Experimental Determination of Cancer Drug Targets with Independent Mechanisms of Resistance.

Mathematical modelling of tumour mutation dynamics has suggested that cancer drug targets that have different resistance mechanisms should be good candidates for combination treatment. This is because the development of mutations that cause resistance to all drugs at once should arise relatively infrequently. However, it is difficult to identify drug targets fulfilling this requirement for particular cancers. Here we present four experimental criteria that we argue are necessary (but not sufficient) conditions that drug combinations should meet in order to be considered for combination drug treatment aimed at delaying or overcoming cancer drug resistance. We present the results of our own experiments - guided by these criteria - using anaplastic lymphoma kinase mutated lung cancer cells. Each set of experiments demonstrate results for different drug combinations. We conclude that the combination of ALK and MEK inhibitors come closest to meeting all our criteria.

The cardioprotective actions of statins in targeting mitochondrial dysfunction associated with myocardial ischaemia-reperfusion injury.

During cardiac reperfusion after myocardial infarction, the heart is subjected to cascading cycles of ischaemia reperfusion injury (IRI). Patients presenting with this injury succumb to myocardial dysfunction resulting in myocardial cell death, which contributes to morbidity and mortality. New targeted therapies are required if the myocardium is to be protected from this injury and improve patient outcomes. Extensive research into the role of mitochondria during ischaemia and reperfusion has unveiled one of the most important sites contributing towards this injury; specifically, the opening of the mitochondrial permeability transition pore. The opening of this pore occurs during reperfusion and results in mitochondria swelling and dysfunction, promoting apoptotic cell death. Activation of mitochondrial ATP-sensitive potassium channels (mitoKATP) channels, uncoupling proteins, and inhibition of glycogen synthase kinase-3ß (GSK3ß) phosphorylation have been identified to delay mitochondrial permeability transition pore opening and reduce reactive oxygen species formation, thereby decreasing infarct size. Statins have recently been identified to provide a direct cardioprotective effect on these specific mitochondrial components, all of which reduce the severity of myocardial IRI, promoting the ability of statins to be a considerate preconditioning agent. This review will outline what has currently been shown in regard to statins cardioprotective effects on mitochondria during myocardial IRI.

Considerations for Whole-Slide Analysis of Murine Xenografts Experiments.

Histochemistry of tumor sections is a widely employed technique utilized to examine cell death in preclinical xenograft animal models of cancer. However, this is under the assumption that tumors are homogeneous, leading to practices such as automatic cell counting across the entire section. We have noted that in our experiments the core of the tumor is largely or partially necrotic, and lacks evidence of vascularization (in contrast to the outer areas of the tumor). We note that this can bias and confound immunohistochemical analyses that do not take care to sample areas of interest in a way to take this into account. Design-based stereology with image analysis techniques is an alternative process that could be used to measure the volume of the necrotic region compared to the volume of the whole tumor.

Inflammasomes and Colorectal Cancer.

Inflammasomes are important intracellular multiprotein signaling complexes that modulate the activation of caspase-1 and induce levels of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 in response to pathogenic microorganisms and molecules that originated from host proteins. Inflammasomes play contradictory roles in the development of inflammation-induced cancers. Based on several findings, inflammasomes can initiate and promote carcinogenesis. On the contrary, inflammasomes also exhibit anticancer effects by triggering pyroptosis and immunoregulatory functions. Herein, we review extant studies delving into different functions of inflammasomes in colorectal cancer development.

Curcumin: A small molecule with big functionality against amyloid aggregation in neurodegenerative diseases and type 2 diabetes.

Amyloidosis is a concept that implicates disorders and complications that are due to abnormal protein accumulation in different cells and tissues. Protein aggregation-associated diseases are classified according to the type of aggregates and deposition sites, such as neurodegenerative disorders and type 2 diabetes mellitus. Polyphenolic phytochemicals such as curcumin and its derivatives have anti-amyloid effects both in vitro and in animal models; however, the underlying mechanisms are not understood. In this review, we summarized possible mechanisms by which curcumin could interfere with self-assembly processes and reduce amyloid aggregation in amyloidosis. Furthermore, we discuss clinical trials in which curcumin is used as a therapeutic agent for the treatment of diseases linking to protein aggregates.

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.

Curcumin: an inflammasome silencer.

Curcumin is the major bioactive polyphenolic ingredient of turmeric. Increasing evidence indicates that the health benefits of curcumin are mediated through its anti-inflammatory and antioxidant effects. Inflammasomes are essential components of inflammatory pathways that activate caspase-1 leading to pyroptosis and stimulate maturation and secretion of the proinflammatory cytokines, interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) through nuclear factor kappa-B (NF-κB) signaling. The current review outlines the mechanisms of curcumin as an inflammasome modulator in inflammatory-related diseases. Regulation of NF-κB signaling and interleukins secretion is the most prominent functional mechanism of curcumin in modulating inflammasomes. More importantly, curcumin can exert its anti-inflammatory role mainly through the down-regulation of NLRP3 inflammasomes. Given the fundamental role of inflammation in diseases, such as arthritis, cancer and cardiorenal disease, curcumin may have a pivotal therapeutic role through its ability to produce beneficial anti-inflammatory effects.

Does Crizotinib Auto-Inhibit CYP3A in vivo?

Crizotinib is a tyrosine kinase inhibitor used to treat anaplastic lymphoma kinase-positive lung cancer. There is in vitro evidence that crizotinib may auto-inhibit cytochrome P450 3A (CYP3A) activity, with important implications for crizotinib pharmacokinetics. In order to test whether crizotinib treatment alters CYP3A activity in vivo, mice were treated with 5 and 25 mg/kg crizotinib (p.o.) daily for 14 days. Results showed that crizotinib treatment did not alter CYP3A activity as determined by erythromycin N-demethylation. In addition, CYP3A polypeptide expression as measured by Western blot was unchanged. Therefore, our results do not support CYP3A inhibition by crizotinib in vivo.

Cytotoxicity of curcumin derivatives in ALK positive non-small cell lung cancer.

Non-small cell lung cancer with ALK rearrangements can be targeted effectively with ALK inhibitors such as crizotinib. However, cancer progression typically occurs within a year as drug resistance develops. One strategy to overcome this drug resistance is to determine if novel cytotoxic agents retain the ability to kill lung cancer cells that have developed ALK inhibitor resistance. We therefore examined curcumin, a drug with anticancer properties, and 2 s-generation curcumin derivatives (1-methyl-3,5-bis[(E)-4-pyridyl) methylidene]-4-piperidone (RL66) and 1-isopropyl-3,5-bis[(pyridine-3-yl) methylene]piperidin-4-one (RL118)) in lung cancer cell lines. The cytotoxicity of curcumin, RL66, and RL118 were tested in both ALK+ lung cancer cells (H3122), crizotinib resistant ALK+ cells (CR-H3122) and ALK- lung cancer cells (A549), both alone and in combination with crizotinib. ALK+ cells were 2-3x more sensitive to RL66 and RL118 than ALK- cells, with the drugs' eliciting IC50 values in the range of 0.7-1 µM in H3122 cells. Retained cytotoxic potency of the curcumin derivatives in crizotinib resistant cells indicated that mechanisms of resistance to the two drug types are independent, with resistance to ALK inhibitors not necessarily causing cross-resistance to curcumin derivatives. This was further corroborated by drug combination analysis where the effect of the drugs in combination was consistent with Bliss additivity, consistent with independent targets for crizotinib and curcumin derivatives. Results from Western blotting showed that RL118 (2 µM) inhibited p-ALK/ALK by ~50%, which was not as potent as the 90% inhibition elicited by crizotinib (0.25 µM). Since this is the primary mechanism of crizotinib cytotoxicity this provides further evidence of independent mechanisms of toxicity.

Does the mouse tail vein injection method provide a good model of lung cancer?

Lung cancer drug development requires screening in animal models. We aimed to develop orthotopic models of human non-small lung cancer using A549 and H3122 cells delivered by tail vein injection. This procedure has been used previously for a mouse lung cancer (Lewis lung carcinoma) and as a model of human breast cancer metastasis to lung. We report that the procedure led to poor animal condition 7-8 weeks after injection, and produced lesions in the lungs visible at necropsy but we were unable identify individual cancer cells using immunohistochemistry. We conclude that if this method is to produce a model that can be used in drug experiments, improvements are required for cancer cell detection post mortem, such as by using of a fluorescently tagged human lung cancer cell line.