Lung Cancer Attracts Greater Stigma than Other Cancer Types in Aotearoa New Zealand.

Background: Lung cancer is the leading cause of cancer death in Aotearoa New Zealand, killing over 1,700 people each year. Despite the burden of lung cancer in Aotearoa New Zealand, the popular press has referred to it as the cancer type that no one talks about. Here, we investigate one factor that may contribute to this state of affairs: lung cancer stigma. Methods: Participants were university students and members of the general public. University students were recruited via an online experiment participation system in 2021. Members of the public were recruited via social media. All participants completed the Cancer Stigma Scale (CSS) for one of five cancer types (lung, cervical, breast, skin, or bowel). The CSS is a 25-item scale with six subscales: awkwardness, avoidance, severity, policy opposition, personal responsibility, and financial discrimination. Results: The mean age of participants was 24.3 (Standard Deviation = 10.4). Data from each subscale were submitted to an analysis of covariance (ANCOVA), with cancer type as a between-participant factor (5: lung, cervical, breast, skin, or bowel) and stigma as the dependent variable. Relative to most other cancer types, people were more likely to avoid someone with lung cancer, view interacting with someone with lung cancer as more awkward, and view people with lung cancer as being responsible for their condition. Conclusion: The Health Research Council of New Zealand recently funded the very first trial of lung cancer screening in Aotearoa New Zealand. The current study suggests that addressing stigma will be essential for the success of such programs, with stigma likely influencing those who engage in such trials.

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.

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.

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.

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.

The Rationale for Repurposing Sildenafil for Lung Cancer Treatment.

There is now a considerable body of evidence for sildenafil possessing anticancer properties. In this article, we argue the case for testing sildenafil as a lung cancer therapy chemoadjuvant. Currently, lung cancer is a disease with insufficient treatment options, with only 20% of patients responding to systemic chemotherapy, and even incremental potential improvements should be explored. We review the literature concerning the biochemical, physiological and metabolic effects on cancer cells by sildenafil alone, and when combined with chemotherapeutic agents. Most studies have shown that sildenafil is cytotoxic to cancer cells, both as a monotherapy and as a chemoadjuvant. Sildenafil enhances cancer cell apoptosis when used as a chemoadjuvant both in vitro and in vivo. In particular, in rodent experiments sildenafil has decreased tumour size compared to chemotherapy alone. Sildenafil has also been proven as an agent to decrease drug-efflux by cancer cells and increases blood perfusion to lung tissue, which can potentially increase the dosage of chemotherapeutic agents delivered to lung cancer cells compared to healthy tissue. In addition, the proven clinical effects of sildenafil on other lung diseases suggest that it could improve other patient outcomes, such as right ventricular function and quality of life. Sildenafil may also extend the half-life of docetaxel and some small molecule inhibitors used in lung cancer treatment by acting as an inhibitor of CYP3A4. We conclude that the evidence strongly warrants clinical investigation into the use of sildenafil as an agent for the treatment of lung-cancer.

ALK and IGF-1R as independent targets in crizotinib resistant lung cancer.

ALK positive non-small cell lung cancer is highly responsive to ALK inhibitors such as crizotinib, but drug resistance typically develops within a year of treatment. In this study we investigated whether IGF-1R is an independent druggable target in ALK-positive lung cancer cells. We confirmed that combination ALK and IGF-1R inhibitor treatment is synergistically cytotoxic to ALK-positive lung cancer cells and that this remains the case for at least 12 days after initial exposure to crizotinib. ALK-positive cells with acquired resistance to crizotinib did not acquire cross-resistance to IGF-1R inhibition, though combination treatment in the resistant cells gave additive rather than synergistic cytotoxicity. We concluded that IGF-1R is an independent druggable target in ALK-positive lung cancer and support the trial of combination treatment.

The atypical cannabinoid O-1602 increases hind paw sensitisation in the chronic constriction injury model of neuropathic pain.

O-1602 is an atypical cannabinoid that acts as an agonist at GPR55, a g protein-coupled receptor that previous studies have indicated may have a pronociceptive role in neuropathic pain. We administered O-1602 to both naive rats and rats that had undergone chronic constriction injury surgery. O-1602 did not cause any changes in hind paw responses to Von Frey hair testing in naive rats. However, O-1602 reversed the desensitising effects of ETOH, which was used as an active and opposing vehicle. Our results are consistent with the hypothesis that GPR55 has a pronociceptive role in neuropathic pain.

Neuroprotective effect of hydroxypropyl-ß-cyclodextrin in hypoxia-ischemia.

Neonatal cerebral ischemic injury is a common and debilitating pathology for which there is currently no known purely pharmacological treatments that are effective when delivered immediately after injury. Cyclodextrins are cyclic oligosaccharides that can remove cholesterol from cell membranes and thereby affect receptor function. Cyclodextrins have previously been shown to be neuroprotective in vitro. We showed that hydroxypropyl-ß-cyclodextrin is neuroprotective in rats in vivo when delivered by intraperitoneal injection 30 min following hypoxia-ischemia, when assessed 15 days after surgery. A single dose of 1 g/kg hydroxypropyl-ß-cyclodextrin reduced brain infarction size by 28.57% compared with control (P<0.001). We also report that the same compound reduces neuronal excitability in hippocampal slices and propose that hydroxypropyl-ß-cyclodextrin is neuroprotective by reducing excitotoxicity in the delayed phase of brain damage.

Characterization of a rat hypoxia-ischemia model where duration of hypoxia is determined by seizure activity.

Perinatal and early childhood asphyxia is common, debilitating and has few efficacious treatments. A hypoxia ischemia (HI) rat model that involves a unilateral ligation of the common carotid artery followed by a 60 min period of 8% oxygen hypoxia is often used to test proposed treatments. However, this HI protocol produces inconsistent infarction volumes due to the variability of individual rats to compensate for the ligated artery and hypoxia. Therefore, this HI model is problematic for experiments that prevent measurement of infarction volume, such as those that require analysis of homogenised brain tissue. We therefore aimed to find a simple and non-invasive predictor of infarction volume. Observations made prior, during and following HI in p26 rats showed that weight change 24 h following surgery was a strong predictor of infarction volume. The occurrence of a tonic clonic seizure during hypoxia was highly correlated with success of inducing an infarction, and for this reason we assessed whether ceasing the hypoxia for each rat following a tonic clonic seizure would produce a more consistent infarction volume. Using this procedure, infarction volumes measured at 3 and 15 days after surgery were significantly less variable, resulting in considerable improvements in statistical power compared with the original model.

Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain.

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.

Characterization of mitochondrial respiratory chain energetics in the vestibular nucleus complex.

CONCLUSIONS: Despite having very high neuronal firing rates, the VNC does not have unusually high mitochondrial activity in vitro. This study is the first in which functionally active mitochondria from the hindbrain have been isolated and characterized. OBJECTIVE: Neurons in the vestibular nucleus complex (VNC) have exceptionally high spontaneous firing rates. Neuronal mitochondria generate adenosine triphosphate critical for maintaining the membrane potentials required for axon firing. We therefore hypothesized a high rate of mitochondrial activity in the VNC. MATERIAL AND METHODS: To test this hypothesis, we compared mitochondrial activity in the VNC with mitochondrial activity from another area of the hindbrain, the cerebellum. Mitochondrial respiratory activity was assessed by measuring oxidative phosphorylation and mitochondrial respiratory enzyme complex activity. RESULTS: Assay results were not significantly different in the VNC compared to those obtained with the cerebellum or with rat brain mitochondria in previous studies.

Cannabinoid CB1 receptor protein expression in the rat choroid plexus: a possible involvement of cannabinoids in the regulation of cerebrospinal fluid.

Cannabinoid CB1 receptors in the brain are expressed on axon terminals presynaptic to neurons that express fatty acid amide hydrolase (FAAH). Postsynaptic FAAH catabolizes endocannabinoids which act as short-range transmitters. It has been previously shown that FAAH is also expressed in the epithelial cells of the choroid plexus. Using immunohistochemisty, we found that CB1 receptor protein is also expressed in choroid plexus epithelia. This is consistent with the hypothesis that FAAH in choroid plexus epithelial cells catabolizes endocannabinoids close to their site of action. Cannabinoids may then act directly on choroid plexus cells, and thereby contribute to the regulation of the composition of the CSF.

Effect of low body temperature during unilateral labyrinthectomy on vestibular compensation in the guinea pig.

OBJECTIVES: Vestibular compensation, the recovery that follows unilateral vestibular deafferentation (UVD), is a model for central nervous system plasticity. Recovery from the static symptoms of UVD may involve temperature-dependent processes that modulate the immediate effects of UVD and/or the capability of the central nervous system to undergo adaptive plasticity. In this study we investigated changes in oculomotor and postural vestibular symptoms resulting from low body temperature during UVD. MATERIAL AND METHODS: To study the effect of low temperatures at the time of UVD on vestibular compensation, we compared the rate of compensation and peak values for postural [roll head tilt (RHT) and yaw head tilt (YHT)] and oculomotor [spontaneous nystagmus (SN)] symptoms in three groups of guinea pigs. Animals in Group 1 (n = 6) were maintained at 38 degrees C throughout unilateral labyrinthectomy (UL). Animals in Group 2 (n = 6) were not temperature-controlled and animals in Group 3 (n = 4) were cooled with ice to 25 degrees C throughout UL. RESULTS: Cooled animals showed significantly higher rates of SN upon recovery from anaesthesia and took a significantly longer time to compensate. Cooled animals were also slower to compensate for postural symptoms (RHT and YHT), with 2 animals showing no compensation for RHT 52 h after UL. CONCLUSION: Hypothermia (25 degrees C) during UVD surgery exacerbates postural and oculomotor symptoms following UL and significantly slows recovery.