Wai M. Liu

Pre-treatment with chemotherapy can enhance the antigenicity and immunogenicity of tumours by promoting adaptive immune responses

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.

Effect of haemopoietic growth factors on cancer cell lines and their role in chemosensitivity

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 antimalarial agent artesunate possesses anticancer properties that can be enhanced by combination strategies

Artemisinins are a class of compounds that are first‐line treatment options for malaria. They also have potent antiproliferative activity, which makes them potential anticancer drugs. We have previously demonstrated anticancer activity of a number of these compounds in vitro; however, cytotoxic activities were compromised by drug‐induced cell cycle arrests. In this study, we have explored further the activity of the clinical lead artemisinin‐drug artesunate (ART), used either alone or in combination with established chemotherapy. Also, by using a cell line expressing polyploidy character, have also explored the impact of cell cycle arrest in determining overall drug activity. Results showed that ART caused dose‐dependent decreases in cell number, which were associated with either increased cytotoxicity or cytostasis. Cytostasis appeared to be a consequence of a simultaneous arrest at all phases of the cell cycle, a deduction that was supported by molecular profiling, which showed reductions in cell cycle transit proteins. ART appeared to maintain cells in this arrested state; however, reculturing these treated cells in drug‐free medium resulted in significant reductions in viabilities. We also showed that ART maintained activity in polyploidy cells, and that an impressive enhancement to its activity was achievable through a combination with the immunomodulatory drug lenalidomide. Taken together, these observations indicate ART and its related compounds may be effective for the treatment of tumours, and that activity is related to schedule. Therefore, it is important to carefully select the most appropriate schedule to maximise ART efficacy.

The Combination of Cannabidiol and Δ9-Tetrahydrocannabinol Enhances the Anticancer Effects of Radiation in an Orthotopic Murine Glioma Model

High-grade glioma is one of the most aggressive cancers in adult humans and long-term survival rates are very low as standard treatments for glioma remain largely unsuccessful. Cannabinoids have been shown to specifically inhibit glioma growth as well as neutralize oncogenic processes such as angiogenesis. In an attempt to improve treatment outcome, we have investigated the effect of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) both alone and in combination with radiotherapy in a number of glioma cell lines (T98G, U87MG, and GL261). Cannabinoids were used in two forms, pure (P) and as a botanical drug substance (BDS). Results demonstrated a duration- and dose-dependent reduction in cell viability with each cannabinoid and suggested that THC-BDS was more efficacious than THC-P, whereas, conversely, CBD-P was more efficacious than CBD-BDS. Median effect analysis revealed all combinations to be hyperadditive [T98G 48-hour combination index (CI) at FU50, 0.77–1.09]. Similarly, pretreating cells with THC-P and CBD-P together for 4 hours before irradiation increased their radiosensitivity when compared with pretreating with either of the cannabinoids individually. The increase in radiosensitivity was associated with an increase in markers of autophagy and apoptosis. These in vitro results were recapitulated in an orthotopic murine model for glioma, which showed dramatic reductions in tumor volumes when both cannabinoids were used with irradiation (day 21: 5.5 ± 2.2 mm3 vs. 48.7 ± 24.9 mm3 in the control group; P < 0.01). Taken together, our data highlight the possibility that these cannabinoids can prime glioma cells to respond better to ionizing radiation, and suggest a potential clinical benefit for glioma patients by using these two treatment modalities. Mol Cancer Ther; 13(12); 2955–67. ©2014 AACR.

Inhibition of metastatic potential in colorectal carcinoma in vivo and in vitro using immunomodulatory drugs (IMiDs)

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.

Enhancing the in vitro cytotoxic activity of Δ9-tetrahydrocannabinol in leukemic cells through a combinatorial approach

Δ9-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 μM) sensitised cells to these cytotoxic agents, by reducing IC50 values by ∼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.

Cannabis-Derived Substances in Cancer Therapy – An Emerging Anti-Inflammatory Role for the Cannabinoids

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.

The gene expression profile of unstimulated dendritic cells can be used as a predictor of function.

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.

Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis

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.

MTT assays can underestimate cell numbers

Sir, The use of the yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) as an indicator of cell proliferation and viability in in vitro studies have increased. The simplicity of the methodology and the rapidity at which results are attained, make the MTT assay a useful operator-independent method of studying cell number [1]. The principle premise of the assay is the intrinsic ability of mitochondrial dehydrogenases to reduce MTT to a purple-coloured formazan, which is detected colorimetrically. This renders the assay a choice for many groups studying drug activity in high-throughput screening studies. Therefore, we have read with interest the report by Simms and Plattner [2] who describe interference of the MTT assay when using imatinib. SpeciWcally, the eVects of imatinib were studied in vitro using tritiated thymidine and by the MTT assay. Most worryingly, their data reported that although imatinib reduces cell numbers and increases apoptosis in a dose-dependent manner, the MTT assay showed a dose-dependent increase in cell number. The authors concluded that the MTT assay was a inappropriate method for testing the activity of the drug in solid tumours. Indeed, this is our experience, and the reason why we did not employ the assay in our studies with imatinib [3]. Another consideration that was not mentioned in the report was that the reduction of MTT may not be entirely dependent upon cell number, but also upon the mitochondrial function and proliferative state. To highlight this possible limitation, non-adherent cell lines CEM, HL60 and