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INTRODUCTION: Naltrexone was designed to inhibit opioid receptors without activating them and hence used to block the stimulatory effects of morphine and heroin. It was noted that in certain patients being treated with naltrexone for an opioid addiction many reported significant secondary benefit when being weaned off naltrexone. This group of patients had chronic inflammatory and autoimmune conditions and reported improvements whilst using the lower dosages of naltrexone. There have also been recent anecdotal reports of cancer resolution following the use of low doses of naltrexone (LDN). However, the mechanism of action is unclear. AREAS COVERED: We review three mechanisms through which LDN can influence cancer progression; namely, (a) antagonism of receptors to which LDN binds, which include toll-like receptors 7-9 that lead to IL-6 suppression b) modulation of immune function in patients; and c) direct inhibition of signaling pathways involved in cancer cell control, including the priming of pro-apoptotic pathways. EXPERT OPINION: Considering the increase in the number of anecdotal reports of activity, there will likely be a bigger drive toward using LDN in the oncological setting. These reports support clinical trials of LDN in cancer, especially when given in combination with certain chemotherapy.

We previously reported that both cannabidiol (CBD) and low‑dose naltrexone (LDN) exhibit complex effects on G‑protein coupled receptors, which can impact the expression and function of other members of this superfamily. These receptors feed into and interact with central signalling cascades that determine the ease by which cells engage in apoptosis, and can be used as a way to prime cancer cells to other treatments. The present study was designed to investigate the effect of combining these two agents on cancer cell lines

Determining the expression of genes in response to different classes of chemotherapeutic drugs may allow for a better understanding as to which may be used effectively in combination. In the present study, the human colorectal cancer cell line HCT116 was cultured with equi-active concentrations of a series of anti-cancer agents. Gene expression profiles were then measured by whole-genome microarray. Although each drug induced a unique signature of gene expression in tumour cells, there were marked similarities between certain drugs, even in those from different classes. For example, the antimalarial agent artesunate and the platinum-containing alkylating agent, oxaliplatin, produced a very similar mRNA expression pattern in HCT116 cells with ~14,000 genes being affected by the two drugs in the same way. Furthermore, the overall correlation of gene responses between two agents could predict whether their use in combination would lead to a greater or lesser effect on cell number, determined experimentally, than predicted by single agent experiments. The results indicated that even when working through different mechanisms, combining drugs that initiate a similar transcriptional response may constitute the best option for determining drug-combination strategies for the treatment of cancer.

Phytocannabinoids possess anticancer activity when used alone, and a number have also been shown to combine favourably with each other in vitro in leukaemia cells to generate improved activity. We have investigated the effect of pairing cannabinoids and assessed their anticancer activity in cell line models. Those most effective were then used with the common anti-leukaemia drugs cytarabine and vincristine, and the effects of this combination therapy on cell death studied in vitro. Results show a number of cannabinoids could be paired together to generate an effect superior to that achieved if the components were used individually. For example, in HL60 cells, the IC50 values at 48 h for cannabidiol (CBD) and tetrahydrocannabinol (THC) when used alone were 8 and 13 µM, respectively; however, if used together, it was 4 µM. Median-effect analysis confirmed the benefit of using cannabinoids in pairs, with calculated combination indices being

It has been reported that lower doses of the opioid antagonist naltrexone are able to reduce tumour growth by interfering with cell signalling as well as by modifying the immune system. We have evaluated the gene expression profile of a cancer cell line after treatment with low-dose naltrexone (LDN), and assessed the effect that adapting treatment schedules with LDN may have on enhancing efficacy. LDN had a selective impact on genes involved with cell cycle regulation and immune modulation. Similarly, the pro-apoptotic genes BAD and BIK1 were increased only after LDN. Continuous treatment with LDN had little effect on growth in different cell lines; however, altering the treatment schedule to include a phase of culture in the absence of drug following an initial round of LDN treatment, resulted in enhanced cell killing. Furthermore, cells pre-treated with LDN were more sensitive to the cytotoxic effects of a number of common chemotherapy agents. For example, priming HCT116 with LDN before treatment with oxaliplatin significantly increased cell killing to 49±7.0 vs. 14±2.4% in cultures where priming was not used. Interestingly, priming with NTX before oxaliplatin resulted in just 32±1.8% cell killing. Our data support further the idea that LDN possesses anticancer activity, which can be improved by modifying the treatment schedule.

Cannabinoids, the active components of the cannabis plant, have some clinical merit both as an anti-emetic and appetite stimulant in cachexic patients. Recently, interest in developing cannabinoids as therapies has increased following reports that they possess anti-tumour properties. Research into cannabinoids as anti-cancer agents is in its infancy, and has mainly focussed on the pro-apoptotic effects of this class of agent. Impressive anti-cancer activities have been reported; actions that are mediated in large part by disruptions to ubiquitous signalling pathways such as ERK and PI3-K. However, recent developments have highlighted a putative role for cannabinoids as anti-inflammatory agents. Chronic inflammation has been associated with neoplasia for sometime, and as a consequence, reducing inflammation as a way of impacting cancer presents a new role for these compounds. This article reviews the ever-changing relationship between cannabinoids and cancer, and updates our understanding of this class of agent. Furthermore, the relationship between chronic inflammation and cancer, and how cannabinoids can impact this relationship will be described.

Background: Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. Methods: We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media. Results: Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function. Conclusion: There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

The increased understanding of the pathogenetic and oncogenic pathways underlying cancer has allowed the successful development of novel approaches to treating the disease. The use of drugs to correct specific genetic defects responsible for the biological behaviour of cancer cells has been successfully applied to the clinic. These have included agents that interfere with cell proliferation and cell cycle signalling, neo-vascularisation, and DNA integrity. As a number of these processes also determine cellular survival, these novel agents can work to lower the cytotoxic threshold for conventional drugs. Consequently, a combinatorial drug approach could potentially be a valuable strategy for improving therapy. Unfortunately, targeting and inactivating a single pathway can adversely promote redundant parallel signalling pathways, which then maintain the aberrant status quo. Indeed, the intrinsic heterogeneity of tumours can preclude the successful application of a single targeted therapy. However, combinatorial drug approaches can be employed to surmount this multi-faceted characteristic of cancer. This article will highlight a number of novel targeted therapies that have been developed, some of which are currently undergoing clinical evaluation, and will also appraise the potential beneficial interactions that they may have with conventional cytotoxic drugs.

Delta(9)-Tetrahydrocannabinol (THC) is the active metabolite of cannabis, which has demonstrable cytotoxic activity in vitro. In support of our previously published data, we have investigated the interactions between THC and anti-leukemia therapies and studied the role of the signalling pathways in mediating these effects. Results showed clear synergistic interactions between THC and the cytotoxic agents in leukemic cells. Additionally, exposure of cells to sub lethal levels of THC (1 microM) sensitised cells to these cytotoxic agents, by reducing IC(50) values by approximately 50%. Sensitisation appeared to be dependent upon the ability of THC to down regulate phosphorylated ERK, as cells dominantly expressive of MEK were not sensitised to the cytotoxic drugs by equi-molar amounts of THC. Overall, these results demonstrate for the first time that a combination approach with THC and established cytotoxic agents may enhance cell death in vitro. Additionally the MAPK/ERK pathway appears responsible in part for these effects.

We sought to establish if methylene homologues of artemisone are biologically more active and more stable than artemisone. The analogy is drawn with the conversion of natural O- and N-glycosides into more stable C-glycosides that may possess enhanced biological activities and stabilities. Dihydroartemisinin was converted into 10β-cyano-10-deoxyartemisinin that was hydrolyzed to the α-primary amide. Reduction of the β-cyanide and the α-amide provided the respective methylamine epimers that upon treatment with divinyl sulfone gave the β- and α-methylene homologues, respectively, of artemisone. Surprisingly, the compounds were less active in vitro than artemisone against P. falciparum and displayed no appreciable activity against A549, HCT116, and MCF7 tumor cell lines. This loss in activity may be rationalized in terms of one model for the mechanism of action of artemisinins, namely the cofactor model, wherein the presence of a leaving group at C10 assists in driving hydride transfer from reduced flavin cofactors to the peroxide during perturbation of intracellular redox homeostasis by artemisinins. It is noted that the carba analogue of artemether is less active in vitro than the O-glycoside parent toward P. falciparum, although extrapolation of such activity differences to other artemisinins at this stage is not possible. However, literature data coupled with the leaving group rationale suggest that artemisinins bearing an amino group attached directly to C10 are optimal compounds. A brief critique is made of proteomic studies purporting to demonstrate the alkylation of intraparasitic proteins by alkyne- and azide-tagged artemisinins and synthetic peroxides in relation to mechanism of action.

The process of nucleocytoplasmic exchange of various macromolecules and metabolites between the nuclear and cytoplasmic compartment is crucial for cell function and survival. It is also closely involved with several pathogeneses including cancer and viral infections. Here, we will discuss the current understanding of the classical nuclear import pathway and the factors that are essential for this process.

The cAMP response element consensus sequence directs the transcription of a wide range of genes. A 24-mer single-stranded cAMP response element decoy oligonucleotide (CDO) has been shown to compete with these sequences for binding transcription factors and therefore interferes with cAMP-induced gene transcription. We have examined the effect of this CDO alone and in combination with a range of common chemotherapeutic agents in colorectal cancer cell lines. CDO had a potent anti-proliferative effect in colorectal cell lines, yet, a similar enhancement of cell death was not observed. Simple drug–drug interaction studies showed that combining CDO with chemotherapy resulted in an enhancement of the antiproliferative effects. Furthermore, this cytostatic effect was protracted and associated with an increase in senescence-associated β-galactosidase activity at pH 6. There is a possible role for p21waf1 in mediating this effect, as the enhancement of cell growth inhibition was not observed in cells lacking the ability to correctly upregulate this protein. Additionally, significant decreases in cyclin-dependent kinase (CDK) 1 and CDK 4 function were seen in the responsive cells. These data provide a possible model of drug interaction in colorectal cell lines, which involves the complex interplay of the molecules regulating the cell cycle. Clinically, the cytostatic ability of CDO could improve and enhance the antiproliferative effects of conventional cytotoxic agents.

s-Thalidomide has proven efficacy in multiple myeloma. Although it has both antiangiogenic and pro-apoptotic effects, its primary mode of therapeutic action remains unclear. We have investigated the changes to the expression of genes involved with these cellular processes following culture with s-thalidomide in the U266 MM cell line. Cells were cultured with s-thalidomide (0-1000 microM), and cell parameters, including apoptosis, were assessed on day 3. RNA was extracted from cells cultured for 24 h at the IC(50) concentration of s-thalidomide, and changes to gene expression were investigated by microarray methodologies. A reduction in cell viability was observed in U266 cells cultured with s-thalidomide (IC(50): 362 microM), which were mirrored by significant increases in apoptosis (for example, 200 microM on day 3: 40.3+/-3.1% vs. 3.2+/-0.4% on day 0; P

Etoposide is a commonly used anticancer agent that is highly schedule-dependent. The in vitro activity of etoposide (0-10 microM) was investigated in a panel of leukaemic cell lines.Cells were cultured with etoposide in drug schedules of equal exposure duration (ED, durationxconcentration), and the effects of drug exposure on cell parameters, including cell cycle distribution, were assessed over an 8-day period.Proliferation assays indicated a concentration- and duration-dependent induction of cell death by etoposide in CEM and HL60 cells, and flow cytometric analysis indicated that this cell kill was by apoptosis. Efficacy was also dependent upon schedule, with more cell kill seen in schedules of longer duration. As an example, accumulative percent cell kill resulting from a continuous exposure to 0.05 microM etoposide was significantly greater than that in cultures involving either a 4-day exposure to 0.1 microM or a single-day exposure to 0.4 microM etoposide (193.4+/-15.9% vs 125.2+/-5.4% vs 42.3+/-5.9%, respectively; P0.001 in all cases; equi-ED 0.4 microM.days). Efficacy was also dependent upon the ED of the schedule. At very low concentrations, the initial enhancement of cytotoxicity mediated by increasing duration would gradually and paradoxically be lost in the more protracted schedules (e.g. accumulative percent cell kill 66.4+/-7.4%, 158.3+/-12.0% and 40.1+/-6.0% with 100 n M for 2 days, 33 n M for 6 days and 25 n M for 8 days, respectively; P0.001 in all cases; equi-ED 0.2 microM x days).Our results confirm the schedule-dependency of etoposide in vitro, highlighting the importance of total duration of drug exposure in determining cytotoxicity, and emphasizing the requirement to achieve a cytotoxic concentration in longer exposures. It is therefore crucial to ensure that etoposide regimens used clinically involve doses that are effectively cytotoxic.

We have combined in vitro clonogenic culture and a highly sensitive stain for haemoglobin to compare the influence of EPO, IL-3, SCF, TGFbeta1, MIP-1alpha and IFNgamma, to directly stimulate cells in the progenitor compartment to develop towards the erythroid lineage. Three cell lines were chosen, as they exist developmentally arrested in the progenitor compartment, yet in a pliant state of maturation. HEL (erythroleukaemia) and K562 (CML-derived) cell lines, may, under appropriate stimuli, develop erythroid characters, whilst the third, U937 (as control cell line), may be stimulated by DMSO to differentiate to myeloid cells. After in vitro semi-solid methylcellulose culture with these cytokines, resulting colonies were stained with 2,7-diaminofluorene (DAF), which sensitively stains haemoglobin blue. Haemoglobin production was low in HEL and K562 cells and absent in U937. Cytokine analysis showed varying levels of influence depending on the starting level of cell line maturation. EPO and TGFbeta1 maximally stimulated haemoglobin production in the HEL and K562 cell lines. This differential cytokine stimulation analysis combined with sensitive DAF haemoglobin detection could be applied in the study of many erythropoiesis-deficient patients or primitive erythropoiesis.

K562 leukaemic cells are known to be less sensitive to etoposide than other cell lines, despite having similar topo II mRNA levels and cleavable complex formation. We have investigated the effect of etoposide schedule on cell cycle distribution, apoptosis and p21(waf1) and cdk1(p34) status in two bcr-abl-positive chronic myeloid leukaemia (CML) cell lines (K562 and KU812) and two small cell lung cancer (SCLC) cell lines (H69 and GLC4). During a continuous 5-day exposure, the SCLC cell lines showed a time and concentration-dependent loss of cell viability, with an initial block in the G2/M phase of the cell cycle followed by apoptosis. In contrast, the two CML cell lines showed no significant apoptosis or loss of viability after a similar block in G2/M. However, when K562 or KU812 cells were placed in drug-free medium following a 3-day drug exposure there was marked, concentration-dependent apoptosis (% apoptosis after release at 1 microM etoposide in K562, 10% at 24 h, 30% at 48 h). Our data also show that p21(waf1) does not increase after etoposide treatment in either H69 or GLC4 (both with mutated-p53). Although K562 and KU812 cells are null-p53, the arrest in G2/M during drug exposure was associated with increased p21(waf1) and a decrease in cdk1 (both P

Prior to work on the influence of dosing and scheduling of the drug etoposide in bone marrow cells, the DNA-damaging effects of three haemopoietic growth factors, either alone or in combination with etoposide, were investigated. Sister chromatid exchange (SCE) frequencies in phytohaemagglutinin-stimulated mononuclear cells of six normal volunteers were used as an indicator of DNA damage. The effects of three growth factors on SCEs were investigated at concentrations ranging between 0 and 100 ng/ml and those of etoposide alone, at concentrations varying between 0 and 2 microM. The effect of combinations of growth factor (GF) and etoposide were assessed at a 40-ng/ml concentration of each cytokine and at 0.4 microM etoposide. Results showed not only a dose-dependent rise in SCE frequency in cells treated with etoposide but also a cytokine effect. Stem-cell factor did not cause a significant change in SCE numbers. However, cytokines with activity at the progenitor cell level induced small but significant increases in SCE numbers at concentrations of 50 and 100 ng/ml (P0.001). Results of combination studies indicated a significant 60% increase in SCE numbers in cells treated with GF and etoposide as compared with etoposide alone (P0.00001). This finding suggests a sensitivity of peripheral blood mononuclear cells to SCE induction by GFs given either as single agents or in combination with etoposide.

Dendritic cells (DCs) possess the capacity to elicit immune responses against harmful antigens and have been used in DC-vaccines to stimulate the immune system to engage cancer cells. However, a lack of an appreciation of the quality of the DC that is used and/or the monocyte from which it is derived has limited their successful incorporation into treatment strategies.In the current study, we explored the relationship between cytokine receptor expression on the monocytes and its subsequent development into DCs. The significance of p21 expression in DCs during differentiation was also studied, as was the effect that manipulating this with chemotherapy may have on DC quality.DCs separated into two groups based on their ability to respond to a maturation stimulus. This quality correlated with a particular receptor profile of granulocyte-macrophage colony-stimulating factor and interleukin 4 expressed on the monocytes from which they were derived. DC quality was also associated with p21 expression, and artificially increasing their levels in DCs by using some chemotherapy improved function.Overall, these studies have highlighted a role for common chemotherapy in activating p21 in DCs, which is a prerequisite for good DC function.

In addition to their cytotoxic/cytostatic properties, some chemotherapeutics have shown a remarkable capacity to modulate immune response. One such chemotherapy is the antimetabolite, gemcitabine (GEM). GEM has been shown to influence the quantity and activation of certain immune cell subsets when used in vivo. It has also been demonstrated that administration of GEM in conjunction with immunotherapy, such as dendritic cell vaccination, is beneficial in terms of overall survival and progression-free survival. The purpose of this study is to investigate if GEM can influence the way that tumor cells surviving treatment are regarded by the immune system. Our group has found that short-term in vitro culture with GEM enhances expression of human leukocyte antigen (HLA) class I, CD95, NKG2D ligands and TRAIL receptors on tumor cell lines. These molecules are all cell surface proteins important for the efficient surveillance and effector function of the immune system. The HLA class I increase is beta-2-microglobulin dependent and changes are underpinned by ERK, JNK and RelB-NFkB signalling pathways. Furthermore, culture with GEM induces immunoproteasomal components LMP2 and MECL-1 in tumor cells and, subsequently, the peptide antigen repertoire displayed on HLA class I is altered. The changes in peptide characteristics include amino acid anchor residue modifications which render the peptides likely to favor binding alternative HLA-allotypes. The renovation of the peptidome displayed by HLA class I may reveal sub-dominant immunogens to T-cells. Taken together, these changes may render tumor cells more sensitive to immune cell-mediated lysis. These data help explain observations made in previous in vivo studies, advise as to which novel antigens could be utilised in future vaccination protocols and lend further credence to the idea that chemotherapy and immunotherapy could be used in combination. Citation Format: Andrew M. Gravett, John P. Copier, Wai M. Liu, Martin P. Cranage, Angus G. Dalgleish. Non-cytotoxic effects of the chemotherapeutic agent gemcitabine can alter the immunophenotype of tumor cells in in vitro culture. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B38.

Many immunotherapeutic agents in phase II cancer studies have given optimistic results, which were not confirmed in larger randomized studies. Here we explore the evidence that, contrary to previous opinion, many chemotherapeutic agents and other classes of drugs may enhance the response to therapeutic vaccines by reducing inflammation and/or by inhibiting regulatory T lymphocytes or myeloid-derived suppressor cells. In addition, some of these agents, such as the immunomodulatory drugs, may produce marked costimulatory activities as in the case of lenalidomide, which also has marked anti-inflammatory properties. With the first approval for a vaccine-based therapy for prostate cancer, we propose that many more vaccines will be able to achieve approval, especially when combined with the optimal chemotherapy and/or immunomodulatory drug schedule.

Background: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. Methods: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. Results: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. Conclusion: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

Background: Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis. Methods: In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines. Results: BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th1-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th1-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement. Conclusion: These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

Dendritic cells (DCs) represent a subset of professional antigen presenting cell (APC) whose role is to elicit immune responses against harmful antigens. They have been used in DC vaccines to stimulate the immune system to kill cancer cells. However, successes in clinical trials have been limited, which may be attributed to a lack of appreciation of the quality of DCs used. In the present study, whole human genome microarrays were used to examine alterations in gene expression of monocyte-derived DCs after stimulation with supernatants derived from tumours. Our primary aim was to investigate the possibility of a gene signature for DCs that could be used to forecast responsiveness to tumour stimuli. Results showed that DCs are divided into two groups based on their ability to increase costimulatory markers and to trigger T-cell responses. The gene profiles of the immature DCs from these two groups were distinct, with particular divergence in genes from the interleukin (IL) 8 and thrombospondin-1 hubs. A subpanel of genes was identified, whose signature of expression was capable of predicting DC-stimulatory capacity. Overall, these studies have highlighted a gene-based screen that predicts DC function, which could be used to guide DC-vaccine trials.

Artemisinins are now established drugs for treatment of malaria. These agents have been shown to possess impressive anti-cancer properties. We have investigated the role of artemisone (ATM), a novel derivative of artemisinin (ART) in a cancer setting both alone and in combination with established chemotherapeutic agents. The anti-proliferative effects of ART and ATM were tested on a panel of human cancer cells in vitro using the methylthiazoletetrazolium assay, and the effect on cell cycling established by flow cytometry. Immunoblot analyses were performed to determine effects at the molecular level. Finally, ART and ATM were combined with the common anti-cancer agents oxaliplatin, gemcitabine and thalidomide. ART and ATM caused dose dependent decreases in cell number. ATM was consistently superior to ART, with IC50 s significantly lower in the former. Neither drug caused significant changes to the cell viability (%viable cells >95%), but arrested cell cycling. Blockade was either exclusively at the level of G1, or at all phases of the cell cycle, and associated with reductions in cyclin D1, CDK4 and pRb. Combination studies showed the anti-proliferative effect of ATM was often enhanced by addition of the other drugs, whilst ART exhibited antagonistic properties. ART and ATM are active in cancer cell lines, with ATM displaying the greater anti-proliferative effect when used alone. ATM also enhances the effects of the above drugs, with ART being less likely to improve activities. Taken together, ATM should be thought of as the ART-derived compound next in line for further study.

Background: Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma and, along with pomalidomide, are being investigated in various other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about their primary mode of therapeutic action in patients with cancer. Methods: As part of a continuing research effort, we have investigated the effects of these agents on the metastatic capacity of murine colorectal cancer cell lines both in vivo and in vitro. Allied to these, we have studied their effects on the molecular pathways associated with metastasis. Results: Results indicate that thalidomide, lenalidomide and pomalidomide significantly inhibit the metastatic capability of colorectal carcinoma cells. Anchorage-independent growth, used as a coarse indicator of transformation, was significantly reduced, as were migratory capacity and invasive competence. In addition, an in vivo experimental metastasis model also showed that treatment with the drugs resulted in a significantly lower number of metastatic pulmonary nodules relative to control mice. Allied to these cellular and phenotypic changes were alterations in molecular markers of metastasis and in intracellular signalling competency. Conclusions: These results provide evidence that in addition to their immunomodulatory effects, thalidomide, lenalidomide and pomalidomide can impair the metastatic capacity of tumours, and that this mechanism may involve alterations to cell signalling functionality.

The proportion of people surviving pancreatic cancer is extremely low, with just 10% of patients diagnosed with any stage of the disease living beyond 1 year and a 5-year relative survival rate of

Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC50) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)0.5 = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu0.5 = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu50 = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC50 concentrations. A non–growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT. [Mol Cancer Ther 2006;5(5):1197–208]

Δ9-Tetrahydrocannabinol (THC) is the active metabolite of cannabis. THC causes cell death in vitro through the activation of complex signal transduction pathways. However, the role that the cannabinoid 1 and 2 receptors (CB1-R and CB2-R) play in this process is less clear. We therefore investigated the role of the CB-Rs in mediating apoptosis in 3 leukemic cell lines and performed microarray and immunoblot analyses to establish further the mechanism of cell death. We developed a novel flow cytometric technique of measuring the expression of functional receptors and used combinations of selective CB1-R and CB2-R antagonists and agonists to determine their individual roles in this process. We have shown that THC is a potent inducer of apoptosis, even at 1 × IC50 (inhibitory concentration 50%) concentrations and as early as 6 hours after exposure to the drug. These effects were seen in leukemic cell lines (CEM, HEL-92, and HL60) as well as in peripheral blood mononuclear cells. Additionally, THC did not appear to act synergistically with cytotoxic agents such as cisplatin. One of the most intriguing findings was that THC-induced cell death was preceded by significant changes in the expression of genes involved in the mitogen-activated protein kinase (MAPK) signal transduction pathways. Both apoptosis and gene expression changes were altered independent of p53 and the CB-Rs.

The cAMP response element consensus sequence directs the transcription of a wide range of genes. A 24-mer single-stranded cAMP response element decoy oligonucleotide (CDO) has been shown to compete with these sequences for binding transcription factors and therefore interferes with cAMP-induced gene transcription. We have examined the effect of this CDO alone and in combination with a range of common chemotherapeutic agents in colorectal cancer cell lines. CDO had a potent anti-proliferative effect in colorectal cell lines, yet, a similar enhancement of cell death was not observed. Simple drug-drug interaction studies showed that combining CDO with chemotherapy resulted in an enhancement of the antiproliferative effects. Furthermore, this cytostatic effect was protracted and associated with an increase in senescence-associated beta-galactosidase activity at pH 6. There is a possible role for p21(waf1) in mediating this effect, as the enhancement of cell growth inhibition was not observed in cells lacking the ability to correctly upregulate this protein. Additionally, significant decreases in cyclin-dependent kinase (CDK) 1 and CDK 4 function were seen in the responsive cells. These data provide a possible model of drug interaction in colorectal cell lines, which involves the complex interplay of the molecules regulating the cell cycle. Clinically, the cytostatic ability of CDO could improve and enhance the antiproliferative effects of conventional cytotoxic agents.

The recombinant growth factors (GFs) erythropoietin (Epo) and granulocyte–macrophage colony stimulating factor (GM-CSF) have important roles in the management of cancer patients. However, the effects of these GFs at a cellular level are not well understood. We examined the effect of GFs alone, and in combination with cytotoxic chemotherapy, in a panel of seven cell lines. Flow cytometric analysis showed varying levels of receptor expression, which correlated with phosphorylated MAPK expression. Additionally, there were also concomitant increases in BCL-2 protein levels in those cells with high levels of MAPK activation. Although culturing cells with Epo or GM-CSF did not alter cell viability by themselves, GF pretreatment in cell lines expressing higher receptor levels resulted in a reduced magnitude of cell kill following exposure to cytotoxic IC50 concentrations of cisplatin. Subsequent co-culture with either the MEK inhibitor U0126 or the GM-CSF antagonist E21R negated this induced resistance to cytotoxic chemotherapy, confirming the importance of the GF receptor as well as MAPK in mediating these effects. These results suggest that the use of GFs during chemotherapy may be detrimental in those cancers expressing higher levels of the specific receptor. Conversely, our results also suggest that GFs are safe to use in chemotherapeutic regimens if the cancer cells do not overexpress the particular receptor.

The use of topoisomerase inhibitors has been associated with the development of secondary malignancies, suggesting that these agents can induce DNA damage that may be persistent. We have investigated the effect of short exposures (3 days) to low etoposide concentrations (LC-etoposide, 0.01-0.04 microM) on the ability of leukaemic cells to initiate apoptosis. Results showed that although LC-etoposide had no effect on cell growth characteristics, the pre-culture of cells with LC-etoposide conferred resistance to subsequent exposure to cytotoxic concentrations of etoposide (0.3 microM etoposide in HL60 on day 3: %V: 95.2 +/- 1.6% vs 60.3 +/- 12.1% in control cells with no pre-culture, and %A: 5.1 +/- 0.2 vs 19.0 +/- 0.7%; P0.001). This effect was still observed 4 weeks after the initial drug exposure. Associated with these observations was a three-fold increase in genetic instability and a reduction in induced bax protein levels. The anti-cytotoxic effect was also shown to be specific to topoisomerase II (topo II) inhibitors, as the pre-culture of cells with a low doxorubicin concentration also induced resistance, while low cisplatin concentrations did not. The persistence of these alterations in cellular processes following an initial exposure to topo II inhibitors suggests a DNA-based mechanism, and highlights the existence of drug/target interactions even at very low drug concentrations.

Chronic myeloid leukaemia is typically characterised by the presence of dysregulated BCR–ABL tyrosine kinase activity, which is central to the oncogenic feature of being resistant to a wide range of cytotoxic agents. We have investigated whether the inhibition of this tyrosine kinase by the novel compound STI571 (formerly CGP57148B) would render K562, KU812 cell lines and chronic myeloid leukaemia-progenitor cells sensitive to induction of cell kill. Proliferation assays showed STI571 to be an effective cytotoxic agent in chronic myeloid leukaemia-derived cell lines (IC50 on day 5 of 4.6 μg ml−1 and 3.4 μg ml−1 for K562 and KU812 respectively) and in leukaemic blast cells (per cent viability on day 3 at 4 μg ml−1: 55.5±8.7 vs 96.4±3.7%). STI571 also appeared to specifically target bcr–abl expressing cells, as results from colony forming assays using the surviving cell fraction from STI571-treated peripheral CD34+ chronic myeloid leukaemia blast cells, indicated a reduction in the expansion of colonies of myeloid lineage, but no effect on normal colony formation. Our data also showed synergy between STI571 and other anti-leukaemic agents; as an example, there were significant increases in per cent cell kill in cell lines cultured with both STI571 and etoposide compared to the two alone (per cent cell kill on day 3: 73.7±11.3 vs 44.5±8.7 and 17.8±7.0% in cultures with STI571 and etoposide alone respectively; P

Meeting abstracts Chemotherapy and Immunotherapy were always considered incompatible in the treatment of cancer. However, analysis of sequential use has revealed that there may be significant additive potential if not synergistic, in enhancing the immune response to cancer. There are many reasons

Progenitor cell failure in the erythroid lineage is a particular problem in bone marrow failure. To provide insight into early erythopoietic development we used sensitive techniques to examine the effects of SCF, IL-3 and MIP-1 alpha on two developmentally arrested progenitor cell lines, HEL and K562. Quantitative flowcytometric analysis showed that both expressed receptors (SCF > MIP-1 alpha > IL-3). Qualitative analysis revealed HEL cells expressed more receptors than K562 cells. Clonogenic assays with sensitive haemoglobin detection showed that SCF and IL-3 did not support HEL development and reduced haemoglobin production. MIP-1 alpha reduced partially developed HEL colonies and haemoglobin in developed colonies. SCF increased development, but not haemoglobin in K562 cells, with IL-3 being more effective in both. MIP-1 alpha increased the proportion of well-developed K562 colonies but not haemoglobin. This suggests SCF, IL-3 and MIP-1 alpha all have a role to play in early erythroid cellular development, with differing actions depending on the stage of development.

Stem cell factor (SCF) is the ligand for the dimeric c-kit tyrosine kinase receptor. Binding of SCF to c-kit is a crucial element in the developmental stimulus of late stem cells and early progenitor cells. In the erythroid lineage the SCF stimulus is important not only for proliferation and differentiation, but is also known to enhance later haemoglobin production. In an earlier report we described a rapid non-radioactive technique using the extended ester-attached labelled SCF protein itself for detecting c-kit expression in marrow and peripheral blood mononuclear populations. In the present study we have taken this a step further to analyse c-kit expression in developing erythroid cells in vitro, principally using normal donor samples. This was designed for use as a foundation for the comparison of haematological disorders. In this case we tested 4 patients with the congenital disorder of erythropoiesis, Diamond-Blackfan anaemia (DBA), finding that in all cases DBA c-kit expression was elevated over normal, in 1 case as high as 348% of the normal average. This may be indicative of the reduced state of progenitor development in these patients. These results show that the described technique is beneficial for analysis in the stem and progenitor compartment.

Cancer patients are typically immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. We have assessed the impact of chemotherapy agents (cyclophosphamide, oxaliplatin and gemcitabine (GEM)) on the potential immunogenicity of tumour cells (A549, Caki2, HCT116, MCF7 and PC3), and investigated the interactions between supernatants derived from these stressed cells and cells of the immune system, by studying the activities between dendritic cells and T-cells. Results showed that some chemotherapy agents can increase total MHC class I expression on tumour cells, even when expression is low. For example, expression on MCF7 cells was increased by ∼4-fold upon treatment with GEM (133 ± 31 vs. 33 ± 7.8 in untreated cells; p This work was supported by the Cancer Vaccine Institute. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5612.

e14635 Background: Lenalidomide is an immunomodulatory and anti-angiogenic agent that has demonstrated activity against a range of hematological malignancies. Despite evidence of direct anti-proliferative activity against hematological cells in vitro, there is no evidence of single agent direct activity against solid tumor cells in vitro. To take advantage of its known immune-enhancing properties alongside direct anti-tumor agents, lenalidomide is being advanced in solid tumor indications in combination with other agents. There are few data regarding the combination of lenalidomide and standard of care chemotherapeutic agents, such as gemcitabine. Methods: Here, we assess the effects of lenalidomide alone, and in combination with gemcitabine, on pancreatic cancer cell growth and survival, and the ability of lenalidomide to enhance the ability of human PBMC to kill allogeneic pancreatic tumor cells (BxPC3, PANC-1 and MiaPaCa) in a PBMC:tumor cell co-culture model. Results: Lenalidomide alone had no effect on the proliferation of pancreatic cancer cells (BxPC-3 and Panc-1) whereas gemcitabine had moderate anti-proliferative activity. With combination therapy there was clear synergistic enhancement of anti-proliferative activity in both cell lines and additive effects were observed in a BxPC-3 xenograft mouse model of pancreatic cancer. About 20% of tumor cells were sensitive to immune-mediated cell death and, for BxPC3, this was increased significantly in the presence of lenalidomide. Lenalidomide significantly and dose-dependently enhanced immune-mediated killing (both T and NK cells are required for tumor cell killing in this model). For PANC-1 and MiaPaCa, immune-mediated killing was also increased by lenalidomide, albeit non-significantly. Conclusions: These results suggest that, in addition to anti-angiogenic and other effects within the tumor microenvironment, lenalidomide may act as an immune adjuvant to enhance the recognition and apoptosis of tumor cells by host T and NK cells. These studies support the potential utility of lenalidomide in combination with chemotherapeutic agents, gemcitabine in particular, in the treatment of patients with solid tumors including pancreatic cancer. [Table: see text]

CSF) after allogeneic bone marrow (BM) transplantation. In this retrospective analysis, patients received bone marrow or peripheral blood stem cells (PBSC), and were administered phrophylatic G-CSF post-transplantation. An increase in GVHD and treatment-related mortality (TRM) was observed only in patients receiving G-CSF after BM transplantation. Similarly, overall and leukemia-free survival rates were significantlyreduced(relativetocontrolrisks:0.59,P.0001;0.64, P.0003, respectively). The results of this study differ from the recently published meta-analyses examining the effect of growth factors on GVHD after allogeneic transplantation. 2 In addition, as discussed in the accompanying editorial, there were significant differences between the two arms in terms of patient characteristics, GVHD prophylaxis, and disease stage. However, although the authors emphasized the possibility of center effect or bias and discussed their rationale in disregarding interpatient or interstudy variability, no clear hypotheses to explain the negative effects of G-CSF in the GVHD-sensitive cohort were mooted. There are fundamental differences in the leukocyte