Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 µM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.

Anti-tumor necrosis factor-a for the treatment of steroid-refractory acute graft-versus-host disease

Allogeneic stem cell transplantation has been increasingly performed for a variety of hematologic diseases. Clinically significant acute graft-versus-host disease (GVHD) occurs in 9 to 50% of patients who receive allogeneic grafts, resulting in high morbidity and mortality. There is no standard therapy for patients with acute GVHD who do not respond to steroids. Studies have shown a possible benefit of anti-TNF-a (infliximab)for the treatment of acute GVHD. We report here on the outcomes of 10 recipients of related or unrelated stem cell transplants who received 10 mg/kg infliximab, iv, once weekly for a median of 3.5 doses (range: 1-6) for the treatment of severe acute GVHD and who were not responsive to standard therapy. All patients had acute GVHD grades II to IV (II = 2, III = 3, IV = 5). Overall, 9 patients responded and 1 patient had progressive disease. Among the responders, 3 had complete responses and 6 partial responses. All patients with cutaneous or gastrointestinal involvement responded, while only 2 of 6 patients with liver disease showed any response. None of the 10 patients had any kind of immediate toxicity. Four patients died, all of them with sepsis. Six patients are still alive after a median follow-up time of 544 days (92-600) after transplantation. Considering the severity of the cases and the bad prognosis associated with advanced acute GVHD, we find our results encouraging. Anti-TNF-a seems to be a useful agent for the treatment of acute GVHD.

The husk fiber of Cocos nucifera L. (Palmae) is a source of anti-neoplastic activity

In the present study, we investigated the in vitro anti-tumoral activities of fractions from aqueous extracts of the husk fiber of the typical A and common varieties of Cocos nucifera (Palmae). Cytotoxicity against leukemia cells was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (2 x 104/well) were incubated with 0, 5, 50 or 500 µg/mL high- or low-molecular weight fractions for 48 h, treated with MTT and absorbance was measured with an ELISA reader. The results showed that both varieties have almost similar antitumoral activity against the leukemia cell line K562 (60.1 ± 8.5 and 47.5 ± 11.9% for the typical A and common varieties, respectively). Separation of the crude extracts with Amicon membranes yielded fractions with molecular weights ranging in size from 1-3 kDa (fraction A) to 3-10 kDa (fraction B) and to more than 10 kDa (fraction C). Cells were treated with 500 µg/mL of these fractions and cytotoxicity was evaluated by MTT. Fractions ranging in molecular weight from 1-10 kDa had higher cytotoxicity. Interestingly, C. nucifera extracts were also active against Lucena 1, a multidrug-resistant leukemia cell line. Their cytotoxicity against this cell line was about 50% (51.9 ± 3.2 and 56.3 ± 2.9 for varieties typical A and common, respectively). Since the common C. nucifera variety is extensively cultured in Brazil and the husk fiber is its industrial by-product, the results obtained in the present study suggest that it might be a very inexpensive source of new antineoplastic and anti-multidrug resistant drugs that warrants further investigation.

In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus amazonicus (Rhamnaceae)

Ampelozizyphus amazonicus Ducke is a tree commonly found in the Amazon region and an extract of its stem bark is popularly used as an antimalarial and anti-inflammatory agent and as an antidote to snake venom. Ursolic acid; five lupane type triterpenes: betulin, betulinic acid, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid, and three phytosteroids: stigmasterol, sitosterol and campesterol, have been isolated from stem extracts of A. amazonicus Ducke. Their structures were characterized by spectral data including COSY and HMQC. In an in vitro biological screening of the isolated compounds, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid was cytotoxic against the SKBR-3 human adenocarcinoma cell line (1 to 10 mg/mL), while 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid exhibited cytotoxicity against both SKBR-3 human adenocarcinoma and C-8161 human melanoma tumor cell lines (>0.1 mg/mL). In the present study, different extracts and some fractions of this plant were also investigated for trypanocidal activity due to the presence of pentacyclic triterpenes. The triterpene classes are potent against Trypanosoma cruzi. The bioassays were carried out using blood collected from Swiss albino mice by cardiac puncture during the parasitemic peak (7th day) after infection with the Y strain of T. cruzi. The results obtained showed that A. amazonicus is a potential source of bioactive compounds since its extracts and fractions isolated from it exhibited in vitro parasite lysis against trypomastigote forms of T. cruzi at concentrations >100 µg/mL. Fractions containing mainly betulin, lupenone, 3ß-hydroxylup-20(29)-ene-27,28-dioic acid, and 2a,3ß-dihydroxylup-20(29)-ene-27,28-dioic acid showed more activity than crude extracts.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

Oxysterols are 27-carbon atom molecules resulting from autoxidation or enzymatic oxidation of cholesterol. They are present in numerous foodstuffs and have been demonstrated to be present at increased levels in the plasma of patients with cardiovascular diseases and in atherosclerotic lesions. Thus, their role in lipid disorders is widely suspected, and they might also be involved in important degenerative diseases such as Alzheimer's disease, osteoporosis, and age-related macular degeneration. Since atherosclerosis is associated with the presence of apoptotic cells and with oxidative and inflammatory processes, the ability of some oxysterols, especially 7-ketocholesterol and 7β-hydroxycholesterol, to trigger cell death, activate inflammation, and modulate lipid homeostasis is being extensively studied, especially in vitro. Thus, since there are a number of essential considerations regarding the physiological/pathophysiological functions and activities of the different oxysterols, it is important to determine their biological activities and identify their signaling pathways, when they are used either alone or as mixtures. Oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever. Moreover, a substantial accumulation of polar lipids in cytoplasmic multilamellar structures has been observed with cytotoxic oxysterols, suggesting that cytotoxic oxysterols are potent inducers of phospholipidosis. This basic knowledge about oxysterols contributes to a better understanding of the associated pathologies and may lead to new treatments and new drugs. Since oxysterols have a number of biological activities, and as oxysterol-induced cell death is assumed to take part in degenerative pathologies, the present review will focus on the cytotoxic activities of these compounds, the corresponding cell death signaling pathways, and associated events (oxidation, inflammation, and phospholipidosis).

Effect of cyhalothrin on Ehrlich tumor growth and macrophage activity in mice

Cyhalothrin, a pyrethroid insecticide, induces stress-like symptoms, increases c-fos immunoreactivity in the paraventricular nucleus of the hypothalamus, and decreases innate immune responses in laboratory animals. Macrophages are key elements in cellular immune responses and operate at the tumor-host interface. This study investigated the relationship among cyhalothrin effects on Ehrlich tumor growth, serum corticosterone levels and peritoneal macrophage activity in mice. Three experiments were done with 10 experimental (single gavage administration of 3.0 mg/kg cyhalothrin daily for 7 days) and 10 control (single gavage administration of 1.0 mL/kg vehicle of cyhalothrin preparation daily for 7 days) isogenic BALB/c mice in each experiment. Cyhalothrin i) increased Ehrlich ascitic tumor growth after ip administration of 5.0 x 106 tumor cells, i.e., ascitic fluid volume (control = 1.97 ± 0.39 mL and experimental = 2.71 ± 0.92 mL; P < 0.05), concentration of tumor cells/mL in the ascitic fluid (control = 111.95 ± 16.73 x 106 and experimental = 144.60 ± 33.18 x 106; P < 0.05), and total number of tumor cells in the ascitic fluid (control = 226.91 ± 43.22 x 106 and experimental = 349.40 ± 106.38 x 106; P < 0.05); ii) increased serum corticosterone levels (control = 200.0 ± 48.3 ng/mL and experimental = 420.0 ± 75.5 ng/mL; P < 0.05), and iii) decreased the intensity of macrophage phagocytosis (control = 132.3 ± 19.7 and experimental = 116.2 ± 4.6; P < 0.05) and oxidative burst (control = 173.7 ± 40.8 and experimental= 99.58 ± 41.7; P < 0.05) in vitro in the presence of Staphylococcus aureus. These data provide evidence that cyhalothrin simultaneously alters host resistance to Ehrlich tumor growth, hypothalamic-pituitary-adrenocortical (HPA) axis function, and peritoneal macrophage activity. The results are discussed in terms of data suggesting a link between stress, HPA axis activation and resistance to tumor growth.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

Classification of brain tumor extracts by high resolution ¹H MRS using partial least squares discriminant analysis

High resolution proton nuclear magnetic resonance spectroscopy (¹H MRS) can be used to detect biochemical changes in vitro caused by distinct pathologies. It can reveal distinct metabolic profiles of brain tumors although the accurate analysis and classification of different spectra remains a challenge. In this study, the pattern recognition method partial least squares discriminant analysis (PLS-DA) was used to classify 11.7 T ¹H MRS spectra of brain tissue extracts from patients with brain tumors into four classes (high-grade neuroglial, low-grade neuroglial, non-neuroglial, and metastasis) and a group of control brain tissue. PLS-DA revealed 9 metabolites as the most important in group differentiation: γ-aminobutyric acid, acetoacetate, alanine, creatine, glutamate/glutamine, glycine, myo-inositol, N-acetylaspartate, and choline compounds. Leave-one-out cross-validation showed that PLS-DA was efficient in group characterization. The metabolic patterns detected can be explained on the basis of previous multimodal studies of tumor metabolism and are consistent with neoplastic cell abnormalities possibly related to high turnover, resistance to apoptosis, osmotic stress and tumor tendency to use alternative energetic pathways such as glycolysis and ketogenesis.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

The cytotoxicity of methacryloxylethyl cetyl ammonium chloride, a cationic antibacterial monomer, is related to oxidative stress and the intrinsic mitochondrial apoptotic pathway

Antibacterial monomers incorporated in dentin bonding systems may have toxic effects on the pulp. Thus, the cytotoxicity of antibacterial monomers and its underlying mechanisms must be elucidated to improve the safety of antibacterial monomer application. The influence of an antibacterial monomer, methacryloxylethyl cetyl ammonium chloride (DMAE-CB), on the vitality of L929 mouse fibroblasts was tested using MTT assay. Cell cycle progression was studied using flow cytometry. Production of intracellular reactive oxygen species (ROS) after DMAE-CB treatment was measured using 2,7-dichlorodihydrofluorescein diacetate staining and flow cytometry analysis. Loss of mitochondrial membrane potential, disturbance of Bcl-2 and Bax expression, as well as release of cytochrome C were also measured using flow cytometry analysis or Western blot to explore the possible involvement of the mitochondrial-related apoptotic pathway. DMAE-CB elicited cell death in a dose-dependent manner and more than 50% of cells were killed after treatment with 30 µM of the monomer. Both necrosis and apoptosis were observed. DMAE-CB also induced G1- and G2-phase arrest. Increased levels of intracellular ROS were observed after 1 h and this overproduction was further enhanced by 6-h treatment with the monomer. DMAE-CB may cause apoptosis by disturbing the expression of Bcl-2 and Bax, reducing the mitochondrial potential and inducing release of cytochrome C. Taken together, these findings suggest that the toxicity of the antibacterial monomer DMAE-CB is associated with ROS production, mitochondrial dysfunction, cell cycle disturbance, and cell apoptosis/necrosis.

A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex)1.3(DOX)20. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K+ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K+ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E2- (PGE2, 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE2) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE2) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE2; 8 µg/paw) and the ATP-sensitive K+ channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE2; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

In vitro and in vivo studies of pirarubicin-loaded SWNT for the treatment of bladder cancer

Intravesical chemotherapy is an important part of the treatment for superficial bladder cancer. However, the response to it is limited and its side effects are extensive. Functional single-walled carbon nanotubes (SWNT) have shown promise for tumor-targeted accumulation and low toxicity. In the present study, we performed in vivo and in vitro investigations to determine whether SWNT-based drug delivery could induce high tumor depression in rat bladder cancer and could decrease the side effects of pirarubicin (tetrahydropyranyl-adriamycin, THP). We modified SWNT with phospholipid-branched polyethylene glycol and constructed an SWNT-THP conjugate via a cleavable ester bond. The cytotoxicity of SWNT-THP against the human bladder cancer cell line BIU-87 was evaluated in vitro. Rat bladder cancer in situ models constructed by N-methyl-N-nitrosourea intravesical installation (1 g/L, 2 mg/rat once every 2 weeks for 8 weeks) were used for in vivo evaluation of the cytotoxicity of SWNT and SWNT-THP. Specific side effects in the THP group including urinary frequency (N = 12), macroscopic hematuria (N = 1), and vomiting (N = 7) were identified; however, no side effects were observed with SWNT-THP treatment. Flow cytometry was used to assess the cytotoxicity in vitro and in vivo. Results showed that SWNT alone did not yield significant tumor depression compared to saline (1.74 ± 0.56 and 1.23 ± 0.42%) in vitro. SWNT-THP exhibited higher tumor depression than THP-saline in vitro (74.35 ± 2.56 and 51.24 ± 1.45%) and in vivo (52.46 ± 2.41 and 96.85 ± 0.85%). The present findings indicate that SWNT delivery of THP for the treatment of bladder cancer leads to minimal side effects without loss of therapeutic efficacy. Therefore, this nanotechnology may play a crucial role in the improvement of intravesical treatment of bladder cancer.