Sarah L. Perry

Dichloroacetate induces apoptosis and cell-cycle arrest in colorectal cancer cells

Background:Cancer cells are highly dependent on glycolysis. Our aim was to determine if switching metabolism from glycolysis towards mitochondrial respiration would reduce growth preferentially in colorectal cancer cells over normal cells, and to examine the underlying mechanisms.Methods:Representative colorectal cancer and non-cancerous cell lines were treated with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase.Results:Dichloroacetate (20 mM) did not reduce growth of non-cancerous cells but caused significant decrease in cancer cell proliferation (P=0.009), which was associated with apoptosis and G2 phase cell-cycle arrest. The largest apoptotic effect was evident in metastatic LoVo cells, in which DCA induced up to a ten-fold increase in apoptotic cell counts after 48 h. The most striking G2 arrest was evident in well-differentiated HT29 cells, in which DCA caused an eight-fold increase in cells in G2 phase after 48 h. Dichloroacetate reduced lactate levels in growth media and induced dephosphorylation of E1α subunit of pyruvate dehydrogenase complex in all cell lines, but the intrinsic mitochondrial membrane potential was reduced in only cancer cells (P=0.04).Conclusions:Pyruvate dehydrogenase kinase inhibition attenuates glycolysis and facilitates mitochondrial oxidative phosphorylation, leading to reduced growth of colorectal cancer cells but not of non-cancerous cells.

Growth factor release following femoral nailing

The aim of this study was to investigate whether growth factors essential for fracture healing are substantially increased in the immediate aftermath following reaming of the intramedullary cavity for stabilisation of femoral shaft fractures. Consecutive adult patients whose femoral shaft fractures stabilised with either reamed (10 patients) or unreamed (10 patients) intramedullary nailing were studied. Peripheral blood samples and samples from the femoral canal before and after reaming and nail insertion were collected. Serum was extracted and using Elisa colorimetric assays the concentration of Platelet Derived Growth Factor-BetaBeta (PDGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-I (IGF-I), Transforming Growth Factor beta 1 (TGF-beta1) and Bone Morphogenetic Protein-2 (BMP-2) was measured. The mean age of the twenty patients who participated in the study was 38 years (range 20-63). Reaming substantially increased all studied growth factors (p<0.05) locally in the femoral canal. VEGF and PDGF were increased after reaming by 111.2% and 115.6% respectively. IGF-I was increased by 31.5% and TGF-beta1 was increased by 54.2%. In the unreamed group the levels of PDGF-BB, VEGF, TGF-beta1 remained unchanged while the levels of IGF-I decreased by 10%. The levels of these mediators in the peripheral circulation were not altered irrespectively of the nail insertion technique used. BMP-2 levels during all time points were below the detection limit of the immunoassay. This study indicates that reaming of the intramedullary cavity is associated with increased liberation of growth factors. The osteogenic effect of reaming could be secondary not only to grafting debris but also to the increased liberation of these molecules.

Activated mesothelial cells produce heparin-binding growth factors: implications for tumour metastases.

Curative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF121and VEGF165. Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bgc) IL-2 receptor. Cytokine-induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum.

Carcinoembryonic antigen is the preferred biomarker for in vivo colorectal cancer targeting

Background:Colorectal cancer-specific biomarkers have been used as molecular targets for fluorescent intra-operative imaging, targeted PET/MRI, and selective cytotoxic drug delivery yet the selection of biomarkers used is rarely evidence-based. We evaluated sensitivities and specificites of four of the most commonly used markers: carcinoembryonic antigen (CEA), tumour-associated glycoprotein-72 (TAG-72), folate receptor-α (FRα) and Epithelial growth factor receptor (EGFR).Methods:Marker expression was evaluated semi-quantitatively in matched mucosal and colorectal cancer tissues from 280 patients using immunohistochemistry (scores of 0–15). Matched positive and negative lymph nodes from 18 patients were also examined.Results:Markers were more highly expressed in tumour tissue than in matched normal tissue in 98.8%, 79.0%, 37.1% and 32.8% of cases for CEA, TAG-72, FRα and EGFR, respectively. Carcinoembryonic antigen showed the greatest differential expression, with tumours scoring a mean of 10.8 points higher than normal tissues (95% CI 10.31–11.21, P<0.001). Similarly, CEA showed the greatest differential expression between positive and negative lymph nodes. Receiver operating characteristic analyses showed CEA to have the best sensitivity (93.7%) and specificity (96.1%) for colorectal cancer detection.Conclusion:Carcinoembryonic antigen has the greatest potential to allow highly specific tumour imaging and drug delivery; future translational research should aim to exploit this.

The omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits mouse MC-26 colorectal cancer cell liver metastasis via inhibition of PGE2-dependent cell motility

The omega‐3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti‐CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E‐type PG synthesis.

Targeting glucose metabolism: an emerging concept for anticancer therapy.

Mortality from locally advanced and metastatic cancer remains high despite advances in our understanding of the molecular basis of the disease and improved adjuvant therapies. Recently, there has been an increased interest in cancer metabolomics, and in particular, the potential for targeting glucose metabolism, for therapeutic gain. This interest stems from the fact that cancer cells metabolize glucose very differently from normal cells. Cancer cells preferentially switch to aerobic glycolysis rather than oxidative phosphorylation as their means of glucose metabolism. This metabolic switch is believed to enhance cancer cell survival. Several therapeutic agents that target tumor metabolism have shown significant cancer cell cytotoxicity in preclinical studies, and some have progressed to clinical trials. In this review, we discuss the alteration of carbohydrate metabolism seen in cancer cells, the underlying mechanisms, and opportunities for targeting cancer metabolism for therapeutic purposes.

Increased colorectal epithelial cell proliferation and crypt fission associated with obesity and roux-en-Y gastric bypass.

Background and Aims: The relationship between obesity, weight reduction, and future risk of colorectal cancer is not well understood. Therefore, we compared mucosal biomarkers in normal weight individuals [body mass index (BMI), 18.5-24.9 kg/m2] with those in morbidly obese patients (BMI >40 kg/m2) before and 6 months after Roux-en-Y gastric bypass (RYGB). Methods: Rectal epithelial cell mitosis, crypt area, and crypt branching were measured following whole crypt microdissection. Apoptosis was measured by immunohistochemistry for neo-cytokeratin 18 on fixed tissue sections. Serum levels of C-reactive protein and cytokines were assayed in combination with quantification of mucosal proinflammatory gene expression by real-time RT-PCR. Results: Twenty-six morbidly obese patients (mean BMI, 54.4 kg/m2) had significantly increased mitosis, crypt area, and crypt branching (all P < 0.01) compared with 21 age- and sex-matched normal weight individuals (mean BMI, 22.5 kg/m2). Morbidly obese patients underwent a mean excess weight loss of 41.7% at a mean of 26 weeks after RYGB. Surprisingly, this was associated with a further increase in mitosis and decreased apoptosis of epithelial cells. At the same time, lower levels of serum C-reactive protein and interleukin-6 following RYGB were accompanied by a reduction in mucosal IL-6 protein content but elevated mucosal expression of other proinflammatory genes such as cyclooxygenase-1 and cyclooxygenase-2. Conclusions: Mucosal biomarkers, accepted as indicators of future colorectal cancer risk, are increased in morbidly obese patients compared with normal weight controls. The hyperproliferative state that exists 6 months after RYGB may have important implications for long-term colorectal cancer risk in bariatric surgery patients. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1401–10)

Neoadjuvant Chemotherapy Induces Expression Levels of Breast Cancer Resistance Protein That Predict Disease-Free Survival in Breast Cancer

Three main xenobiotic efflux pumps have been implicated in modulating breast cancer chemotherapy responses. These are P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1), and Breast Cancer Resistance Protein (BCRP). We investigated expression of these proteins in breast cancers before and after neoadjuvant chemotherapy (NAC) to determine whether their levels define response to NAC or subsequent survival. Formalin-fixed paraffin-embedded tissues were collected representing matched pairs of core biopsy (pre-NAC) and surgical specimen (post-NAC) from 45 patients with invasive ductal carcinomas. NAC regimes were anthracyclines +/− taxanes. Immunohistochemistry was performed for Pgp, MRP1 and BCRP and expression was quantified objectively using computer-aided scoring. Pgp and MRP1 were significantly up-regulated after exposure to NAC (Wilcoxon signed-rank p = 0.0024 and p<0.0001), while BCRP showed more variation in response to NAC, with frequent up- (59% of cases) and down-regulation (41%) contributing to a lack of significant difference overall. Pre-NAC expression of all markers, and post-NAC expression of Pgp and MRP1 did not correlate with NAC response or with disease-free survival (DFS). Post-NAC expression of BCRP did not correlate with NAC response, but correlated significantly with DFS (Log rank p = 0.007), with longer DFS in patients with low post-NAC BCRP expression. In multivariate Cox regression analyses, post-NAC BCRP expression levels proved to predict DFS independently of standard prognostic factors, with high expression associated with a hazard ratio of 4.04 (95% confidence interval 1.3–12.2; p = 0.013). We conclude that NAC-induced expression levels of BCRP predict survival after NAC for breast cancer, while Pgp and MRP1 expression have little predictive value.

The systemic inflammatory response following femoral canal reaming using the reamer-irrigator-aspirator (RIA) device

We evaluated the peripheral release of inflammatory mediators after femoral fracture and subsequent intramedullary reaming using the RIA reamers. IL-6 was elevated after trauma, and reaming with RIA induced a measurable second hit response. However, despite a higher ISS, the levels of IL-6 in the RIA group were similar to the levels measured in a group of patients where reaming of the femoral canal was performed using conventional reamers. There was one death related to fat embolism syndrome in the conventional reamers group. However, the overall incidence of complications was low and similar between the 2 groups of studied patients. In polytrauma patients, large scale studies are desirable to evaluate further the immuno-inflammatory response using the RIA reamers prior to the instrumentation of the femoral canal.

Expression of cyclin D2 is an independent predictor of the development of hepatic metastasis in colorectal cancer

Introduction  Cyclin D1 has been implicated in the progression of several cancers by virtue of its influence on progression of the G1/S phase of the cell cycle. However, little is known about the possible roles of cyclin D2 and D3 in colorectal cancers (CRCs).