Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P-30 protein, a novel protein possessing anti-tumour activity

Abstract. P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.     

Are breast tumours resistant to tamoxifen also resistant to pure antioestrogens?

A substantial proportion of patients with breast cancer are treated with the antioestrogen tamoxifen. As with other endocrine therapies, clinical experience has shown that some tumours in which growth is initially attenuated by tamoxifen treatment become resistant to continued drug treatment and resume growth. The mechanisms underlying the development of tamoxifen resistance have yet to be described but represent an important focus of research with the aim of defining what other therapies might be effective following tamoxifen treatment. Secondly, an understanding of tamoxifen resistance might suggest means to develop more effective agents for primary treatment of the disease. The development of pure antioestrogens, for example ICI 164,384 and ICI 182,780, which differ pharmacologically from tamoxifen in being entirely free of oestrogen partial-agonist activity, together with cell and animal models of tamoxifen resistant human breast cancer, has revealed one mechanism which might be of considerable clinical significance. Pure antioestrogens were shown to inhibit the proliferation of a greater proportion of tumor cells than tamoxifen in vitro, a differential effect that was attributed to the oestrogenic activity of tamoxifen. Subsequently, cell culture studies have shown that breast cancer cell lines selected for resistance to tamoxifen can still remain sensitive to the growth inhibitory action of pure antioestrogens. Similarly, the growth of human breast tumours in nude mice, which is initially attenuated by tamoxifen but then resumes, can be inhibited by pure antioestrogens. Both types of experiment are consistent with the view that tamoxifen resistance in these model systems is due to the oestrogenic action of tamoxifen. Thus, it can be predicted that in some patients whose tumours recur during tamoxifen therapy, a further response to pure antioestrogen treatment might occur. Studies to examine this hypothesis are currently being undertaken with ICI 182,780. One mechanism which might account for the experimental observations is an intrinsic heterogeneity amongst breast tumour cells in their response to tamoxifen, i.e. that there are at least two different populations of cells; one population which responds to tamoxifen as an antioestrogen and one which “reads” tamoxifen as an oestrogen. The growth advantage thus conferred on the latter population would lead to its predominance. If this is what actually happens in a proportion of human tumours, it can be argued that primary treatment of the tumour with a pure antioestrogen, rather than tamoxifen, would be preferred since a more complete and longer-lasting response would be predicted. Recent comparative studies with human breast tumours grown in nude mice support these predictions.     

Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents

Cancer contributes to 50% of deaths worldwide and new anti-tumour therapeutics with novel mechanisms of actions are essential to develop. Metabolic inhibitors represent an important class of anti-tumour agents and for many years, agents targeting the nutrient folate were developed for the treatment of cancer. This is because of the critical need of this factor for DNA synthesis. Similarly to folate, Fe is an essential cellular nutrient that is critical for DNA synthesis. However, in contrast to folate, there has been limited effort applied to specifically design and develop Fe chelators for the treatment of cancer. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) group of ligands that demonstrate marked and selective anti-tumour activity in vitro and also in vivo against a wide spectrum of tumours. Indeed, administration of these compounds to mice did not induce whole body Fe-depletion or disturbances in haematological or biochemical indices due to the very low doses required. The mechanism of action of these ligands includes alterations in expression of molecules involved in cell cycle control and metastasis suppression, as well as the generation of redox-active Fe complexes. This review examines the alterations in Fe metabolism in tumour cells and the systematic development of novel aroylhydrazone and thiosemicarbazone Fe chelators for cancer treatment.     

Dihydroartemisinin induces apoptosis and sensitizes human ovarian cancer cells to carboplatin therapy

The present study was designed to determine the effects of artemisinin (ARS) and its derivatives on human ovarian cancer cells, to evaluate their potential as novel chemotherapeutic agents used alone or in combination with a conventional cancer chemotherapeutic agent, and to investigate their underlying mechanisms of action. Human ovarian cancer cells (A2780 and OVCAR-3), and immortalized non-tumourigenic human ovarian surface epithelial cells (IOSE144), were exposed to four ARS compounds for cytotoxicity testing. The in vitro and in vivo antitumour effects and possible underlying mechanisms of action of dihydroartemisinin (DHA), the most effective compound, were further determined in ovarian cancer cells. ARS compounds exerted potent cytotoxicity to human ovarian carcinoma cells, with minimal effects on non-tumourigenic ovarian surface epithelial (OSE) cells. DHA inhibited ovarian cancer cell growth when administered alone or in combination with carboplatin, presumably through the death receptor- and, mitochondrion-mediated caspase-dependent apoptotic pathway. These effects were also observed in in vivo ovarian A2780 and OVCAR-3 xenograft tumour models. In conclusion, ARS derivatives, particularly DHA, exhibit significant anticancer activity against ovarian cancer cells in vitro and in vivo , with minimal toxicity to non-tumourigenic human OSE cells, indicating that they may be promising therapeutic agents for ovarian cancer, either used alone or in combination with conventional chemotherapy.     

Liposomes targeting tumour stromal cells

Liposomes have found clinical application in cancer therapy in the delivery of cytostatic agents. As a result of the targeted delivery of these toxic molecules to the tumour cells coupled to avoidance of toxicity-sensitive tissues, the therapeutic window is widened. Over the past years the focus of cancer therapy has shifted towards the stromal cells that are present in the tumour. It appears that clinically relevant tumours have acquired the ability to modulate the microenvironment in such a way that a chronic pro-inflammatory and pro-angiogenic state is achieved that contributes to invasion and metastasis and continued proliferation. Over the past years, liposomal formulations have been designed that target key stromal cell types that contribute to tumour growth. At the same time, many promising cell types have not been targeted yet and most of the studies employ drugs that aim at depleting stromal cells rather than modulating their activity towards an anti-tumour phenotype. In this review these target cell types will be addressed. Complementing these targeted formulations with the appropriate drugs to optimally suppress tumour-promoting signals while preserving anti-tumour action will be the challenge for the future.     

Design, synthesis and biological evaluation of novel quinazoline derivatives as potential anti-cancer agents

Twenty-two quinazoline derivatives have been synthesised and examined for their anti-tumour activity against three tumour cell lines, namely human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human hepatoma cell line (HepG2). Twelve of the tested compounds have shown promising anti-tumour activity with an IC(50) range of 5.0-9.7 μg/mL. Regarding the spectrum of activity, five compounds exhibited interesting anti-proliferative properties against the three tested cell lines comparable to the reference drug (dasatinib).     

Liposomes targeting tumour stromal cells

Abstract

Liposomes have found clinical application in cancer therapy in the delivery of cytostatic agents. As a result of the targeted delivery of these toxic molecules to the tumour cells coupled to avoidance of toxicity-sensitive tissues, the therapeutic window is widened. Over the past years the focus of cancer therapy has shifted towards the stromal cells that are present in the tumour. It appears that clinically relevant tumours have acquired the ability to modulate the microenvironment in such a way that a chronic pro-inflammatory and pro-angiogenic state is achieved that contributes to invasion and metastasis and continued proliferation. Over the past years, liposomal formulations have been designed that target key stromal cell types that contribute to tumour growth. At the same time, many promising cell types have not been targeted yet and most of the studies employ drugs that aim at depleting stromal cells rather than modulating their activity towards an anti-tumour phenotype. In this review these target cell types will be addressed. Complementing these targeted formulations with the appropriate drugs to optimally suppress tumour-promoting signals while preserving anti-tumour action will be the challenge for the future.     

Design,synthesis and biological evaluation of novel quinazoline derivatives as potential anti-cancer agents

Twenty-two quinazoline derivatives have been synthesised and examined for their anti-tumour activity against three tumour cell lines, namely human breast cancer cell line (MCF-7), human cervical cancer cell line (HeLa) and human hepatoma cell line (HepG2). Twelve of the tested compounds have shown promising anti-tumour activity with an IC₅₀ range of 5.0–9.7 µg/mL. Regarding the spectrum of activity, five compounds exhibited interesting anti-proliferative properties against the three tested cell lines comparable to the reference drug (dasatinib).     

Effect of methylglyoxal on tumour microtubular protein

Methylglyoxal inhibits cell division, exerting an antiproliferative action on tumour cells. Supernatants from ascites hepatoma cell homogenate, previously incubated with the aldehyde, showed a decrease in colchicine binding activity dependent on methylglyoxal concentration. In contrast, in vivo treatment of tumour-bearing rats apparently did not cause a significant impairment of microtubular protein, suggesting that the aldehyde interaction with microtubules cannot be considered responsible for its carcinostatic action.     

Ridaifen B,a tamoxifen derivative,directly binds to Grb10 interacting GYF protein 2

Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.     

穿膜素TAT介导的KDR-siRNA慢病毒载体的构建及其靶向肺癌A549细胞的抗肿瘤作用

为了构建TAT与KDR-siRNA慢病毒载体,观察其对肺癌细胞株 A549的体外靶向抗肿瘤作用,利用重组技术构建TAT-KDR siRNA慢病毒载体并转染人肺癌细胞株 A549。实时荧光定量PCR、Western blot检测KDR基因水平变化;流式细胞仪、MTT 法、集落形成试验检测其对A549细胞株细胞凋亡、细胞增殖和克隆形成的影响;细胞黏附实验评价其肿瘤靶向性。其抗癌作用主要表现为可有效地抑制A549细胞KDR基因表达、细胞增殖和克隆形成,促进细胞凋亡,并具有肿瘤靶向性作用。因而认为,TAT与KDR靶向siRNA慢病毒载体具有显著的肿瘤靶向性和抗肿瘤活性。     

Interaction of the breast cancer resistance protein with plant polyphenols

Multidrug transporters influence drug distribution in vivo and are often associated with tumour drug resistance. Here we show that plant-derived polyphenols that interact with P-glycoprotein can also modulate the activity of the recently discovered ABC transporter, breast cancer resistance protein (BCRP/ABCG2). In two separate BCRP-overexpressing cell lines, accumulation of the established BCRP substrates mitoxantrone and bodipy-FL-prazosin was significantly increased by the flavonoids silymarin, hesperetin, quercetin, and daidzein, and the stilbene resveratrol (each at 30 microM) as measured by flow cytometry, though there was no corresponding increase in the respective wild-type cell lines. These compounds also stimulated the vanadate-inhibitable ATPase activity in membranes prepared from bacteria (Lactococcus lactis) expressing BCRP. Given the high dietary intake of polyphenols, such interactions with BCRP, particularly in the intestines, may have important consequences in vivo for the distribution of these compounds as well as other BCRP substrates.     

Decreased monocyte-mediated cytostasis of human cancer cell in patients with lung cancer

Summary In vivo animal studies support the concept that monocytes and macrophages are important in the immune surveillance of oncogenesis and that in vitro activated murine macrophages are cytocidal for tumour cells. In this study, the tumour cell cytotoxic activity of human peripheral blood monocytes was examined by measuring the inhibition of ³H-thymidine uptake in the human cancer cell line, established in our laboratory from human squamous cell lung cancer. The monocytes from 8 of the 31 lung cancer patients (26%) showed a percentage growth inhibition of less than 69.8%, which exceeded the 95% confidence limits of the percentage growth inhibition observed with healthy control monocytes. On the other hand, among the 16 sarcoidosis and the 8 tuberculosis cases no value was below 69.8%. However, there was no significant difference between the growth inhibition and the clinical stages or histological type. When OK-432, a Streptococal agent, was administered in vivo to patients with lung cancer, an elevation of the growth inhibition was observed in 7 out of 8 patients. It was confirmed that the tumour cell cytostatic activity of the monocyte is suppressed in patients with lung cancer, and these monocyte deficits hinder the inhibition of tumour growth and metastasis.     

Nonsteroidal antiestrogen suppresses protein kinase C--its inhibitory effect on interaction of substrate protein with membrane.

The mechanism by which nonsteroidal antiestrogen inhibits Ca(2+)- and phospholipid-dependent protein kinase (PKC) activity was investigated. Antiestrogenic agents, clomiphene and tamoxifen, inhibited the PKC-dependent phosphorylation of histone and r-annexin I in a dose-dependent manner. Ki values for the agents were different for two substrate proteins. The inhibitory action of the agents depended on the membrane-substrate protein interaction. Phosphorylation of cytoplasmic proteins obtained from rat uterus and mammary gland, including annexin I, by endogenous PKC was also inhibited by low concentrations of these agents. These results suggest that the inhibitory action of nonsteroidal antiestrogens occurs through their inhibitory effect on the membrane-substrate protein interaction.     

AL3810, a multi-tyrosine kinase inhibitor, exhibits potent anti-angiogenic and anti-tumour activity via targeting VEGFR, FGFR and PDGFR

Angiogenesis plays an important role in neoplastic transformation and progression as well as in the metastasis process of most human cancers. Herein, we identified AL3810 as a novel and orally bioavailable small molecular inhibitor with potent inhibitory activity against multiple tyrosine kinases involved in the process of angiogenesis. We found that AL3810 substantially inhibited the autophosphorylation of VEGFR2, PDGFRβ and FGFR1 in endothelial cells. Moreover, AL3810 exhibited potent anti-angiogenesis activity, manifested by significant inhibition of microvessel outgrowth of rat arterial ring and chickallantochorion membrane (CAM) in ex vivo angiogenesis models. Daily dosing of AL3810 has shown broad-spectrum anti-tumour activity in human kidney, pancreas, liver cancer xenograft models. Importantly, immunohistochemistry results demonstrated that the anti-tumour activity of AL3810 was closely correlated with its anti-angiogenesis activity, as demonstrated by a decreased microvessel area and reduced microvessel numbers in tumour tissues. The overall pharmacological profiles of AL3810 are superior to sorafenib. The clinical trials of AL3810 will soon be launched in China.     

The cytotoxic activity of the bacteriophage lambda-holin protein reduces tumour growth rates in mammary cancer cell xenograft models

BACKGROUND: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy. METHODS: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model. RESULTS: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene. CONCLUSIONS: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.     

Monoclonal antibody Daratumumab promotes macrophage-mediated anti-myeloma phagocytic activity via engaging FC gamma receptor and activation of macrophages

Daratumumab (DAR) is novel human anti-CD38 IgG1, high-affinity human monoclonal antibody having broad-spectrum killing activity. The antibody is recommended to treat multiple myeloma. Recently Antibody-dependent cellular phagocytosis (ADCP) have been identified as the potential mechanism of DAR in addition to complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In the present study we evaluated the effect of Daratumumab on other effector cells of multiple myeloma. Luciferase⁺ MM.1R GFP cells were selected for the study. For immune-compromised multiple myeloma tumour xenograft mouse model we used severe combined immunodeficient beige (SCID-beige), NOD SCID gamma (NSG) and C57Bl/6j mice. Bioluminescence imaging was carried by injecting luciferin, and in vivo confocal microscopy was done for tracing bone marrow niches. Spleen and tumours were submitted to immunophenotypic analysis. MTT assay was done for cell proliferation studies. We established tumour xenograft mouse model. It was found that DAR showed significant anti-tumour effect in tumour xenograft multiple myeloma mice. We found that DAR showed anti-tumour activity via Fc–FcγR interaction with macrophages. DAR induced phenotypic activation of macrophages in mice and resulted in ADCP of cancerous cells via interacting Fc-FcγR in vitro. The study suggested that DAR exerted anti-tumour activity in multiple myeloma by interacting with Fc-FcγR.

     

Ecology-based screen identifies new metabolites from a Cordyceps-colonizing fungus as cancer cell proliferation inhibitors and apoptosis inducers

Objectives:  This study aims to identify new anti-cancer agents from Cordyceps -colonizing fungi, using an ecology-based approach. It also aims to explore their anti-cell proliferative mechanisms, and to evaluate their anti-tumour effects in vivo .
Materials and methods:  Extracts from Cordyceps -colonizing fungi were tested on HeLa cells, and active extracts were separated to obtain anti-tumour metabolites; their structures were elucidated by mass and nuclear magnetic resonance spectroscopy. Cell cycle analysis was evaluated using flow cytometry. Tumour formation assays were performed using C57BL/6J mice.
Results:  Based on ecological considerations, the selected extracts were subjected to initial anti-tumour screening. Bioassay-guided fractionation of the active extract afforded two new epipolythiodioxopiperazines, named gliocladicillins A ( 1 ) and B ( 2 ). (A) 1 and B ( 2 ) inhibited growth of HeLa, HepG2 and MCF-7 tumour cells. Further study demonstrated that both preparations arrested the cell cycle at G₂/M phase in a dose-dependent manner, and induced apoptosis through up-regulation of expression of p53, p21, and cyclin B, and activation of caspases-8, -9 and -3. These data imply that gliocladicillins A ( 1 ) and B ( 2 ) induce tumour cell apoptosis through both extrinsic and intrinsic pathways. In addition, in vivo studies showed that they displayed significant inhibitory effects on cell population growth of melanoma B16 cells imlanted into immunodeficient mice.
Conclusions:  Gliocladicillins A ( 1 ) and B ( 2 ) are effective anti-tumour agents in vitro and in vivo and should be further evaluated for their potential in clinical use.