► We selected one propolis extract with highest cytotoxic effects on cancer cells. ► We have investigated the chemical composition by using HPLC–ESI-MS. ► We have elucidated the cell cycle ...perturbations induced by propolis compound M. ► This is the first study that investigated propolis by using BrdU/DNA assay.
The study was designed to evaluate anti-tumour properties of Iraqi propolis collected from Mosul region (M) on HL-60 and HCT-116 cell lines and on HCT-116 in vivo. M induced an inhibitory effect against the proliferation of HL-60 and colony potential of HCT-116 cells. The apoptosis in HL-60 cells was associated with down-regulation of Bcl-2 and activation of Bax, while in HCT-116 cells, necrotic features were observed; size of cells was dramatically increased by swelling of cytoplasm and loss of membrane integrity, cell rupture and release of cellular contents. Analysis of BrdU/DNA cell cycle in both cell lines showed that M induced cell cycle perturbations in both BrdU positive and BrdU negative cells. The exposure of HL-60 to M caused γ-H2AX in a dose dependent manner and was associated with induction of apoptosis. The experiments in HCT-116 tumor-bearing mice showed that oral administration of propolis at doses that caused no detectable toxicity was associated with a decrease in mitotic cells and an increase in endoreduplications, increased p53 and decreased Ki-67 expression of cells in tumor sections. This study provides the rationale to investigate the potential beneficial effect of propolis in the diet of patients receiving anti-cancer therapies.
Innovative strategies that utilize nanoparticles (NPs) for a better delivery of drugs and to improve their therapeutic efficacy have been widely studied in many clinical fields, including oncology. ...To develop safe and reliable devices able to reach their therapeutic target, a hierarchical characterization of NP interactions with biological fluids, cells, and whole organisms is fundamental. Unfortunately, this aspect is often neglected and the development of standardized characterization methods would be of fundamental help to better elucidate the potentials of nanomaterials, even before the loading of the drugs. Here, we propose a multimodal in vitro/in vivo/ex vivo platform aimed at evaluating these interactions for the selection of the most promising NPs among a wide series of materials. To set the system, we used non-degradable fluorescent poly(methyl-methacrylate) NPs of different sizes (50, 100, and 200 nm) and surface charges (positive and negative). First we studied NP stability in biological fluids. Then, we evaluated NP interaction with two cell lines of triple-negative breast cancer (TNBC), 4T1, and MDA-MB231.1833, respectively. We found that NPs internalize in TNBC cells depending on their physico-chemical properties without toxic effects. Finally, we studied NP biodistribution in terms of tissue migration and progressive clearance in breast-cancer bearing mice. The use of highly stable poly(methyl-methacrylate) NPs enabled us to track them for a long time in cells and animals. The application of this platform to other nanomaterials could provide innovative suggestions for the development of a systematic method of characterization to select the most reliable nanodrug candidates for biomedical applications.
Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert ...direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t-test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent.
Background/Aims: Cholangiocarcinoma is a devastating tumour with a poor prognosis. An efficient therapy is unavailable in unoperable patients and new drugs are widely sought for and required. ...Resveratrol (RES) is a natural molecule with a reported anticancer effect, evaluated on different tumour cell lines. We tested the efficacy of RES on a cholangiocarcinoma cell line for the first time.
Methods: We used the human SK‐ChA‐1 cell line, cultured in the classical two‐dimensional model and in the three‐dimensional spheroids. After RES exposure morphology, cell viability (colony‐forming assay), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and cancer antigen (CA) 19‐9 medium releases, cellular transglutaminase activity, karyotype and cell cycle were evaluated.
Results: Resveratrol inhibited cell growth in both the cell culture systems used (from −15 to −80% vs untreated controls) and induced a 40‐fold increase of LDH and ALP activities in the culture medium. Also, transglutaminase (TG) activity increased in the cell lysates, together with a cell cycle perturbation characterised by an accumulation in the G1/S phase. Karyotype and CA 19‐9 expression were not influenced by the treatment.
Conclusions: The observed cytotoxic effect of RES on the human cholangiocarcinoma SK‐ChA‐1 cell line cultured two‐ and three‐dimensionally suggests to further analyse its chemotherapic/chemopreventive possibilities for this kind of cancer.
Abstract Resveratrol exerts a drastic growth inhibitory effect on human breast cancer MDA-MB-231 cells grown both in vitro and in vivo . Here we show that resveratrol affects the aggregation ...properties of MDA-MB-231 cells into multicellular tumor spheroids (MCTSs), in association with induction of de novo synthesis of ceramide. After 9 days of 3D growth in the presence of resveratrol (64 μM), MDA-MB-231 cells formed significantly smaller MCTSs. Further, cells from these aggregates failed to form colonies. Addition of resveratrol (64 μM) to preformed MDA-MB-231 MCTSs caused no significant size change, consistent with lack of ceramide induction. Only some apoptotic blebs were found on the MCTSs surface. Altogether these findings indicate that resveratrol might be effective for prevention of breast cancer cell growth.
Variolin B (VAR-B) is a natural product isolated from the sponge
Kirkpatrickia variolosa, found in Antarctica. VAR-B has been shown previously to possess potent pro-apoptotic activity. This study was ...undertaken to investigate the mechanism of action of chemically synthesised VAR-B and its analogue deoxy-variolin B (dVAR-B). In different human cancer cell lines both compounds inhibited colony formation, caused cell cycle perturbations and induced apoptosis at concentrations ranging from 0.1 to 2
μM. LoVo/Dx cells over-expressing Pgp were equally sensitive as the parental cell line to VAR-B and dVAR-B, indicating that variolins are not substrates of Pgp. Although variolins induced an increase in the levels of
p53 with an increase in
p21, their cytotoxicities did not appear to be dependent on
p53 status as their potency was comparable in cells with wild-type
p53, or in sub-lines with inactivated
p53. Both VAR-B and dVAR-B prevent the cells from entering S phase, blocking cells in G1 and cause an accumulation of cells in G2. The apoptosis induced by VAR-B and dVAR-B occurs very rapidly in some cell lines (
e.g., Jurkat leukaemia cells) and is already evident 4
h after the beginning of treatment. Although intercalation of dVAR-B in DNA has been demonstrated, neither VAR-B nor dVAR-B produce detectable breaks in DNA. These results are consistent with the
in vitro biochemical assays that also demonstrated that dVAR-B is not topoisomerase I or II poison. Instead, each of these variolins appears to inhibit cyclin-dependent kinases (CDKs) in the μM range. CDK1-cyclin B, CDK2-cyclin A and CDK2/cylin E complexes were inhibited in a range of concentrations lower than those required to inhibit the activity of CDK4/cyclin D or CDK7/cyclin H complexes. In conclusion, these variolins are a new class of CDK inhibitors that activate apoptosis in a
p53-independent fashion and thus they may be effective against tumours with
p53 mutations or deletions.
The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8
T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord ...microglia and motor axons of transgenic SOD1
mice.
To assess the role of MHCI in the disease, we examined transgenic SOD1
mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8
T cells.
The lack of MHCI and CD8
T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8
T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1
mice.
We provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the clinical responses of ALS mice. These findings offer a possible explanation for the failure of systemic immunomodulatory treatments and suggest new potential strategies to prevent the progression of ALS.
Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other ...chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (gamma-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity.
The ubiquitin−proteasome system plays a critical role in many diseases, making it an attractive biomarker and therapeutic target. However, the impact of results obtained in vitro using purified ...proteasome particles or whole cell extracts is limited by the lack of efficient methods to assess proteasome activity in living cells. We have engineered an internally quenched fluorogenic peptide with a proteasome-specific cleavage motif fused to TAT and linked to the fluorophores DABCYL and EDANS. This peptide penetrates cell membranes and is rapidly cleaved by the proteasomal chymotrypsin-like activity, generating a quantitative fluorescent reporter of in vivo proteasome activity as assessed by time-lapse or flow cytometry fluorescence analysis. This reporter is an innovative tool for monitoring proteasomal proteolytic activities in physiological and pathological conditions.
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