Anti-metastatic activity of the recombinant disintegrin, r-mojastin 1, from the Mohave rattlesnake

Cancer is the uncontrollable growth of cell, which may spread to other parts of the body. The interaction of cancer cells with extracellular matrix (ECM) is essential for metastasis, which is the principal cause of death in cancer patients. Disintegrins are naturally occurring low molecular weight peptides found in the venoms of many snakes. Disintegrins were first used to inhibit platelet aggregation, but more recently have been used to inhibit cancer cell growth, adhesion, migration, invasion and/or angiogenesis. The purpose of this study was to determine the anti-tumor properties of recombinant mojastin 1 (r-mojastin 1) and r-mojastin-GST, cloned from the venom glands of the Mohave rattlesnake (Crotalus scutulatus scutulatus). Human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-ML-28) and murine skin melanoma (B16F10) cell lines were used. r-Mojastin 1 inhibited SK-MEL-28 cell adhesion to fibronectin, but was not able to inhibit T24 cell adhesion to fibronectin. However, r-Mojastin-GST inhibited SK-MEL-28 and T24 cells adhesion to fibronectin. r-Mojastin-GST and r-mojastin 1 decreased the ability of SK-MEL-28 cells to migrate after 24 h of incubation but were not able to inhibit T24 cell migration. r-Mojastin 1 and r-mojastin-GST inhibited invasion of T24 and SK-MEL-28 cancer cells in vitro, and r-Mojastin 1 inhibited lung tumor colonization of B16F10 cells in mice in vivo. In conclusion, our studies suggest that r-mojastin could be a useful tool to develop novel anti-tumor agents by virtue of its ability to inhibit tumor cell adhesion, migration and invasion.     

Novel Suppressive Effects of Ketotifen on Migration and Invasion of MDA-MB-231 and HT-1080 Cancer Cells

The high mortality rates associated with cancer reflect the metastatic spread of tumor cells from the site of their origin. Metastasis, in fact, is the cause of 90% of cancer deaths. Therefore, considerable effort is being made to inhibit metastasis. In the present study, we screened ketotifen for anti-migratory and anti-invasive activities against MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer cells. Cancer cell migration and invasion were measured using multi-well chambers. Additionally, western blots were used to examine the effects of ketotifen on the expressions of CDC42, Rho, Rac, and matrix metalloproteinase 9 (MMP-9). The results showed that ketotifen dose-dependently suppressed the migration and invasion of MDA-MB-231 and HT-1080 cells. Ketotifen also suppressed the expressions of CDC42, Rac, and Rho, which, significantly, are involved in MDA-MB-231 and HT-1080 cancer cell migration. Moreover, ketotifen suppressed the expression and activity of MMP-9, which is involved in degradation of the extracellular matrix leading to invasion. The overall data suggested that ketotifen suppresses the migration and invasion of MDA-MB-231 and HT-1080 cancer cells via inhibition of CDC42, Rac, Rho, and MMP-9 expression.     

Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC50s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.     

Differential cytostatic and cytotoxic action of Metallocorroles against human cancer cells: potential platforms for anticancer drug development

A gallium(III)-substituted amphiphilic corrole noncovalently associated with a targeting protein was previously found by us to confer promising cytotoxic and antitumor activities against a breast cancer cell line and a mouse xenograft breast cancer model. To further explore potential anticancer applications, the cytostatic and cytotoxic properties of six nontargeted metallocorroles were evaluated against seven human cancer cell lines. Results indicated that toxicity toward human cancer cells depended on the metal ion as well as corrole functional group substitution. Ga(III)-substituted metallocorrole 1-Ga inhibited proliferation of breast (MDA-MB-231), melanoma (SK-MEL-28), and ovarian (OVCAR-3) cancer cells primarily by arrest of DNA replication, whereas 2-Mn displayed both cytostatic and cytotoxic properties. Confocal microscopy revealed extensive uptake of 1-Ga into the cytoplasm of melanoma and ovarian cancer cells, while prostate cancer cells (DU-145) displayed extensive nuclear localization. The localization of 1-Ga to the nucleus in DU-145 cells was exploited to achieve a 3-fold enhancement in the IC(50) of doxorubicin upon coadministration. Time-course studies showed that over 90% of melanoma cells incubated with 30 μM 1-Ga internalized metallocorrole after 15 min. Cellular uptake of 1-Ga and 1-Al was fastest and most efficient in melanoma, followed by prostate and ovarian cancer cells. Cell cycle analyses revealed that bis-sulfonated corroles containing Al(III), Ga(III), and Mn(III) induced late M phase arrest in several different cancer cell lines, a feature that could be developed for potential therapeutic benefit.     

Antitumor effect of chondroitin AC lyase (PsPL8A) from Pedobacter saltans on melanoma and fibrosarcoma cell lines by in vitro analysis

Background

PGs are involved in cellular communication and cancer biology. The role of CS in melanoma and fibrosarcoma cell lines was explored by using chondroitin AC lyase (PsPL8A).

Vixapatin (VP12), a C-Type Lectin-Protein from Vipera xantina palestinae Venom: Characterization as a Novel Anti-angiogenic Compound

A C-type lectin-like protein (CTL), originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2β1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC₅₀ of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC); 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin’s ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2β1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.     

A cytotoxic secocycloartenoid fromAbies koreana

Two triterpenoids, 24-methylene-3,4-seco-cycloart-4(28)-en-3-oic acid (1) and 3-oxo-9beta-lanosta-7,22Z,24-trien-26,23-olide (6) were isolated from Abies koreana, together with beta-sitosterol (2), maltol (3), beta-sitosterol-O-beta-D-glucoside (4), and hexacosylferulate (5). The structures of the compounds were established based on the spectroscopic data. The cytotoxic activities of triterpenoids have been evaluated using the sulforhodamine B (SRB) method. Compound 1 showed moderate cytotoxicities against human lung carcinoma (A549), ovarian carcinoma (SK-OV-3), malignant melanoma (SK-MEL-2), and colon carcinoma (HCT-15) cell lines.     

VI-14, a novel flavonoid derivative, inhibits migration and invasion of human breast cancer cells

It has been well characterized that flavonoids possess pronounced anticancer potentials including anti-angiogenesis, anti-metastasis, and pro-apoptosis. Herein, we report, for the first time, that VI-14, a novel flavonoid derivative, possesses anti-cancer properties. The purpose of this study is to investigate the anti-migration and anti-invasion activities of VI-14 in breast cancer cells. Our data indicate that VI-14 inhibits adhesion, migration and invasion of MDA-MB-231 and MDA-MB-435 human breast cancer cells. MDA-MB-231 cells treated with VI-14 display reduced activities and expressions of ECM degradation-associated proteins including matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) at both the protein and mRNA levels. Meanwhile, VI-14 treatment induces an up-regulated expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and 2 (TIMP-2) in MDA-MB-231 cells. Western blotting results show that phosphorylation levels of critical components of the MAPK signaling pathway, including ERK, JNK and P38, are dramatically decreased in VI-14-treated MDA-MB-231 cells. Furthermore, treatment of VI-14 significantly decreases the nuclear levels and the binding ability of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). Taken together, our data suggest that VI-14 treatment suppresses migration and motility of breast cancer cells, and VI-14 may be a potential compound for cancer therapy.     

Chemopreventive effects of Ginkgo biloba extract in estrogen-negative human breast cancer cells

Excessive level of estrogen is considered as a main cause of breast cancer, therefore, many studies have focused on estrogen receptor (ER)-positive breast cancer, even though ER-negative cancer has a poor prognosis than ER-positive breast cancer. We evaluated the anti-cancer effects of Ginkgo biloba extract (GBE) in estrogen-independent breast cancer. GBE has been traditionally used as a platelet activating factor, a circulatory stimulant, a tonic, and anti-asthmatic drug, and anti-cancer agent. However, anti-cancer effects of GBE on ER-negative breast cancer have not been proved yet. In this study, we tested chemotherapeutic potential of GBE in the MDA-MB-231 (ER-negative) human breast cancer cell line. Our results showed that cytotoxicity effects of GBE in MDA-MB-231 lead to DNA fragmentation at high concentrations (500 and 1,000 μg/ml). Caspase-3 was significantly activated and mRNA levels of apoptosis-related genes (Bcl-2 and Bax) were altered. These results indicate that GBE induces apoptosis in MDA-MB-231 cells. It is presumed that GBE has chemopreventive effects in ER-independent breast cancer through anti-proliferation and apoptosis-inducing activities.     

A New Class of 5-Fluoro-2′-deoxyuridine Prodrugs Conjugated with a Tumor-Homing Cyclic Peptide CNGRC by Ester Linkers: Synthesis,Reactivity, and Tumor-Cell–Selective Cytotoxicity

No HeadingPurpose. Tumor-targeting prodrugs of 5-fluoro-2-deoxyuridine (5-FdUrd), which are chemical conjugations of 5-FdUrd with a tumor-homing cyclic peptide CNGRC by succinate and glutarate linkers, were synthesized to investigate the structural effects of linkers on the hydrolytic release of 5-FdUrd and the tumor-cell–selective cytotoxicity.Methods. A solid phase synthesis method was used to produce 5-FdUrd prodrugs. The kinetics and efficiency of hydrolytic 5-FdUrd release from the prodrugs were investigated in phosphate buffer (PB), fetal bovine serum (FBS), HT-1080 cell lysate, MDA-MB-231 cell lysate, and MEM containing 10% FBS. The tumor-cell–selective cytotoxicity of prodrugs was evaluated by an MTT method.Results. Two tumor-targeting prodrugs CNF1 and CNF2 bearing 5-FdUrd conjugated with a common cyclic peptide CNGRC by succinate and glutarate linkers, respectively, and their control compounds CN1 and CN2 without 5-FdUrd moiety were synthesized and identified. CNF1 underwent hydrolysis to release 5-FdUrd more rapidly and efficiently than CNF2. Both prodrugs were of lower cytotoxicity compared to 5-FdUrd, showing more selective cytotoxicity toward APN/CD13 positive cells (HT-1080) than toward APN/CD13 negative cells (HT-29, MDA-MB-231).Conclusions. A new class of tumor-targeting 5-FdUrd prodrugs CNF1 and CNF2 were successfully synthesized. These prodrugs targeted a tumor marker APN/CD13 to cause tumor-cell–selective cyctotoxicity due to 5-FdUrd release, the rate of which could be controlled by the structure of ester linker.     

Cytotoxicity,cell cycle arrest,and apoptosis in breast cancer cell lines exposed to an extract of the seed kernel of Mangifera pajang (bambangan)

An extract of Mangifera pajang kernel has been previously found to contain a high content of antioxidant phytochemicals. The present research was conducted to investigate the anticancer potential of this kernel extract. The results showed that the kernel crude extract induced cytotoxicity in MCF-7 (hormone-dependent breast cancer) cells and MDA-MB-231 (non-hormone dependent breast cancer) cells with IC50 values of 23 and 30.5 μg/ml, respectively. The kernel extract induced cell cycle arrest in MCF-7 cells at the sub-G1 (apoptosis) phase of the cell cycle in a time-dependent manner. For MDA-MB-231 cells, the kernel extract induced strong G2-M arrest in cell cycle progression at 24 h, resulting in substantial sub-G1 (apoptosis) arrest after 48 and 72 h of incubation. Staining with Annexin V-FITC and propidium iodide revealed that this apoptosis occurred early in both cell types, 36 h for MCF-7 cells and 24 h for MDA-MB-231cells, with 14.0% and 16.5% of the cells respectively undergoing apoptosis at these times. This apoptosis appeared to be dependent on caspase-2 and -3 in MCF-7 cells, and on caspase-2, -3 and -9 in MDA-MB-231 cells. These findings suggest that M. pajang kernel extract has potential as a potent cytotoxic agent against breast cancer cell lines.     

Antiproliferative activity of Vietnamese medicinal plants

Methanol, methanol-water (1:1) and water extracts were prepared from seventy-seven Vietnamese medicinal plants and tested for their antiproliferative activities against human HT-1080 fibrosarcoma cells. Among them, fifteen extracts including seven methanol extracts of Caesalpinia sappan, Catharanthus roseus, Coscinium fenestratum, Eurycoma longifolia, Hydnophytum formicarum and Streptocaulon juventas (collected at two areas), six methanol-water (1:1) extracts of Cae. sappan, Cat. roseus, Co. fenestratum, H. formicarum and S. juventas (at two areas), and two water extracts of Cae. sappan and S. juventas exhibited antiproliferative activities in a concentration-dependent manner. Their antiproliferative activities against human cervix HeLa adenocarcinoma, human lung A549 adenocarcinoma, murine colon 26-L5 carcinoma, murine Lewis lung carcinoma (LLC) and murine B16-BL6 melanoma cells were then examined. Co. fenestratum showed selective activity against lung carcinoma and/or lung metastatic cell lines, A549, LLC and B16-BL6, while H. formicarum and S. juventas showed selective activity against human tumor cell lines, HeLa and A549. Characteristic morphological change and DNA fragmentation indicated the antiproliferative activity to be due to the induction of apoptosis.     

Organoammonium hydroselenites: antitumor action through radical balance regulation

Organoammonium hydroselenites were synthesized and investigated as potential selective, anticancer prodrugs. These compounds were studied in vitro on human fibrosarcoma (HT-1080), hamster kidney endothelial (BHK 21) and normal mouse embryonic fibroblasts (NIH 3T3). Most of them were very active against HT-1080 (0.6-5.3 g/ml). Amino acid hydroselenites readily increased the nitric oxide (NO) concentration in the culture medium of HT-1080 cells (up to TG(100)=1500%); however, 4-amidohydroximinomethylpyridinium hydroselenite (TG(100)=24%) and o-phenanthrolinium hydroselenite (TG(100)=50%) were free radical inhibitors. All compounds were glutathione peroxidase inhibitors; some of them could also prevent hydrogen peroxide degradation by inhibition of catalase. The influence of the investigated ammonium hydroselenites on tumor cell (HT-1080) morphology was examined. The substances studied were also active in vivo against sarcoma S-180. The role of organoammonium hydroselenites as free radical regulators and their therapeutic antitumor are discussed.     

Isolation and identification of novel selective antitumor constituents,sidrin and sidroside,from Zizyphus spina-christi

BackgroundThe leaves of Zizyphus spina-christi (L.) Willd contain several compounds exhibiting different pharmacologic activities. However, studies on the cytotoxic activity of these compounds are limited.ObjectivesWe aimed to investigate and isolate cytotoxic compounds with selective antitumor effects from the leaves of Z. spina-christi using bioassay-guided fractionation of methanol extract.MethodsPowdered, dried leaves were subjected to methanol extraction and fractionated using n-hexane, chloroform, ethyl acetate, and n-butanol. Fractions with positive cytotoxicity against HeLa and THP-1 cell lines were further fractionated and eluted using various concentrations of organic solvents. Active compounds were isolated using different chromatographic methods and their chemical structures were determined using extensive spectroscopic methods, such as 1D NMR (¹H NMR, ¹³C NMR, and DEPT), 2D NMR (COSY, HMBC, and HMQC), HRFAB-MS, and IR. Furthermore, the cytotoxic effects of the isolated compounds were evaluated against 62 tumor cell lines (including HeLa and THP-1) in addition to normal bone marrow cells.ResultsThe chloroform and aqueous methanol fractions of the leaves showed cytotoxic activity. Two compounds were successfully isolated and named “sidrin” (13-β-hydroxy-lup-20(30)-ene-2,3-β-epoxy-28-carboxylate) and “sidroside” (3-O-β-D-glucopyranosyl-(1–3)-α-L-arabinopyranosyl-jujubogenin-20-O-α-L-rhamnopyranoside). Sidrin exhibited cytotoxic activity against the human leukemia (Hl-60, RPMI-8226), lung cancer (A549, EKVX), breast cancer (BT-549, MDA-MB-231/ATCC), colon cancer (KM12), melanoma (M14, SK-MEL-5), and central nervous system (CNS) cancer (SF-295) cell lines, and selectivity was observed against the Hl-60, EKVX, BT-549, KM12, and SF-295 cell lines. In addition, sidrin was more active than sidroside and doxorubicin against the Hl-60 and EKVX cell lines. In contrast, sidrin had a similar effect to doxorubicin against the BT-549 and renal cancer (UO-31) cell lines. Sidroside was more selective against the leukemia (CCRF-CEM, MOLT-4), lung cancer (HOP-92, NCI-H322M), breast cancer (MDA-MB-468), melanoma (LOX IMVI), CNS cancer (SNB-19), ovarian cancer (OVCAR-8), renal cancer (UO-31, RXF 393), and prostate cancer (PC-3) cell lines. Both compounds exhibited similar activity against the breast cancer (MDA-MB-231, T-47D), colon cancer (HCC-2998, HCT-116), ovarian cancer (OVCAR-3), renal cancer (UO-31, 786–0, and SN 12C) cell lines. Normal bone marrow cells were unaffected at the same concentrations of sidrin and sidroside applied to tumor cells.ConclusionsThese results suggest tumor-selective cytotoxicity of sidrin and sidroside.     

Preclinical efficacy of Virulizin in human breast,ovarian and prostate tumor models

Virulizin is a novel biological response modifier (BRM) approved for the treatment of melanoma and is currently in a phase III clinical trial against advanced pancreatic cancer. The purpose of this study was to define the anti-cancer activity of Virulizin against a number of solid human tumors. The therapeutic effect of Virulizin was evaluated in mouse xenograft models, and the results demonstrate that Virulizin has high efficacy against breast, ovarian and prostate tumor xenografts. Seventy-seven percent inhibition, with an optimal T/C value of 24.8%, was observed in human beast MDA-MB-231 xenografts treated with Virulizin as compared to saline-treated controls (p=0.0004). In human ovarian SK-OV-3 tumor xenografts, administration of Virulizin inhibited tumor growth by 77.6% compared to saline controls (p=0.0439). Furthermore, high anti-tumor activity was also demonstrated in DU145 and PC-3 prostate tumor xenografts, as indicated by 72.6 and 49.1% suppression of tumor growth (versus saline controls, p=0.0007 or p=0.0049), respectively. Direct comparisons with the anti-tumor activities of conventional drugs demonstrated that Virulizin has higher or equal efficacy against all four tumors tested. Finally, addition of Virulizin into co-cultures of tumor cells and macrophages stimulated the cytolytic activity of the macrophages against the tumor cells in a dose-dependent manner. This result suggests that stimulation of immune cells is at least part of the anti-tumor mechanism of action of Virulizin. These results clearly demonstrate that Virulizin inhibits the growth of human breast, ovarian and prostate tumors, indicating great potential for expansion of the clinical indications for this novel BRM.     

Anti-skin cancer properties of phenolic-rich extract from the pericarp of mangosteen (Garcinia mangostana Linn.)

Skin cancers are often resistant to conventional chemotherapy. This study examined the anti-skin cancer properties of crude ethanol extract of mangosteen pericarp (MPEE) on human squamous cell carcinoma A-431 and melanoma SK-MEL-28 lines. Significant dose-dependent reduction in% viability was observed for these cell lines, with less effect on human normal skin fibroblast CCD-1064Sk and keratinocyte HaCaT cell lines. Cell distribution in G₁ phase (93%) significantly increased after 10 μg/ml of MPEE versus untreated SK-MEL-28 cells (78%), which was associated with enhanced p21^WAF1 mRNA levels. In A-431 cells, 10 μg/ml MPEE significantly increased the sub G₁ peak (15%) with concomitant decrease in G₁ phase over untreated cells (2%). In A-431 cells, 10 μg/ml MPEE induced an 18% increase in early apoptosis versus untreated cells (2%). This was via caspase activation (15-, 3- and 4-fold increased caspse-3/7, 8, and 9 activities), and disruption of mitochondrial pathways (6-fold decreased mitochondrial membrane potential versus untreated cells). Real-time PCR revealed increased Bax/Bcl-2 ratio and cytochrome c release, and decreased Akt1. Apoptosis was significantly increased after MPEE treatment of SK-MEL-28 cells. Hence, MPEE showed strong anti-skin cancer effect on these two skin cancer cell lines, with potential as an anti-skin cancer agent.     

几种植物来源不同作用机制的抗癌药抗侵袭作用

用细胞培养法和癌细胞侵袭实验,观察几种植物来源不同作用机理的抗癌药如紫杉醇、三尖杉酯碱、高三尖杉酯碱及喜树碱^[1],对黑色素瘤高转移株B16-BL6细胞及人纤维肉瘤细胞HT-1080的细胞毒作用和抗侵袭作用。结果表明紫杉醇、三尖杉酯碱、高三尖杉酯碱及喜树碱对B16-BL6和HT-1080细胞增殖均有很强的抑制作用。紫杉醇、三尖杉酯碱及高三尖杉酯碱对B16-BL6细胞侵袭和运动也有明显的抑制作用,而喜树碱在同样浓度下对B16-BL6细胞侵袭和运动均无明显抑制。     

Biochemical mechanism of acetaminophen (APAP) induced toxicity in melanoma cell lines

In this work, we investigated the biochemical mechanism of acetaminophen (APAP) induced toxicity in SK-MEL-28 melanoma cells using tyrosinase enzyme as a molecular cancer therapeutic target. Our results showed that APAP was metabolized 87% by tyrosinase at 2 h incubation. AA and NADH, quinone reducing agents, were significantly depleted during APAP oxidation by tyrosinase. The IC(50) (48 h) of APAP towards SK-MEL-28, MeWo, SK-MEL-5, B16-F0, and B16-F10 melanoma cells was 100 microM whereas it showed no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 nonmelanoma cells, demonstrating selective toxicity towards melanoma cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, enhanced APAP toxicity towards SK-MEL-28 cells. AA and GSH were effective in preventing APAP induced melanoma cell toxicity. Trifluoperazine and cyclosporin A, inhibitors of permeability transition pore in mitochondria, significantly prevented APAP melanoma cell toxicity. APAP caused time and dose-dependent decline in intracellular GSH content in SK-MEL-28, which preceded cell toxicity. APAP led to ROS formation in SK-MEL-28 cells which was exacerbated by dicoumarol and 1-bromoheptane whereas cyslosporin A and trifluoperazine prevented it. Our investigation suggests that APAP is a tyrosinase substrate, and that intracellular GSH depletion, ROS formation and induced mitochondrial toxicity contributed towards APAP's selective toxicity in SK-MEL-28 cells.     

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.     

Synthesis and evaluation of several new (2-chloroethyl)nitrosocarbamates as potential anticancer agents

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC(50) values in the range of 1-10 microg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4, 6-tetra-O-acetyl-1-alpha,beta-D-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.