Caterina Malcontenti-Wilson

Effect of ACE inhibitors and angiotensin II receptor antagonists in a mouse model of colorectal cancer liver metastases

Background and Aims:  Angiotensin converting enzyme inhibitors (ACE‐I) and angiotensin II type I receptor (AT1R) antagonists are commonly used as a treatment for hypertension. Recent experimental and population studies have suggested that these agents may exert an inhibitory effect on malignancy, possibly through anti‐angiogenic pathways. The aim of this study was to investigate the effect of an ACE‐I (captopril) and an AT1R antagonist (irbesartan) in colorectal cancer liver metastases.

Long-term survival of patients with unresectable colorectal liver metastases treated by percutaneous interstitial laser thermotherapy

In situ ablation of colorectal cancer (CRC) liver metastases is an accepted form of treatment for selected patients. It is associated with low morbidity and mortality and increases the number of patients who may benefit from therapy compared to resection alone. This study assesses the impact of interstitial laser thermotherapy (ILT) on local tumor control and long-term survival in patients with unresectable CRC liver metastases. Percutaneous ILT was performed in patients with unresectable CRC liver metastases between January 1992 and December 1999 using a bare-tip quartz fiber connected to an Nd:YAG laser source. This was prior to the routine use of a diffusing fiber for ablative therapy. Treatment was monitored with real-time ultrasonography. Tumors were considered unresectable based on their anatomic location or the extent of liver involvement. Patients with extrahepatic disease, more than five liver metastases, or tumors larger than 10 cm in diameter were excluded from this study. Local tumor control was assessed by dynamic computed tomography (CT) 6 months after therapy. Long-term follow-up was undertaken, and the impact of various factors on survival was analyzed. Eighty patients with a mean age of 63.8 years were suitable for ILT. In total, 168 liver tumors with a median diameter of 5 cm (range 1–10 cm) were so treated. There were no procedure-related deaths. The overall complication rate was 16%, with all cases managed conservatively. Bradycardia (n = 5), pneumothorax (n = 3), and persistent pyrexia (n = 3) were the most common complications. Complete tumor ablation was noted in 67% of patients assessed by CT 6 months following the initial therapy. Median follow-up was 35 months (range 4–96 months), with 10 patients alive at the end of this period. Altogether there were 67 deaths, which were related to hepatic disease in 55 cases and to extrahepatic disease in 9; they were unrelated to malignancy in 3 others. Three patients were excluded from follow-up after ILT down-staging of tumors that allowed complete surgical resection. The median disease-free survival of patients treated by ILT was 24.6 months, with a 5-year survival of 3.8%. Poor tumor differentiation and the presence of more than two hepatic metastases were associated with lower overall survival (p < 0.01). Fourteen patients treated by ILT for postoperative hepatic recurrences had the best outcome, with a median overall survival of 36.3 months and a 5-year survival of 17.2%. Percutaneous ILT is a minimally invasive, safe, effective technique that appears to improve overall survival in specific patients with unresectable CRC liver metastases, compared to the natural history of untreated disease reported in the literature.

In vitro and in vivo evaluation of tumor targeting styrene-maleic acid copolymer-pirarubicin micelles: Survival improvement and inhibition of liver metastases

Pirarubicin is a derivative of doxorubicin with improved intracellular uptake and reduced cardiotoxicity. We have prepared a micellar formulation of pirarubicin using styrene‐maleic acid copolymer (SMA) of mean molecular weight of 1.2 kDa, which exhibits a mean diameter of 248 nm in solution. Being a macromolecule, SMA‐pirarubicin micelles exhibit excellent tumor targeting capacity due to the enhanced permeability and retention (EPR) effect. Here we report the antitumor activity of SMA‐pirarubicin micelles on human colon and breast cancer cell lines in vitro, and a murine liver metastasis model in vivo. Metastatic tumor microvasculature, necrosis, apoptosis, proliferation, and survival were also investigated using immunohistochemistry for Ki‐67, active caspase‐3, and CD34, respectively. Drug cytotoxicity in vitro was assessed using MTT (3‐[4,5‐dimethyl‐2‐thiazolyl]‐2, 5‐diphenyl‐2H‐tetrazolium bromide) assay. In vivo, SMA‐pirarubicin was administered at 100, 150, or 200 mg/kg (pirarubicin equivalent). Tumor microvasculature was also assessed using scanning electron microscopy. Styrene‐maleic acid copolymer (SMA)‐pirarubicin micelles were toxic against human colorectal and breast cancer cells in vitro. IC50 was at or below 1 μM, free pirarubicin equivalent. In vivo, SMA‐pirarubicin at 100 mg/kg reduced tumor volume by 80% and achieved a survival rate of 93% at 40 days after tumor inoculation. Styrene‐maleic acid copolymer (SMA)‐pirarubicin micelles demonstrated potent antitumor activity in this liver metastases model, contributing to prolonged survival. Histological examination of tumor nodules showed significant reduction and proliferation of tumor cells (>90%). The present results suggest that investigation of the effect of multiple dosing at later time points to further improve survival is warranted. (Cancer Sci 2010)

P-21 activated kinase 1 knockdown inhibits β-catenin signalling and blocks colorectal cancer growth

The p21-activated kinase 1 (PAK1) plays important roles in cell growth, motility, and transformation. The aims of this study were to delineate the signalling mechanisms downstream of PAK1, and to investigate the importance of PAK1 for colorectal cancer (CRC) growth and metastasis in vivo. PAK1 knockdown in human CRC cell lines inhibited β-catenin expression, β-catenin/TCF4 transcriptional activity, and the expression of c-Myc. In mouse models PAK1 knockdown suppressed the growth and metastasis of human CRC cells by decreasing proliferation and increasing apoptosis. Our findings demonstrate for the first time the crucial role of PAK1 in CRC progression in vivo.

Focal Hyperthermia Produces Progressive Tumor Necrosis Independent of the Initial Thermal Effects

Focal hyperthermia, produced using laser, radio frequency, and microwave, is used to treat liver tumors. The exact mechanisms of tissue destruction using focal hyperthermia are, however, unknown. Clinical and experimental studies suggest a progression of injury after cessation of the initial heat stimulus. This study investigates the mechanisms and time sequence of progressive tissue necrosis induced using focal hyperthermia in a murine model of colorectal liver metastases. Focal hyperthermia produced using a neodymium-yttrium aluminum garnet (Nd-YAG) laser source was applied to the normal liver and colorectal cancer liver metastases in inbred male CBA strain mice. The extent of direct lethal thermal injury was assessed histochemically using vital stain for nicotinamide adenine dinucleotide (NADH) diaphorase immediately after laser application. Tissue injury at subsequent time points was assessed using both NADH diaphorase staining and routine histology to determine the temporal relationship between tissue necrosis and time. Thermal injury occurring immediately after the application of 100 joules of energy was greater in the tumor tissue than in the normal liver (mean [standard error of the mean (SEM)]), measuring 23.5 (3.4) and 16.3 (2.6) mm3, respectively (P = 0.046), despite similar tissue temperature profiles. There was a significant increase in tissue necrosis after initial injury that was greater in the normal liver than in the tumor tissue. In the normal liver, the peak volume of necrosis was 137.4 (9.8) mm3 and occurred at 3 days, whereas in the tumor tissue the peak was 49.0 (3.5) mm3 at 4.5 days (P < 0.001). Focal hyperthermia produces tissue necrosis that occurs in two phases. The first phase is caused by the direct lethal thermal injury followed by a second phase involving a progression of necrosis beyond the initial thermal effects. The normal liver and the tumor tissue responded differently to focal hyperthermia. In the tumor tissue, the direct injury is more pronounced, whereas the progression of injury is more rapid and extensive in the normal liver.

5-Aminosalicylic acid and olsalazine inhibit tumor growth in a rodent model of colorectal cancer

The ability of 5-aminosalicylic acid and olsalazine to inhibit colonic aberrant crypts and tumors was investigated in 1,2-dimethylhydrazine-treated rats. The effect of these drugs on the rates of tumor apoptosis and proliferation was studied as potential mechanisms for their action. 5-Aminosalicylic acid reduced the number of aberrant crypt foci by over one third, while olsalazine had no effect on this parameter. However, both agents effectively reduced tumor number and load, increased the rate of tumor apoptosis, and reduced the rate of tumor cell proliferation. In conclusion, 5-aminosalicylic acid and olsalazine are both ultimately effective chemopreventive agents in this model; however, only 5-aminosalicylic acid inhibited the formation of aberrant crypt foci. The inhibitory effect of these agents in tumors is related to the inhibition of proliferation and the induction of apoptosis.

Patterns of heat shock protein (HSP70) expression and Kupffer cell activity following thermal ablation of liver and colorectal liver metastases

The time course and extent of thermal ablative injury differs in liver compared to tumour tissue. This may be influenced by differences in the expression of heat shock proteins (HSP) and the response of Kupffer cells to thermal injury. This study determines the expression and response of HSP70 and Kupffer cells to thermal ablative injury in a Murine model of colorectal liver metastases. Thermal ablation by laser (Nd-YAG wavelength 1064 nm) was induced in liver and colorectal cancer liver metastases in CBA strain mice. Laser energy was applied at 2 W for 50 s and produced incomplete tumour ablation. Established tissue injury was assessed in separate groups of animals at time points ranging from 12 h to 21 days following therapy. HSP70 and Kupffer cell expression at the margins of coagulated tissue was determined by immunohistochemical staining for HSP70 and F4/80 antigens, respectively. HSP70 was faintly expressed in the cytoplasm of all tumour cells, with distinct clusters exhibiting intense cytoplasmic and nuclear HSP70 staining (130 ± 19 cells mm−2). Comparatively, HSP70 expression was uncommon in untreated control liver specimens (2 ± 2 cells mm−2, p < 0.001). Thermal ablation increased expression of HSP70 at coagulated tissue margins. The peak response in tumours occurred at 2 days post-ablation and was significantly greater than the peak response in liver, occurring at 12 h (809 ± 80 cells mm−2 vs. 454 ± 52 cells mm−2, p < 0.001). HSP70 expression remained significantly elevated for 7 days following therapy in tumour tissue, compared to 3 days in liver. Kupffer cell numbers in untreated control tumours were significantly lower than in untreated control livers (285 ± 23 cells mm−2 vs. 451 ± 30 cells mm−2, p < 0.001). Following thermal ablation, there was an initial decrease in Kupffer cell numbers at the margin of coagulation with subsequent persistent increases thereafter. In liver tissue, the peak Kupffer cell response occurred at 5 days post-therapy and was significantly greater than the peak response in tumour tissue 3 days post-thermal ablation (1074 ± 34 cells mm−2 vs. 860 ± 53 cells mm−2, p = 0.007). Thermal ablation produces a greater and more prolonged HSP70 response in colorectal liver metastases than in liver tissue. It also induces persistent increases in Kupffer cell activity in liver and tumour tissue.

A model of partial hepatectomy in mice.

A versatile, simple, and reproducible model of hepatectomy is essential for the study of liver regeneration and its effects on various pathological processes. A murine model of liver resection and regeneration suitable for research is described. Male inbred CBA mice 6–8 wk old were used in all experiments. The contribution of the hepatic lobes to the total liver mass was determined by wet weight measurements. Resection of 37% (n = 10) and 70% (n = 10) liver volume was performed using hemostatic clips to ligate the hepatic lobe pedicles. Animals were recovered and subsequently killed 21 days postoperatively Liver mass was determined and compared to control animals (n = 17) to assess the completeness of liver regeneration. There were no operative deaths in animals undergoing either 37% or 70% hepatectomy. The procedures could be performed expediently, and animal recovery was complete. Liver mass (grams) assessed 21 days postoperatively [mean (SE)] in both the 37% resection, 1.76 g (0.07), and 70% resection, 1.56 g (0.05), groups was not significantly different from control animals, 1.64 g (0.07) (p =. 265). Thus, partial hepatectomy can be performed safely and rapidly in mice using haemostatic clip ligation of hepatic lobes, with no impairment to the subsequent process of liver regeneration.

Evaluation of the effect of SMA-pirarubicin micelles on colorectal cancer liver metastases and of hyperbaric oxygen in CBA mice

Tetrahydropyranyladriamycin (THP or pirarubicin) destroys tumors via several mechanisms; one of which involves the production of ROS that requires molecular oxygen for its generation. SMA forms stable self-assembled associated micelles with pirarubicin (SMA–pirarubicin), and confers macromolecular characteristics to pirarubicin. This micellar macromolecular drug is selectively delivered to solid tumors via the EPR effect and its preferential tumor accumulation suppresses the systemic toxicity whilst its prolonged high concentration at the site of tumor enhances its efficacy much higher compared to free pirarubicin. Administration of SMA–pirarubicin micelle under HBO can further enhance the delivery of molecular oxygen that facilitates tumor selective generation of ROS, thus augmenting its antitumor potency. In this study, we evaluated the efficacy of SMA–pirarubicin micelles either as single drug or in combination with HBO in a mouse metastatic colorectal cancer model. At or below the maximum tolerated dose, SMA–pirarubicin remarkably reduced metastatic tumor nodules and it was far more effective than free pirarubicin. The data also suggests a potential benefit of combined therapy of HBO with micellar anthracyclins.

Spatial morphological and molecular differences within solid tumors may contribute to the failure of vascular disruptive agent treatments

BackgroundTreatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor destruction. However, a thin rim of viable cells persists in the tumor periphery following treatment, contributing to subsequent recurrence. This study investigates inherent differences in the microenvironment of the tumor periphery that contribute to treatment resistance.MethodsUsing a murine colorectal liver metastases model, spatial morphological and molecular differences within the periphery and the center of the tumor that may account for differences in resistance to OXi4503 treatment were investigated. H&E staining and immunostaining were used to examine vessel maturity and stability, hypoxia and HIF1α levels, accumulation of immune cells, expression of proangiogenic factors/receptors (VEGF, TGF-β, b-FGF, and AT1R) and expression of EMT markers (ZEB1, vimentin, E-cadherin and β-catenin) in the periphery and center of established tumors. The effects of OXi4503 on tumor vessels and cell kinetics were also investigated.ResultsSignificant differences were found between tumor periphery and central regions, including association of the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased evidence of EMT. OXi4503 treatment resulted in collapse of vessels in the tumor center; however vasculature in the periphery remained patent. Similarly, tumor apoptosis and proliferation were differentially modulated between centre and periphery after treatment.ConclusionsThe molecular and morphological differences between tumor periphery and center may account for the observed differential resistance to OXi4503 treatment and could provide targets for drug development to totally eliminate metastases.