, whereas normal resting or activated T lymphocytes were resistant. EAPB0203 treatment significantly down-regulated the antiapoptotic proteins c-IAP-1 and Bcl-XL and resulted in a significant loss of mitochondrial membrane potential, cytoplasmic release of cytochrome c, and caspase-dependent apoptosis. Moreover, in HTLV-I-transformed cells only, EAPB0203 treatment stabilized p21 and p53 proteins but had no effect on NF-kappaB activation. These results support a potential therapeutic role for EAPB0203 in ATL and HTLV-I-negative T-cell lymphomas, either as a systemic or topical therapy for skin lesions.

Topics: Aminoquinolines; Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspases; Cell Division; Cytochromes c; G2 Phase; Human T-lymphotropic virus 1; Humans; Imiquimod; Inflammation; Inhibitor of Apoptosis Proteins; Jurkat Cells; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Lymphoma, T-Cell; Membrane Potential, Mitochondrial; NF-kappa B; Quinoxalines; Skin Neoplasms; Tumor Suppressor Protein p53

Apoptotic elimination of UV-damaged cells from the epidermis is an important step in preventing both the emergence and expansion of cells with carcinogenic potential. A pivotal event in apoptosis is the release of apoptogenic factors from the mitochondria, although the mechanisms by which the different proteins are released are not fully understood. Here we demonstrate that UV radiation induced the mitochondrial to nuclear translocation of apoptosis inducing factor (AIF) in normal skin. The human papillomavirus (HPV) E6 protein prevented release of AIF and other apoptotic factors such as cytochrome c and Omi from mitochondria of UV-damaged primary epidermal keratinocytes and preserved mitochondrial integrity. shRNA silencing of Bak, a target for E6-mediated proteolysis, demonstrated the requirement of Bak for UV-induced AIF release and mitochondrial fragmentation. Furthermore, screening non-melanoma skin cancer biopsies revealed an inverse correlation between HPV status and AIF nuclear translocation. Our results indicate that the E6 activity towards Bak is a key factor that promotes survival of HPV-infected cells that facilitates tumor development.

Topics: Active Transport, Cell Nucleus; Apoptosis; Apoptosis Inducing Factor; bcl-2 Homologous Antagonist-Killer Protein; Cell Line; Cytochromes c; DNA-Binding Proteins; High-Temperature Requirement A Serine Peptidase 2; Humans; Mitochondria; Mitochondrial Proteins; Oncogene Proteins, Viral; Serine Endopeptidases; Skin Neoplasms; Ultraviolet Rays

TR3 was originally recognized for its role in the regulation of cell survival and differentiation, however, it was recently found to be a potent pro-apoptotic protein. In order to characterize the role of TR3 in melanoma cell apoptosis, we studied expression of TR3 in melanoma cell lines and tissues, its subcellular distribution and function during apoptosis using various expression and RNA interference vectors. We found that TR3 was constitutively expressed in both cultured melanoma cells and melanoma tissues. TR3 expression was significantly decreased in advanced melanomas comparing to benign nevi. Over-expression of wild type TR3 or mutant TR3 lacking the DNA binding domain resulted in massive apoptosis in melanoma cells, whereas stable knockdown of TR3 using RNA interference resulted in melanoma cell resistance to apoptosis induced by chemotherapeutic agents ATRA and cisplatin. We further demonstrated that apoptosis in melanoma cells was mediated, at least partially, through TR3 mitochondrial translocation but not alteration in TR3 expression levels. Our results suggest that TR3 is an important apoptosis inducing factor in melanoma cells. Decreased expression of TR3 in metastatic melanoma cells may contribute to their reduced apoptotic potential and increased resistance to chemotherapy.

Topics: Apoptosis; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA-Binding Proteins; Humans; Melanoma; Mitochondria; Nuclear Receptor Subfamily 4, Group A, Member 1; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Skin Neoplasms; Transcription Factors; Tretinoin

Loss of pigment in hamster amelanotic melanoma line is accompanied by a faster growth rate, higher tumorigenicity and shorter animal survival time. Thus, the malignancy of melanoma increases during the alteration of melanotic (Ma) into amelanotic (Ab) line. As changes in the ability to undergo a spontaneous or induced apoptosis, and the role of caspases in this process during melanoma progression are not well defined, they were investigated in this work. Our results show that the proportion of spontaneously early apoptotic (caspase+/PI-) cells in the Ab line decreased in comparison to the Ma line. Cytochrome c release into cytosol, and the activation of effector caspases, estimated by PARP degradation clearly showed that during the spontaneous death in the cells from both melanoma lines intrinsic way of apoptosis was activated. Confocal and cytometric flow analyses indicate that camptothecin (CPT) induced apoptosis with caspase activation by the intrinsic way only in the amelanotic melanoma cells, even though cells of the Ma line also underwent CPT-induced apoptosis (the content of TUNEL-positive cells increased). Thus, our results suggest that melanoma progression, associated with a decreased ability to undergo spontaneous apoptosis but an increased susceptibility to CPT-induced apoptosis, relates to different levels of caspase activation; they also show that intrinsic way of apoptotis depends on the phenotype of melanoma cells, being more pronounced in the melanotic melanoma cells. On the other hand, melanotic melanoma cells resistance to camptothecin-induced apoptosis suggests that the melanogenic apparatus or melanin itself may have the protective effect on the ability of the melanoma cells to undergo apoptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Camptothecin; Caspases; Cell Line, Tumor; Cricetinae; Cytochromes c; Disease Progression; Flow Cytometry; Male; Melanins; Melanoma, Amelanotic; Melanoma, Experimental; Mesocricetus; Microscopy, Confocal; Poly(ADP-ribose) Polymerases; Skin Neoplasms

Chronic exposure to UV light is a risk factor for skin cancer in which signature mutations in the p53 tumor suppressor gene occur due to DNA damage and contribute to cancer development. In this issue of the JCI, Tang et al. report on their study of a nonimmunodeficient mouse model of UVB-induced skin cancer and human skin carcinoma cells and show that the mutant p53 conformation-modifying drug CP-31398 not only treats these tumors but also prevents them (see the related article beginning on page 3753). These studies have important implications for chemoprevention as well as therapy of common, mutant p53-driven tumors.

Topics: Animals; Apoptosis; bcl-X Protein; Caspase 3; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D; Cyclins; Cytochromes c; Environmental Exposure; Female; Humans; Male; Mice; Mice, Hairless; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mutation; Neoplasms, Radiation-Induced; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Protein Transport; Pyrimidines; Skin Neoplasms; Tumor Suppressor Protein p53; Ultraviolet Rays

Mutations in the tumor suppressor p53 are detectable in over 50% of all human malignancies. Mutant p53 protein is incapable of transactivating its downstream target genes that are required for DNA repair and apoptosis. Chronic exposure to UVB induces p53 mutations and is carcinogenic in both murine and human skin. CP-31398, a styrylquinazoline compound, restores the tumor suppressor functions of mutant forms of p53 in tumor cells. However, its effectiveness in vivo remains unclear. Here, we demonstrate that CP-31398 blocked UVB-induced skin carcinogenesis and was associated with increases in p53, p21, and BclXs. CP-31398 downregulated Bcl2, proliferating nuclear cell antigen, and cyclin D1. Activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase also occurred in both tumor and perilesional skin following treatment. CP-31398 induced the expression of p53-dependent target proteins, and this was followed by apoptosis in UVB-irradiated wild-type mice but not in their p53-deficient littermates. Similar effects were observed in human skin carcinoma A431 cells expressing mutant p53. In addition, CP-31398 induced mitochondrial translocation of p53, leading to changes in mitochondrial membrane permeability pore transition (MPT) and consequent cytochrome c release in these cells. Blocking MPT diminished p53 translocation and apoptosis. These studies indicate that reconstituting p53 tumor suppressor functions in vivo by small molecular weight compounds may block the pathogenesis and progression of skin cancer.

Topics: Animals; Apoptosis; bcl-X Protein; Caspase 3; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D; Cyclins; Cytochromes c; Environmental Exposure; Female; Humans; Male; Mice; Mice, Hairless; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mutation; Neoplasms, Radiation-Induced; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Protein Transport; Pyrimidines; Skin Neoplasms; Tumor Suppressor Protein p53; Ultraviolet Rays

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

alpha-Santalol, an active component of sandalwood oil, has been studied in detail in recent years for its skin cancer preventive efficacy in murine models of skin carcinogenesis; however, the mechanism of its efficacy is not defined. Two major biological events responsible for the clonal expansion of transformed/initiated cells into tumors are uncontrolled growth and loss of apoptotic death. Accordingly, in the present study, employing human epidermoid carcinoma A431 cells, we assessed whether alpha-santalol causes cell growth inhibition and/or cell death by apoptosis. Treatment of cells with alpha-santalol at concentrations of 25-75 microM resulted in a concentration- and a time-dependent decrease in cell number, which was largely due to cell death. Fluorescence-activated cell sorting analysis of Annexin V/propidium iodide (PI) stained cells revealed that alpha-santalol induces a strong apoptosis as early as 3 h post-treatment, which increases further in a concentration- and a time-dependent manner up to 12 h. Mechanistic studies showed an involvement of caspase-3 activation and poly(ADP-ribose) polymerase cleavage through activation of upstream caspase-8 and -9. Further, the treatment of cells with alpha-santalol also led to disruption of the mitochondrial membrane potential and cytochrome c release into the cytosol, thereby implicating the involvement of the mitochondrial pathway. Pre-treatment of cells with caspase-8 or -9 inhibitor, pan caspase inhibitor or cycloheximide totally blocked alpha-santalol-caused caspase-3 activity and cleavage, but only partially reversed apoptotic cell death. This suggests involvement of both caspase-dependent and -independent pathways, at least under caspase inhibiting conditions, in alpha-santalol-caused apoptosis. Together, this study for the first time identifies the apoptotic effect of alpha-santalol, and defines the mechanism of apoptotic cascade activated by this agent in A431 cells, which might be contributing to its overall cancer preventive efficacy in mouse skin cancer models.

Topics: Apoptosis; Carcinoma; Caspase Inhibitors; Caspases; Cell Proliferation; Cycloheximide; Cytochromes c; Enzyme Inhibitors; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Plant Oils; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Polycyclic Sesquiterpenes; Sesquiterpenes; Skin Neoplasms; Tumor Cells, Cultured

Solid tumors are often placed under stress conditions, such as glucose starvation which may result in topoisomerase II drug resistance. In this study, we investigated whether glucose deprivation or substitution by fructose regulates tumor cell apoptosis induced by 2-acetyl furanonaphthoquinone (FNQ). We now show that FNQ exerts much greater antitumor activity than either 7-methoxy 2-ethyl FNQ or 2-ethyl FNQ. Whereas 0.8 microM FNQ induces apoptosis after 16 hours in glucose-supplemented conditions irrespective of bcl-2 overexpression in K1735 melanoma, 0.5 microM FNQ is also effective within 12 hours in low glucose or in fructose-supplemented medium. Under the latter conditions, apoptosis-associated PARP cleavage and cytosolic cytochrome C are increased, together with induction and partial translocation to mitochondria of phosphorylated Jun-N-terminal kinase and massive upregulation of mitochondrial Mn superoxide dismutase. We propose that mitochondrial colocalization of these activities is important in this synergistic anti-tumor effect of FNQ and glucose depletion. Since glucose limitation slows proliferation and decreases efficacy of some genotoxic drugs that trigger apoptosis in rapidly dividing cells, we propose evaluating FNQ as a novel therapeutic anti-cancer adjuvant against slowly proliferating tumors.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cytochromes c; Furans; Glucose; Glycolysis; Humans; MAP Kinase Kinase 4; Melanoma; Mitochondria; Naphthoquinones; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms; Superoxide Dismutase; Transcriptional Activation; Tumor Cells, Cultured

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Topics: Adenosine Triphosphate; Animals; Body Weight; Carcinogenicity Tests; Carcinogens; Cell Proliferation; Cytochromes c; DNA Adducts; Genes, ras; Hydroxyl Radical; Hyperplasia; Inflammation; Leukocytes; Luminescent Measurements; Mice; Mice, Inbred SENCAR; Oxidation-Reduction; Oxidative Stress; Peroxidase; Salicylates; Skin; Skin Neoplasms; Smoke; Superoxides

Hypothermia is known to retard mammalian cell growth, however, BC-8 cells, which have originated from AK-5 tumor after single cell cloning, were triggered into apoptotic pathway when grown at 30 degrees C. Cell death process showed typical apoptotic features like DNA fragmentation, cytochrome c release, etc. Introduction of Bcl-2 gene in BC-8 cells inhibited hypothermia-induced apoptotic process, which is ascribed to reduced ROS generation and higher glutathione production. Thus, Bcl-2 seems to control the apoptotic induction process at the level of redox regulation, in addition to its known effects at the mitochondrial dysregulation. These observations suggest that tumors, which are low in Bcl-2 expression, are sensitive to hypothermic shock and make hypothermia an interesting inducer of apoptosis in tumor cells.

Topics: Animals; Annexin A5; Apoptosis; Cell Division; Cytochromes c; Glutathione; Hypothermia, Induced; Oxidative Stress; Propidium; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Skin Neoplasms; Transfection; Tumor Cells, Cultured

Topics: Cytochromes c; Electron Transport Complex IV; Epidermis; Humans; NADH Dehydrogenase; Nucleotides; Oxidoreductases; Pyridines; Skin Neoplasms