Anti-PSMA antibody-coupled gold nanorods detection by optical and electron microscopies.

While cancer is one of the greatest challenges to public health care, prostate cancer was chosen as cancer model to develop a more accurate imaging assessment than those currently available. Indeed, an efficient imaging technique which considerably improves the sensitivity and specificity of the diagnostic and predicting the cancer behavior would be extremely valuable. The concept of optoacoustic imaging using home-made functionalized gold nanoparticles coupled to an antibody targeting PSMA (prostate specific membrane antigen) was evaluated on different cancer cell lines to demonstrate the specificity of the designed platform. Two commonly used microscopy techniques (indirect fluorescence and scanning electron microscopy) showed their straightforwardness and versatility for the nanoparticle binding investigations regardless the composition of the investigated nanoobjects. Moreover most of the research laboratories and centers are equipped with fluorescence microscopes, so indirect fluorescence using Quantum dots can be used for any active targeting nanocarriers (polymers, ceramics, metals, etc.). The second technique based on backscattered electron is not only limited to gold nanoparticles but also suits for any study of metallic nanoparticles as the electronic density difference between the nanoparticles and binding surface stays high enough. Optoacoustic imaging was finally performed on a 3D cellular model to assess and prove the concept of the developed platform.

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis.

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO(2), whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC-MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼600 individual proteins) and quantified proteins (>95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins.

Cytotoxic aporphine alkaloids from Cassytha filiformis.

Purification of a cytotoxic crude alkaloid extract of Cassytha filiformis led to the isolation of four known aporphine alkaloids: neolitsine, dicentrine, cassythine (= cassyfiline) and actinodaphnine. Their structures were determined by analysis of spectroscopic data. All isolated alkaloids were tested for their cytotoxic activities on cancer and non-cancer cell lines in vitro. Neolitsine was the most active against HeLa and 3T3 cells (IC 50 :21.6 microM, and 21.4 microM, respectively). Cassythine and actinodaphnine showed the highest activity against Mel-5 (IC 50 : 24.3 microM and 25.7 microM, respectively) and HL-60 (IC 50 : 19.9 microM and 15.4 microM, respectively). This is the first report on the cytotoxic activity of C. filiformis extract and of neolitsine and cassythine. Furthermore, the complete NMR data of cassythine and actinodaphnine are given here for the first time.

ent-trachyloban-3beta-ol, a new cytotoxic diterpene from Croton zambesicus.

The dichloromethane extract of leaves of Croton zambesicus (Euphorbiaceae) showing in vitro cytotoxicity against human cervix carcinoma cells was investigated in order to identify its active compounds. A bio-guided fractionation by HSCCC followed by MPLC led us to isolate a trachylobane diterpene, ent-trachyloban-3beta-ol, with cytotoxic properties (IC50 on HeLa cells = 7.3 microg/ml). This is the first report on the cytotoxicity of a trachylobane diterpene.

DNA intercalation, topoisomerase II inhibition and cytotoxic activity of the plant alkaloid neocryptolepine.

Cryptolepine and neocryptolepine are two indoloquinoline alkaloids isolated from the roots of the African plant Cryptolepis sanguinolenta. Both drugs have revealed antibacterial and antiparasitic activities and are strongly cytotoxic to tumour cells. We have recently shown that cryptolepine can intercalate into DNA and stimulates DNA cleavage by human topoisomerase II. In this study, we have investigated the mechanism of action and cytotoxicity of neocryptolepine, which differs from the parent isomer only by the orientation of the indole unit with respect to the quinoline moiety. The biochemical and physicochemical results presented here indicate that neocryptolepine also intercalates into DNA, preferentially at GC-rich sequences, but exhibits a reduced affinity for DNA compared with cryptolepine. The two alkaloids interfere with the catalytic activity of human topoisomerase II but the poisoning activity is slightly more pronounced with cryptolepine than with its isomer. The data provide a molecular basis to account for the reduced cytotoxicity of neocryptolepine compared with the parent drug.

Stimulation of topoisomerase II-mediated DNA cleavage by three DNA-intercalating plant alkaloids: cryptolepine, matadine, and serpentine.

Cryptolepine, matadine, and serpentine are three indoloquinoline alkaloids isolated from the roots of African plants: Cryptolepis sanguinolenta, Strychnos gossweileri, and Rauwolfia serpentina, respectively. For a long time, these alkaloids have been used in African folk medicine in the form of plant extracts for the treatment of multiple diseases, in particular as antimalarial drugs. To date, the molecular basis for their diverse biological effects remains poorly understood. To elucidate their mechanism of action, we studied their interaction with DNA and their effects on topoisomerase II. The strength and mode of binding to DNA of the three alkaloids were investigated by spectroscopy. The alkaloids bind tightly to DNA and behave as typical intercalating agents. All three compounds stabilize the topoisomerase II-DNA covalent complex and stimulate the cutting of DNA by topoisomerase II. The poisoning effect is more pronounced with cryptolepine than with matadine and serpentine, but none of the drugs exhibit a preference for cutting at a specific base. Cryptolepine which binds 10-fold more tightly to DNA than the two related alkaloids proves to be much more cytotoxic toward B16 melanoma cells than matadine and serpentine. The cellular consequences of the inhibition of topoisomerase II by cryptolepine were investigated using the HL60 leukemia cell line. The flow cytometry analysis shows that the drug alters the cell cycle distribution, but no sign of drug-induced apoptosis was detected when evaluating the internucleosomal fragmentation of DNA in cells. Cryptolepine-treated cells probably die via necrosis rather than via apoptosis. The results provide evidence that DNA and topoisomerase II are the primary targets of cryptolepine, matadine, and serpentine.

Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro. Preliminary study on rats.

Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.

Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds.

Characterization of non pigmented B16 melanoma cell-derived cytotoxic factors.

We analyzed and tried to characterize substance(s) responsible for cytotoxic activities detected in culture media conditioned by non pigmented B16 melanoma cells (NPB16). The different cytological tests used showed that ultrafiltrated conditioned media (CM U1 fraction) contained several cytotoxic factors with a Mw lower than 1000 Da. These factors seemed to act either directly or indirectly on cell membranes, mitochondria, on the cell cycle and on protein and DNA synthesis. A cytotoxic activity could be found even after high dilution of CM U1. These cytotoxic factors were rapidly released by B16 cells in culture, independently of cell confluence. Their activities in the treated cells were also very fast and the cytotoxic effects were irreversible after only a few hours of treatment. These factors were not intermediate products during melanogenesis, neither polyamines, nor proteases. At least one of them seemed to be a small acidic and basic stable peptide without disulfide bounds but not heat stable. The synthesis of at least one of these cytotoxic factors was inhibited by cycloheximide and the cytotoxic activity was partially destroyed by pronase and trypsin, but not by pepsin. The cytotoxicity was not modified by copper complexants or free radical inhibitors (bovine serum albumin (BSA), tyrosine, superoxyde dismutase (SOD), catalase, vitamin E). Furthermore the levels of glutathione peroxydase activity and reduced glutathione did not change after treatment by CM U1 as compared to controls.

In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos: strychnopentamine and usambarensine.

The cytotoxicity and the selective antiprotozoal activity of some Strychnos alkaloids, namely strychnopentamine (SP) and usambarensine (US) (7) led us to analyze and compare their effects with emetine (EM) by using mouse B16 melanoma cells cultivated in vitro. We observed by cytological analysis and proliferation rate studies that these substances induce analogous cytotoxic effects in B16 cells, but at different concentrations i.e. formation of lamellar bodies in the cytoplasm, the which contain pre-melanosomes in the case of SP and US, vacuoles and blebs. At concentrations near their respective IC50, SP and US, but not EM, decreased colony formation. We showed by incorporation of labelled precursors that SP and US first inhibit RNA synthesis while EM initially acts on protein synthesis. These alkaloids increased melanin synthesis. Furthermore, only EM and SP caused hemolysis of sheep red blood corpuscles. This could explain why the rate of antiplasmodial activity is higher for SP and EM.

Evaluation of matrix metalloproteinases and serine proteases activities in three B16 melanoma cell lines with distinct tumorigenic potential.

Mouse B16 melanoma cells (B16, parental line) and two derived clones either pigmented (B16P) or non pigmented (B16NP) were cultured as monolayers (2D) or on agar, as aggregates (3D). The productions of gelatinases A and B (72 kDa and 92 kDa type IV collagenases) and their inhibitors (TIMP1 and TIMP2), plasminogen activators (PAs) and plasminogen activator inhibitors (PAI) were investigated. The B16 cell lines did not secrete any gelatinase, but they secreted TIMP2, tissue-type (t-PA), urokinase-type (u-PA) plasminogen activators and PAI-1 like activities. High levels of PAI activity were determined in conditioned media and cellular extracts of B16NP, which could account for the lower tumorigenic potential of these cells. In 3D cultures, the cellular extracts of the three cell lines contained essentially u-PA activity. This activity could contribute to the greater tumorigenic and invasive capacities of B16, B16P and B16NP when cultured in 3D.

Characterization and tumorigenicity of spheroids composed of pigmented or non pigmented B16 melanoma cells.

A parental line of mouse B16 melanoma cells (B16) and two derived cloned lines, either pigmented (B16P) or non pigmented (B16NP), were cultured in vitro as spheroids. After 48 hrs, the pigmented cells (B16, B16P) formed smaller and looser aggregates, with higher rates of cell proliferation and lower amounts of extracellular matrix as compared to B16NP spheroids. The three lines were more tumorigenic when inoculated subcutaneously as spheroids than as isolated cells. Furthermore, B16P or B16 spheroids developed richly vascularized subcutaneous tumors and metastases more rapidly than B16NP aggregates. After intravenous injection of spheroids, the measurement with an image analyzer of the area of sections in lung colonies indicated that B16P colonies were larger and more numerous than those induced by B16NP cells.

Effects of alpha-hederin, a saponin extracted from Hedera helix, on cells cultured in vitro.

In this work, we have analysed the effects of alpha-hederin, a monodesmosidic triterpenoid saponin isolated from Hedera helix, on mouse B16 melanoma cells and non-cancer mouse 3T3 fibroblasts cultured in vitro. Our results indicate that, in a serum-free medium, alpha-hederin is cytotoxic and inhibits proliferation in both cell lines at rather low concentrations (< 5 micrograms/ml) after only 8 hours of treatment. Its cytotoxicity decreases in the presence of serum in which BSA seems to be able to bind the saponin. alpha-Hederin also induces vacuolization of the cytoplasm and membrane alterations leading to cell death.

Effect of selenium compounds on murine B16 melanoma cells and pigmented cloned pB16 cells.

The effects of selenium compounds such as sodium selenite, sodium selenate, seleno-DL-cystine and seleno-DL-methionine (100 microM and 10 microM) on B16 and pigmented cloned pB16 murine melanoma cells were investigated in vitro. At the tested concentrations, B16 cells showed a greater sensitivity to the toxic effects of sodium selenite and seleno-DL-cystine than pB16 cells, whereas no decrease of B16 and pB16 cell number was observed after incubation with sodium selenate or seleno-DL-methionine. Glutathione (GSH) percentages were strongly decreased only by selenite and seleno-DL-cystine; it was marked more in B16 than in pB16 cells. The pretreatment of B16 cells with a GSH depleting agent (10 microM buthionine-[S,R]-sulfoximine) did not significantly influence the cytotoxic effects of selenite and seleno-DL-cystine. On both cell populations, GSH preincubation (50 microM) enhanced the cytotoxicity of selenite whereas the survival of seleno-DL-cystine treated cells was increased. Glutathione peroxidase (GSH-Px) activity in B16 cells was more sensitive than in pB16 cells to the activating effect of selenite, and particularly of seleno-DL-cystine: however, cell-free controls indicated that activation was mainly due to glutathione reductase. The rate of 75Se (as sodium selenite) uptake in both cell populations was maximal within the first hour of incubation, with a preferential accumulation in the cytosol; after 24 h of incubation, the amount of 75Se in cytosol and pellet was approximately the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of tumorigenicity of human breast adenocarcinoma cells in nude mice by matrigel and fibroblasts.

The failure of MCF7 cells to induce the formation of tumours after sub-cutaneous inoculation into athymic nude mice can be obviated by the simultaneous injection of an extract of basement membrane proteins (matrigel). Tumour growth is promoted and the latency period is low (2 to 4 weeks). In the absence of matrigel, the simultaneous inoculation of fibroblasts and MCF7 cells also resulted in the development of tumours, but with a longer latency period (about 2 months). The tumorigenic synergy between matrigel and fibroblasts was evidenced by co-inoculating MCF7 cells MDA-MB 231 cells with fibroblasts and matrigel. This co-inoculation decreased the delay of appearance of the tumours and/or accelerated the tumour growth, depending upon the number of fibroblasts injected. Repeated injections of fibroblasts conditioned medium, at the site of inoculum of tumour cells also enhanced tumour growth, suggesting the involvement of soluble factors secreted by fibroblasts. Histologically, tumours induced by co-inoculation of tumour cells and fibroblasts contained more stromal structures including vimentin-positive cells, fibronectin and interstitial collagens. These data suggest that human tumours may be reconstituted and grown in athymic nude mice using basement membrane components and fibroblasts as inductors.

Effects of trace metals on mouse B16 melanoma cells in culture.

The effects of fourteen metal ions (As3+, As5+, Cd2+, Co2+, Cr3+, Cr6+, Hg2+, Li+, Mg2+, Mn2+, Ni2+, Se4+, V5+, VO2+) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10(-4)M, metal ions such as As3+, As5+, Cd2+, Cr6+, Se4+, V5+, VO2+, and, to a minor extent, Li+, Hg2+, and Co2+ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg2+ > Cr6+ = Cd2+ > As3+, As5+, > V5+, VO2+ > Se4+ = Ni2+ = Co2+ = Li+. An increased synthesis of melanin in B16 cells was noted after incubation with Co2+, Ni2+, Cd2+, and Li+, whereas Se4+ had, on the contrary, an inhibiting effect on melanogenesis.

Reevaluation by image analysis of the effects of fibroblasts, fibronectin or laminin upon colony formation in mouse lungs by B16 melanoma cells.

A recently described personal method based on image analysis of histological sections was used in order to quantify lung colony formation by B16 melanoma cells injected intravenously into the mouse. These tumor cells were preincubated in vitro either with fibronectin (FN), laminin (LN) or fibroblasts (FB), which are implicated in the process of invasion and metastasis. Thanks to this method, a more accurate analysis of lung colonies (section area and number) formed by tumor cells was realized. By image analysis, we show that when FB were mixed with B16 cells, a drastic increase of tumor sections number and area was induced. LN increased the tumor sections area, but not their number. No effect of FN on B16 cells was observed. LN and FN promoted tumor anchorage in the depth of the lungs while FB reduced the latter. These facts could explain the contradictory results obtained by simply counting macroscopically superficial lung colonies. When cultured in vitro, these B16 melanoma cells did not produce any type of IV collagenase, either alone or in the presence of LN or FN, but in cocultures (B16 with 3T3) and in fibroblasts cultures, this enzyme was present. This could explain, among other factors, why the rate of invasiveness exerted by B16 cells is higher when the latter are coinjected with FB.

Fibronectin promotes lung colony formation in the mouse by B16 melanoma cells spheroids.

By microscopical observation and using an original method of automatic image analysis, we studied on histological sections the rate of lung colony formation after intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as pure or mixed spheroids (B16 + 3T3 fibroblasts). The preincubation in vitro of pure spheroids with fibronectin significantly increased the percentages of lung section area occupied by tumors and the relative number of internal lung colonies. This effect of fibronectin was even more obvious when mixed spheroids were injected.

Influence of laminin or fibroblasts upon colony formation in the mouse by B16 melanoma cell spheroids: a morphometric analysis.

By microscopical observation and using an original morphometric method, we analyzed on histological sections the rate of lung colony formation after the intravenous injection into the mouse of B16 melanoma cells previously cultivated in vitro as aggregates. After the injection of B16 pure spheroids, superficial lung colonies were more numerous than internal lung colonies. After the injection of mixed spheroids (B16 + 3T3 fibroblasts), the size of colony sections was increased. Addition of laminin to pure or mixed spheroids decreased the size of colony sections but increased the number of internal lung colonies.

Cytotoxic and mitogenic activities in culture media conditioned by mouse B16 melanoma cells and 3T3 fibroblasts.

Cytotoxic and mitogenic soluble factors are released into media conditioned by pure or mixed populations of mouse 3T3 fibroblasts and B16 melanoma cells cultivated in vitro. These activities are demonstrated by the use of MTT cell survival test and 3HTDR incorporation. Mitogenic (M.W. greater than 10,000) and cytotoxic factors (M.W. less than 1,000) are present and are generally more active on B16 cells than on fibroblasts. Their release into conditioned media is related to the rate of pigmentation in B16 cells and to the mode of cultivation (monolayers or cell aggregates).